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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 MT AD ............................................................................................... 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
3
more sensitive methods for microbiological investigations such as real-time quantitative
polymerase chain reaction (RTQ-PCR) which is based on the amplification and detection
of bacterial nucleic acids (8, 45). Another method to overcome the problem is the direct
enumeration of bacteria using epifluorescence microscopy. This method has recently
been introduced to the endodontic research field (34, 46, 47). Not only is the
epifluorescence microscopy technique more sensitive than the culture method in
quantifying bacteria, but it is also capable of distinguishing live and dead bacteria in a
sample ( 48-53).
The current study represents an attempt to investigate the efficacy of a novel irrigating
solution (MTAD) and 2% chlorhexidine gel as an intra canal medication in infected root
canals by enumerating the live and dead bacteria before and after the application of these
two medications. For the enumeration of bacteria in this study, both a traditional
anaerobic culture method based on colony forming units, and a direct enumeration of
bacteria based on epifluorescence microscopy were implemented. For the second method,
two bacterial staining methods, LIVE/DEAD® BacLight™ Bacterial Viability Kit
(Molecular Probes, Eugene, OR), and dihydroethidium (Molecular Probes, Eugene, OR)
used in combination with DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich,
Oakville, Canada) were selected.
4
2. REVIEW OF LITERATURE
2.1. Bacteria in Endodontic Disease
2.1.1. Microbial Flora in Infected Root Canals with Apical Periodontitis
Antonin van Leeuwenhoek in the 1 ih century made a note of his microscopic observation
of a severely decayed tooth and reported the presence of many living creatures inside the
hollow branches of the roots (54). Since then, scientific and technological advancements
have resulted in a better understanding of root canal infections. More than 700 different
bacterial species have been identified in the oral cavity (55), and they have the same
opportunity to invade the root canal system., A limited number of bacterial species,
however, have been detected in the necrotic root canal environment (29, 56, 57). The
microbial flora is shaped by the unique ecological factors in the necrotic root canal such
as anaerobic milieu, available nutrients, interaction between species and the presence of
host immunological system (58-62). As a result, the typical microbial flora in infected
root canals has been reported to be poly-microbial, consisting of predominately two to ten
species, dominated by Gram negative obligate anaerobes (12, 29, 57, 63-72). The total
number of bacterial cells residing in root canals is reported to be from 102 to 108 per
sample (2, 6, 26, 27, 32, 34, 59, 66, 73, 74). However, due to the limitations of sampling
and detection methods, these reported numbers may only represent a proportion of the
actual numbers of bacteria present in the root canal system.
2.1.2. Responses of Pulp and Periapical Tissues to Invasion of Bacteria
2.1.2.1. Invasion of Bacteria
5
Healthy pulp and periradicular tissues are bacteria-free, in contrast with the rest of the
oral cavity. Therefore, the presence of microorganisms in these areas is indicative of a
disease. For bacteria to reach and establish in the root canal system, they must
successfully breach hard and soft tissue barriers on and within the tooth (59).
Microorganisms present in dental caries are the main source of pulpal and periradicular
infection. The most common pathway of invasion for these bacteria is through the
dentinal tubules. There are several other ways that the microorganisms can get to the pulp
space, such as: through enamel and dentinal cracks, direct pulp exposure, direct
communication with the periodontal membrane through lateral and accessory canals (75-
77), and anachoresis (78). For bacteria to participate in the pathogenesis of periradicular
disease, they must establish in sufficient numbers in the root canal system, and express an
array of virulence factors. Moreover, the host reaction to bacteria is necessary at the
periradicular tissues to inhibit the spread of the infection, which results in tissue damage
(79).
2.1.2.2. Pulpal Response
Like any other soft tissue in the body, healthy pulp responds to irritants such as bacteria
and their by-products. It has been reported that cellular reactions can be detected in the
pulpal tissue as soon as caries affects more than a quarter of the total enamel width.
6
Progression of caries can trigger changes in the pulp-dentin complex, rangmg from
alterations in the functional aspects and the number of odontoblasts to intratubular and
tertiary dentin formation (80). Pulp responses to bacteria and their by-products can lead
to non-specific and specific immunological reactions. The non-specific inflammatory
response can be manifested by polymorphonuclear leukocyte (PMN), monocyte and
natural killer cell (NK) infiltrations (81-85). Dental pulp also has the capacity to respond
through an array of specific immunological reactions with more intense cellular
infiltrates, including T helper, T cytotoxic/suppressor cells, B cells, and plasma cells, as
well as by complement activation and vascular changes.
Pulp is surrounded by a hard tissue, and is therefore a unique low compliance
environment without collateral circulation (86); its immunological mechanisms usually
are not able to overcome infection. As the inflammatory process continues, the intra
pulpal pressure increases. This results in the impediment of the normal circulation and
function, which may lead to localized abscess formation, and if this persists, the pulp
becomes partially or completely necrotic (76, 82).
2.1.2.3. Periapical Response, Apical Periodontitis
Apical periodontitis is a localized inflammatory process in the periradicular tissues in
response to intra-radicular infection (1 ). It is an effective barrier against the spread of the
infection to the alveolar bone and to other body sites, (58, 79, 87). Localized periapical
bone destruction is a consequence of this immune response. It can even be apparent
before complete pulpal necrosis with vital pulp tissue still present in the apical portion of
7
the tooth (88, 89). This may imply that the periapical tissue destruction starts before
bacteria reach the area. It may be induced by indirect stimulation of host-derived
mediators as a result of bacterial by-products reaching the area rather than by direct
interaction of bacteria with osteoclasts or other cells in the periapical tissues (88, 89).
In response to infection, a consortium of inflammatory cells, mediating both the specific
and non-specific immune responses, infiltrates the periapical tissues. T lymphocytes and
macrophages are reported to be the predominant inflammatory cells (90-94). PMNs are
also present in large numbers (95). B lymphocytes, plasma cells, NK cells, eosinophils,
and mast cells can also be found (96-98). The host derived mediators including pro
inflammatory and regulatory cytokines, arachidonic acid metabolites, kinins and
neuropeptides also play an important role in the development of apical periodontitis
elicited by microorganisms (76, 99).
2.1.3. Etiological Role of Bacteria
In one of the first publications on the presence of bacteria in infected dental pulps, Miller
(59, 100) reported his microscopic observation that many microorganisms could be found
in the infected pulp space. He noted that some of those microorganisms were not
cultivable. He also reported that the flora was different in the apical, middle and coronal
portions of the dental pulp. In 1965, Kakehashi et al. (28)using an animal model, clearly
demonstrated that the presence of bacteria is an essential element in developing apical
periodontitis in exposed dental pulps of rats. Within their model, germ-free rats did not
develop apical periodontitis even though the pulps were exposed to the oral environment.
8
Later in 1976 Sundqvist (29) in his bacteriological studies of necrotic dental pulps in
humans observed that the necrotic pulps associated with apical periodontitis contained
bacteria. In contrast, dental pulps of teeth without apical periodontitis were not
contaminated with bacteria. These investigations were followed and expanded by Dahlen
1980 (31), Moller 1981 (101), Fabricius 1982 (24, 102), and several others who have
proven the etiological role of bacteria in the development and persistence of apical
periodontitis. As a result, bacterial reduction to the point of elimination from the root
canal systems is the primary goal to achieve successful healing of apical periodontitis.
2.1.4. Outcome Investigations
The ultimate outcome of endodontic treatment is the health of the periradicular tissues
(103). This means an absence of signs and symptoms in clinical, radiographic and
histological evaluations. Although a clear correlation has been demonstrated between
healing after endodontic treatment, and the presence of bacteria in the root canal before
root filling (11, 104-107), this correlation has not been universally confirmed (108). In
the study of Sjogren et al. (106), the complete healing rate was 94% for teeth with
negative cultures, compared to 68% for teeth with positive cultures. On the other hand in
the study by Peters et al. (108) the presence of positive cultures at the time of root filling
did not influence the outcome of the treatment. However, there is universal agreement
that elimination of bacteria is essential for successful healing (109).
Although still methodologically challenging, studies assessing the removal of microbial
load in the root canal system after different treatment protocols are shorter in duration
9
and require smaller populations than follow-up clinical outcome studies. Therefore, these
measurements might be considered a "surrogate outcome" of endodontic treatment,
which is correlated with the ultimate outcome of healing and retention of the treated tooth
(110).
2.1.5. Reduction of Bacteria and Disinfection of the Root Canal System
Elimination of endodontic infections is different from the elimination of infections from
most other parts of the body, because of the specific anatomical and physiological
characteristics of the root canals of teeth. Although host defense plays a major role in
localizing infection and later in the healing steps of apical periodontitis, it alone is not
able to eliminate the infection from root canals. Thus far, elimination of infection from
root canals is mainly achieved through the reduction of bacteria by debridement of the
root canal system utilizing mechanical preparation of the canals combined with chemical
interventions.
2.1.5.1. Mechanical Preparation
Optimum mechanical preparation of the root canals is challenging, that is why a
considerable amount of endodontic investigation has focused on the technical aspects of
the instrumentation of the root canal. The technical objectives of mechanical preparation
include removing the vital or necrotic pulp tissue and infected dentin from the canals and
creating an ideal shape for further cleansing and predictive placement of the intra-canal
medicaments or filling materials. Many instrumentation techniques for root canal
preparation have been introduced such as step-back (111, 112) and crown-down
10
techniques (113, 114). The latter technique has been reported to produce less debris
extrusion from the apical foramen than the former technique (115-117). Traditionally
stainless steel files and reamers were probably the most commonly used hand
instruments. Introduction of nickel-titanium alloy (118) and rotary engine-driven
instruments were an invaluable addition to the endodontic field. Several criteria have
been used for the assessment of the efficacy of the techniques such as the cleanliness of
the root canal walls and a reduction of the number of microorganism in the canals.
Clinical trials have shown that instrumentation alone appears to yield negative cultures in
only 17% to 53% of cases (32, 33, 73, 119) demonstrating the limited antibacterial
efficacy of the mechanical preparation.
It is reasonable to conclude that mechanical preparation alone is not able to reach and
clean all the confined areas of the root canal system such as isthmuses, fins and lateral
canals. Even in the main root canal space, it has been shown that 30-35% of root canal
walls remain untouched after completion of instrumentation (120). Moreover, bacteria
which have invaded the deep layers of the dentinal tubules are not accessible by
mechanical preparations. Therefore, the limited ability of mechanical preparation in the
reduction of bacteria is to be expected. Moreover, after mechanical preparation, the
instrumented canal walls are covered with a smear layer.
The smear layer is a layer of organic and inorganic substances composed of dentine,
remnants of pulp tissue and odontoblastic processes, and sometimes bacteria and their
11
byproducts (121, 122). It has been investigated using scanning electron microscopy and
described as a 1 to 2 µm thick superficial layer which could also be packed up to 40µm
into the dentinal tubules (123, 124). The removal of smear layer is an issue of great
debate, and to date there is no solid clinical evidence that its removal can favour a better
outcome for treatment. However, as reported by Bystrom et al. the combined use of
ethylene diamine tetra-acetic acid (EDTA), a chelating agent, and 5% sodium
hypochlorite was more efficient than sodium hypochlorite solution alone in the reduction
of bacteria (74). Thus, there is a general belief among endodontists that the removal of
the smear may help in the reduction of bacterial load from the root canal system which is
the primary objective of root canal treatment. For the purpose of removing the smear
layer, chelating agents such as EDTA or citric acid have been recommended in
conjunction with sodium hypochlorite (122-125).
2.1.5.2. Chemomechanical Preparation
To increase the efficacy of mechanical preparation in reducing the bacterial load,
instrumentation needs to be supplemented with active irrigating solutions.
The ideal endodontic irrigant should possess the following characteristics (126):
1. Be an effective germicide and fungicide.
2. Be non-irritating to the periapical tissues.
3. Remain stable in solution.
4. Have a prolonged antimicrobial effect.
5. Be active in the presence of blood, serum, and protein derivates of tissue.
12
6. Have low surface tension.
7. Should not interfere with repair of periapical tissues.
8. Not stain tooth structure.
9. Be capable of inactivation in a culture medium.
10. Should not induce a cell mediated immune response.
In addition, according to Torabinejad et al. (122) to effectively disinfect the root canal
system, an irrigant should be able to completely remove the smear layer, allow the
penetration of antimicrobial agents into the dentinal tubules and disinfect them, and
sustain its antibacterial effect after use without having any adverse effects on the physical
properties of the exposed dentin or on the sealing ability of filling materials. Furthermore,
an ideal irrigant should be non-antigenic, nontoxic, and non-carcinogenic to tissue cells
surrounding the tooth as well as be easily applicable and relatively inexpensive. An
irrigant should also dissolve organic and inorganic tissues, while lubricating the canal
during instrumentation (127).
Sodium hypochlorite (NaOCl) is the most commonly used irrigating solution. It is an
excellent antibacterial agent, able to dissolve necrotic and vital pulp tissue and the
organic components of dentin (37, 128, 129). Sodium Hypochlorite as a buffered 0.5%
solution was recommended for the irrigation of wounds during world war I by Dakin
(130), however, as a disinfectant it was recommended long before that time (110).
Coolidge introduced NaOCI to endodontics (131). As an endodontic irrigant NaOCl is
used in concentrations between 0.5 and 5.25% solutions. There has been controversy over
13
the use of different concentrations of sodium hypochlorite during root canal treatment.
Some in vitro studies have shown that NaOCl in higher concentrations is more effective
against E. faecalis and Candida albicans (132-134). In contrast, clinical studies have
shown both low and high concentrations to be equally effective in reducing bacteria from
the root canal system (74, 119). NaOCl in higher concentrations has a better tissue
dissolving ability (39), however, even in lower concentrations when used in high volumes
it can equally be effective (38, 135). Higher concentrations ofNaOCl are more toxic than
lower concentrations (136); however, due to the confined anatomy of the root canal
system, higher concentrations have successfully been used during root canal treatment,
with a low incidence of mishaps. Altogether, if lower concentrations are to be used for
intra-canal irrigation, it is recommended that the solution be used in higher volume and in
more frequent intervals to compensate for the limitations of low concentrations.
Instrumentation coupled with an antimicrobial irrigant, such as sodium hypochlorite
(NaOCl), has been shown to yield more negative cultures than instrumentation alone and
reach 37% to 63% of cases (2, 25, 26, 34, 36). However, even with the use of NaOCl,
removal of bacteria from the root canal systems following instrumentation remains an
elusive goal.
2.2.MTAD
MT AD as the first irrigating solution which is capable of removing the smear layer and
disinfecting the root canal system at the same time was introduced by a group from Loma
Linda University (137).
14
MTAD is a mixture of 3% doxycycline hyclate, 4.25% citric acid and 0.5% polysorbate
(Tween) 80 detergent (138). It has been commercialized as BioPure MTAD (BioPure,
Dentsply, Tulsa Dental, Tulsa, OK, U.S.A.) and is available as a two-part set, liquid in a
syringe and powder in a bottle, which should be mixed before application. MT AD has
been recommended in clinical practice as a final rinse after completion of a conventional
chemo-mechanical preparation (137, 139, 140).
2.2.1. Protocol for Use
The MTAD protocol was developed on the basis of a pilot project (141). The results of
this project showed that the consistent disinfection of the infected root canals could occur
after chemomechanical preparation using 1.3% NaOCl as a root canal irrigant and a 5
min exposure time to MT AD as a final rinse.
2.2.2. Properties of MT AD
Several in vitro studies have been conducted to evaluate the different characteristics of
MTAD (13 7, 139-166), but there is only one in vivo study (167) that evaluates the effect
ofMTAD on post operative discomfort in a randomized clinical trial.
2.2.2.1. Mechanism of Action
There is no detailed information on the exact mechanism of action of MT AD in the
removal of the smear layer and in the killing of bacteria. In most studies, its effect on the
smear layer is attributed to the existence of doxycycline and citric acid. These two
components of MTAD have been separately reported as effective smear layer removal
15
solutions (125, 168, 169). Its antibacterial effect is mostly attributed to the doxycycline
which is an isomer of tetracycline. Tetracycline, including tetracycline-HCl, minocycline,
and doxycycline, are broad-spectrum antibiotics that are effective against a wide range of
microorganisms. Tetracycline is a bacteriostatic antibiotic which exerts its effect through
the inhibition of protein synthesis. According to Torabinejad et al. (2003) this property
may be advantageous because in the absence of bacterial cell lysis, antigenic by-products
(i.e. endotoxin) are not released (137). In high concentrations, tetracycline may also have
a bactericidal effect (170, 171). The role of citric acid in killing bacteria is not well
known (156). Tween 80, the other component of MTAD, seems to have limited
antibacterial activity, yet it may increase the antibacterial effect of some substances (172)
by directly affecting the bacterial cell membrane (156). It may facilitate the penetration of
MTAD into dentine (137, 140). On the other hand, Tween 80 may also be used as a
nutrient by some bacteria (173), and it may inactivate the antibacterial properties of some
disinfecting agents such as chlorhexidine and povidone-iodine (174-176). Doxycycline,
citric acid, and Tween 80, together might have a synergistic effect on the bacterial cell
wall and on the cytoplasmic membrane (177).
2.2.2.2. Cytotoxicity of MT AD
Using a MMT -Tetrazolium method, Zhang et al. (166) compared the cytotoxicity of
MTAD to that of eugenol, 3% hydrogen peroxide, REDTA Aqueous Irrigant, Peridex
(CHX 0.12%), Pulpdent Ca(OH)2 paste, and four concentrations of NaOCl (5.25%,
2.63%, 1.31 %, and 0.66%). They concluded that MTAD appeared to be less cytotoxic
than eugenol, 3% H202, Ca(OH)2 paste, 5.25% NaOCl, Peridex, and EDTA and more
16
cytotoxic than 2.63%, 1.31 %, and 0.66% NaOCl solutions. The authors suggested further
investigation was needed to determine if the results from their in vitro study could be
applied to a clinical situation.
2.2.2.3. Surface Tension
According to Grossman (126), low surface tension is one of the ideal characteristics of an
irrigant. Lower surface tension may help in better penetration of the irrigating solutions
into the dentinal tubules and inaccessible areas of the root canal system (178). In order to
be more effective in debris removal and to penetrate more readily into the root canal
system, irrigants must be in contact with the dentin walls. The closeness of this contact is
directly related to its surface tension (149). Irrigants with a low surface tension are more
suitable as endodontic irrigants. To decrease the surface tension, Tween 80 has been
added to the MT AD solution. It is reported that MT AD has lower surface tension than
5.25% NaOCl, 17% EDTA and water (149). Although it seems that lowering surface
tension may help the penetration of the irrigants deeper into the dentinal tubules or other
confined areas of the root canal system, and consequently improve the antibacterial
effectiveness of the irrigants (13 7), there is no clinical evidence to support this
speculation (179).
2.2.2.4. Smear Layer Removal
Scanning electron microscopy has been used to determine the effectiveness of various
irrigants to remove the smear layer. The paper that introduced MT AD addressed its
potential in removal of the smear layer in extracted human teeth (137). The authors
17
reported that MT AD performed better than EDTA in cleaning dentinal tubules from
debris and smear layer in the apical third of root canals; however, there was no significant
difference in the middle and coronal portions of the root canals. In the same study, the
results also indicated that MT AD created less erosion than EDT A in the coronal and
middle thirds of the root canals. The better efficacy of MT AD in removal of the smear
layer was attributed to the combination of citric acid, doxycycline and Tween 80 in the
MT AD solution. In two other studies, the efficacy of MT AD or EDT A in the removal of
the smear layer was confirmed, however, no significant difference between these two
solutions was reported (161, 163).
2.2.2.5. Antibacterial Efficacy
Reported results regarding the antibacterial properties of MT AD are conflicting.
The studies measuring zones of inhibition on agar plates have shown consistently that
MTAD was an effective antibacterial agent against E. faecalis (139, 145, 152). Tay et al.
(160) also found larger zones of bacterial inhibition using dentin cores irrigated with
MTAD compared to NaOCl-irrigated dentin cores; however, when they applied MTAD
to the dentin that was already irrigated with 1.3 % NaOCl, they noticed a contradictory
result. The diameters of zones of inhibition were significantly smaller than those of
MTAD alone and while comparable to those irrigated with 1.3% NaOCl alone. They
concluded that the antimicrobial effect of MT AD was lost due to oxidation of the MT AD
byNaOCl.
18
A study using extracted human teeth contaminated with saliva showed that MT AD was
more effective than 5.25% NaOCl in disinfection of the teeth (140). In contrast, Krause et
al. (152) using bovine tooth sections showed that 5.25% NaOCl was more effective than
MT AD in disinfection of the dentin discs inoculated with E. faecalis.
In another study performed on extracted human teeth inoculated with E. faecalis, a
protocol of 1.3% NaOCl followed by 5 min MTAD was more effective in the
disinfection of canals than a protocol of 5 .25% N aOCl followed by 1 min 17% EDT A
and then 5 min 5.25% NaOCl as a final rinse (141).
Using a culture method and extracted human teeth inoculated with E. faecalis, the
opposite was found, i.e. the latter protocol was significantly superior to the 1.3% NaOCl/
5 min MTAD protocol in disinfection of the root canals (142). The same group in another
study using the same model, disinfected the canals with the same two protocols, and then
resected and pulverized the last 5 mm of the root ends in liquid nitrogen. After
inoculation of the samples on BHI agar culture plates, the results of the study indicated
that there was no significant difference in the antimicrobial efficacy of those two
protocols in disinfection of the apical 5 mm of the infected canals (151).
In a series of studies MT AD has failed to show a superior antibacterial efficacy against
bacterial biofilms.
19
Bacteria collected from the teeth of patients diagnosed with apical periodontitis were
grown as a biofilm on hemisections of root apices. MTAD was shown to be an effective
antibacterial agent in this model; however, it was not able to completely disrupt the
bacterial biofilm compared to 6% NaOCl (144).
NaOCl (5.25%) was the most effective irrigant against a biofilm of E. faecalis generated
on cellulose nitrate membrane filters, while the bacterial load reduction using MT AD was
not significant ( 150).
MTAD was the least effective irrigant when compared to 6% and 1 % NaOCl,
SmearClear™, 2% CHX, and REDTA, when tested in flow cell generated biofilms of E.
faecalis (146).
When efficacy of four irrigants including MT AD was tested in teeth inoculated with
Candida albicans, it was demonstrated that 6% NaOCl and 2% CHX were equally
effective and superior to MTAD and 17% EDTA (158).
According to Portenier et al. (2006), although the antibacterial effect of MT AD is
comparable to that of chlorhexidine, calcium hydroxide, iodine potassium iodide, and
sodium hypochlorite, the presence of dentin or bovine serum albumin causes a marked
reduction in the antibacterial efficacy of MTAD against E. faecalis (156, 177).
20
The results of testing the antibacterial efficacy of medicaments obtained from in vitro
studies should be analyzed with caution, as they may be influenced by factors such as the
test environment, bacterial susceptibility and the different methodologies used to evaluate
the results (180).
2.2.3. In vivo Clinical Trial
With the exception of the study (167) that evaluated the effect of MTAD on post
operative discomfort, there has been no other in vivo study to address the other
characteristics of MTAD. Based on the results of their study, the clinical protocol for the
removal of the smear layer and the disinfection of the root canal system using 1.3%
NaOCl and MTAD does not result in an increased incidence of postoperative pain
compared to biomechanical instrumentation using 5.25% NaOCl and 17% EDTA.
2.3. Intracanal Medication
When treatment cannot be completed in one appointment, the surviving bacteria often
proliferate in the time interval until the next treatment appointment (11, 36, 73, 74, 181,
182). To curtail bacterial regrowth, and possibly even improve bacterial suppression,
intracanal medication can be applied between appointments (5, 40, 182). Inter
appointment antimicrobial medication acts to (i) inhibit proliferation of surviving
bacteria, and (ii) further eliminate surviving bacteria and minimize ingress through
leaking restoration (2, 5, 40, 74). The most commonly used intracanal medicament is
calcium hydroxide (Ca (OH)z). It has been shown to exert a potent bactericidal effect on
most of the root canal bacteria (5). Most importantly, the effectiveness of Ca(OH)2 has
21
been confirmed when cleaning and shaping coupled with inter-appointment medication
with Ca(OH)z yielded negative cultures in 75% to 97% of teeth (2, 5, 26, 33).
2.3.1. Limitations of Calcium Hydroxide
There are, however, some concerns regarding the use of Ca(OH)2• Firstly, the handling
and proper placement of Ca(OH)2 present a challenge to the average clinician (183, 184).
Secondly, removal of Ca(OH)z is frequently incomplete, resulting in a residue covering
20% to 45% of the canal wall surfaces, even after copious irrigation with saline, NaOCl
or EDTA (185). Residual Ca(OH)z can shorten the setting time of zinc-oxide eugenol
based endodontic sealers (186). Most notably, it may interfere with the seal of the root
filling (187), and compromise the quality of treatment (185, 187). Thirdly, Ca(OH)2 is
not totally effective against several endodontic pathogens including E. faecalis (180) and
Candida albicans (188). Finally, Peters et al. demonstrated that after 4 weeks of
intracanal medication with Ca(OH)z, the number of bacteria and proportion of positive
cultures increased in the samples taken from root canals. This observation has been
confirmed recently by another clinical trial (11).
2.3.2. Chlorhexidine (CHX)
A possible alternative to Ca(OH)z as an intracanal medication is chlorhexidine gluconate
(CHX) (189).
22
2.3.2.1. History
CHX was developed more than 50 years ago at Imperial Chemical Industries in England,
and was first marketed in the United Kingdom in 1953 as an antiseptic cream (190).
Since 1957 it has been used for general disinfection purposes, also for the treatment of
skin, eye and throat infections in both humans and animals (191, 192).
2.3.2.2. Molecular Structure
CHX belongs to the polybiguanide antibacterial family, consisting of two symmetric 4-
chlorophenyl rings and two bisguanide groups connected by a central hexamethylene
chain. CHX is a strongly basic molecule and is stable as a salt. CHX digluconate salt is
easily soluble in water (191, 193).
2.3.2.3. Mode of Action
CHX is a wide-spectrum antimicrobial agent, active against Gram-positive and Gram
negative bacteria, and yeasts (194). Due to its cationic nature, CHX is capable of
electrostatically binding to the negatively charged surfaces of bacteria (195), damaging
the outer layers of the cell wall and rendering it permeable (196-198). Depending on its
concentration, CHX can have both bacteriostatic and bactericidal effects. At high
concentration CHX acts as a detergent, and by damaging the cell membrane (199) it
causes precipitation of the cytoplasm and thereby exerts a bactericidal effect (191, 193,
200). At low sub-lethal concentrations, chlorhexidine is bacteriostatic, causing low
molecular weight substances, i.e. potassium and phosphorous, to leak out of the cell
which is irreversibly damaged (193, 200). It also can affect bacterial metabolism in
23
several other ways such as abolishing the activity of the PTS sugar transport system and
inhibiting acid production in some bacteria (199).
2.3.2.4. Substantivity
Due to the cationic nature of the CHX molecule, it can be absorbed by anionic substrates
such as the oral mucosa (201, 202). Chlorhexidine has the ability to bind to proteins such
as albumin present in serum or saliva, pellicle found on the tooth surface, salivary
glycoproteins and mucous membranes (195, 203, 204). This reaction is reversible (205).
CHX can also be adsorbed onto hydroxyapatite and teeth (195, 203, 206, 207). Studies
have shown that the uptake of CHX onto teeth also is reversible (195, 207, 208). This
reversible reaction of uptake and release of CHX leads to long lasting antimicrobial
activity and is referred to as "substantivity" (191, 193, 209-214). This effect depends on
the concentration of CHX (207, 211, 213). At low concentrations of 0.005-0.01 %, a
stable monolayer of CHX is adsorbed and formed on the tooth surface, which might
change the physical and chemical properties of the surface and may prevent or reduce
bacterial colonization. At higher concentrations (>0.02% ), a multilayer of CHX is formed
on the surface providing a reservoir of chlorhexidine which can rapidly release the excess
into the environment (207) as the concentration of the CHX in the surrounding
environment decreases (192, 203, 208).
2.3.2.5. Cytotoxicity
In the medical field, chlorhexidine is normally used at concentrations between 0.12% and
2.0%. According to Loe, at these concentrations, CHX has a low level of tissue toxicity,
24
both locally and systemically (192). In another report, when 2% CHX was used as a
subgingival irrigant, no apparent toxicity was noted on gingival tissues (215, 216).
Moreover, CHX rinse was reported to promote the healing of periodontal wounds (217).
Based on these reports, Jeansonne et al. (1994) assumed that the periapical tissues would
be as tolerant to chlorhexidine as gingival tissues (212). In two studies when CHX and
NaOCl were injected into subcutaneous tissues of guinea pigs and rats, an inflammatory
reaction developed; however, the toxic reaction to CHX was less than that to NaOCl
(218, 219). Furthermore, when CHX was applied as a rinse in the extraction sites of the
third molars on the day of surgery and several days after, it was reported to reduce the
incidence of alveolar osteitis (220). In addition, there are only a few allergic and
anaphylactic reactions reported to CHX (221, 222).
Conversely, some studies have reported an unfavourable effects of CHX on the tissues.
Hidalgo demonstrated that CHX is cytotoxic to some lines of cultured human skin
fibroblasts (223). Recently, the behaviour of osteoblastic human alveolar bone cells in the
presence of CHX and povidone-iodine (PI) has been investigated. It has been reported
that CHX has a higher cytotoxicity profile than povidone-iodine (224). Faria et al. also
demonstrated that CHX injected in the hind paw of mice could induce severe toxic
reactions. In addition, they reported that CHX induced apoptosis at lower concentrations
and necrosis at higher concentrations when added to cultured L929 fibroblast cells (225).
Another interesting observation has been reported recently when CHX is in contact with
other agents such as NaOCl. The by-product of the reaction of CHX with NaOCl is the
25
formation of toxic breakdown products such as para-chloroaniline (PCA) that may have a
negative impact on tissues (226). The toxicity level of CHX on periapical tissues when
applied in the root canals needs to be further investigated.
2.3.2.6. Chlorhexidine Application in Dentistry
Chlorhexidine has several applications in dentistry (191, 227). It has been used for
prevention of dental caries, plaque formation and gingivitis (193, 215), especially in
elderly and senile patients, as well as those with handicapping conditions such as cerebral
palsy and patients with immunocompromising diseases (191). It has also been
recommended for prevention of alveolar osteitis after extraction of third molars (220).
Another application of CHX is in the treatment and management of periodontal diseases
(193), as well as in the reduction of the incidence, severity and duration of aphthous
ulceration (228). In addition, it has been advocated as a denture disinfectant in patients
susceptible to oral candidiasis (191).
CHX can be prepared in the form of mouthrinses, gels (229), varnishes (230), and
controlled-release devices (231 ).
2.3.2.7. Chlorhexidine Application in Endodontics
In endodontics, CHX has been studied as an irrigant and intracanal medication, both in
vivo (6, 34, 232, 233) and in vitro (180, 210, 211, 214, 234, 235). In vitro, CHX has at
least as good, or even better antimicrobial efficacy than Ca(OH)z (180, 210, 214, 234).
Notably, 2% CHX was very effective in eliminating a biofilm of E. faecalis (236). In
26
vivo, it inhibits experimentally-induced inflammatory external root resorption when
applied for 4 weeks (233). In infected root canals, it reduces bacteria as effectively as
Ca(OH)2 when applied for one week (6, 232). Unlike Ca(OH)2, CHX has substantive
antimicrobial activity (193, 210, 211, 213, 214) that, if imparted onto the root dentin, has
the potential to prevent bacterial colonization of root canal walls for prolonged periods of
time. This effect depends on the concentration of CHX (211, 213), but not on its mode of
application, which maybe either as liquid, gel or a controlled release device (210, 214).
2.3.2.8. Chlorhexidine as an Endodontic Irrigant
Chlorhexidine (CHX) in liquid and gel form has been recommended as an irrigant
solution, and its different properties have been tested in several studies, both in vitro
(132, 158, 209, 212, 213, 218, 237-257) and in vivo (8, 14, 15, 258-265).
Many investigations have been conducted to study the antibacterial effectiveness of CHX
in different concentrations. It has been demonstrated that 2% CHX as an irrigant has a
better antibacterial efficacy than 0.12% CHX in vitro. Thus, it is concluded that the
antibacterial efficacy (266) of CHX depends on its concentration (213, 219, 238, 241).
Another antibacterial property of CHX termed as substantivity was demonstrated to be
present up to 72h, in teeth treated with 2.0% chlorhexidine, and up to 24h in those treated
with 0.12% CHX (213). Moreover, in another study the antibacterial property was
retained in root canals up to 12 weeks when bovine teeth were treated for 10 min with 2%
CHX (266).
27
Since NaOCl is still the most commonly used irrigant, the antibacterial efficacy of CHX
is tested against that of NaOCl. The results from these studies are not conclusive, but in
general no significant difference between these two solutions has been reported (132,
212,218,237,238,240,244,245,252,255-257).
Unlike NaOCl, chlorhexidine lacks a tissue dissolving property (267). Therefore, NaOCl
is still considered the main irrigating solution.
The cleanliness of root canals by CHX in gel and liquid forms was evaluated using
scanning electron microscopy in two separate experiments. In an in vitro study, the canals
treated with 2% CHX gel were cleaner than those treated with 2% CHX liquid or 5.25%
NaOCl, and it was suggested that the mechanical action of the gel might have facilitated
the cleansing of the canals (250). Another in vitro study showed that the 2% CHX liquid
was inferior to 2.5% NaOCl in cleaning of the canals (268). However, in vitro studies
may not properly reflect the actual in vivo situations, which are more clinically relevant
(59).
The antibacterial effectiveness of CHX in the reduction of bacteria in infected root canals
has been investigated in several studies. Ringel et al. reported that 2.5% sodium
hypochlorite was significantly more effective than 0.2 % chlorhexidine when the infected
root canals were irrigated for 30 minutes by either of the solutions (258).
28
In a controlled and randomized clinical trial, the efficacy of 2% CHX liquid was tested
against saline using the culture technique. All the teeth were initially instrumented and
irrigated using 1 % sodium hypochlorite. Then, either 2% chlorhexidine liquid or saline
was applied as a final rinse. The authors reported a further reduction in the proportion of
positive cultures in the CHX group. Their results showed a better disinfection of the root
canals using chlorhexidine compared to saline as a final rinse (262).
In a recent study the efficacy of 2% CHX gel was tested against 2.5% NaOCl in teeth
with apical periodontitis and the bacterial load was assessed using real-time quantitative
polymerase chain reaction (RTQ-PCR) and colony forming units (CFU). The bacterial
reduction in the NaOCl group was significantly greater than the CHX-group when
measured by RTQ-PCR. Based on the culture technique, bacterial growth was detected in
50% of the CHX-group cases compared to 25% in the NaOCl group (8).
On the other hand, a more recent study also based on the culture technique revealed no
significant difference between the antibacterial efficacy of 2.5% NaOCl and 0.12% CHX
liquid when used as irrigants during the treatment of infected canals (14).
2.3.2.9. Chlorhexidine as an Intracanal Medication
Chlorhexidine (CHX) in liquid, gel, or in a controlled-release device has been suggested
as an alternative intra-canal medication to replace calcium hydroxide. This has been the
focus of many in vitro (180, 189, 210, 211, 214, 234-236, 269-284) and in vivo studies (6,
34, 232, 233, 285-288).
29
The results of in vitro experiments were mostly in favour of CHX regardless of its mode
of application (180, 236, 271, 280, 281, 283). The results have also demonstrated the
potential antibacterial substantivity of CHX in root canals (210, 211, 214, 234).
Haapasalo et al. developed an experimental model using dentin powder particles to
investigate the possible inactivation of some antibacterial medicaments with the dentine
(272). The medicaments tested were Ca(OH)2, 1 % NaOCl, 0.5% and 0.05% CHX acetate,
and different concentrations of iodine potassium iodide (IKI). They showed that dentine
powder had an inhibitory effect on all medicaments tested. The effect was dependent on
the concentration of the medicament and the contact time period. The effect of Ca(OH)z
was totally abolished by the presence of dentine powder. The effect of 0.05% CHX and
1 % NaOCl was reduced but not totally eliminated by the presence of dentine. No
inhibition could be measured when full strength solutions of CHX and IKI were used
(272, 275, 289).
Contradicting results have also been reported, when the effect of different intracanal
medications including CHX gel on the sealing of root canals was evaluated. In an in vitro
study using extracted human teeth, after 10 days of intracanal medication, all root canals
were filled and then tested for microbial leakage. The root canals medicated with CHX
gel demonstrated less resistance against bacterial leakage compared to the root canals
medicated with Ca(OH)z. (235). In contrast, Wuerch et al. (2004) did not find any
significant difference in leakage between the canals medicated with CHX or Ca(OH)2 and
30
reported that 2% chlorhexidine gel and calcium hydroxide paste did not adversely affect
the apical seal of the root-canal system (290).
The antibacterial efficacy of 0.2% CHX liquid applied for 24h was tested against a saline
solution in a human ex-vivo model. After extraction, the infected teeth were
endodontically treated using CHX or saline for irrigation and then further medicated with
either of the solutions. Samples were obtained and evaluated using the culture technique
at each treatment step. In both groups the numbers of bacteria were decreased after the
instrumentation and irrigation of the canals. However, after 24h medication it was
reported that the numbers of bacteria were further decreased in the CHX group, in
contrast to the saline group in which an increase was noted (189).
CHX may also have an effect on the reduction of inflammatory external root resorption
which is caused by infection. Lindskog et al. assessed the therapeutic effect of a four
week intra-canal application of chlorhexidine gel on inflammatory root resorption in
replanted infected teeth of monkeys. They reported that the extent of inflammatory
resorption was significantly reduced compared to non-medicated teeth, suggesting that
chlorhexidine may have been a useful adjunct in the treatment of inflammatory root
resorption (233).
The effects of 2% chlorhexidine gel and Ca(OH)2 paste on the regeneration of apical
periodontitis in rats have been studied. Apical periodontitis was induced by leaving the
root canals exposed to the oral cavity for 2 weeks. In the positive control group, the root
31
canals were not treated, but the coronal access openings were sealed. In the negative
control group, partial pulpotomies were performed and the coronal access openings were
sealed immediately. In a third control group the canals were instrumented, left empty, and
the coronal access openings were sealed. They reported that CHX and Ca(OHh were
equally effective and performed better than those of control groups. They suggested that
chlorhexidine gel could be a good alternative to calcium hydroxide as an intracanal
medication (286).
These in vitro and animal studies suggest that CHX has the potential to replace Ca(OHh
as an intracanal medicament; however, due to the limitations of the in vitro experiments
the conclusions obtained from these studies cannot be extrapolated to the actual clinical
situations. In vivo human experiments are, therefore, required in order to test the efficacy
of CHX as intracanal medication.
Compared to the number of in vitro studies, very few in vivo studies have been conducted
to assess the effectiveness of chlorhexidine as an intracanal medication. Barbosa et al.
(1997) assessed in vivo the antibacterial efficacy of three different intracanal medications:
camphorated paramonochlorophenol, calcium hydroxide and 0.12% CHX liquid by
applying them for one week in single-rooted teeth of patients. When using a culture
method it was reported that the proportions of positive cultures were not significantly
different among the tested medications; however, they were slightly lower in teeth
medicated with chlorhexidine (0.12%) liquid than those medicated with camphorated
paramonochlorophenol or calcium hydroxide (232).
32
Paquette et al. in an in vivo study evaluated the antibacterial effectiveness of 2% CHX
liquid as an intracanal medication in teeth with apical periodontitis (34). The results
showed a moderate increase in bacterial counts during a medication period of 7-14 days,
that was similar to that seen and reported for Ca(OH)2 by Peters et. al. (26). It was
speculated that the CHX liquid may have partially escaped from the apical foramen, and
that a gel form might have been better suited for intracanal medication than liquid (34).
Furthermore, Manzur et al. (2007) demonstrated that intracanal medication with
Ca(OH)2, 2% chlorhexidine gel, or a mixture of Ca(OH)2/CHX applied for 7 days did not
reduce the bacterial concentration beyond what was achieved after chemomechanical
preparation using 1 % N aOCl. The results were not significantly different among the
medication groups ( 6).
During the last few years researchers have studied the combination of calcium hydroxide
and chlorhexidine, with the idea that their antimicrobial properties interact in a
synergistic fashion thus enhancing their efficacy. However, the results have not been
conclusive. Some in vitro studies have reported an improved antibacterial action when
both agents were combined (280, 291, 292), while other studies reported the opposite
result (278, 284). Recent animal studies have evaluated the tissue reactions to a mix of
Ca(OH)2/CHX, showing that the combination exerts good antimicrobial properties (293)
and improves healing of the periapical tissues (288). In vivo studies have shown that the
mix is at least as good as both agents applied separately in necrotic teeth with apical
periodontitis (6), as well as in previously treated cases with persistent apical periodontitis
33
(285, 287). A more recent study utilizing a chlorhexidine based protocol of 0.12% CHX
as an irrigant, followed by a 7-day intracanal medication of Ca(OH)2/0.12% CHX have
shown promising results. The authors concluded that chemomechanical preparation with
0.12% CHX solution as an irrigant significantly reduced the number of intracanal
bacteria, however failed to render the canals bacteria-free. Further intracanal medication
with a Ca(OH)2/CHX paste significantly improved the results by reducing the number of
bacteria (15).
2.3.3. Root Canal Microbial Investigations
Since bacteria are the most important etiological factors involved in the development and
persistence of apical periodontitis, a substantial body of endodontic research has focused
on understanding of the nature of the micro-biota in infected root canals through
identification and quantification of bacteria (24, 28, 29, 57, 73, 102, 294). Bacterial
investigations have also been carried out for other purposes such as to study the presence
of bacteria in periapical tissues, (295-300), to study the effectiveness of different
antibacterial agents (5, 6, 26, 34, 40) or to evaluate different endodontic techniques (2,
25, 27, 32, 33).
Traditionally, in order to assess the microbial status of root canals, samples have been
obtained with paper points and transferred to culture media (2, 6, 26, 27, 73, 106).
34
These sampling and culture techniques have provided a valuable understanding of the
nature of the micro-biota in the root canals; however, the accuracy of these methods is
dependent on how carefully the samples have been obtained (59).
Ideally, samples taken from root canals should reflect the type, number and diversity of
the actual micro flora (59). Due to the complex anatomy of the root canal system, these
samples primarily represent the flora in the main canal; therefore, it is highly possible
that bacteria in confined areas of the canals are not recovered. Another disadvantage of
these methods is the occurrence of false negative and false positive results.
Although every living cell is expected to be cultivable, not all the bacteria present in the
samples can be grown and detected by the culture technique. It has been stated that 50%
of bacteria present in root canals though viable are yet uncultivable (16, 301 ). The term
viable but not cultivable (VBNC) has been coined for the bacteria that are present in the
environment but not able to grow on culture media (302). There are several reasons for
bacterial unculturability (303). For example, while a number of bacteria depend on the
metabolic activities of other species to grow, growth of some others could be inhibited by
specific metabolic substances produced by certain microorganisms. The culture medium
itself can be toxic for some bacterial species. Moreover, not all the nutrients required for
growth are present in culture media. In addition, some bacteria under specific
environmental conditions could enter a low metabolic non-dividing state, and in order to
grow on culture media they need to be resuscitated (303).
35
False negative results can also occur when antibacterial agents used during the root canal
therapy are carried over onto the culture media, inhibiting the growth of some bacteria
(304). To overcome this problem, it has been recommended that all antibacterial agents
should be inactivated before sampling. Sodium thiosulfate has been suggested for the
inactivation of NaOCl and iodine compounds (305). A combination of L-a-lecithin and
Tween-80 has been recommended for the inactivation of chlorhexidine (175).
False positive results may occur due to contamination of the samples and culture media
through a contaminated operative field or during laboratory processing (304-306).
To avoid false positives, Moller in his thesis clearly demonstrated the importance of
proper isolation, field disinfection and sampling strategies (305). He provided a protocol
that has been adopted by many other researchers since then (5, 6, 26, 27, 40, 73). In
Moller's protocol paper points were used for sampling from root canals (305).
Over the years some modifications have been made to the sampling technique. In
addition to paper points, 0rstavik et al. (33) has used files or reamers to obtain dentin
shavings from canals. Recently an aspirating technique has been implemented for root
canal sampling replacing paper points. This technique has been used for the collection of
samples from extracted animal teeth (307). The efficacy of this technique has been
assessed by Dagher (308) in an in vitro study. He reported recovery of more bacteria
using this technique. Paquette applied this technique in a human in vivo study and
reported higher numbers of recovered bacteria. Furthermore, Paquette et al. pointed out
36
that due to the specific technical aspects of the aspiration using fine needles under high
magnification of operating microscope, there is less chance for contamination of the
samples (34).
Several techniques have been used to explore bacteria other than the culture method:
Imageless detection methods such as flow cytometry (309) and molecular methods such
as polymerase chain reaction (PCR) (3, 310) and checkerboard DNA-DNA hybridization
(311 ); imaging methods based on microscopy such as scanning electron microscopy
(312), confocal microscopy (313), light and dark-field microscopy (314), transmitted
light microscopy and epifluorescence microscopy (51, 52). In novel microscopic
methods, instead of original staining methods, fluorescent dyes are used (315, 316).
These dyes can be conjugated to RNA and DNA probes such as fluorescent in situ
hybridization (FISH) assays (300, 317) or monoclonal (318) and polyclonal antibodies
(319, 320) to visualize the bacterial cells.
Epifluorescence microscopy is more sensitive and specific in detecting bacteria than the
culture method (48, 49, 51). It has been reported that it can be used in the endodontic
field as well (46, 47). It was used successfully for the first time by Paquette et al. in a
clinical study for the detection and direct enumeration of bacteria in samples obtained
from infected root canals (34). This endeavor has opened a new era for further the study
of endodontic infections and the assessment of the efficacy of materials and methods
used in the field.
37
2.3.4. Previous Work and Background of the Project
MTAD has demonstrated the following properties in vitro: (i) ability to remove the smear
layer, with lesser erosion of dentin than EDTA (137), (ii) antibacterial effect against E.
faecalis (139, 141), (iii) lesser cytotoxicity than eugenol, Ca(OHh paste, and 5.25%
NaOCl (166).
For several years our group has been involved in studies investigating the in vitro
efficacy and the in vivo effectiveness of CHX as an intracanal medication (34, 187, 210,
211, 214, 273). Our results have demonstrated: (i) substantial antibacterial activity in root
dentin (210, 211, 214), (ii) the ability of sealers to bond to CHX-treated dentin (187), (iii)
the ability to dispense controlled amounts of CHX overtime from an endodontics-specific
controlled release device (273), and (iv) the ability to curtail regrowth of bacteria in
infected canals between treatment appointments (34). These and other studies form a
solid basis for the proposed in vivo investigation.
In addition, our earlier in vitro investigation into microbiological culture testing of root
canals revealed an improved accuracy of sampling with the aspiration technique than
with the paper point technique (308). The aspiration technique as well as an improved
enumeration with a combination of Colony Forming Unit (CFU) counts and
epifluorescence microscopy were used in a previous clinical investigation into the
effectiveness of CHX liquid intracanal medication (34). The latter forms the basis for the
proposed study methodology, and provides the rationale for using both the aspiration
38
sampling technique and the combined CFU and epifluorescence microscopy enumeration
methods.
The results of Paquette's study showed a moderate increase in bacterial counts during a
medication period of 7-14 days, that was similar to that seen and reported for Ca(OH)z
(26). It was speculated that the CHX liquid may have partially escaped from the apical
foramen, and that a gel form might be better suited for intracanal medication than liquid
(34). A CHX gel may create a better physical barrier against bacteria, and sustain its
effect in the canal for a longer period of time. Furthermore, CHX gel may clean the canal
walls better than the liquid form (250). The early concern of the potential effect of the gel
residue on the apical seal of root fillings (235) has recently been dismissed (290). Thus
2% CHX gel appears to be a viable alternative to Ca(OH)2 and CHX liquid as an
intracanal medication, offering easier placement and removal, comparable or better
antibacterial effectiveness, and a potential for substantive antibacterial activity.
AIMS AND HYPOTHESIS
39
40
3. AIMS OF THE STUDY
3.1. Principle Aim
The principle aim of this study was:
To assess in vivo the effectiveness of a final rinse with MTAD.
3.2. Secondary Aims
1- To assess the efficacy of 2% CHX gel used for intracanal medication in reduction of
bacteria between appointments regardless of whether MT AD rinse was applied or not.
2- To evaluate an automated, microscopy-based method for the enumeration of bacteria
present in infected root canals before and after endodontic treatment.
3.3. Specific Aims
The specific aims of this study were to compare bacterial counts:
1. Between samples taken at the same treatment step in MT AD and control group using
both enumeration techniques.
2. Between samples taken at the different treatment steps within the same group using
both enumeration techniques.
3. Between the enumeration techniques, for each sample.
3.4. Null Hypothesis
The final rinse with MTAD at the end of the first appointment will not significantly
decrease the number of bacteria from root canals beyond that achieved by conventional
chemo-mechanical cleaning and shaping procedures performed in the first appointment.
MATERIALS AND
METHODS
41
42
4. MATERIALS AND METHODS
4.1. Design
The study was designed as a randomized-controlled and double-blinded clinical trial.
4.1.1. Randomization
Subjects were randomly assigned into experimental (MTAD) or control (coloured saline)
groups by drawing from a pool oflots prior to the investigation.
4.1.2. Blinding
Saline was administered in place of MT AD to the control group. In order to meet the
requirements of a double-blinded clinical trial, saline was spiked with a food colouring
solution that mimicked the clear yellowish colour of MTAD. The test solutions were
delivered in identical 5 mL syringes with 30 gauge needles. The operator and patients
were blinded as to which were in the test and control groups.
4.2. Sample Size Calculation
In the absence of conclusive in vivo data regarding the effectiveness of MT AD as a final
rinse, the initial sample size was calculated to test the proposed effectiveness of 2% CHX
gel applied as an intracanal medication. The calculation was based on Dr. Paquette's
study results where the effectiveness of 2% CHX liquid applied as an intracanal
medication was tested (34). According to Dr. Paquette's study results, the means of the
bacterial counts obtained after staining with DAPI were the most normally distributed;
43
therefore, the means and standard deviations obtained before and after treatment with 2%
CHX liquid were selected for initial sample size calculation (SamplePower™ 2.0, SPSS).
To test the null hypothesis that the mean difference within pairs, before and after
application of CHX in gel form, would be 0.00, with an alpha level at 0.05 (two sided), a
sample size of 11 teeth would be adequate to have a power of 83.2% to yield a
statistically significant result. However, the application of MT AD before CHX and its
potential effect on the results necessitated the addition of another group with a minimum
of 11 teeth, where MT AD was replaced with an inert solution such as saline.
The computation assumed that the population from which the sample was drawn had a
mean difference of 8.4 (natural logarithm transformed DAPI counts) with a standard
deviation of 8.6. The observed value would be tested against a theoretical value
(constant) of 0.00. The effect was selected as the smallest effect that would be important
to detect, in the sense that any smaller effect would not be of clinical or substantive
significance. It was also assumed that the effect size was reasonable, in the sense that an
effect of this magnitude could be expected in this research.
Due to the absence of in vivo data regarding the effectiveness of MT AD as a final rinse
and also to account for loss to follow-up, the initial sample size was increased by 36%
from 22 to a feasible number of 30 teeth (divided into two equal groups) resulting in a
power of 94% to yield a statistically significant result.
44
4.3. Study Cohort and Final Sample Size of the Study
The study cohort included patients who attended the Graduate Endodontics Clinic in the
Faculty of Dentistry, University of Toronto, and were selected in accordance with
specific inclusion and exclusion criteria (Table 1 ).
The final sample size for this clinical study was 30 teeth from 30 human subjects,
experimental (n=15) and control (n=15). The cohort included 15 male and 15 female
patients, with 15 maxillary teeth (6 anteriors and 9 premolars) and 15 mandibular teeth (4
anteriors, 7 premolars, and 4 molars). Detailed supplementary characteristics of the study
cohort can be found in Appendix 2.
4.4. Ethics and Scientific Merit Reviews
Before data collection, the research protocol was approved by the Health Sciences I
Research Ethics Board and the Research Committee of the Faculty of Dentistry,
University of Toronto, Canada. The informed consent of all human subjects who
participated in the experimental investigation was obtained after the nature of the
procedure and possible discomforts and risks had been fully explained.
4.5. Pre-Clinical Procedures
4.5.1. Laboratory Procedures
Reduced transport fluid (RTF) was mixed with dithiothreitol (DTT; Sigma-Aldrich, St.
Louis, MO) and used as the transport medium as described by Paquette (34). After filter
sterilization, aliquots of 1.0 mL were placed into 4 labeled microtubes (1.5 mL) for root
45
canal sampling during the clinical procedures. Two empty microtubes were also prepared
(only for the first treatment session) for measuring the volume of the treated root canal(s)
before and after chemomechanical preparation. Each of the prepared microtubes was
weighed three times using a precision balance (Sartorious, Gottingen, Germany). The
average weight of each was calculated and applied in calculations. After the samples
were collected, all the microtubes were re-weighed following the same protocol.
4.5.2. Preparation of 2% Chlorhexidine Gel
The 2% Chlorhexidine gel was prepared specifically for the study by the pharmacist of
the Faculty of Dentistry, University of Toronto. Methylcellulose powder (3g) (Wiler Fine
Chemicals, London, ON, Canada) was added to 60mL heated sterile distilled water
(SDW) (Abbott Laboratories, Ltd, Saint Laurent, Quebec, Canada) and stirred for 2-5
minutes. After cooling for about 3 minutes, 1 OmL chlorhexidine digluconate (20%
aqueous solution; Wiler-PCCA, London, ON, Canada) was added and stirred. The
remaining SDW in sufficient quantity (qs) was added to have lOOmL of solution, then
well stirred and allowed to stand about 1 hour. After storage in the refrigerator overnight,
the mixture was poured into 50mL container(s) with a squeeze top and then labeled. One
mL was drawn up in to a 3mL disposable syringe for clinical use. The expiry date was 6
months from the date of manufacture.
46
4.6. Clinical Procedures
All patients were treated in two appointments which were spaced 7 days apart with the
exception of two patients (5 days and 6 days). All subjects were treated following a strict
infection control protocol (305).
4.6.1. Isolation and Field Disinfection
Teeth were cleaned with pumice and a rubber cup, and patients were asked to rinse their
mouth with Listerine® Antiseptic mouthwash (Pfizer Canada Inc., Markham, ON,
Canada) for 30 seconds. After administration of local anaesthetic, the tooth was isolated
with a rubber dam (DermaDam; Ultradent, South Jordan, Utah) and the tooth/rubber dam
interface was sealed with OpalDam® (Ultradent Products, South Jordan, Utah). The tooth
and rubber dam were disinfected with 30% hydrogen peroxide (Sigma Aldrich
Laborchemikalien GmbH, Germany) and 5% iodine tincture (Wiler-PCCA Fine
chemicals, London, ON, Canada) for one minute each. After inactivation, a sterility
control sample was obtained from the rubber dam and the tooth. Old, inadequate or
leaking coronal restorations and caries were removed without penetration into the pulp
chamber using sterile burs (Brassier, USA) under sterile saline (Hospira, Inc. Lake
Forest, Illinois, USA) irrigation. Whenever required, the peripheral walls were restored
with glass ionomer (GI) filling material. The access cavity and the tooth were then
disinfected with 30% hydrogen peroxide and 5% iodine tincture following the same
protocol for another 1 min each. After inactivation, an access cavity sample was obtained
with the aspirating technique. New sterile burs were used to penetrate the pulp chamber
under saline irrigation.
47
4.6.2. Inactivation of Disinfecting Solutions
Disinfecting solutions (hydrogen peroxide, iodine tincture and sodium hypochlorite) were
inactivated with 5% sodium thiosulfate (J.T. Baker, Phillipsburg, N.J.) for 1 min (305)
and then rinsed with sterile saline.
Chlorhexidine gel was inactivated with 3mL of 0.3 % L-a-lecithin (Sigma Aldrich, St.
Louis MO) and 3% Tween 80 (175) and rinsed out with 3mL of sterile saline.
Test solutions (MTAD and coloured saline) were not inactivated. However, an effort was
made to thoroughly rinse them out to limit any effect on the culture process.
Sodium thiosulfate, coloured saline, 0.3 % L-a-lecithin and 3% Tween 80 were filter
sterilized.
4.6.3. Sample Acquisition
4.6.3.1. Sterility Control Samples:
Sterility control samples were obtained at the beginning of each treatment appointment
after rubber dam isolation, surface disinfection and inactivation. The tooth surface was
rubbed with 4 pieces of sterile cotton pellets which were then used to inoculate blood
agar plates and brain-heart infusion (BHI) tubes by: 1- inoculating directly on the surface
of a blood agar plate, 2- transferring into a microtube filled with transport fluid (RTF)
and then inoculating 52 µL of the fluid onto blood agar using a Spiral Plater (Spiral
48
system, Cincinnati, Ohio), 3- transferring into a tube containing BHI, and the last cotton
pellet from the same pouch was directly transferred to BHI to serve as a negative control.
All of the cultures were incubated aerobically at 37° C, for 14 days, and inspected at days
7 and 14 for bacterial growth.
4.6.3.2. Access Cavity and Root Canal Samples:
Access cavity and root canal samples were acquired by aspiration as described by others
(34, 307, 308). In this technique the access cavity or root canal was filled completely with
RTF prior to sampling. Using a fine needle (27 gauge) (Monoject, Sherwood Medical, St.
Louis, MO) all the contents were aspirated into a syringe containing 0.3ml of RTF and
then emptied into a microtube containing 1.0 mL of RTF.
During root canal sampling an attempt was made to dislodge residual bacteria from root
canal walls or from recesses of the canals by using the pumping motion of a file as
recommended by Moller (pumping movement with a file and maximum removal of fluid
method) (305). Moreover, the canal contents were agitated by gentle pumping (pulling
and pushing the piston of the needle) of the canal fluids during sampling before aspirating
the whole contents into the syringe.
The vials were immediately transferred and stored at 4°C until laboratory processing.
The samples collected during the treatment steps are summarized in Table 2.
49
4.6.4. Root Canal Volume Measurement:
In order to calculate the bacterial concentration inside the root canals, the root canal
volume was measured before (lA) and after (lB) cleaning and shaping of the canals
during the first appointment according to the method of Paquette et al. (34).
4.6.5. First Treatment Appointment
All treatment procedures were standardized. The final diagnoses were established based
on clinical and radiographic exams. After isolation, field disinfection, obtaining initial
sterility control samples, caries removal, access cavity sample collection (ACl) as
described above, new sterile burs were used to penetrate the pulp chamber. An ISO size
10 and/or 15 stainless steel K-file (Flexofile; Dentsply Maillefer, Balleigue, Switzerland)
was used to negotiate the canal apically to the estimated working length (WL) minus 1-2
mm measured on a pre-operative radiograph. An electronic apex locator (EAL) (Root ZX
II; J. Morita, Irvine, CA) was also used to prevent extension beyond the WL. The initial
bacteriological sample (lA) was obtained from the root canal(s) as described in the
sampling protocol. No active disinfecting solution was used to this point. The canals were
dried and the volume (lA) was obtained. Each canal was cleaned and shaped to the
working length using hand stainless steel k-file instruments to an apical size of #15 or 20.
Coronal portions were preflared with Gates-Gliden drills (Dentsply Maillefer N.A.,
Tulsa, OK) number 2 and 3 followed by rotary Ni-Ti ProTaper files (Dentsply, Tulsa
Dental, Tulsa, OK, U.S.A.) with a crown down technique according to the manufacturer's
recommendations, while irrigating with 1.5 mL of 1.3% NaOCl after each instrument.
The final size was one size larger than the first gauging file. The second root canal
50
sample (lB) was taken after completion of root canal cleaning and shaping as described
above and the lB volume was measured. After completion of the chemomechanical
cleaning and shaping of the root canals the test solutions (MT AD or coloured saline)
were delivered in identical 5 ml syringes with a 30 gauge needle. Each canal was
irrigated with lmL of assigned solution, and was flushed out after 5 minutes with the
remaining 4mL of the solution following the protocol provided by the manufacturer of
the MTAD. In order to minimize the potential carry-over of any residual antibacterial
effect of the MTAD, canal(s) were flushed again with 3 mL of sterile saline before the
third root canal sample (1 C) was obtained by aspiration as described above. Canals were
dried again with sterile paper points. Each canal was filled with 2% CHX gel using a
preloaded syringe and by introducing a Lentulo spiral in the canal. The access opening
was sealed with sterile sponge pellets, and two layers of light curing glass ionomer
cement (GI) (Photac™ Fil Quick Aplical, 3M ESPE, St. Paul, MN).
4.6.6. Second Treatment Appointment
The tooth was isolated, disinfected and sterility control samples were obtained as
described above. After partial removal of the GI cement without penetrating the chamber,
an access cavity sample was obtained (AC2). The root canal system containing 2% CHX
gel was rinsed with 3ml saline and the residue was inactivated (175) and then rinsed
again with 3mL of sterile saline. The canals were dried and a bacteriological sample (2A)
was obtained. The canal was irrigated again with 1.3% sodium hypochlorite and root
canal instrumentation was finalized as required with either the previous master apical file
size or one size larger. The irrigant was inactivated and the final sample (2B) was
51
obtained. Finally, canals were dried and filled and the access cavities sealed with Cavit
and glass ionomer cement. A final periapical radiograph was taken.
4.7. Post-clinical Procedures
4. 7 .1. Bacteriological Procedures
Samples (comprising approximately 0.3mL when withdrawn into the syringe) were
placed in pre-weighed microtubes containing lmL of RTF and transferred to the
microbiology laboratory. They were processed within 2-3 hrs to obtain colony forming
unit (CFU) counts and direct epifluorescence microscopy (EFM) counts based on two
vital staining techniques. To this end, each sample was divided into three sub-samples as
outlined in Table 3. The final volume used for processing was constant (lmL for each
microscopy technique and 0.5mL for CFU).
4.7.1.1. CFU
Aliquots of 52 µL of one sub-sample were inoculated on blood agar plates (Difeo, Becton
Dickinson, Sparks, MD) using a Spiral System Plater (Spiral system, Cincinnati, Ohio).
Samples obtained from root canals were incubated anaerobically while access cavity
samples were incubated separately for both aerobic and anaerobic bacteria. Anaerobic
plates were incubated under strict anaerobic conditions in sealed jars with a gas mixture
of 10% hydrogen, 10% carbon dioxide, and 80% nitrogen at 37 °C for 14 days. Aerobic
plates were incubated in an ambient atmosphere at 37 °C for the same time period. A
count template was used to establish CFU counts on the Tth and 14-th days.
52
4.7.1.2. Microscopy
The other two sub-samples were processed with two different viability staining methods
followed by enumeration using epifluorescence microscopy (EFM). One sub-sample was
stained with LIVE/DEAD® BacLight™ Bacterial Viability Kit (BacLight) (Molecular
Probes /Invitrogen, Eugene, OR) and the other with dihydroethidium (DHET) (Molecular
Probes, Eugene, OR) and DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich,
Oakville, Canada) using a modification of the method of Paquette (53).
4.7.1.2.1. BacLight vs. DHET and DAPI
The BacLight viability kit was expected to distinguish between viable and non-viable
bacterial cells based on the integrity of the cell wall. With this method, live bacteria
fluoresce green and dead bacteria fluoresce red. With the DHET and DAPI method,
dihydroethidium relies on bacterial cell dehydrogenases for vital staining (309, 321, 322),
while DAPI is used as a counter stain that labels all bacterial cells regardless of their
viability (316). With this method, live bacteria fluoresce red, and the total bacterial
population fluoresces blue. The concentration of dead bacteria was calculated as the
difference of the two counts.
4.7.1.3. Baclight
The original Molecular probes protocol for Baclight staining, requires both Syto 9 and
propidium iodide emissions to be visualized in the same microscopic field as one single
image. It requires a broad band excitation filter (BP 450-490 nm), emission filter in the
53
range 520-560 run with a FT510 chromic beam splitter, and an RGB camera such as the
one used in Paquette's study.
In the present study, the signals were captured in two separate channels using a high
sensitivity Hamamatsu CCD black and white camera (emission 510-540 run for green and
610-680 run for red, with excitation 460-500 run (L5 cube) and 540-580 run (TX2 cube)
respectively) (Leica Microsystems, Wetzlar GmbH, Germany).
In order to attain comparable signals in terms of the same cell viability status, the
luminance of the pixels had to be processed by image analysis. Programs in Openlab
allowed the reconstruction of composite (merged) images based on the highest luminance
of two superimposed frames thus allowing for a "total" count of bacteria.
In order to compare the results obtained for the enumeration of live bacteria using either
one or two channels, a limited comparison experiment (Table 12) representing about 20%
of the IA samples was conducted.
4.7.1.4. DAPI and Dihydroethidium
According to the protocol used in Paquette's thesis study for DAPI and DHET staining,
both blue and red emissions are visualized for the same microscopic field as two separate
images. DAPI-stained blue emitting cells were visualized by UV excitation (G365) and
blue emission (LP420) filters with a FT395 chromatic beam splitter, red-emitting bacteria
54
stained with dihydroethidium were visualized with green excitation (BP546/12) and
emission (LP590) filters with a FT580 chromatic beam splitter.
In the present study, the signals were captured in two separate channels using a high
sensitivity Hamamatsu CCD black and white camera (emission 450-490 nm for blue and
608-682 nm for red, with excitation 340-380 nm (A4 cube) and 540-580 nm (TX2 cube)
respectively) (Leica Microsystems, Wetzlar GmbH, Germany).
4.7.2. Adopting the LEICA DMIRE2 Microscopy System
In Paquette's study, images were observed with a Carl Zeiss Universal Research
microscope, captured and then stored in a computer. The microscope was equipped with
three different sets of optical filters which were switched manually between individual
images to distinguish between viable and non-viable cells. In the current study, images
were acquired using a Leica DMIRE2 (Leica Microsystems, Wetzlar GmbH, Germany)
epifluorescence microscope with Modular Imaging Software (Open/ab 4.0.2.,
Improvision®, Quorum Technologies Inc. Lexington, MA. USA). After manual focusing,
the microscope was programmed first to capture and save one of the paired images, then
automatically switch to the appropriate filter cube sets to capture and save the second
image. The captured paired images were saved in LIFF format (Layered Image File
Format).
The automation significantly enhanced the efficiency of the procedure, almost doubling
the number of captured images.
55
Moreover, the automated filter changer allowed an exact superimposition of the images.
Such superimposition was not possible with the Zeiss microscope because the manual
changing of filters moved the stage preventing superimposition. The superimposed
images of the Lei ca microscope allowed more accurate counts in the current study.
In addition, the Leica microscope was equipped with a high sensitivity Hamamatsu CCD
black and white camera. The resolution of the images obtained with this camera
(I344xI024) was 4 times higher than that of the RGB camera used in Paquette's study.
This improved the quality of the captured images making it easier for the automated
programs to detect and enumerate bacteria.
The images were copied and converted to a TIF format (Tag Image File), necessary for
further image processing on a personal computer. On average I 400 digital images were
captured and saved for each tooth, approximately 130 from each of the ACI, AC2 and
IA samples and 250 from each of the IB, IC, 2A and 2B samples.
The Optimas image analysis package (Optimas 6.5; Media Cybernetics, Silver Spring,
MD) was used to enumerate bacteria. The results of the analyses were stored in Excel®
spreadsheets (Microsoft office, Microsoft Corporation, USA). The results of both
methods (culture and microscopy) were expressed as the number of bacteria per µL of
root canal volume.
56
4.7.3. Modifications to the Microscopic Counting Technique of Paquette
In Paquette's study bacteria at low densities were counted based on visual counting,
whereas at high densities an interactive image analysis software was used.
In the current study, due to the larger sample size, the higher number of samples and the
greater number of captured images, visual counting would have been extremely
labourious. Automated programs without human interaction have been successfully tested
for the enumeration of fluorescently labeled microorganisms in different environments
such as marine picoplankton, human gut flora, and human dental plaque (323-328).
In the current study, to facilitate the labourious task of enumerating bacteria in captured
images, an automated procedure was developed in our laboratory.
Automated counts of bacterial images stored in TIF files of microscopic fields were made
with a macro written in the Analytical Language for Images (ALI) available in the
Optimas image processing package from Media Cybernetics (Optimas 6.5;Media
Cybernetics Inc MD). Pseudo colour images were converted to 32 bit grayscale images,
background noise was reduced with a 3x3 median filter, edges were enhanced with an
eight point compass filter and an attempt was made to separate images of individual
bacteria from images of clumped or overlapping bacteria with a water-shed algorithm.
Particles were then automatically located by edge detection and counted. Counts, areas,
circularity, mean width and breadth of each particle were calculated. Data were exported
for storage in an Excel spreadsheet prior to statistical analysis.
57
In order to examine the performance of the macro, bacteria from some samples were also
counted visually and the results were compared to those obtained with the automated
procedure.
4.8. Effects of Food Colouring
In order to emulate the visual characteristics of MT AD a yellow food colouring solution
was gradually added to a sterile saline solution until no difference in colour could be
observed. Absorbance (A,i80) of this concentration was recorded using a
spectrophotometer (Ultrospec III, Pharmacia LKB). A setting of 480 nm was chosen as a
complementary wavelength to the yellow colour and, consequently, expected to
maximize absorbance. Concentrations of food colouring ranging from 4x to 64x the
value established as MTAD equivalent (5 tubes) were prepared with a final volume of
9mL each and tested in order to ensure that the food colourant had no effect on bacterial
growth. In two additional tubes, BHI broth undiluted and diluted 10 times was used as a
positive control. As a negative control, a suspension of bacteria plus 9 mL RTF was
used. Bacteria were harvested with an inoculation loop from blood agar plates after 14
days of anaerobic incubation and dispersed in 10 mL of RTF. The suspension was
vortexed and one mL was added to each of eight prepared tubes. Tubes were incubated
under anaerobic conditions at 37° C in the dark for two weeks. Values of OD6oo
(estimated using the same spectrophotometer as above with a 1 cm light path) were
measured on days 0, 7 and 14. The optical density at 600 nm was regarded as the best
approximation of bacterial biomass based on cell turbidity. As an additional test, 52-µl
58
aliquots were taken aseptically from all tubes at days zero, seven and fourteen and
inoculated on blood agar using a Spiral Plater.
4.9. Analysis
All statistical analyses were performed using SAS version 9.1 (Cary, NC), using a 0.05
alpha level of significance with the appropriate correction for multiple testing
(Bonferoni). The means of bacterial densities per µL of root canal volume were
transformed to their natural logarithms to normalize the data and analysed. Due to the
dependent nature of the multiple treatment steps, repeated measures analysis of
covariance was used to detect significant differences in bacterial densities between
samples at different treatment steps for the microscopic methods. Since the treatment
difference occurs after step 1 B, bacterial densities at 1 A and 1 B steps were used as
baseline measurements and were applied as covariates in this analysis.
Logistic regression (SAS: PROC LOGISTIC) analysis was used to detect significant
differences in the proportion of positive cultures between samples after treatment step
lB.
Three separate two-way ANOV A analyses were used at steps 1 C, 2A, and 2B to compare
the differences between the two microscopic methods in detecting live or dead bacterial
cells within the respective samples.
59
The similarity between visual (manual) and automated (macro) counting methods was
evaluated by Pearson's correlation coefficient analysis using SPSS software® (SPSS 15.0
for Windows, SPSS Inc Chicago, Illinois, USA).
RESULTS
60
6I
5. RESULTS
Results recorded at different stages of the study with the three methods of assessment are
summarized in Table 4 (access cavity samples) and Table 5 (root canal samples). The raw
data from all the samples in all the studied subjects are presented in Appendices 3-I 6.
The data obtained at the IA and IB samples were used as baseline to control differences
within and between the MT AD and Saline groups at subsequent samples.
The CPU counts from subject I 8 were excluded, because no bacterial growth occurred in
the IA sample. The IB sample CPU count from subject 5 was also excluded, because of
contamination of the culture plate. As the I 4-day incubation CPU counts appeared
consistently higher than the 7-day incubation densities, only the former are presented
below for simplicity. The distributions of CPU counts after conversion to natural
logarithms are plotted in Figure 1. Abundant CPUs were recorded in IA samples, in
sharp contrast to the few CPUs recorded in all subsequent samples. Because the data in
all the subsequent samples frequently included zero values (below detection threshold) a
parametric statistical test could not be used to analyze this dataset.
All samples examined by epifluorescence microscopy (EFM) showed presence of live
bacteria, except one sample stained with DHET (2A sample, subject I4). EFM also
allowed detection of dead bacteria, in all the samples stained with Baclight except one
(2B sample, subject I2). In IO samples stained with DAPI/DHET, the live bacterial count
(DHET stain) exceeded the total bacterial count (total DAPI-DHET count minus DHET
count). For these samples, the total counts were replaced by the live counts, and the dead
62
counts were considered zero. The mean live bacterial counts obtained with both EPM
methods were consistently higher than the CPU counts in each treatment step. The
discrepancy was more evident in lB sample and the subsequent ones.
5.1. Access Cavity Samples
Proportions of positive cultures obtained from access cavity samples are presented in
Table 4. A minority of the samples showed bacterial growth in the first treatment session
(ACl samples) and in the second treatment session (AC2 samples). Apparent differences
were observed between samples incubated aerobically and anaerobically, and ACl and
AC2 samples; however, this dataset was not analyzed statistically.
The CPU counts obtained from access cavity samples after 7 and 14 days of aerobic and
anaerobic incubation are also presented in Table 4. These counts were consistently low.
The EPM bacterial counts obtained from the access cavity samples are also presented in
Table 4. Notably, the live bacterial counts were at least 5 orders of magnitude higher than
the CPU counts in the same samples. These counts followed the same pattern observed in
the CPU counts with an apparent decrease in AC2 samples relative to ACl samples. The
dead bacterial counts in the Baclight-stained and DAPVDHET-stained samples were
inconsistent, with apparently higher counts obtained with the latter staining method than
with the former.
63
5.2. Sterility Control Samples
Bacteria were only detected in 3/60 samples (5%) with all three methods of assessment,
two samples from the first treatment session and one from the second treatment session.
In 7/60 samples (12%) bacteria were detected with two methods, while in 24/60 samples
(40%) bacteria were detected with one of the methods. In 26/60 samples no bacteria were
detected with any of the assessment methods (Table 14).
5.3. Root Canal Samples Before and After Preparation
At the outset (IA samples), 29/30 root canal samples (96.6%) showed bacterial growth in
culture plates (Figure 4), while live and dead bacteria were detected with EFM in 100%
of the samples. The bacterial densities (counts per µL of root canal volume) obtained
with the three methods of assessment are presented in Table 5. The differences in CFU
and EFM live bacterial densities between the MT AD and Saline groups were not
statistically significant (Table 6). The difference in dead bacterial densities obtained with
EFM methods between the MT AD and Saline groups was significant when counted with
PI (p= 0.0397) and was not significant when counted with DAPI-DHET (p=0.9177)
(Table 6). However, the significance disappears after Bonferroni correction.
After chemomechanical preparation (IB samples), 4/14 root canal samples (29%) in the
MTAD group showed bacterial growth in culture plates, compared to 6/14 samples (43%)
in the Saline group (Figure 4). The difference in the proportion of positive growth
cultures between both groups was not statistically significant.
64
The CPU densities in lB samples decreased sharply from those recorded in IA samples,
in both the MTAD group (5 orders of magnitude) and Saline group (4 orders of
magnitude). The difference between the lB sample densities of the two groups was not
significant. With EFM, both the Baclight-stained (Syto9) and the DHET-stained live
bacterial densities decreased substantially (2 orders of magnitude) in both the MT AD and
Saline groups. The difference between 1 B sample densities of two groups was not
significant.
Also the dead bacterial densities in lB samples decreased substantially. Baclight-stained
(PI) dead densities were reduced by 2 orders of magnitude in both the MT AD and Saline
groups, and a similar reduction in DAPI-DHET stained dead densities was recorded in
the MT AD group, while a one order of magnitude reduction was recorded in the Saline
group. The difference between the lB sample densities of the two groups was not
significant. Except for DHET-stained live bacterial densities and PI-stained dead
bacterial densities, the densities recorded in 1 B samples were the only explanatory
variable for the densities in all subsequent samples (Table 7). The higher the densities in
1 B sample, the higher they were in the subsequent samples. The group (MT AD or Saline)
had no statistically significant effect for any of the EFM densities (Table 7).
5.4. Root Canal Samples After Final Irrigation/immersion
After final irrigation/immersion of the canals (lC samples), 8/15 root canal samples
(53%) in the MTAD group showed bacterial growth in culture plates, compared to 8/14
samples (57%) in the Saline group (Figure 4). The slight increase in the proportion of
65
positive cultures in both groups was not statistically significant, and neither was the
difference between both groups (Tables 8 and 9). With EFM, live bacteria were detected
in all of the 1 C samples.
The CFU densities in the 1 C samples increased by one order of magnitude in the MT AD
group, while a slight increase was recorded in the Saline group. With EFM, a decrease by
one order of magnitude was recorded in the DHET-stained live bacterial densities in the
MTAD group, while a slight decrease was recorded in Baclight-stained (Syto9) live
bacterial densities. In the Saline group, a slight decrease in live bacterial counts was
recorded with both EFM methods. In both groups, the decrease was not statistically
significant, and neither was the difference between the densities in both groups.
The densities of dead bacteria showed a different pattern with both EFM methods. The
Baclight-stained (PI) densities decreased by one order of magnitude in both MTAD and
Saline groups, while the DAPI-DHET stained densities increased slightly in both MTAD
and Saline groups. None of these changes were statistically significant, and nor were the
differences between the densities in both groups.
5.5. Root Canal Samples After Intracanal Medication
After medication of the canals with 2% chlorhexidine gel (2A samples), the proportion of
samples showing bacterial growth in culture plates remained unchanged from 1 C samples
in both MTAD and Saline groups (Figure 4). With EFM, live bacteria were detected in all
of the 2A samples.
66
The CFU densities in 2A samples, decreased slightly in both the MT AD and Saline
groups. With EFM, an increase by one order of magnitude was recorded in the Baclight
stained (Syto9) and DHET stained live bacterial densities in the MTAD group, while only
minor changes were recorded in the Saline group. In neither groups were the changes
statistically significant, and nor was the difference between the densities in both groups.
The densities of dead bacteria stained with Baclight (PI) were significantly increased by
one order of magnitude in both MTAD and Saline groups (p= 0.0215) (Table 7).
However, there was no significant difference between the two groups. A similar increase
was recorded in the DAPI-DHET stained densities in the MTAD group, while only a
minor change was recorded in the Saline group. These changes were not statistically
significant, and nor were the differences between the densities in both groups.
5.6. Root Canal Samples After Final Preparation Before Root Filling
After final chemomechanical preparation (2B samples), 6/15 root canal samples (40%) in
the MT AD group showed bacterial growth in culture plates, compared to 3/14 samples
(21 %) in the Saline group (Figure 4). The decreases in the proportion of positive cultures
in both groups were not statistically significant, and nor were the differences between
both groups (Tables 8 and 9). With EFM, live bacteria were detected in all of the 2B
samples.
The CFU densities in 2B samples decreased by one order of magnitude in the Saline
group, while a slight decrease was recorded in the MTAD group. With EFM, a decrease
67
by one order of magnitude was recorded in the Baclight-stained (Syto9) live bacterial
densities in the MT AD group, while only minor changes were recorded in the Saline
group and in DHET stained live bacterial densities in both groups. In both groups, the
changes were not statistically significant, and nor were the differences between the
densities in both groups.
The densities of dead bacteria in 2B samples stained with Baclight (PI) were significantly
decreased by one order of magnitude in both MTAD and Saline groups (p= 0.0215)
(Table 7). However, there was no significant difference between the two groups. A
similar decrease was recorded in the DAPI-DHET stained densities in the Saline group,
while only a minor change was recorded in the MTAD group. These changes were not
statistically significant, and neither were the difference between the densities in both
groups.
5.7. Comparison of Microscopic Techniques for the Detection of Live and Dead
Bacteria
The densities of live bacteria in samples 1 C, 2A and 2B were consistently and
significantly higher when stained with Baclight (Syto9) than with DHET (Table 10,
Figure 2). In the same samples the densities of dead bacteria were consistently and
significantly higher when stained with DAPI-DHET than with Baclight (PI) (Table 11,
Figure 3).
68
5.8. Comparison of Unmerged vs. Merged (Composite) images in Baclight Staining
Comparison of means and standard deviations suggested that there was no major
difference between the merged images (two channels) versus the unmerged (single green
channel). These results have also been compared to the results obtained from the DHET
staining for the same samples and the same subjects (Table I2). The mean bacterial
counts were higher for live bacteria stained with Baclight than those stained with DHET
regardless of whether or not the images were merged.
5.9. Comparison of Automated (Macro) vs. Visual (manual) Enumeration Methods
The results of statistical analysis demonstrated a good correlation between bacterial
counts obtained with automated and visual techniques. Correlation coefficients for
different microscopic methods ranged between 0.803 and 0.977 (p<O.OI) (Tables I5a and
I5b).
In general, the automated counts appeared to be lower than the visual counts. On average,
macro counts were 63% of visual counts (Figure 5); however, they were within different
ranges when different microscopy techniques were used. They were also different when
bacteria were in high (1 A) or low densities. In cases when bacterial densities were
expected to be higher (IA samples), there appeared to be a wider difference between the
two methods of enumeration. For example, in IA samples stained with DAPI, macro
counts were 56% of the visual counts and with DHET, the macro counts were 39% of
visual counts. In IA samples stained with Syto9, macro counts were 45% of the visual
counts and with PI they were 83 % of the visual counts.
69
The above comparisons suggested a higher accuracy of the automated counting program
in the fields with lower bacterial densities.
5.10. Effects of Food Colouring
An absorbance A480 of 0.202 was found for the food-colouring spiked saline that was
indistinguishable from MTAD. The spiked saline was tested to rule out any effect it
might have on enhancing bacterial growth. No change was found in the OD600 after a two
week incubation at any of the concentrations of food colouring in the tested series (Table
13). In the positive control tubes (undiluted and lOx diluted BHI medium), there was a
noticeable increase in OD600 after one and two weeks of incubation. Undiluted BHI gave
a substantially greater increase (Table 13). No differences were observed on plates
inoculated from individual tubes containing bacteria. It was concluded that neither a
positive nor a negative effect on bacterial growth was detected with food colouring.
DISCUSSION
70
71
6. DISCUSSION
6.1. Study Methodology
The methodology of this study was based mainly on two previous studies. In 1966,
Moller (305) established the widely used protocol for bacteriological sampling of root
canals, including tooth surface and rubber dam decontamination, sterility controls,
acquisition of root canal samples by means of paper points, transportation of the samples
to the laboratory, and anaerobic culturing of the samples. The main quantitative outcome
measures established by Moller for monitoring bacteria in root canals comprised
calculation of the proportion of cultures that yielded bacterial growth (positive cultures)
versus those that did not (no-growth cultures). Although various modifications of
Moller's protocol have been used over the years, the basic steps and procedures remained
largely unchanged. Forty years after Moller's thesis, Paquette et al. (34) used a modified
protocol that included access cavity controls, acquisition of samples by means of
aspiration, enumeration by means of epifluorescent microscopy using vital dye staining in
addition to CFU's, and expression of bacterial cell densities per root canal volume.
6.1.1. Design
This study was designed as a randomized, double-blinded clinical trial. MT AD was tested
and compared to sterile saline (negative control). In addition, 2% CHX gel was used as
intracanal medication. Although to test its efficacy would necessitate a control group (e.g.
no or alternative medication), the increased sample size that would be required was
considered unattainable. Therefore, the design precludes a definitive conclusion
regarding the efficacy of CHX.
72
6.1.2. Inclusion/exclusion Criteria
The study cohort included subjects with all types of teeth except maxillary molars, with
and without symptoms, and with clinical and radiographic signs of apical periodontitis.
Restriction of the study to single-rooted teeth might have reduced the variability of the
results; nevertheless, inclusion of the different tooth types was believed to closer
represent the general population. Mandibular molars were included only if apical
periodontitis was associated with both the mesial and distal roots, and pulp necrosis was
confirmed upon access of all canals. Maxillary molars were excluded to avoid the
challenges they would present in terms of anatomy, variability of canal volumes and
treatment time. Although symptomatic teeth may harbor higher concentrations of
cultivable bacteria than asymptomatic teeth (29, 33), exclusion of the symptomatic teeth
would have restricted the recruitment of subjects for this study. To facilitate analysis and
interpretation of the data, only one tooth per subject was included (329).
6.1.3. Prevention of False Positive Root Canal Samples
Because root canal samples are obtained through an access cavity drilled through the
occlusal surface of the tooth, thorough decontamination of the tooth surface is required to
prevent false positive root canal cultures (305). In addition to surface decontamination,
the access cavity is also recommended to be decontaminated before root canal samples
are acquired (305). Thus, the protocol includes a two-step decontamination procedure, of
the surface and the access cavity after removal of caries and old restorations and before
penetrating into the pulp chamber. At each of these steps, control samples are obtained by
pressing a cotton pellet against the tested surface, to ascertain its sterility. Moller (305)
73
tested different decontamination protocols, and concluded that the isolation of the tooth
with a rubber dam, removal of organic material from the tooth surface using 30%
hydrogen peroxide for 1-2 minutes, preparation of the preliminary access cavity, removal
of caries and defective restorations and repeated decontamination using 30% hydrogen
peroxide for 10-15 seconds followed by 5% orl 0% iodine tincture for 2 minutes gave the
best results.
Over the years, different researchers have modified Moller's protocol. Ng et al. (306)
compared the efficacy of surface decontamination with 2.5% NaOCl and 10% tincture of
iodine, and found them comparable when a culture method was used; however, a superior
effectiveness of the NaOCl in surface decontamination was demonstrated when PCR
methods were used for evaluation. Ng's protocol using NaOCl, was used with some
modifications in several studies (6, 14, 330).
In the present study the operative field including rubber dam, clamp and tooth, were
decontaminated during each treatment session. Two decontamination steps were
performed, at the beginning of treatment and after caries removal, before penetrating the
pulp chamber, in accordance with Moller's protocol (305) and the majority of previous
studies (2, 5, 25, 27, 29, 32, 34, 36, 73, 74, 107).
Although several researchers have followed a strict field disinfection protocol, they have
not reported on surface or access cavity control samples (2, 25, 27, 32, 40, 119, 232).
74
In this study, the surface control samples were obtained by rubbing the tooth and rubber
dam surface with three sterile cotton pellets which were then inoculated separately in
three different ways on culture media, in an attempt to compare the recovery level of
bacteria with each method. At the second step the access cavity control samples were
obtained with aspiration technique following Paquette's protocol .
Many of the previous studies have reported no growth in the surface or access cavity
control samples (5, 6, 8, 11, 26, 33, 36, 73, 74, 262, 331). In contrast, in the present study
bacteria were recovered in 10% to 40% of the surface control samples, and in 3% to 43%
of the access cavity samples, in agreement with several other studies reporting 7% to 50
% of positive surface or access cavity control cultures (10, 34, 306, 332, 333). According
to Moller (305), the ability to decontaminate the surface depends on the restorative
condition of the tooth, with the highest chance for no-growth cultures (98%) in teeth with
intact crowns, and the lowest (83%) in teeth with fillings, surface roughness and crevices
present. For the latter, surface controls are more critical than for the former (305).
In some of the previous studies (14, 15, 334), subjects whose surface or cavity control
samples were positive have been excluded, on the premise of preventing false positive
root canal samples. In the present study, these subjects were not excluded, as in several
other studies (10, 34, 332, 333). Since, after removal of the caries and defective
restorations, an additional step of decontamination was attempted, none of the subjects
with initial positive surface controls were excluded. Moreover, following Paquette's
protocol the subjects with positive access cavity samples were not excluded either.
75
Paquette et al. (34) have suggested that when root canal samples are acquired with an
aspiration needle, as in the present study, there is less risk of false positive samples
because the needle does not contact the access cavity wall and tooth surface.
The discrepancies in the recovery of the bacteria from the decontaminated surfaces
obtained with three cotton pellets precluded any definitive conclusion on the superiority
of one method over the others and reflected the limitations of sampling techniques.
Moreover, the occurrence of different positive access cavity control samples obtained
with a more effective method (aspiration) than that used in the majority of previous
studies (paper points or cotton pellets), highlighted the limitations of the topically applied
antiseptics in killing all bacteria on the exposed surface, although that surface was rather
regular and easily accessible. By extension, this finding indicated that in the irregular and
largely inaccessible root canal space, topical antiseptics should not be expected to kill all
the bacteria.
To summarize the prevention attempts of false positive samples, it should be noted that
the tooth surface and access cavity can be decontaminated at an early stage, but they may
then become re-contaminated when the infected pulp chamber is entered, or when root
canal preparation is initiated without antibacterial agents. To address the repeated
contamination, the surface and access cavity should be decontaminated again after each
one of the above steps, or better still, before each root canal sample is acquired. This
protocol would be very laborious, and it would still not preclude the possibility of false
positive samples (305, 306).
76
6.1.4. Prevention of False Negative Root Canal Samples
Bacteria may not be readily recoverable from the root canal system. When harbored in
the dentinal tubules (335-337) and in inaccessible niches within the root canal system
(338, 339), bacteria are difficult to retrieve by in situ sampling procedures. When bacteria
are recovered, in order to grow on culture plates they must survive not only the effects of
antiseptic agents used in treatment, but also the stresses caused by changes in
environment consequent to exposure of the root canals, depletion of substrate, sampling
and transportation (59). When severely stressed, viable bacteria in culture plates may not
grow at all (340, 341), or grow very slowly exceeding the usual incubation period of 7-10
days (34, 262), particularly when the samples are excessively diluted in the laboratory.
Furthermore, over 50% of root canal bacteria are not cultivable in culture plates (16,
301). In addition, antibacterial agents used during treatment may be carried over into the
samples and prevent bacterial growth in culture plates, unless inactivating agents are used
prior to acquisition of the samples (304, 305). The above considerations suggest the
importance of the methods of sample acquisition and enumeration, and inactivation of
antibacterial agents, for the prevention of false negative samples.
In contrast to sample acquisition with paper points used in the majority of studies, root
canal samples in this study were acquired by aspiration, originally described by Le Goff
et al. (12). Paquette's method (34) was followed with minor modifications. Because the
canal space is immersed in the sampling fluid (RTF) that is later aspirated, the aspiration
method may recover bacteria from areas that would be inaccessible to paper points.
Indeed, it has been shown to recover higher numbers of bacteria than the paper point
77
method (308); thus, the aspiration method might reduce the risk of false negative
samples. In addition, specific steps were performed to maximize bacterial recovery,
including stirring of the canal content with a file (305), and back and forth pumping of
the injected transport medium with the piston of the syringe.
All the studies have reported dramatic reductions in bacterial cell densities after
chemomechanical preparation of infected root canals. With low bacterial numbers, the
least feasible dilution of the acquired samples is recommended to minimize the risk of
false negative samples (59, 61, 304, 342). Less dilution would also have an impact on the
accuracy of bacterial quantification. In this study, samples lB, IC, 2A and 2B, which
comprised the main core of the study but where bacterial numbers were low, were diluted
25% less than in Paquette et al. (34). This was believed to improve the accuracy of
enumeration and to reduce the risk of false negative samples.
The maJor enumeration method used in this study was epifluorescent microscopy,
compared to CFU counts in all of the previous studies except one (34). Direct
microscopic bacterial enumeration has opened a new era for the detection and
quantification of bacteria, and its value has been well demonstrated (46, 47, 50).
However, the microscopy method is very time consuming because samples must be
processed immediately and images captured on the same day of the experiment, requiring
over 12 hours. Moreover the visual enumeration of bacteria on a screen is also arduous.
The enumeration process can be greatly facilitated, and errors can be minimized by use of
an automated counting program. Such a program was used in the present study, whereas
78
manual and semi-automated counting was used in Paquette et al. (53). Over 240 digital
images from each root canal sample were captured and analyzed, compared to 120
images in Paquette et al. (53). Thus, a minimum of 1400 digital images was processed
from each tooth, to improve the accuracy of bacterial enumeration. In addition to
microscopy, cultures were processed as in all previous studies, and incubated for 14 days.
CFU's were counted both on the 7-day and 14-day of incubation, to allow comparison of
the results with other studies.
Because bacterial growth on culture plates can be inhibited by antibacterial agents carried
from the root canal into the samples, inactivation of these agents before sampling is
required to minimize false negative cultures (59, 175, 305). Tincture of iodine and NaOCl
are inactivated using 5% sodium thiosulfate (305), and CHX is inactivated with a mixture
of I-a-Lecithin and Tween 80 (175). These inactivation protocols were applied in the
present study. However, MTAD was not inactivated, because there is no suitable
compound available for its inactivation (140, 141, 144, 146, 150, 152). To overcome this
limitation, the root canals were thoroughly rinsed with 3mL of saline before sampling, in
an attempt to minimize residual MT AD and its carry over into the samples. The efficacy
of such rinse, and the potential substantive effect of the doxycycline component of
MTAD (141), were not addressed in this study. A period without antibacterial medication
following the MT AD regimen could be used to circumvent the inability to inactivate it,
by allowing surviving bacteria to regrow (10, 304, 333). However, this approach was
considered unethical, as it would have added a treatment session and potentially
jeopardized the efforts to maximize bacterial elimination during treatment.
79
Notwithstanding the potential effect of residual MTAD on bacteria in cultures, it may
have no effect on enumeration by microscopy that detects live bacteria without reliance
on their cultivability. Although theoretically MTAD, if carried-over into the samples,
might still affect the counts, it is unlikely that bacteria that have survived 5-minute
exposure to fully concentrated MTAD would be killed by a shorter exposure of highly
diluted residue of MT AD in the samples, particularly in the favorable transport fluid
environment of the sample.
6.1.5. Quantification of Bacterial Densities
In this study, root canal volume was measured at the different stages of sampling, and
bacterial densities were reported per 1 µL of canal volume, as opposed to densities per
volume of transport medium or per tooth as reported in all previous studies but one (34).
The mean root canal volume increased by 60% from the beginning of treatment (sample
IA), to the completion of canal preparation (samples lB, 1 C, 2A and 2B). Consideration
of this change in canal volume, and the anatomic variation among canals even within the
same tooth, suggests that expression of bacterial densities in reference to canal volume
better reflects the reality of the infected root canal than reference to an arbitrary sample
unit volume or the tooth. It also may become critically important if a threshold of
bacterial density that correlates with healing should ever become established. Such
correlation has thus far been suggested for positive or no-growth cultures (106, 343), but
in the future attempts may be made to establish a maximum bacterial density that may not
impede healing.
80
The in situ measurement of canal volume in the present study may be questioned, in view
of the smaller volume after than before canal preparation found in 10% of the study
sample. This inconsistency might have resulted from entrapment of air bubbles in the
canal during vol 1 B measurement, or from the difficulty associated with pre and post
measurement of the orifice levels, especially in molar teeth. The in situ measurement has
also been challenged by comparison to canal volumes measured in vitro using
microcomputed tomography (MCT) (344-346). The MCT-measured mean increase in
volume after canal preparation was 1.21 µL, while in the present study it was 7.64 µL.
However, the discrepancy between the MCT-measured and the in situ measured volumes
most likely resulted from differences in tooth types and canal preparation strategies used
in the in vitro studies (344-346) and in the present study. It would be sufficient to
consider the extent of coronal flaring of the canals (minimal in the in vitro studies and
extensive in this study), as accounting for much of the discrepancy. The accuracy of in
situ volume measurement especially at the early stage of treatment (IA) may also be
questioned because the presence of necrotic pulp tissue, and draining exudates from the
periapical area into the canal would interfere with filling the canal space with liquid,
which is the basis for measuring the canal volume in situ. None of these factors would
affect the MCT measured volume. The measurements carried out after canal preparation,
in particular, are less subject to error because none of the aforementioned factors that
interfere with measurement accuracy before preparation exist. In fact, the post
preparation canal volumes measured in this study were rather close to those of single
rooted teeth in a recent study (34 7), where the method of measurement was not described.
They were also close to the values reported in the only study where canal volume was
81
measured in situ (34) as in this study (16.13 µLand 18.75 µL, respectively). Importantly,
the post-preparation volumes are critical for realistically expressing the low bacterial
densities at this stage of treatment and their potential relation with healing or non-healing,
whereas the initial canal volumes are less critical when the bacterial densities are
abundant.
6.1.6. Clinical Procedures and Materials
To assess the antibacterial efficacy of a final rinse/immersion of the root canal with
MTAD, a control group was introduced where canals were finally rinsed with saline.
Following the manufacturer-recommended regimen for application of MTAD, canals
were primarily irrigated with 1.3% NaOCl. NaOCl is an effective antibacterial agent (36,
248), and concentrations from 0.5% to 6% appear to be equally effective (74, 110, 119).
Although its tissue dissolution effect is enhanced with higher concentrations (129),
increased volume and exposure time can compensate for lower concentration (135). In a
regular course of canal preparation, 1 % NaOCl is sufficient to affect total dissolution of
pulp remnants (38, 129). In this study, 1.5 mL of irrigant was used after each
instrumentation step, for a minimum of 10.5 mL for each canal.
Although MT AD is intended to allow completion of treatment of infected teeth in one
session, it is unrealistic to expect all dentists to be able to do so; therefore, use of
intracanal medication may still be necessary after the use of MTAD. In this study, canals
were medicated with CHX gel, following the suggestion from Paquette's study where
CHX liquid was used (34). CHX gel has shown an excellent performance in vitro (210,
82
214, 236), and its physical properties satisfy the requirements for intracanal medication
(279). It was also suggested to prevent bacterial regrowth in vivo ( 6).
Further root canal irrigation with 1.3% NaOCl and the final sample 2B were implemented
following the observation of bacterial regrowth during the intracanal medication period in
Paquette's study (34). This regimen was consistent with that performed in the second half
of the subjects in Paquette's study (34), and with previous studies (2, 36, 119).
6.1.7. Outcome Measures
Two outcome measures were used in the present study. Both the proportions of positive
cultures and bacterial counts are measures of the bacterial load in canals (2, 6, 14, 15, 25,
26, 33, 34). Correlation of the outcomes recorded with both measures is usually expected.
6.2. The Findings
6.2.1. Differences in Enumeration Methods
For the cultures method, the 14-day CFU counts were consistently higher than the 7-day
counts, suggesting the presence of bacteria that require more than one week to grow on
blood agar plates. For the microscopy method, only the live bacterial counts can be
compared with the CFU counts that can only detect live bacteria (50, 56). The higher 14-
day CFU counts were used for this comparison.
Across all the samples analyzed, the mean live bacterial densities obtained with
microscopy were consistently higher than the mean CFU counts. At the beginning of
83
treatment (sample lA), the microscopic densities were 6 to 10-fold higher. In contrast, in
all the subsequent steps of treatment where bacteria were less abundant, the microscopic
densities were 1,600 times (sample 1 C, MTAD group, DHET staining) to 68,000 times
(sample 2B, saline group, BacLight staining) higher than the respective CFU counts. This
consistent difference between the two methods of enumeration was in agreement with
previous reports ( 46, 48-50, 52). It might be explained by the ability of the microscopy
method to detect bacteria, that may be viable but not cultivable (VBNC) (16, 56, 340)
partly due to severe stress and possible damage. Such would be the case after the bacteria
are exposed to antibacterial agents used for canal preparation (NaOCl - sample lB), final
rinse (MTAD - sample 1 C) and intracanal medication (CHX - samples 2A and 2B).
The marked difference in results obtained with the two enumeration methods underlined
the limitations and possible unsuitability of the culture method for monitoring root canal
bacteria during endodontic treatment. This limitation was well demonstrated by the
finding of one lA sample (3%) that showed no bacterial growth in culture, a consistent
finding with the 2% to 16% of no-growth cultures in the initial samples of several
previous studies (2, 10, 11, 25, 27, 29, 32, 33, 304, 331, 332). In contrast, abundant
bacteria in the same sample were detected by microscopy. Furthermore, over 50% of no
growth samples were recorded in each of the subsequent samples 1 B, 1 C, 2A and 2B,
whereas microscopy detected bacteria in all these samples.
On the other hand, the difference between microscopic and CFU counts might have been
heightened by false-positive microscopic detection of autofluorescent and detrital
84
material, that may be present in the samples and be mistaken as bacteria by the automated
programs, particularly when bacterial cells are very few or none (324, 326).
Comparing the two staining methods used with microscopy, more live bacteria were
consistently detected when stained with Baclight (Syto 9) than with DHET, in agreement
with all the samples except lA in Paquette's study (34). In contrast, more dead bacteria
were consistently detected when stained with DAPI/DHET than with Baclight (propidium
iodide), in agreement with Paquette's study (53). The difference between the two staining
methods is explained by their different mode of action. Bacteria must have an intact
membrane to be stained as live by Syto 9 (BacLight), whereas they must be metabolically
active to be detected by DHET. Thus, the DHET staining appeared to be more stringent,
as non-metabolizing bacteria with an intact membrane would still be detected as live with
BacLight, resulting in higher counts compared with DHET.
Because of the systematic differences in results recorded with the different enumeration
methods, and in order to streamline the discussion of the results, bacterial densities
obtained with DHET staining only are used to discuss the antibacterial efficacy of the
various treatment steps. CFU's are used where comparison with other studies is
applicable, particularly in regards to the proportion of positive and no-growth cultures.
When appropriate, 14-day rather than 7-day culture results are discussed.
85
6.2.2. Efficacy of Canal Preparation with 1.3% NaOCl Irrigation
At the beginning of canal preparation, bacteria in culture were observed in 97% of root
canal samples, confirming the association of apical periodontitis with root canal
infection, while also highlighting the limitations of the culture method. Root canal
preparation with copious 1.3% NaOCl irrigation affected a decrease of the positive
cultures to 7% after 7-day incubation. Except for one study where no positive cultures
were found at this stage of treatment in canine and premolar teeth (27), this proportion
was lower than the range of 11 % to 63% of positive cultures reported after canal
preparation using various concentrations of NaOCl in other studies (2, 6, 8, 25, 27, 34,
40, 74, 119, 332). After 14-day incubation, the proportion of positive cultures was 36%,
compared to 48% to 63% in other studies (14, 34, 262, 334). In spite of the apparent
better performance in the present study, canal preparation clearly failed in eliminating all
viable bacteria. This limitation was underlined by the microscopy finding of viable
bacteria after canal preparation in all of the teeth. Furthermore, the fact that nearly 40%
of bacteria remained cultivable indicated that they were not severely damaged.
The bacterial densities at the beginning and end of canal preparation are discussed mainly
for reference to other studies, even though the methodological differences among the
studies preclude most direct comparisons. The mean CFU counts per µL of canal volume
in 14-day cultures were comparable to those in Paquette's study (34), both before
(2.08x105 and 2.29x105, respectively) and after canal preparation (1.62 and 5.38,
respectively), even though a lower concentration ofNaOCl was used in the present study.
For reference to all other studies, bacterial densities are expressed per tooth. Before canal
86
preparation, the mean bacterial density in 7-day culture was 1.47x106 per tooth, within
the range of6.31x104 to 6x107 reported in previous studies (2, 6, 25-27, 32-34, 331). The
mean CFU value could not be compared to the median value reported in several other
studies (5, 40, 73, 74). The mean density in 14-day culture in the present study was
1.59x106 per tooth, lower than the range of 3.85x106 to 1.99x107 reported in several
studies (13-15, 334) (In the four studies by Siqueira and co-workers, the mean CFU
counts were calculated based on their reported CFU counts for individual samples).
As expected, the mean CFU count after root canal preparation with copious 1.3% NaOCl
was reduced from 1.47x106 to 5.44 per tooth in 7-day culture. This value was lower than
the range of 1.86x 101 to 6.60x106 reported in all previous studies (2, 6, 25, 26, 34). Also
the mean count of 7.62 x 101, calculated from the only two positive cultures, was lower
than the 1.8x103 reported after similar calculation in another study (26). Similarly, the
mean CFU count of 2.l 8x 101 in 14-day culture was lower than the range of l.14x 102 to
9.36x105 reported in previous studies with similar incubation periods (13-15, 334). It is
noteworthy that the apparently better antibacterial efficacy of canal preparation in the
present study was achieved with a lower concentration of NaOCl than in the majority of
the previous studies (13-15, 25, 26, 34).
Densities of live bacteria obtained by epifluorescence microscopy can only be related to
those of Paquette's study (34) and, as stated above, only DHET-stained counts are
considered for this comparison. Before canal preparation, the mean density of l.30x106
per µL of canal volume was lower in the present study, compared with l.87x106 in
87
Paquette's study. After canal preparation, the mean density of 4.00x104 was higher,
compared with 9.91x103 in Paquette's study, possibly because of the lower concentration
of NaOCl used. The reduction in counts affected by canal preparation, as observed by
microscopy, was less pronounced than that observed by culture, but was considered to
better reflect the antibacterial efficacy of canal preparation.
The live bacterial density after canal preparation was somewhat different in the MT AD
and saline groups. As suggested by the statistical analysis, the density of bacteria at this
stage of treatment was the only determinant of bacterial densities at the subsequent stages
in both groups.
6.2.3. Efficacy of Final Irrigation/immersion with MT AD
MT AD was introduced in response to the trend in endodontics to complete treatment of
all teeth, including those that are infected, in a single treatment session. This trend
developed in spite of research that highlighted the limited antibacterial efficacy of canal
preparation in one session, and the improved efficacy achieved by use of intracanal
medication between subsequent treatment sessions (5, 40). MTAD, introduced as a final
irrigation/immersion solution, was expected to enhance the antibacterial effect of canal
preparation so that treatment could be completed in one treatment session. Preliminary in
vitro studies (139-141) suggested that a brief exposure of the canal to MTAD reduced the
number of surviving bacteria beyond the levels achieved with conventional canal
preparation, possibly due to substantive activity of doxycycline. Accordingly, the
hypothesis of this study was that a final rinse with MT AD at the end of the first
88
appointment would significantly decrease the number of bacteria from root canals beyond
that achieved by conventional chemo-mechanical cleaning and shaping procedures
performed in the first appointment.
Application of MTAD according to the manufacturer's protocol in the present study, did
not affect a decrease in positive cultures. In fact, the proportion of positive 14-day
cultures was not significantly different before (29%) and after (53%) application of
MTAD, and it did not differ from the control saline group (43% and 57%, respectively).
Similarly, there was no significant decrease in density of live bacteria as shown with
DHET staining, as well as with the other methods of enumeration. The low numbers of
live bacteria before MTAD was applied (4.08x104) certainly undermined the power of
analysis, and made prospects of significant improvement in the subsequent step unlikely;
however, the relatively small difference in the mean densities of live bacteria before
(4.08x104) and after (9.58x103
) application of MTAD suggested that it did not affect a
meaningful antibacterial effect. An added insight into the antibacterial effect of MT AD
was offered by the comparison of the counts of dead bacteria. A significant increase in
the count after application of MT AD would be indicative of a strong antibacterial effect.
The almost similar dead bacterial counts before (3.85x104) and after (5.70x104
)
application of MT AD were not suggestive of a strong antibacterial efficacy either.
Other antibacterial regimens than MT AD have been assessed for their efficacy as a final
rinse in one-session treatment; their results are compared with those of the present study.
In a one study (332), after a 10-minute final rinse with iodine potassium iodide, 29% of
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7-day cultures were positive, compared to 27% after application of MTAD in the present
study. In another study (262), after a 30-second final rinse with 2% CHX liquid only 8%
of 28-day cultures were positive, whereas 58% of the saline control cultures were still
positive. Notably, the saline control results in that study (58%) and the present one (57%)
were similar, while the 2% CHX liquid in the former study (8%) appeared to be more
efficacious than the MTAD in the present study (53%).
Taken together, the results of the present study did not support the hypothesized
antibacterial efficacy of MT AD when applied as a final rinse/immersion in the first
treatment session.
6.2.4. Efficacy of Intracanal Dressing with 2 % Chlorhexidine Gel
In the absence of a control group to assess the efficacy of canal medication with CHX
gel, the results of the present study could only be discussed in relation to Paquette's study
(34), where canals were medicated with CHX liquid, and the study by Manzur et al. (6),
where 2% CHX gel was used as intracanal medication in 11 teeth. Paquette et al. (34)
reported a significant increase in the proportion of positive 14-day cultures, from 48% at
the end of canal preparation, to 68% after medication. In the present study, 55% of all 14-
day cultures obtained after canal medication with CHX gel were positive, similar to the
proportion recorded after canal preparation in the first treatment session. The results
obtained from 7-day cultures were 33% positive which were slightly lower than those of
Manzur et al. (6), who reported 45% of positive post-medication 7-day cultures.
90
Because the mean post-medication CFU counts were close in the MT AD and saline
groups, both groups were pooled to facilitate the discussion. The non-significant decrease
recorded in the present study in mean CFU counts before (6.94 µL- 1 canal volume) and
after (2.94 µL- 1 canal volume) canal medication, was in agreement with the non
significant increase (from 1.46 x 102, to 7.84x102 per tooth) reported by Manzur et al. (6).
In contrast, Paquette et al. (34) reported a significant increase in the mean CFU count
(from 5.38, to l.08x102 µL- 1 canal volume) after medication with 2% CHX liquid.
The microscopy counts of DHET-stained live bacteria in the present study, before
(l.39x104) and after (l.75x104
) medication, were not much different from those in
Paquette's study (9.9lxl03 and 2.24x104, respectively) (34). Also the minor non
significant increase in mean counts of dead bacteria was comparable, for the present
study (9.10x104 and l.8lxl05, respectively) and Paquette et al. (8.23x104 and 3.39x105
,
respectively) (34)
An ideal intracanal medication should possess long-term antimicrobial and suitable
physico-chemical properties, so as to prevent root canal recontamination for extended
periods of time (214, 279). Paquette et al. (34) noted that often, at the beginning of the
second treatment session, the CHX liquid in the canals was depleted, potentially allowing
bacterial growth between treatment sessions. In the past, concerns have been raised
regarding the potential interference of CHX gel residue with the seal of the root canal
filling (210, 214). These concerns have been disputed by an in vitro study (290), where
canals medicated with CHX gel did not demonstrate greater leakage than other canals.
91
Taken together, the results of canal medication with CHX gel in the present study and
those of Manzur et al. (6) appeared to be better relative to Paquette's study (34), in
regards to all outcome measures except the microscopy counts. These results suggested
that CHX gel might be more effective at curtailing bacteria, and thus be better suited as
intracanal medication than CHX liquid. Notwithstanding the apparent ability of CHX gel
to effectively prevent regrowth of bacteria, it did not impart the significant antibacterial
effect expected of an effective intracanal medication. Possibly, the interaction of CHX
gel with root dentin and canal contents resulted in its inactivation or buffering, as
suggested by several in vitro studies (272, 275, 289).
6.2.5. Efficacy of Additional 1.3% NaOCl Irrigation in the Second Treatment
Session
Having access to the root canals at the second treatment session affords an opportunity to
repeat irrigation with an effective antibacterial agent. After such additional irrigation with
1.3% NaOCl, a non-significant decrease in the proportion of positive 14-day cultures was
noted in both groups. With an average of 31 %, the proportion of positive cultures was
similar to the 30% reported by Paquette et al. (34), and close to the 36% recorded in the
present study at the end of the first treatment session.
The mean CFU count at the end of the second treatment sess10n was close to that
recorded at the end of the first treatment session. At 1.20 per µL of canal volume, the 14-
day incubation CFU count was lower than the 7.67 per µL of canal volume reported by
92
Paquette et al. (53). When the mean was calculated for positive 7-day cultures only, the
3.8lx101 CPU per tooth in the present study was lower than the l .4x102 per tooth
reported by Peters et al. (26).
The density of DHET-stained live bacteria obtained with microscopy across all the teeth
at the end of this study, was higher than in Paquette et al. (34), with l .47x104 and
4.1Ox103 per µL of canal volume, respectively. The density of dead bacteria, on the other
hand, was higher in the present study (l.33x105) than in Paquette's study (6. l 7x104
).
Taken together, the results indicated that the second treatment session did not improve
the overall antibacterial efficacy of treatment beyond the end of the first treatment
session. First and foremost, the differences in proportions of positive cultures and live
bacterial counts, regardless of the incubation period and enumeration method, were not
statistically significant among the samples acquired at the end of the first session, the
beginning of the second session, and the end of the second session. Whatever minor
changes may have occurred in the numerical values, mainly increase in positive cultures
and live bacterial counts from the end of the first session to the beginning of the second
session, were merely reversed by the additional irrigation during the second treatment
sess10n.
6.3. General Discussion
The antibacterial effect of MTAD is attributed to its doxycycline component (140).
Doxycycline has also the ability to remove organic and inorganic substances from the
93
root canal walls (140, 168), and this action is enhanced by the presence of citric acid and
a detergent that aids diffusion into the canal and dentinal tubules (140). Doxycycline,
belonging to the tetracycline antibiotic family, is a bacteriostatic agent that prevents
multiplication of susceptible bacteria, and consequently inhibits their growth (348).
Nevertheless, it was speculated that in high concentrations, doxycycline might be
bactericidal (177), and that a local topical application of doxycycline might be different
from systemic administration (141).
The rationale for the present study was based on the excellent antibacterial efficacy of
MTAD reported in early in vitro studies (140, 141), where canals inoculated with
Enterococcus faecalis served as the testing model. In such a model, the canal is populated
mostly with bacteria in a planktonic state; planktonic bacteria are easier to kill than
bacteria organized in resistant biofilms, such as are found in infected teeth. In the 2-year
period between the initiation and completion of this study, several additional in vitro
studies have become available on the antibacterial efficacy of MTAD (142, 145, 146,
150, 151). In several of these studies (144, 146, 150), the antibacterial efficacy of MTAD
was tested on laboratory-grown biofilms of E. faecalis, a more stringent test model than
planktonic bacteria, although still easier than infected teeth in vivo. The majority of those
recent studies have not supported the antibacterial efficacy of MT AD. MTAD in these
studies was reportedly inferior to 1 % to 6% NaOCl (146, 150), and 2% CHX (146). The
manufacturer-recommended irrigation sequence of 1.3% NaOCl/MTAD was found to be
inferior to a sequence of 5.25% NaOCl/15% EDTA in a culture-based study (142, 151). It
has been suggested that exposure of dentin to 1.3% NaOCl before interaction with
94
MTAD may reduce the MTAD-imparted substantive antimicrobial activity by 30%,
relative to unexposed dentin (160). Treating the dentin with ascorbic acid or another anti
oxidant prior to interaction with MT AD may minimize the inhibitory effect of prior
exposure to NaOCl. In addition, the antimicrobial activity of MTAD may be partly
inhibited by the buffering effect of dentin and serum albumin present in the root canal
(156, 177).
Another potential concern for the antibacterial efficacy of MT AD may be the Tween-80
component, added to decrease surface tension of the compound (140). While it was
suggested that Tween-80, citric acid and doxycycline - all three components of MT AD -
might have a synergistic effect on the bacterial cell wall and the cytoplasmic membrane
(177), a concern was raised that Tween detergents, including Tween-80, might act as a
nutrient for specific bacteria (173). In another study (36), where bacteria were isolated
from infected root canals and identified after cultivation using different culture media,
"one group of organisms ... grew poorly in PYG broth unless this was supplemented with
Tween-80". At this stage of research of MTAD, it is essentially unknown whether
inclusion ofTween-80 in the formula is beneficial or has an adverse effect.
The ability of MTAD to remove the smear layer (137) suggests an additional concern for
its antibacterial efficacy. The smear layer - covering the inner surface of canals following
instrumentation - consists of inorganic and organic substances ( 123, 124, 349), including
bacteria and their by-products. While removal of the smear layer may eliminate
95
embedded bacteria (349, 350), it may also allow bacteria within the dentinal tubules to
recolonize the main canal space (335), offsetting the former, beneficial effect.
Although in vitro models are useful when evaluating potency and spectrum of activity,
testing antimicrobial agents under in vivo conditions is required to establish their efficacy
(146). Set against the background of previous in vitro studies with conflicting
conclusions, the present in vivo study results questioned the antibacterial efficacy of
MTAD, and thus the added benefit of its application. These results, the first reported in
an in vivo clinical trial designed specifically to test the antibacterial efficacy of MTAD,
did not support the conclusions of the early in vitro studies (140, 141), but rather those of
the later ones (142, 145, 146, 150, 151). An argument could be made that MTAD should
be used as a final rinse just before root filling, whereas in this study, MTAD was rinsed
out with sterile saline, which may have mitigated the antimicrobial effect of MTAD. If
indeed this rinse - required by the methodology used in this study- had any effect, this
would have been on the substantive effect of MT AD as assessed at the beginning of the
second session, but a mitigating effect was unlikely to affect the sampling results at the
end of the first session, when the MT AD regimen was just completed.
Our result once again emphasized the importance of carrying out in vivo assessment
before any root canal disinfection regimen can be established as having superior
antimicrobial efficacy. The ultimate test, however, should be a randomized controlled
trial to assess healing after treatment with and without application of MTAD. Taking into
consideration the good prognosis of treatment without MTAD, with a probability of
96
complete healing over 80% (351), such a randomized controlled trial would require a
minimal sample of 400 subjects in each group (without factoring attrition) to have
adequate statistical power (90%). An extensive follow-up period of four to six years
would be required. Considering these logistical obstacles, it is unlikely that such a
randomized controlled trial will become available in the near future. In the absence of
such research, the results of the present study provide the only surrogate outcome
assessment of the antibacterial efficacy of MT AD available to date.
Tetracycline, as well as macrolides and chloramphenicol, is known to have an
antagonistic effect on penicillin, and it has been shown to reduce the antibacterial
effectiveness of penicillin in the treatment of pneumococcal and other infective diseases
(352-357). As the mechanism of penicillin's action is by stopping bacterial cell wall
synthesis, it is mostly effective against actively growing cells. A bacteriostatic agent such
as tetracycline acts to suppress bacterial growth, and hence also the action of penicillin
(348, 353). The extent of antagonistic effects among other anti-infective agents, if any,
remains uncertain (348).
A potential concern, of an antagonistic effect of MT AD on the bactericidal activity of
CHX, may be unfounded considering the mode of action of CHX. In high concentrations
like those used in this study, CHX is bactericidal (193). It acts to damage and
permeabilize the bacterial cell wall (196-199), then penetrates into the cell and causes
precipitation of the cytoplasm, preventing repair of the cell membrane and leading to
bacterial death (192, 193, 195). Thus, the action of CHX is unlikely to be inhibited by
97
doxycycline, and to the best of our knowledge, there is no indication in the literature of
such an antagonistic effect.
This study and the previous ones have highlighted the methodological challenges in the
area of clinical endodontic research focused on microbiology. Not only that over 50% of
root canal bacteria are non-cultivable (16, 301 ), and those that are may be stressed and no
longer grow in culture (16, 358-361), but also the widely used sampling techniques are
inadequate for recovering bacteria from the complex root canal space. Because root canal
disinfection is the main focus of endodontic treatment, it would appear appropriate to
develop a universal standard for conducting studies aimed at assessing the antibacterial
efficacy of endodontic treatment regimens. As a first step, ex vivo studies should be
conducted to establish correlations between patterns observed by different root canal
sampling methods (paper points, aspiration), enumeration methods (culture, microscopy,
PCR, flow cytometry), and transmission electron microscopy. The latter can visually
confirm or exclude presence of bacteria, as well as provide insight into their viability by
identifying cells in state of division (4). Such correlative research would provide the basis
for designing a research protocol, and also tools for better interpretation of results
obtained from sampling of root canal bacteria.
On the basis of the results of this study, it is suggested that the methodology for future
studies should include the following: (1) minimum of 14-day incubation if culture is
used, to allow recovery of slow growing bacteria (13-15, 34, 262, 334); (2) reduced
dilutions of the samples, especially in treatment stages where low numbers of bacteria are
98
expected (59); and (3) addition of a non-germicidal surfactant to the medium placed into
canals for sample acquisition, to improve distribution throughout the canal space. If
epifluorescent microscopy should be used in future studies, it may be standardized to
include the following suggestions: (1) attempts should be made to count all the bacteria
on filters, which best reflect the concentration of the bacteria in the samples; (2) the
diameter of the funnel and filter should be reduced by half or by quarter, to concentrate
the same number of bacteria on a X4 or X16 smaller surface, and thus improve the
precision of the counts, which would be closer to the real number of bacteria in the
samples; (3) more sensitive staining methods should be developed, that target the bacteria
without detecting extraneous substances in the samples; ( 4) improved automated methods
for acquiring the microscopic images should be used, to be applied in randomization of
field selection, focusing, capturing and saving multiple images on drives. Such may
include automated stages and deconvolution programs to enhance the number and quality
of captured images, and to minimize labour and potential human error. Ultimately, such
methods may capture images from the entire surface of the filters, count all the bacteria
present, perform statistical analysis and establish a baseline for future studies; and (5)
automated image analysis programs and enumerating techniques should be used to
distinguish bacteria from interfering fluorescing objects, and to detect individual bacterial
cells within overlapping bacterial aggregates.
In summary, the relative plateau observed in the antibacterial effect exerted by the
endodontic treatment regimen used in the present study confirmed the critical role of
canal preparation - instrumentation and irrigation with NaOCl - in canal disinfection.
99
Beyond this critical step, it remains questionable whether additional procedures, if any,
add a significant antibacterial advantage.
CONCLUSIONS
100
101
7. CONCLUSIONS
1- A final rinse with MT AD did not reduce bacterial counts, nor did it decrease the
proportion of positive cultures in teeth with apical periodontitis beyond levels achieved
by chemomechanical canal preparation using 1.3% NaOCl irrigation.
2- A 7-day intracanal medication with 2 % chlorhexidine gel between treatment
appointments with prior rinse with MT AD or without MT AD curtailed bacterial
regrowth, but did not further decrease the proportion of teeth with positive cultures, nor
did it reduce the bacterial counts beyond that achieved after chemomechanical
preparation.
3- Chlorhexidine in gel form seems better suited as intra canal medication compared to
the liquid form. All medicated teeth showed the presence of gel at the beginning of the
second appointment in contrast to a Paquette's study. As opposed to the same study
where a significant increase in number of bacteria after application of 2% chlorhexidine
liquid as intracanal medication was noted, there was no significant difference after
application of2% chlorhexidine in gel form.(34).
4- Bacterial quantification using epifluorescence microscopy yielded considerably higher
cell counts and lower negative samples than the culture method. Given the widespread
use of the culture method, a combination of both should still be considered in future
research.
102
5- The counts of dead bacteria obtained with the dihydroethidium and DAPI staining
method were consistently higher than those obtained by propidium iodide of the BacLight
staining method. And vice versa, the group representing live bacteria obtained with
Baclight staining method was consistently higher than that enumerated with
dihydroethidium and DAPI.
6- When using the culture method in this study, the 14-days incubation period resulted in
higher CFU counts than the 7-days period, suggesting the presence of slowly growing
bacteria in the samples.
7- The automated, microscopy-based method was reasonably accurate in enumeration of
bacteria in infected root canals.
REFERENCES
103
104
8. REFERENCE LIST
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TABLES
125
9. TABLES
Table 1. Inclusion and exclusion criteria
Patient
I-ASA I or ASA II 2-Age above 18 years old 3-Consent to participate in the study 4-Cotnpliance 5-Good oral hygiene
I-Adequate coronal structure 2-No infrabony defects deeper than 5tntn
. requit~ment ·· 3-Radiographic evidence of apical periodontitis 4- Negative response to cold test
Exclusion
I-ASA III and above 2-Pregnant\Votnen 3-Nursing tnothers 4-Cognitively itnpaired
126
5-Kno\Vn allergy to chlorhexidine, tetracycline ( doxycycline ), citric acid, T\Veen80
I-Previous endodontically treated 2-Abnormal anatotnical variation 3-Maxillary Molars 4-Marked internal or external root resorption
127
Table 2. Treatment sampling steps from root canal/s of teeth in each treatment
session
· 2nd session
lA: before instrumentation lB: after instrumentation and 1.3 % NaOCl 1 C: after final rinse (MT AD or saline)
2A: after removal of CHX 2B: before root filling, after final instrumentation and 1.3 % NaOCl
128
Table 3. Aliquots taken from original sample (approximately 1.3mL in microtube)
and used to prepare final subsamples
< .···
Ji) APl/DH£T BacLight CFU anaerobic or
1", aerobic .. < .• . <· ...
aliquot RTF aliquot RTF aliquot RTF µL µL µL µL µL µL
lA 200 800 200 800 250 250
1B,iC~2A,2B 400 600 400 600 250 250 ..
AC1,.AC2 300 700 300 700 250 250
The final volume of subsamples prepared for each microscopy staining method was lOOOµL (volume of aliquot plus volume of added RTF} and for each culture method was 500µL (volume of aliquot plus volume of added RTF).
i
129
Table 4. Access Cavity samples: Means and standard deviations of the bacterial counts per sample and proportion of positive cultures obtained from Access Cavities at two treatment sessions with different enumeration techniques
.. Coµ.IJ.tper acc~ss::c~vity ~.Proportion of positives cultures
iti . ... .. •i i
A Cl AC2
Aerobic 5.97 x101 1.27 ±2.92x102 ± 6.96
7d
Anaerobic 2.29 Xl01 1.02 xl01
±6.22x101 ±2.99 x101
€FU. . 7.llx101 8.89
Aerobic ±2.92 x102 ± 2.17 x101 i
14d ·.·
Anaerobic 3.30 x101 2.41 x101
i
±6.23 x101 ±3.81 x101
i
Live 2.62 x 105 1.08 x 105
±5.77 x 105 ±1.49 x 105
1lA,:rJ4>JIET Dead 1.75 x 106 2.24 x 106
±2.81x106 ±5.96 x 106 '\I • h
i 2.01 x 106 2.35 x 106
Total ±2.76 x 106 ±6.06 x 106 ·.
. 1.15 x 106 3.12 x 105
Live ±3.41 x 106 ±2.39 x 105
..
Baclig~t Dead 1.21x105 7.83 x 104
±3.58 x 105 ±9.17 x 104
Total 1.27 x 106 3.90 x 105
±3.75 x 106 ±2.69 x 105
A Cl= Access cavity sample at the first treatment session AC2= Access cavity sample at the second treatment session
.·
A Cl AC2
13.3% 3.3%
23.3% 16.7%
30.0% 16.7%
43.3% 40.0%
'
...
130
Table 5: Root canal samples: Means and standard deviations of the bacterial counts per µ,L of root canal volume obtained at different treatment steps and enumerated with different techniques for MT AD and Saline groups
tA tB '" tc · .. 2A 2B :.
MTAD 3.26 x105 1.72 x10·1 2.55 1.63 2.00 x10·1
±5.69 x105 ±6.44 x10"1 ±5.20 ±3.35 ±7.75 xlO·l 7d
Saline 4.61 x104 6.08 xl0-1 5.49 1.73 1.63 xl0-1
±9.62 x104 ±2.27 ±9.26 ±2.58 ±6.11 x10·1
CFU
MTAD 3.52 x105 9.37 xlQ·l 6.04 3.71 1.59 ±5.83 x105 ±1.74 ±1.13 x101 ±4.69 ±2.32
14d
Saline 5.41 xl04 2.30 6.66 2.11 7.82 x10·1
±1.04 x105 ±3.45 ±1.01 x101 ±2.50 ±1.59
MTAD 1.51x106 4.08 x 104 9.58 x 103 1.79 x 104 1.86 x 104
±1.98 x 106 ±7.75 x 104 ±8.75 x 103 ±2.76 x 104 ±2.29 x 104
•<: Live 1.01 x 106 3.66 x 104 1.73 x 104 1.72 x 104 1.30 x 104
Saline ±1.21 x 106 ±6.67 x 104 ±2.71x104 ±2.70 x 104 ±2.29 x 104
. MTAD 4.40 x 106 3.85 x 104 5.70 x 104 2.27 x 105 2.13 x 105
·:. ±3.93 x 106 ±5.60 x 104 ±4.95 x 104 ±4.56 x 105 ±7.00 x 105
DAPIIDRET. Dead
Saline 1.95 x 106 1.03 x 105 1.22 x 105 1.32 x 105 4.79 x 104
±1.75 x 106 ±2.75 x 105 ±3.25 x 105 ±2.03 x 105 ±6.47 x 104
>· 5.90 x 106 7.93 x 104 6.66 x 104 2.45 x 105 2.32 x 105
MTAD ±4.23 x 106 ±8.39 x 104 ±5.24 x 104 ±4.65 x 105 ±6.96 x 105
Total
Saline 2.96 x 106 1.40 x 105 1.39 x 105 1.50 x 105 6.09 x 104
±2.18 x 106 ±3.24 x 105 ±3.31x105 ±2.13 x 105 ±7.82 x 104
..
MTAD 2.50 x 106 9.76 x 104 2.07 x 104 1.57 x 105 3.88 x 104
. ±3.11x106 ±1.29 x 105 ±1.68 x 104 ±3.56 x 104 ±5.33 x 104
Live 5.33 x 104
Saline 1.41x106 7.90 x 104 5.53 x 104 3.56 x 104
±2.60 x 106 ±1.46 x 105 ±9.34 x 104 ±4.33 x 104 ±5.74x 104
2.97 x 106 1.13 x 104 4.46 x 103 8.18 x 104 5.28 x 103
' MTAD ±3.30 x 106 ±1.43 x 104 ±3.93 x 103 ±2.41x105 ±4.21x103
Baclight Dead 2.72 x 104 6.18 x 103
Saline 1.11 x 106 6.53 x 104 6.30 x 103
I . ±1.10 x 106 ±2.19 x 105 ±1.08 x 104 ±7.81x104 ±9.93 x 103
MTAD 5.47 x 106 1.09 x 105 2.51 x 104 2.39 x 105 4.40 x 104
±5.44 x 106 ±1.34 x 105 ±1.89 x 104 ±7.14x 105 ±6.48 x 104
Total :•. Saline
2.52 x 106 1.44 x 105 6.16x 104 6.29 x 104 5.95 x 104
±2.96 x 106 ±2.61 x 105 ±1.04 x 105 ±1.11 x 105 ±6.48 x 104
:
131
Table 6. Comparison between number of bacteria per µL of root canal volume after natural logarithm transformation for MTAD and Saline treatment steps lA and lB
".rreatllienf Enhrne~.ation N Mean Meitn . Nt!i'Xl>-
step te~~nlij~e • . . . M:J'AD Control Saline
1A ''';
· CF:U7d. 15-14 10.25 6.95
ti\ 'CFU14d 15-14 10.46 8.02
1A. Syto9Uve 15-15 13.68 12.62
lA Pl1dead 15-15 14.27 12.94
lA . BI;~:totaJ 15-15 14.99 13.95
lA ·DHE".rUve 15-15 13.27 12.56 >L': ,, i'i' ,,~":<
lA< DAflfotai 15-15 15.25 14.52
IA DD11idead 15-15 13.66 13.81
1B CFU7d 14-14 -4.23 -4.12
1B CFU14d 14-14 -2.98 -1.96
1B Syto9 live 15-15 11.05 10.26
1B Pl dead 15-15 8.84 8.26
1B BL total 15-15 11.18 10.64
1B DHETlive 15-15 9.34 8.87
1B DAPltotal 15-15 10.88 10.88
1B DD dead 15-15 7.38 8.58
CFU7d= Colony forming units after 7 days of incubation CFU14d= Colony forming units after 14 days of incubation Syto9= live bacteria PI= Propidium Iodide, dead bacteria BL= Baclight, live and dead bacteria DHET= Dihydroethidium, live bacteria DAPI= live and dead bacteria DD= DAPI- DHET= dead bacteria N= number of cases
(Control) groups at
Equal/ Unequal Pr> ltl
Variances
E 0.0409
E 0.0599
E 0.1376
E 0.0397
E 0.0486
E 0.3140
E 0.0627
u 0.9177
E 0.8633
E 0.3672
E 0.0720
u 0.3294
E 0.2556
E 0.5000
E 0.9962
E 0.5774
132
Table 7. Repeated measures analysis of covariance for microscopy techniques
:.En~m~rati'.on technique
,, i(' ,,
Syto9 live
Pl dead
BL total
DHET live
DAPI total
DD dead
Bold numbers= p< 0.05 Group= MT AD or Saline
0.19 0.6703
2.43 0.1305
0.06 0.8134
0.02 0.8849
0.99 0.3271
1.48 0.2336
Time= treatment steps 1 C, 2A and 2B
T·im~ F
Pri>F
0.56 0.5768
4.11 0.0215
1.85 0.1660
0.62 0.5413
3.27 0.0452
1.45 0.2430
Group, Time and Group*Time are considered as an effect lB has been considered as a covariate in the analysis Syto9= live cells PI= propidium iodide, dead cells BL= Baclight, live and dead cells DHET= dihydroethidium, live cells DAPI= live and dead cells DD= DAPI- DHET, dead cells
Group*Time . F
Pr>F
3.39 0.0407
3.64 0.0327
lB F
Pr>F
6.34 0.0147
3.38 0.0711
4.57 0.0369
8.84 0.0043
5.16 0.0269
The group * time effect is confounded for Syto9live (p= 0.0407) and BLtotal (p= 0.0327) enumeration techniques; in particular, the numerical densities in the MTAD group are lower than the Saline group at sample 1 C, but are higher than the Saline group at sample 2A (Table 4). The effects and covariates that are statistically significant at levels of significance between 0.03 to 0.05, lose significance after the Bonferoni correction for multiple testing, therefore they should probably be considered trends rather than significant effects.
133
Table 8. Results of logistic regression for proportion of positive cultures (CFU14 d)
Type 3 Analysis of Effects
·. Effect
Group
Treatment step
DF ·
1
3
Group= Saline or MT AD Treatment step= lB, lC, 2A, 2B
· ~ald Chi-&quare
0.0385
6.3231
Pr> CbiSq
0.8444
0.0969
Table 9. Contrast Test Results for proportion of positive cultures (CFU 14d)
Contras·t l>F · :Wald ~lil'"S'qua~.e ,. Pr>ChiSq
lB vs. lC 1 2.1474 0.1428
lC vs. 2A 1 0.0000 1.0000
lB vs. 2B 1 0.1393 0.7090
lC vs. 2B 1 3.3675 0.0665
134
Table 10. Statistical analysis of the two staining techniques for the detection of live
bacteria (Syto91ive to DHETlive)
Live - Treatment step 1 C
::. :.n:F· Soq~~e •. . : Mean Square FValue Pr>F ·:
Method 1 18.73018225 12.35 0.0009
SalMTAD 1 2.00419361 1.32 0.2552
Live - Treatment step 2A
Source DF :. Mean::Square FValue Pr>F
Method 1 48.42437844 8.77 0.0045
SalMTAD 1 0.99251395 0.18 0.6732
Live - Treatment step 2B
Source DF ·Mean Square FValue Pr>F
Method 1 22.58334699 12.33 0.0009
SalMTAD 1 0.61793609 0.34 0.5636
Method = Syto9live vs. DHETlive SalMT AD = Saline vs. MTAD
135
Table 11. Statistical analysis (ANOV A) of the two staining techniques for the
detection of dead bacteria (Pldead vs. DAPI-DHETdead)
Dead-Treatment step lC
· Sou:rce D.F :M~an Sqp,are FValue Pr>F
Method 1 62.11359249 12.16 0.0009
SalMTAD 1 2.95249163 0.58 0.4502
Dead - Treatment step 2A
Sou:rce UF Mean Square FValue Pr>F
Method 1 43.35110471 6.87 0.0112
SalMTAD 1 24.18729640 3.83 0.0551
Dead - Treatment step 2B
DF' 1'f ean" Squ~:re FValue Pr>F
Method 1 51.45237659 4.43 0.0398
SalMTAD 1 8.09815641 0.70 0.4074
Method= Pldead vs. DAPI-DHETdead SalMTAD = Saline vs. MTAD
136
Table 12. The results obtained from a limited experiment (representing about 20%
of the lA Baclight-stained samples) for comparison of the enumeration of live
bacteria using either one or two channels (unmerged vs. merged), and compared to
the number of live bacteria stained with DHET
i \
:Ba~liglit DHET
Method One channel Two channel DHET
Mean± SD Mean± SD Mean± SD
Live counts 2.25 x107± 2.50 x107 1.90 x107± 2.82 xl07 5.97 xl06± 6.58 xl06
One channel= live bacteria enumerated with L5cube only Two channel= images of bacteria visualized with the cubes L5 and TX2 merged and the number obtained with TX2 cube (dead) subtracted
137
Table 13. Mixed bacteria with food colouring solution (OD) at 600 nm
.. Concentrations WeekO ·Week 1 Week2
4x 0.01 0.00 0.00
8x 0.00 0.00 0.00
16x 0.01 0.00 0.00
32x 0.00 0.00 0.00
64x 0.05 0.00 0.00
Diluted BHI 1110 0.05 0.149 0.171
Undiluted BHI 0.1395 1.55325 1.509
Concentrations= multiples of the MT AD equivalent BHI= positive control
138
Table 14. Proportion of positive cultures with different sterility control sampling
(SC) techniques at two treatment sessions after 7 and 14days of incubation.
7 da s 14 days
,i 2 4 f 2 3 4
S~f 40.00% 10.00% 13.33% 3.33% 40.00% 20.00% 13.33% 3.33%
SC2 1 23.33% 26.67% 13.33% 0.00% 40.00% 36.67% 13.33% 0.00%
1 = Brushing a sterile cotton pellet ( CP) directly on the surface of a blood agar plate. 2= Transferring into a microtube filled with transport fluid. 3= Transferring into a tube containing BHI. 4= A fourth sterile Cotton Pellet from the same pouch was transferred to BHI to serve as a negative control. SCl= Sterility control sample obtained at the beginning of the first treatment session SC2= Sterility control sample obtained at the beginning of the second treatment session
139
Table 15a. Pearson's correlation coefficient of the similarity between visual (manual) and automated (macro) counting methods for BacLight-stained bacteria
Descriptive Statistics
ManualSytb~
Macro syto9 .
Correlations
· Macro. Syfo91
;
'M:ea-0.····
8.231387
4.172231
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Std. J1>eviation
15.3194096
7.9743094
.Manua\$yto9
1
33
.803(**)
.000
33
** Correlation is significant at the 0.01 level (2-tailed).
Descriptive Statistics
.Manu~lPIH i
·Macro,~Pi.•···
Correlations
ManuiflPI
Ma{(ro:PI
.Mean
4.623442
3.826786
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Std. Deviation
10.7077215
9.4884044
Manual PI
1
33
.964(**)
.000
33
**Correlation is significant at the 0.01 level (2-tailed).
N
33
33
Macro Syto9
.803(**)
.000
33
1
33
N
33
33
Macro PI
.964(**)
.000
33
1
33
140
Table15b. Pearson's correlation coefficient of the similarity between visual (manual) and automated (macro) counting methods for DAPI/DHET-stained bacteria
Descriptive Statistics
M·attl.J.aIDA.PI
~acroDiPr
Correlations
Manual D~fl.
Mean
13.365549
8.213589
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Std. Deviation
26.9327844
14.9498333
M~nualDAPI
I
33
.977(**)
.000
33
** Correlation is significant at the 0.01 level (2-tailed).
Descriptive Statistics
ManualDHET
Correlations
iVl~m1al I>HET
MacroDHET
:Mea.n ..
4.024797
2.593212
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Std .. Deviation
8.2658812
3.5285425
ManualDHET
I
33
.810(**)
.000
33
**Correlation is significant at the 0.01 level (2-tailed).
N
33
33
MacroDAPI
.977(**)
.000
33
I
33
N
33
33
MacroDBET
.810(**)
.000
33
1
33
FIGURES
141
142
10. FIGURES
20
15
,-..... MTAD ~ 10 c::::=J Saline ~ u 0
"-" 0
5 ~
0 l -5 1A 18 1C 2A 28
Sample
Figure 1: The distribution of CFU counts per µL of root canal volume after 14 days of incubation in all samples converted to natural logarithms.
The box-and-whisker plots or "boxplots" show the overall "shape" of the distribution of the underlying data for a particular group. The boxplots here have the 25th and 75th percentiles shown by the lower and upper boundaries of the box, the 5th and 95th percentiles shown by the ends of the "whiskers", the 1st and 99th percentiles shown by stars, the minimum and maximum values (if different from the 1st and 99th percentiles) shown by short horizontal lines, the median shown by a line across the box, and the mean shown by a small square.
20
C:=J Syto9 Live ,,-..._ 15 DHET Live
00 " § ~
" " "
+ "
~ $ 0 u 10 0,) " > " ·~ " ~ "' "' '-' 5 " ~ ~
0
1A 18 1C 2A 28
Sample
Figure 2: The distribution of the live bacterial counts per µL of root canal volume for comparison of two microscopy methods for the live bacteria, converted to natural logarithm at five treatment steps. n = 30 in each group at each treatment step.
143
20
)(
~ ,-..._
r.r.:i
E )(
~ 0 u
"'O clj Q)
Q "-"" ~ ~
0
1A 18
Pl Dead c:::=J DDDead
)(
)( $
1C
Sample
)(
"
~
2A
144
10
)( 8
)(
6
4
2
28
Figure 3: The distribution of the dead bacterial counts per µL of root canal volume for comparison of two microscopy methods for the dead bacteria, converted to natural logarithm at five treatment steps. n = 30 in each group at each treatment step.
145
Proportion of positive cultures ---·-··----.... -··--·---··--···--·---
··-t.··MTAD7d
40 +-----__.'..._~-'='--~=----~P·_·_··_··_··_··_··_·_··_·-0-'-.--~--=.ill._--H . · o. -Saline 7d
-MTAD14
Ir ......• ...... A. -saline14d
20 +------------.-_ . .....,.....-------~-.-."'-T--.~~---t;.,_ ___ ~ .. ·
\,.:'."-· . : . c
0 ~------------------~-------< lB IC 2A
··A·· MTAD 7d
· · o · ·Saline 7d
_.,_MTAD 14d
-Saline14d
Sample
Figure 4: Proportion of positive cultures after 7 d and 14 d of anaerobic incubation of two groups (MT AD and Saline) at different treatment steps.
146
Total counts Macro vs. Visual
120%
100%
80%
60%
40%
20%
0% ' visual macro
Figure 5: Comparison of total counts obtained visually and with macro (automated)
APPENDICES
147
148
11. APPENDICES
Appendix 1. Clinical Information
Title of Project: "Antibacterial effectiveness ofa final rinse with MTAD and intracanal medication with 2% chlorhexidine gel in teeth with apical periodontitis"
Investigator: Dr. Gevik Malkhassian, Graduate Endodontics resident
Supervisor: Dr. Bettina Basrani, Assistant Professor
Name of Contact Person: Dr. Bettina Basrani (for address and phone numbers, see the end of the form)
Subjects Name: ________________ Patient file# ___ _
Periapical periodontitis is an inflammation in the bone surrounding the tip of the tooth root. It is caused by bacteria in the root canal systems of infected teeth. The usual treatment for the periapical periodontitis is root canal treatment. When the root canal system is infected, the root canal treatment is often done in more than one appointment to help reduce as much bacteria as possible, ideally all of the bacteria. In the first appointment, the root canals are cleaned and prepared to eliminate the majority of bacteria. However, there are often residual bacteria hiding in more complicated areas of the root canal system. To help eliminate these bacteria, an antibacterial (antibiotic) medication is placed in the root canals for a few days in between appointments, usually one week. At the second appointment, the medication is rinsed from the canal system and the root canal treatment is completed.
Calcium hydroxide is the antibacterial medication routinely used by dentists and specialist. However, calcium hydroxide has some disadvantages: it is difficult to place in the root canals, difficult to rinse completely from the root canals and does not destroy all the types of bacteria.
For these reasons, we are studying other kinds of antibacterial agent: MTAD* and chlorhexidine. MTAD (Biopure) is a novel anti bacterial agent and is a mixture of citric acid (the same acid found in lemon juice), doxycycline (an antibiotic from tetracycline group), and Tween80 (a detergent). All of these ingredients are in the market and used separately or in combination with other medications for a long time. Several studies have shown that it is effective against root canal bacteria, meanwhile is less toxic compared to full strength 5.25% sodium hypochlorite and Pulpdent (a paste of calcium hydroxide) and eugenol (a very common solution used in dental medications). MTAD is planned to be used only in half of the subjects who are randomly selected. It is used as an irrigating (cleaning) solution at the end of the first appointment for 5 minutes, in order to achieve an additional antibacterial effect from cleaning procedures. Chlorhexidine has shown successful results from different studies in eliminating bacteria as an intracanal medication. Chlorhexidine has been use for more than 50 years in different medical and dental applications: in the bladder, the nasal cavity, on burns, on skin and in throat lozenges, toothpastes, gum, mouthwash, cavity fighting gel, etc. Chlorhexidine is very efficacious in killing all types of bacteria.
The root canal treatment will be carried out as usual except that (1) MT AD will be used as a final root canal irrigant at the end of the first appointment, in half of the subject population. Sterile saline will serve as the final irrigant in the other half of the subject population (control group), (2) we will use chlorhexidine 2% gel instead of calcium hydroxide as the intracanal medication, and (3) we will need to take some samples of the root canals liquid content to look for the presence or absence of bacteria. The way we will take samples from the root canals is by aspirating the root canals liquid content with a small pipette. The sample collection is totally painless, there are no physical risks involved, and the sampling
149
takes less than a few minutes to perform. At the end of the first appointment, the chlorhexidine medication will be applied in the root canal system and the tooth will be temporarily closed. At the next appointment, chlorhexidine will be rinsed from the root canals, another sample will be taken and the treatment will be completed.
* MTAD (BioPure, Dentsply, Tulsa Dental, Tulsa, OK, U.S.A.) is an FDA-approved irrigant marketed in the U.S. and in Canada for clinical application
Risks The main possible risk is an allergic reaction to MT AD or chlorhexidine, which is very rare. MT AD contains citric acid (the same acid which is found in tomato, orange, and lemon juice), a well-known antibiotic (doxycycline from tetracycline family) that is being prescribed for a long time, and Tween 80 (a detergent). If there is any known allergy or contraindications to any of these agents by patients they will be excluded from the study. Its toxicity also is investigated and shown that is less toxic compared to full strength 5.25% sodium hypochlorite and Pulpdent (a paste of calcium hydroxide) and eugenol ( a very common solution used in dentistry). Chlorhexidine is very often prescribed as an oral rinse in concentration from 0.12% to 2%. Here we will be using chlorhexidine 2% only inside the root canal, isolated from the mouth and in contact with extremely small amounts of tissue. If a patient has a known allergy to chlorhexidine, she/he will not be included in the study. CHX has been used in previous study in the same clinic in 2% liquid form as an intracanal medication. No known complications for patients have been reported. In this study, the same concentration (2%) but in a gel form will be used.
Benefits MT AD is effective against E. faecalis (one of the most resistant bacterial strains in infected canals). It is able to remove the smear layer (a layer which is produced during mechanical cleaning of the canals and can harbour bacteria and their by-products). In addition it has specific penetrability into complex areas of the canals where some bacteria could be hidden. More over its special characteristic allows it to bind to the canal walls and continue its antimicrobial effect for a longer period of time. Chlorhexidine is an efficient antibacterial medication that is easy to place and to remove from the root canals. One advantage that chlorhexidine has is that even after it is rinsed from the root canals, the antibacterial effect is still present for many days after.
Confidentiality Any information learned about you during the study will be confidential and neither your name, nor any other identifying information will be made available to anyone other than the investigators.
Subject's rights You are encouraged to ask any questions about the study that you may have and all your inquiries will be answered.
You may refuse to participate in this study or withdraw from it at any time. If you do not participate in the study, or if you withdraw at any time, the quality of care provided to you and to other members of your family will not be affected in any way.
Any new significant findings which become available during the course of this study and which may relate to your willingness to participate or continue to participate in this study will be provided to you.
For questions or more information please contact the primary investigator:
Dr. Gevik Malkhassian
Phone:
Or the principal supervisor:
Dr. Bettina Basrani Department ofEndodontics, Faculty of Dentistry, University of Toronto 124 Edward Street, Toronto, Ontario MSG 1G6
Phone: 416-979-4911 Ext. 1-4402
150
151
Informed Consent Form (in two copies, one copy for the participant, the other copy for the investigator)
Title of Project: "Antibacterial effectiveness ofa final rinse with MTAD and intracanal medication with 2% chlorhexidine gel in teeth with apical periodontitis"
Investigator and contact persons: Dr. Gevik Malkhassian, Graduate Endodontics student and Dr. Bettina Basrani, Assistant Professor
Subjects Name: _______________ Patient file# ___ _
I have been asked to participate in a research study conducted by Dr. Gevik Malkhassian, and treated by him
Dr. Gevik Malkhassian (name of the treating dentist)
I have fully discussed and understand the purpose, procedures and potential risks involved in the study which have been explained to me. I have been invited to ask any questions which I may have about the study and all of my questions have been answered. I have been given a clinical information sheet that describes the research procedures.
I understand that my participation is voluntary and that I am under no obligation to participate in this study. I also understand that I may withdraw from the study at any time. I further understand that if I do not participate in the study, or if I withdraw from the study, the quality of care provided to me or to other members of my family will not be affected in any way.
Signature
I acknowledge that I have been provided with a copy of this consent form and the attached clinical information sheet.
Having thoroughly read, understood and had full explanation of this consent form and the attached clinical information sheet, I voluntarily consent to participate in this research study.
Patient Date
Dentist Date
Appendix 2. Characteristics of the study cohort (see also the next page)
.. · .. .c;···· .. ~Igus and Symptoms
I Root Canal .
Patl¢D.l'; I
0i\i'aster Apic:at File Y oluip.~ <e-L,~ ?: Group .. ·· Age <;;ep.d~r .· Tooth* :<::,~') , ~: ': ',< : Number ofRoorCanals0 I· . .·. ... ... p s ST STP .. · . I :: .. . . lA 1B . . . .·•. I: . ... • : •· ·.: .. · • I· : .. .• : 00•
l Saline 69 M 2.5 0 0 x x 2 B40,L40 5.4 11.9 :·· ..
2 .•. MTAD 63 F 1.4 x x 0 x 2 B andL 35 8.4 16.5
3 MTAD 68 F 3.5 0 0 0 0 1 35 3.9 8.0
4 Saline 77 M 1.2 0 0 0 0 1 35 6.2 9.8
s M'I'A1J 65 M 4.5 0 0 0 0 1 40 5.4 16.9
6 MTAD 76 M 3.5 0 0 0 0 1 40 5.1 18.7
7 Saline 46 M 1.2 0 0 0 x 1 35 9.3 21.2
8 Saline 43 F 4.5 0 0 0 x 1 50 19.2 14.9
9 Mr.AI> 52 M 2.5 0 0 0 x 2 45 7.9 19.7
10 Saline 27 F 1.1 x x 0 x 1 60 12.7 24.5
11 Saline 53 F 3.2 0 0 0 0 1 40 6.1 12.2
12 Saline 42 F 3.5 x x x x 1 40 16.9 17.9
13 MTAD 51 M 1.1 0 x 0 0 1 55 12.2 14.3 ..
14 MTAD 57 M 1.4 0 0 0 x 2 B40,L45 4.8 22.6
15 Saline 35 F 3.1 x x 0 x 1 45 11.1 16.4
16 Saline 27 F 4.6 x 0 0 0 4 Ms40,Ds60 6.8 5.7
17 MTAD 38 M 4.1 0 0 x x 1 40 8.7 20.2
18 Saline 50 F 2.5 0 0 0 x 2 B40, L35 8.9 11.0
19 Saline 47 M 4.6 x x 0 0 3 30 39.7 34.l
152
20 Saline 39 M 1.5 0 0
21 MTAD: 67 M 3.5 0 0
22 .MTAD:; 25 F 4.7 0 0 ,... ;
23 MT.Al}. ;; •... ;.: ::• .; . 48 F 1.5 x 0
24 MI:AP · .. 67 F 4.3 x 0
25 M'rAD.· 78 M 4.4 x 0 .. ····.·;·; ...
26 MT.AD .. 70 F 1.4 x 0 ':;.~
27 Saline 45 F 1.4 0 0
28 MTAP 71 M 2.1 0 0
29 Saline 25 M 1.1 0 0
30 Saline 37 F 4.6 0 0
Mean
SD
*=Tooth enumeration with the International Nomenclature. P= Spontaneous pain S= Swelling ST= Sinus tract STP= Sensitivity on percussion IA= Volume of root canal before chemomechanical preparation lB= Volume of root canal after chemomechanical preparation µL= Microlitre
0
0
0
0
x
0
0
0
0
x
0
B= buccal canal, L= lingual canal, M= mesial canal, D= distal canal SD= Standard deviation O= Absence of sign or symptom x= Presence of sign or symptom
x 2 B40, L30 7.6 22.5
x 2 B40, L35 4.7 14.8
x 4 Ms30,Ds45 18.9 20.5
x 1 40 11.1 16.1
x 1 45 10.9 16.9
x 1 40 7.0 7.5
x 2 Band L 35 7.1 14.3
0 2 Band L 35 8.2 21.2
x 1 45 2.2 12.6
x 1 80 9.8 16.6
0 4 30 9.9 24.4
9.9 16.8
7.0 5.9
153
Appendix 3. Data from root canal(s) at lA treatment step with all methods
··' ... .·
pt 1· group .. .: 1ACFU7ds. MCFU14d 1AGr/itoGr7d .. '
' .: .
1 Saline l.76E+04 2.01E+04 1 ..
2 MT#)< .. 2.35E+04 2.62E+04 1
3 MTAD 9.28E+04 2.12E+05 1
4 Saline l.54E+Ol 2.30E+Ol 1 .. ·
5 M:fAD l.01E+05 l.45E+05 1
6 '.MTAb' 9.74E+03 l.02E+04 1 . . .....
7 Saline O.OOE+OO 9.31E+Ol 0
8 Saline 5.76E+04 8.87E+04 1
9 MTAD l.57E+06 l.68E+06 1 ' ,,
10 Saline 3.53E+05 3.56E+05 1
11 Saline 2.67E+02 3.88E+02 1
12 Saline 1.41E+05 2.09E+05 1
13 MTAD l.76E+06 l.76E+06 1
14 MTAD 5.14E+04 5.14E+04 1
15 Saline l.40E+02 2.26E+02 1
Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
. ' 1AGr/noGrl4d lASyto9J:..ive •'·· ... ·.·
1 7.96E+06
1 2.21E+06
1 3.29E+06
1 5.64E+04
1 8.52E+06
1 3.27E+05
1 7.45E+06
1 1.49E+06
1 6.88E+06
1 l.98E+05
1 2.07E+05
1 9.60E+05
1 l.78E+06
1 3.24E+05
1 l.07E+06
" •"" > ·• ' . ·1AJ}Idead l~Ltotal tADHLive l~apitOtal lADDd~!ld
·+·,·.· f: .. I• . . :·:·, ....
l.63E+06 9.59E+06 2.48E+06 7.02E+06 4.54E+06
3.98E+05 2.61E+06 3.74E+05 2.19E+06 l.81E+06
l.96E+06 5.25E+06 4.39E+06 l.02E+07 5.77E+06
l.24E+04 6.88E+04 9.08E+03 l.06E+05 9.69E+04
l.31E+07 2.16E+07 l.67E+06 1.32E+07 l.16E+07
l.13E+06 1.46E+06 2.22E+04 l.47E+06 1.45E+06
l.13E+06 8.57E+06 5.05E+04 1.69E+06 1.64E+06
l.79E+06 3.28E+06 5.48E+05 5.58E+06 5.03E+06
3.39E+06 l.03E+07 5.41E+06 9.62E+06 4.21E+06
6.97E+05 8.95E+05 2.58E+06 7.21E+06 4.63E+06
2.08E+04 2.28E+05 5.76E+03 3.67E+05 3.61E+05
2.75E+05 l.23E+06 4.74E+05 l.09E+06 6.16E+05
2.32E+06 4.10E+06 l.56E+05 4.30E+06 4.14E+06
6.53E+05 9.76E+05 2.49E+05 2.13E+06 l.88E+06
6.93E+05 l.77E+06 5.11E+05 3.16E+06 2.65E+06
154
Appendix 3. continued, (lA)
' ' ,,, pf group 1ACFU7d 1ACFUl4d 1AGr/noGr7d , 1AGr/noG::r14d
16 Saline 6.10E+02 9.04E+02 1 .
17 MTAD 9.78E+02 l.62E+03 1
18 Saline -------------- -------------- -----------
19 Saline 3.26E+04 3.37E+04 1
20 Saline 7.llE+Ol 9.69E+Ol 1 ..
21 MTAD 5.16E+03 8.54E+03 1
22 w~:w, 3.51E+Ol 3.51E+Ol 1
23 !\1J:AD 7.15E+04 8.69E+04 1 . ?.
24 MTAD 5.67E+05 6.25E+05 1 ,, ·.·
25 MTAD 2.86E+Ol 3.57E+Ol 1
26 MTAD. 2.83E+05 3.06E+05 1
27 Saline 1.19E+Ol 1.60E+02 1
28 MTAD 3.44E+05 3.60E+05 1
29 Saline 1.29E+04 1.36E+04 1
30 Saline 2.98E+04 3.45E+04 1
---------- was not included in the study. Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
1
1
-----------
1
1
1
1
1
1
1
1
1
1
1
1
.. ,. lASyto9Live lAPidead lABElotal lADHUve lADapitotal lADDdead .,
1.39E+05 2.01E+05 3.39E+05 3.07E+05 l.61E+06 1.31E+06
2.46E+05 5.55E+06 5.79E+06 l.16E+05 9.54E+06 9.42E+06
1.65E+05 l.OOE+04 l.75E+05 l.41E+04 3.59E+06 3.58E+06
3.85E+05 l.90E+06 2.28E+06 1.52E+05 2.27E+06 2.11E+06
9.81E+05 2.88E+06 3.86E+06 2.44E+06 2.48E+06 4.46E+04
8.93E+06 l.74E+06 l.07E+07 7.66E+05 8.89E+06 8.13E+06
l.17E+04 l.10E+05 l.22E+05 l.44E+05 8.88E+05 7.44E+05
4.73E+05 5.24E+06 5.72E+06 4.53E+05 l.17E+07 1.12E+07
l.03E+05 3.19E+06 3.30E+06 l.75E+06 4.11E+06 2.36E+06
6.21E+05 l.27E+05 7.48E+05 3.29E+05 7.37E+05 4.08E+05
3.12E+06 l.63E+06 4.75E+06 l.11E+06 3.92E+06 2.81E+06
l.59E+04 2.84E+05 3.00E+05 l.67E+06 2.48E+06 8.10E+05
6.88E+05 4.03E+06 4.72E+06 5.66E+06 5.66E+06 O.OOE+OO
1.34E+04 3.58E+06 3.59E+06 3.67E+06 3.82E+06 1.49E+05
4.26E+04 1.52E+06 1.56E+06 3.06E+05 1.97E+06 1.66E+06
155
Appendix 4. Data from root canal(s) at lB treatment step with all methods
" ... : •. 17:. •• ·• .T ,.< •· I • • ~··> .. ,.. ·:·· 0' ~· '·'
1, Pl , group 1BCFU7d 1BCFU14it 1BGr/noGr7d 1BGr/noGr14d 1BSyto9Live . laPidead lBBLtotal lBDHLive: lBDapitotal lBDDdeait '· :._ .· ·:>· .. ·.. .. < .·
1 Saline O.OOE+OO O.OOE+OO 0 0 2.43E+04 5.35E+03 2.97E+04 6.04E+03 5.91E+04 5.30E+04
2 MTAD ~ O.OOE+OO O.OOE+OO 0 0 l.51E+05 4.53E+03 1.56E+05 2.87E+04 5.18E+04 2.31E+04 . ?
3 MTAU O.OOE+OO O.OOE+OO 0 0 9.84E+04 l.21E+04 1.11E+05 3.88E+04 7.59E+04 3.72E+04 . 4 Saline O.OOE+OO 4.98E+OO 0 1 3.37E+04 5.14E+03 3.88E+04 3.86E+03 2.65E+04 2.26E+04
•••• ·~· '· '%'
5 MTAD' -------------- -------------- ----------- ------------ 3.81E+04 l.42E+04 5.23E+04 3.21E+03 4.03E+04 3.71E+04 .. ,. ;.
6 MTAD O.OOE+OO O.OOE+OO 0 0 l.74E+04 1.57E+03 l.90E+04 l.44E+03 1.25E+04 1.11E+04 ....•...
7 Saline O.OOE+OO 4.54E+OO 0 1 2.0IE+04 2.27E+03 2.24E+04 2.50E+03 2.12E+04 l.87E+04
8 Saline O.OOE+OO 6.58E+OO 0 1 l.67E+04 2.47E+03 l.91E+04 8.63E+02 4.69E+04 4.61E+04
9 I MTAI> O.OOE+OO 2.54E+OO 0 1 l.16E+05 5.92E+04 l.75E+05 2.02E+04 9.51E+04 7.49E+04
10 Saline O.OOE+OO l.98E+OO 0 1 l.99E+04 5.59E+02 2.05E+04 5.52E+03 2.79E+04 2.24E+04
11 Saline O.OOE+OO O.OOE+OO 0 0 2.83E+04 5.70E+02 2.89E+04 4.18E+02 2.80E+04 2.76E+04
12 Saline O.OOE+OO 2.73E+OO 0 1 4.43E+04 2.05E+03 4.63E+04 2.72E+03 8.69E+04 8.42E+04
13 MTAD O.OOE+OO O.OOE+OO 0 0 6.07E+04 6.80E+03 6.75E+04 5.75E+03 5.34E+04 4.76E+04
14 MTAD O.OOE+OO O.OOE+OO 0 0 l.79E+04 l.73E+03 l.96E+04 8.04E+03 2.34E+05 2.26E+05
15 Saline O.OOE+OO O.OOE+OO 0 0 2.58E+05 7.69E+03 2.66E+05 l.01E+05 l.50E+05 4.99E+04
---------- was not included in the study. Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
156
Appendix 4. continued, (lB)
. .. . •, pt ,gr;oup• 1BCFU7d•' 1BCFU14d 1BGr/noGr7d
16 Saline O.OOE+OO O.OOE+OO 0
17 .• mAf) 2.41E+OO 2.41E+OO 1
18 Saline -------------- -------------- -----------
19 Saline O.OOE+OO O.OOE+OO 0
20 Saline O.OOE+OO O.OOE+OO 0
·T.MF~ 21 O.OOE+OO O.OOE+OO 0 . .
22 ·• M~X:n O.OOE+OO 2.36E+OO 0 .... ·;• .. ·,'
23 •. M:f,W O.OOE+OO O.OOE+OO 0 .. . .......
24 .. Ml_'~ O.OOE+OO 5.81E+OO 0
25 ·MtAD O.OOE+OO O.OOE+OO 0 .
'• .·
26 MTAD. O.OOE+OO O.OOE+OO 0 .... 27 Saline O.OOE+OO O.OOE+OO 0
28 M'f AD O.OOE+OO O.OOE+OO 0
29 Saline 8.51E+OO 1.13E+Ol 1
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
: > '~ . . . . . . . ,' ,, ,,, .
1BGr/noGrl4d lBSyto,9Live '?lBPidead IBBLtOtaL lBIJIHi,iy,ei: lBDapitotaf lBDDdead ·.· ..
0 5.47E+05 8.44E+04 6.31E+05 2.04E+05 1.30E+06 l.09E+06
1 2.05E+04 l.95E+03 2.24E+04 2.41E+02 2.70E+04 2.68E+04
------------ 2.24E+04 8.54E+05 8.77E+05 1.11E+03 7.61E+04 7.50E+04
0 l.38E+05 4.31E+03 l.43E+05 2.97E+04 2.97E+04 O.OOE+OO
0 l.49E+04 7.06E+02 l.56E+04 4.66E+03 2.50E+04 2.03E+04
0 3.48E+04 3.42E+03 3.83E+04 2.66E+04 3.87E+04 l.21E+04
1 5.82E+04 3.99E+03 6.22E+04 3.17E+03 3.05E+04 2.73E+04
0 8.61E+04 l.33E+04 9.93E+04 5.80E+04 1.02E+05 4.42E+04
1 5.72E+04 l.09E+04 6.81E+04 1.48E+03 1.15E+04 l.OOE+04
0 5.45E+05 2.09E+04 5.65E+05 3.l 1E+05 3.l 1E+05 O.OOE+OO
0 8.24E+04 6.79E+03 8.92E+04 5.38E+04 5.38E+04 O.OOE+OO
0 3.47E+03 5.43E+02 4.02E+03 2.75E+03 9.31E+03 6.57E+03
0 8.06E+04 8.61E+03 8.93E+04 5.14E+04 5.14E+04 O.OOE+OO
1 6.39E+03 8.92E+03 l.53E+04 l.34E+04 4.21E+04 2.87E+04
0 6.59E+03 7.64E+02 7.36E+03 l.72E+05 l.72E+05 O.OOE+OO
157
Appendix 5. Data from root canal(s) at lC treatment step with all methods
{ ..... . . . . .7
' 1CCFU7d • ,., ·. ..7• '·
1CGl"/noGr7d .,· f gioup .1CCFtf.14d p ,, .' ;,. i! " ... . .· ·. . · ..
1 Saline O.OOE+OO O.OOE+OO 0
2 ·... 'M'J'<ArzL ' 9.02E+OO 9.02E+OO 1 ' ·' •. ''· 7 . • •.
f'' ...•
3 . . ·.;,;, ~ · ... O.OOE+OO O.OOE+OO 0 . 4 Saline O.OOE+OO O.OOE+OO 0
5 . mf\.If, 8.63E+OO 4.03E+Ol 1 • '·7'• '
6 ,;::.=;.,, • .\.n. ,. O.OOE+OO O.OOE+OO 0 ... , > .. ••
7 Saline O.OOE+OO 4.57E+OO 0
8 Saline 3.26E+OO 3.26E+OO 1 .
9 MTAD O.OOE+OO 2.40E+OO 0 ..
10 Saline O.OOE+OO 5.97E+OO 0
11 Saline l.20E+Ol l.20E+Ol 1
12 Saline 2.17E+Ol 2.17E+Ol 1
13 MTAD O.OOE+OO O.OOE+OO 0 ...
14 MTAD O.OOE+OO O.OOE+OO 0
15 Saline 8.99E+OO 8.99E+OO 1
Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
1CGr/noGr14d lCSyto9Live;
0 6.97E+04
1 5.78E+04
0 5.99E+04
0 3.19E+04
1 l.70E+04
0 1.20E+04
1 l.66E+04
1 9.74E+04
1 1.60E+04
1 1.82E+04
1 l.17E+04
1 3.71E+05
0 2.89E+04
0 l.43E+04
1 6.22E+04
. ' ' ·.·/
lCDQdead 1C£Id,ead· l'cBLtqtal' 1CDHJ,ivti t€Dapltqtal .. . .
6.50E+03 7.62E+04 l.23E+04 4.19E+04 2.96E+04
4.66E+03 6.25E+04 1.30E+04 l.47E+05 l.34E+05
l.08E+04 7.07E+04 l.91E+04 8.66E+04 6.75E+04
5.68E+03 3.76E+04 4.64E+03 l.44E+04 9.78E+03
6.08E+02 1.76E+04 3.37E+03 2.21E+04 1.87E+04
l.04E+03 1.31E+04 l.82E+03 6.16E+03 4.34E+03
2.94E+03 l.95E+04 l.92E+03 2.08E+04 l.88E+04
l.19E+04 1.09E+05 7.53E+03 3.27E+04 2.52E+04
6.09E+03 2.21E+04 l.22E+04 5.73E+04 4.52E+04
1.49E+03 1.97E+04 5.53E+03 3.96E+04 3.41E+04
1.32E+03 1.30E+04 4.35E+03 4.68E+04 4.25E+04
4.36E+04 4.15E+05 l.09E+05 3.03E+05 l.94E+05
2.73E+03 3.17E+04 5.66E+03 5.29E+04 4.72E+04
1.11E+03 1.54E+04 1.13E+02 5.59E+04 5.58E+04
3.40E+03 6.56E+04 2.97E+04 9.14E+04 6.17E+04
158
Appendix 5. continued, (1 C)
; ···. pt group·••· 1CCFU7d' 1CCFU14d 1CGf/noGr7d
7 • ... .. "-.:'z :> :.
16 Saline O.OOE+OO O.OOE+OO 0
17 M'*FAD·· O.OOE+OO O.OOE+OO 0
18 Saline -------------- -------------- ------------
19 Saline O.OOE+OO O.OOE+OO 0
20 Saline 2.21E+OO 2.21E+OO 1
21 1 .~rn~~ O.OOE+OO 3.33E+OO 0 7. ;~ <: ""
• ••; Xx ';•; •
22 O.OOE+OO 2.38E+OO 0
23 f!rm••x 3.03E+OO 3.03E+OO 1 ... ·
24 MT .· l.76E+Ol 2.35E+Ol 1 . .
25 MTAD·· O.OOE+OO O.OOE+OO 0 ..
26 M:;fAD .. O.OOE+OO 6.69E+OO 0 ; ...
27 Saline O.OOE+OO O.OOE+OO 0
28 MTAD O.OOE+OO O.OOE+OO 0
29 Saline 2.87E+Ol 3.45E+Ol 1
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
.. . ; ··• ....
1CGr/noGrl4d' ... ;rcsyb}9Live, tCPidead lCBLtofal lCDHEive \L CD21)Jitotal lCDDdead .. 0 9.77E+04 7.91E+03 1.06E+05 2.36E+04 l.31E+06 1.28E+06
0 8.75E+03 2.43E+02 9.00E+03 4.73E+03 l.35E+05 l.31E+05
------------ 1.44E+04 1.40E+03 1.58E+04 4.61E+02 4.75E+04 4.71E+04
0 9.76E+03 5.20E+02 1.03E+04 2.82E+04 4.69E+04 1.87E+04
1 3.81E+03 2.31E+03 6.12E+03 1.32E+03 4.55E+04 4.42E+04
1 l.21E+04 3.18E+03 1.53E+04 1.83E+03 4.93E+04 4.75E+04
1 8.94E+03 3.69E+03 1.26E+04 3.50E+03 2.50E+04 2.15E+04
1 l.93E+04 1.25E+04 3.18E+04 9.08E+03 4.00E+04 3.09E+04
1 1.21E+04 6.92E+03 1.90E+04 2.16E+03 1.00E+04 7.86E+03
0 l.33E+04 l.74E+03 l.50E+04 2.87E+04 9.27E+04 6.40E+04
1 3.50E+03 l.83E+03 5.33E+03 1.54E+04 1.82E+05 l.66E+05
0 1.52E+04 1.43E+03 l.66E+04 1.26E+04 1.26E+04 O.OOE+OO
0 2.59E+04 9.82E+03 3.57E+04 2.31E+04 3.69E+04 l.38E+04
1 5.26E+03 2.16E+03 7.42E+03 1.56E+04 2.78E+04 1.21E+04
0 4.47E+03 1.90E+03 6.38E+03 3.09E+03 1.03E+04 7.21E+03
159
Appendix 6. Data from root canal(s) at 2A treatment step with all methods
.. . · .
pt + group ... 2ACFU7<1 2ACFU14<1 lAGr/n:oGr7d . 2AG:r/noGd4d .. •. . ~·· "" .. .
1 Saline 4.07E+OO 4.07E+OO 1
2 r::. MT.A. n·· ·. O.OOE+OO O.OOE+OO 0 :c:;;;;:c· - ·. , ...
3 .tN:ITAD : ... : ·~~.
5.75E+OO 1.15E+Ol 1
4 Saline O.OOE+OO O.OOE+OO 0
5 1:..r''Mt ·. O.OOE+OO O.OOE+OO 0 •• . J •• •••
6 r••• MTAD'z O.OOE+OO 5.14E+OO 0
7 Saline O.OOE+OO O.OOE+OO 0
8 Saline O.OOE+OO 3.27E+OO 0 .
9 Mi'AD O.OOE+OO 0.00E+OO 0 .
10 Saline O.OOE+OO 2.03E+OO 0
11 Saline O.OOE+OO O.OOE+OO 0
12 Saline 2.68E+OO 2.68E+OO 1
13 MTAD O.OOE+OO 3.41E+OO 0 .. 14 MTAD 2.17E+OO 2.17E+OO 1
15 Saline 2.92E+OO 2.92E+OO 1
Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
1
0
1
0
0
1
0
1
0
1
0
1
I
1
1
2ASyto9J:,ive: .... 1.04E+05
5.43E+04
4.68E+04
l.31E+05
6.09E+04
1.79E+04
2.31E+04
l.50E+04
5.37E+04
l.33E+04
1.43E+04
1.43E+04
3.72E+04
1.76E+04
4.56E+04
. ...
:2'.M>Idead 2:ABLtotal 2ADllliiV/" 2ADapitotal 21\DDdea:d
6.31E+03 1.10E+05 1.93E+04 5.12E+04 3.19E+04
3.41E+03 5.77E+04 3.75E+03 2.61E+04 2.24E+04
1.84E+04 6.52E+04 8.58E+03 5.49E+04 4.63E+04
3.07E+05 4.38E+05 1.10E+05 4.28E+05 3.18E+05
l.55E+04 7.64E+04 6.10E+02 2.00E+04 l.94E+04
8.88E+03 2.68E+04 l.92E+03 2.49E+04 2.30E+04
6.47E+03 2.96E+04 2.49E+03 3.47E+04 3.22E+04
7.68E+02 l.58E+04 l.05E+04 9.25E+04 8.21E+04
l.83E+04 7.21E+04 4.90E+03 1.42E+05 l.37E+05
7.24E+03 2.06E+04 9.45E+o2 5.66E+04 5.57E+04
5.30E+02 l.48E+04 8.02E+02 3.21E+04 3.13E+04
3.44E+04 4.88E+04 6.50E+03 7.98E+05 7.92E+05
5.69E+03 4.29E+04 5.19E+03 4.94E+04 4.42E+04
3.06E+03 2.06E+04 O.OOE+OO 2.56E+04 2.56E+04
9.57E+03 5.52E+04 2.06E+04 1.81E+05 l.60E+05
160
Appendix 6. continued, (2A)
- ·. . ' • · •. ··,, ? .• .. ""' ::·,····
pt ' group ~ACFU1& 2.A'.CFtrl4d 2AGr/noGr7d 2AGr/noGr14d 2ASyto9Live 2APidead , .2ABLtotal 2~HLive 2.ADapitotal 2ADDdead
16 Saline 8.51E+OO 8.51E+OO 1 1 l.12E+05 2.21E+04 1.34E+05 3.58E+04 2.44E+05 2.08E+05 ·.
17 MTAD ··.· 4.99E+OO 1.25E+Ol 1 1 1.87E+06 9.47E+05 2.82E+06 8.83E+04 9.82E+05 8.94E+05 ··.
18 Saline -------------- -------------- ----------- ------------ 1.03E+04 8.21E+02 1.11E+04 2.82E+04 1.78E+05 l.50E+05
19 Saline 1.43E+OO 1.43E+OO 1 1 9.62E+03 1.62E+03 1.12E+04 7.15E+Ol 1.57E+04 1.56E+04
20 Saline O.OOE+OO O.OOE+OO 0 0 5.00E+02 4.00E+02 8.99E+02 2.20E+02 2.17E+04 2.14E+04
21 •• MTAD O.OOE+OO O.OOE+OO 0 0 7.50E+03 1.27E+05 1.34E+05 7.98E+04 1.57E+05 7.71E+04 .· ·.
22 < MiAD O.OOE+OO O.OOE+OO 0 0 4.25E+04 2.83E+04 7.08E+04 8.72E+03 2.04E+05 1.95E+05 •.
·· .. 23 NfTAD O.OOE+OO 3.05E+OO 0 1 3.62E+04 1.21E+04 4.83E+04 2.46E+04 7.97E+04 5.51E+04
24 •···•· MTAD O.OOE+OO O.OOE+OO 0 0 5.26E+04 1.30E+04 6.56E+04 5.65E+03 1.42E+05 1.37E+05 .... ·.:··-
25 M.rAD O.OOE+OO 6.35E+OO 0 1 1.62E+04 1.24E+04 2.86E+04 l.19E+04 1.68E+06 1.67E+06
26 MTAD O.OOE+OO O.OOE+OO 0 0 l.13E+04 9.29E+03 2.06E+04 l.54E+04 3.55E+04 2.01E+04
27 Saline 4.68E+OO 4.68E+OO 1 1 2.53E+03 2.20E+02 2.74E+03 1.33E+03 1.43E+04 1.29E+04
28 MTAD l.16E+Ol l.16E+Ol 1 1 3.12E+04 4.93E+03 3.61E+04 9.10E+03 5.30E+04 4.39E+04
29 Saline O.OOE+OO O.OOE+OO 0 0 3.38E+04 9.18E+03 4.30E+04 2.08E+04 8.81E+04 6.73E+04
30 Saline O.OOE+OO O.OOE+OO 0 0 5.26E+03 1.66E+03 6.91E+03 1.08E+04 1.08E+04 O.OOE+OO
----------was not included in the study. Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
161
Appendix 7. Data from root canal(s) at 2B treatment step with all methods
· .. . .
p:t ·~··.· group .. 2BCFU7d 1 2BCFU14d 2BGr/noGfi7(I ·2BGr/no©rl4d: .... •. .
1 Saline O.OOE+OO O.OOE+OO 0 .·
2 MT.AD O.OOE+OO O.OOE+OO 0 . :.
3 .• MTAD O.OOE+OO 6.22E+OO 0 .·
4 Saline O.OOE+OO O.OOE+OO 0
5 MTAD· O.OOE+OO 2.89E+OO 0 .
6 MT~: O.OOE+OO 2.61E+OO 0
7 Saline 2.29E+OO 4.57E+OO 1
8 Saline O.OOE+OO 3.40E+OO 0
9 : .
MTAD:· O.OOE+OO O.OOE+OO 0
10 Saline O.OOE+OO O.OOE+OO 0
11 Saline O.OOE+OO O.OOE+OO 0
12 Saline O.OOE+OO O.OOE+OO 0
13 MTAD O.OOE+OO O.OOE+OO 0 ... 14 MTAD O.OOE+OO O.OOE+OO 0
15 Saline O.OOE+OO 2.98E+OO 0
Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
: .
0
0
1
0
1
1
1
1
0
0
0
0
0
0
l
;2JlSyt~9Tuive
l.30E+05
6.19E+04
2.57E+05
2.98E+04
2.31E+04
2.77E+04
8.51E+04
l.51E+05
l.38E+04
l.69E+04
l.86E+04
l.30E+04
2.46E+04
l.97E+04
8.64E+04
.. . ... .. 2BPldead ... 2BBLto(al ·~JJDHLive 2Bnapitotal 2BD:Pdead
.·· .
6.28E+03 1.36E+05 2.72E+04 9.79E+04 7.07E+04
9.67E+03 7.16E+04 l.74E+04 2.47E+04 7.37E+03
l.24E+o4 2.69E+05 8.00E+04 1.50E+05 7.03E+04
7.46E+03 3.73E+04 l.87E+04 4.28E+04 2.41E+04
5.89E+03 2.90E+04 l.54E+03 l.82E+04 1.66E+04
l.08E+04 3.84E+04 2.09E+03 2.57E+04 2.37E+04
7.30E+03 9.24E+04 l.77E+03 2.15E+04 1.97E+04
3.94E+04 l.90E+05 5.04E+04 3.09E+05 2.59E+05
4.38E+03 l.82E+04 2.69E+03 6.51E+04 6.24E+04
l.18E+03 l.81E+04 3.08E+03 3.45E+04 3.14E+04
3.77E+02 1.90E+04 4.21E+02 3.39E+04 3.35E+04
O.OOE+OO l.30E+04 l.43E+03 3.62E+04 3.48E+04
2.05E+03 2.66E+04 l.04E+03 5.13E+04 5.02E+04
2.81E+03 2.25E+04 l.11E+04 4.58E+04 3.47E+04
3.52E+03 8.99E+04 2.97E+03 6.63E+04 6.33E+04
162
Appendix 7. continued, (2B)
...• .. . :
pt group 2BeFU1d 2BCFU14d ·2~Gr/n0Gr7d h . ·
16 Saline O.OOE+OO O.OOE+OO 0 --""
17 MTA? ... O.OOE+OO 2.43E+OO 0
18 Saline -------------- -------------- ------------
19 Saline O.OOE+OO O.OOE+OO 0
20 Saline O.OOE+OO O.OOE+OO 0
21 ~,p~·fi' O.OOE+OO O.OOE+OO 0
22 ···• ·w~n ·~· O.OOE+OO O.OOE+OO 0 -·-~ .. c -:;:::: · .
•... 23 , .. M:'.Jl0.Af>' 3.00E+OO 3.00E+OO 1
,; .. ·:•
24 ··. iffAD O.OOE+OO O.OOE+OO 0 '
25 M1'AP O.OOE+OO 6.65E+OO 0 ...
.· .· 26 ·MTA® O.OOE+OO O.OOE+OO 0
·.• 27 Saline O.OOE+OO O.OOE+OO 0
.
28 MTA® O.OOE+OO O.OOE+OO 0 .. '
29 Saline O.OOE+OO O.OOE+OO 0
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per µL of root canal volume. 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
·· .... . :
2BGt/noGrl4d •:iBS:Jto9Live 2Bl>Idead 2BBl..fotal ~BDHLive. ]lBD3:~!tQ~ .~BDDdead . · ·. . . .. .. ..... . ····· .·
0 l.70E+05 l.21E+04 l.82E+05 2.33E+04 1.18E+05 9.43E+04
1 7.77E+03 3.43E+02 8.11E+03 4.70E+03 3.97E+04 3.50E+04
----------- 5.06E+04 8.84E+03 5.95E+04 4.72E+04 l.06E+05 5.88E+04
0 2.60E+03 8.66E+02 3.46E+03 2.42E+03 l.11E+04 8.72E+03
0 3.50E+03 3.89E+02 3.89E+03 2.03E+03 l.70E+04 1.50E+04
0 3.60E+03 3.00E+02 3.90E+03 1.81E+03 2.75E+06 2.74E+06
0 2.37E+04 1.16E+04 3.53E+04 3.91E+04 6.93E+04 3.02E+04
1 9.56E+03 2.46E+03 1.20E+04 1.47E+04 5.83E+04 4.36E+04
0 2.02E+04 2.92E+03 2.31E+04 2.16E+04 4.58E+04 2.42E+04
1 2.42E+04 4.53E+03 2.87E+04 2.16E+04 5.19E+04 3.03E+04
0 5.10E+04 8.17E+03 5.91E+04 5.48E+04 5.48E+04 O.OOE+OO
0 1.15E+03 3.13E+02 1.46E+03 8.03E+03 8.03E+03 O.OOE+OO
0 l.41E+04 8.67E+02 1.49E+04 5.34E+03 3.47E+04 2.94E+04
0 8.60E+03 9.56E+02 9.56E+03 3.82E+03 6.56E+03 2.75E+03
0 3.21E+04 3.74E+03 3.58E+04 1.76E+03 4.25E+03 2.49E+03
163
Appendix 8. Data from access cavities obtained at first treatment session, (ACl) with culture methods
... ·. '•'= pt. ··'· group 7daerobic 14daerobic
<- -· ----____ :;_-
1 Saline l.60E+03 l.60E+03 •·· .. · .
2 MTAD O.OOE+OO O.OOE+OO . ... .. .. · .. 3 MTAD' O.OOE+OO O.OOE+OO ..... 4 Saline O.OOE+OO O.OOE+OO
5 NITAD .. O.OOE+OO O.OOE+OO
6 -, :~::;~~ <> --- _--
MTAD.:s O.OOE+OO O.OOE+OO . . .
7 Saline O.OOE+OO 3.81E+Ol
8 Saline O.OOE+OO 0.00E+OO
9 MTAD' O.OOE+OO O.OOE+OO
10 Saline O.OOE+OO O.OOE+OO
11 Saline O.OOE+OO O.OOE+OO
12 Saline O.OOE+OO O.OOE+OO
13 MTAD O.OOE+OO O.OOE+OO
14 MTAD O.OOE+OO O.OOE+OO
15 Saline O.OOE+OO O.OOE+OO
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
>
74(;r/no=growth ··14d Gr/noGrowth· =7d anaerobic 14danaerooic
1 1 3.05E+02 3.05E+02
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO 7.62E+Ol
0 1 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO 7.62E+Ol
0 0 O.OOE+OO 3.81E+Ol
0 0 3.81E+Ol 3.81E+Ol
0 0 O.OOE+OO 3.81E+Ol
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 0.00E+OO 3.81E+Ol
0 0 O.OOE+OO O.OOE+OO
. 7d Gr/no Gi;owth 14d· «rtnoGrowth
·. .. .•..
1 1
0 0
0 0
0 0
0 0
0 1
0 0
0 1
0 1
1 1
0 1
0 0
0 0
0 1
0 0
164
Appendix 8. continued, (ACl)
....... ... 14d;:.erobic · pt
..... 1···· .. 7da~robic ... grQup .,
16 Saline O.OOE+OO O.OOE+OO
17 MTAD O.OOE+OO 3.81E+Ol
18 Saline O.OOE+OO O.OOE+OO
19 Saline O.OOE+OO 0.00E+OO
20 Saline O.OOE+OO O.OOE+OO
21 !'... MTA:D 3.81E+Ol 3.81E+Ol ·.
1· ..
22 M'f:At0'.· O.OOE+OO l.14E+02 ;.
I< .... 23 MTAliJ .. O.OOE+OO 3.81E+Ol
. ··. ;.; . ·.·
24 i• MTAD l.14E+02 l.90E+02
25 MTAD O.OOE+OO 3.81E+Ol .
26 MTAD O.OOE+OO 0.00E+OO
27 Saline O.OOE+OO O.OOE+OO
28 MTAD O.OOE+OO O.OOE+OO
29 Saline O.OOE+OO O.OOE+OO
30 Saline 3.81E+Ol 3.81E+Ol
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
;.
7d Gr/no ... growth 14d Gr/no Growth 7d anaerobic 14'd1anaero~ic 7d<Grlno Growth 14d Grlno Growth . .; . .
0 0 O.OOE+OO O.OOE+OO 0 0
0 1 O.OOE+OO O.OOE+OO 0 0
0 0 7.62E+Ol 7.62E+Ol 1 1
0 0 3.81E+Ol 3.81E+Ol 1 1
0 0 O.OOE+OO O.OOE+OO 0 0
1 1 O.OOE+OO O.OOE+OO 0 0
0 1 O.OOE+OO O.OOE+OO 0 0
0 1 3.81E+Ol 3.81E+Ol 1 1
1 1 O.OOE+OO 3.81E+Ol 0 1
0 1 O.OOE+OO O.OOE+OO 0 0
0 0 O.OOE+OO O.OOE+OO 0 0
0 0 3.81E+Ol 3.81E+Ol 1 1
0 0 O.OOE+OO O.OOE+OO 0 0
0 0 l.52E+02 1.52E+02 1 1
1 1 O.OOE+OO O.OOE+OO 0 0
165
Appendix 9. Data from access cavities obtained at second treatment session, (AC2) with culture methods
': '' ; .,,
• l4d aerobiC pt I'+ group .. 7d ·aerobic ' ;'
1 Saline 3.81E+Ol 3.81E+Ol .. ., '
2 MTAD O.OOE+OO O.OOE+OO ;
•;;
3 MTAD O.OOE+OO O.OOE+OO ;
4 Saline O.OOE+OO O.OOE+OO
5 ' MTAD O.OOE+OO O.OOE+OO '•
'· .. :.""
6 Mt AD O.OOE+OO O.OOE+OO .,,..,
7 Saline O.OOE+OO 3.81E+Ol
8 Saline O.OOE+OO O.OOE+OO
9 MTAD O.OOE+OO O.OOE+OO '
10 Saline O.OOE+OO O.OOE+OO
11 Saline O.OOE+OO O.OOE+OO
12 Saline O.OOE+OO O.OOE+OO
13 MTAD O.OOE+OO O.OOE+OO '''''
14 MTAD O.OOE+OO O.OOE+OO
15 Saline O.OOE+OO O.OOE+OO
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
' .·· •; ~·".,, ~" ""
· 7d Gr/no gro,wih t4d Gr/no. Growth 7d anaerobic 14d.anaerobic ; 5 '
; ' ·;
1 1 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO 7.62E+Ol
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO 1.14E+02
0 1 1.52E+02 l.52E+02
0 0 O.OOE+OO 7.62E+Ol
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO 3.81E+Ol
0 0 O.OOE+OO O.OOE+OO
0 0 O.OOE+OO 3.81E+Ol
0 0 O.OOE+OO O.OOE+OO
0 0 3.81E+Ol 3.81E+Ol
7d.Gr/no.GroWth 14d Gr/no Growth ; 5,' ' ,, ..
0 0
0 0
0 1
0 0
0 0
0 1
1 1
0 1
0 0
0 0
0 1
0 0
0 1
0 0
1 1
166
Appendix 9. continued, (AC2)
·:.· . .. .. .. . pt group' 'id:?aerobic :14d aerobic
16 Saline O.OOE+OO O.OOE+OO
17 MTAO O.OOE+OO O.OOE+OO .. ?.':'
18 Saline O.OOE+OO O.OOE+OO
19 Saline O.OOE+OO O.OOE+OO
20 Saline O.OOE+OO O.OOE+OO
21 MT.AD O.OOE+OO 7.62E+Ol
22 .•..•. •Mr~ O.OOE+OO O.OOE+OO
23 ·MrAD O.OOE+OO O.OOE+OO
24 ·'Mr.AB· O.OOE+OO 3.81E+Ol . ·.·
25 MTAO. 0.00E+OO 7.62E+Ol
26 MT.AB O.OOE+OO O.OOE+OO .
27 Saline O.OOE+OO O.OOE+OO
28 MT.AD O.OOE+OO O.OOE+OO
29 Saline O.OOE+OO O.OOE+OO
30 Saline O.OOE+OO O.OOE+OO
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
7d=Gr/no growth
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
y • ··• . .... ·· .. .·
.l4dGr/no Growth," 7d anaerobic 14d anaerobic 7d Gr/no Growtfr lr4tt=Gt/ll:o=Growth . ·. .. .. .
0 O.OOE+OO O.OOE+OO 0 0
0 O.OOE+OO O.OOE+OO 0 0
0 O.OOE+OO O.OOE+OO 0 0
0 3.81E+Ol 3.81E+Ol 1 1
0 O.OOE+OO O.OOE+OO 0 0
1 3.81E+Ol 3.81E+Ol 1 1
0 3.81E+Ol 3.81E+Ol 1 1
0 O.OOE+OO 3.8IE+Ol 0 1
1 O.OOE+OO O.OOE+OO 0 0
1 O.OOE+OO 3.81E+Ol 0 1
0 O.OOE+OO O.OOE+OO 0 0
0 O.OOE+OO O.OOE+OO 0 0
0 O.OOE+OO O.OOE+OO 0 0
0 O.OOE+OO O.OOE+OO 0 0
0 O.OOE+OO O.OOE+OO 0 0
167
Appendix 10. Data from access cavities obtained at first treatment session, (ACl) with microscopy methods
.. ....• ·.
pt group AC1Syto9Live .
1 Saline 5.02E+04
2 MTAD 7.84E+05
• .·
3 MTAD l.05E+06
4 Saline 4.35E+05 .. .
5 MIAD 7.59E+05 ..... 6 MIAD 2.96E+05
·. >•• ..
7 Saline 4.27E+05
8 Saline 6.74E+05 ·.
9 MIAD 2.10E+05 .•
10 Saline l.04E+06
11 Saline 2.12E+05
12 Saline l.40E+06 ..
13 MIAD 8.69E+05
14 MIAD 2.72E+05
15 Saline 2.11E+05
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
. .. . I ACI:Pldea:d· AClBLtotal · AClDHLive .• .. ..
O.OOE+OO 5.02E+04 l.10E+05
5.58E+04 8.40E+05 4.59E+04
7.71E+04 l.13E+06 4.34E+05
7.05E+04 5.05E+05 O.OOE+OO
2.77E+05 l.04E+06 l.76E+05
2.65E+05 5.62E+05 l.12E+05
6.80E+04 4.95E+05 5.34E+05
l.22E+04 6.86E+05 6.20E+04
l.05E+05 3.15E+05 l.45E+05
4.49E+04 l.08E+06 6.58E+04
3.llE+03 2.15E+05 7.15E+04
l.02E+05 l.51E+06 5.96E+04
l.76E+04 8.86E+05 O.OOE+OO
4.73E+04 3.19E+05 4.73E+04
3.51E+04 2.46E+05 l.76E+04
. . *ClDapitotal . AClDDdea'.d .. · .. ..
4.28E+05 3.18E+05
7.49E+06 7.45E+06
l.06E+06 6.24E+05
l.47E+05 l.47E+05
8.38E+05 6.62E+05
7.24E+05 6.12E+05
6.91E+05 l.57E+05
l.31E+06 l.25E+06
l.12E+06 9.73E+05
5.77E+05 5.11E+05
4.98E+05 4.26E+05
3.89E+06 3.83E+06
6.98E+05 6.98E+05
3.29E+06 3.25E+06
l.48E+06 l.46E+06
168
...
Appendix 10. continued, (ACl)
pt groy.p ACJSyto9Live . .. 16 Saline 5.89E+06
..
17 I MTAD l.83E+07 . ... ..
18 Saline l.01E+05
19 Saline 4.09E+05
20 Saline l.27E+05 .....
21 MT'IID . 4.86E+04 ·····
22 c iMrA® 1.35E+05 ...... . . .. . . . 23 Ml''IID 5.03E+04 ... 24 MTAD 3.36E+04 .. 25 MFA® l.98E+05
... 26 ·MT'IID 1.18E+05
27 Saline 7.04E+04
28 MT.A® l.32E+05
29 Saline 4.65E+04
30 Saline 7.10E+04
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
•. ,•?. :··· .· .
·· AClPidead ,. . ;t\ClBLtqtal ....... ACJDBLive . AClDi(pitqt~. ••·• ·· A.CJ.DDdeacl
· . . ·. . ... 2.88E+04 5.92E+06 2.30E+04 9.12E+06 9.10E+06
1.99E+06 2.03E+07 2.73E+06 3.83E+06 l.10E+06
O.OOE+OO l.01E+05 O.OOE+OO l.11E+06 l.11E+06
3.50E+04 4.44E+05 6.42E+04 7.35E+05 6.71E+05
l.21E+04 1.39E+05 1.l 7E+04 3.71E+06 3.70E+06
9.l 1E+03 5.77E+04 l.83E+06 l.83E+06 O.OOE+OO
7.05E+04 2.06E+05 l.03E+05 8.78E+05 7.75E+05
4.47E+04 9.50E+04 3.69E+05 l.43E+06 l.07E+06
1.12E+04 4.49E+04 O.OOE+OO l.29E+05 1.29E+05
7.91E+04 2.77E+05 2.09E+05 5.20E+05 3.11E+05
6.19E+04 1.80E+05 l.12E+05 l.26E+06 l.15E+06
2.35E+04 9.38E+04 8.80E+04 2.23E+05 1.35E+05
6.89E+04 2.01E+05 3.79E+05 3.79E+05 O.OOE+OO
5.81E+03 5.23E+04 5.23E+04 l.14E+07 l.14E+07
O.OOE+OO 7.10E+04 5.92E+03 2.19E+05 2.13E+05
169
Appendix 11. Data from access cavities obtained at second treatment session, (AC2) with all methods
... .··.7 · ..
ptc gr0up ACZSyto9Livec .. .·· I•
1 Saline 6.75E+05
2 MTAD 3.30E+05 •.
3 .. ··c .. MTAI> 8.47E+04
4 Saline 3.44E+05
5 MTtID 7.27E+05 ....
6 MTtID l.32E+05 ..
7 Saline l.11E+06
8 Saline 3.21E+05 ... ..
9 MTAD 4.09E+05
10 Saline 5.58E+05
11 Saline 3.45E+05
12 Saline 3.85E+05
13 MTtID 4.70E+05 ..
14 MTtID 5.99E+05
15 Saline 2.96E+05
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
... . . AC2Pldead AC2BLtotat
.. AC2DHLive !. .
l.03E+05 7.78E+05 7.50E+04
9.05E+04 4.20E+05 2.52E+05
3.39E+05 4.23E+05 5.68E+04
l.75E+04 3.61E+05 6.41E+04
2.59E+05 9.86E+05 3.20E+04
2.35E+05 3.66E+05 5.32E+04
l.84E+05 1.30E+06 l.07E+05
9.98E+04 4.21E+05 l.25E+05
l.23E+04 4.21E+05 l.84E+04
l.83E+04 5.76E+05 2.08E+05
1.20E+04 3.57E+05 3.60E+04
l.18E+04 3.96E+05 l.77E+04
7.13E+04 5.41E+o5 l.19E+04
1.35E+04 6.12E+05 2.37E+04
4.06E+04 3.36E+05 5.22E+04
. . .. AC2Dapitotal .. , :7·ccAC2DDdead
1.30E+06 l.22E+06
5.66E+05 3.14E+05
8.52E+05 7.95E+05
1.46E+06 1.40E+06
3.33E+05 3.01E+05
7.36E+05 6.83E+05
6.17E+05 5.10E+05
l.04E+06 9.14E+05
1.38E+06 1.36E+06
8.67E+05 6.60E+05
5.71E+05 5.35E+05
6.51E+05 6.33E+05
l.02E+06 l.OOE+06
7.63E+05 7.39E+05
2.76E+06 2.71E+06
170
I
Appendix 11. continued, (AC2)
. pt ... ...
group .... ACZSyto9Live
16 Saline 2.25E+05
17 MTAD 2.04E+05 ........ 18 Saline 2.71E+05
19 Saline 9.67E+04
20 Saline 9.17E+04 .··
21 MTAD 9.27E+04 .. .......
. ...
22 MJ'AP l.65E+05 . .....
23 MTAP .. .... 3.39E+05 ... ..
MfAD 24 ... 2.10E+05 . ... ......
25 MTAD 8.88E+04 · .....
26 MTAD 6.73E+04 ... ....
27 Saline 7.04E+04
28 MTAD 3.75E+05
29 Saline 2.16E+05
30 Saline 6.00E+04
Bacterial densities are expressed per access cavity 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
· . .. ·. . ·< •
AC2Pfdead AC2BLtotal AC2DHLive AJ.?2Dapitotal AC2DDdead ... . . · ... . .
5.77E+03 2.31E+05 4.79E+05 2.00E+06 l.52E+06
4.80E+04 2.52E+05 1.20E+04 l.36E+07 l.36E+07
3.01E+04 3.01E+05 l.20E+04 l.02E+06 l.01E+06
1.48E+05 2.45E+05 l.14E+04 6.77E+05 6.66E+05
5.73E+03 9.74E+04 l.77E+04 l.51E+06 l.49E+06
O.OOE+OO 9.27E+04 l.74E+04 3.36E+05 3.19E+05
7.40E+04 2.39E+05 2.73E+05 l.19E+06 9.16E+05
6.43E+04 4.04E+05 2.34E+05 9.94E+05 7.60E+05
3.97E+04 2.50E+05 1.47E+05 6.58E+05 5.10E+05
7.11E+04 l.60E+05 6.61E+05 3.18E+07 3.12E+07
l.68E+04 8.41E+04 l.01E+05 4.04E+05 3.03E+05
l.17E+04 8.22E+04 1.17E+04 l.23E+05 l.12E+05
5.27E+04 4.27E+05 8.83E+04 9.16E+05 8.28E+05
2.75E+05 4.91E+05 4.09E+04 l.05E+05 6.43E+04
O.OOE+OO 6.00E+04 O.OOE+OO l.14E+05 l.14E+05
171
Appendix 12. Data from root canal(s) at lA treatment step with all methods
.. ..
pt group lACFl.J7d· 1ACFU14d lA(fi.r/noGr7d . ..
' ,, ',,:;, ·,
1 Saline 7.20E+04 8.24E+04 1 ''••c. .
2 MTAD • 1.51 E+05 1.68E+05 1 ....
3 M'.f>AD ... ·· 2.78E+05 6.37E+05 1 . 4 Saline 7.62E+01 1.14E+02 1
. 5 MTAJ) 4.39E+05 6.29E+05 1
.. 1 ..
6 MT@ 3.86E+04 4.03E+04 1
7 Saline O.OOE+OO 6.86E+02 0
8 Saline 8.67E+05 1.33E+06 1 .· ..
9 Ml'AD 9.67E+06 1.03E+07 1
10 Saline 3.45E+06 3.48E+06 1
11 Saline 1.26E+03 1.83E+03 1
12 Saline 1.83E+06 2.72E+06 1 .
13 MTAD 1.67E+07 1.67E+07 1
14 MTAD 1.95E+05 1.95E+05 1
15 Saline 1.18E+03 1.90E+03 1
Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
. . . 1AGr/noGr14d lASyto?Uive
., ~ <,,.,;,,, ' ' ''
1 4.28E+07
1 1.86E+07
1 1.30E+07
1 3.50E+05
1 4.60E+07
1 1.66E+06
1 6.92E+07
1 2.87E+07
1 5.43E+07
1 2.51E+06
1 1.25E+06
1 1.62E+07
1 2.17E+07
1 1.56E+06
1 1.19E+07
. . lAPldead lABLtotat l'A:DHLive · . .. JMJapitotal lADDdead .. . : ,·;'}:
8.75E+06 5.16E+07 1.33E+07 3.77E+07 2.44E+07
3.35E+06 2.20E+07 3.14E+06 1.84E+07 1.53E+07
7.75E+06 2.07E+07 1.73E+07 4.01E+07 2.28E+07
7.68E+04 4.26E+05 5.63E+04 6.57E+05 6.00E+05
7.09E+07 1.17E+08 9.03E+06 7.14E+07 6.24E+07
5.74E+06 7.39E+06 1.12E+05 7.45E+06 7.34E+06
1.05E+07 7.97E+07 4.69E+05 1.57E+07 1.53E+07
3.44E+07 6.30E+07 1.05E+07 1.07E+08 9.67E+07
2.67E+07 8.09E+07 4.26E+07 7.58E+07 3.32E+07
8.82E+06 1.13E+07 3.27E+07 9.13E+07 5.86E+07
1.26E+05 1.38E+06 3.49E+04 2.22E+06 2.19E+06
4.64E+06 2.08E+07 8.00E+06 1.84E+07 1.04E+07
2.84E+07 5.01E+07 1.91 E+06 5.25E+07 5.06E+07
3.15E+06 4.71E+06 1.20E+06 1.03E+07 9.08E+06
7.70E+06 1.96E+07 5.67E+06 3.51E+07 2.94E+07
172
Appendix 12. continued, (lA)
. . pt ,, group. 1ACFV7d 1ACFU14d. ·.lAGr/noGr7d
·• .. : : /'
16 Saline 3.24E+03 4.80E+03 1
17 MTAD' 6.71E+03 1.11 E+04 1 .,
18 Saline -------------- -------------- -----------
19 Saline 1.00E+06 1.03E+06 1
20 Saline 4.19E+02 5.71 E+02 1
•• 21 MTAD
. •.. . . ..... 1.84E+04 3.05E+04 1
22 MTNJ 5.33E+02 5.33E+02 1 .... ..., . 23 MTAD .. 6.17E+05 7.50E+05 1
.
24 MTAI>'' 4.87E+06 5.37E+06 1 . ' .
25 MTAD 1.52E+02 1.90E+02 1 ·.· .
26 MTAD· 1.56E+06 1.69E+06 1
27 Saline 7.62E+01 1.03E+03 1 .
28 MTAD 5.90E+05 6.17E+05 1
29 Saline 9.76E+04 1.02E+05 1
30 Saline 2.34E+05 2.71E+05 1
---------- was not included in the study. Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
,., .. . lADDdead 1AGr/noGtl4d t~yto9Hve lAl'l<lead·· lABLtotal lADHLive lADagi~otal . . . . .:
1 9.40E+05 1.36E+06 2.30E+06 2.08E+06 1.09E+07 8.87E+06
1 2.15E+06 4.83E+07 5.05E+07 1.01E+06 8.31E+07 8.21E+07
----------- 1.47E+06 8.90E+04 1.56E+06 1.25E+05 3.20E+07 3.18E+07
1 1.53E+07 7.54E+07 9.07E+07 6.06E+06 9.01E+07 8.40E+07
1 7.42E+06 2.18E+07 2.92E+07 1.85E+07 1.88E+07 3.37E+05
1 4.19E+07 8.16E+06 5.01 E+07 3.60E+06 4.18E+07 3.82E+07
1 2.20E+05 2.07E+06 2.29E+06 2.72E+06 1.68E+07 1.40E+07
1 5.23E+06 5.80E+07 6.32E+07 5.01E+06 1.29E+08 1.24E+08
1 1.13E+06 3.49E+07 3.60E+07 1.91 E+07 4.49E+07 2.58E+07
1 4.32E+06 8.82E+05 5.21E+06 2.29E+06 5.13E+06 2.84E+06
1 2.23E+07 1.16E+07 3.39E+07 7.96E+06 2.80E+07 2.00E+07
1 1.30E+05 2.32E+06 2.45E+06 1.36E+07 2.03E+07 6.63E+06
1 1.50E+06 8.76E+06 1.03E+07 1.23E+07 1.23E+07 O.OOE+OO
1 1.31 E+05 3.49E+07 3.51 E+07 3.58E+07 3.73E+07 1.46E+06
1 4.20E+05 1.50E+07 1.54E+07 3.02E+06 1.94E+07 1.64E+07
173
Appendix 13. Data from root canal(s) at lB treatment step with all methods
. · · .. . ; . .. . • . I· pt 1 : group, 1BCFU7d 1BCFU14d· lBGr/rioGr7d
::::. •0 "" ...
1 Saline O.OOE+OO O.OOE+OO 0
2 "d .. •·.
:. ..N11'A1.J •. O.OOE+OO O.OOE+OO 0
3 NnA8. O.OOE+OO O.OOE+OO 0
4 Saline O.OOE+OO 3.81E+01 0
I'·'' , •.
5 M1'AD -------------- -------------- -----------; . . ;; ..
.....•.•. '·. '· .
0 6 M1'AD O.OOE+OO O.OOE+OO . . · ..
7 Saline O.OOE+OO 7.62E+01 0
8 Saline O.OOE+OO 7.62E+01 0 ,. ..
9 MTAD O.OOE+OO 3.81 E+01 0
10 Saline O.OOE+OO 3.81 E+01 0
11 Saline O.OOE+OO O.OOE+OO 0
12 Saline O.OOE+OO 3.81E+01 0
13 MTAD O.OOE+OO O.OOE+OO 0
14 MTAD O.OOE+OO O.OOE+OO 0
15 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
;
"," "
1BGrtnoGr14d 1BSyto9Live .
0 2.89E+05
0 2.50E+06
0 7.83E+05
1 3.31E+05
------------ 6.44E+05
0 3.26E+05
1 4.28E+05
1 2.49E+05
1 2.29E+06
1 4.88E+05
0 3.46E+05
1 7.92E+05
0 8.66E+05
0 4.05E+05
0 4.23E+06
I' .. · .· .·
lBPfdead ··lBBLtotal · lBDHLive: lBDapjtotal lB:QDdea<l .:;·
6.36E+04 3.53E+05 7.18E+04 7.02E+05 6.30E+05
7.48E+04 2.57E+06 4.74E+05 8.55E+05 3.81E+05
9.67E+04 8.80E+05 3.09E+05 6.04E+05 2.96E+05
5.05E+04 3.81E+05 3.79E+04 2.60E+05 2.22E+05
2.39E+05 8.84E+05 5.43E+04 6.82E+05 6.27E+05
2.94E+04 3.56E+05 2.70E+04 2.35E+05 2.08E+05
4.83E+04 4.76E+05 5.31E+04 4.50E+05 3.97E+05
3.69E+04 2.86E+05 1.29E+04 7.00E+05 6.88E+05
1.17E+06 3.45E+06 3.97E+05 1.87E+06 1.48E+06
1.37E+04 5.01E+05 1.35E+05 6.83E+05 5.48E+05
6.97E+03 3.53E+05 5.11 E+03 3.43E+05 3.38E+05
3.66E+04 8.28E+05 4.86E+04 1.56E+06 1.51 E+06
9.70E+04 9.63E+05 8.19E+04 7.60E+05 6.79E+05
3.91E+04 4.44E+05 1.82E+05 5.30E+06 5.12E+06
1.26E+05 4.36E+06 1.65E+06 2.47E+06 8.18E+05
174
Appendix 13. continued, (lB)
;. Pt group 1BCFU7d 1BCFU14d iBGr/noGr7d ... ~; ~ " L ·.
16 Saline O.OOE+OO O.OOE+OO 0
..
17 MTAD·· 3.81E+01 3.81 E+01 1 :.·.:.· .. '"'.:.· ·<,. .
18 Saline -------------- -------------- -----------
19 Saline O.OOE+OO O.OOE+OO 0
20 Saline O.OOE+OO O.OOE+OO 0 .•. •;
21 .MTAD O.OOE+OO O.OOE+OO 0
22 Mt.AD O.OOE+OO 3.81 E+01 0 " ....
..
' ~ 23 ;• O.OOE+OO O.OOE+OO 0 ·. ;· :.;
24 MTAf) •· O.OOE+OO 7.62E+01 0 ....
25 MTAf) O.OOE+OO O.OOE+OO 0 ..
..
26 MTAf) O.OOE+OO O.OOE+OO 0 " ;
27 Saline O.OOE+OO O.OOE+OO 0
28 MTAf) O.OOE+OO O.OOE+OO 0
29 Saline 1.14E+02 1.52E+02 1
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
" ..
1BGr/noGr14d: l~~yto9Live lBPidead lBBLtotaI lBDHLive lBDapitoJal .lBDDdead . · .. ::. ;; .·.: . .. . ...
0 1.17E+06 7.43E+06 6.26E+06 1.17E+06 7.43E+06 6.26E+06
1 4.88E+03 5.46E+05 5.41 E+05 4.88E+03 5.46E+05 5.41 E+05
------------ 1.22E+04 8.36E+05 8.24E+05 1.22E+04 8.36E+05 8.24E+05
0 1.01 E+06 1.01 E+06 O.OOE+OO 1.01 E+06 1.01 E+06 O.OOE+OO
0 1.05E+05 5.61E+05 4.57E+05 1.05E+05 5.61E+05 4.57E+05
0 3.95E+05 5.75E+05 1.80E+05 3.95E+05 5.75E+05 1.80E+05
1 6.50E+04 6.25E+05 5.60E+05 6.50E+04 6.25E+05 5.60E+05
0 9.33E+05 1.64E+06 7.11 E+05 9.33E+05 1.64E+06 7.11 E+05
1 2.50E+04 1.95E+05 1.70E+05 2.50E+04 1.95E+05 1.70E+05
0 2.34E+06 2.34E+06 O.OOE+OO 2.34E+06 2.34E+06 O.OOE+OO
0 7.70E+05 7.70E+05 O.OOE+OO 7.70E+05 7.70E+05 O.OOE+OO
0 5.81E+04 1.97E+05 1.39E+05 5.81E+04 1.97E+05 1.39E+05
0 6.48E+05 6.48E+05 O.OOE+OO 6.48E+05 6.48E+05 O.OOE+OO
1 2.23E+05 6.99E+05 4.77E+05 2.23E+05 6.99E+05 4.77E+05
0 4.19E+06 4.19E+06 O.OOE+OO 4.19E+06 4.19E+06 O.OOE+OO
175
Appendix 14. Data from root canal(s) at lC treatment step with all methods
-- --pt· --- g~oup 1CCFU7d.- 1GCFUl4d 1CGr/n~Gr7d
·-1 Saline O.OOE+OO O.OOE+OO 0
---2 MTAD 1.14E+02 1.14E+02 l
3 :Ml'Th O.OOE+OO O.OOE+OO 0
4 Saline O.OOE+OO O.OOE+OO 0
5 .Ml':AD 1.14E+02 5.33E+02 l
6 :MF;M) O.OOE+OO O.OOE+OO 0 -
7 Saline O.OOE+OO 7.62E+01 0
8 Saline 3.81 E+01 3.81E+01 1
9 :Mf;M) O.OOE+OO 3.81 E+01 0 --
10 Saline O.OOE+OO 1.14E+02 0
11 Saline 1.14E+02 1.14E+02 1
12 Saline 3.05E+02 3.05E+02 1
13 MT:AD O.OOE+OO O.OOE+OO 0
14 MT:AD O.OOE+OO O.OOE+OO 0
15 Saline 1.14E+02 1.14E+02 l
Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
1GGr/noGr14d 1 cSyto9Li¥e --- ... _.
0 8.28E+05
1 9.55E+05
0 4.77E+05
0 3.13E+05
1 2.88E+05
0 2.26E+05
1 3.52E+05
1 1.45E+06
1 3.15E+05
1 4.45E+05
1 1.43E+05
1 6.64E+06
0 4.12E+05
0 3.22E+05
1 1.02E+06
- ··- - -- -- _--_ ---~- lCPidead lCBLtotal lCDHLive 1CDapitotal lCDDdead
7.73E+04 9.05E+05 1.46E+05 4.98E+05 3.52E+05
7.70E+04 1.03E+06 2.14E+05 2.43E+06 2.22E+06
8.58E+04 5.63E+05 1.52E+05 6.90E+05 5.38E+05
5.57E+04 3.69E+05 4.56E+04 1.42E+05 9.60E+04
1.03E+04 2.98E+05 5.70E+04 3.73E+05 3.16E+05
1.94E+04 2.45E+05 3.41 E+04 1.15E+05 8.13E+04
6.25E+04 4.14E+05 4.08E+04 4.41 E+OS 4.00E+OS
1.78E+05 1.63E+06 1.12E+05 4.88E+05 3.75E+05
1.20E+05 4.35E+05 2.39E+05 1.13E+06 8.89E+05
3.66E+04 4.82E+05 1.35E+05 9.70E+05 8.35E+05
1.61 E+04 1.59E+05 5.32E+04 5.73E+05 5.20E+05
7.80E+05 7.42E+06 1.95E+06 5.42E+06 3.47E+06
3.89E+04 4.51E+05 8.06E+04 7.54E+05 6.73E+05
2.52E+04 3.48E+05 2.56E+03 1.26E+06 1.26E+06
5.57E+04 1.08E+06 4.87E+05 1.50E+06 1.01E+06
176
Appendix 14. continued, (lC)
. ..• .. ;···::< j·? -- ,/,:. ; . ·'· .· ,-,-._ ..
pt I• . . ·•• .cc; ·•• gro.up .. 1.CCFU7d·· 1CCFU14d 1CGr/lioGr7q. 1Q~r/noGr14d
16 Saline O.OOE+OO O.OOE+OO 0
17 MTAD O.OOE+OO O.OOE+OO 0 ·.··•
18 Saline -------------- -------------- ------------
19 Saline O.OOE+OO O.OOE+OO 0
20 Saline 3.81 E+01 3.81 E+01 1
21 MTAD O.OOE+OO 3.81 E+01 0 ;
;•
22 MT~ O.OOE+OO 3.81 E+01 0
23 ~Ar(~ 3.81E+01 3.81 E+01 1
24 MTAD 2.29E+02 3.05E+02 1 .
25 MTAD O.OOE+OO O.OOE+OO 0 ·· .. ·:·-
26 l\1TAD·· O.OOE+OO 7.62E+01 0
27 Saline O.OOE+OO O.OOE+OO 0
28 MTAD O.OOE+OO O.OOE+OO 0
29 Saline 3.81E+02 4.57E+02 1
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
0
0
------------
0
1
1
1
1
1
0
1
0
0
1
0
I ······· .. .. J ·. . ..
1 CSyto9Live ,. JCPidead 1CBLt9tal lCDHLive 1 CDapitotal fCDDdeaa ; .... ; ; ;
5.59E+05 4.53E+04 6.04E+05 1.35E+05 7.48E+06 7.35E+06
1.77E+05 4.92E+03 1.82E+05 9.57E+04 2.73E+06 2.64E+06
1.58E+05 1.54E+04 1.74E+05 5.07E+03 5.22E+05 5.17E+05
3.33E+05 1.77E+04 3.50E+05 9.61E+05 1.60E+06 6.39E+05
8.58E+04 5.19E+04 1.38E+05 2.98E+04 1.02E+06 9.93E+05
1.80E+05 4.72E+04 2.27E+05 2.72E+04 7.32E+05 7.05E+05
1.83E+05 7.56E+04 2.59E+05 7.17E+04 5.12E+05 4.41 E+OS
3.11E+05 2.00E+OS 5.11E+05 1.46E+05 6.44E+05 4.98E+05
2.04E+05 1.17E+05 3.21E+05 3.65E+04 1.69E+05 1.33E+05
1.00E+OS 1.31 E+04 1.13E+05 2.17E+05 6.99E+05 4.82E+05
5.01E+04 2.61E+04 7.62E+04 2.20E+05 2.60E+06 2.38E+06
3.22E+05 3.03E+04 3.52E+05 2.67E+05 2.67E+05 O.OOE+OO
3.26E+05 1.24E+05 4.50E+05 2.91E+05 4.64E+05 1.73E+05
8.74E+04 3.59E+04 1.23E+05 2.60E+05 4.61E+05 2.01 E+OS
1.09E+05 4.64E+04 1.55E+05 7.53E+04 2.51E+05 1.76E+05
177
Appendix 15. Data from root canal(s) at 2A treatment step with all methods
.·. > .
pt , .. group 2ACFU7d 2ACFU14d 2AGr/lloGr7d ·. ..
1 Saline 3.81E+01 3.81 E+01 1 , ..
2 MEAD O.OOE+OO O.OOE+OO 0
3 MTAD 3.81 E+01 7.62E+01 1
4 Saline O.OOE+OO O.OOE+OO 0
5 MTAD O.OOE+OO O.OOE+OO 0 >•
6 MTAD O.OOE+OO 7.62E+01 0
7 Saline O.OOE+OO O.OOE+OO 0
8 Saline O.OOE+OO 3.81 E+01 0 .,
9 MTAD O.OOE+OO O.OOE+OO 0
10 Saline O.OOE+OO 3.81E+01 0
11 Saline O.OOE+OO O.OOE+OO 0
12 Saline 3.81 E+01 3.81 E+01 1
13 MTAD O.OOE+OO 3.81 E+01 0 ..
14 MTAD 3.81 E+01 3.81E+01 1
15 Saline 3.81 E+01 3.81 E+01 1
Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates 0= undetectable bacterial growth on agar plates
.
.·
2AGr/noGrl4d • 2ASyto9Live
1 1.23E+06
0 8.96E+05
1 3.73E+05
0 1.29E+06
0 1.03E+06
1 3.35E+05
0 4.90E+05
1 2.25E+05
0 1.06E+06
1 3.27E+05
0 1.75E+05
1 2.56E+05
1 5.31E+05
1 3.97E+05
1 7.48E+05
- > · ....
2APidead 2ABLfotat lADIILivce. ~ADapi~tal 2ADDdea:d .. . ; · .... ·
7.50E+04 1.31E+06 2.29E+05 6.08E+05 3.79E+05
5.63E+04 9.52E+05 6.19E+04 4.31E+05 3.69E+05
1.46E+05 5.19E+05 6.83E+04 4.37E+05 3.68E+05
3.02E+06 4.30E+06 1.08E+06 4.20E+06 3.12E+06
2.62E+05 1.29E+06 1.03E+04 3.38E+05 3.27E+05
1.66E+05 5.02E+05 3.59E+04 4.67E+05 4.31E+05
1.37E+05 6.28E+05 5.29E+04 7.36E+05 6.83E+05
1.15E+04 2.36E+05 1.56E+05 1.38E+06 1.22E+06
3.61E+05 1.42E+06 9.66E+04 2.79E+06 2.70E+06
1.77E+05 5.04E+05 2.31E+04 1.39E+06 1.36E+06
6.48E+03 1.81 E+OS 9.81E+03 3.92E+05 3.83E+05
6.16E+05 8.72E+05 1.16E+05 1.43E+07 1.42E+07
8.11E+04 6.12E+05 7.39E+04 7.04E+05 6.30E+05
6.91E+04 4.66E+05 O.OOE+OO 5.78E+05 5.78E+05
1.57E+05 9.05E+05 3.39E+05 2.97E+06 2.63E+06
178
Appendix 15. continued, (2A)
/ .. 2ACFU14d~
~·--
pt >gJ'oup 1. 2ACFu7d •.2AGr/noGr7d· . · ........ . . ....... . 16 Saline 3.81 E+01 3.81 E+01 1
17 ·MTM>· 7.62E+01 1.90E+02 1
18 Saline -------------- -------------- -----------
19 Saline 3.81E+01 3.81E+01 1
20 Saline O.OOE+OO O.OOE+OO 0
21 ·.·· MtAD O.OOE+OO O.OOE+OO 0 . .
22 1. ~iEM> O.OOE+OO O.OOE+OO 0
23 MTAD. .· .. O.OOE+OO 3.81 E+01 0
•.
24 MTM> O.OOE+OO O.OOE+OO 0 /
25 ·Mtl\D O.OOE+OO 3.81 E+01 0
26 MTAD+·•··· O.OOE+OO O.OOE+OO 0 .··.
27 Saline 7.62E+01 7.62E+01 1
28 MTAD 1.14E+02 1.14E+02 1
29 Saline O.OOE+OO O.OOE+OO 0
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
. . 2AGr/noGrl4d 2ASyto9Live 2APidead 2ABUotal 2ADHLive· M.l>apit()tal · iADDdead+
r .. .. . 1 6.40E+05 1.26E+05 7.66E+05 2.05E+05 1.39E+06 1.19E+06
1 3.78E+07 1.91 E+07 5.69E+07 1.78E+06 1.99E+07 1.81 E+07
------------ 1.13E+05 9.02E+03 1.22E+05 3.09E+05 1.96E+06 1.65E+06
1 3.28E+05 5.54E+04 3.83E+05 2.44E+03 5.34E+05 5.31E+05
0 1.12E+04 8.99E+03 2.02E+04 4.95E+03 4.87E+05 4.82E+05
0 1.11E+05 1.89E+06 2.00E+06 1.18E+06 2.33E+06 1.14E+06
0 8.71E+05 5.79E+05 1.45E+06 1.79E+05 4.18E+06 4.00E+06
1 5.83E+05 1.95E+05 7.77E+05 3.95E+05 1.28E+06 8.86E+05
0 8.88E+05 2.20E+05 1.11E+06 9.55E+04 2.41E+06 2.31E+06
1 1.22E+05 9.37E+04 2.16E+05 8.97E+04 1.27E+07 1.26E+07
0 1.62E+05 1.33E+05 2.95E+05 2.21E+05 5.08E+05 2.88E+05
1 5.34E+04 4.65E+03 5.81E+04 2.81 E+04 3.02E+05 2.73E+05
1 3.93E+05 6.21E+04 4.55E+05 1.15E+05 6.69E+05 5.54E+05
0 5.62E+05 1.52E+05 7.15E+05 3.45E+05 1.46E+06 1.12E+06
0 1.28E+05 4.03E+04 1.68E+05 2.62E+05 2.62E+05 O.OOE+OO
179
Appendix 16. Data from root canal(s) at 2B treatment step with all methods
,.,., . , ...• ·. . ., ···•·•···· .·.
pt ... I gl'.Q.Up 2BCFU7d: 2BCFlH4d 2BGrlnoGr7d 2BGr/noGl'.14d
1 Saline O.OOE+OO O.OOE+OO 0 ,., .. '·!· ...
2 MT.AO O.OOE+OO O.OOE+OO 0 .... '
3 MTAD O.OOE+OO 3.81 E+01 0 ·. _<· .< ·.'
4 Saline O.OOE+OO O.OOE+OO 0
5 · .. ~~· •..... O.OOE+OO 3.81E+01 0
~·· .. ·.
6 MTAD O.OOE+OO 3.81E+01 0 ., ..
7 Saline 3.81E+01 7.62E+01 1
8 Saline O.OOE+OO 3.81E+01 0
9 ... MTAD O.OOE+OO O.OOE+OO 0 . 10 Saline O.OOE+OO O.OOE+OO 0
11 Saline O.OOE+OO O.OOE+OO 0
12 Saline O.OOE+OO O.OOE+OO 0
13 MTAD O.OOE+OO O.OOE+OO 0
14 MTAD O.OOE+OO O.OOE+OO 0
15 Saline O.OOE+OO 3.81 E+01 0
Bacterial densities are expressed per individual tooth 1 = detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
0
0
l
0
l
1
1
1
0
0
0
0
0
0
1
... . ..
2BSyto9Live
1.54E+06
1.02E+06
2.04E+06
2.93E+05
3.91E+05
5.19E+05
1.81 E+06
2.25E+06
2.73E+05
4.15E+05
2.28E+05
2.32E+05
3.50E+05
4.44E+05
1.42E+06
'. .. . . 2BPidead 2B:QLtotal 2BDHLiv~ 2BDapitotal 2BDDdead
7.47E+04 1.62E+06 3.23E+05 1.16E+06 8.40E+05
1.60E+05 1.18E+06 2.86E+05 4.08E+05 1.22E+05
9.84E+04 2.14E+06 6.36E+05 1.20E+06 5.60E+05
7.32E+04 3.66E+05 1.84E+05 4.20E+05 2.37E+05
9.95E+04 4.90E+05 2.61E+04 3.07E+05 2.81E+05
2.01E+05 7.20E+05 3.91E+04 4.82E+05 4.43E+05
1.55E+05 1.96E+06 3.76E+04 4.56E+05 4.19E+05
5.87E+05 2.84E+06 7.52E+05 4.62E+06 3.87E+06
8.62E+04 3.59E+05 5.30E+04 1.28E+06 1.23E+06
2.88E+04 4.44E+05 7.53E+04 8.44E+05 7.69E+05
4.61E+03 2.33E+05 5.15E+03 4.15E+05 4.09E+05
O.OOE+OO 2.32E+05 2.55E+04 6.48E+05 6.23E+05
2.92E+04 3.79E+05 1.48E+04 7.31E+05 7.16E+05
6.35E+04 5.08E+05 2.52E+05 1.03E+06 7.83E+05
5.78E+04 1.47E+06 4.87E+04 1.09E+06 1.04E+06
180
Appendix 16. continued, (2B)
P·
. · .· . .. ..
pt~ • gro,µp 2BCFU7d 2BQFU14d 2BGr/noGr1d .. . . . ;· . '
16 Saline O.OOE+OO O.OOE+OO 0
17 MTAD , O.OOE+OO 3.81 E+01 0 .·
18 Saline -------------- -------------- ------------
19 Saline O.OOE+OO O.OOE+OO 0
20 Saline O.OOE+OO O.OOE+OO 0
21 j MT~.r· O.OOE+OO O.OOE+OO 0 ';•, .. .. ..
22 O.OOE+OO O.OOE+OO 0 ; . · .. '·
'
23 •MT~ 3.81 E+01 3.81 E+01 1 ; '
24 MT..A!D O.OOE+OO O.OOE+OO 0 ·. ,,.
25 ,. M't~x O.OOE+OO 3.81E+01 0 ·.
; ·.·· .;,
26 .,. MTAD O.OOE+OO O.OOE+OO 0 '' '' '
27 Saline O.OOE+OO O.OOE+OO 0
28 MTAD O.OOE+OO O.OOE+OO 0
29 Saline O.OOE+OO O.OOE+OO 0
30 Saline O.OOE+OO O.OOE+OO 0
---------- was not included in the study. Bacterial densities are expressed per individual tooth 1 =detectable bacterial growth on agar plates O= undetectable bacterial growth on agar plates
. 2BGr/noGF14d 2BSyto9Live 2BPidead 2BBLfotaI;• 2BDHY¥e 2BDapitotal .2BDDdean'; ; . . .·
' ·;;, . ' ".:,; ,_;; ''· : ~> ' ,,
0 9.74E+05 6.94E+04 1.04E+06 1.33E+05 6.72E+05 5.39E+05
1 1.57E+05 6.93E+03 1.64E+05 9.50E+04 8.03E+05 7.08E+05
----------- 5.56E+05 9.72E+04 6.54E+05 5.19E+05 1.17E+06 6.47E+05
0 8.86E+04 2.95E+04 1.18E+05 8.24E+04 3.80E+05 2.97E+05
0 7.87E+04 8.75E+03 8.75E+04 4.57E+04 3.82E+05 3.37E+05
0 5.34E+04 4.45E+03 5.79E+04 2.69E+04 4.08E+07 4.07E+07
0 4.85E+05 2.38E+05 7.23E+05 8.01E+05 1.42E+06 6.20E+05
1 1.54E+05 3.95E+04 1.93E+05 2.37E+05 9.37E+05 7.01E+05
0 3.41 E+OS 4.93E+04 3.90E+05 3.65E+05 7.75E+05 4.10E+05
1 1.82E+05 3.42E+04 2.17E+05 1.63E+05 3.91E+05 2.28E+05
0 7.30E+05 1.17E+05 8.47E+05 7.85E+05 5.20E+05 -2.65E+05
0 2.43E+04 6.62E+03 3.09E+04 1.70E+05 1.07E+05 -6.31E+04
0 1.77E+05 1.09E+04 1.88E+05 6.73E+04 4.38E+05 3.70E+05
0 1.43E+05 1.59E+04 1.59E+05 6.34E+04 1.09E+05 4.57E+04
0 7.82E+05 9.10E+04 8.73E+05 4.30E+04 1.04E+05 6.07E+04
181
