Proliferation of Pathogenic Biofilms within Sealer-Root Dentin Interfaces is Affected by

Sealer Type and Aging Period

by

Karina Adriana Roth

A thesis submitted in conformity with the requirements

for the degree of Masters of Science (Endodontics)

Graduate Department of Dentistry

University of Toronto

© Copyright by Karina Adriana Roth 2011

ii

Proliferation of Pathogenic Biofilms within Sealer-Root Dentin Interfaces is Affected by Sealer Type and

Aging Period

Karina Roth

Masters of Science

Discipline of Endodontics

University of Toronto

2011

Abstract

Objective: To assess biofilm proliferation within the sealer-dentin interfaces of methacrylate

resin-based sealers, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP).

Methods: Standardized human root specimens were filled with the test materials and were aged

for 1 week, 1, 3 or 6 months in saline (n=3/group). Monoclonal biofilms of Enterococcus

faecalis were grown on the specimens for 7 days in continuous media reactor. The extent of

biofilm proliferation of E. faecalis within the sealer-dentin interface for each material at each

incubation period was assessed using fluorescence microscopy of dihydroethidium-stained

specimens. Results: TE had less biofilm proliferation than EP and SE (p<0.01). Deeper biofilm

proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1

week or 6 months (p<0.05). Conclusion: Self-etch and epoxy resin-based sealers were more

susceptible to interfacial biofilm proliferation than total-etch system at shorter incubation

periods.

iii

Acknowledgments

I would like to thank my principal supervisor, Dr. Yoav Finer to whom I wish to express my

deepest gratitude for his guidance, numerous hours of dedication and invaluable feedback

throughout this research project and during the preparation of the manuscript. I will be forever

grateful and thankful for having had the privilege of working with him, learning from his wealth

of knowledge, and most importantly for his immeasurable kindness and generosity throughout

my time at U of T, both at the professional level and for understanding and helping me during

personal hardships.

Next, thank you to Dr. Shimon Friedman, Head of the Discipline of Endodontics, for the

opportunity to be part of the Graduate program at the University of Toronto. Dr. Friedman has

been instrumental in bridging the endodontic and restorative/biomaterials aspect of the project

and contributed numerous hours to the correction and perfection of the manuscript.

Thank you to Dr. Céline Lévesque who taught me the ABC’s of her lab, supported me and was

always there for me providing constant moral and technical support in many aspects of the

project together with all of the wonderful people working with her.

I furthermore wish to extend my appreciation and thanks to Dr. Bettina Basrani and to Stephanie

Koyanagi, Richard Mair, Dr. Milos Legner, Dr. Babak Shokati and Dr. Jian Wang who helped

me with all of the technical aspects of my project.

Finally and most importantly, I would like to thank my dear husband Gustavo and amazing son

Max for their constant support and encouragement throughout my years of education. Their

constant love and patience in the numerous hours that I was away working and studying are what

made it possible for me to complete my work. To them, I owe the greatest debt of gratitude.

Karina A Roth, June 2011

iv

Table of Contents

Proliferation of Pathogenic Biofilms within Sealer-Root Dentin Interfaces is Affected by

Sealer Type and Aging Period ................................................................................................... ii

Abstract ...................................................................................................................................... ii

Acknowledgments ........................................................................................................................ iii

Table of Contents ........................................................................................................................... iv

List of Appendices ........................................................................................................................ iix

List of Abbreviations .......................................................................................................................x

Chapter 1 ..........................................................................................................................................1

1 Introduction .................................................................................................................................1

1.1 Purpose and Hypothesis .......................................................................................................3

1.1.1 Purpose of the study .................................................................................................3

1.1.2 Hypothesis................................................................................................................4

1.1.3 Objectives ................................................................................................................4

Chapter 2 ..........................................................................................................................................5

2 Literature review .........................................................................................................................5

2.1 The rationale for root canal treatment ..................................................................................5

2.2 Bacterial penetration into the dentin and along the dentin-sealer interface .........................6

2.3 Enterococcus faecalis ..........................................................................................................7

2.4 Biofilms and their role in disease progression .....................................................................9

2.5 The smear layer ..................................................................................................................11

2.6 Materials for root filling.....................................................................................................11

2.6.1 Epoxy-resin based sealers ......................................................................................13

2.6.2 Methacrylate resin-based root canal sealers ..........................................................14

2.7 Degradation of the bond between methacrylate resins and dentin ....................................21

v

2.8 Quality Assessment of the sealer-dentin interface .............................................................22

2.8.1 In vitro models .......................................................................................................22

2.8.2 In vivo models ........................................................................................................23

Chapter 3 ........................................................................................................................................24

3 Materials and Methods ..............................................................................................................24

3.1 Specimen Preparation ........................................................................................................24

3.2 Degradation media incubation of specimens .....................................................................27

3.3 Incubation of Specimens in Chemostat-Based Biofilm Fermentor (CBBF) .....................28

3.4 Reflected Light Microscopy (RLM) Analysis ...................................................................29

3.5 Scanning Electron Microscopy (SEM) Analysis ...............................................................30

3.6 Microbiological controls ....................................................................................................31

3.7 Statistical Analysis .............................................................................................................32

Chapter 4 ........................................................................................................................................33

4 Article ........................................................................................................................................33

Chapter 5 ........................................................................................................................................47

5 Discussion .................................................................................................................................47

Chapter 6 ........................................................................................................................................54

6 Conclusions ...............................................................................................................................54

Chapter 7 ........................................................................................................................................55

7 Recommendations .....................................................................................................................55

Chapter 8 ........................................................................................................................................57

8 Appendices ................................................................................................................................57

APPENDIX A: MEDIA AND SOLUTIONS ................................................................................58

APPENDIX B: GAMMA IRRADIATION ...................................................................................59

APPENDIX C: SAMPLE PREPARATION ..................................................................................60

APPENDIX D: STERILITY ASSAYS OF SPECIMEN PREPARATION ..................................62

vi

APPENDIX E: MICROBIOLOGY TECHNIQUES .....................................................................63

APPENDIX F: CHEMOSTAT-BASED BIOFILM FERMENTOR SET-UP ..............................66

APPENDIX G: STATISTICS........................................................................................................69

APPENDIX H: MICROSCOPIC IMAGES ..................................................................................71

APPENDIX I: SEM IMAGES .......................................................................................................75

APPENDIX J: BACTERIAL CELL PENETRATION .................................................................77

APPENDIX K: ETHICS APPROVAL ..........................................................................................78

References ......................................................................................................................................79

vii

List of Figures

Figure 1: Top images- Representative three-dimensional reconstruction of select Z-stack series

of E faecalis biofilms captured from sealer-dentin interfaces for the different materials.

Bottom images- Representative Z-stack images of E. faecalis captured from the sealer-dentin

interface of specimens aged for 1 month

Figure 2: E faecalis biofilm formation along the sealer-dentin interfaces for the different

materials.

Figure 3: Samples in sterile vial after being gamma irradiated.

Figure 4: Illustration of the sample preparation procedure.

Figure 5: Photograph of the sample preparation procedure.

Figure 6: Illustration and photograph of the 5 mm specimens.

Figure 7: Bacterial viability assessed using the plate counting technique.

Figure 8: Gram stain of E. faecalis cultured in experiment.

Figure 9: Analysis of immersing solution obtained from the vials.

Figure 10: Individual components of the chemostat-based biofilm fermentor (CBBF).

Figure 11: Image of the CBBF set-up within the laminar flow hood.

Figure 12: Microscopic image of a sample (10 X original magnification) denoting demarcation

of cardinal points to be subsequently analyzed (N = north, S = south, E = east, W = west). Four

points were randomly identified for each sample.

Figure 13: Mapping of selected points to be analyzed.

Figure 14: Photographic images through microscope’s optics of samples from different groups,

at 5X (original magnification).

viii

Figure 15: Microscopic images (5X original magnification) showing gap formation along the

dentin-sealer interface in specimens from different groups.

Figure 16: SEM images at 10 kV, 200 X original magnification, demonstrating gap formation in

samples from different groups.

Figure 17: Mean values for biofilm formation, for each sealer at each incubation time point.

ix

List of Appendices

Appendix A: MEDIA AND SOLUTIONS

Appendix B: GAMMA IRRADIATION

Appendix C: SAMPLE PREPARATION

Appendix D: STERILITY ASSAYS OF SPECIMEN PREPARATION

Appendix E: MICROBIOLOGY TECHNIQUES

Appendix F: CHEMOSTAT-BASED BIOFILM FERMENTOR SET-UP

Appendix G: STATISTICS

Appendix H: MICROSCOPIC IMAGES

Appendix I: SEM IMAGES

Appendix J: BACTERIAL CELL PENETRATION

Appendix K: ETHICS APPROVAL

x

List of Abbreviations

Bis-GMA Bisphenol A-glycidyl methacrylate

BHI Brain Heart Infusion broth

CBBF Chemostat-based Biofilm Fermentor

EDTA Ethylenediaminetetraacetic acid

EP Epoxy-resin based sealer

EBPADMA Ethoxylated bisphenol A dimethacrylate

HEMA Hydroxyethil methacrylate

MMPs Matrix metalloproteinases

MPa Megapascal

NaOCl Sodium hypochlorite

PBS Phosphate Buffer Saline

ROI Region of interest

SE Self-Etch system

SEM Scanning electron microscopy

SL Smear layer

TE Total-Etch system

TSB Trypticase Soy Broth

1

Chapter 1

1 Introduction

Bacterial invasion of the root canal space most frequently results in infection of the root canal

and periapical tissues of the affected tooth (1). To resolve infection, root canals are cleaned,

shaped and medicated, with the aim of reducing the bacterial load to a level that bacteriological

samples obtained from the canals yield no visible growth in culture. When no-growth cultures

are obtained, the probability of healing is high, in the range of 94%, compared to 68% when

cultures yield bacterial growth (2). Because of the invasion of bacteria into the dentinal tubules,

and because of anatomic irregularities of root canal systems, the conventional disinfection

regimens used clinically are only partially effective, resulting in residual bacteria and positive-

growth cultures in 10% to 70% of canals (3-5). Under favorable conditions in unfilled canals,

residual bacteria can proliferate to pre-treatment numbers within 2 to 4 days (6).

To eliminate residual bacteria or at least prevent their proliferation, the canal is filled after the

disinfection procedure (7). Root canal sealers should prevent the growth of microorganisms in

unfilled areas of the root canal system (isthmus, lateral canals, etc), and if any residual

microorganisms have remained after cleaning and shaping procedures, filling materials should

prevent their passage into periapical tissues (8). However, the currently used root filling

materials do not completely fulfill these requirements (9, 10). One of the factors influencing the

invasion of bacteria is the adaptation of the root filling to the canal wall (11). The standard root

filling is a combination of core material and sealer cement. The core acts as a piston on the

flowable sealer, causing the sealer to closely adapt to the dentin walls. The sealer layer should be

thin to minimize dimensional changes during and after setting. For resin-based sealers, in

particular, contraction/shrinkage after polymerization might lead to separation of the sealer from

the dentin, creating a potential pathway for future bacterial invasion.

2

In recent years, great emphasis has been placed on sealers that can bond to root dentin. This

trend, following those in restorative dentistry (10), is based on the premise that the bonded

interface may resist bacterial invasion. However, studies have consistently noted the difficulty to

establish a reliable bond between resin-based materials and dentin (12, 13). In the root canal, in

particular, bonding is undermined because the unfavorable cavity configuration causes increased

shrinkage stresses that de-bond the sealer from the dentin (14). Bonding can also be undermined

because of the dentin exposure to sodium hypochlorite, a potent oxidant producing an oxygen

rich layer on the dentin surface that inhibits polymerization (15, 16).

Eventual breakdown of the resin-dentin interface and subsequent penetration by oral fluids,

bacteria and their products might jeopardize the long-term outcome of the treated tooth. It has

already been well established that the primary cause of endodontic treatment failure is directly

related to the development of intraradicular infections in the form of biofilms (17).

The current gold standard for endodontic sealers, against which all new sealers are measured, are

epoxy-resin based sealers (18), such as AH Plus (Dentsply De Trey, Konstanz, Germany) which

adhere but do not bond to root dentin (19). Recently, methacrylate resin-based sealers have

gained popularity (20). The bond of these sealers to root dentin depends on the penetration of

hydrophilic resin monomers, incorporated to facilitate resin invasion into the wet dentinal

tubules (21), into the conditioned dentin surface to create micromechanical interlocking between

the dentin collagen and resin, forming a hybrid layer (22). Several types of adhesives for

methacrylate resin-based systems are available: (1) “Etch and rinse” (total-etch) systems

conventionally involve three steps with successive application of an acid etchant, primer and

bonding agent (23), and more recently two steps incorporating the primer and bonding agent into

one. The three-step approach produces the most durable bond (24) and is the gold standard for all

current bonded restorative systems. There are no current commercial total-etch endodontic

sealers available. “Self-etch” systems involve one step to etch, prime and bond, incorporating

the smear layer into the hybrid layer. Self-etch commercial endodontic sealers are available;

however, concerns have surfaced about inadequacy of their bond in the presence of a thick smear

layer (25, 26). The resin-dentin interface can undergo degradation over time, allowing salivary

3

and tissue fluid movement between the hybrid layer and dentin (24, 27-29) with consequent

breakdown of the covalent bonds within collagen fibrils and resin polymers (30). Products of

degradation eluting from composites can have an effect on bacterial cells by affecting their

intracellular functions and virulence factors (31, 32). As a result, interfacial bacterial penetration

and proliferation may occur (27), potentially resulting in endodontic failure.

1.1 Purpose and Hypothesis

1.1.1 Purpose of the study

Several commercially available products are currently available for root canal filling. Many have

undergone multiple modifications in their formulas due to unfavorable outcomes and due to the

emergence of evidence highlighting deleterious effects of some of their components. There

seems to be a continuous search for a material with ideal properties, that forms a single unity

between the dentin interface and the material itself, and that its long-term seal would not be

affected by some ulterior degradation or additional bacterial challenge. If subsequently the

dentin-sealer interface degrades over time, and if bacteria are present, they may form biofilms

which have been linked to endodontic failures.

The aim of my thesis was to compare the quality of seal of the sealer-dentin interface of two

methacrylate resin-based systems, self-etch (SE; RealSeal SE, Sybron Endo) and total-etch (TE;

Scotchbond MP, 3M, Bisfil 2B, Bisco), and an epoxy resin-based sealer (EP; AH Plus,

DENTSPLY/DeTrey) after aging the interfaces for up to 6 months using interfacial bacterial

invasion and biofilm proliferation as the biological markers for quality of the sealer-dentin

interface.

4

1.1.2 Hypothesis

The null hypothesis was that there will be no difference in the interfacial biofilm proliferation

and bacterial penetration of Enterococcus faecalis between root dentin and three test materials

(total-etch resin, self-etch sealer, epoxy resin sealer) following aging of the interfaces for up to 6

months.

1.1.3 Objectives

The objectives of the proposed study are:

To establish a physiologically relevant in vitro model for characterization of the sealer-

dentin interface following aging in physiological media, using biofilm proliferation and

bacterial invasion as the biological markers for the quality of the sealer-dentin interface.

To compare the quality of the sealer-dentin interface and the effect of aging of two

methacrylate resin-based systems, self-etch (SE; RealSeal SE, Sybron Endo) and total-

etch (TE; Scotchbond MP, 3M, Bisfil 2B, Bisco), and an epoxy resin-based sealer (EP;

AH Plus, DENTSPLY/DeTrey).

5

Chapter 2

2 Literature review

2.1 The rationale for root canal treatment

Bacterial invasion of the root canal space most frequently results in infection of the root canal

and periapical tissues; in the absence of bacteria, pulpal or periradicular pathoses would not

develop (1, 33, 34). Cleaning and shaping of the root canal is performed in order to reduce

bacterial concentrations within the root canal. Follow-up studies of endodontic treatment have

shown a higher rate of healing when bacterial culture obtained prior to root filling yielded no

visible growth (35); when samples were positive only 68% of the teeth healed, when samples had

no growth, as many as 94% of the teeth healed (2).

It has been established that mechanical (instrumentation) and chemical (irrigating solutions)

methods for cleansing root canals do not completely eliminate bacteria from the root canal (4, 5,

36-38). Several authors have demonstrated that under favorable conditions residual bacteria in

unfilled canals can proliferate to pre-treatment numbers within 2 to 4 days (6, 39). Therefore, one

of the goals of filling root canals is to prevent re-growth of bacteria. The second goal is to

prevent recontamination of the treated root canal system by ingress of endogenous bacteria

through the coronal pathway. Such ingress has the potential to re-infect the canal and periapical

tissues (40). This goal is achieved by combining the root canal seal with an impervious coronal

seal.

The ideal root canal filling should provide a seal impermeable to bacterial penetration to prevent

ingress of bacteria and bacterial by-products (41). Also, it should prevent percolation of substrate

to bacteria that survive treatment (7).

6

2.2 Bacterial penetration into the dentin and along the dentin-

sealer interface

Most of the radicular dentinal tubules run perpendicular to the pulp and the periphery in the root

canal. Their size and number differ along the root, with diameters ranging from 1 to 3 μm and

density from 4900 to 90,000 tubules per square millimeter (42). In the coronal dentin near the

pulp there can be as many as 42,000 tubules/mm2

(43) and the density decreases towards the root

apex, to just over 8,000 tubules/mm2

(44) at the apical level. Intratubular dentin is highly

mineralized (approximately 95 volume % mineral phase) compared with the less-mineralized

collagen matrix (about 30 volume % mineral phase) of intertubular dentin, and mineralization

increases with age resulting in a reduction in size of the tubules which can lead to a complete

obliteration. There is also a decrease in number of tubules due to physiologic aging of patients,

reaching to a reduction of up to 40% in 80 year-old patients compared to 20 year-old ones (44,

45).

Scanning electron micrographs of dentinal tubules in the coronal (A), mid-root (B), and

apical (C) root dentin of a human maxillary central incisor from a 40-yr-old individual.

Extracted from Carrigan P. Scanning electron microscopic evaluation of human dentinal tubules

according to age and location (44).

The diameter of the dentinal tubules is large enough to allow penetration by different bacterial

species (46-48), whose size may not exceed 0.3 µm. Bacterial penetration into dentinal tubules

can extend to different depths. Ando & Hoshino (49) using sampling methods, reported on

predominantly obligate anaerobes invading the deep dentin layers to a depth of 50 to 200 µm

from the surface of the root canal wall. Horiba et al. (50) using an abrasive micro sampling

7

method, reported that the endotoxin from Gram-negative bacteria may be distributed mainly in

dentin from the pulpal surface of dentin up to a depth of 300 μm. Sen et al. (51) found bacteria

and yeasts in the dentinal tubules from 10 to 150 μm using scanning electron microscopy. Peters

et al. (52), using culturing methods and histological sections, found that the deepest penetration

from the canal towards the cementum was 375 μm. The degree of bacterial invasion depends on

the type of bacterial species, the time of incubation (51, 53) and the age of the patient: bacterial

infection of dentinal tubules occurs to a lesser extent in older patients (54) probably due to an

increase in mineral content within the dentinal tubules which in turn results in their occlusion.

2.3 Enterococcus faecalis

Periapical endodontic disease is strictly related to presence of microorganisms (34). In primary

endodontic infections, microorganisms that initially invade and colonize necrotic pulp tissue are

found. In persistent endodontic infections, primary or secondary microorganisms that resisted

intracanal antimicrobial procedures and endured periods of nutrient deprivation can colonize the

endodontic milieu, striving and multiplying causing disease (7, 55). Finally, even in some cases

where treatment has followed the highest standards, extraradicular infections might have been

established (56), microbial invasion of inflamed periradicular tissues ensues and is a sequel to the

intraradicular infection. Usually there is evidence of biofilm formation, with well-established

microbial communities that are more resistant and harder to eliminate through standard intracanal

endodontic procedures (17).

Bacteria invading coronal dentinal tubules, due to optimal environmental conditions, can further

multiply and invade radicular dentin (57) which has been proven to have a negative impact on the

outcome of endodontic treatment (58). Failed endodontic cases demonstrate only one or two

species of Gram-positive facultative anaerobic microorganisms per canal, and Enterococcus,

Streptococcus, Peptostreptococcus and Actinomyces are the most frequently identified (59).

Recently, Vagococcus fluvialis was detected in root canals for the first time, and Solobacterium

8

moorei and Fusobacterium nucleatum were the most prevalent species in root filled teeth

exhibiting periradicular lesions (60).

Enterococcus faecalis strains are rarely found in primary infections but frequently found in

previously endodontically treated teeth (7, 61, 62) which would indicate that they could gain

access into the root canal after treatment completion probably due to coronal leakage (63). In

failed cases, these microorganisms gain access into the root canal after treatment completion

probably as a result of coronal leakage (63) and proliferate due to their ability to compete with

other microorganisms, invade dentinal tubules, and resist nutritional deprivation.

Several static in vitro (64), in vivo (65) and ex-vivo (66) investigations have used Enterococcus

faecalis to study their behavior and aptitude to strive and survive in endodontically treated teeth,

showing that they can maintain viability for twelve months ex vivo (67). The choice of this strain

was based on their ability to penetrate into dentinal tubules in as little as 48 hours of inoculation

(67) adhering and forming communities of organized biofilms as monospecies over root canal

surfaces and because this organism is frequently found in failed endodontic treatments (63, 68).

When E. faecalis were grown on abiotic surfaces such as microtiter plates, it was found that their

ability to develop into biofilms was dependant on the surface attributes of the substratum and

that these could vary according to the environmental and nutritional conditions present (69).

These authors demonstrated that when E. faecalis was grown aerobically under nutrient-rich

medium, they demonstrated both biofilm formation and deeper penetration of bacteria into

dentinal tubules.

9

2.4 Biofilms and their role in disease progression

A biofilm is a mode of microbial growth where dynamic communities of interacting sessile cells

are irreversibly attached to a solid substratum as well as each other and are embedded in a self-

made matrix of extracellular polymeric substances (70). The basic structural unit of a biofilm is a

microcolony or cell clusters (discrete units of densely packed bacterial cells, single or

multispecies, aggregates). A glycocalix matrix made up of EPS surrounds the microcolonies and

anchors the bacterial cells to the substrate.

Biofilms are of concern in endodontics and can be found as:

1) Intracanal biofilms: (71) These biofilms are formed by multiple species of microorganisms.

They degrade the dentin substrate as a consequence of the interaction of bacteria and their

metabolic products on dentin (internal resorption can be a consequence of bacteria-mediated

substrate dissolution).

2) Extraradicular or root surface biofilms: Formed by multiple species of microorganisms

(cocci and short rods with cocci attached to the tooth substrate), plus filamentous and fibrillar

forms. Calcified biofilms over root surfaces have been reported by Ricucci (72) and Harn

(73) in teeth refractory to conventional root canal therapy.

3) Periapical biofilms: Actinomyces and Propionibacterium propionicum producing

sulphur granules. This granular biofilms structure consists of a central mass of intertwined

branching bacterial filaments held together by an extracellular matrix with periapical

radiating clubs. PMN’s and macrophages patrolling the periapex area are unable to engulf

bacteria in a matrix-enclosed biofilm structure.

4) Biomaterial-centered infection: bacteria adhere to an artificial material (for example: to

gutta-percha) (74).

10

There are different stages observed in biofilm formation:

STAGE 1: formation of a conditioning layer by adsorption of inorganic and organic molecules

to the solid surface (which takes places from minutes to hours).

STAGE 2: adhesion of microbial cells to this layer (days to weeks).

STAGE 3: bacterial growth and biofilm expansion / organization (months).

When biofilms are formed, their structure protects the residing bacteria from environmental

threats. Biofilms show higher resistance to both antimicrobial agents and host defense

mechanisms when compared with planktonic cells (75-77) due to the inability of chemicals to

penetrate the full depth of the biofilm.

There are several mechanisms by which the resistance to antimicrobial agents is created in

biofilms:

1) Collective metabolic activity: multiplying effects according to number / diversity.

2) Planktonic cells are eliminated by antimicrobial challenges (ones are sacrificed in expense of

others).

3) Persister cells accumulate in a biofilm since they revert less readily and are physically

retained by the biofilm matrix.

4) Collective neutralizing power of groups of cells leads to slow or incomplete penetration of

the antimicrobial into the biofilm.

The increase resistance to antimicrobial agents and host defense mechanisms provide biofilms

with increased virulence as compared with planktonic cells. Therefore, biofilms forming

processes are regarded as major contributors to disease pathogenesis

11

2.5 The smear layer

Instrumentation of the root canal during endodontic cleaning and shaping procedures results in

the production of a smear layer (78, 79). This layer is composed of organic and inorganic

substances- dentin, necrotic and viable tissue, remnants of odontoblastic processes, pulp tissue

and bacteria (79) which can be forced into the dentinal tubules to form smear plugs up to

different depths. Mader (78) reported that the smear layer was composed of two sections- a

superficial layer on the surface of the canal wall which is 1 to 2 μm thick and a more profound

layer which is condensed into the dentinal tubules reaching a depth of up to 40 μm.

There is much controversy regarding the removal or maintenance of this layer (80). This

controversy exists because the removal of the smear layer prior to sealing of the canal may affect

the following:

Bacterial penetration: the smear layer might disintegrate due to coronal or apical

microleakage thus providing a patent pathway for bacterial ingress.

It may shield remaining bacteria within the dentinal tubules, creating a protective micro-

environment and promoting their proliferation.

The ability of root canal irrigants and medications to penetrate into dentinal tubules.

The ability of sealers to adhere or have an intimate contact with the root canal walls.

2.6 Materials for root filling

The standard root filling is a combination of core material and sealer cement. The core acts as a

piston on the flowable sealer, causing the sealer to closely adapt to the dentin walls. The sealer

layer should be thin to minimize dimensional changes during and after setting. For resin-based

12

sealers, in particular, contraction / shrinkage after polymerization might lead to separation of the

sealer from the dentin, creating a potential pathway for future bacterial invasion.

In recent years, great emphasis has been placed on sealers that can bond to root dentin. This trend,

following tendencies in restorative dentistry (10), is based on the premise that the bonded interface

may resist bacterial invasion. However, studies have consistently noted the difficulty to establish a

reliable bond between resin-based materials and dentin (12, 13). In the root canal, in particular,

bonding is undermined because the unfavorable cavity configuration causes increased shrinkage

stresses that de-bond the sealer from the dentin (14). Bonding can also be undermined because of

the dentin exposure to sodium hypochlorite, a potent oxidant producing an oxygen rich layer on the

dentin surface that inhibits polymerization (15, 16). Eventual breakdown of the resin-dentin

interface and subsequent penetration by oral fluids, bacteria and their products might jeopardize

the long-term outcome of the treated tooth.

The current gold standard for sealers, against which all new sealers are measured, are epoxy-

resin based sealers (18). Epoxy resin sealers, such as AH Plus (Dentsply De Trey, Konstanz,

Germany) adhere but do not bond to root dentin (19). Nevertheless, they have been widely used

for many years with acceptable clinical outcomes.

In the beginning of the 21st century, methacrylate-based sealers were reintroduced with the

objective to bond directly to the root canal walls, and since then, they have increasingly gained

popularity (20). The bond of these sealers to root dentin depends on the penetration of

hydrophilic resin monomers, incorporated to facilitate resin invasion into the wet dentinal

tubules (21), into the conditioned dentin surface to create micromechanical interlocking between

the dentin collagen and resin, forming a hybrid layer (22). Several types of resin-based systems

are available:

(1) “Etch and rinse” (total-etch) systems conventionally involve three steps with successive

application of an acid etchant, primer and bonding agent (23), and more recently two steps

incorporating the primer and bonding agent into one. The three-step approach produces the most

13

durable bond (24) and is the gold standard for all current bonded restorative systems. Currently,

there are no commercial total-etch endodontic sealers available, possibly because of application

challenges within the root canal configuration.

(2) “Self-etch” systems involve one step to etch, prime and bond, incorporating the smear layer

into the hybrid layer. Self-etch commercial endodontic sealers are available; however, concerns

have surfaced about inadequacy of their bond in the presence of a thick smear layer (25, 26).

A number of studies have demonstrated that leakage occurs between the root canal wall and the

filling material (81-83). Factors influencing the adaptation of the root filling to the canal wall are

of great significance in determining the degree and extent of leakage (11).

2.6.1 Epoxy-resin based sealers

Epoxy-resin based sealers have been used for many years with clinical success and are widely

used due to their good mechanical properties and compatibility with subsequent restoration of

endodontically treated teeth with adhesive systems. Common materials of this group are AH 26

and AH Plus sealers (Dentsply, Tulsa Dental Specialties, Tulsa, OK, US). AH Plus consists of a

two-paste components system; one component is the catalyst (an epoxide) and the other is the

base (an amine paste). The two pastes are mixed in equal lengths until a uniform and

homogenous consistency with a single color is obtained. According to the manufacturer, the

epoxide paste contains diepoxide, calcium tungstate, zirconium oxide, aerosol and pigments; and

the amine paste contains 1-adamantane amine, N,N'-dibenzyl-5-oxa-nonandiamine-1,9, TCD-

diamine, calcium tungstate, zirconium oxide, aerosol and silicone oil (MSDS, Dentsply).

AH 26 and AH Plus sealers are thought to be able to react with any exposed amino groups in

collagen to form covalent bonds between the resin and collagen when the epoxide ring opens

14

during polymerization. Their bond strength to dentin is 2.06 MPa (Megapascal), and 2.93 MPa to

gutta-percha, suggesting that the resin can adhere to both substrates (84). Similarly, McComb

and Smith (85) found that AH26 had tensile bond strength to dentin of 1.62 MPa; Wennberg and

Ørstavik (86) reported 2.5 MPa bond strength.

Other studies compared bond strengths in the presence or absence of the smear layer (SL) with

contradictory results: 1.19 MPa and 0.30 MPa when the dentin had been pretreated with EDTA

(87) as opposed to 1.22 MPa with smear layer and 2 MPa without (88). In another study, Eldeniz

et al (89) tested the shear bond strength of three resin-based sealers (Diaket, AH Plus and Endo-

Rez) in presence or absence of smear layer, and found that AH Plus had the highest shear bond

strength among all of them both with or without smear layer present, and the highest values were

recorded when the smear layer had been removed.

2.6.2 Methacrylate resin-based root canal sealers

Methacrylate resin-based root canal sealers are gaining popularity amongst practitioners as they

may be used with dentin adhesives for bonding to intraradicular dentin (20). Hydrophilic resin

monomers are incorporated into the endodontic sealers to facilitate better resin penetration into

the wet dentinal tubules after the removal of the smear layer (21). The bond between the

adhesive systems and the dentine will depend on the penetration of the monomers into the

conditioned dentine surface to create micromechanical interlocking between the dentin collagen

and resin and thus to form a hybrid layer (22). It has been well established that it is impossible to

completely dry the root canals prior to their filling, and residual moisture could affect the seal of

the obturation. However, drying the canals with paper points or after using a low vacuum, and

despite the fact that some moisture was still present, methacrylate-based sealers demonstrated

significantly less leakage than zinc oxide-eugenol based sealers (90).

15

Historically, there have been four different generations of methacrylate resin-based sealers for

endodontic use (91):

The first generation appeared during the 70’s and was used until the 80’s with poor outcomes. It

contained a poly[2-hydroxyethyl methacrylate] (poly[HEMA]) as the principal component in

Hydron (Hydron Technologies, Inc, Pompano Beach, Fl, USA).

The second generation consisted of a non-etching hydrophilic resin that did not require the

adjunct utilization of a dentin adhesive (EndoRez, Ultradent Products Inc, South Jordan, UT,

USA).

The third generation consisted of self-etching sealers that contained a self-etching primer and a

dual-cured composite resin which was based on the concept of incorporating the smear layer

created during rotary preparation into the sealer-dentin interface. Materials comprised within this

generation are: FibreFill R.C.S. (Pentron Clinical Technologies, Wallingford, CT, USA), Resilon

(Resilon Research LLC, Madison, CT, USA), Epiphany (Pentron Clinical Technologies),

RealSeal (SybronEndo, Orange, CA), Resinate (Obtura Spartan Corp, Fenton, MO), and Smart

(Discus Dental, CulverCity, CA).

The fourth generation, are self-adhesive resins that have eliminated the separate

etching/bonding step and are represented by MetaSEAL (Parkell Inc, Edgewood, NY, USA),

Hybrid Bond SEAL (Sun Medical Co Ltd, Shiga, Japan) and RealSeal SE (SybronEndo, Orange,

CA, USA).

16

Current methacrylate-based systems require the use of adhesive systems for optimal

performance. These adhesive systems can be divided into two major categories:

1. Self-etch systems (SE)

2. Total-etch bonding agents (TE)

2.6.2.1 Self-etch systems (SE)

These materials contain an acidic resin which etches and primes and sometimes bonds

simultaneously, incorporating the smear layer and any residual irrigant components into the

hybrid layer. Sealers like RealSeal SE (SybronEndo, Orange, CA) combine in a single product a

self-etching primer and a moderately filled flowable composite thus eliminating the use of

separate self-etching primers (25). This approach potentially eliminates over etching and its

potential deleterious effects on the integrity of the resin-dentin interface (92).

According to the manufacturer (SybronEndo, Orange, CA), RealSeal SE is a self-etch

methacrylate/epoxy resin root canal sealer in a catalyst/base paste-paste formulation, which

combines a modified methacrylate chemistry based upon SE Epiphany Root Canal Sealer and

epoxy resin chemistry similar to AH Plus root canal sealer. Both pastes are contained within 2

separate chambers that after extrusion through an auto mix syringe, provides the sealer in its final

adequate consistency for delivery into the root canal system. According to the manufacturer, it

contains a mixture of EBPADMA, HEMA, BisGMA and acidic methacrylate resins, silane-

treated bariumborosilicate glasses, silica hydroxylapatite, Ca-Al-F-silicate, bismuth oxychloride

with amines, peroxide, photo initiator, stabilizers and pigments.

17

2.6.2.2 Clinical Performance of self-etch systems in endodontics

The quality of the adaptation of the sealer to the dentin wall is affected by the presence of the

smear layer. Concerns have been raised when using self-adhesive sealers, for multiple reasons:

first, due to the fact that these materials might not be aggressive enough to be able to etch

through thick smear layers thus creating micromechanical retention via dentin hybridization (26)

or in cases where there is incomplete removal of the smear layer in hard to reach areas (such as

the apical third). In these cases, EDTA (ethylenediaminetetraacetic acid) is used in order to

remove the smear layer (25). Kim et al (93) tested Real Seal versus Real Seal SE in dentin

covered with smear layer, intact dentin irrigated with sterile deionised water and dentin that had

been treated with 6.15% NaOCl and EDTA. They reported that Real Seal SE was not able to etch

the radicular dentin thus there was no bonding between the smear layer and the intact dentin.

When sterile water had been used, the demineralised dentin layer was very thin (< 100 nm-thick)

and when EDTA had been used, some apatite crystals remained within the partially

demineralised dentin after the self-adhesive resin had been used. They concluded that Real Seal

SE (with a pH of 3.9 versus the pH of 2.5 of Real Seal) was not acidic enough to etch through

smear layers to produce micromechanical retention to improve adhesion of the sealers to the

canal walls.

Another concern is that several studies have demonstrated that endodontic irrigants can produce

erosion of root canal walls, decrease the microhardness of the dentine by removing its organic

components and altering its mineral composition. Sodium hypochlorite which is widely used as

an endodontic irrigant is a deproteinizing agent and a potent biological oxidant, leaving behind

an oxygen rich layer on the dentin surface that reduces bond strength and increased microleakage

(15, 16, 94, 95). This oxidizing effect may be reversed with the use of reducing agents such as

sodium ascorbate or ascorbic acid (15, 16, 94-97) so that it would be possible to acid-etch and

bond immediately to endodontically treated teeth where sodium hypochlorite had been used as

an irrigating solution. Additionally, the manufacturer also claims that previous irrigation of the

root canal with sodium hypochlorite might negatively affect the bonding strength of the primer,

thus suggesting that the last irrigant to be used should be sterile water (2% chlorhexidine

18

gluconate does not affect the bond strength). Also lubricants containing peroxide might delay the

setting of the resins, so they should be rinsed with sterile water as well.

In vitro studies have shown that specimens where self-etching adhesives had been used

experienced a quick loss of structural integrity after aqueous aging (98). This was due to the

water content that was present within the dentinal tubules that might have inhibited the

polymerization of the acidic monomers. Also, the components that constitute the self-adhesive

systems are hydrophilic which enhances water sorption and hydrolytic breakdown in the mouth

(99) so after their application, the hybrid layers that are formed behave as semi-permeable

membranes that allow water movement across the bonded interface even after adhesive

polymerization (100).

A sealer should favor the reorganization of injured structures and should not interfere with tissue

repair. Numerous studies have reported that hydrophilic methacrylate resins can absorb water

largely in the resin matrix (101) and elute unreacted monomers (mostly released during the first

few days) which might promote cytotoxic reactions (102-104) and promote bacterial growth

(31).

Concerns over the toxicity of resin-based endodontic sealers were raised by several investigators.

Ames et al. (105) conducted a study where MetaSEAL, RealSeal SE and EndoREZ were tested

in self-cured mode, which is the mode of setting relevant to the apical third of the canal walls

and within the perirradicular tissues, thus representing the worst possible situation in which a

sealer would perform in a clinical scenario. The cytotoxicity of the methacrylate resin-based

sealers was investigated by the 3-(4, 5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide

assay, which measures cell viability by assessing its succinate dehydrogenase activity. All sealers

were severely cytotoxic at 72 hours after mixing. RealSeal SE was moderately cytotoxic during

the first two weeks, mildly cytotoxic at weeks 3-4 and nontoxic after the fifth week. The above

studies highlight the importance of a prolonged testing period in order to be able to detect long

19

term release of toxic un- polymerized components from the material due to the reduced degree of

conversion at the apex.

2.6.2.3 Total-etch bonding agents

These systems use an acid etching process (most commonly 30-40% phosphoric acid) in an

attempt to completely remove the smear layer, open the dentinal tubules and demineralise the

dentin leaving an exposed collagen matrix (106). Rinsing will remove the dissolved mineral

component of dentine and the remaining irrigating solutions or interaction by-products. This step

is followed by application of primer and a bonding agent, provided in 2 separate bottles (in

contrast to the SE system where all components are dispensed simultaneously). According to the

manufacturer (3M ESPE, St Paul, MN, USA), Scotchbond multi-purpose primer contains: water,

methacrylic acid, 2-hydroxyethyl ester, (2-hydroxyethyl-methacrylate) and ploycarboxylic acid.

The catalyst contains: bisphenol A diglycidyl ether dimethacrylate, methacrylic acid, 2-

hydroxyethyl ester and benzoyl peroxide. During the priming, the hydrophilic monomers that

diffuse across the demineralised dentin stabilize the hydrated collagen network and displace

water with polymerizable monomers (107). Then, the adhesive resins are applied to the primed

dentin and later polymerized.

After applying the adhesive system, the methacrylate based resin composites are applied. These

can be either light-cured (one component system) or self-cured (two component systems). One

self cure system is Bisfil II (BISCO, Schaumburg, IL, USA) t. According to the manufacturer,

Bisfil II composite is composed of a base paste. This resin system is mainly composed of Bis-

GMA, triethyleneglycol dimethacrylate (TEGDMA), glass fillers and amorphous silica.

The bonding mechanism of etch-and-rinse adhesives to dentin is primarily diffusion-based and

depends upon hybridization or infiltration of resin within the exposed collagen scaffold. The

adhesive resin fills the porosities between the collagen fibers forming resin tags that seal the

20

dentinal tubules that have been opened; they initiate the polymerization reaction, stabilize the

hybrid layer and provide enough methacrylate double bonds for copolymerization with the resin.

As the demineralised collagen fibril mesh is used as the bonding substrate, a wet bonding

technique is required to insure its full expansion (108). But even if the resin monomers are able

to penetrate the dentin, if the polymerization is not adequate, the resin-dentin bond might be

compromised. Several factors influence the degree of conversion inside the hybrid layer: the

mode of polymerization of the material (light-cured, chemically cured or a combination); the

area where the polymerization is initiated; the number of available double-carbon bonds and the

presence of substances that might inhibit the polymerization (traces of irrigation solutions,

lubricants, etc).

Even though most of the available systems that use the three bonding steps can produce high

resin bonding strengths, excessive etching of the dentin can produce a weak bonding because the

collagen fibers at the base are not completely impregnated by the resin (109). Also if the area is

dried in excess the collagen network can collapse. Another factor that must be taken into

consideration is the unfavorable cavity configuration present in a root canal system, where the

volume of monomer is reduced creating sufficient shrinkage stresses to debond the material from

the dentin decreasing retention thus increasing leakage (14).

Methacrylate resin-based total-etch systems are widely used in restorative dentistry as they

produce the most durable bond (24). However, no total-etch endodontic sealers are commercially

available, possibly because of application challenges within the root canal configuration.

Ceballos et al (108) evaluated the bond strength of total-etch versus self-etch adhesives to caries-

free versus normal dentine, and found that the total-etch systems yielded higher bond strength

values. Similarly, Bouillaguet et al (107) evaluated the microtensile bond strength of eight

different adhesive systems in vitro in bovine teeth and reported that Scotchbond 1 exhibited the

third highest tensile bond values (18.9 ± 3.2 MPa). This three step ethanol-water-based etch-and-

21

rinse adhesion strategy is the most conventional and effective approach to obtain an efficient,

durable and stable bond (24) and remains the gold standard technique in restorative dentistry.

2.7 Degradation of the bond between methacrylate resins and

dentin

The resin-dentin interface can undergo degradation over time, allowing salivary and tissue fluid

movement between the hybrid layer and dentin (24, 27, 28, 92) with consequent breakdown of

the covalent bonds within collagen fibrils and resin polymers (30). This process begins when the

dentin is acid-etched (110). Collagen degradation is enhanced by enzymes, particularly dentinal

matrix metalloproteinases (MMPs) (28) which degrade the collagen component of the hybrid

layer. Incorporation of MMP inhibitors, such as chlorhexidine (99) into the endodontic treatment

regimen and into future methacrylate-based sealers may arrest degradation of the hybrid layer

(20).

Methacrylate adhesives can also degrade in aqueous solution and salivary esterases can catalyze

this process and increase the degradation of the resin-dentin interface at a greater rate (27, 29). It

has been proven that after prolonged exposure of a restoration to the fluids present in the oral

cavity, water begins to penetrate the resin (101) promoting chemical hydrolysis of ester bonds in

the material. If the pH is neutral, the process will be slow, but in the presence of bacteria where

the pH might significantly drop, the process might be accelerated. “The carboxylate and alcohol

degradation products of ester hydrolysis are more hydrophilic than the parent ester, further

enhancing the local ingress of water” (92). These ester linkages are the weakest link, thus

considered one of the main reasons for resin degradation within the hybrid layer (111).

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2.8 Quality Assessment of the sealer-dentin interface

Researchers have characterized the interface between sealers and root dentin using different

models.

2.8.1 In vitro models

When assessment of the quality of the root filling is investigated, in vitro microleakage studies

have been the most widely used approach due to their ease, reproducibility and cost. Initially,

dyes and tracers had been used (112); saliva (113-115); fluid filtration models (116, 117);

bacteria and endotoxins (9, 118, 119); glucose filtration (120) among many others with

questionable clinical relevance (83).

Bond strength has been used as a measure of adhesion quality (25, 121). However, it only

assesses mechanical properties and not interfacial porosity. It has been demonstrated that the

push-out strength for EndoREZ (in MPa) was 8.7±4.3, 9.1±2.9 and 7.6±2.1 for the coronal,

middle and apical thirds; for MetaSEAL 18.6±4.8, 18.2±4.8 and 16.1±4.6 and for RealSeal SE

12.6±4.3, 14.9±5.5 and 14.4±8.1 (25), but it should be noted that in this study a final rinse with

EDTA was performed following manufacturer’s instructions, thereby not being able to assess the

true self-etching potential of RealSeal and MetaSEAL, the two self-adhering resin-based sealers.

All push-out strengths values were independent of the location of the radicular dentin. The

authors concluded that the self-adhesive sealers exhibited higher push-out strengths than the non-

etching sealer and that the variations in tubular density or sclerotic dentin along the canal wall

would not be factors that would alter the mechanical retention of the studied sealers.

More recently, using the non-invasive confocal laser scanning microscopy (CLSM),

Kermanshahi et al (27) characterized the salivary enzyme catalyzed degradation of restorative

23

resin-dentin interfaces by measuring the extent of interfacial bacterial cells and biofilms of a

major species associated with dental caries, Streptococcus mutans. They used a constant media

model simulating in-vivo pathogenic oral conditions (27). Where hybrid layer disruption and

marginal gaps were present, bacterial biofilms proliferated into the resin-dentin interface and

invaded the dentinal tubules (27). This system utilizes both biologically relevant and

microbiological components, making it highly relevant to in vivo settings.

2.8.2 In vivo models

In vivo research encompasses both animal studies and results stemming from outcomes in

clinical practice. Several studies were conducted using dog models, where teeth with endodontic

fillings were challenged through bacterial ingress and later histological analysis was performed

to evaluate the response of the periradicular tissues to different tested materials (40, 122, 123).

However, correlation between these results and clinical outcomes is not straightforward as

demonstrated by Pitt Ford (124) where he showed a lack of correlation between dye penetration

and periapical tissue response in dogs’ teeth. This is further demonstrated by the fact that dye

penetration through root-filled teeth was shown to occur in teeth that had been clinically

successful (125). In clinical practice, only the patient’s signs and symptoms can be considered

as outcome predictors, as their treated teeth cannot be extracted and evaluated for

presence/absence of flaws. Conventional two-dimensional radiographs have limited ability in

assessing three-dimensional objects (126). More recently, cone beam CT (CBCT) has been

employed to assess the quality of endodontic treatment. Much speculation has been surrounding

this new diagnostic modality, and some concerns were raised about the amount of radiation that

patients would receive. Many studies were conducted in this regard, but analysis of their results

is difficult due to the multiple differences among them (different machines, technical parameters,

measurement methods, etc). Overall, it has been demonstrated that CBCT results in doses that

are three to seven times those of panoramic doses and 40% less than conventional CT doses

(127).

24

Chapter 3

3 Materials and Methods

3.1 Specimen Preparation

Human caries-free teeth with single canals were collected after extractions from anonymous

patients in an Oral Surgery practice (University of Toronto Human Ethics Protocol #24315). The

teeth were kept frozen until used. The teeth were inspected for cracks under the operating

microscope at 10X magnification. Cracked teeth were discarded and replaced with new ones,

until 45 suitable teeth were selected. To disinfect and prevent further bacterial contamination,

teeth were sterilized with Gamma irradiation (4080 Gy), shown not to alter the structure and

permeability of dentin (128). Endodontic treatment procedures leading to root filling were

performed in a sterility-controlled environment to avoid contamination (Labculture® Class II

Type A2 Biohazard Safety Cabinet, Esco Micro Pte Ltd, Singapore).

The tooth crowns were severed at the cemento-enamel junction. Canals were negotiated to the

apical foramen with K-files (Lexicon Flex SSK, Dentsply Tulsa Dental Specialties, Tulsa, OK,

USA) and cleaned and shaped with ProTaper rotary instruments (Dentsply Tulsa Dental

Specialties, Tulsa, OK, USA) up to a size F4 at the foramen, while being intermittently irrigated

with 5 mL of 5.25% of sodium hypochlorite (NaOCl) using a 30 gauge needle. The last rinse

with 5 mL of 5.25% NaOCl was activated with the EndoActivator (Dentsply Tulsa Dental

Specialties, Tulsa, OK, USA) to sonically agitate the irrigation solution, two cycles of 30

seconds each. Smear layer was removed with 5 mL of 17% EDTA solution (Vista Dental,

Racine, WI, USA), followed with 5 mL of 5.25% NaOCl and a final flush with 10 mL of

25

distilled water. Canals were dried with paper points. After the completion of cleaning and

shaping, teeth were randomly divided into three experimental groups and the canals filled.

A ProTaper #40 gutta-percha master cone was fitted in the canal to working length with

excellent tug-back. The selected sealer for each group was placed in the canal, master cone

inserted, seared off with an Elements System B plugger handpiece (SybronEndo, Orange, CA,

USA) for down-pack leaving the apical 3 mm of gutta-percha in the canal.

Group 1 (n=18): Self-etch sealer: RealSeal SE sealer (SybronEndo, Orange, CA) was

dispensed from the auto mix syringe and placed onto a sterile glass slab, then introduced into

the canal lumen using a Lentulo spiral filler (Dentsply Tulsa Dental Specialties, Tulsa, OK,

USA). Excess sealer was removed with paper points to ascertain uniform coating of the canal

walls with only a thin layer of sealer. The canal lumen was then filled with thermo-plasticized

RealSeal SE injectable gutta-percha (SybronEndo, Orange, CA, USA) using an Elements

obturation unit extruder handpiece (SybronEndo, Orange, CA, USA) with a 23 gauge needle

tip and at a temperature setting of 115°C. The coronal end of the root filling was condensed

with Schilder pluggers (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) to offset

shrinkage of the filling core mass and additionally light cured the coronal aspect for 40

seconds with Spectrum curing light unit (Dentsply Tulsa Dental Specialties, Tulsa, OK,

USA). According to the manufacturer, the sealer in the canal will self-cure within 45 minutes.

Group 2 (n=18): Total-etch system: 37% phosphoric acid without benzalkonium chloride

(BAC) (Bisco, Schaumburg, IL, USA) was used to etch the root canal walls for 15 seconds,

then rinsed with sterile water for 15 seconds and dried with compressed air (Memorex Air

Duster, Imation Enterprises Corp, Oakdale, MN, USA) for 5 seconds. Adper Scotchbond

multi-purpose primer (3M ESPE, St Paul, MN, USA) was applied to the etched dentin

surface and was dried gently for 5 seconds. Adper Scotchbond multi-purpose adhesive (3M

ESPE, St Paul, MN, USA) was applied to the primed dentin and was lightcured for 10

seconds. Equal amounts of base and catalyst of Bisfil 2B self-cured resin (BISCO,

26

Schaumburg, IL, USA) were dispensed on a sterile glass slab and mixed until paste was

uniform (15 seconds) and was placed in the canal with a K-file #40 (Lexicon Flex SSK,

Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) to coat the walls uniformly. RealSeal SE

gutta-percha points were added passively until the canal lumen was filled. Excess was

removed with Elements System B plugger handpiece (SybronEndo, Orange, CA, USA) and

condensed with Schilder pluggers (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) to

offset shrinkage of the filling core mass.

Group 3 (n=18): Epoxy-resin sealer (AH Plus, Dentsply De Trey, Konstanz, Switzerland) and

gutta-percha. AH 26 sealer was dispensed on a sterile glass slab (equal amounts of both pastes

were mixed to a homogeneous consistency which broke when lifted 2 cm above the glass

slab), then introduced into the canal lumen using a Lentulo spiral filler (Dentsply Tulsa Dental

Specialties, Tulsa, OK, USA ). Excess sealer was removed with paper points to ascertain

uniform coating of the canal walls with only a thin layer of sealer. The canal lumen was then

filled with thermo-plasticized RealSeal SE injectable gutta-percha (SybronEndo, Orange, CA,

USA) using an Elements obturation unit extruder handpiece (SybronEndo, Orange, CA,

USA) with a 23 gauge needle tip and at a temperature setting of 115°C. The coronal end of the

root filling was condensed with Schilder pluggers (Dentsply Tulsa Dental Specialties, Tulsa,

OK, USA) to offset shrinkage of the filling core mass. This combination was selected after

results from a previous study that demonstrated that a more cohesive “monoblock” could be

obtained in a root canal when combining Resilon core material with the epoxy resin–based

sealer (AH-26) rather than Epiphany sealer (129).

All filled roots were stored for 72 hours in a 100% humid environment at 37°C (Hera Cell 150,

Heraeus, Newton, CT, USA) to allow the sealers to completely set. Subsequently, each root was

sectioned horizontally at 5 mm from its coronal end using a low-speed water-cooled rotary

diamond disc (Brasseler, Savannah, GA, USA) under sterile conditions. The remaining portion

of the roots was discarded. In this manner, standardized 5 mm-thick root dentin blocks with

filled canal lumens were obtained. Use of only the coronal portion of the roots was intended to

standardize specimens avoiding canal irregularities frequently encountered in the middle and

apical portions. Also, the coronal portion of root dentin is the most critical for investigation,

27

being the first challenged by bacteria invading through the pulp chamber (130). An indentation

with a round bur was performed on the coronal aspect of each specimen to clearly distinguish it

from the apical, so as to standardize the positioning for microscopic observation as described

below.

In all block specimens, the cementum periphery and exposed dentin on the coronal and apical

surfaces adjacent to the filled root canal margins was sealed with two layers of clear nail varnish

(Revlon, Mississauga, ON, Canada; no formaldehyde) to block cut dentinal tubules from access

to the sealer-dentin interface.

3.2 Degradation media incubation of specimens

Specimens underwent different incubation periods to expose the sealer-dentin interface to

potential degradation, before being incubated with E. faecalis (131).

Experimental groups: Three block specimens from each sealer group (Groups 1, 2, 3) were

incubated in sterile phosphate-buffered saline (Dulbecco's Phosphate Buffered Saline, containing

no calcium or magnesium; Invitrogen, Molecular Probes, Carlsbad, CA, USA) at 37°C, pH 7.0,

for each of the following periods:

7 days (n = 3/group)

1 month (n = 3/group)

3 months (n = 3/group)

6 months (n = 3/group)

28

3.3 Incubation of Specimens in Chemostat-Based Biofilm

Fermentor (CBBF)

At the end of their respective degradative incubation period, the specimens were suspended in a

chemostat-based biofilm fermentor (CBBF) (Appendix F). The continuous flow system consisted

of a 20 liter glass flask used as a reservoir to contain 16 liters of sterile TSB (BD Biosciences,

Sparks, MD, USA) that was supplemented with 200 cc of a 0.25% glucose (BDH Inc, Toronto,

Canada) solution that was placed on top of a hot plate stirrer (Corning stirrer, Corning, NY,

USA). The 0.25% glucose had been freshly prepared and filter-sterilized (Nalgene filters,

Thermo Fischer Scientific, Rochester, NY, USA). The tubing (LS25, Masterflex Precision pump

tubing, Cole Parmer instruments Co., Vernon Hills, IL, USA) coming from the flask was

attached to a drop counter to avoid any backflow and possible contamination due to negative

pressure back into the flask. Media flowed through the silicone tubing through a peristaltic pump

(Bio Rad Econo column pump; Hercules, CA, USA) that regulated the inflow rate into the glass

vessel that contained the samples in 400 cc sterile TSB in which 5 mL glucose had been added.

Another silicone tubing (LS16, Masterflex Precision pump tubing, Cole Parmer instruments Co.,

Vernon Hills, IL, USA, pumped out media through another peristaltic pump (Masterflex L/S,

Cole Parmer, Niles, IL, USA) and emptied into a waste vessel. All components of the system

were sterilized prior to each run by autoclaving at 121°C for 45 min (PRIMUS sterilizer Mc

Davis; North York, ON, Canada).Temperature in the fermentor’s vessel was set at 37°C and was

controlled through a temperature controller unit (Cole Parmer, Niles, IL, USA).

A mono-culture biofilm of E. faecalis (ATCC 47077) was established on the specimens for 7

days, through continuous flow of fresh medium (TSB supplemented with 0.25% glucose) which

was pumped into the vessel at a flow rate of 0.72 L/day (132) at a dilution rate of D=0.075/hour,

mimicking the resting flow rate of saliva over human oral tissues. E. faecalis was selected

because it is non-fastidious, biofilm-forming microorganism commonly found in the endodontic

flora of infected root-filled teeth (62, 133). The specific, non-pathogenic ATCC 47077 strain was

selected to meet the laboratory biosafety Level 1 requirement. After incubation in the CBBF, the

specimens were aseptically removed, gently rinsed with sterile phosphate-buffered saline and

29

stained using dihydroethidium (Invitrogen, Molecular Probes, Carlsbad, CA, USA). They were

then subjected to microscopic examination within 1 hour after staining, while maintained wet in

sterile PBS buffer.

Controls: To provide a baseline for bacterial presence and morphology for the subsequent

microscopic examination, one specimen from each sealer group (Groups 1, 2, 3) was subjected to

each one of the following procedures:

A. No pre-incubation, incubation in the CBBF without inoculation, staining.

B. No pre-incubation, incubation in the CBBF with inoculation, staining.

C. No pre-incubation, no incubation in the CBBF, staining.

3.4 Reflected Light Microscopy (RLM) Analysis

Biofilm proliferation and individual bacterial cell penetration were measured in specimens

stained with dihydroethidium (Invitrogen, Molecular Probes, Carlsbad, CA) using an

epifluorescence setup of the Leica DMIRE2 inverted microscope (Leica Microsystems; Wetzlar,

Germany). Red fluorescence of dihydroethidium was visualized by the TX2 filter cube

(excitation BP560/40, dichroic 595 nm, emission BP645/75). The object positioning was

controlled by an XY piezo Z stage (MS-2000, Applied Scientific Instrumentation Inc.).

Fluorescence imaging was performed by a black and white CCD camera (Hamamatsu) and

processed by a Power PC G5 (Apple Computer, Inc.Cupertino, CA, USA) using Openlab 4.0.2.

Software (Improvision Inc., Waltham, Massachussets, USA.). Selection of the regions of interest

(ROI) were performed by randomly assigning a “north” in the uppermost portion of the

specimen, and then establishing the corresponding opposite “south”, “east” and “west” for each

(all performed at 5X original magnification) (Fig. 12). Subsequently, those ROI were plotted into

a “map” and processed by a Power PC G5 (Apple Computer, Inc. Cupertino, CA, USA) using

Openlab 4.0.2 software (Improvision Inc., Waltham, Massachussets, USA.) (Fig. 13). The

30

imaging operations were programmable (using Openlab Automation) which substantially sped

up repetitive procedures and made them less invasive by reducing the irradiation of specimens.

Additionally, the software allows for re-evaluation of an area of interest multiple times, focusing

on the exact same analyzed point, as long as samples are not moved from the stage. Analysis of

the samples was performed in a corono-apical direction, establishing the zero at the base of the

sample (where it was contacting the base of the well) and progressing in an apical direction for

up to 500 µm, which was the maximum threshold available. Z stacks were obtained, establishing

the surface of the specimen as the zero, and in 5 µm incremental slices up to a maximum depth

of 500 µm. The depth of field was ± 1 µm.

Outcome measures: The depth of biofilm formation and bacterial invasion along the sealer-

dentin interface was recorded in microns from coronal to apical direction of the specimens.

Pictures were taken through the microscope’s eyepiece (Fig. 14) at 5X original magnification

and further analyzed at 10 X original magnification (Fig. 15).

3.5 Scanning Electron Microscopy (SEM) Analysis

Specimens were subjected to increasing concentrations of ethanol (30, 50, 70, 90, 95 and 100%)

for dehydration. Specimens were dried using a critical point dryer (Polaron CPD7501 critical

point dryer; Fisons instruments; Structure Probe Inc / SPI Supplies; West Chester, PA, USA).

Samples were sputter coated with platinum in a SEM coating system (Polaron coater SC515;

Fisons instruments; Structure Probe Inc / SPI Supplies; West Chester, PA, USA) and examined

by SEM (Hitachi S-2500; Sapporo, Japan) at 10 kV, (Fig. 16, 17).

31

3.6 Microbiological controls

Assessment of bacterial viability was conducted with the plate count method. Serial dilutions

(10-1

to 10-7

) were conducted both for the media obtained for the overnight culture and for the

media collected from the CBBF after the 1 week inoculation for each group aged for different

periods and for the controls, and plated onto BHI agar plates (and repeated in duplicates) (Fig.

7). Numbers of viable cells for the different time points (1 week, 1 month, 3 months and 6 month

samples) were comparable.

Gram staining was conducted for characterization of type of bacterial cells present in the media

extracted from the fermentor after 1 week inoculation and to confirm that a monospecies had

been growing. Results indicated homogeneity (Fig. 8).

PBS contained in the sterile vials where samples had been stored for the different degradation

times was analyzed. Undiluted media was plated in triplicate for each sample, and no growth was

observed for all the tested samples (Fig. 9).

• Samples were prepared with each sealer, stored individually in sterile vials for 72 hours

in a 100% humid environment at 37°C in a tri-gas cell culture incubator (HeraCell 150,

Heraeus, Newtown, CT, USA) to allow the sealers to completely set. They were then

placed in BHI, incubated at 37°C w/ 5% CO2 and OD600 readings were taken at 24, 48

and 72 hs. Results: no growth confirming sterility of samples.

32

3.7 Statistical Analysis

All study groups were run in parallel, with three independent samples in each group. One-way

ANOVA and LSD’ post-hoc analysis was conducted to determine the effect of sealer type and

incubation time on biofilm proliferation and bacterial penetration along the sealer-dentin

interface. The independent variables are the sealer type and the incubation time before

inoculation with bacteria. The dependent variables were biofilm proliferation and total bacterial

cells observed within the resin-sealer interface. The level of confidence was set to 95%.

33

Chapter 4

4 Article

Note: The following was submitted to the Journal of Endodontics

Proliferation of Pathogenic Biofilms within Sealer-Root Dentin

Interfaces is Affected by Sealer Type and Aging Period

Karina A Roth 1, DDS, Shimon Friedman

1 DMD, Céline M Lévesque PhD

2, Bettina R

Basrani DDS, PhD 1

and Yoav Finer DMD, PhD, FRCD(C)3

From the 1Discipline of Endodontics,

2Oral Microbiology and

3Biomaterials, Faculty of

Dentistry, University of Toronto, Toronto, Ontario, Canada.

Address requests for reprints to Dr Yoav Finer; Discipline of Biomaterials, Department of

Biological Sciences, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada

Fax number: (416) 979-4760

Phone number: (416) 979-4903 ext. 4554

E-mail address: yoav.finer@dentistry.utoronto.ca

34

Acknowledgements: Stephanie Koyanagi; Richard Mair; Milos Legner, Babak Shokati, Jian

Wang.

Grants: The American Association of Endodontists Foundation, Alpha Omega Fraternity, Endo

Tech, Canadian Association of Endodontists Endowment Fund.

Sources of support: Dentsply Tulsa Dental Specialties; SybronEndo; Endo Tech.

Abstract

Background: Root canal fillings are intended to prevent microbial proliferation over time in the

canal after treatment. Objective: To assess biofilm proliferation within the sealer-dentin

interfaces of two methacrylate resin-based systems, self-etch (SE) and total-etch (TE), and an

epoxy resin-based sealer (EP), aged for up to 6 months. Methods: Standardized specimens

(n=45) comprising the coronal 5 mm of human roots were filled with the test materials and gutta-

percha. Specimens were either not pre-incubated (control; n=9), or incubated in sterile saline for

1 week, 1 month, 3 months or 6 months days (n=3/group). Monospecies biofilms of

Enterococcus faecalis were grown on the specimens for 7 days in a chemostat-based biofilm

fermentor mimicking pathogenic oral conditions. The extent of E. faecalis proliferation within

the sealer-dentin interface for each material and incubation period group was assessed using

fluorescence microscopy of dihydroethidium-stained specimens. Results: TE had less biofilm

proliferation than both EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and

EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05).

Maximum depth of biofilm penetration was recorded for SE at 1 month (p<0.05). Conclusion:

Within the test model used, the self-etch and epoxy resin-based sealers were more susceptible to

interfacial biofilm proliferation than the total-etch restorative material. This susceptibility

diminished after aging the materials’ interfaces for 6 months.

Key words: resin-dentin interface, endodontic sealer, resin-composite, biofilm, E faecalis,

fluorescence microscopy

35

Introduction

Root canal fillings, comprising a core and a flowable sealer, should prevent bacterial ingress into

the canal after treatment (1). Sealers that adhere or bond to root dentin are expected to resist

bacterial proliferation within the sealer-dentin interface (2). Epoxy resin (ER)-based sealers

adhere to dentin and are considered the “gold standard” (3). Methacrylate resin (MR)-based

sealers bond to conditioned dentin (4) by penetrating the tubules (5) and interlocking with dentin

collagen forming a hybrid layer (6, 7). Two main MR-based systems are currently available: i)

“Total-etch” systems, requiring acid-etching, priming and bonding to form a hybrid layer (7).

Although they are the benchmark for bonded restorative systems (6), they are not available as

commercial endodontic sealers. ii) “Self-etch” systems employ one-step etching, priming and

bonding, incorporating the smear layer into the hybrid layer (8). They are available as endodontic

sealers, but concerns have recently emerged about inadequacy of their bond (8).

Bonding MR-based sealers to root dentin is challenging (9). Resin polymerization is inhibited by

dentin exposure to sodium hypochlorite (10), shrinkage-related debonding occurs due to

unfavorable cavity configuration (11), and interfacial degradation over time, allowing salivary

and tissue fluid movement between the hybrid layer and dentin (12), may lead to bacterial

proliferation and re-infection of the tooth (13).

Sealer-dentin interfaces have been studied using in-vitro static models measuring penetration of

dyes (14), endotoxins (15), inoculated bacteria (16) or saliva (17), with questionable clinical

relevance (18). Recently, our group has introduced the use of the chemostat-based biofilm

fermentor (CBBF) for assessment of interfacial bacterial biofilm proliferation of the cariogenic

biofilm organism Streptococcus mutans after aging of MR-dentin specimens (12). The purpose

of the present study was to assess biofilm proliferation within the sealer-dentin interface of two

MR-based systems, self-etch and total-etch, and an ER-based sealer, using the CBBF model.

36

Material and Methods

Specimen preparation and aging

Intact, human teeth with single canals (University of Toronto Human Ethics Protocol #24315)

were sterilized by gamma-irradiation (4080Gy) (19) and decoronated at the cemento-enamel

junction. Canals were prepared with ProTaper rotary instruments (Dentsply, Tulsa, OK), to size

F4 with intermittent 5.25% sodium-hypochlorite irrigation, flushed with 10mL distilled water

and dried with paper points. Roots were randomly divided into three sealer type groups

(n=18/group): EP, an ER-based sealer (AH Plus Dentsply, Konstanz, Switzerland); SE, a MR-

based self-etch sealer (RealSeal, SybronEndo, Orange, CA); TE, a MR-based total-etch

restorative material (Adper Scotchbond multi-purpose, 3M, St Paul, MN and Bisfil 2B self-cured

resin, Bisco, Schaumburg, IL).

In EP and SE, sealers were applied with a Lentulo (Dentsply), canals filled with injectable gutta-

percha (Elements, SybronEndo) compacted with Schilder pluggers (Dentsply), and the coronal

end light-cured (SE only). Canals in TE were etched with 37% phosphoric acid (Bisco) for 15

sec, rinsed with sterile water for 15 sec, lightly air-dried, treated with Scotchbond primer and

adhesive, light-cured, coated with Bisfil 2B, and filled with RealSeal SE gutta-percha points

(SybronEndo) using passive lateral compaction.

Filled roots were stored for 72hrs at 37°C and 100% humidity (Hera Cell 150, Heraeus, Newton,

CT), and sectioned horizontally 5mm from the coronal end with a slow-speed water-cooled

rotary diamond disc (Brasseler, Savannah, GA) under sterile conditions, obtaining standardized

5mm-long specimens. Peripheral cementum, apical surfaces and exposed coronal dentin adjacent

to root fillings were coated with nail varnish to prevent bacterial access to the sealer-dentin

interface through cut dentinal tubules. Specimens were subjected to aging in vials with sterile

37

phosphate-buffered saline (PBS), incubated (37°C, pH 7.0) for 7 days, 1, 3 or 6 months

(n=3/material group/time).

Biofilm cultivation

Aged specimens were suspended in CBBF (37°C) to cultivate monospecies biofilms of

Enterococcus faecalis (ATCC 47077) over interfacial margins (12), under continuous flow of

fresh Tryptic Soy Broth (BD Bioscience, Sparks, MD) with 0.25% (wt/vol) glucose, at 0.72L/d

(20) and dilution rate D=0.075/hr mimicking the resting salivary flow rate (13).

Specimens were aseptically removed after 7 days and gently rinsed with sterile PBS. To assess

bacterial viability, a 10 mL sample was collected from each vial, serially-diluted, and spot-plated

in triplicate onto Brain Heart Infusion agar for bacteria colony forming unit (CFU) counting after

24hr of incubation at 37°C. Bacterial counts in the order of 109

CFU/mL were obtained for all

tested samples.

Outcome assessment

Specimens were stained with dihydroethidium (Invitrogen, Molecular Probes, Carlsbad, CA) and

examined with an epifluorescence microscope (DMIRE2, Leica Microsystems; Wetzlar,

Germany). Red fluorescence was visualized with TX2 filter cube (excitation BP560/40, dichroic

595nm, emission BP645/75).

Biofilm proliferation and individual bacterial cell penetration were analyzed at 4 cardinal points

(100X magnification). Captured images (CCD; Hamamatsu, Shizuoka, Japan) were processed

38

(Openlab 4.0.2, Improvision, Waltham, MA) and Z-stack series established in a corono-apical

direction for each specimen in 5µm increments up to 500µm. Three-dimensional images of

biofilm formation were reconstructed from Z-stack series by Image J software (NIH).

Analysis

We performed one-way ANOVA and LSD post-hoc analysis (p<0.05) to determine the effects of

material and aging period on biofilm proliferation and bacterial cell penetration along the sealer-

dentin interfaces.

Results

Representative three-dimensional image reconstructions from Z-stack series captured from 1

month-aged specimens revealed different patterns of interfacial E. faecalis biofilm formation for

EP, SE and TE (Fig. 1, top). Z-stack images depict individual bacterial cells at different depths

within the interfaces of all test materials (Fig.1, bottom).

Mean interfacial biofilm proliferation depth ranged across test materials and aging periods (Fig.

2). Lumping aging periods together, biofilm proliferation depth differed significantly (p<0.005)

among test materials; TE had significantly less biofilm proliferation than EP and SE (p<0.01),

which did not differ significantly from each other.

SE and EP revealed biofilm proliferation for all aging periods with increased levels for

specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Maximum

39

depth of biofilm proliferation was recorded for SE at 1 month (p<0.05). No biofilms were

detected for TE at 3 and 6 months periods.

Individual bacterial cells within the sealer-dentin interfaces were detected consistently deeper

than biofilm aggregates for all test materials at all aging periods (See Appendix J, Fig. 17). Their

mean depth ranged from 198±66µm for 6 month-aged TE specimens, to 431±21µm for 3 month-

aged EP specimens. TE showed less individual bacterial cell penetration depth than SE and EP

(p<0.05).

Discussion

Novel endodontic sealers require assessment with physiologically relevant models. Our

previously used experimental model (12) was adapted for assessment of root canal sealers’

interface with root dentin. Interfaces were post-aged in CBBF to simulate pathogenic intraoral

conditions, and subsequently challenged by E. faecalis, an endogenous oral bacterial species

frequently isolated in root canal infections (21). E. faecalis forms monospecies biofilms over

root dentin, survives disinfection regimens and nutritional deprivation, and invades filled canals

and dentinal tubules (22). Coronal-third root dentin was used, being the first challenged by

bacteria invading through the pulp chamber (13). Its structure differs from that of apical root

dentin where abundant branching and irregularities (23) preclude standardization of specimens.

Sealers that adhere or bond to dentin, a desirable property (24) expected to curtail interfacial

bacterial ingress and proliferation, were tested. ER-based AH Plus is widely used and frequently

tested against novel sealers (3). Scotchbond and Bisfil 2B are typical components of MR-based

total-etch systems, producing durable dentin bonds (6). This design allowed comparison of

RealSeal SE, representing the new direction of MR-based self-etch sealers, to the ER-based

40

sealer and MR-based total-etch restorative, both extensively tested and used clinically for many

years.

Interfacial bacterial ingress occurred consistently, with less biofilm proliferation than individual

bacterial cell penetration. Biofilm proliferation overall was deeper for SE than for TE, peaking in

specimens aged for 1 month when it was also deeper than for EP. The susceptibility of SE to

biofilm proliferation suggested that it did not fully satisfy the requirements for root filling

materials (24). After aging for 6 months, biofilm proliferation declined for SE and EP and was

undetected for TE, suggesting that prolonged aging of the materials’ interfaces might have

changed the ecological milieu in a manner that curtailed biofilm proliferation despite invasion of

individual bacterial cells. This change may be related to altered sealing capacity of the materials

(12) and to the materials' degradation by-products released into the interfacial margins (25).

Previous study showed that free-floating planktonic cells of E. faecalis readily invaded all

interfaces within 7 days of inoculation prior to formation of biofilms (26). This observation

supported the aforementioned hypothesis that the extent of biofilm proliferation could be

influenced by the ecological milieu within the interfaces, which in turn might be affected by

aging-related degradation.

Despite the ability of AH Plus to adhere to root dentin, its ability to resist bacterial invasion has

been disputed (27, 28). Adhesive and mixed failures of its bond with root dentin (29) might lead

to gaps where bacteria can invade and proliferate. Indeed, our results indicate that AH Plus was

susceptible to interfacial bacterial invasion, even though biofilm proliferation appeared to

diminish with prolonged aging of the sealer-dentin interface.

MR-based sealers are expected to form a micromechanical bond with root dentin, but undergo

biodegradation over time (12, 30) allowing bacterial invasion and biofilm formation (12, 16).

Interfacial areas where the resin does not completely infiltrate the dentin substrate, or where it

does not polymerize within the hybrid layer, are especially susceptible (31). RealSeal SE was

previously reported to be inferior to AH Plus (32). Our results indicated that after aging for 1

41

month, RealSeal SE was more susceptible to interfacial biofilm proliferation to a greater extent

than the total-etch and AH Plus systems. This phenomenon could be attributed to the greater

hydrophilicity of the self-etch MR-based system that promotes water sorption and hydrolytic

breakdown of the interface (33).

MR-based total-etch systems are widely used in restorative dentistry as they produce the most

durable bond (6). Acid etching enhanced bonding to cervical root canal dentin up to 200% (34).

Our results indicated that the total-etch restorative material was only minimally susceptible to

interfacial biofilm proliferation, and significantly less than the self-etch sealer. However, no

total-etch endodontic sealers are commercially available, possibly because of application

challenges within the root canal configuration. Thus, even though the self-etch sealers represent

an innovation (35), developing a sealer that would be impervious to interfacial biofilm

proliferation remains an elusive goal.

Conclusions

This study presented a non-invasive assessment of interfacial biofilm proliferation between

sealers and root dentin simulating pathogenic oral conditions. The self-etch and epoxy-resin

based endodontic sealers were more susceptible at early stages to interfacial biofilm proliferation

than the total-etch restorative material, but this susceptibility diminished after aging of the

materials’ interfaces for 6 months. Given the importance of biofilm forming processes in

protecting bacteria, and their relevance to disease pathogenesis, the experimental model

presented herein could be instrumental for testing novel strategies aimed at improving the

resistance of the sealer-dentin interface to bacterial invasion and biofilm proliferation.

42

REFERENCES

1. Nair PN. Apical periodontitis: a dynamic encounter between root canal infection

and host response. Periodontol 2000 1997;13:121-148.

2. Barthel CR, Zimmer S, Wussogk R, Roulet JF. Long-Term bacterial leakage along

obturated roots restored with temporary and adhesive fillings. J Endod 2001;27(9):559-

562.

3. Brackett MG, Martin R, Sword J, Oxford C, Rueggeberg FA, Tay FR, et al.

Comparison of seal after obturation techniques using a polydimethylsiloxane-based root

canal sealer. J Endod 2006;32(12):1188-1190.

4. Schwartz RS, Fransman R. Adhesive dentistry and endodontics: materials, clinical

strategies and procedures for restoration of access cavities: a review. J Endod

2005;31(3):151-165.

5. Tay FR, Loushine RJ, Monticelli F, Weller RN, Breschi L, Ferrari M, et al.

Effectiveness of resin-coated gutta-percha cones and a dual-cured, hydrophilic

methacrylate resin-based sealer in obturating root canals. J Endod 2005;31(9):659-664.

6. De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M, et

al. A critical review of the durability of adhesion to tooth tissue: methods and results. J

Dent Res 2005;84(2):118-132.

7. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al.

Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future

challenges. Oper Dent 2003;28(3):215-235.

8. Babb BR, Loushine RJ, Bryan TE, Ames JM, Causey MS, Kim J, et al. Bonding of

self-adhesive (self-etching) root canal sealers to radicular dentin. J Endod 2009;35(4):578-

582.

9. Van Meerbeek B, Perdigao J, Lambrechts P, Vanherle G. The clinical performance

of adhesives. J Dent 1998;26(1):1-20.

10. Santos JN, Carrilho MR, De Goes MF, Zaia AA, Gomes BP, Souza-Filho FJ, et al.

Effect of chemical irrigants on the bond strength of a self-etching adhesive to pulp chamber

dentin. J Endod 2006;32(11):1088-1090.

11. Tay FR, Loushine RJ, Lambrechts P, Weller RN, Pashley DH. Geometric factors

affecting dentin bonding in root canals: a theoretical modeling approach. J Endod

2005;31(8):584-589.

12. Kermanshahi S, Santerre JP, Cvitkovitch DG, Finer Y. Biodegradation of resin-

dentin interfaces increases bacterial microleakage. J Dent Res 2010;89(9):996-1001.

43

13. Torabinejad M, Ung B, Kettering JD. In vitro bacterial penetration of coronally

unsealed endodontically treated teeth. J Endod 1990;16(12):566-569.

14. Stratton RK, Apicella MJ, Mines P. A fluid filtration comparison of gutta-percha

versus Resilon, a new soft resin endodontic obturation system. J Endod 2006;32(7):642-645.

15. Carratu P, Amato M, Riccitiello F, Rengo S. Evaluation of leakage of bacteria and

endotoxins in teeth treated endodontically by two different techniques. J Endod

2002;28(4):272-275.

16. Shipper G, Orstavik D, Teixeira FB, Trope M. An evaluation of microbial leakage

in roots filled with a thermoplastic synthetic polymer-based root canal filling material

(Resilon). J Endod 2004;30(5):342-347.

17. Khayat A, Lee SJ, Torabinejad M. Human saliva penetration of coronally unsealed

obturated root canals. J Endod 1993;19(9):458-461.

18. Wu MK, Wesselink PR. Endodontic leakage studies reconsidered. Part I.

Methodology, application and relevance. Int Endod J 1993;26(1):37-43.

19. White JM, Goodis HE, Marshall SJ, Marshall GW. Sterilization of teeth by gamma

radiation. J Dent Res 1994;73(9):1560-1567.

20. Pratten J, Andrews CS, Craig DQ, Wilson M. Structural studies of microcosm

dental plaques grown under different nutritional conditions. FEMS Microbiol Lett

2000;189(2):215-218.

21. Kaufman B, Spangberg L, Barry J, Fouad AF. Enterococcus spp. in endodontically

treated teeth with and without periradicular lesions. J Endod 2005;31(12):851-856.

22. Zehnder M, Guggenheim B. The mysterious appearance of enterococci in filled root

canals. Int Endod J 2009;42(4):277-287.

23. Mjor IA, Nordahl I. The density and branching of dentinal tubules in human teeth.

Arch Oral Biol 1996;41(5):401-412.

24. Grossman L. Antimicrobial effect of root canal cements. J Endod 1980;6(6):594-597.

25. Singh J, Khalichi P, Cvitkovitch DG, Santerre JP. Composite resin degradation

products from BisGMA monomer modulate the expression of genes associated with biofilm

formation and other virulence factors in Streptococcus mutans. J Biomed Mater Res A

2009;88(2):551-560.

26. Estrela C, Sydney GB, Figueiredo JA, Estrela CR. A model system to study

antimicrobial strategies in endodontic biofilms. J Appl Oral Sci 2009;17(2):87-91.

27. Hirai VH, da Silva Neto UX, Westphalen VP, Perin CP, Carneiro E, Fariniuk LF.

Comparative analysis of leakage in root canal fillings performed with gutta-percha and

Resilon cones with AH Plus and Epiphany sealers. Oral Surg Oral Med Oral Pathol Oral

Radiol Endod 2010;109(2):e131-135.

44

28. Nawal RR, Parande M, Sehgal R, Rao NR, Naik A. A comparative evaluation of 3

root canal filling systems. Oral Surg Oral Med Oral Pathol Oral Radiol Endod

2011;111(3):387-393.

29. Teixeira CS, Alfredo E, Thome LH, Gariba-Silva R, Silva-Sousa YT, Sousa-Neto

MD. Adhesion of an endodontic sealer to dentin and gutta-percha: shear and push-out

bond strength measurements and SEM analysis. J Appl Oral Sci 2009;17(2):129-135.

30. Shokati B, Tam LE, Santerre JP, Finer Y. Effect of salivary esterase on the integrity

and fracture toughness of the dentin-resin interface. J Biomed Mater Res B Appl Biomater

2010;94(1):230-237.

31. Hashimoto M. A review--micromorphological evidence of degradation in resin-

dentin bonds and potential preventional solutions. J Biomed Mater Res B Appl Biomater

2010;92(1):268-280.

32. Saleh IM, Ruyter IE, Haapasalo M, Orstavik D. Bacterial penetration along

different root canal filling materials in the presence or absence of smear layer. Int Endod J

2008;41(1):32-40.

33. Yiu CK, King NM, Pashley DH, Suh BI, Carvalho RM, Carrilho MR, et al. Effect of

resin hydrophilicity and water storage on resin strength. Biomaterials 2004;25(26):5789-

5796.

34. Ferrari M, Mannocci F, Vichi A, Cagidiaco MC, Mjor IA. Bonding to root canal:

structural characteristics of the substrate. Am J Dent 2000;13(5):255-260.

35. Teixeira FB, Teixeira EC, Thompson J, Leinfelder KF, Trope M. Dentinal bonding

reaches the root canal system. J Esthet Restor Dent 2004;16(6):348-354; discussion 354.

45

Figure Legends

Figure 1. Top images- Representative three-dimensional reconstruction of select Z-stack series

of E faecalis biofilms captured from sealer-dentin interfaces of epoxy resin-based (EP), self-etch

(SE) and total-etch (TE) sealers, aged for 1 month. Specimens were dihydroethidium-stained and

examined using epifluorescence microscopy (excitation in the range of 560/40, dichroic 595 nm

and emission of 645/75 nm, 100X magnification). Analysis of the samples was performed in a

corono-apical direction, establishing the zero at the base of the sample (where it was contacting

the base of the well) and progressing in an apical direction for up to 500 µm, which was the

maximum threshold available. The depth of field was ± 1 µm. Three-dimensional Images were

reconstructed using Image J software (National Institutes of Health, Bethesda, MD). Levels of

luminescence demonstrate different patterns of biofilm formation for the different materials.

Bottom images- Representative Z-stack images of E. faecalis captured from the sealer-dentin

interface of specimens aged for 1 month. Images show differences in trends of biofilm presence

within the interface (µm from the specimen’s surface in an apical direction) for each material

group.

Figure 2: E faecalis biofilm formation along the sealer-dentin interfaces of epoxy resin-based

(EP), self-etch (SE) and total-etch (TE) methacrylate-based sealers. Specimens were aged in PBS

(37ºC, pH=7.0) for 1 week, 1, 3 and 6 months, then incubated with E. faecalis in biofilm forming

constant media conditions for 7 days. Mean ± SE values with same superscripts denote

statistically not-significant differences between groups (p > 0.05, one-way ANOVA, LSD post-

hoc test). Mean biofilms depths (from the specimen’s surface, in a corono-apical direction)

ranged from 0 to 355 ± 21 µm from the specimen’s surface. TE showed lower biofilm

proliferation than EP and SE (p<0.01). No biofilm were detected for TE specimens pre-incubated

for 3 and 6 months. Maximum depth of biofilm proliferation was recorded for SE at 1 month

(p<0.05).

46

Figure 1

Figure 2

10 µm 10 µm 10 µm

47

Chapter 5

5 Discussion

Current and novel endodontic sealer systems require assessment with physiologically relevant

models. Our experimental model, based on previous research with S. mutans biofilms, coronal

dentin and MR-based restorative material (27), was adapted for assessment of root canal sealers’

interface with root dentin. Samples were individually kept in sterile glass vials containing PBS to

expose the interfaces to the different assigned study time points, after which, they were

asceptically removed and challenged with E faecalis in the chemostat-based biofilm fermentor to

simulate pathogenic intraoral conditions. These two separate phases were established in order to

separate the aging phase of the specimens (up to 6 months) from the exposure to bacteria in the

CBBF (all specimens being exposed for 7 days).

The previous timelines were chosen due to the fact that:

1. It is very demanding to run the CBBF for up to 6 months, with a high risk of contamination of

the samples.

2. There is limited space within the CBBF to accommodate all specimens from the different

groups and time frame for such a long period.

3. Different aging groups cannot be run in parallel due to the availability of a single vessel for

this purpose. Until complete run of the experiment for one group is completed, a subsequent one

48

cannot be studied. Use of a single vessel, eliminates the possibility of variation in the set-up

which could affect the outcomes.

4. Analysis of degradation products cannot be done in specimens from the CBBF due to the

continuous replacement of the media.

After sample shad been exposed to the different time points for degradation, they were

subsequently challenged by E. faecalis, a common constituent of the endodontic microflora in

root-filled teeth (59). E. faecalis forms monospecies biofilms over root dentin, survives

disinfection regimens and nutritional deprivation, and invades the filled canal and dentinal

tubules (63-67).

Specimens were prepared from the coronal portion of roots, the first challenged by bacteria

invading through the pulp chamber (130). Structure of root dentin differs from that of coronal

dentin, with abundant branching and irregularities in the apical area of the root (42) precluding

standardization of apical root segments.

We investigated sealers that can adhere or bond to root dentin, a desirable property (8) expected

to curtail microbial ingress. AH Plus is a widely used, epoxy-resin-based dentin-adhering sealer

(89), frequently used as the "gold standard" for comparison of novel sealers (18). RealSeal SE is

a typical MR-based self-etch sealer, while Scotchbond and Bisfil 2B comprise a typical MR-

based total-etch "gold standard" for dentin bonding restorative materials. This design allowed

49

comparison of the self-etch sealer, representing a new direction in root filling materials (134) , to

the ER-based sealer and MR-based total-etch restorative, both extensively tested and used

clinically for many years.

Biofilms develop preferentially on inert surfaces, or on dead tissue. A root canal system after

endodontic treatment has no defences against bacteria or their by-products. When biofilms are

formed, their structure protects the residing bacteria from environmental threats and host defense

mechanisms. Microorganisms in biofilm form show higher resistance to both antimicrobial

agents and host defense mechanisms when compared with planktonic cells (75-77). From an

endodontic perspective, it is possible to eradicate bacteria in planktonic state, and much harder to

eliminate well established biofilms.

Interfacial bacterial penetration was observed with all tested materials after all aging periods.

Biofilm proliferation consistently extended to less depth than that of planktonic bacteria.

Comparing the three test materials, SE allowed for significantly deeper biofilm proliferation than

TE, the “gold standard” for MR-based restorative materials. After aging for 1 month, biofilm

proliferation for SE was also deeper than for EP, the commonly used epoxy based-based sealer.

These findings suggested that after aging for 1 month, SE was more susceptible to biofilm

proliferation than the two widely used materials, and in this regard, it did not fully satisfy the

requirements for root filling materials (8). After aging for 6 months, biofilm proliferation for SE

and EP was comparable to aging for 1 week, and it was zero for TE, suggesting that with

prolonged aging, all test materials’ interfaces might have changed in a manner that curtailed

50

biofilm proliferation despite the invasion of planktonic bacteria. This suggestion was

substantiated by the comparison of the four aging periods, with 1 and 3 months showing

significantly deeper biofilm proliferation than 1 week and 6 months. The aging might have

affected changes in the ecological milieu, related to altered sealing capacity of the materials (27)

and to the materials' degradation by-products (32).

Interfacial invasion of planktonic bacterial cells in all materials and aging periods was expected,

as a stage preceding the formation of biofilms (135). Indeed, E. faecalis readily invaded all

interfaces within 7 days of inoculation, regardless of aging or material. This observation

supported the aforementioned suggestion that the extent of biofilm proliferation was affected by

the ecological milieu within the interfaces, which in turn was affected by aging-related

degradation.

Singh et al (32) reported a potential influence of resin monomer-derived biodegradation products

on biofilm formation. Hydrolysis of methacrylate monomers (such as TEGDMA) produces

biodegradation by-products such as methacrylic acid (MA) and triethylene glycol (TEG). These

by-products affect various cellular activities such as nutrient uptake, signal transduction, and

gene expression when directly contacting oral bacteria (31). For example, triethylene glycol

(TEG) proved to be a growth stimulant (136) being utilized as a carbon source by anaerobic

bacteria, and MA had an inhibitory effect on bacterial growth. Similarly, other degradation by-

product may affect bacterial growth and gene expression.

51

Comparable levels of bacterial cell penetration were measured for TE and SE specimens.

However, TE group had significantly lower biofilm formation vs. SE throughout the aging

periods, with no biofilm detected for 3 and 6 months aging groups. These findings lead to the

hypothesis that the material itself or possible breakdown products could perturb the local micro-

environment within the interfacial gap and inhibit bacterial growth (31, 32, 136). Concentration

of eluted components and degradation products from the sealer system could be relatively high in

such a small interfaces and in sufficient levels to affect biofilm formation. It is possible

therefore, that the difference in biofilm formation observed between SE and TE specimens, is

related to the materials’ different formulations and biostability.

Despite the ability of AH Plus to adhere to root dentin (89), its ability to resist bacterial invasion

has been disputed (137, 138). Adhesive and mixed failures of its bond with root dentin (19)

might lead to gaps where bacteria can proliferate. Indeed, our results indicate that AH Plus was

susceptible to interfacial bacterial invasion, even though biofilm proliferation appeared to

diminish with prolonged aging of the sealer-dentin interface.

MR-based sealers are expected to form a micromechanical bond with root dentin (91), but that

bond may undergo biodegradation over time (110) allowing bacterial invasion (10). Interfacial

areas where the resin did not completely infiltrate the dentin substrate, or where it did not

polymerize within the hybrid layer, are especially susceptible (139). In regards to bacterial

invasion, RealSeal SE was previously reported to be inferior to AH Plus (87). Indeed, our results

52

indicated that RealSeal SE was susceptible to interfacial biofilm proliferation to a greater extent

than the total-etch system and, after aging for 1 month, also the AH Plus sealer.

MR-based total-etch systems are widely used in restorative dentistry as they produce the most

durable bond (24). Acid etching enhanced bonding to cervical-level root canal dentin up to 200%

(140). Indeed, our results indicated that the total-etch restorative material was only minimally

susceptible to interfacial biofilm proliferation, and significantly less than the self-etch sealer.

However, as yet no total-etch endodontic sealers are commercially available, possibly because of

application challenges within the root canal configuration. Thus, even though the self-etch

sealers represent an innovation (134), developing a sealer that would be impervious to interfacial

biofilm proliferation remains an elusive goal.

The differences in dentin bonding mechanisms among the three test materials notwithstanding, it

should be taken into account that the high ratio of the bonded surface area in a cavity to the

unbounded surface area (C factor) present in root canals could affect shrinkage-related

debonding (14), potentially leading to bacterial ingress. During polymerization, the volume of

monomers is reduced, which creates sufficient shrinkage stresses to debond the material from

dentin, thereby decreasing retention and increasing leakage. Bonding of adhesive root-filling

materials to root canals is challenging due to the configuration of the root canal space. SEM

analysis revealed interfacial gaps along the sealer dentin interface of all specimens studied.

These gaps provided pathways for bacterial invasion and subsequent proliferation and biofilm

formation. A distinct pattern of gap formation amongst the different materials was found, with

53

larger gaps for SE, followed up EP, while the smallest gaps were found for TE. These findings

were consistent for all specimens at all time points. It can therefore be hypothesized that the

magnitude of the interfacial gap affected bacterial cell penetration, and even more significantly

affect biofilm formation within the interface as evidenced by the statistical results.

54

Chapter 6

6 Conclusions

This study presented a non-invasive assessment of interfacial biofilm proliferation between

sealers and root dentin simulating pathogenic oral conditions. The self-etch and epoxy-resin

based endodontic sealers were more susceptible at early stages to interfacial biofilm proliferation

than the total-etch restorative material, but this susceptibility diminished after aging of the

materials’ interfaces for 6 months. Given the importance of biofilm forming processes in

protecting bacteria, and their relevance to disease pathogenesis, the experimental model

presented herein could be instrumental for testing novel strategies aimed at improving the

resistance of the sealer-dentin interface to bacterial invasion and biofilm proliferation.

Overall, the total-etch system demonstrated less interfacial biofilm proliferation and penetration

of individual E. faecalis cells. Therefore, the null hypothesis that “that there will be no difference

in the interfacial bacterial penetration and biofilm proliferation of E. faecalis between root dentin

and three test materials (total-etch resin, self-etch sealer, epoxy resin sealer) following aging of

the interfaces for up to 6 months” is rejected.

55

Chapter 7

7 Recommendations

Improvements / modifications to the established protocol / future directions:

• Use of confocal microscopy to analyze bacterial cell and biofilm formation within the

resin-dentin interface. This will increase the optical resolution and contrast of the

acquired images due to its ability to eliminate out-of-focus light in specimens that are

thicker than the focal plane. Additionally, this technique is able to generate three

dimensional images by superimposing the obtained images without the need of additional

software or image transfer.

• Standardize to cylindrical samples: if further analysis could be conducted with the use of

confocal laser microscopy, due to the pattern of light reflection, samples must have

parallel walls to allow light transmission / reflection. Human roots, are tapered, thus

analysis through this modality is not possible unless modifications are made.

Alternatively, use of bovine teeth would not only have the benefit of more parallel root

canals, but also, due to their larger size, a wider area for analysis could be obtained.

• Use of more specific fluorescent dyes and/or markers. This will allow for better analysis

of bacterial cells and biofilms within the interface and reduce optical interference from

materials and tooth structures. For example utilization of the dead/live (Baclight) in

combination with confocal microscopy will allow for bacterial live/dead ratio and further

investigate if the eluted products exert any effects on bacteria at the different evaluated

time points.

56

• Human saliva and periapical tissues contain enzymes which are capable of degrading the

resin-dentin at accelerated manner compared with buffer alone. Evaluate performance

difference between the materials with relevant enzyme containing-degradative media will

provide better simulation of in-vivo conditions.

• Analysis of incubation media for the presence of degradation by-products. This will

allow for quantitative measurement of the degradation of the sealer materials and possible

correlation with actual degradation of the interface and interfacial biofilm proliferation.

57

Chapter 8

8 Appendices

Appendix A: MEDIA AND SOLUTIONS

Appendix B: GAMMA IRRADIATION

Appendix C: SAMPLE PREPARATION

Appendix D: STERILITY ASSAYS OF SPECIMEN PREPARATION

Appendix E: MICROBIOLOGY TECHNIQUES

Appendix F: CHEMOSTAT-BASED BIOFILM FERMENTOR SET-UP

Appendix G: STATISTICS

Appendix H: MICROSCOPIC IMAGES

Appendix I: SEM IMAGES

Appendix J: BACTERIAL CELL PENETRATION

Appendix K: ETHICS APPROVAL

58

APPENDIX A: MEDIA AND SOLUTIONS

Sterile glucose solutions

Stock solutions of glucose (1% (wt/vol), 20% (wt/vol)) were prepared by dissolving D-Glucose

Anhydrous (BDH Inc.) in sterile distilled deionised water (Milli-Q Gradient Millipore filter,

Fisher Scientific). The final volume was adjusted to 100 ml with sterile distilled deionised water

prior to filter sterilization. For bacterial growth, filter-sterilized glucose was added to the media

at a final concentration of 0.25% (vol/vol).

Trypticase Soy Broth (TSB)

Trypticase Soy Broth (TSB) was prepared by dissolving 30.0 g of Bacto Tryptic Soy Broth

powder (BD Biosciences) in 1.0 litre of distilled deionised water, and sterilized by autoclaving.

Brain Heart Infusion (BHI)

Brain Heart Infusion (BHI) broth and BHI agar were prepared by dissolving 37.0 g of Bacto BHI

or BHI agar (BD Biosciences) in 1.0 litre of distilled deionised water, and sterilized by

autoclaving.

59

APPENDIX B: GAMMA IRRADIATION

Human caries-free teeth, even if extracted for orthodontic reasons, are not free of bacteria. In

order to prepare samples that will be later inoculated with a specific type of microorganism, and

due to infection control concerns regarding the handling of teeth for research purposes, teeth

need to be sterilized by a process that does not affect their natural properties. Gamma irradiation

has been effectively used for sterilization purposes and has proven to be an effective system

(141) but there is the potential for changes in the dentin substrate after its utilization (142). De

Wald (143) in a literature review, concluded that Gamma radiation did not produce structural

changes in dentin as well as Brauer (144) or White et al. (128) that also found that gamma

radiation had no effect on dentin permeability, nor on structural changes of the dentin as

measured by Fourier transform infrared spectroscopy (FTIR) or ultraviolet-visible-near-infrared

(UV NISINIR) spectra.

Doses ranging between 7 kGy and 35 kGy are safe to use without causing alterations in the

dentin surface morphology. Most studies use doses of 25 kGy with an exposure time of 6 hours

which is capable of inactivating most microorganisms and showing no deleterious effect to

dentin (142) or enamel (145).

Consequently, samples were sent to the Gamma Irradiation services of the University of

Toronto’s Department of Environmental Nuclear Science, for processing.

Figure 3: Samples in sterile vial after being gamma irradiated.

60

APPENDIX C: SAMPLE PREPARATION

Figure 4. Illustration of the sample preparation procedure. Freshly extracted human lower

premolar teeth (a) were kept frozen until use. The crowns were severed at the cemento-enamel

junction (b). Teeth were sent for gamma irradiation and kept in sterile vials until processing.

Root canal treatment was performed (c) and all filled roots were stored for 72 hours in a 100%

humid environment at 37°C to allow the sealers to completely set. Subsequently, each root was

sectioned horizontally at 5 mm from its coronal end (d) using a low-speed water-cooled rotary

diamond disc (Brasseler, Savannah, GA, USA) under sterile conditions. The coronal portion of

the root was kept for analysis and the remaining portion of the roots was discarded. In this

manner, standardized 5 mm-thick root dentin blocks with filled canal lumens were obtained.

61

Figure 5. Photograph of the sample preparation procedure: freshly extracted teeth (a) and after

their decoronation (b).

Figure 6. Illustration of the 5 mm specimens with their cementum periphery and exposed dentin

on the coronal and apical surfaces adjacent to the filled root canal margins sealed with two layers

of nail varnish (a, b). Photograph of one of the samples (c).

a, b

c

a b

62

APPENDIX D: STERILITY ASSAYS OF SPECIMEN PREPARATION

INTRODUCTION

Pilot studies were run to assess the sterility of the chemostat-based biofilm fermentor (CBBF).

METHOD

The CBBF was run continuously for 7 days inside a laminar flow hood. Visual inspection of the

CBBF vessel and the inflow flask were performed on a daily basis to ensure that there could be

no problems and that biofilms were established and grown under carefully controlled conditions.

RESULTS

Contamination of the CBBF vessel occurred twice during pilot studies. For the first

contamination, we suspected that the problem might be due to backflow of media into the inflow

pump. A drop count mechanism was added to the system to overcome this problem. The second

contamination was most probably due to a problem with the outflow pump. The pump was

replaced and the outflow tubing was set accordingly to maintain the media levelled at 400 cc.

CONCLUSION

After the changes were implemented, the CBBF system was successfully run twice during a 7-

day period without any contamination.

63

APPENDIX E: MICROBIOLOGY TECHNIQUES

AGAR PLATE STOCK CULTURE

The wild-type strain Enterococcus faecalis ATCC 47077 was generously donated by Dr. Celine

Levesque from the Department of Oral Microbiology, Faculty of Dentistry. E. faecalis strain was

routinely cultured on BHI agar at 37°C. Bacteria were stored for up to one week on BHI-agar

plate at 4°C.

PREPARATION OF CULTURES FOR CBBF

The precultures were prepared as follows:

Day 1: Few colonies of E. faecalis grown on the surface of a BHI-agar plate were used to

inoculate 10 mL of TSB broth. The culture was incubated statically overnight at 37°C.

Day 2: The overnight preculture was diluted (1:20) into 30 ml of fresh TSB and incubated under

agitation at 37°C for 18 h.

Day 3: The overnight culture was diluted (1:30) into 400 ml of fresh TSB broth supplemented

with 0.25% sterile glucose inside the CBBF vessel.

SERIAL DILUTIONS

Bacterial viability was assessed by plate counting on BHI-agar. Briefly, serial dilutions (10-1

to

10-7

) were performed with sterile phosphate-buffered saline and 20 µl of diluted cells were spot-

plated in triplicates onto BHI-agar for enumeration. The colonies were counted after 24 h of

incubation at 37°C (Fig. 7). The results are given as colony forming units (CFU) per ml and are

presented at Table 1.

64

Fig. 7. Bacterial viability assessed using the plate counting technique.

Table 1. Bacterial viability expressed as CFU/ml for the overnight preculture and CBBF system

after 1 week.

Experiment Preculture (CFU/ml) CBBF (CFU/ml)

1 week 7.03 108 1.86 10

9

1 month 1.49 109 1.86 10

9

3 months 1.71 109 6.49 10

8

6 months 2.26 109 2.98 10

8

CONTROL

65

GRAM STAINING

Gram staining was performed on biofilm culture after one week to confirm culture purity.

Typical results are shown at Fig. 8.

Fig. 8. Gram stain of E. faecalis cultured into the CBBF after 1 week.

STERILITY CONTROL

Sample preparations were immersed into sterile phosphate-buffered saline (PBS) and kept into

vials at 37°C. To ensure that no contamination occurred during the PBS incubation, 20 µl of

immersing solution was spot-plated in triplicates onto BHI-agar for enumeration (Fig. 3). No

colonies were detected after 24 h of incubation at 37°C confirming the sterility of our

preparations.

Fig. 9. Analysis of immersing solution obtained from the vials.

66

APPENDIX F: CHEMOSTAT-BASED BIOFILM FERMENTOR SET-UP

Fig. 10. Individual components of the chemostat-based biofilm fermentor (CBBF)

Fig. 11. Image of the CBBF set-up within the laminar flowhood.

Prior to use, all components of the system were sterilized prior to each run by autoclaving at

121°C for 45 min (PRIMUS sterilizer Mc Davis; North York, ON, Canada). The system was

67

assembled within the confines of a laminar flowhood (Labculture® Class II Type A2 Biohazard

Safety Cabinet, Esco Micro Pte Ltd, Singapore).

The continuous flow system consisted of a 20-liter glass flask used as a reservoir to contain 16

liters of sterile TSB that was supplemented with 200 cc of a 0.25% glucose solution that was

placed on top of a hot plate stirrer (Corning stirrer, Corning, NY, USA). Within the flask, a

magnetic stirrer had been placed to continuously stir the liquid within the vessel and create low

levels of physical stress within the environment.

The 0.25% glucose had been freshly prepared and filter-sterilized (Nalgene filters, Thermo

Fischer Scientific, Rochester, NY, USA). The tubing coming from the flask was attached to a

drop counter to avoid any backflow and possible contamination due to negative pressure back

into the flask.

Prior to inoculation, the CBBF was operated in for 24 hours to make sure that media was

pumping in and out of the vessel at the pre-set parameters.

Media flowed through silicone tubing (LS25, Masterflex Precision pump tubing, Cole Parmer

instruments Co., Vernon Hills, IL, USA) through a peristaltic pump (Bio Rad Econo column

pump; Hercules, CA, USA ) that regulated the inflow rate into the glass vessel that contained the

samples in 400 cc sterile TSB in which 5 mL glucose had been added. Another silicone tubing

(LS16, Masterflex Precision pump tubing, Cole Parmer instruments Co., Vernon Hills, IL, USA,

pumped out media through another peristaltic pump (Masterflex L/S, Cole Parmer, Niles, IL,

USA) and emptied into a waste vessel. Temperature in the fermentor’s vessel was set at 37°C

and was controlled through a temperature controller unit (Cole Parmer, Niles, IL, USA).

68

A mono-culture biofilm of E. faecalis (ATCC 47077) was established on the specimens for 7

days, through continuous flow of fresh medium (TSB supplemented with 0.25% glucose) which

was pumped into the vessel at a flow rate of 0.72 L/day (132) at a dilution rate of D=0.075/hour,

mimicking the resting flow rate of saliva over human oral tissues (146-148).

E. faecalis was selected because it is non-fastidious, biofilm-forming microorganism commonly

found in the endodontic flora of infected root-filled teeth (62, 133). The specific, non-pathogenic

ATCC 47077 strain was selected to meet the laboratory biosafety Level 1 requirement. After

incubation in the CBBF, the specimens were aseptically removed, gently rinsed with sterile

phosphate-buffered saline, and stained using dihydroethidium (Invitrogen, Molecular Probes,

Carlsbad, CA, USA). They were then subjected to microscopic examination within 1 hour after

staining, while maintained wet in sterile PBS buffer.

69

APPENDIX G: STATISTICS

Biofilm depth, Univariate

Time point Post-hoc: Dependent Variable:Biofilm Depth Total

70

Material Post-hoc: Dependent Variable:Biofilm_Depth_Total

71

72

APPENDIX H: MICROSCOPIC IMAGES

Selection of cardinal points for microscopic analysis

Fig. 12. Fluorescence imaging was performed by a black and white CCD camera (Hamamatsu)

and processed by a Power PC G5 (Apple Computer, Inc.Cupertino, CA, USA) using Openlab

4.0.2. Software (Improvision Inc.,Waltham, Massachussets, USA.). Microscopic image of a

sample (10 X original magnification) denoting demarcation of cardinal points to be subsequently

analyzed (N = north, S = south, E = east, W = west). Four points were randomly identified for

each sample. At each point, analysis was performed in a corono-apical direction, starting at the

surface of the sample and progressing through the interface when present.

W E

N

S

73

Fig. 13. Selected points to be analyzed, were mapped and processed by a Power PC G5 (Apple

Computer, Inc.Cupertino, CA, USA) using Openlab 4.0.2. Software (Improvision Inc.,Waltham,

Massachussets, USA.). The imaging operations are programmable (using Openlab Automation)

which substantially speeds up repetitive procedures and makes them less invasive by reducing

the irradiation of specimens. Multiple samples and areas of interest can be mapped

simultaneously (in this image, 6 samples have been mapped). Additionally, the software allows

for re-evaluation of an area of interest multiple times, focusing on the exact same analyzed point,

as long as samples are not moved from the stage. Magnified view of the representative four

cardinal points (red square).

74

Microscopic images

Fig. 14. Photographic images taken with a digital camera (Olympus Stylus 5010, 7.1 megapixels,

5 X zoom, Olympus, Tokyo, Japan) through microscope’s optics of samples from EP (a), SE (b)

and TE (c) groups, at 5X (original magnification).

Fig. 15. Microscopic images (5X original magnification) showing gap formation along the

dentin-sealer interface in specimens from EP group (a), SE (b) and TE (c) group. All specimens,

for all groups at all incubation periods exhibited an open interface which could potentially allow

for bacterial penetration.

75

APPENDIX I: SEM IMAGES

Scanning electron microscopy produces high resolution images of a sample surface which allows

visualization of details which are smaller than 1 µm. Due to its large depth of field, a three-

dimensional appearance is obtained, which is useful for analyzing the surface structure of a

sample.

Specimens were subjected to increasing concentrations of ethanol (30, 50, 70, 90, 95 and 100%)

for dehydration. Specimens were dried using a critical point dryer (Polaron CPD7501 critical

point dryer; Fisons instruments; Structure Probe Inc / SPI Supplies; West Chester, PA, USA).

Samples were sputter coated with platinum in a SEM coating system (Polaron coater SC515;

Fisons instruments; Structure Probe Inc / SPI Supplies; West Chester, PA, USA) and examined

by SEM (Hitachi S-2500; Sapporo, Japan) at 10 kV, Fig. 16.

After SEM analysis, a different and distinct pattern of gap formation was observed for specimens

filled with the different test materials, which held true and consistent for all specimens within a

group at all time points analyzed. Wider gaps were consistently observed for SE specimens,

followed by EP and finally TE specimens revealed the smallest. It can therefore be hypothesized

that the magnitude of the interfacial gap affected bacterial cell penetration, and even more

significantly affect biofilm formation within the interface. .

76

Fig. 16. Images obtained through SEM (Hitachi S-2500; Sapporo, Japan) at 10 kV, 200 X

original magnification, demonstrating gap formation in samples from EP group (a), SE group (b)

and TE group (c). Comparison of the size and type of gap observed was consistent among

samples from the same group at all time points, and was related to the different levels of biofilm

formation.

77

APPENDIX J: BACTERIAL CELL PENETRATION

Fig. 17. Mean values for bacterial cell penetration, for each sealer at each incubation time

point.

78

APPENDIX K: ETHICS APPROVAL

79

References

1. Nair PN. Apical periodontitis: a dynamic encounter between root canal infection

and host response. Periodontol 2000 1997;13:121-148.

2. Sjogren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of

root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int

Endod J 1997;30(5):297-306.

3. Paquette L, Legner M, Fillery ED, Friedman S. Antibacterial efficacy of

chlorhexidine gluconate intracanal medication in vivo. J Endod 2007;33(7):788-795.

4. Peters LB, Wesselink PR. Periapical healing of endodontically treated teeth in one

and two visits obturated in the presence or absence of detectable microorganisms. Int

Endod J 2002;35(8):660-667.

5. Waltimo T, Trope M, Haapasalo M, Orstavik D. Clinical efficacy of treatment

procedures in endodontic infection control and one year follow-up of periapical healing. J

Endod 2005;31(12):863-866.

6. Gomes BP, Lilley JD, Drucker DB. Variations in the susceptibilities of components

of the endodontic microflora to biomechanical procedures. Int Endod J 1996;29(4):235-241.

7. Siqueira JF, Jr., Rocas IN. Clinical implications and microbiology of bacterial

persistence after treatment procedures. J Endod 2008;34(11):1291-1301 e1293.

8. Grossman L. Antimicrobial effect of root canal cements. J Endod 1980;6(6):594-597.

9. Carratu P, Amato M, Riccitiello F, Rengo S. Evaluation of leakage of bacteria and

endotoxins in teeth treated endodontically by two different techniques. J Endod

2002;28(4):272-275.

10. Shipper G, Orstavik D, Teixeira FB, Trope M. An evaluation of microbial leakage

in roots filled with a thermoplastic synthetic polymer-based root canal filling material

(Resilon). J Endod 2004;30(5):342-347.

11. Cobankara FK, Adanr N, Belli S. Evaluation of the influence of smear layer on the

apical and coronal sealing ability of two sealers. J Endod 2004;30(6):406-409.

12. Murray PE, Hafez AA, Smith AJ, Cox CF. Bacterial microleakage and pulp

inflammation associated with various restorative materials. Dent Mater 2002;18(6):470-

478.

13. Van Meerbeek B, Perdigao J, Lambrechts P, Vanherle G. The clinical performance

of adhesives. J Dent 1998;26(1):1-20.

14. Tay FR, Loushine RJ, Lambrechts P, Weller RN, Pashley DH. Geometric factors

affecting dentin bonding in root canals: a theoretical modeling approach. J Endod

2005;31(8):584-589.

80

15. Lai SC, Mak YF, Cheung GS, Osorio R, Toledano M, Carvalho RM, et al. Reversal

of compromised bonding to oxidized etched dentin. J Dent Res 2001;80(10):1919-1924.

16. Santos JN, Carrilho MR, De Goes MF, Zaia AA, Gomes BP, Souza-Filho FJ, et al.

Effect of chemical irrigants on the bond strength of a self-etching adhesive to pulp chamber

dentin. J Endod 2006;32(11):1088-1090.

17. Ricucci D, Siqueira JF, Jr. Biofilms and apical periodontitis: study of prevalence

and association with clinical and histopathologic findings. J Endod 2010;36(8):1277-1288.

18. Brackett MG, Martin R, Sword J, Oxford C, Rueggeberg FA, Tay FR, et al.

Comparison of seal after obturation techniques using a polydimethylsiloxane-based root

canal sealer. J Endod 2006;32(12):1188-1190.

19. Teixeira CS, Alfredo E, Thome LH, Gariba-Silva R, Silva-Sousa YT, Sousa-Neto

MD. Adhesion of an endodontic sealer to dentin and gutta-percha: shear and push-out

bond strength measurements and SEM analysis. J Appl Oral Sci 2009;17(2):129-135.

20. Schwartz RS, Fransman R. Adhesive dentistry and endodontics: materials, clinical

strategies and procedures for restoration of access cavities: a review. J Endod

2005;31(3):151-165.

21. Tay FR, Loushine RJ, Monticelli F, Weller RN, Breschi L, Ferrari M, et al.

Effectiveness of resin-coated gutta-percha cones and a dual-cured, hydrophilic

methacrylate resin-based sealer in obturating root canals. J Endod 2005;31(9):659-664.

22. Nakabayashi N, Kojima K, Masuhara E. The promotion of adhesion by the

infiltration of monomers into tooth substrates. J Biomed Mater Res 1982;16(3):265-273.

23. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al.

Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future

challenges. Oper Dent 2003;28(3):215-235.

24. De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M, et

al. A critical review of the durability of adhesion to tooth tissue: methods and results. J

Dent Res 2005;84(2):118-132.

25. Babb BR, Loushine RJ, Bryan TE, Ames JM, Causey MS, Kim J, et al. Bonding of

self-adhesive (self-etching) root canal sealers to radicular dentin. J Endod 2009;35(4):578-

582.

26. Monticelli F, Osorio R, Mazzitelli C, Ferrari M, Toledano M. Limited

decalcification/diffusion of self-adhesive cements into dentin. J Dent Res 2008;87(10):974-

979.

27. Kermanshahi S, Santerre JP, Cvitkovitch DG, Finer Y. Biodegradation of resin-

dentin interfaces increases bacterial microleakage. J Dent Res 2010;89(9):996-1001.

81

28. Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al. Collagen

degradation by host-derived enzymes during aging. J Dent Res 2004;83(3):216-221.

29. Shokati B, Tam LE, Santerre JP, Finer Y. Effect of salivary esterase on the integrity

and fracture toughness of the dentin-resin interface. J Biomed Mater Res B Appl Biomater

2010;94(1):230-237.

30. Hashimoto M, Ohno H, Endo K, Kaga M, Sano H, Oguchi H. The effect of hybrid

layer thickness on bond strength: demineralized dentin zone of the hybrid layer. Dent

Mater 2000;16(6):406-411.

31. Khalichi P, Cvitkovitch DG, Santerre JP. Effect of composite resin biodegradation

products on oral streptococcal growth. Biomaterials 2004;25(24):5467-5472.

32. Singh J, Khalichi P, Cvitkovitch DG, Santerre JP. Composite resin degradation

products from BisGMA monomer modulate the expression of genes associated with biofilm

formation and other virulence factors in Streptococcus mutans. J Biomed Mater Res A

2009;88(2):551-560.

33. Bergenholtz G. Micro-organisms from necrotic pulp of traumatized teeth. Odontol

Revy 1974;25(4):347-358.

34. Kakehashi S, Stanley HR, Fitzgerald RJ. The Effects of Surgical Exposures of

Dental Pulps in Germ-Free and Conventional Laboratory Rats. Oral Surg Oral Med Oral

Pathol 1965;20:340-349.

35. Sjogren U, Hagglund B, Sundqvist G, Wing K. Factors affecting the long-term

results of endodontic treatment. J Endod 1990;16(10):498-504.

36. Dalton BC, Orstavik D, Phillips C, Pettiette M, Trope M. Bacterial reduction with

nickel-titanium rotary instrumentation. J Endod 1998;24(11):763-767.

37. Shuping GB, Orstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria

using nickel-titanium rotary instrumentation and various medications. J Endod

2000;26(12):751-755.

38. Siqueira JF, Jr., Lima KC, Magalhaes FA, Lopes HP, de Uzeda M. Mechanical

reduction of the bacterial population in the root canal by three instrumentation techniques.

J Endod 1999;25(5):332-335.

39. Bystrom A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root

canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89(4):321-328.

40. Friedman S, Torneck CD, Komorowski R, Ouzounian Z, Syrtash P, Kaufman A. In

vivo model for assessing the functional efficacy of endodontic filling materials and

techniques. J Endod 1997;23(9):557-561.

41. Schilder H. Filling root canals in three dimensions. 1967. J Endod 2006;32(4):281-

290.

82

42. Mjor IA, Nordahl I. The density and branching of dentinal tubules in human teeth.

Arch Oral Biol 1996;41(5):401-412.

43. Garberoglio R, Brannstrom M. Scanning electron microscopic investigation of

human dentinal tubules. Arch Oral Biol 1976;21(6):355-362.

44. Carrigan PJ, Morse DR, Furst ML, Sinai IH. A scanning electron microscopic

evaluation of human dentinal tubules according to age and location. J Endod

1984;10(8):359-363.

45. Tronstad L. Ultrastructural observations on human coronal dentin. Scand J Dent

Res 1973;81(2):101-111.

46. Love RM, Jenkinson HF. Invasion of dentinal tubules by oral bacteria. Crit Rev

Oral Biol Med 2002;13(2):171-183.

47. Sen BH, Wesselink PR, Turkun M. The smear layer: a phenomenon in root canal

therapy. Int Endod J 1995;28(3):141-148.

48. Siqueira JF, Jr., De Uzeda M, Fonseca ME. A scanning electron microscopic

evaluation of in vitro dentinal tubules penetration by selected anaerobic bacteria. J Endod

1996;22(6):308-310.

49. Ando N, Hoshino E. Predominant obligate anaerobes invading the deep layers of

root canal dentin. Int Endod J 1990;23(1):20-27.

50. Horiba N, Maekawa Y, Matsumoto T, Nakamura H. A study of the distribution of

endotoxin in the dentinal wall of infected root canals. J Endod 1990;16(7):331-334.

51. Sen BH, Piskin B, Demirci T. Observation of bacteria and fungi in infected root

canals and dentinal tubules by SEM. Endod Dent Traumatol 1995;11(1):6-9.

52. Peters LB, Wesselink PR, Buijs JF, van Winkelhoff AJ. Viable bacteria in root

dentinal tubules of teeth with apical periodontitis. J Endod 2001;27(2):76-81.

53. Akpata ES, Blechman H. Bacterial invasion of pulpal dentin wall in vitro. J Dent

Res 1982;61(2):435-438.

54. Kakoli P, Nandakumar R, Romberg E, Arola D, Fouad AF. The effect of age on

bacterial penetration of radicular dentin. J Endod 2009;35(1):78-81.

55. Nair PN. On the causes of persistent apical periodontitis: a review. Int Endod J

2006;39(4):249-281.

56. Siqueira JF, Jr. Aetiology of root canal treatment failure: why well-treated teeth can

fail. Int Endod J 2001;34(1):1-10.

57. Fouad AF. Endodontic microbiology. 1st edition. ed: Wiley-Blackwell.; 2009.

83

58. Imura N, Pinheiro ET, Gomes BP, Zaia AA, Ferraz CC, Souza-Filho FJ. The

outcome of endodontic treatment: a retrospective study of 2000 cases performed by a

specialist. J Endod 2007;33(11):1278-1282.

59. Gomes BP, Pinheiro ET, Gade-Neto CR, Sousa EL, Ferraz CC, Zaia AA, et al.

Microbiological examination of infected dental root canals. Oral Microbiol Immunol

2004;19(2):71-76.

60. Schirrmeister JF, Liebenow AL, Pelz K, Wittmer A, Serr A, Hellwig E, et al. New

bacterial compositions in root-filled teeth with periradicular lesions. J Endod

2009;35(2):169-174.

61. Chavez De Paz LE, Dahlen G, Molander A, Moller A, Bergenholtz G. Bacteria

recovered from teeth with apical periodontitis after antimicrobial endodontic treatment.

Int Endod J 2003;36(7):500-508.

62. Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth

with apical periodontitis. Int Endod J 1998;31(1):1-7.

63. Zehnder M, Guggenheim B. The mysterious appearance of enterococci in filled root

canals. Int Endod J 2009;42(4):277-287.

64. Baumgartner G, Zehnder M, Paque F. Enterococcus faecalis type strain leakage

through root canals filled with Gutta-Percha/AH plus or Resilon/Epiphany. J Endod

2007;33(1):45-47.

65. Kaufman B, Spangberg L, Barry J, Fouad AF. Enterococcus spp. in endodontically

treated teeth with and without periradicular lesions. J Endod 2005;31(12):851-856.

66. Chivatxaranukul P, Dashper SG, Messer HH. Dentinal tubule invasion and

adherence by Enterococcus faecalis. Int Endod J 2008;41(10):873-882.

67. Sedgley CM, Lennan SL, Appelbe OK. Survival of Enterococcus faecalis in root

canals ex vivo. Int Endod J 2005;38(10):735-742.

68. Rocas IN, Siqueira JF, Jr., Santos KR. Association of Enterococcus faecalis with

different forms of periradicular diseases. J Endod 2004;30(5):315-320.

69. George S, Kishen A, Song KP. The role of environmental changes on monospecies

biofilm formation on root canal wall by Enterococcus faecalis. J Endod 2005;31(12):867-

872.

70. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of

persistent infections. Science 1999;284(5418):1318-1322.

71. Nair PN, Henry S, Cano V, Vera J. Microbial status of apical root canal system of

human mandibular first molars with primary apical periodontitis after "one-visit"

endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod

2005;99(2):231-252.

84

72. Ricucci D, Martorano M, Bate AL, Pascon EA. Calculus-like deposit on the apical

external root surface of teeth with post-treatment apical periodontitis: report of two cases.

Int Endod J 2005;38(4):262-271.

73. Harn WM, Chen YH, Yuan K, Chung CH, Huang PH. Calculus-like deposit at apex

of tooth with refractory apical periodontitis. Endod Dent Traumatol 1998;14(5):237-240.

74. Takemura N, Noiri Y, Ehara A, Kawahara T, Noguchi N, Ebisu S. Single species

biofilm-forming ability of root canal isolates on gutta-percha points. Eur J Oral Sci

2004;112(6):523-529.

75. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, et al.

Bacterial biofilms in nature and disease. Annu Rev Microbiol 1987;41:435-464.

76. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G.

Biofilms, the customized microniche. J Bacteriol 1994;176(8):2137-2142.

77. Gilbert P, Das J, Foley I. Biofilm susceptibility to antimicrobials. Adv Dent Res

1997;11(1):160-167.

78. Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopic

investigation of the smeared layer on root canal walls. J Endod 1984;10(10):477-483.

79. McComb D, Smith DC. A preliminary scanning electron microscopic study of root

canals after endodontic procedures. J Endod 1975;1(7):238-242.

80. Pashley DH, Michelich V, Kehl T. Dentin permeability: effects of smear layer

removal. J Prosthet Dent 1981;46(5):531-537.

81. Hovland EJ, Dumsha TC. Leakage evaluation in vitro of the root canal sealer

cement Sealapex. Int Endod J 1985;18(3):179-182.

82. Saunders WP, Saunders EM. Coronal leakage as a cause of failure in root-canal

therapy: a review. Endod Dent Traumatol 1994;10(3):105-108.

83. Wu MK, Wesselink PR. Endodontic leakage studies reconsidered. Part I.

Methodology, application and relevance. Int Endod J 1993;26(1):37-43.

84. Lee KW, Williams MC, Camps JJ, Pashley DH. Adhesion of endodontic sealers to

dentin and gutta-percha. J Endod 2002;28(10):684-688.

85. McComb D, Smith DC. Comparison of physical properties of polycarboxylate-based

and conventional root canal sealers. J Endod 1976;2(8):228-235.

86. Wennberg A, Orstavik D. Adhesion of root canal sealers to bovine dentine and

gutta-percha. Int Endod J 1990;23(1):13-19.

87. Saleh IM, Ruyter IE, Haapasalo M, Orstavik D. Bacterial penetration along

different root canal filling materials in the presence or absence of smear layer. Int Endod J

2008;41(1):32-40.

85

88. Gettleman BH, Messer HH, ElDeeb ME. Adhesion of sealer cements to dentin with

and without the smear layer. J Endod 1991;17(1):15-20.

89. Eldeniz AU, Erdemir A, Belli S. Shear bond strength of three resin based sealers to

dentin with and without the smear layer. J Endod 2005;31(4):293-296.

90. Zmener O, Pameijer CH, Serrano SA, Vidueira M, Macchi RL. Significance of

moist root canal dentin with the use of methacrylate-based endodontic sealers: an in vitro

coronal dye leakage study. J Endod 2008;34(1):76-79.

91. Kim YK, Grandini S, Ames JM, Gu LS, Kim SK, Pashley DH, et al. Critical review

on methacrylate resin-based root canal sealers. J Endod 2010;36(3):383-399.

92. Spencer P, Ye Q, Park J, Topp EM, Misra A, Marangos O, et al. Adhesive/Dentin

interface: the weak link in the composite restoration. Ann Biomed Eng 2010;38(6):1989-

2003.

93. Kim YK, Mai S, Haycock JR, Kim SK, Loushine RJ, Pashley DH, et al. The self-

etching potential of RealSeal versus RealSeal SE. J Endod 2009;35(9):1264-1269.

94. Ari H, Yasar E, Belli S. Effects of NaOCl on bond strengths of resin cements to root

canal dentin. J Endod 2003;29(4):248-251.

95. Perdigao J, Lopes M, Geraldeli S, Lopes GC, Garcia-Godoy F. Effect of a sodium

hypochlorite gel on dentin bonding. Dent Mater 2000;16(5):311-323.

96. Morris MD, Lee KW, Agee KA, Bouillaguet S, Pashley DH. Effects of sodium

hypochlorite and RC-prep on bond strengths of resin cement to endodontic surfaces. J

Endod 2001;27(12):753-757.

97. Yiu CK, Garcia-Godoy F, Tay FR, Pashley DH, Imazato S, King NM, et al. A

nanoleakage perspective on bonding to oxidized dentin. J Dent Res 2002;81(9):628-632.

98. Wang Y, Spencer P. Continuing etching of an all-in-one adhesive in wet dentin

tubules. J Dent Res 2005;84(4):350-354.

99. Hebling J, Pashley DH, Tjaderhane L, Tay FR. Chlorhexidine arrests subclinical

degradation of dentin hybrid layers in vivo. J Dent Res 2005;84(8):741-746.

100. Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R, De Stefano Dorigo E.

Dental adhesion review: aging and stability of the bonded interface. Dent Mater

2008;24(1):90-101.

101. Ito S, Hashimoto M, Wadgaonkar B, Svizero N, Carvalho RM, Yiu C, et al. Effects

of resin hydrophilicity on water sorption and changes in modulus of elasticity. Biomaterials

2005;26(33):6449-6459.

102. Ferracane JL. Elution of leachable components from composites. J Oral Rehabil

1994;21(4):441-452.

86

103. Finer Y, Santerre JP. Influence of silanated filler content on the biodegradation of

bisGMA/TEGDMA dental composite resins. J Biomed Mater Res A 2007;81(1):75-84.

104. Ortengren U, Wellendorf H, Karlsson S, Ruyter IE. Water sorption and solubility of

dental composites and identification of monomers released in an aqueous environment. J

Oral Rehabil 2001;28(12):1106-1115.

105. Ames JM, Loushine RJ, Babb BR, Bryan TE, Lockwood PE, Sui M, et al.

Contemporary methacrylate resin-based root canal sealers exhibit different degrees of ex

vivo cytotoxicity when cured in their self-cured mode. J Endod 2009;35(2):225-228.

106. Buonocore MG. A simple method of increasing the adhesion of acrylic filling

materials to enamel surfaces. J Dent Res 1955;34(6):849-853.

107. Bouillaguet S, Gysi P, Wataha JC, Ciucchi B, Cattani M, Godin C, et al. Bond

strength of composite to dentin using conventional, one-step, and self-etching adhesive

systems. J Dent 2001;29(1):55-61.

108. Ceballos L, Camejo DG, Victoria Fuentes M, Osorio R, Toledano M, Carvalho RM,

et al. Microtensile bond strength of total-etch and self-etching adhesives to caries-affected

dentine. J Dent 2003;31(7):469-477.

109. Paul SJ, Welter DA, Ghazi M, Pashley D. Nanoleakage at the dentin adhesive

interface vs microtensile bond strength. Oper Dent 1999;24(3):181-188.

110. Sano H. Microtensile testing, nanoleakage, and biodegradation of resin-dentin

bonds. J Dent Res 2006;85(1):11-14.

111. Tay FR, Pashley DH. Have dentin adhesives become too hydrophilic? J Can Dent

Assoc 2003;69(11):726-731.

112. Wilcox LR, Diaz-Arnold A. Coronal microleakage of permanent lingual access

restorations in endodontically treated anterior teeth. J Endod 1989;15(12):584-587.

113. Khayat A, Lee SJ, Torabinejad M. Human saliva penetration of coronally unsealed

obturated root canals. J Endod 1993;19(9):458-461.

114. Madison S, Swanson K, Chiles SA. An evaluation of coronal microleakage in

endodontically treated teeth. Part II. Sealer types. J Endod 1987;13(3):109-112.

115. Swanson K, Madison S. An evaluation of coronal microleakage in endodontically

treated teeth. Part I. Time periods. J Endod 1987;13(2):56-59.

116. Stratton RK, Apicella MJ, Mines P. A fluid filtration comparison of gutta-percha

versus Resilon, a new soft resin endodontic obturation system. J Endod 2006;32(7):642-645.

117. Wu MK, Wesselink PR, Boersma J. A 1-year follow-up study on leakage of four

root canal sealers at different thicknesses. Int Endod J 1995;28(4):185-189.

87

118. Alves J, Walton R, Drake D. Coronal leakage: endotoxin penetration from mixed

bacterial communities through obturated, post-prepared root canals. J Endod

1998;24(9):587-591.

119. Trope M, Chow E, Nissan R. In vitro endotoxin penetration of coronally unsealed

endodontically treated teeth. Endod Dent Traumatol 1995;11(2):90-94.

120. Xu Q, Fan MW, Fan B, Cheung GS, Hu HL. A new quantitative method using

glucose for analysis of endodontic leakage. Oral Surg Oral Med Oral Pathol Oral Radiol

Endod 2005;99(1):107-111.

121. Rahimi M, Jainaen A, Parashos P, Messer HH. Bonding of resin-based sealers to

root dentin. J Endod 2009;35(1):121-124.

122. Leonardo MR, Barnett F, Debelian GJ, de Pontes Lima RK, Bezerra da Silva LA.

Root canal adhesive filling in dogs' teeth with or without coronal restoration: a

histopathological evaluation. J Endod 2007;33(11):1299-1303.

123. Shipper G, Teixeira FB, Arnold RR, Trope M. Periapical inflammation after

coronal microbial inoculation of dog roots filled with gutta-percha or resilon. J Endod

2005;31(2):91-96.

124. Pitt Ford TR. Relation between seal of root fillings and tissue response. Oral Surg

Oral Med Oral Pathol 1983;55(3):291-294.

125. Oliver CM, Abbott PV. Correlation between clinical success and apical dye

penetration. Int Endod J 2001;34(8):637-644.

126. Grondahl H, Huumonen S. Radiographic manifestations of periapical inflammatory

lesions. Endodontic Topics 2004;8:55-67.

127. Hirsch E, Wolf U, Heinicke F, Silva MA. Dosimetry of the cone beam computed

tomography Veraviewepocs 3D compared with the 3D Accuitomo in different fields of

view. Dentomaxillofac Radiol 2008;37(5):268-273.

128. White JM, Goodis HE, Marshall SJ, Marshall GW. Sterilization of teeth by gamma

radiation. J Dent Res 1994;73(9):1560-1567.

129. Gogos C, Theodorou V, Economides N, Beltes P, Kolokouris I. Shear bond strength

of AH-26 and Epiphany to composite resin and Resilon. J Endod 2008;34(11):1385-1387.

130. Torabinejad M, Ung B, Kettering JD. In vitro bacterial penetration of coronally

unsealed endodontically treated teeth. J Endod 1990;16(12):566-569.

131. Zapata RO, Bramante CM, de Moraes IG, Bernardineli N, Gasparoto TH, Graeff

MS, et al. Confocal laser scanning microscopy is appropriate to detect viability of

Enterococcus faecalis in infected dentin. J Endod 2008;34(10):1198-1201.

88

132. Mohamed JA, Huang W, Nallapareddy SR, Teng F, Murray BE. Influence of origin

of isolates, especially endocarditis isolates, and various genes on biofilm formation by

Enterococcus faecalis. Infect Immun 2004;72(6):3658-3663.

133. Sundqvist G, Figdor D, Persson S, Sjogren U. Microbiologic analysis of teeth with

failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral

Med Oral Pathol Oral Radiol Endod 1998;85(1):86-93.

134. Teixeira FB, Teixeira EC, Thompson J, Leinfelder KF, Trope M. Dentinal bonding

reaches the root canal system. J Esthet Restor Dent 2004;16(6):348-354; discussion 354.

135. Estrela C, Sydney GB, Figueiredo JA, Estrela CR. A model system to study

antimicrobial strategies in endodontic biofilms. J Appl Oral Sci 2009;17(2):87-91.

136. Khalichi P, Singh J, Cvitkovitch DG, Santerre JP. The influence of triethylene

glycol derived from dental composite resins on the regulation of Streptococcus mutans gene

expression. Biomaterials 2009;30(4):452-459.

137. Hirai VH, da Silva Neto UX, Westphalen VP, Perin CP, Carneiro E, Fariniuk LF.

Comparative analysis of leakage in root canal fillings performed with gutta-percha and

Resilon cones with AH Plus and Epiphany sealers. Oral Surg Oral Med Oral Pathol Oral

Radiol Endod 2010;109(2):e131-135.

138. Nawal RR, Parande M, Sehgal R, Rao NR, Naik A. A comparative evaluation of 3

root canal filling systems. Oral Surg Oral Med Oral Pathol Oral Radiol Endod

2011;111(3):387-393.

139. Hashimoto M. A review--micromorphological evidence of degradation in resin-

dentin bonds and potential preventional solutions. J Biomed Mater Res B Appl Biomater

2010;92(1):268-280.

140. Ferrari M, Mannocci F, Vichi A, Cagidiaco MC, Mjor IA. Bonding to root canal:

structural characteristics of the substrate. Am J Dent 2000;13(5):255-260.

141. Reid BD. Gamma processing technology: an alternative technology for terminal

sterilization of parenterals. PDA J Pharm Sci Technol 1995;49(2):83-89.

142. Sperandio M, Souza JB, Oliveira DT. Effect of gamma radiation on dentin bond

strength and morphology. Braz Dent J 2001;12(3):205-208.

143. DeWald JP. The use of extracted teeth for in vitro bonding studies: a review of

infection control considerations. Dent Mater 1997;13(2):74-81.

144. Brauer DS, Saeki K, Hilton JF, Marshall GW, Marshall SJ. Effect of sterilization by

gamma radiation on nano-mechanical properties of teeth. Dent Mater 2008;24(8):1137-

1140.

89

145. Rodrigues LK, Cury JA, Nobre dos Santos M. The effect of gamma radiation on

enamel hardness and its resistance to demineralization in vitro. J Oral Sci 2004;46(4):215-

220.

146. Guyton A, Hall J. Secretary functions of the alimentary tract. Philadelphia:

Saunders; 1992.

147. Lamb J, Ingram C, Johnston I, Pitman R. Essentials of Physiology. Oxford:

Blackwell Scientific Publications; 1991.

148. Pratten J, Andrews CS, Craig DQ, Wilson M. Structural studies of microcosm

dental plaques grown under different nutritional conditions. FEMS Microbiol Lett

2000;189(2):215-218.