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SOURCE, OR PART OF THE FOLLOWING SOURCE:

Type Dissertation
Title New insights into the root canal wall
Author H. Shemesh
Faculty Faculty of Dentistry
Year 2009
Pages 83

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Vo l u m e 0 1 issue 01 October 2009 www.shemesh.nl

SHEMESH
New insights into the root canal wall

THE GLUCOSE PENETRATION MODEL 15-47

Feasibility, limitations and results

NOVEL IMAGING METHODS 49-65

Optical coherence tomography, Ultrasound

DENTINAL DEFECTS 67-77

Incidence, causes and development endodontic procedures
NEW INSIGHTS INTO THE ROOT CANAL WALL

Hagay Shemesh

This thesis is supplemented by a website:

www.shemesh.nl
NEW INSIGHTS INTO THE ROOT CANAL WALL

ACADEMISCH PROEFSCHRIFT

Ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
Prof. dr. D.C van den Boom
ten overstaan van een door het college voor promoties
ingestelde commissie, in het openbaar te verdedigen
in de Agnietenkapel
op vrijdag 23 oktober 2009, te 14:00 uur

door

Hagay Shemesh

Geboren te Beer Yaakov, Israel

Promotor: Prof. dr. P.R. Wesselink

Copromotors: dr. M.– K. Wu

dr. L.W.M van der Sluis

Faculteit der Tandheelkunde

This thesis was prepared at the Department of Endodontology

Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam
and VU University Amsterdam, the Netherlands.
This thesis is dedicated to Paul Wesselink and Min-Kai Wu for their
unprecedented contribution to endodontology in the last 3 decades.
 Contents

9 Chapter I General introduction

Chapter II Permeability of the root canal wall and leakage

17 Leakage along apical root fillings using two different leakage models
26 Glucose and fluid transport through coronal root structure and filled roots
33 Influence of passive ultrasonic irrigation on the seal of root canal fillings
39 Comparability of results from two leakage models
44 Glucose reactivity with different filling materials

Chapter III Novel imaging methods

51 High frequency ultrasound imaging of a single-species biofilm

57 The ability of optical coherence tomography to characterize the root canal walls
62 Diagnosis of vertical root fracture with optical coherence tomography

Chapter IV Inspection of dentinal defects in the root canal wall

69 Effects of canal preparation and filling on the incidence of dentinal defects

75 The ability of different Ni-Ti rotary instruments to induce dentinal damage

79 Chapter V Summary and conclusions

Samenvatting en conclusies

Acknowledgements
 9

Chapter I

Introduction
10

New insights into the root canal wall
Introduction
Endodontology is concerned with the study of
the form, function and health of, injuries to and
diseases of the dental pulp and periradicular region,
and the prevention and treatment of apical
periodontitis, caused by infection (European Society
of Endodontology, 2006). The technical aim of the
endodontic treatment is the shaping and cleaning of
the root canal system and the filling of the canals as
if to prevent coronal leakage and entomb remaining
microorganisms, preventing them from irritating the
periapical tissues (Sundqvist et al.1998). Recent
evidence show that endodontic therapy may not
always result in favorable and predictable outcomes.
Moreover, it seems that clinical endodontic research
neglected important issues concerning the outcome
of the treatment like low sensitivity of conventional
radiographs in detecting periapical lesions and low
recall rates (Wu et al. in print).
In order to investigate the effectiveness of
procedures during endodontic therapy, two major
approaches could be taken: randomized clinical
studies comparing the results of treatments and in
vitro / ex vivo investigations. Randomized clinical
studies have a higher level of evidence when
compared to other studies but are difficult to
perform not only because of the need to standardize
conditions but also because experimenting on
human beings requires a set of preconditions which
are nowadays hard to meet. When human subjects
are difficult to investigate and control, animals may
provide more relevant information on the outcomes
of different treatments. Experimenting with animals
is controversial in most countries and strict rules and
requirements make these investigations time
consuming and expensive. The next step in
considering an investigation method is in vitro
studies. These laboratory tests aim at investigating
different materials and methods in laboratory
conditions using models, extracted teeth and special
machinery.
The common substrate for all endodontic
procedures is the root canal itself, but different
aspects of the root canal treatment from the root
canal wall perspective were seldom addressed. The
root canal wall is made of dentine which has a
complex relationship between morphology and
11
function (Kishen et al. 2000). This could possibly
affect procedures we use during root canal treatment,
which are roughly divided into two important steps:
Cleaning of the root canal system (including
mechanical instrumentation) and filling. These pose
challenges to the clinician and researcher while there
is still no consensus as to the preferred method or
material of choice.
Cleaning and instrumentation of the canal
The objective of mechanical instrumentation is
to remove the main bulk of infected material and its
nutritional supply, facilitate irrigation and
appropriately shape the canal for the filling
procedure (Hülsmann et al. 2005). There are
numerous problems associated with the preparation
procedures and their effect on the root canal walls:
1. A debate exists as to the formation, importance
and faith of the smear layer which is created
during the preparation procedures (Şen et al.
1995). This debate is addressed in a few studies
in this thesis checking the influence of the
removal of the smear layer on the effectiveness
of the filling to prevent leakage, and the
propagation of light through the dentinal tubules.
2. In infected root canals microorganisms could be
attached to the root canal walls in the form of
biofilms (Svensäter & Bergenholtz 2004,
Chavez de Paz 2007) which are resistant to
cleaning procedures and disinfectants. Biofilms
are difficult to image and investigate, and
existing investigation methods involve
preparation procedures that damage the structure
and vitality of the biofilm. This thesis checks the
ability of an ultrasound scan to image the 3D
structure of biofilms nondestructively.
3. The canal anatomy is complex and often long-
oval in form (Wu et al. 2000). Efficient
instrumentation and cleaning of such canals is
extremely challenging. It is important to
visualize and to have knowledge of internal
anatomy relationships before undertaking
endodontic therapy. This thesis explores the

feasibility of optical coherence tomography as a
non destructive method to look at the anatomy
of the root canals and associated structures.
The root canal filling
The purpose of filling the root canal is to prevent
the growth of bacteria remaining after canal
preparation and the passage of fluids and bacterial
elements from the pulp cavity into the periapical
tissues (Sundqvist et al.1998). However, it seems
that current filling materials and techniques fail to
provide a leak-free seal (Wu & Wesselink 1993,
Eldeniz & Ørstavik 2009).
A variety of laboratory-based experimental
models are used to detect and measure leakage along
root fillings. The fluid transport set-up is often used
to measure leakage of water through root canal
fillings (Wu et al. 1993). Xu et al. (2005) proposed
and used a new model that measures the leakage of
glucose molecules. The glucose leakage model is
relatively easy to assemble and use and could give
quantitative leakage measurements which are based
on a chemical reaction between specific enzymes
and glucose. This reaction is very sensitive,
measured by a spectrophotometer and could detect
minute concentration changes of glucose. This thesis
uses the glucose penetration model in order to
compare the sealing ability of various materials and
methods and to further assess the advantages and
limitations of this model.
Both mechanical instrumentation and obturation
of the root canal could introduce defect in the root
canal wall (Wilcox et al. 1997). These defects could
eventually propagate into vertical root fractures and
might have clinical significance. The procedures that
mostly cause these defects are debatable
(Lertchirakarn et al. 1999). Evidence exists as to the
role of lateral compaction of gutta percha in the
formation of vertical root fracture (Dang and Walton
1989), but the ability of other endodontic procedures
to cause dentinal defects was not studied yet. The
recent introduction of different Ni-Ti files used with
engine-driven mechanical forces to endodontics
could be a contributing factor in the formation of
such defects. This thesis explores the influence of
different endodontic procedures on the formation of
defects like craze lines and vertical root fractures in
the root canal wall.
12
Objectives of the thesis
The aim of this thesis was to suggest new
methodologies to measure, image and explore the
root canal walls and related interfaces and to present
new insights into the root canal wall associated with
endodontic treatment procedures. Specifically, 3
approaches were taken:
1. Leakage measurements using the penetration of
glucose through root canal fillings as a new
model for assessing the sealing ability of
different materials and methods.
2. The application of novel imaging technologies
for non destructive high resolution
characterization of the root canal wall and
related structures.
3. Studies into the ability of different types of files
and filling methods to inflict defects on the root
canal wall.
Outline of the thesis
- In chapter II, the glucose penetration model for
leakage tests is tried. Different experiments are
presented using this model and its ability to detect
leakage patterns through different materials, set ups
and conditions. Comparison between performance
of different materials and methods as observed with
this new model and the already established fluid
transport model were also made. The limitations of
this model to reliably detect leakage through root
canal fillings are discussed.
- In chapter III new endodontic applications for
novel imaging techniques are suggested. Ultrasound
scans for imaging live biofilm without damaging its
delicate 3D structures and vitality. Optical
coherence tomography for intra-canal imaging,
showing the root canal walls and related structures
nondestructively and without using ionized radiation.
- In chapter IV the formation and incidence of
dentinal defects in the root canal wall after different
preparation and filling procedures are discussed.
Comparison is made between the lateral compaction
technique and a non compaction technique, and
between the use of hand files and different rotary
Ni-Ti systems.
 13

References:

Quality guidelines for endodontic treatment: consensus

Wu MK, Wesselink PR (1993) Endodontic leakage
report of the European Society of
studies reconsidered. Part I. Methodology,
Endodontology.(2006) International
applications and relevance. International
Endodontic Journal 39, 921-930.
endodontic journal 26, 203-8.
Chavez de Paz LE. (2007) Redefining the persistent
Wu MK, R'oris A, Barkis D, Wesselink PR (2000)
infection in root canals: possible role of biofilm
Prevalence and extent of long oval canals in the
communities. Journal of endodontics 33,652-62.
apical third. Oral Surg Oral Med Oral Pathol
Dang DA, Walton RE (1989) Vertical root fracture and Oral Radiol Endod 89,739-43.
root distortion: effect of spreader design.
Wu MK, Shemesh H, Wesselink PR (2009)
Journal of Endodontics 15, 294-301.
Limitations of previously published systematic
Eldeniz AU, Ørstavik D (2009) A laboratory reviews evaluating the outcome of endodontic
assessment of coronal bacterial leakage in root treatment. International Endodontic Journal in-
canals filled with new and conventional sealers. print
International Endodontic Journal 42:303-12.
Xu Q, Fan MW, Fan B, Cheung GS, Hu HL (2005) A
Hülsmann M, Peters OA, Dummer PMH (2005) new quantitative method using glucose for
Mechanical preparation of root canals: shaping analysis of endodontic leakage. Oral Surg Oral
goals, techniques and means. Endodontic Topics Med Oral Pathol Oral Radiol Endod 99,107-11.
10, 30-76.
Kishen A, RamamurtyU, Asundi A (2000)
experimental studies on the nature of property
gradients in the human dentine. Journal of
Biomarials research 51: 650-9.
Lertchirakarn V, Palamara JE, Messer HH (1999) Load
and strain during lateral condensation and
vertical root fracture. Journal of Endodontics 25
99-104.
Şen BH, Wesselink PR, Türkün M (1995) The smear
layer: a phenomenon in root canal therapy.
International Endodontic Journal 28,141-8.
Sundqvist G, Figdor D, Persson S, Sjögren U (1998)
Microbiologic analysis of teeth with failed
endodontic treatment and the outcome of
conservative re-treatment. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 85,86-93.
Svensäter G, Bergenholtz G (2004) Biofilms in
endodontic infections. Endodontic Topics 9, 27-
36.
Wilcox LR, Roskelley C, Sutton T (1997) The
relationship of root canal enlargement to finger
spreader induced vertical root fracture. Journal
of Endodontics 23, 533-4.
Wu MK, De Gee AJ, Wesselink PR, Moorer WR
(1993) Fluid transport and bacterial penetration
along root canal fillings. International
Endodontic Journal 26:203-8.
14
 15

Chapter II

Permeability of the root canal wall and leakage

This chapter is enhanced by additional high-resolution images available on

www.shemesh.nl

1. Leakage along apical root fillings with and without smear layer using two
different leakage models: a two-month longitudinal ex vivo study. Shemesh H,
Wu MK, Wesselink PR. Int Endod J. 2006 Dec;39(12):968-76.
2. Glucose penetration and fluid transport through coronal root structure and filled
root canals. Shemesh H, van den Bos M, Wu MK, Wesselink PR. Int Endod J.
2007 Nov;40(11):866-72.
3. An evaluation of the influence of passive ultrasonic irrigation on the seal of root
canal fillings. van der Sluis LW, Shemesh H, Wu MK, Wesselink PR. Int Endod
J. 2007 May;40(5):356-61.
4. Comparability of results from two leakage models. Souza EM, Wu MK,
Shemesh H, Bonetti-Filho I, Wesselink PR. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod. 2008 Aug;106(2):309-13.
5. Glucose reactivity with filling materials as a limitation for using the glucose
leakage model. Shemesh H, Souza EM, Wu MK, Wesselink PR. Int Endod J.
2008 Oct;41(10):869-72.
16

Leakage along apical root fillings with and without
smear layer using two different leakage models: a
two-month longitudinal ex vivo study
H. Shemesh, M.-K. Wu & P. R. Wesselink
Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, The
Netherlands
Abstract
Shemesh H, Wu M-K, Wesselink PR. Leakage along apical
root fillings with and without smear layer using two different
leakage models: a two-month longitudinal ex vivo study.
International Endodontic Journal, 39, 968–976, 2006.
Aim To compare two different experimental models
when measuring leakage along root fillings with or
without smear layer.
Methodology One hundred and twenty single-
rooted teeth were prepared to size 50 and allocated to
two groups: fluid transport model (n ¼ 60) and glucose
penetration model (n ¼ 60). The roots in each group
were divided into three subgroups of 20 teeth each.
Smear layer was left in place in group 1 but removed in
groups 2 and 3. In groups 1 and 2 canals were filled
with laterally compacted gutta-percha cones and AH
26. Group 3 was laterally compacted with Resilon
cones and Epiphany sealer. The coronal portion of the
filling was removed to assure only 4 mm of filling
remained in the canal. Leakage of glucose was evalu-
ated by measuring its concentration once a week for a
total period of 56 days using a glucose penetration
model. Fluid transport was evaluated by measuring the
movement of an air-bubble using a fluid transport
model, 1 and 8 weeks after canal filling. Differences
Introduction
The purpose of a root filling is to prevent bacterial
growth and penetration of fluid and antigenic agents
Correspondence: H. Shemesh, Department of Cariology Endo-
dontology Pedodontology, ACTA, Louwesweg 1, 1066 EA
Amsterdam, The Netherlands (Tel.: 3120 518 8549; fax
3120 669 2881; e-mail: h.shemesh@acta.nl).
968 International Endodontic Journal, 39, 968–976, 2006
17
doi:10.1111/j.1365-2591.2006.01181.x
between the groups in glucose concentrations and fluid
transport were statistically analysed with the Kruskal–
Wallis and the Mann–Whitney tests. The level of
significance was set at a ¼ 0.05.
Results Glucose penetration was significantly differ-
ent between the three groups after the first 8 days
(P < 0.05). Resilon leaked the most throughout the
experiment period. No significant difference (P > 0.05)
existed between the two gutta-percha groups at all time
intervals (Mann–Whitney test). In the fluid transpor-
tation model, no statistically significant differences
were observed between all three experimental groups
(P > 0.05) at either 1 or 8 weeks after filling (Kruskal–
Wallis test).
Conclusions Under the conditions of this study, the
glucose penetration model was more sensitive in
detecting leakage along root fillings. Removing the
smear layer before filling did not improve the sealing of
the apical 4 mm of filling. Resilon allowed more glucose
penetration but the same amount of fluid transport as
the gutta-percha root fillings.
Keywords: gutta-percha, leakage, Resilon, root
canal filling, smear layer.
Received 20 March 2006; accepted 13 June 2006
between the canal and periapical tissues (Sundqvist
et al. 1998).
A variety of laboratory-based experimental models
are used to detect and measure leakage along root
fillings. Dye leakage, fluid transport and bacterial
penetration are currently the methods used most often.
Recently Xu et al. (2005) discussed a new model that
measures the leakage of glucose molecules. The model
consists of a tube containing concentrated glucose
ª 2006 International Endodontic Journal

solution that is connected to the coronal aspect of the
tooth, whilst the apical region is dipped in water.
Glucose that accumulates in the apical chamber is
measured with a spectrophotometer following an
enzymatic reaction. Glucose has a low molecule weight
of 180 Da, and may be used as an indication for toxins
that might penetrate the canal (Xu et al. 2005).
Leakage studies consistently show bacterial penetra-
tion through root fillings. Torabinejad et al. (1990)
reported that 50% of filled single-rooted teeth were
contaminated along the whole length of the canal after
19 and 42 days of exposure depending on the infecting
microorganism. Khayat et al. (1993) reported that all
root canals filled with laterally or vertically condensed
gutta-percha were contaminated in less than 30 days
after exposure to human saliva.
One of the methods previously described for improv-
ing the seal and for minimizing leakage is the removal
of the smear layer before filling (Clark-Holke et al.
2003). This has been claimed to improve sealer
penetration inside the dentinal tubules, achieving a
potentially greater adherence to the canal wall (Kokkas
et al. 2004). Indeed, some studies that investigated the
removal of the smear layer concluded that a better seal
was achieved when the smear layer was removed
(Kennedy et al. 1986, Cergneux et al. 1987, Taylor
et al. 1997, Clark-Holke et al. 2003, Çobankara et al.
2004). Other studies have suggested that removing the
smear layer increases dentine permeability and might
impair the sealing ability, and even allow bacteria to
grow inside the dentinal tubules (Pashley et al. 1981,
Drake et al. 1994, Galvan et al. 1994, Love 1996). Two
review articles on the clinical implications of the smear
layer in endodontics (Şen et al. 1995, Torabinejad et al.
2002) confirmed the uncertainty and debate relating to
the removal of smear layer before filling. More recently
Gulabivala et al. (2005) discussed the effects of
mechanical and chemical procedures including the
removal of the smear layer on the seal and stated that
the mechanisms leading to successful root canal
treatment remained to be determined.
Current filling materials and techniques fail to
provide a leak-free seal (Wu & Wesselink 1993, Wu
et al. 1993). Gutta-percha is the most popular filling
material and has been used for this purpose for many
years. Systems like warm injection and carrier-coated
root fillings have been developed but have been shown
to leak to a certain extent (Mannocci et al. 1999,
Abarca et al. 2001, Wu et al. 2003, Chu et al. 2005).
Recently, a new thermoplastic synthetic polymer-
based root filling material was introduced (Resilon;
ª 2006 International Endodontic Journal
18
Shemesh et al. Leakage of apical root fillings
Pentron Clinical Technologies, Wallingford, CT, USA).
This material resembles gutta-percha in appearance,
has similar handling properties and is available both in
cone format and in pellets for warm injection. The
corresponding sealer (Pentron Clinical Technologies) is
a dual curable dental resin composite. This so-called
‘Epiphany’ system (Resilon and sealer combined with
self-etching of the canal wall) is claimed to form a
‘monoblock’ which adheres to the dentine walls,
prevents leakage and increases resistance to fracture
(Shipper et al. 2004, Teixeira et al. 2004).
The purpose of this study was to compare two
different experimental models in measuring leakage
along apical root fillings with and without the smear
layer.
Materials and methods
Selection and preparation of teeth
One hundred and sixty recently extracted single-rooted
human teeth were selected and stored in 0.2% sodium
azide, NaN3 (E. Merck, Darmstadt, Germany) at +4 C
until use. Mandibular incisors were excluded because of
their morphological diversity (Kaffe et al. 1985). Pre-
molars were used only when a radiograph indicated a
single canal. Teeth with open apices or large carious
lesions were excluded.
The coronal portions of all teeth were removed so that
each root specimen was 15 mm long. A diamond bur
(FG 173 Horico, Berlin, Germany) was used to
gain straight-line entry to the root canal. A size 20
K-Flexofile (Dentsply Maillefer, Ballaigues, Switzerland)
was inserted into the canal to verify patency (Kuttler
1955). All samples were examined under a microscope
(Zeiss Stemi SV6, Jena, Germany) to exclude cracks. The
coronal 4 mm of the root specimens were then embed-
ded in acryl (Vertex; Dentimex BV, Zeist, the Nether-
lands) to form an acrylic cylinder around the root and
enable intimate contact between the rubber tube used to
connect the specimen during the leakage phase of the
study and the root specimen. All procedures and
treatments were preformed by one individual.
Instrumentation and obturation of root canals
The working length was determined by subtracting
1 mm from the total length of the root. The apical
portion of the canal was instrumented to a size 50
master file using the balanced force technique (Roane &
Sabala 1985) with K-Flexofiles (Dentsply Maillefer). A
International Endodontic Journal, 39, 968–976, 2006 969

Leakage of apical root fillings Shemesh et al.
step-back flaring technique was then performed at
1 mm increments with Gates Glidden burs number 2–6
(Dentsply Maillefer) making the taper 0.2 mm mm)1
(Wu et al. 2002). The purpose of this preparation
regimen was to create a uniform size of canal and to
overcome the variation in natural morphology. Each
canal was irrigated with freshly prepared 2% NaOCl
with a 27-gauge needle after every instrument and
ensuring patency by extrusion of the solution beyond
the apical foramen. A minimum of 10 mL NaOCl
solution was used for each root. The prepared roots
were randomly divided into three experimental groups
of 40 roots, and two control groups of 20 roots each.
Group 1
After preparation was completed, canals were rinsed
with an additional 5 mL 2% NaOCl solution and then
with 5 mL deionized water. Each canal was dried using
paper point size 50.
A size 50 gutta-percha master cone coated with AH
26 sealer (Dentsply Maillefer) was inserted into the
canal. Light pumping motions were used to fill the
canal with sealer and bring the cone to full working
length. Lateral compaction was achieved using a size C
finger spreader (Dentsply Maillefer) and size 25 acces-
sory gutta-percha cones that initially reached to within
1 mm of the working length. The tip of each accessory
cone was lightly coated with sealer, placed and
compacted laterally. The process was repeated until
cones could not be inserted more than 10 mm into the
canal. An estimation of the total amount of sealer used
was achieved by using a 0.5 cm · 0.5 cm square of
mixed sealer for each tooth.
The coronal gutta-percha was removed with a hot
plugger (0.5 mm diameter, Dentsply Maillefer) and
vertically packed, leaving the apical 4 mm of root filling
subjected to the leakage test (Fan et al. 1999).
Group 2
After completion of preparation canals were rinsed with
5 mL 17% EDTA for 3 min to remove the smear layer
(Hülsmann et al. 2003) and then rinsed with 5 mL
deionized water. The filling was completed in the same
way as group 1.
Group 3
All canals were rinsed with 5 mL 17% EDTA for 3 min
and then with 5 mL deionized water. After drying, a
self-etching primer (Epiphany primer; Pentron Clinical
Technologies) was placed into the canal with a 26-
gauge needle. Two drops of primer were used for each
970 International Endodontic Journal, 39, 968–976, 2006
19
root. Three paper points size 50 were used to remove
excess primer after 1 min from each root. Roots were
then filled with lateral compaction of Resilon cones and
Epiphany sealer (Pentron Clinical Technologies) in the
same way as in group 1. The filling was removed from
the coronal portion of the canal in the same manner as
group 1, leaving 4 mm of the apical filling intact.
Positive control group
Canals were filled using lateral compaction of gutta-
percha cones without any sealer. No warm vertical
forces were used and the whole length of the filling
remained.
Negative control group
All roots were sealed with laterally compacted gutta-
percha and AH 26 for the whole length of the canal
and completely covered with nail varnish.
After filling all specimens were maintained for
1 week at 37 C and 100% humidity to allow the
materials to set. Specimens in each group were then
divided equally between the two different models,
glucose penetration and fluid transport.
Glucose penetration model – preparation and
measurements
The difference between the current version of the
glucose penetration model and the original model
introduced by Xu et al. (2005) lies mainly in the
environment in which the equipment was stored: in
order to overcome evaporation of fluids, specimens
were placed in a closed jar with 100% humidity. From
a pilot study it was concluded that this method would
eliminate the effect of fluid evaporation on glucose
concentration measurements.
The resin block around the coronal part of each root
was connected to a rubber tube with stainless steel
wires, which was in itself connected to a 16 cm long
pipette (Pyrex, Acton, MA, USA). The assembly was
then placed in a sterile glass bottle with a screw cap
and sealed with sticky wax. A uniform hole was drilled
in the screw cap with a diamond bur (No.173 Horico,
Berlin, Germany) to assure an open system at all times
(Fig. 1). Two millilitres of 0.2% NaN3 solution were
inserted into the glass bottle, such that the root samples
were immersed in the solution. NaN3 was used to
inhibit the growth of microorganisms that might
influence the glucose readings. The tracer used in the
present study was 1 mol L)1 glucose solution (pH 7.0).
Glucose has a low molecular weight and is hydrophilic
ª 2006 International Endodontic Journal
 20

Shemesh et al. Leakage of apical root fillings

Glucose solution 33, 40, 48 and 56 days. The same amount of fresh
0.2% NaN3 was added to the glass bottle reservoir to
maintain a constant volume of 2 mL. The sample was
then analysed with a Glucose kit (Megazyme, Wicklow,
Ireland) in a spectrophotometer (Molecular Devices,
Glass tube
Spectra max 384 plus) at a wavelength of 340 nm.
Concentrations of glucose in the lower chamber were
presented in mmol L)1 at each time interval following
filling. The lowest glucose level for which the current
Open system procedure is believed to be accurate is 0.003 mmol L)1
which derives from an absorbance difference of 0.02
14 cm
(d-Glucose-HK assay procedure; Megazyme, 2004).
Below this level, the absorbance readings become
relatively small, and results are subject to greater error
Rubber tube from technique variables. Concentrations smaller than
this were thus ignored. Similarly, once leakage excee-
Acrylic cylinder ded 21 mmol L)1 samples were no longer observed as
the glucose concentration in the lower chamber
Metal wire suggested substantial leakage had occurred.
Root specimen
Fluid transport model – preparation and
0.2% NaN3 solution measurements
Roots were mounted in the fluid transport device
Figure 1 Glucose penetration model. (Fig. 2) previously described by Wu et al. (1993). The
pipettes used were 22 mm long 1 mL glass pipettes
(Witeg, Wertheim, Germany). All connections were
and chemically stable. About 4.5 mL of the glucose tightly closed by twisting pieces of stainless steel wire in
solution, containing 0.2% NaN3, was injected into the a water bath at 20 C. Fluid transport along the root
pipette until the top of the solution was 14 cm higher filling was measured under a headspace pressure of
than the top of gutta-percha in the canal, which 30 kPa (0.3 atm) and after 3 h the volume of fluid
created a hydrostatic pressure of 1.5 kPa or 15 cm H2O transport was recorded. The results were expressed as
(Xu et al. 2005). All specimens were then returned to lL min)1. After measurements teeth were carefully
the incubator at 37 C for the duration of the obser- disconnected from the assembly, placed in 0.2% NaN3
vation period. A total of 25 lL of solution was drawn solution and returned to the incubator for a period of
from the glass bottle using a micropipette at 8, 13, 20, 8 weeks. The medium was changed with a fresh NaN3

Headspace pressure
Silicon tube Water bath (20°)
Filled root Standard capillary
Air bubble

Silicon tube

Figure 2 Fluid transport model. Wire connections

ª 2006 International Endodontic Journal International Endodontic Journal, 39, 968–976, 2006 971
 21

Leakage of apical root fillings Shemesh et al.

solution every week. After 8 weeks the roots were GP+AH26, GP+AH26, SL Resilon-Epiphany
SL present removed

Glucose concentration mmol L–1

mounted again and checked for fluid transport in the Negative control Positive control

same way. 26
22
18
Statistical analysis
14
The differences between the groups with regard to 10
glucose concentrations and fluid transport were statis- 6
tically analysed with the Kruskal–Wallis and the 2
Mann–Whitney tests (version 12.0.1, SPSS, Chicago,
IL, USA). The level of significance was set at a ¼ 0.05. 0 10 20 30 40 50 60
Days after obturation

Figure 3 Median glucose penetration in mmol L)1 after

Results
2 months.
The results for the glucose model are shown in Table 1
and Figs 3 and 4. The positive control group had
substantial leakage of glucose from the first day which
GP+AH26, GP+AH26, SL Resilon-
increased over time. After 2 weeks all samples had SL present
Negative control
removed
Positive control
Epiphany

maximum leakage (21 mmol L)1). In the negative

Percent leakaing samples

control group no glucose was detected in the apical 100
90
reservoirs throughout the experiment. Glucose concen- 80
70
trations in the experimental groups revealed that after 60
the first 8 days the difference between the three groups 50
40
was significant (Kruskal–Wallis test, P < 0.05). 30
Resilon laterally compacted had the most leakage at 20
10
all time intervals. However, no significant difference 0
existed between the two gutta-percha groups (Mann– –10 0 10 20 30 40 50 60
Whitney test, P > 0.05) at all time intervals. The Day after obturation
statistical significance of the differences between all
Figure 4 Percentage of leaking samples in the glucose penet-
three groups is summarized in Table 2. ration model – after 2 months.
The results of the fluid transport model are shown in
Table 3. The positive control group had bubble move-
ment that exceeded the pipette length after 3 h and was (P > 0.05) existed between the three experimental
impossible to measure. The negative control group had groups at both time intervals, 1 and 8 weeks, after
no movement of the bubble. No significant difference filling (Kruskal–Wallis test).

Table 1 Mean and median of glucose leakage in mmol L)1 at different times after obturation

Day

Group 8 13 20 33 40 48 56

GP AH 26 (smear layer present)

Mean (SD) 3.1 (6.0) 4.1 (7.3) 4.6 (7.9) 5.1 (7.9) 5.5 (8.0) 6.0 (8.4) 7.3 (8.7)
Median (range) 0 (0–21) 0 (0–21) 0 (0–21) 0 (0–21) 0.2 (0–21) 0.8 (0–21) 2.2 (0–21)
Percentage leaking 30 40 40 45 50 50 70
GP AH 26 (smear layer removed)
Mean (SD) 3.2 (6.6) 3.6 (6.8) 4.5 (7.3) 4.8 (7.5) 6.3 (8.7) 6.8 (9.0) 7.0 (9.0)
Median (range) 0 (0–21) 0 (0–21) 0 (0–21) 0 (0–21) 1.5 (0–21) 2.0 (0–21) 2.5 (0–21)
Percentage leaking 30 35 40 40 55 55 55
Resilon–Epiphany (smear layer removed)
Mean (SD) 3.5 (5.6) 6.2 (7.0) 6.6 (7.0) 8.0 (7.2) 9.6 (7.4) 12.0 (8.0) 12.8 (7.9)
Median (range) 1.4 (0–13.5) 2.4 (0–21) 2.9 (0–21) 4.4 (0–21) 7.2 (0–21) 12.3 (0–21) 12.9 (0–21)
Percentage leaking 55 90 90 90 90 90 90

972 International Endodontic Journal, 39, 968–976, 2006 ª 2006 International Endodontic Journal

Table 2 P values – statistical signifi-
cance of the difference in glucose con-
centrations between the groups at Time after
specific time intervals filling (days)
8 0.415
13
20
33
40
48
56
Table 3 Average fluid transportation and percentage of leak-
ing samples 1 and 8 weeks after obturation
Average fluid transport
in lL (percentage
leaking samples)
Group 1 week 8 weeks
GP/AH26 (smear layer 0.5 (20)
present; n ¼ 20)
GP/AH26 (smear layer 0.1 (15) 0.05 (5)
removed; n ¼ 20)
Resilon/Epiphany (smear 0.0 (0)
layer removed; n ¼ 20)
Discussion
Several test methods have been described to evaluate
the sealing quality of filled root canals. In the present
study, two different models were used: the fluid
transport model (Wu et al. 1993) and the glucose
penetration model (Xu et al. 2005). The latter can be
seen as a further development of the fluid transporta-
tion concept: both measure passage of fluid along root
filled teeth after subjecting them to constant pressure.
However, the glucose model allows measurements of
diffusion of the marker molecules as well. The glucose
test might be more sensitive than the measurement of
air-bubble movement, not only because the detected
threshold measurement by eye is higher than that of
the spectrophotometer, but also because the convective
fluid transport was combined with glucose molecule
diffusion.
Time difference is an important factor when com-
paring the results from the two different models. In the
glucose penetration model the tooth is continuously
subjected to the pressure of the glucose solution in the
coronal chamber for a period of 2 months. The fluid
penetration model detects leakage after subjecting the
filling to pressure for 3 h. This enormous time differ-
ence might result in detection of smaller voids in the
ª 2006 International Endodontic Journal
22
Shemesh et al. Leakage of apical root fillings
Kruskal–Wallis test Mann–Whitney test
P (groups P (groups 1 P (groups 1 P (groups 2
1–3) and 2) and 3) and 3)
– – –
0.012 0.799 0.015 0.013
0.026 0.989 0.020 0.033
0.034 0.799 0.038 0.026
0.031 0.799 0.023 0.030
0.020 0.820 0.013 0.026
0.035 0.738 0.035 0.024
filling, making the glucose test more sensitive. Fur-
thermore, summated glucose leakage during 2 months
was measured whereas fluid transportation was meas-
ured for 3 h and observed at two different time
intervals, 1 and 8 weeks after filling.
Evaporation of fluids during the 56 days experiment
0.2 (10) duration could alter the glucose concentrations both in
the apical and the coronal chambers. Evaporation will
inevitably occur as these two compartments have to
have an opening to release pressure build-up and
0.0 (0)
cannot be closed hermetically in order to allow leakage
to occur. Xu et al. (2005) refers only to evaporation
from the apical chamber, compensating it with water
according to a representative sample. The method used
here, storing the models in a closed humid jar,
addresses the evaporation factor from both chambers
and proved to be effective in initial pilot studies.
The effect of the removal of smear layer before
obturation has been the subject of extensive debate.
According to the current findings, the smear layer did
not affect the seal with gutta-percha and AH 26 in the
apical 4 mm, when checked with the fluid transport or
the glucose penetration models. These results are in
agreement with those of Saunders & Saunders (1994a)
who found no significant difference in dye leakage after
4 months between root fillings when the smear layer
was removed or present. Saunders & Saunders (1994b)
assessed dye leakage of Thermafil fillings and laterally
condensed gutta-percha with glass–ionomer sealer no
significant difference was observed after 4 months
between any of the groups. Madison & Krell (1984)
and Evans & Simon (1986) also found no difference in
leakage when the smear layer was removed or not.
Although dye-leakage results have debatable relevancy
(Wu & Wesslink 1993) they were the most frequently
used to assess the influence of smear layer. In contrast
to these findings, Clark-Holke et al. (2003) checked a
mixed culture of bacteria penetrating through root
fillings. A total of 30 teeth were used, amongst which
International Endodontic Journal, 39, 968–976, 2006 973

Leakage of apical root fillings Shemesh et al.
10 served as controls. During the 2-month observation
period, leakage was not observed in the group where
smear layer was removed, whilst 60% of the specimen
having a smear layer leaked. In spite of the small
number of specimen and the authors’ own claim that
leakage could occur after the 2-month observation
period, it was suggested that removing the smear layer
decreased bacterial leakage. Similarly, Kennedy et al.
(1986) and Cergneux et al. (1987) observed that smear
layer removal reduced dye leakage. These conflicting
reports might be attributed to difference in the type of
sealer and filling technique, the method of producing
and removing the smear layer and different laboratory
procedures to check the leakage (Şen et al. 1995). More
recently, Paqué et al. (2006) found no effect of the
smear layer on dye penetration through root dentine,
and suggested that tubular sclerosis rather than the
smear layer that influences penetrability.
Resilon is a new root filling material that consists of a
composite that may be bonded to the wall of the canal.
Results from the current experiment indicate that the
apical 4 mm of Resilon–Epiphany root canal fillings
allowed more glucose penetration than gutta-percha.
Shipper et al. (2004) detected more rapid bacterial
leakage in gutta-percha and AH 26 fillings when
compared with Resilon–Epiphany during a period of
31 days when the whole length of the root canal was
filled with laterally or vertically compacted material. In
the current experiment only the 4 apical mm of filling
were checked. The dentinal tubules configuration
which is less dense in the apical part than the coronal
part (Fogel et al. 1988) might lead to compromised
bonding apically. Tay et al. (2005a) observed in a
transmission electron micrograph gaps of about 2 lm
between the root dentine and the Resilon primer. These
imperfections in the bonding might be too small to be
detected by bacterial penetration models, as the aver-
age length of bacteria varies from 0.2 to more than
10 lm, the width from 0.2 to 1.5 lm (Hobot 2002).
The influence of the geometric variables involved in
the use of adhesive sealers was previously discussed by
Feilzer et al. (1993) and more recently by Tay et al.
(2005a). The latter study simulated different scenarios
and appraised the C-factors that arise from thin resin
films. In a Class I cavity, the bonded surface area is five
times more than the unbonded surface area (C-factor is
5). As the unbonded surface area becomes smaller, as
in a root canal, the C-factor becomes much higher,
there is insufficient stress relief by flow and a high
probability that one or more bonded areas will debond.
The probability of imperfect dentine bonding in a root
974 International Endodontic Journal, 39, 968–976, 2006
23
canal during polymerization might exceed the bond
strength, and a high volumetric shrinkage of the sealer
might occur when it polymerizes. In cross-sections of
filled roots, gaps were observed between the dentine
and the Epiphany layer (Tay et al. 2005a). These
imperfections in the bonding to the walls of the canal
might be too small to be detected by bacterial penet-
ration models.
The dimensional stability of Resilon should also be
addressed. Preliminary unpublished studies have
shown that Resilon cones discharged a coloured
substance to the surrounding medium it, that may
affect the measurements of optical density. As this
colour (pink) is not absorbed at the same wavelength
that is assessed by the glucose kit, the results were not
compromised. However, every new material that is
about to be checked with this method, should be
assessed for its colour properties when it is immersed in
fluid for an extended period. Gutta-percha and AH 26
on the other hand, did not show any colour discharge
when soaked in water.
Tay et al. (2005b,c) discussed the susceptibility of
Resilon to degradation in two different studies: in the
first, 15 mm diameter Resilon and gutta-percha discs
were immersed in sodium etoxide for 20 and 60 min.
The treated discs were then examined with a
scanning electronic microscope and dispersive X-ray
analysis. The surface of the Resilon discs was
hydrolysed after 20 min exposing the filler, whilst
gutta-percha discs were unaffected. The second
experiment examined 15 mm diameter discs of Resi-
lon, gutta-percha and polycaprolactone that were
incubated with phosphate-buffered saline, Lipase PS
or cholesterol esterase. Resilon and polycaprolactone
discs had significant weight loss and surface thinning
when compared with the gutta-percha discs. The
influence of this phenomenon on glucose penetration
may be greater in the current setting than in that of
Shipper et al. (2004) because of the longer observa-
tion period. These results challenge the claims of the
manufacturer (‘Epiphany Newsletter’, July 2005,
Pentron Clinical Technologies) that the colour dis-
charge from Resilon cones is only food grade dye
‘leaching out into the tooth’. However, it may
provide an explanation for the increased leakage in
the Resilon group.
Conclusions
• The glucose penetration model is a sensitive method
to detect leakage along root fillings.
ª 2006 International Endodontic Journal

• Under the conditions of this study, no statistically
significant difference in glucose penetration or fluid
transportation was observed along the 4 mm apical
root filling with gutta-percha and AH 26 whether or
not the smear layer was removed prior to filling.
• Canals filled with Resilon had more glucose penetra-
tion than gutta-percha and AH 26 during a period of
56 days, whilst no statistically significant difference
was observed between the Resilon and gutta-percha
filled teeth in the fluid transportation model either at 1
or 8 weeks.
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ª 2006 International Endodontic Journal

Glucose penetration and fluid transport through
coronal root structure and filled root canals
H. Shemesh, M. van den Bos, M.-K. Wu & P. R. Wesselink
ACTA, Amsterdam, The Netherlands
Abstract
Shemesh H, van den Bos M, Wu M-K, Wesselink PR.
Glucose penetration and fluid transport through coronal root
structure and filled root canals. International Endodontic Journal,
40, 866–872, 2007.
Aim To measure glucose penetration and fluid
transport through coronal root structure and com-
pare it with leakage along the coronal region of root
fillings.
Methodology A total of 50 single-rooted teeth were
selected and divided into three groups. Ten roots were
sectioned longitudinally and the apical portion was
removed leaving a total length of 9 mm. These 20 half-
roots served as group 1: root structure (n ¼ 20). The
canals of the remaining 40 roots were prepared to size
50 and filled with vertically compacted injectable filling
material and sealer. Group 2: Resilon + Epiphany
(n ¼ 20) and group 3: gutta-percha + AH26
(n ¼ 20). The apical portion of the root was removed.
Glucose penetration through the coronal root structure
and coronal root fillings was checked over a period of
Introduction
A variety of laboratory-based experimental models are
used to detect and measure leakage along root fillings.
Whilst dye leakage, fluid transport and bacterial
penetration were the most frequently used, other
methods such as radio-labelled isotopes (Haikel et al.
1999), electromechanical tests (von Fraunhofer et al.
2000) and glucose penetration (Xu et al. 2005) have
also been described. These models check penetration of
Correspondence: Hagay Shemesh, DMD Department of Cariol-
ogy Endodontology Pedodontology, ACTA, Louwesweg 1,
1066 EA Amsterdam, The Netherlands (Tel.: +31 20
5188367; fax: +31 20 6692881; e-mail: hshemesh@acta.nl).
866 International Endodontic Journal, 40, 866–872, 2007
26
doi:10.1111/j.1365-2591.2007.01302.x
4 weeks and fluid transport was measured after com-
pletion of the glucose penetration test. Differences
between the groups were statistically analysed with
the Kruskal–Wallis test and the Mann–Whitney test.
Results The three groups presented significantly
different glucose penetration (P < 0.05). The two
groups of filled canals showed significant glucose
leakage whilst the root structure group did not show
any leakage. In the fluid transport model, the root
structure group also did not show any leakage. No
significant difference in leakage existed between the
two vertically compacted filling materials, Resilon with
Epiphany sealer and gutta-percha with AH26 in both
models (P > 0.05).
Conclusion Under the conditions of this study, in
both models used, no leakage was observed through
root structure. Filled canals were associated with
penetration of glucose regardless of the material used.
Keywords: coronal microleakage, fluid transport
model, glucose penetration model, root structure.
Received 1 March 2007; accepted 25 April 2007
different tracers through the root canal, assuming it
travels along the canal and reaches the apical region.
The clinical relevancy of these studies has been
debated. Pitt Ford (1983) showed no correlation
between dye penetration through human teeth and
periapical tissue response to four different filling mate-
rials in dogs. Oliver & Abbott (2001) reported dye
penetration through root-filled teeth that had been
clinically successful. Karagenç et al. (2006) demon-
strated a poor correlation between different leakage
model results and concluded that their clinical rele-
vancy was questionable. Pommel et al. (2001) claimed
that filtration, diffusion or electrical phenomena
governed the outcome of leakage studies in the
corresponding models and thus raised doubt on the
relevancy of these findings. The size of the tracer might
ª 2007 International Endodontic Journal

also influence the results (Barthel et al. 1999) as well as
the potential of the tracer to react or affect the filling
material itself. Methylene blue, for example, frequently
used in dye leakage studies, might react with different
filling materials and calcium hydroxide, making the
results from some of these tests unreliable (Wu et al.
1998). A different problem with leakage tests might be
that different leakage tracers could penetrate through
root dentine and not through the canal. This claim is
supported by the findings that dentinal tubules are
permeable to bacteria (Love et al. 1996, Perez et al.
1996), adhesive agents (Pashley et al. 1993), cements
(Çalt & Serper 1999, Weis et al. 2004), and fluids (Ozok
et al. 2002). Furthermore, dentinal tubules are oriented
perpendicular to the root canal walls (Ferrari et al.
2000) and the contact surface area may affect the seal
provided by the filling material. As the smear layer is
usually removed prior to obturation (Torabinejad et al.
2002), the tubules might be open and allow the tracer
used in leakage studies to penetrate through them.
Previously performed leakage studies did not address
this issue and used a negative control group of similar
filled roots to those checked in the experimental groups,
usually coated with nail varnish or sticky wax (Khayat
et al. 1993, Taylor et al. 1997, Abarca et al. 2001).
These control groups prevent the tracer from penetrat-
ing the canal, but also the dentine itself. If, however,
small molecules such as glucose or water can leak
through root dentine, it would make results from such
leakage studies questionable.
(c)
(a)
(b)
Figure 1 Preparation of the experimental groups. Root vertically sectioned (a) to result in two halves where the root canal was
polished away (b); apical part removed and embedded in acrylic resin block to form group 1 specimens (c); root canal prepared and
filled (d); apical part removed, prepared and embedded in acrylic resin block to from group 2, 3 specimens (e). Both the length of
the root canal filling and the halved roots measured 9 mm.
ª 2007 International Endodontic Journal
27
Shemesh et al. Glucose penetration and fluid transport
The purpose of this experiment is to compare leakage
through root structure with leakage through the
coronal part of root-filled canals using both the
glucose penetration model and the fluid transport
model.
Materials and methods
Selection and preparation of teeth
Seventy freshly extracted single-rooted teeth were
selected and stored in water. Teeth with open apices
or large carious lesions were excluded. The roots were
randomly divided into two control groups and three
experimental groups. The coronal portions of all teeth
were removed so that each root specimen was 16 mm
long.
Group 1 (n ¼ 20)
Ten roots were cut vertically with a diamond-coated
bur (H-327, Horico, Berlin, Germany), so 20 half-roots
were formed (Fig. 1). The canal area was flattened with
a diamond disc and the apical area was removed,
leaving a total of 9 mm root structure. A 7-mm thick
acrylic cylinder was prepared around the root, which
left 1 mm root protruding from each side of the acrylic
cylinder. The contact between the acrylic and the root
was covered with methacrylate glue (Permacol, Ede,
The Netherlands).
9 mm
(e) (d)
International Endodontic Journal, 40, 866–872, 2007 867

Glucose penetration and fluid transport Shemesh et al.
Preparation of root canals
All samples were examined under a microscope (Zeiss
Stemi SV6, Jena, Germany) to exclude cracks. A
diamond bur (FG 173 Horico, Berlin, Germany) was
used to gain straight-line access to the root canal. An
ISO size 20 K-Flexofile (Dentsply Maillefer, Ballaigues,
Switzerland) was inserted into the canal to verify
patency. The coronal 7 mm of the root specimens was
embedded in acrylic (Vertex, Dentimex BV, Zeist, The
Netherlands) to form a cylinder around the root and
enable intimate contact between the root and the
rubber tube used to connect the specimen during the
leakage phase of the study.
Instrumentation and filling of root canals
All teeth were treated by the same operator. The
working length was determined by subtracting 1 mm
from the total length of the root (16 mm). The apical
portion of the canal was instrumented to an ISO size 50
master file using the balanced force technique (Roane
et al. 1985) with K-Flexofiles. Step-back flaring was
performed at 1 mm increments with Gates Glidden burs
numbers 2–6 (Dentsply Maillefer) making the taper
0.2 mm mm)1 (Wu et al. 2002). The purpose of this
preparation was to create a uniform size of canal and to
overcome variation in natural morphology. Each canal
was irrigated with freshly prepared 2% sodium hypo-
chlorite solution (NaOCL) with a 27-gauge needle after
every instrument and ensuring patency by extrusion of
the solution beyond the apical foramen. A minimum of
10 mL NaOCl solution was used for each root. After
preparation, canals were ultrasonically irrigated for
20 s and rinsed with 2 mL of 2% NaOCl. This proce-
dure was repeated thrice (van der Sluis et al. 2006). All
canals were then rinsed with 5 mL 17% ethylene-
diamine tetraacetic acid (EDTA) for 3 min (Scelza et al.
2003) and with 5 mL de-ionized water. Each canal was
dried with an ISO size 50 paper point and filled
according to the relevant group.
Group 2 (n ¼ 20)
Canals were filled with gutta-percha and AH26 (Dents-
ply Maillefer) using the continuous-wave technique
with a System B device and Obtura II system (Obtura
Corporation, Fenton, MO, USA). An ISO size 50 gutta-
percha cone coated with AH26 sealer was inserted into
the canal. Light pumping motions were used to fill the
868 International Endodontic Journal, 40, 866–872, 2007
28
canal with sealer and bring the cone to full working
length.
The coronal part of the gutta-percha was removed
with a heated System B plugger reaching the apical
5 mm and compacted with a pre-fitted hand plugger.
The coronal section was compacted vertically with
gutta-percha using the Obtura II system leaving the
coronal 2 mm of the canal empty.
Group 3 (n ¼ 20)
Canals were filled with the Resilon–Epiphany system
(Pentron Clinical Technologies, Wallingford, CT, USA).
A self-etching primer was placed into the canal with a
26-gauge needle. Two drops of primer were used for
each root. Three paper points ISO size 50 were used to
remove the excess primer after 1 min from each root.
Roots were filled with Resilon cones and Epiphany
sealer with the continuous-wave technique using the
System B device and compacted vertically with the
Obtura II system. An ISO size 50 Resilon cone coated
with Epiphany sealer was inserted into the canal. Light
pumping motions were used to fill the canal with sealer
and bring the cone to full working length. The coronal
part of the filling was removed with a heated System B
spreader extending to the apical 5 mm and compacted
with a pre-fitted hand plugger. The coronal part was
filled with Resilon using the Obtura system, leaving the
coronal 2 mm of the canal empty.
Controls
The Positive control group (n ¼ 10) consisted of canals
that were filled using lateral compaction of gutta-
percha cones without sealer. No warm vertical forces
were used and the whole length of the filling remained
intact. The negative control group (n = 10) consisted of
roots that were sealed with laterally compacted gutta-
percha and AH26 for the whole length of the canal and
completely covered with nail varnish.
Storage and apical preparation
The filled roots were stored for 1 week at 37 C and
100% humidity to allow the materials to set. The apical
2 mm of the roots were cut with a diamond bur (FG
109 Horico, Berlin, Germany) and 3 mm of the apical
root fillings were removed mimicking apical surgery
preparation, leaving a total of 9 mm root canal filling
(Fig. 1).
ª 2007 International Endodontic Journal
 29

Shemesh et al. Glucose penetration and fluid transport

Model assembly and measurements: glucose Fluid transport model

penetration model
All samples were mounted on a glucose leakage model.
This model was first used by Xu et al. (2005) and was
further developed and described in detail in other
publications (Shemesh et al. 2006, van der Sluis et al.
2007). The acrylic resin block around the root was
connected to a rubber tube with stainless steel wires,
which were connected to a 16-cm long pipette (Pyrex,
Acton, MA, USA). The assembly was placed in a sterile
glass bottle with a screw cap and sealed with sticky
wax. A uniform hole was drilled in the screw cap with a
diamond bur (No.173 Horico, Berlin, Germany) to
assure an open system at all times. Sterile water (2 mL)
was injected into the glass bottle, such that the root
samples were immersed in the solution. The tracer used
in the present study was 1 mol L)1 glucose solution.
Glucose has a low molecular weight and is hydrophilic
and chemically stable. About 4.5 mL of the glucose
solution was injected into the pipette until the top of the
solution was 14 cm higher than the top of the root
canal filling, which created an hydrostatic pressure of
1.5 kPa or 15 cm H2O (Xu et al. 2005). All specimens
were returned to the incubator at 37 C for the
duration of the observation period. A total of 100 lL
of the solution was drawn from the glass bottle using a
micropipette every week for 4 weeks. The same amount
of fresh de-ionized water was added to the glass bottle
reservoir to maintain a constant volume of 2 mL. All
assemblies were stored in a closed plastic container at
100% humidity to prevent evaporation.
The sample taken every week was analysed with a
Glucose kit (Megazyme, Wicklow, Ireland) in a
spectrophotometer (Molecular Devices, Spectra max
384 plus) at a wavelength of 340 nm. Concentra-
tions of glucose in the lower chamber were presented
in mg L)1 at each time interval. The lowest glucose
After completion of the glucose penetration, test roots
were mounted on a fluid transport model (Wu et al.
1993). Fluid transport was measured under a head-
space pressure of 30 kPa (0.3 atm), and after 3 h the
movement of an air bubble was recorded and expressed
in microlitre.
Statistical analysis
The differences between the groups with regard to
glucose concentrations and fluid transport were
analysed statistically with the Kruskal–Wallis and
the Mann–Whitney tests (spss, version 12.0.1, Chi-
cago, IL, USA). The level of significance was set at
a ¼ 0.05.
Results
The negative control group showed no leakage in either
model throughout the experiment period. The positive
control group leaked immediately. The results obtained
from the experimental groups are summarized in
Table 1.
The difference in glucose leakage between the three
experimental groups was highly significant
(P < 0.0001). Group 1 (root structure) had no leakage,
whilst the two other groups (gutta-percha, Resilon) did
leak (Fig. 2). Although more roots in the Resilon group
showed glucose penetration, there was no statistical
significance between them (P ¼ 0.174). In the fluid
transport model, no samples leaked at the end of
4 weeks in the Resilon–Epiphany group, and one
sample leaked (4 lL) in the gutta-percha group. No
Glucose concentration (mg L–1)

level for which the current procedure is believed to 50

be accurate is 0.6 mg L)1 which derives from an
40
absorbance difference of 0.02 (d-Glucose HK assay GP
procedure, Megazyme International Limited, 2004). RESILON
30
Below this level, the absorbance readings become
relatively small, and results are subjected to greater 20
error from technique variables. Concentrations smal-
10
ler than this were thus ignored. Similarly, once the
optical density reading exceeded 1.2 (corresponds to 0
1 week 2 week 3 week 4 week
a glucose concentration of 79.6 mg L)1), samples
Time
were no longer observed as the glucose concentration
in the lower chamber suggested substantial leakage Figure 2 Mean glucose penetration in mg L)1 after
had occurred. 1–4 weeks.

ª 2007 International Endodontic Journal International Endodontic Journal, 40, 866–872, 2007 869
 30

Glucose penetration and fluid transport Shemesh et al.

Table 1 Mean glucose leakage in mg L)1 when compared with gutta-percha (Shipper et al.
1 week 2 week 3 week 4 week 2004) and an increase in the fracture resistance of
root-filled teeth (Teixeira et al. 2004). However, Most of
GP and AH26
Mean 0.96 3.14 5.58 9.05 the more recent studies comparing Resilon–Epiphany
SD 1.23 4.61 12.22 20.73 root fillings to more conventional gutta-percha tech-
Median 0.50 1.62 1.13 1.23 niques concluded that there is no apparent advantage
Range 0–4.97 0.20–19.30 0–41.46 0–79.60 of using Resilon–Epiphany over gutta-percha with
% Leaking samples 0 15 20 20
different sealers (Biggs et al. 2006, Onay et al. 2006,
Resilon–Epiphany
Mean 8.75 12.69 16.04 16.85 Pitout et al. 2006, Sagsen et al. 2006, Baumgartner et
SD 20.45 24.78 27.76 27.67 al. 2007). Consistent with these reports, the current
Median 0.13 2.88 4.97 5.67 experiment indicates no statistically significant differ-
Range 0–79.60 0–79.60 0–79.60 0–79.60 ence between the two different materials: Resilon with
% Leaking samples 15 40 50 50
Epiphany sealer or gutta-percha with AH26 in terms of
Root structure
Mean 0.0 0.0 0.0 0.0 fluid movement and glucose penetration.
SD 0.0 0.0 0.0 0.0 The apical portion of root fillings with Resilon or
% Leaking samples 0 0 0 0 gutta-percha allow glucose penetration when checked
with the glucose model (Shemesh et al. 2006). How-
statistical significance was observed between groups in ever, the coronal portion of the root canal is wider,
the fluid transport model (P ¼ 0.368). more accessible, and it is assumed that the filling of this
part of the canal is more efficient than the apical
portion. In the case of Resilon, it could be hypothesized
Discussion
that the bond between the filling material and the
At the end of 4 weeks, both tracers penetrated all 10 conditioned canal walls may be stronger in the coronal
roots of the positive control group and none of the portion. However, this has not been substantiated. For
negative control group. Root structure showed no example, Wu et al. (2003) assessed the coronal two-
penetration of any of the tracers. Overall, 14 of the 40 thirds of gutta-percha and RoekoSeal root fillings with
filled roots allowed glucose to pass through the canal the fluid transport model. Comparing their results to a
and reach the apical portion. previously published study (Wu et al. 2002) on the
Most studies in this field found fluid movement or same materials and at similar conditions, the coronal
tracer penetration through root-filled teeth regardless portion allowed more fluid movement than the apical
of the filling material or filling method used (Abarca et portion. In the current experiment, leakage along the
al. 2001, Wu et al. 2003). The current finding that root coronal portion of the canal was assessed: Resilon
dentine did not allow fluid movement or glucose allowed more glucose penetration than gutta-percha,
penetration is important when assessing the relevancy but this difference was not statistically significant
of such studies. It proves that the tracer passes through because of the small group size (n = 20). However,
the canal and the measurements reflect the ability of comparing previous reports on the sealing ability of the
the canal filling to prevent tracer penetration through apical 4 mm of canal filling (Shemesh et al. 2006) to
it. Nevertheless, we cannot exclude the possibility that the current findings, Resilon performed better in the
parts of the root dentine might be permeable to the coronal part of the canal. This difference could be
tracer: only if the whole length of the checked root explained by methodological differences between the
structure (9 mm long) could allow the tracer to pass two experiments, and possibly by the different location
through it, would we detect leakage at the apical side. of the filling along the canal. In the present study, the
The possibility that certain parts of the root dentine length of the root filling was significantly longer (9 mm
might be permeable is of little concern to the present as compared with 4 mm in the previous experiment)
study, however, as it does not affect the detection of and the longer the root filling, the less leakage observed
tracer at the other side of the root. (Mattison et al. 1984).
Advances in bonding techniques have led to the There were differences between results in the two
development of composite resin root filling materials experimental models used. The glucose test may be
such as Resilon. Early studies with Resilon have more sensitive than the measurement of the fluid
presented a decrease in the amount of bacterial transport observations: measurements of fluid trans-
penetration through root canals treated using Resilon portation are carried out by observing an air bubble

870 International Endodontic Journal, 40, 866–872, 2007 ª 2007 International Endodontic Journal

and its movement along a capillary whilst the glucose
concentration is determined by a highly sensitive
enzymatic reaction measured by the spectrophotome-
ter. Furthermore, the glucose concentration is meas-
ured continuously for a period of 4 weeks, whilst
specimens in the fluid transport model are subjected to
pressure for only 3 h.
Concerns have been raised over the possibility that
different filling materials might react with the tracer
used. A pilot study was conducted where all filling
materials involved in the current experiment were
immersed in a glucose solution for up to a month and
glucose concentrations were checked periodically.
Resilon, Epiphany, gutta-percha and AH26 did not
influence the concentration of glucose over time.
Conclusion
Under the conditions of this study, in both models used,
samples of coronal root structure did not show any
leakage. Root canals filled with vertically compacted
Resilon and Epiphany or gutta-percha and AH26
allowed penetration of glucose with no statistical
difference between them. However, there was a trend
for Resilon–Epiphany-filled roots to allow more penet-
ration of glucose.
Acknowledgement
This work was supported, in part, by the ESE research
award 2006.
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ª 2007 International Endodontic Journal

An evaluation of the influence of passive ultrasonic
irrigation on the seal of root canal fillings
L. W. M. van der Sluis, H. Shemesh, M. K. Wu & P. R. Wesselink
Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, The
Netherlands
Abstract
van der Sluis LWM, Shemesh H, Wu MK, Wesselink PR.
An evaluation of the influence of passive ultrasonic irrigation on
the seal of root canal fillings. International Endodontic Journal,
40, 356–361, 2007.
Aim To evaluate the influence of passive ultrasonic
irrigation (PUI) on the seal of root canal fillings.
Methodology A total of 40 mandibular premolars
were distributed equally into two groups and the root
canals were cleaned and shaped; they were then filled
with gutta-percha and AH26 (sealer) using the warm
vertical compaction technique with the System B
(Analytic Technology, Redmond, WA, USA) device. In
one group PUI was applied, after completion of instru-
mentation and hand-irrigation. In the other group, PUI
was not applied. Thereafter, leakage of glucose was
Introduction
During passive ultrasonic irrigation (PUI) a small
ultrasonically oscillating file or smooth wire (e.g. size
15) is placed in the centre of the root canal following
canal shaping (Ahmad et al. 1987). Because the root
canal has been enlarged, the irrigant can flow in the
root canal and the file or wire can vibrate relatively
freely, which will result in more powerful acoustic
streaming (Ahmad et al. 1987). PUI with sodium
hypochlorite (NaOCl) as the irrigant, removes more
dentine debris, planktonic bacteria and pulp tissue from
the root canal than the syringe irrigation (Huque et al.
1998, Lee et al. 2004, Gutarts et al. 2005). During PUI,
Correspondence: L.W.M. van der Sluis, Department of Cariol-
ogy Endodontology Pedodontology, ACTA, Louwesweg 1,
1066 EA Amsterdam, The Netherlands (Tel.: +31 20
5188651; fax: +31 20 6692881; e-mail: l.vd.sluis@acta.nl).
356 International Endodontic Journal, 40, 356–361, 2007
33
doi:10.1111/j.1365-2591.2006.01227.x
evaluated by measuring its concentration once a week
for a total period of 56 days using a glucose penetration
model. Differences between the groups in terms of
glucose concentrations were statistically analysed with
the Mann–Whitney test; the level of significance was
set at P ¼ 0.05.
Results After the first month the root fillings in teeth
where PUI had been used, sealed the root canal
significantly better than in teeth where no PUI had
been used (P ¼ 0.017).
Conclusion Root fillings sealed the root canal better
when PUI had been used.
Keywords: irrigation, passive, root fillings, sealing,
ultrasonic.
Received 21 August 2006; accepted 18 October 2006
more dentine debris can be removed from the isthmus,
oval extensions in the root canal and irregularities from
the root canal wall (Goodman et al. 1985, Lee et al.
2004).
It is generally accepted that relatively few dentists
use PUI. Remaining dentine debris, which cannot be
removed during hand-irrigation, will be packed into
oval extensions and irregularities of the root canal (Wu
& Wesselink 2001). If oval extensions are filled with
dentine debris, the filling material cannot adapt to the
canal wall and leakage may ensue (Wu & Wesselink
2001, van der Sluis et al. 2005a) because the filling
material and sealer are not compacted against a clean
root canal wall but against dentine debris particles, no
matter which root filling technique or materials are
used.
It is unclear whether root fillings, placed after PUI,
seal the root canal better because more dentine debris is
removed from the root canal. In the study of Wu et al.
ª 2007 International Endodontic Journal

van der Sluis et al. Seal of root fillings after passive ultrasonic irrigation
(2001), the quality of root fillings in oval canals was
evaluated using the percentage of gutta-percha (PGP)
in the root canal. The mean PGP was 86.6% varying
from 47.1–100%. Many oval extensions were not
prepared and became filled with dentine debris. PUI
was not used for irrigation of the root canals. In
another study, where a similar methodology was used,
but with PUI, the mean PGP was 98.9% and varied
from 94.5–100%. This indicated that PUI removed
effectively dentine debris from the oval extensions and
thus allowed the gutta-percha to fill more completely
the root canal (Ardila et al. 2003).
Healing following root canal treatment is assured
through effective infection control (Sjögren et al.
1990). However, it is not clear whether a better sealing
root filling leads to less periapical inflammation.
Yamauchi et al. (2006) demonstrated, in an animal
experiment, that less periapical inflammation occurred
when root fillings were protected against coronal
leakage by application of an additional coronal plug
of filling material. Because an additional coronal plug
leads to less coronal leakage (Chailertvanitkul et al.
1997), these data indicate that less coronal leakage
leads to less periapical inflammation.
Different leakage models were used to detect and
measure leakage along root fillings (Wu & Wesselink
1993). Leakage tests are improving and recently Xu
et al. (2005) reported a new model that measures the
leakage of glucose molecules. This model consists of a
tube where a concentrated glucose solution is placed
and connected to the test element, whilst the apical side
of the specimen is dipped in water. Glucose, which leaks
through the root canal accumulates in the apical
chamber and is measured using an enzymatic reaction
with a spectrophotometer. Glucose has a low molecular
weight, and may be used as an indication for toxins
that might penetrate the canal.
The aim of this study was to evaluate the influence of
PUI on the seal of root fillings.
Materials and methods
Digital radiographs from bucco-lingual and mesial-
distal directions were made of extracted mandibular
premolars that had been stored in water with added
NaOCl for not more than 1 month. A total of 40 teeth
with a single canal that had a long, oval shape at 5 mm
coronal to the working length (WL), where the bucco-
lingual diameter was more than twice as large as the
mesio-distal diameter, were selected. The curvature of
the roots was measured following Schneider’s
ª 2007 International Endodontic Journal
34
methodology (Schneider 1971). The 40 specimens
were divided equally in two anatomically comparable
groups taking into account the curvature and the oval
shape of the root canals. Two teeth were used as
negative controls (the roots were completely covered
with nail varnish) and two as positive controls (canals
were filled using lateral compaction of Gutta-percha
cones without sealer).
The teeth were decoronated and all roots were reduced
to a length of 12 mm. WL was determined by inserting a
size 15 K-file into the canal until the tip of the file was just
visible at the apical foramen. The canals were accessed
and prepared to the apical foramen. The coronal aspect of
each canal was flared using Gates Glidden drills (Dents-
ply Maillefer, Ballaigues, Switzerland) sizes 2–4. All the
root canals were prepared with the GT rotary system
(Dentsply Maillefer) in a crown-down sequence using a
series of size 20 and 30, 0.10–0.04 taper. Between the
instruments, each canal was irrigated with 2 mL of a 2%
NaOCl solution, freshly prepared each day, using a
syringe and a 27-gauge needle that was placed 1 mm
short of the WL. The NaOCl solution was prepared by
diluting a 10% NaOCl solution (Merck, Darmstadt,
Germany) and its pH adjusted to 10.8 with 1 N HCl.
The concentration of the NaOCl solution was measured
iodometrically (Moorer & Wesselink 1982). The final file
[master apical file (MAF)] for each root canal at the
foramen was size 30, 0.06 taper. After preparation of the
root canal, PUI was performed in group 1 (n ¼ 18) with
a piezoelectronic unit (PMax:Satelec, Meriganc Cedex,
France). After the root canal was filled with 2% NaOCl,
using a syringe and a needle, an ultrasonically activated
smooth wire of stainless steel size 15, taper 0.02 was
inserted in the root canal 1 mm short of WL (van der
Sluis et al. 2005b). The irrigant was ultrasonically
activated for 1 min. The root canal was then rinsed with
2 mL of 2% NaOCl, using a syringe and a 27-gauge
needle that was placed 1 mm short of WL, and the
irrigant was again activated ultrasonically for 1 min.
This sequence was repeated thrice resulting in a total
irrigation time of 3 min and a total irrigation volume of
6 mL. The oscillation was performed in bucco-lingual
direction at power setting ‘blue’ three. According to the
manufacturer, the frequency used under these condi-
tions was approximately 30 kHz and the displacement
amplitude was 28 lm. The teeth in group two (n ¼ 18)
were irrigated with 6 mL of 2% NaOCl by syringe
irrigation in place of PUI for the same time period.
After irrigation, the canals were dried and filled with
a warm vertical compaction technique. AH26 silver-
free root canal sealer (De Trey Dentsply, Konstanz,
International Endodontic Journal, 40, 356–361, 2007 357

Seal of root fillings after passive ultrasonic irrigation van der Sluis et al.
Germany) was mixed manually according to the
recommendations of the manufacturer. The sealer
was placed in the root canals using an EZ-Fill
bi-directional spiral (EDS, Hackensack, NJ, USA),
1 mm short of WL in a pumping motion for 5 s. The
complete spiral was coated with sealer. Before the filling
procedure the tip of a medium sized non-standardized
gutta-percha cone (Autofit, Analytic Endodontics,
Glendora, CA, USA) was trimmed until tug-back was
achieved 0.5 mm short of the full WL. The trimmed
gutta-percha cone, lightly coated with sealer, was
placed into the canal 0.5 mm short of the WL. At the
level of the cementum–enamel junction the gutta-
percha was seared off with the tip of an activated heat
carrier at 260 (System B, Analytic Technology,
Redmond, WA, USA) by placing it in the coronal
section of the root canal. After deactivating the heat
carrier, the cooled instrument was removed from the
canal, bringing out an increment of gutta-percha.
Vertical force was then applied with a cold size 11
handplugger (1.1 mm diameter, Dentsply Maillefer) to
compact the gutta-percha in the coronal section of the
canal. During the application of the plugger care was
taken not to contact the canal wall. This procedure was
repeated twice, first to a level 3–4 mm deeper than the
cementum–enamel junction, vertically compacting the
gutta-percha in the middle section of the canal using a
cold size 7 plugger (0.7 mm diameter, Dentsply Mail-
lefer), and secondly to the level 4 mm short of the full
WL, vertically compacting the gutta-percha in the
apical section of the canal using a cold size 5 plugger
(0.5 mm diameter, Dentsply Maillefer). After the apical
section the middle and the coronal section were filled
using the same technique. During the filling procedure
two roots of each group were fractured and discarded.
The remaining teeth were stored in a moist sponge at
37 C for 1 year.
Glucose penetration model – preparation
and measurements
All samples were examined under a microscope (Zeiss
Stemi SV6, Jena, Germany) to exclude those with
cracks. The coronal 4 mm of the root specimens were
then embedded in Acryl (Vertex, Dentimex BV, Zeist,
The Netherlands) to form an acrylic cylinder around
the root and enable leak-free contact between the
rubber tube and root specimen.
The difference between the current version of the
glucose penetration model and the original model
introduced by Xu et al. (2005) lies mainly in the
358 International Endodontic Journal, 40, 356–361, 2007
35
environment where the models were stored: in order to
prevent evaporation of fluids, the models were placed in
a closed jar with 100% humidity. From a pilot study it
was concluded that this method would eliminate the
effect of fluid evaporation on glucose concentration
measurements.
The resin block around the coronal part of each root
was connected to a rubber tube and the adaptation
improved with stainless steel wires. The other end of
the tube was similarly connected to a 16-cm long
pipette. The assembly was then placed in a sterile glass
bottle with a screw cap and sealed with sticky wax, and
a uniform hole drilled in the screw cap with a size 173
diamond bur (Horico, Berlin, Germany) to assure an
open system at all times. Two mL of a 0.2% NaN3
solution was added into the glass bottle, such that the
root samples were immersed in the solution. NaN3 was
used to inhibit the growth of microorganisms that
might influence the glucose readings. The tracer used
in the present study was 1 mol L)1 glucose solution
(pH ¼ 7.0). Glucose has a low molecular weight and is
hydrophilic and chemically stable. About 4.5 mL of the
glucose solution, containing 0.2% NaN3, was injected
into the pipette until the top of the solution was 14 cm
higher than the top of gutta-percha in the canal, which
created a hydrostatic pressure of 1.5 kPa or 15 cm H2O
(Xu et al. 2005)(Fig. 1). All specimens were then
returned to the incubator at 37 C for the duration of
the observation period. A 25 lL increment of solution
was drawn from the glass bottle using a micropipette at
5, 14, 21, 37, 48 and 56 days. The same amount of
fresh 0.2% NaN3 was added to the glass bottle reservoir
to maintain a constant volume of 2 mL. The sample
was then analysed with a Glucose kit (Megazyme,
Wicklow, Ireland) in a spectrophotometer (Molecular
Devices, Spectra max 384 plus, Seattle, Wa, USA) at
340 nm wavelength. Concentrations of glucose in the
lower chamber were presented in mg L)1 at that
particular time after obturation. The lowest glucose
level for which the current procedure is believed to be
accurate is 0.663 mg L)1 which derives from an
absorbance difference of 0.020 (d-Glucose – HK assay
procedure, Megazyme International Limited, 2004).
Below this level, the absorbance readings become
relatively small, and results are subject to greater error
from technique variables. Concentrations smaller than
this were thus ignored. Similarly, once leakage excee-
ded 1.28 g L)1, samples were no longer tested as the
glucose concentration in the lower chamber at this
stage was very high and significant leakage had
occurred.
ª 2007 International Endodontic Journal
 36

van der Sluis et al. Seal of root fillings after passive ultrasonic irrigation

Glucose solution Discussion

Glass tube
Open system
14 cm
Rubber tube
Acrylic cylinder
Metal wire
Root specimen
0.2% NaN3 solution
Figure 1 The glucose penetration model.
The positive control group (n ¼ 2) was filled using
lateral compaction of gutta-percha cones without any
sealer. No warm vertical forces were used.
In the negative control group (n ¼ 2) all roots were
filled with laterally compacted gutta-percha and AH 26
and completely covered with nail varnish.
The data were statistically analysed with the Mann–
Whitney test and P was set at 0.05.
Results
The results are shown in Table 1 and 2 and Fig. 2.
After the first month, the root fillings in teeth where
PUI had been used, sealed the root canal significantly
better than in teeth where PUI had been not used
(P ¼ 0.017).
The root fillings placed after PUI allowed significantly
less leakage of glucose, which indicates it resulted in a
better sealing of the root canal. This can be explained
by the fact that more dentine debris can be removed
from the oval extensions or irregularities (Lee et al.
2004) and/or more smear layer can be removed from
the canal wall using PUI (Cameron 1983, 1987,
Alaçam 1987, Cheung & Stock 1993, Huque et al.
1998). When oval extensions or irregularities of the
root canal wall are free of dentine debris they can be
filled, which is likely to result in a better seal of the root
filling with probability of reduced or no coronal
leakage.
Shaping of the root canal in combination with
irrigation is more efficient in cleaning the canal than
shaping alone (Baugh & Wallace 2005). The present
study indicates, that efficient irrigation can also result
in significant improvement of the sealing of a root
filling. This would suggest that efficient irrigation
could decrease coronal leakage of the root fillings and
thus reduce the nutrition for the biofilm in the root
canal with the potential to reduce the occurrence
and severity of apical periodontitis (Yamauchi et al.
2006).
Glucose as a marker in leakage studies has clinical
relevance because it is an important nutrient for
microorganisms and even at very low concentrations
a biofilm is able to survive (Siqueira 2001). Because it is
impossible to remove completely the biofilm from the
root canal (Ricucci & Bergenholtz 2003, Naı̈r et al.
2005) leakage of very small amounts of glucose could
help the biofilm survive or promote its re-growth when
left in the root canal after preparation (Siqueira 2001).
In this study, during PUI, 2% NaOCl was placed in
the root canal using a syringe in place of a continuous
flow. During 3 min of PUI, the 2% solution NaOCl was
refreshed every minute using further 2 mL volumes of
2% NaOCl. The results of a previous study showed no
significant difference between this method of adminis-
tration and a continuous flow of irrigant when PUI was
used for 3 min (van der Sluis et al. 2006).

Table 1 Mean ± SD of glucose leakage in g L1 at different times

Days

Use PUI 5 14 21 37 48 57

No (n ¼ 18) 0.19 ± 0.34 0.27 ± 0.37 0.35 ± 0.48 0.61 ± 0.46 0.69 ± 0.46 0.86 ± 0.37
Yes (n ¼ 18) 0.05 ± 0.08 0.14 ± 0.21 0.20 ± 0.31 0.30 ± 0.42 0.40 ± 0.47 0.53 ± 0.40

PUI, passive ultrasonic irrigation.

ª 2007 International Endodontic Journal International Endodontic Journal, 40, 356–361, 2007 359

Seal of root fillings after passive ultrasonic irrigation van der Sluis et al.
Table 2 The P-values of statistic analysis after comparing
leakage with and without passive ultrasonic irrigation at the
different time intervals
Time (days)
5 0.440
14
21
37
48
57
without PUI
with PUI
Mean leakage
1.00
0.80
0.60
0.40
0.20
0.00
Day 5 Day 14 Day 21 Day 37 Day 48
Figure 2 The mean leakage of glucose over time.
The standard deviation of the leakage results of both
groups were large. This may have resulted because of
the variation in the oval dimensions of the root canals.
Although the MAF was standardized in all the teeth,
the root canal dimension was not standardized; rather
the specimens were distributed equally. Another
explanation for the large standard deviations in the
PUI group can be explained because it is difficult to
standardize the positioning of the ultrasonically acti-
vated instrument in the centre of the root canal and to
standardize the displacement amplitude as any con-
straint on the wire in the canal will change the
amplitude. This will have a direct effect on the efficacy
of PUI. This could be overcome by increasing the
frequency of the ultrasound, which will reduce the
influence of a variation in the displacement amplitude.
In the present study, the modified glucose penetra-
tion model was used (Xu et al. 2005, Shemesh et al.
2006). This test can be seen as a further development
of the fluid transportation concept (Wu & Wesselink
1993). Both measure penetration of fluid through root
fillings after subjecting them to constant pressure;
however, the glucose model allows measurements of
diffusion of the marker molecules as well. The glucose
test might be more sensitive than the measurement of
air-bubble movement, not only because the threshold
360 International Endodontic Journal, 40, 356–361, 2007
37
measurement detected by the eye is larger than that of
the spectrophotometer, but also because the convective
fluid transport was combined with glucose molecule
P diffusion.
Time difference is an important factor when com-
0.274 paring the two different models. In the glucose penet-
0.970 ration model the tooth is continuously subjected to the
0.017 pressure of the glucose solution in the coronal chamber
0.045
for a period of 2 months. The fluid penetration model
0.014
detects leakage usually after subjecting the filling to
pressure for 3 h (Shemesh et al. 2006). This enormous
time difference might make the glucose test more
sensitive, as it may result in detection of smaller voids
in the filling.
Some authors claim that PUI removes the smear layer
completely (Cameron 1983, 1987, Alaçam 1987,
Huque et al. 1998) or partially from the root canal wall
(Cheung & Stock 1993), whereas syringe irrigation of
NaOCl does not remove the smear layer from the root
canal wall (Cheung & Stock 1993, Huque et al. 1998).
The improved sealing of the root canal filling could also
Day 57 be due to the removal of the smear layer by PUI.
However, the reports in the literature on this subject are
inconclusive (Sen et al. 1995, Torabinejad et al. 2002).
Leakage along root fillings may increase or decrease
with the time following filling. Dissolution of sealer and
the smear layer may result in a rise in leakage, whereas
swelling of GP may result in diminished leakage (Sen
et al. 1995, Kontakiotis et al. 1997). The leakage data
measured some time after filling the root canals may be
clinically more relevant.
Conclusion
Root fillings sealed the root canal better when PUI had
been used.
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International Endodontic Journal, 40, 356–361, 2007 361
 39

Comparability of results from two leakage models

Erick Miranda Souza, MSc,a Min-Kai Wu, MD, MSD, PhD,b Hagay Shemesh, DMD,c
Idomeo Bonetti-Filho, PhD,d and Paul R. Wesselink, DDS, PhD,e São Paulo, Brazil and
Amsterdam, The Netherlands
SÃO PAULO STATE UNIVERSITY AND ACADEMIC CENTRE FOR DENTISTRY AMSTERDAM

Objective. The goal of this study was to check whether leakage results of the same specimens measured by 2 different
leakage models are similar.
Study design. Canine root canals were prepared and filled with cold gutta-percha cones and 1 of 4 sealers (20 canals
for each sealer). The 80 specimens were first connected to a fluid transport model where air-bubble movement was
measured. The same specimens were later connected to a glucose penetration model where the concentration of
glucose was measured. In both models, a headspace pressure of 30 kPa was used to accelerate leakage.
Results. In both models, 4 sealers ranked the same regarding the leakage they allowed, and a significant correlation
between the results of the 2 models was confined (Spearman test coefficient ⫽ 0.65; P ⫽ .000001).
Conclusion. Under the conditions of this study, leakage results of 80 specimens recorded in the fluid transport model
and glucose penetration model were similar. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:309-13)

To achieve periapical healing, root fillings should prevent
coronal reinfection and entomb remaining bacteria.1 In a
study by Felippe et al.,2 roots of dogs’ teeth and periapical
tissues were examined histologically 5 months after dif-
ferent endodontic treatments. Prepared but unfilled canals
were associated with severe chronic periapical inflamma-
tory reaction and severe bone and root resorption.
Defective root fillings, which provide pathways for
bacteria and toxins to the periapex, are not always
identified with 2-dimensional radiographs. A recent
treatment outcome study reported reduced success rates
when the root filling contained radiographically detect-
able voids (poor root filling density or dark lines along
the filling).3 Therefore, root fillings should present as
few voids as possible, and, once present, voids should
be as narrow as possible.
Erick Miranda Souza was supported by MEC/CAPES (Foundation
for the Coordination of Higher Education and Graduate Training)
with an exchange Scholarship. Study conducted at Academic Centre
for Dentistry Amsterdam, The Netherlands.
a
PhD student, Department of Restorative Dentistry, Araraquara Den-
tal School, São Paulo State University.
b
Senior Scientist, Department of Cariology, Endodontology, and Ped-
odontology, Academic Centre for Dentistry Amsterdam.
c
Lecturer, Department of Cariology, Endodontology, and Pedodon-
tology, Academic Centre for Dentistry Amsterdam.
d
Assistant Professor, Department of Restorative Dentistry, Arara-
quara Dental School, São Paulo State University.
e
Professor and Chairman, Department of Cariology, Endodontology,
and Pedodontology, Academic Centre for Dentistry Amsterdam.
Received for publication Jan 22, 2008; returned for revision Feb 17,
2008; accepted for publication Feb 19, 2008.
1079-2104/$ - see front matter
© 2008 Mosby, Inc. All rights reserved.
doi:10.1016/j.tripleo.2008.02.025
Different leakage models, including fluid transport4
and glucose penetration,5 have been used in vitro to
determine the presence of voids along the root filling.
Because new materials are continuously developing, so
is the need for assessing their sealing ability. Results of
different in vitro leakage tests are used to rank various
materials. However, it has rarely been studied whether
the results of the same specimens recorded in different
leakage models are similar.
The purpose of the present study was to investigate
the comparability of leakage results of the same spec-
imens recorded in the fluid transport model and glucose
penetration model.
MATERALS AND METHODS
One hundred recently extracted maxillary and man-
dibular canines were selected, and proximal radio-
graphs were taken to confirm the presence of a single
canal. The coronal parts were removed, leaving roots
15 mm in length.
Instrumentation
Canals were prepared with K-files #15 to #50
(Dentsply Maillefer, Ballaigues, Switzerland) to 1 mm
short of the apical foramen. This enlargement was
chosen following the recommendations of Tronstad.6 A
step-back flaring technique was performed at 2-mm
increments with Gates-Glidden burs #2 to #6. File #50
was used to smooth the irregularities left by this flaring
regimen. Canals were rinsed between each instrument
with 2 mL 2% NaOCl solution. One minute of passive
ultrasonic irrigation was performed using a #15 En-
dosonore file (Dentsply Maillefer).7 Because the apical

309

310 Souza et al.
Fig. 1. Schematic illustration of 2 models. A, Fluid transport. B, Glucose penetration.
diameter of canines could be larger than 0.2 mm,8 a
K-file #30 was used to verify patency and assure that
the apical foramen was not smaller than #30. Canals
were rinsed with NaOCl and dried using paper points.
Roots were randomly divided into 4 experimental
groups (20 each) according to the sealers tested—AH26
(Dentsply Detrey, Konstanz, Germany), AH Plus
(Dentsply Detrey), RSA (Roeko Dental Products, Lan-
genau, Germany), an experimental castor oil polymer
(Polifil; Poliquil Araraquara Polímeros Químicos,
Araraquara, Brazil)—and 2 control groups (10 each).
Obturation
Polifil is commercially available and consists of a
paste (polyester), liquid (biphenyl methane isocyanine),
and zinc oxide. The manufacturer’s instructions were
followed and 2.5 g of supplied zinc oxide was mixed
with 1.0 g of paste and inserted into a plastic syringe.
This was then mixed with the liquid in a 3:1 ratio on a
glass plate. The other 3 sealers were also prepared
following manufacturer’s recommendations.
Sealers were introduced into the canal twice, 5 s
each, using a bidirectional spiral #25 (EDS, Hacken-
sack, NJ). A gutta-percha cone #50 (Henry Schein,
Mexico City, Mexico), coated with sealer, was placed
into the canal followed by 2 accessory cones #25 placed
to a depth where resistance was met. No spreader was
used.
Eleven millimeters of the coronal gutta-percha was
removed immediately after obturation with a heated
plugger, leaving the apical 4 mm to be subjected to the
leakage tests.
40
OOOOE
August 2008
In the positive control group, lateral compaction of
gutta-percha was performed without sealer. Negative
controls were filled with 3 gutta-percha cones and
AH26, and the external root surface was completely
covered with cyanoacrylate.9
To facilitate the leakage setting up, the coronal 8 mm of
each specimen was embedded in acrylic resin to form a
cylinder around the root. Specimens were then stored for
1 month at 370C and 100% humidity for sealers’ setting.
Fluid transport
Roots were mounted on a fluid transport model pre-
viously described4 and shown in Fig. 1, A. A 30-kPa
(360 cm H2O) headspace pressure was applied, and
after 3 h, 6 h, and 24 h, the air bubble movement (in
␮L) in the capillary tube (Fig. 1, A) was recorded.
Glucose penetration
Twenty-four hours after finishing fluid transport
readings, samples were mounted on a glucose penetra-
tion model (Fig. 1, B), where glucose solution was
placed in the coronal reservoir. A headspace pressure of
30 kPa was created by connecting the open orifice of
the pipette to a pressure source (Fig. 1, B). After 24 h,
a sample of 100 ␮L was taken from the apical reservoir
and the glucose concentration was measured.
The samples were analyzed using a Glucose kit
(Megazyme, Wicklow, Ireland) in a spectrophotometer
(Spectra Max 384 Plus; Molecular Devices, Sunnyvale,
CA) at a wavelength of 340 nm.5 Glucose concentra-
tions were presented in mg/mL.
 41
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Volume 106, Number 2 Souza et al. 311

Table I. Leakage of four sealers: fluid transport and glucose penetration

Fluid transport (␮L) Glucose concentration (mg/mL)
Sealer 3h 6h 24 h 24 h
AH26 (n ⫽ 20)
Median (range) 0 (0-1) 0 (0-2) 0 (0-4) 0.01 (0-1.2)
Mean (SD) 0.2 (0.41) 0.25 (0.55) 0.85 (1.73) 0.10 (0.30)
RSA (n ⫽ 20)
Median (range) 0 (0-0) 0 (0-0) 0 (0-1) 0 (0-0.05)
Mean (SD) 0 (0) 0 (0) 0.05 (0.22) 0.01 (0.01)
AH Plus (n ⫽ 20)
Median (range) 0 (0-1) 0 (0-2) 1 (0-4) 0.02 (0-1.3)
Mean (SD) 0.2 (0.41) 0.35 (0.67) 1.20 (1.20) 0.23 (0.43)
Polifil (n ⫽ 20)
Median (range) 0 (0-0) 0 (0-0) 0 (0-2) 0 (0-0.1)
Mean (SD) 0 (0) 0 (0) 0.25 (0.55) 0.02 (0.03)

Kruskal-Wallis and Mann-Whitney tests checked dif-
ferences among the 4 sealer groups and Spearman test
verified the correlation between the results of two models
(SPSS, version 12.0.1; Chicago, IL). Chi-squared test
detected differences in the number of leaking samples
demonstrated by the fluid transport model at different time
intervals (Sigma Stat, version 3.1; San Jose, CA).
RESULTS
In the fluid transport test, no movement of the air
bubble was detected in the negative controls. All pos-
itive controls showed bubble movement that exceeded
the pipette length within 3 h. In the glucose penetration
test, no glucose penetration was detected in the nega-
tive controls. In all positive controls, glucose penetrated
completely to the apical reservoir within 24 h.
Tables I and II show the results of the experimental
groups. After 24 h, in both models, RSA showed the
best sealing, and AH Plus leaked the most (P ⬍ .01); no
significant difference between RSA and Polifil was
observed (P ⫽ 0.15, fluid transport; P ⫽ 0.89, glucose
penetration).
When the fluid transport model was used, signifi-
cantly more leaking samples were detected after 24 h
than after 3 h or 6 h (Table II; P ⬍ 0.01).
A positive correlation was observed between the
results of both models (Fig. 2; coefficient ⫽ 0.65; P ⫽
0.000001).
DISCUSSION
In the original glucose model,5 the headspace pres-
sure was 15 cm of glucose solution. In the present study
a higher headspace pressure of 30 kPa (360 cm H2O),
was applied to accelerate glucose penetration and create
conditions comparable to those for the fluid transport.
Thus, water or glucose solution that moved to the apical
chamber under the same headspace pressure was quan-
Table II. Number of leaking samples detected by the
fluid transport model at different time intervals
Number of leaking samples
Sealer 3h 6h 24 h
AH26 (n ⫽ 20) 4 4 8
RSA (n ⫽ 20) 0 0 1
AH Plus (n ⫽ 20) 4 5 14
Polifil (n ⫽ 20) 0 0 3
Total (n ⫽ 80) 8 9 26
tified. In fluid transport, air-bubble movement was mea-
sured to indicate the volume of water penetration; in
glucose penetration, the concentration of glucose in the
apical chamber was measured to indicate the volume of
glucose solution movement.
Fluid transport and glucose penetration occur only
through voids that are completely open, while cul-de-
sac type voids prevent fluid transport or glucose pene-
tration.10,11 It has been shown that neither water nor
glucose solution penetrate through root dentin.12 Thus,
evidence of fluid transport or glucose penetration dem-
onstrates the existence of at least 1 continuous void
along the root filling. A higher value for fluid transport
or glucose penetration indicates that the total volume of
the voids is bigger.10,11
In this study, 24 h after fluid transport the specimens
were connected to the glucose model, where the con-
centration of glucose was measured. When the same
specimens are tested by 2 models, similarity of the
results could be considered to be a measure of the
models’ reliability. However, one may argue that run-
ning the specimens through the fluid transport system
under 30 kPa pressure had changed the property of the
filling and affected the results of glucose penetration. In
a study by Wu et al., the amount of fluid transport along

312 Souza et al.
Fig. 2. Correlation between the 24 h results of 80 specimens recorded in 2 models (coefficient ⫽ 0.65; P ⫽ .000001).
the same root fillings was examined.13 Fluid transport
under headspace pressure of 60 kPa was measured
every 2 h. All specimens filled by lateral compaction,
vertical compaction, and single cone showed consistent
values, indicating that the 60 kPa headspace pressure
did not result in detectable damage to the root fillings.
In earlier studies where fluid transport was measured,
the headspace pressure was applied for a certain period
of time from a few minutes to 24 h.4,14,15 To investigate
whether the time of fluid transport plays a role in
detection of leaking samples, fluid transport was re-
corded also after 3 h and 6 h. Significantly more leaking
samples were detected after 24 h than after 3 h and 6 h
(Table II). Presumably, samples presented with narrow
voids required longer pressure time to display detect-
able fluid accumulation in the apical reservoir. This is
supported by the observation that only 1 to 2 ␮L of
fluid transport was recorded after 24 h (Table I).
The purpose of this study was to examine whether
the same root fillings displayed a similar amount of
leakage in 2 different leakage models, not to test spe-
cific root filling techniques. Only 3 gutta-percha cones
were used to fill each canal to simplify the procedure.
Spreader was not used because of the difficulty to
42
OOOOE
August 2008
standardize the spreader load and the number and size
of spreader tracks.16,17 Furthermore, using no spreader
may prevent root fracture.
Similar techniques, where no compaction forces are
used, have been included in several recent studies and
in some of them showed a comparable sealing ability to
compaction techniques.18-21 Therefore, the results of
the present study should provide useful information
about the 4 sealers used whenever these noncompaction
techniques are accepted.
AH Plus is considered to be a new generation of
AH26, having a faster setting time.22 The accelerated
setting time may cause shrinkage stress, leading to
debonding of sealer from the root canal wall.23,24 In
addition, silicon oils present in the sealer have been
claimed to affect its sealing properties.24,25 In accor-
dance with other studies, RSA sealer displayed effec-
tive sealing,21,26,27 which may be due to its slight
expansion during setting28,29 and close adaptation to
dentinal walls.30 Polifil is a polyurethane (polyester)31
mixed with zinc oxide. According to our results, Polifil
may become a promising endodontic sealer. Similar
tissue response has been observed to Polifil and to
calcium hydroxide sealer.32

OOOOE
Volume 106, Number 2
Under the conditions in this study, results of the
same specimens recorded in the fluid transport model
and glucose penetration model were similar.
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11. Van den Berg HR, Ten Seldam CA, Van der Gulik PS. Com-
pressible laminar flow in a capillary. J Fluid Mech
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12. Shemesh H, van den Bos M, Wu M-K, Wesselink PR. Glucose
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filled root canals. Int Endod J 2007;40:866-72.
13. Wu MK, van der Sluis LWM, Ardila CN, Wesselink PR. Fluid
movement along the coronal two-thirds of root fillings placed by
three different gutta-percha techniques. Int Endod J
2003;36:533-40.
14. Cobankara FK, Adanir N, Belli S, Pashley DH. A quantitative
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15. Pommel L, Camps J. Effects of pressure and measurement time
on the fluid filtration method in endodontics. J Endod
2001;27:256-8.
16. Budd CS, Weller RN, Kulild JC. A comparison of thermoplas-
ticized injectable gutta-percha obturation techniques. J Endod
1991;17:260-4.
17. Jarrett IS, Marx D, Covey D, Karmazin M, Lavin M, Gound T.
Percentage of canals filled in apical cross sections: an in vitro
study of seven obturation techniques. Int Endod J 2004;37:392-8.
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Souza et al. 313
18. Dalat DM, Spångberg LS. Comparison of apical leakage in root
canals obturated with various gutta-percha techniques using a
dye vacuum tracing method. J Endod 1994;20:315-9.
19. Cohen BI, Pagnillo MK, Musikant BL, Deutsch AS. The evalu-
ation of apical leakage for three endodontic fill systems. Gen
Dent 1998;46:618-23.
20. Pommel L, Camps J. In vitro apical leakage of System B com-
pared with other filling techniques. J Endod 2001;27:449-51.
21. Wu MK, van der Sluis LW, Wesselink PR. A 1-year follow-up
study on leakage of single-cone fillings with RoekoRSA sealer.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2006;101:662-7.
22. Cohen BI, Pagnillo MK, Musikant BL, Deutsch AS. An in vitro
study of the cytotoxicity of the two root canal sealers. J Endod
2000;26:228-9.
23. Zmener O, Spielberg C, Lamberghini F, Rucci M. Sealing prop-
erties of a new epoxy resin-based root-canal sealer. Int Endod J
1997;30:332-4.
24. Miletic I, Anic I, Pezelj-Ribaric S, Jukic S. Leakage of five root
canal sealers. Int Endod J 1999;32:415-8.
25. De Moor RJ, De Boever JG. The sealing ability of an epoxy resin
root canal sealer used with five gutta-percha obturation tech-
niques. Endod Dent Traumatol 2000;16:291-7.
26. Cobankara FK, Adanir N, Belli S, Pashley DH. A quantitative
evaluation of apical leakage of four root-canal sealers. Int Endod
J 2002;35:979-84.
27. Roggendorf MJ, Ebert J, Petschelt A, Frankenberger R. Influence
of moisture on the apical seal of root canal fillings with five
different types of sealer. J Endod 2007;33:31-3.
28. Kazemi RB, Safavi KE, Spångberg LSW. Dimensional changes
of endodontic sealers. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 1993;76:766-71.
29. Ørstavik D, Nordahl I, Tibballs JE. Dimensional change follow-
ing setting of root canal sealer materials. Dent Mater
2001;17:512-9.
30. Gençoglu N, Türkmen C, Ahiskali R. A new silicon-based root
canal sealer (Roekoseal-Automix). J Oral Rehabil 2003;30:
753-7.
31. Pereira-Júnior OCM, Rahal SC, Iamaguti P, Felisbino SL, Pavan
PT, Vulcano LC. Comparison between polyurethanes containing
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treatment of segmental bone defect induced in rabbits. J Bio-
mater Appl 2007;21:283-97.
32. Perassi, FT. Tecidual response of EndoRez sealer and a castor
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logic study (PhD thesis). Araraquara, Brazil: São Paulo State
University; 2004.
Reprint requests:
Dr. Min-Kai Wu
Department of Cariology, Endodontology, and Pedodontology
Academic Centre for Dentistry Amsterdam (ACTA)
Louwesweg 1
1066 EA Amsterdam
The Netherlands
m.wu@acta.nl

Glucose reactivity with filling materials as a
limitation for using the glucose leakage model
H. Shemesh1, E. M. Souza2, M.-K. Wu1 & P. R. Wesselink1
1
Department of Cariology Endodontology Pedodontology, Academic Center for Dentistry Amsterdam (ACTA), Amsterdam, the
Netherlands; and 2Department of Dentistry and Endodontics, Araraquara Dental School, São Paulo State University, São Paulo,
Brazil
Abstract
Shemesh H, Souza EM, Wu M-K, Wesselink PR. Glucose
reactivity with filling materials as a limitation for using the
glucose leakage model. International Endodontic Journal, 41,
869–872, 2008.
Aim To evaluate the reactivity of different endodontic
materials and sealers with glucose and to asses the
reliability of the glucose leakage model in measuring
penetration of glucose through these materials.
Methodology Ten uniform discs (radius 5 mm,
thickness 2 mm) were made of each of the following
materials: Portland cement, MTA (grey and white),
sealer 26, calcium sulphate, calcium hydroxide
[Ca(OH)2], AH26,Epiphany, Resilon, gutta-percha and
dentine. After storing the discs for 1 week at 37C and
humid conditions, they were immersed in 0.2 mg mL)1
glucose solution in a test tube. The concentration of
Introduction
A new leakage model was suggested recently (Xu et al.
2005) using glucose as the tracer. Several studies were
published using this model (Shemesh et al. 2006,
2007, Kaya et al. 2007, van der Sluis et al. 2007,
Zou et al. 2007, Xu et al. 2007, Ozok et al. 2008) and
more are being conducted. This model is based on
measurements of glucose concentrations in an apical
chamber using a sensitive enzymatic reaction. A
coloured substance is produced and optical density
(OD) is determined by a spectrophotometer, translated
Correspondence: H. Shemesh, Department of Cariology En-
dodontology Pedodontology, Academic Center for Dentistry
Amsterdam (ACTA), Louwesweg 1, 1066 EA, Amsterdam, the
Netherlands (Tel.: +31 20 5188651; fax: +31 20 6692881;
e-mail: hshemesh@acta.nl).
ª 2008 International Endodontic Journal
44
doi:10.1111/j.1365-2591.2008.01440.x
glucose was evaluated using an enzymatic reaction
after 1 week. Statistical analysis was performed with
the anova and Dunnett tests at a significant level of
P < 0.05.
Results Portland cement, MTA, Ca(OH)2 and sealer
26 reduced the concentration in the test tube of glucose
significantly after 1 week (P < 0.05). Calcium sulphate
reduced the concentration of glucose, but the difference
in concentrations was not significant (P = 0.054).
Conclusions Portland cement, MTA, Ca(OH)2 and
sealer 26 react with a 0.2 mg mL)1 glucose solution.
Therefore, these materials should not be evaluated for
sealing ability with the glucose leakage model.
Keywords: glucose, leakage model, optical density,
reactivity.
Received 29 January 2008; accepted 10 April 2008
later to concentration units. The advantages of this
model are the relative ease of assembly and operation,
the availability of the materials and equipment and the
great sensitivity of the test (Shemesh et al. 2006).
As leakage studies are constantly being scrutinized
for their clinical relevance and reproducibility (Editorial
Board of the Journal of Endodontics, 2007), a critical
approach to the various leakage models is required.
One of the problems of using a tracer for evaluating
leakage is that the tracer itself could chemically react
with some materials challenging the reliability of such
a test in evaluating sealing ability. For example,
methylene blue, which is often used for dye-leakage
studies, was previously shown to discolour when in
contact with certain filling materials (Wu et al. 1998),
whilst root canal fillings might directly inhibit bacteria
in bacterial leakage studies (Pitout et al. 2006). The
purpose of this study was to determine the reactivity of
International Endodontic Journal, 41, 869–872, 2008 869
 45

Reliability of glucose leakage model Shemesh et al.

glucose solution with different dental materials to
assess whether glucose is an acceptable tracer for all
tested materials.
Materials and methods
The following materials were mixed according to the
manufacturer’s instructions and inserted into round
plastic moulds 2 mm deep with a diameter of 5 mm :
Portland cement (Gamma, Leusden, the Netherlands),
MTA grey (Angelus, Londrina, Brazil), MTA white
(Angelus), Sealer 26 (Dentsply, Petrópolis, Brazil),
calcium hydroxide powder (Merck, Darmstadt, Ger-
many), calcium sulphate (Sigma-Aldrich, Steinheim,
Germany), AH26 (Dentsply-Maillefer, Tulsa, OK, USA)
and Epiphany sealer (Pentron Clinical Technologies,
Wallingford, CT, USA). The following thermoplastic
materials were adapted into similar moulds with a
warm instrument: Resilon cones (Pentron), Gutta-
percha cones (Dentsply De Trey, Konstanz, Germany).
Ten moulds were filled with each material. An
additional 10 dentine discs of similar dimensions were
created from extracted molar teeth. All moulds and
discs were maintained at 37C and humid conditions.
After 1 week, the set materials were carefully removed
from the moulds, forming round discs. Each of the discs
was then inserted to a small test tube with 4 mL of
glucose 0.2 mg mL)1 solution. Additional 10 test tubes
were used as controls and contained only 4 mL of
glucose 0.2 mg mL)1 solution. All test tubes were kept
at 37C. A sample of 0.1 mL of the solution was
removed after 1 week from each test tube and was
analysed with a Glucose kit (Megazyme, Wicklow,
Ireland) in a spectrophotometer (Spectra max 384 plus;
Molecular Devices, Sunnyvale, CA, USA) at a wave-
length of 340 nm. Results were expressed as OD.
Statistical analysis was performed with the anova and
the Dunnett test at significance level of P < 0.05 (spss
version 15.0, SPSS Inc., Chicago, IL, USA).

Table 1 Average optical density (OD), SD and P-value of the

OD measured after 1-week immersion with different materials Results
Average P-value (compared with
The results are presented in Table 1 and Fig. 1.
Material OD ± SD glucose solution)
The glucose kit used has a detectable threshold of OD
Water 0 ± 0.005 <0.001 0.05, so all readings lower than 0.05 were ignored. The
Glucose stand. 0.290 ± 0.010 –
glucose test tubes with no test material showed the
Dentine 0.293 ± 0.007 0.999
Portland cement 0 ± 0.007 <0.001 expected OD from a 0.2 mg mL)1 glucose solution (OD
MTA-grey 0 ± 0.004 <0.001 0.3 ± 0.05).
MTA-white 0 ± 0.007 <0.001 Amongst the materials tested, MTA, Portland
Sealer 26 0 ± 0.027 <0.001 cement, sealer 26 and Ca(OH)2 lowered significantly
Ca(OH)2 0 ± 0.005 <0.001
the OD of the solution after 1 week (P < 0.05) .
Calcium sulphate 0.275 ± 0.007 0.054
AH26 0.282 ± 0.007 0.645 Dentine, Resilon, Epiphany, AH26 and gutta-percha
Epiphany 0.290 ± 0.017 1.000 did not significantly alter the concentration of glucose.
Resilon 0.300 ± 0.015 0.382 Calcium sulphate reduced the concentration of glucose
Gutta-percha 0.297 ± 0.008 0.781 but the difference in concentrations was not significant
The kit used has a detectable threshold of OD 0.05. (P = 0.054).

Calculated absorption from 0.2

0.35 mg mL–1 glucose solution according to
the glucose kit used
0.3
0.25
OD values

0.2
0.15
0.1
0.05
0 Figure 1 The measured optical density
(OD) of 0.2 mg mL)1 glucose solution
er

se

TA .

y
Se hite

a( 6

Su 2
te

Ep 26

ny

ta ilon

a
M dc
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Po ntin

ch
lfa
at

H
co

ha
AH
er

es
an

-w

er
W

lu

ip
al

-p
R
TA
D

rtl

after 1-week immersion with different

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al

ut
C

materials.
G

870 International Endodontic Journal, 41, 869–872, 2008 ª 2008 International Endodontic Journal

Discussion
After 1 week in contact with glucose solution, a
number of the materials tested reduced significantly
the OD of the glucose reaction to the level of water
(Fig. 1).
As all published glucose leakage tests checked the
penetration of glucose for periods ranging from 20 days
(Zou et al. 2007) to 56 days (Shemesh et al. 2006) an
observation period of 1 week seems relevant when
assessing the reactivity of different materials with the
tracer.
Glucose (an aldohexose) in an alkaline solution is
slowly oxidized by oxygen, forming gluconic acid:
1 materials on glucose should always precede glucose
CH2 OH-(CHOH)4  CHO þ O2
2
! CH2 OH-(CHOH)4  CO2 H
In the presence of sodium hydroxide, for example,
gluconic acid is converted to sodium gluconate (Sow-
den & Schaffer 1952). This means that glucose will not
be detected by the glucose kit and could thus mask
leakage in the glucose leakage model.
Interestingly, Sealer 26 releases Ca(OH)2 (Duarte
et al. 2000), whilst MTA contains Ca(OH)2 (Camilleri
2007). It seems that Ca(OH)2 containing products react
directly with glucose. This reaction could also be
initiated by traces of Ca(OH)2 left in the canal when
used as an intra-canal medication. Thus, the leakage
measured by glucose concentrations could be influ-
enced by this chemical reaction.
Zou et al. (2007) used the glucose leakage model and
concluded that calcium sulphate significantly improved
the sealing ability of 1 mm perforations repaired with
composite resin. Although the statistical tests used in
this study resulted in a nonsignificant influence of
calcium sulphate on glucose (P = 0.054), the P-value
is small and it cannot be excluded that calcium
sulphate might have an influence. Thus, calcium
sulphate could be better evaluated for its sealing
properties by another tracer or model.
In the glucose leakage model, a 1 mol L)1 glucose
solution is used as the tracer and placed in the upper
chamber of the model assembly (Xu et al. 2005). This
concentration is much higher than the linear range
detected by the glucose kits: 4–80 lg of glucose per
assay (Glucose–HK assay procedure, Megazyme Inter-
national Limited, 2004). However, the range of glucose
concentrations measured in the apical chamber during
a leakage test is dependent on the amount of glucose
that penetrated through the canal and can demon-
ª 2008 International Endodontic Journal
46
Shemesh et al. Reliability of glucose leakage model
strate a large range of concentrations (Shemesh et al.
2006). In this experiment, a glucose concentration of
0.2 mg mL)1 (20 lg per assay) was chosen as it lies
within the linear range of detection of the glucose kit
used and could give more accurate information on the
variations in glucose concentrations after immersion
with different materials .
Conclusion
The observation that certain materials react with
glucose suggests that evaluating these materials with
the glucose leakage model might lead to erroneous
conclusions. An investigation of the influence of tested
leakage tests to validate the conclusions from such
studies.
References
Camilleri J (2007) Hydration mechanisms of mineral trioxide
aggregate. International Endodontic Journal 40, 462–70.
Duarte MA, Demarchi AC, Giaxa MH, Kuga MC, Fraga SC, de
Souza LC (2000) Evaluation of pH and calcium ion release of
three root canal sealers. Journal of Endodontics 26, 389–90.
Editorial Board of the Journal of Endodontics (2007) Wanted:
a base of evidence. Journal of Endodontics 33, 1401–2.
Kaya BU, Kececi AD, Belli S (2007) Evaluation of the sealing
ability of gutta-percha and thermoplastic synthetic polymer-
based systems along the root canals through the glucose
penetration model. Oral Surgery, Oral Medicine, Oral Pathol-
ogy, Oral Radiology and Endodontics 104, 66–73.
Ozok AR, van der Sluis LWM, Wu MK, Wesselink PR (2008)
Sealing ability of a new polydimethylsiloxane-based root
canal filling material. Journal of Endododontics 34, 204–7.
Pitout E, Oberholzer TG, Blignaut E, Molepo J (2006) Coronal
leakage of teeth root-filled with gutta-percha or Resilon root
canal filling material. Journal of Endodontics 32, 879–81.
Shemesh H, Wu MK, Wesselink PR (2006) Leakage along
apical root fillings with and without smear layer using two
different leakage models: a two-month longitudinal ex vivo
study. International Endodontic Journal 39, 968–76.
Shemesh H, van den Bos M, Wu MK, Wesselink PR (2007)
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root structure and filled root canals. International Endodontic
Journal 40, 866–72.
van der Sluis LW, Shemesh H, Wu MK, Wesselink PR (2007)
An evaluation of the influence of passive ultrasonic irriga-
tion on the seal of root canal fillings. International Endodontic
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Sowden JC, Schaffer R (1952) The reaction of d-Glucose,
d-Mannose and d-Fructose in 0.035 N Sodium Hydroxide at
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Wu MK, Kontakiotis EG, Wesselink PR (1998) Decoloration of
1% methylene blue solution in contact with dental filling
materials. Journal of Dentistry 26, 585–9.
Xu Q, Fan MW, Fan B, Cheung GS, Hu HL (2005) A new
quantitative method using glucose for analysis of endodon-
tic leakage. Oral Surgery, Oral Medicine, Oral Pathology, Oral
Radiology and Endodontics 99, 107–11.
Xu Q, Ling J, Cheung GS, Hu Y (2007) A quantitative
evaluation of sealing ability of 4 obturation techniques
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by using a glucose leakage test. Oral Surgery, Oral
Medicine, Oral Pathology, Oral Radiology and Endodontics
104, 109–13.
Zou L, Liu J, Yin SH et al. (2007) Effect of placement of calcium
sulphate when used for the repair of furcation perforations
on the seal produced by a resin-based material. International
Endodontic Journal 40, 100–5.
ª 2008 International Endodontic Journal
48
 49

Chapter III
Novel imaging methods

This chapter is enhanced by additional high-resolution images and video

available on www.shemesh.nl

1. High frequency ultrasound imaging of a single-species biofilm. Shemesh H, Goertz DE,

van der Sluis LW. De Jong N, Wu MK, Wesselink PR. J Dent. 2007 Aug;35(8) 673-8.
2. The ability of optical coherence tomography to characterize the root canal walls.
Shemesh H, van Soest G, Wu MK, van der Sluis LW, Wesselink PR J Endod.
2007Nov;33 (11):1369-73
3. Diagnosis of vertical root fractures with optical coherence tomography Shemesh H, van
Soest G, Wu MK, Wesselink PR J Endod. 2008 Jun;34(6):739-42
50
 51
journal of dentistry 35 (2007) 673–678

available at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

High frequency ultrasound imaging of a

single-species biofilm

H. Shemesh a,*, D.E. Goertz b, L.W.M. van der Sluis a, N. de Jong b,

M.K. Wu a, P.R. Wesselink a
a
Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA),
Louwesweg 1, 1066 EA Amsterdam, The Netherlands
b
Department of Biomedical Engineering, Erasmus University Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam,
The Netherlands

article info abstract

Article history: Objective: This study evaluated the feasibility of a high frequency ultrasound scan to
Received 27 March 2007 examine the 3D morphology of Streptococcus mutans biofilms grown in vitro.
Received in revised form Methods: Six 2-day S. mutans biofilms and six 7-day biofilms were grown on tissue culture
21 May 2007 membranes and on bovine dentine discs. A sterile growth medium on the membrane and
Accepted 22 May 2007 disc were used as controls. Surfaces were rinsed and then immersed in sterile saline. High-
frequency ultrasound imaging system was used to scan these surfaces at 55 MHz, and a
computer program calculated the average thickness of the biofilm layer from the 3D images.
Keywords: Results: 3D pictures of the biofilm layers were obtained. Different cross-sections and plains
Biofilm are easily demonstrated. The average thickness of the 7-day biofilm was significantly bigger
Ultrasound than the 2-day on both the membranes and dentinal discs. No structures were observed on
Imaging the sterile membrane or disc.
3D Conclusion: Three-dimensional structural imaging in situ is possible without damaging the
biofilm layer in a quick and easy manner and can therefore be used to evaluate biofilms
longitudinally as a function of time.
# 2007 Elsevier Ltd. All rights reserved.

1. Introduction chemically remove it.6 Although various successful attempts

Biofilms are communities of microorganism growing on a
surface or interface.1 One of the best studied biofilms is dental
plaque, which forms on tooth surfaces and causes dental
caries.2,3 It is a multi species biofilm dominated by Streptococcus
species.4 Biofilm structure has a crucial role in its function and
resistance. It has a circulatory system and mixed microbial
community that is highly integrated in terms of nutritional
needs and output.5 Knowledge of the 3D structure of a biofilm
is important in order to understand its special characteristics
as well as to monitor the efficacy of procedures to eliminate or
to quantify biofilm structure were made,7 producing a clear
image of the biofilm or obtaining reliable data about its
thickness, density or biomass still presents a challenge.8,9
Laser scanning microscopy is the method most frequently
employed for 3D biofilm imaging but its disadvantages are the
toxicity of fluorescent markers used, depth of penetration and
resolution.10 Recently, nuclear magnetic resonance (NMR) was
suggested as a non-invasive method to image live biofilms.11
Ultrasound imaging has been extensively used as a non-
invasive diagnostic tool in medicine for over 50 years, and in
the majority of applications, it operates in the frequency range

* Corresponding author.
E-mail address: hshemesh@acta.nl (H. Shemesh).
0300-5712/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2007.05.007

674 journal of dentistry 35 (2007) 673–678
of 1–5 MHz. Ultrasound imaging as a possible diagnostic dental
tool has been investigated as far back as the 1960s. Lees and
Barber12 were the first to describe a sonic echogram of incisor
teeth, and later discussed the difference of echoes received
from the dentine-amalgam interface and demineralized
dentine.13 They were able to estimate the enamel and dentine
thickness at various points, but these measurements could
not be representative for the whole tooth. The researchers also
neglected to take into account the phase change that occurs
when acoustic waves pass through interfaces between
materials of different acoustic impedances, thus allowing
an error to arise in their measurements.14
More recently, relevant clinical dental applications of
ultrasonic scans were discussed,15,16 such as the detection
of caries and cracks in the enamel. These studies were imaging
hard-tissues, while ultrasound imaging has an excellent
capacity to image soft tissues as well. Higher frequency
ultrasound systems (20–100 MHz) have lately been developed
for specialized biomedical applications, often imaging soft
tissues, and offer improved spatial resolution for situations
where the imaging probe (transducer) can be brought to within
<10 mm from the region of interest.17
The aim of the present study was to evaluate the feasibility
of a high frequency ultrasound imaging system to examine the
3D morphology of biofilms. In particular, single-species
Streptococcus mutans biofilms on the order of 100–300 mm were
non-invasively imaged.
2. Materials and methods
2.1. Growth of S. mutans biofilm
S. mutans biofilms were prepared: a frozen stock of S. mutans
C180 was plated on Brain Heart Infusion (BHI) agar and grown
under anaerobic conditions in gas packed jars for 2 days in
37 8C. One colony was inoculated in 25 ml BHI solution and
stored in 37 8C overnight.18 The next morning 0.5 ml of the
bacterial suspension was transferred to 25 ml of half strength
BHI with pipes buffer (50 mM). 0.1 ml sterile sucrose solution
0.2% was added. This bacterial suspension was pipetted into
cell culture inserts (BD Biosciences San Jose, CA, USA) which
have a thin (12 mm) membrane on which the biofilm was
grown and maintained in anaerobic conditions. The super-
natant was carefully removed every day and exchanged with
a fresh sterile growth medium containing sucrose. A white
layer was visible on the insert’s membrane after 24 h of
incubation, indicating biofilm formation. Four different
plates of six inserts each were used to generate different
biofilms or environments: in plate A (six inserts), biofilms
were grown for 7 days before they were ultrasonically
scanned, while in plate B (also six inserts) biofilms were
grown for only 2 days before scanning. Two other sets of
plates were prepared with dentine discs: in plate C, six sterile
bovine dentine discs (diameter: 5 mm, uniform thickness of
2 mm) were placed on the insert’s membrane. They were
inoculated with 4 ml of bacterial culture and growth medium
like the previous plates and the procedure was repeated as in
group A. In plate D biofilms were grown on the dentine discs
for 2 days. The control plate contained four inserts with
52
sterile growth medium supplemented with sucrose, two of
which contained a sterile bovine dentine disc in it.
2.2. Ultrasound scan
A commercially available high frequency ultrasound system,
designed primarily for use with small animal imaging, was
employed in this study (Visualsonics Vevo 770, Toronto,
Canada). The 55 MHz probe (#708) was used, which has a single
element focused transducer with an aperture of 2 mm and a
focal length of 4.5 mm. The estimated 6 dB beam width of this
transducer was 62 mm. The nominal bandwidth of the pulses
was reported to be 100%, giving pulse duration of approxi-
mately 0.02 ms. This type of transducer mechanically scans
laterally over a region of interest, and a 2D image is built up
using echoes from a series of 384 adjacent transducer beam
locations (one pulse per location). 2D images were acquired at
20 frames per second and for an 8 mm lateral image distance,
such as that used in this study, the spacing between pulses
was 21 mm on average. At a given beam location, the received
ultrasound echo is converted into a brightness scale (loga-
rithmically compressed) that is then displayed as one line on
the image. The transducer was mounted to the proprietary
positioning system, which permitted manual control of the
transducer orientation and location relative to an imaging
platform. 3D imaging capabilities were enabled with a
motorized position axis, which scans the imaging probe
through a series of 2D imaging planes to acquire volumetric
data.
Before scanning, growth medium was carefully removed
and the culture inserts were rinsed and filled with saline,
taking care no air bubbles were formed. The inserts were
immediately placed on a stable imaging platform and the
membrane or dentine surface was manipulated to be in the
focal zone of the transducer, and perpendicular to the
transducer beam. The configuration is shown in Fig. 1, which
indicates that the transducer beam travels through saline
before encountering the biofilm. The saline is required as a
coupling medium to permit the ultrasound energy to travel
into the biofilm. The presence of air, which creates larger
echoes, would prohibit ultrasound imaging of the sample.
An image of 8 mm  8 mm  10 mm in the x–y–z directions
(Fig. 2) took approximately 10 s to scan. Taking into account
Fig. 1 – Tissue culture insert, and the setting of the scan. A
transducer is located inside saline perpendicular to the
biofilms’ surface.

journal of dentistry 35 (2007) 673–678
Fig. 2 – Ultrasound scan output screen: (A) cube view, 3D reconstruction, (B) cross view, (C) transverse view, (D) sagittal view.
the beam width of the transducer, each location was exposed
to a maximum of 4 pulses during an individual 2D plane scan
and 12 pulses during the 3D scan. The transmit amplitude was
set to 10% of the maximum setting. The specific pressure
levels employed in this study were not available, but the
manufacturer indicated that the peak negative pressure
measured using the maximum transmit amplitude for
another unit of the same type was 3.3 MPa.
The resulting data was presented as a series of standard 2D
ultrasound ‘b-scan’ (brightness) images. These data sets then
underwent two forms of processing. First, the 3D images were
rendered and examined using the internal Vevo 770 system
software. This resulted in a 3D visualization of the biofilm
surface, which permitted inspection of the biofilm morphol-
ogy. The images could also be manipulated to look at user-
selected slices within the volumetric data sets. Second, to
perform quantitative analyses, the data sets were transferred
to a personal computer and processed using custom software
written in a MatlabTM environment (Mathworks Inc., Natick,
MA, USA).
Differences between thicknesses of 2-day and 7-day
biofilms were statistically analyzed with the Mann–Whitney
53
675
tests (SPSS, version 12.0.1, Chicago, IL, USA). The level of
significance was set at a = 0.05.
3. Results
The 3D output shows four different views (Fig. 2). A specific
sagittal or transverse image could be generated at the specific
plane determined by the cross-view. Sterile dentine discs and
membranes immersed in sterile growth medium showed no
biofilm growth at all. The membrane itself appeared as a very
bright line resulting from a strong echo. In the experimental
groups, biofilm structures were clearly visible (Figs. 2–4) as
heterogeneous layers above the membrane or dentine discs.
The upper boundary of the biofilm could be delineated due to
presence of ‘‘scattered’’ echoes from within the biofilm and
the absence of echoes from the overlying saline. The high
amplitude membrane reflection could be readily used to
identify the lower boundary of the biofilm. Using these
boundaries, the average thickness of the biofilm was
calculated for both substrates: the membrane and the dentine
discs (Table 1). Seven-day biofilms were significantly thicker

676 journal of dentistry 35 (2007) 673–678
Fig. 3 – Seven-day-old Streptococcus mutans biofilm grown
on tissue-culture membrane with mm-scale.
than 2-day biofilms both on the membrane and dentine discs
(P = 0.08, P = 0.016, respectively).
4. Discussion
This study was a preliminary attempt to visualize S. mutans
biofilms with an ultrasound imaging system and to assess its
validity as a new method in the research of these structures.
The assessment of biofilm was conducted at different growth
stages, providing an illustration of its compatibility for
Fig. 4 – Streptococcus mutans biofilm growth on tissue-culture membranes (A–C) and on dentine discs (D–F): (A) sterile
membrane (negative control), (B) 2-day-old biofilm, (C) 7-day-old biofilm, (D) sterile dentine disc (negative control), (E) 2-day
biofilm, (F) 7-day biofilm.
54
Table 1 – Average thickness of biofilms grown on
different substrates (membrane in tissue culture plates
and dentine discs) of 2 and 7 days old
Substrate Biofilm Average S.D.
age days thickness (mm)
Membrane 2 102 11
7 266 56
Dentine discs 2 92 13
7 126 16
longitudinal studies. The approach employed was to use a
commercially available instrument which has rapid scan time
(10 s per sample) and could immediately produce 3D visua-
lization results. These factors are appealing from the
perspective of increasing the accessibility of ultrasound
techniques to the wider dental biofilm research community.
Ultrasound imaging is widely used for a range of medical
applications.19 A short burst of ultrasound is emitted from a
transducer and directed into tissue. Echoes are produced as a
result of the interaction of sound with tissue, and some of these
travel back to the transducer. By timing the period elapsed
between the emission of the pulse and the reception of the echo,
the distance between the transducer and the tissue can be
calculated and an image formed. Echoes are generated when
the propagating ultrasound wave encounters an object with
different acoustic impedance (product of density and speed of
sound). When the ‘object’ is much smaller than the acoustic
wavelength, such as in the case of cells within tissue, echo is
generated through scattering. This is the origin of the echoes
within the biofilm. If the wave encounters an interface that is
relatively large with respect to the wavelength, then reflection
occurs. This is the situation for the substrate/biofilm interface.

journal of dentistry 35 (2007) 673–678
The biofilms thickness estimates were made based on an
assumed ultrasound velocity in biofilm of 1540 m/s.20 It is
important to note however that the velocity of sound in biofilm
was not measured and although expected to be within the
relatively narrow range of velocities found in soft tissues,21 a
calculation of this value will allow more precise measure-
ments within the biofilm itself and not only its borders.
According to the results of this experiment, a 7-day biofilm
is thicker than a 2-day biofilm regardless of the substrate used.
This is in accordance with previous reports, where an increase
in dental plaque thickness was observed, that did not conform
to simple growth pattern models.22 The current results
however show that the 7-day biofilm was significantly thicker
when grown on the membrane. This could be explained by the
method applied to grow the biofilm on the dentine discs. Since
the discs were not attached to the membrane below them,
they constantly moved during the growth medium refresh-
ment every day and it may have dislodged bacteria layering
over the disc. Since this problem was not present in the
membrane group, a thicker biofilm was able to grow.
There have been two other recent reports of using high
frequency ultrasound to image biofilms. Holmes et al.23
imaged non-specific biofilms created in water ponds with a
custom 50 MHz system. Placing an immersed transducer
below a membrane and detecting reflections from an air/
biofilm interface allowed imaging the surface morphology of
these biofilms. The use of air provided a very large reflection
signal due to the difference in acoustic properties between air
and biofilm. This strong echo, in combination with the signal
processing methods applied, permitted the measurement of
films of thickness on the order of 10 mm. Unfortunately, this
approach has the potential drawback of altering the properties
of the biofilm and making longitudinal studies difficult.
Furthermore, such an approach may not be possible to
implement if the substrate is dentine rather than a thin
membrane. Good et al.24 reported biofilm visualization of
bacteria relevant to water pipes using 50 MHz ultrasound. The
scanning approach was to discretely move the transducer
through a grid, while stopping at each point to acquire a series
of signals. Custom software was then used to form 3D images
and measure thicknesses. It was also concluded that sound
energy is an effective way to investigate biofilms non-
invasively.
Microscopy is a highly valuable approach to biofilm
investigations, and especially confocal scanning laser micro-
scopy can create accurate 3D reconstructions. However, it
cannot be used to visualize unstained (non-fluorescent)
material or any other matrix which was not specifically
treated.25 Furthermore, the ultrasonic scan of a biofilm takes a
few seconds to complete which makes it far easier and more
convenient than previous methods. The ultrasound scan can
provide an additional tool as a non-invasive method which
could be applied repeatedly on the same sample after
subjecting is to different treatments or materials without
the need of any staining or fixing procedure.
As the number of pulses exposing any given location of the
biofilm was very low (<12 over 150 ms) and each pulse was of
short duration (<20 ns) and at 20% of the maximum transmit
power, it is not expected that any heating, radiation pressure,
or bioeffects would be induced during measurements. Cucevic
55
677
et al.26 used a Visualsonics system (40 MHz) and found
temperature rises only when exposing at maximum power
with longer pulses (0.4 ms) at high repetition rates (10–20 kHz)
sustained over many seconds at the same location.
While in this study we have focused only on morphology
and thickness, it should also be noted that the received
ultrasound signal may contain more useful information. For
example, it has been shown that high frequency ultrasound
signals can be sensitive to both tissue structure and cells
undergoing apoptosis.27 It may therefore be useful to examine
ultrasound signals coming from different biofilm types or
during growth and after therapy. Another avenue of investi-
gation would be to examine the use of ultrasound molecular
imaging probes which are typically comprised of targeted
micro-bubbles.28,29
5. Conclusion
The present study demonstrated a high frequency ultrasound
imaging system that was successfully used to generate 3D
computer images of S. mutans biofilm. The biofilm thickness
could be calculated from these images. Biofilms on bovine
dentine discs were also imaged, proving the method as an
effective way to investigate biofilms for dental research.
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The Ability of Optical Coherence Tomography to
Characterize the Root Canal Walls
Hagay Shemesh, DMD,* Gijs van Soest, PhD,† Min-Kai Wu, PhD,*
Lucas W.M. van der Sluis, PhD,* and Paul R. Wesselink, PhD*
Abstract
A detailed understanding of the complexity of root
canal systems is imperative to ensure successful root
canal therapy. The aim of this study was to evaluate the
ability of an optical coherence tomography (OCT) sys-
tem in imaging root canal walls after endodontic prep-
aration and to correlate these images to histologic
sections. Ten extracted mandibular incisors were pre-
pared to size 50 with K-files and Gates Glidden drills. A
three-dimensional OCT scan was made with a rotating
optical fiber probe inside the root canal. All teeth were
sectioned at 5 and 7 mm from the apex and viewed
through a microscope. Histologic sections were com-
pared with the corresponding OCT output. All oval
canals, uncleaned fins, risk zones, and one perforation
that was detected by histology were also imaged by
OCT. OCT proves to be a reliable method to image root
canals and root dentin in a nondestructive way. This
technique holds promise for full in vivo endodontic
imaging. (J Endod 2007;33:1369 –1373)
Key Words
Histology, intracanal imaging, optical coherence to-
mography, root canal
From the *Department of Cariology, Endodontology, Ped-
odontology, Academic Centre for Dentistry Amsterdam
(ACTA), Amsterdam, The Netherlands; and †Department of
Biomedical Engineering, Thorax Center, Erasmus University
Medical Center, Rotterdam, The Netherlands.
Address requests for reprints to Hagay Shemesh, Depart-
ment of Cariology, Endodontology, Pedodontology, Academic
Center for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066
EA Amsterdam, The Netherlands.
0099-2399/$0 - see front matter
Copyright © 2007 by the American Association of
Endodontists.
doi:10.1016/j.joen.2007.06.022
JOE — Volume 33, Number 11, November 2007
57
Basic Research—Technology
M odern imaging techniques are clinically applied during root canal treatment, but
important information over inner canal anatomy and dentin thickness is still
limited to in vitro observations. Moreover, more than 50% of lower incisors show
long-oval form (ratio of long/short canal diameter ⱖ2) 5 mm from the apex (1), which
requires special considerations in cleaning and obturation (2). One difficulty in treating
oval or curved canals is the chance of strip perforations because of the short distance
between the inner canal wall and the periodontal ligament. In these so-called “risk
zones,” the clinician is often faced with the challenge to sufficiently clean and enlarge
the root canal space while not perforating the mesial or distal wall (3). Current clinical
imaging techniques cannot give reliable information on this aspect.
Optical coherence tomography (OCT) is a new diagnostic medical imaging tech-
nology that was first introduced in 1991 (4). Since then, it has become a standard tool
in ophthalmology and promising imaging method for intracoronary atherosclerosis
detection (5). For example, most heart attacks are caused by sudden ruptures of
unstable arterial plaques that cannot be detected by using conventional imaging mo-
dalities. OCT has the potential to identify these arterial plaques and differentiate stable
plaque from unstable. In addition, it is an attractive technique for the early identi-
fication of gastrointestinal malignancies, including the esophagus, stomach, and
colon (6). Recently, optical in vivo biopsy, providing microscope-quality images in
which cell function can be distinguished, is one of the most challenging fields of
OCT application (7).
OCT combines the principles of an ultrasound with the imaging performance
of a microscope; although an ultrasound produces images from backscattered
sound “echoes,” OCT uses infrared light waves that reflect off the internal microstruc-
ture within the biological tissues. Using the principle of low-coherence interferometry,
it achieves a depth resolution of the order of 10 ␮m and an in-plane resolution similar
to the optical microscope. By scanning the probe along the imaged specimen while
acquiring image lines, a two-dimensional or three-dimensional image is built up. The
OCT light source has a wavelength of 1300 nm. Visible light that has a shorter wavelength
is prone to a higher level of scattering and absorption and produces a shallower imaging
depth (8). The frequencies and bandwidths of infrared light are significantly higher than
medical ultrasound signals, resulting in increased image resolution (9). In endoscopic
OCT imaging, near-infrared light is delivered to the imaging site (usually blood vessels)
through a thin fiber. The imaging tip contains a lens-prism assembly to focus the beam
and direct it toward the vessel wall. The fiber can be retracted inside a catheter sheath
to perform a so-called “pullback,” allowing the user to make a stack of cross-sections,
scanning the investigated vessel lengthwise. Modern OCT systems reach a 6-mm imaging
depth, with 8-␮m resolution, at 50 to 80 frames per second.
OCT potential in dentistry was not overlooked. OCT images of hard and soft tissues in
the oral cavity were compared with histologic images using an animal model showing an
excellent match (10). In another study, Otis et al (11) discussed the clear depiction of
periodontal tissue contour, sulcular depth, connective tissue attachment, and marginal ad-
aptation of restorative materials to dentin, concluding that OCT is a powerful method for
generating high-resolution, cross-sectional images of oral structures. Amaechi et al (12) and
Baumgartner et al (13) described the recognition of caries with OCT. Recently, Lantis Laser,
Inc (Denville, NJ) gained license to LightLab Imaging’s intellectual property portfolio (West-
wood, MA) related to OCT in the field of dentistry. OCT could provide dentists with an
unprecedented level of image resolution to assist in the evaluation of periodontal disease,
Ability of Optical Coherence Tomography to Characterize Root Canal Walls 1369

Basic Research—Technology
dental restorations, and in the detection of caries. Commercial development
of chair-side OCT dental system is already underway (www.lantislaser.
com). The purpose of this experiment was to evaluate the ability of an OCT
system to provide important information on the root canal walls using his-
tologic sections as control.
Materials and Methods
Preparation of Teeth
Ten extracted single-rooted mandibular incisors were selected. A
radiograph was taken from two angles to verify a single canal. Teeth with
open apices or large carious lesions were excluded. Each tooth was
accessed coronally with a diamond bur (FG 173; Horico, Berlin, Ger-
many), and the canal opening was enlarged with Gates Glidden drills #3
and #4, which were inserted 4 and 3 mm into the canal, respectively.
The canal was instrumented to a size 50 stainless steel K-file (Dentsply
Maillefer, Ballaigues, Switzerland). Irrigation with 2% NaOCl using a
26-G needle followed after every instrument so that a total of 15 mL of
solution was used per tooth. Each canal was then rinsed with sterile
saline. For endodontic imaging, the probe’s tip has to be placed inside
the root canal (Figs. 1 and 2) so that the distal end is inserted through
the apex. The apical constriction was opened thus with #45 K-file to
allow the optic fiber to penetrate through the canal.
Test Setup
OCT pullback scans were performed by using a LightLab Imaging
M2-CV system in combination with an ImageWire 2 catheter. This sys-
tem is designed for intracoronary imaging in atherosclerotic plaque
diagnosis. It is commercially available for clinical use in cardiac cath-
eterization laboratories. The catheter consists of a 2-m long optical
single-mode fiber inside a protective sheath, with a diameter of 0.5 mm.
The imaging depth in water is 3.3 mm. The axial and transverse image
resolutions are 14 and 25 ␮m, respectively. The imaging guidewire is
integrated into an imaging catheter, which is connected to a motor (Fig.
1). Both rotational and horizontal movements of the catheter could be
performed, allowing the fiber to be pulled inside the imaged canal in an
apical-coronal direction while rotating. This movement is also used in
cardiovascular imaging and is commonly referred to as “pullback.”
The tooth was placed in a water bath to improve the optical match
between the catheter and the tooth. Motorized “pullbacks” from the
apex to the coronal opening were performed, with a speed of 1 mm/s
and 10 rotations per second, using 312 lines per frame and 760 samples
per line. The result is a stack of images with a spacing of 100 ␮m. These
images were stored as “audio video interleave” files for visual inspec-
tion. All teeth were sectioned at 5 and 7 mm from the apical area with a
saw microtome (Leica Microsystems SP1600, Wetzlar, Germany). Slices
were then viewed through a stereomicroscope (Zeiss Stemi SV6; Carl
Zeiss, Gottingen, Germany) by using a cold light source (KL 2500 LCD,
Figure 1. (A) The OCT screen and machine. (B) The motor connected to the
catheter. (C) The activated OCT catheter inside the root canal.
1370 Shemesh et al.
58
Figure 2. (A) The OCT catheter inside the root canal. (B) A rotating needle with
a transparent tip is situated inside the catheter and serves both as a light source
and as the receiver, transmitting the reflected beams to the imaging computer.
Carl Zeiss). Pictures were taken with a camera (Axio Cam, Carl Zeiss) at
a magnification of ⫻12 and compared with the corresponding OCT
images at the same level. Canal diameters were measured in cross-
sections and in the OCT images and were identified as oval when the
ratio of long to short canal diameter was ⱖ1.5. “Risk zone” was defined
as dentinal wall thinner than 1 mm.
Results
Results are summarized in Table 1 and Figures 3 to 6. All oval
canals, uncleaned fins, risk zones, and one perforation that was histo-
logically detected were also visualized with OCT.
Discussion
The results show excellent correlation between the histologic im-
ages and the OCT output. Risk zones were created in 30% of the teeth at
5 mm from the apex, and one root was perforated. The large master file
(#50) and the anatomy of the roots that were used (lower incisors)
could explain this large incident.
The use of novel imaging techniques is gaining a lot of attention in
the field of endodontics. New computed tomography methods prove to
be more accurate in the evaluation of bone lesions than conventional
radiography (14). Similarly, canal morphology (15), root fractures
(16), tooth anatomy (17), and the interface between the root canal and
filling materials (18) were successfully shown with different computed
tomography techniques. These methods use ionizing radiation, which
could be harmful at higher doses when used in vivo. Furthermore, two
major disadvantages are limiting the successful application of these
methods for intracanal imaging: First, the resolution is usually not suit-
able for microscopic-level imaging. Digital dental radiography systems
TABLE 1. Various Clinically Relevant Parameter Observed in Histology
and OCT Images at 5 mm and 7 mm from the Apex
Histology OCT
5 mm 7 mm 5 mm 7 mm
Teeth with uncleaned fins 2 1 2 2
Oval canals 7 8 7 8
“Risk zones” (canal wall 3 2 3 2
ⱕ1 mm thick)
Perforation 1 0 1 0
JOE — Volume 33, Number 11, November 2007

Figure 3. The OCT output at 7 mm from the apex shows a cross-section of a prepared canal. (A) The root canal, (B) cementum, and (C) dentinal tubules. (D) “Risk
zone” (canal wall ⫽ 1-mm thick).
have a pixel size approaching 100 ␮m. Second, the probe size is usually
much bigger than a root canal. These methods are also time consuming
and often require the interpretation of thousands of images. In contrast,
OCT combines a very narrow optical fiber measuring 0.5 mm in diam-
eter, with high-resolution capacities, enabling imaging of objects mea-
suring a few micrometers and does not involve ionizing radiation. The
imaging wire can be deployed independently or integrated straightfor-
wardly into existing therapeutic or imaging catheters. Furthermore, it
can easily fit into a prepared root canal and is flexible, allowing pene-
tration through curvatures. The optical probe rotates inside the imaged
vessel so that adjacent lines in each rotation compose a frame showing
a cross-section of the tissue architecture in the wall. The scan is quick
and takes 15 seconds for a 15-mm long root.
The influence of a specific tissue’s microstructure on light propa-
gation is an important factor when considering diagnostic applications
of light. Dentin is a structure with anisotropic optical properties that is
different from most other biological tissues (19) because the tubules
are the primary cause of light scattering (20). In our experiments, the
root dentin was semitransparent, allowing imaging of the outer root
surface as well. However, a thicker dentine wall will not allow sufficient
light penetration, and the outer outline of the root will not be seen.
However, some care has to be taken with interpreting measured dis-
tances in the OCT images of dentin. The OCT system assumes a refractive
index of the imaging medium that is close to that of water (1.33), which
is a reasonable approximation for soft tissues. Dental and bone-like
JOE — Volume 33, Number 11, November 2007
59
Basic Research—Technology
materials have a higher index of refraction, closer to 1.5 (20). As a
result, the distances inside the tooth are approximately 13% shorter
than suggested by the images. Straightforward image processing can
remove this ambiguity. The tooth was placed in a water bath to improve
the refractive index match between imaging medium and dental mate-
rial. A contrast in refractive index causes light to be reflected from the
interface (21), reducing the signal from the tissue. A smaller refractive
index step (achieved by imaging through water, instead of air) leads to
a smaller reflection at the dentin interface and improves image quality.
Furthermore, because clinical application of the OCT system was also
considered, placing the optical fibre in a wet canal is more clinically
relevant. Indeed, one of the big disadvantages of the recently introduced
“endoscope” to endodontic clinical practice (22) is that it requires a
dry environment. The endoscope has a 0.7-mm probe that is inserted
into a dry canal to image the inner anatomy. However, this system is
based on a camera that produces a digital image and not on microscop-
ic-level characterization or light propagation as observed by the OCT.
Furthermore, no penetration of light through the dentinal tubuli is pos-
sible and, hence, no detection of the outer outline of the root. The
smaller diameter and increased flexibility of the OCT probe allows
deeper penetration and easier application in clinical situations.
It is noteworthy that the current experimental setting requires the
catheter to penetrate through the apex, pulling it back through the canal.
Adaptation of the catheter to allow imaging from the fiber’s tip is possible,
which will enable imaging without probing through the apex and thus allow
Ability of Optical Coherence Tomography to Characterize Root Canal Walls 1371
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Figure 4. Oval canals and uncleaned fins at 7 mm from the apex revealed by histology (H) and OCT (O): sample A and sample B.

Figure 5. Cleaned root canals at 7 mm from the apex revealed by histology (H) and OCT (O): sample A and sample B.

1372 Shemesh et al. JOE — Volume 33, Number 11, November 2007

Figure 6. Canal perforation during preparation at 5 mm from the apex revealed by histology (H) and OCT (O).
clinical imaging all the way to the apical part. Another disadvantage of the
current setting is the cost of the OCT catheter. Because these wires are
designed for disposable cardiac use, they are relatively expensive. Nonethe-
less, OCT imaging systems for clinical dental use are under development,
and more affordable versions could be available soon.
In conclusion, this study shows a noninvasive and nondestructive
technique for analyzing the anatomy and cleanliness of root canal walls.
OCT could generate intracanal microscopic images without applying
ionizing radiation and could be used in vivo.
References
1. Wu MK, R’oris A, Barkis D, Wesselink PR. Prevalence and extent of long oval canals
in the apical third. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2000;89:739 – 43.
2. Mauger MJ, Schindler WG, Walker WA 3rd. An evaluation of canal morphology at
different levels of root resection in mandibular incisors. J Endod 1998;24:607–9.
3. Tamse A, Katz A, Pilo R. Furcation groove of buccal root of maxillary first
premolars—a morphometric study. J Endod 2000;26:359 – 63.
4. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science
1991;254:1178 – 81.
5. Regar E, van Beusekom HM, van der Giessen WJ, Serruys PW. Images in cardiovas-
cular medicine. Optical coherence tomography findings at 5-year follow-up after
coronary stent implantation. Circulation 2005;112:345– 6.
6. Pitris C, Jesser C, Boppart SA, et al. Feasibility of optical coherence tomography for
high-resolution imaging of human gastrointestinal tract malignancies. J Gastroen-
terol 2000;35:87–92.
7. Gambichler T, Hyun J, Moussa G, et al. Optical coherence tomography of cutaneous
lupus erythematosus correlates with histopathology. Lupus 2007;16:35– 8.
8. Low AF, Tearney GJ, Bouma BE, Jang IK. Technology insight: optical coherence
tomography— current status and future development. Nat Clin Pract Cardiovasc Med
2006;3:154 – 62.
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9. Schmitt JM. Optical coherence tomography (OCT): a review. IEEE J Sel Top Quantum
Electron 1999;5:1205–15.
10. Colston BW Jr, Everett MJ, Sathyam US, DaSilva LB, Otis LL. Imaging of the oral
cavity using optical coherence tomography. Monogr Oral Sci 2000;17:32–55.
11. Otis LL, Everett MJ, Sathyam US, Colston BW Jr. Optical coherence tomography: a new
imaging technology for dentistry. J Am Dent Assoc 2000;131:511– 4.
12. Amaechi BT, Higham SM, Podoleanu AG, Rogers JA, Jackson DA. Use of optical
coherence tomography for assessment of dental caries: quantitative procedure.
J Oral Rehabil 2001;28:1092–3.
13. Baumgartner A, Dichtl S, Hitzenberger CK, et al. Polarization-sensitive optical coher-
ence tomography of dental tructures. Caries Res 2000;34:59 – 69.
14. Velvart P, Hecker H, Tillinger G. Detection of the apical lesion and the mandibular
canal in conventional radiography and computed tomography. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2001;92:682– 8.
15. Eder A, Kantor M, Nell A, et al. Root canal system in the mesiobuccal root of the
maxillary first molar: an in vitro comparison study of computed tomography and
histology. Dentomaxillofac Radiol 2006;35:175–7.
16. Youssefzadeh S, Gahleitner A, Dorffner R, Bernhart T, Kainberger FM. Dental vertical
root fractures: value of CT in detection. Radiology 1999;210:545–9.
17. Plotino G, Grande NM, Pecci R, Bedini R, Pameijer CH, Somma F. Three-dimensional
imaging using microcomputed tomography for studying tooth macromorphology.
J Am Dent Assoc 2006;137:1555– 61.
18. Jung M, Lommel D, Klimek J. The imaging of root canal obturation using micro-CT.
Int Endod J 2005;38:617–26.
19. Kienle A, Forster FK, Diebolder R, Hibst R. Light propagation in dentin: influence of
microstructure on anisotropy. Phys Med Biol 2003;48:N7–14.
20. Zijp JR, ten Bosch JJ. Theoretical model for the scattering of light by dentin and
comparison with measurements. Appl Opt 1993;32:411–36.
21. Born M, Wolf E. Principles of Optics. Ed 7. Cambridge, UK: Cambridge University
Press; 2002:132– 4.
22. Bahcall JK, Barss JT. Fiberoptic endoscope usage for intracanal visualization. J Endod
2001;27:128 –9.
Ability of Optical Coherence Tomography to Characterize Root Canal Walls 1373

Diagnosis of Vertical Root Fractures with Optical
Coherence Tomography
Hagay Shemesh, DMD,* Gijs van Soest, PhD,† Min-Kai Wu, PhD,* and
Paul R. Wesselink, PhD*
Abstract
The purpose of this experiment was to evaluate the
ability of optical coherence tomography (OCT) to image
vertical root fractures (VRFs). Twenty-five mandibular
premolars were prepared to size 50. Five teeth served
as controls. Group 1 (n ⫽ 10) was treated with ethyl-
enediaminetetraacetic acid and ultrasonic irrigation,
whereas group 2 (n ⫽ 10) received no further treat-
ments. Teeth from groups 1 and 2 were fractured, and
the presence of a fracture line was demonstrated mi-
croscopically. Control group teeth were not subjected
to any force. Teeth were pooled and scanned with an
OCT fiber. The resulting video files were blindly inter-
preted by 2 observers .No fractures were detected in
the control teeth. The overall sensitivity for detection of
VRFs with OCT was 93% for group 1 and 84% group 2,
whereas the specificity was 95% for group 1 and 96%
for group 2. OCT is a promising nondestructive imaging
method for the diagnosis of VRFs. (J Endod 2008;34:
739 –742)
Key Words
Imaging, optical coherence tomography, sensitivity,
specificity, vertical root fracture
From the *Department of Cariology Endodontology Ped-
odontology, Academic Center for Dentistry Amsterdam
(ACTA), Amsterdam, The Netherlands; and the †Department of
Biomedical Engineering, Thorax Center, Erasmus University
Medical Center, Rotterdam, The Netherlands.
Address requests for reprints to Dr Hagay Shemesh, ACTA,
CEP, Louwesweg 1, 1066 EA Amsterdam, The Netherlands.
E-mail address:hshemesh@acta.nl.
0099-2399/$0 - see front matter
Copyright © 2008 by the American Association of
Endodontists.
doi:10.1016/j.joen.2008.03.013
JOE — Volume 34, Number 6, June 2008
62
Basic Research—Technology
V ertical root fracture (VRF) is a considerable threat to the tooth’s prognosis during
and after root canal treatment (1). VRF presents a challenge to the clinician in that
the diagnosis is often difficult and is based on subjective parameters. The current
available methods to clinically diagnose VRF include illumination, x-ray, periodontal
probing, staining, surgical exploration, bite test, direct visual examination, and opera-
tive-microscope examination. All of these have limited success (2– 4). Radiographic
images could reveal VRF only if the x-ray beam is parallel to the fracture line (5), and
thus in many cases radiographic diagnosis is based on other findings like the size and
shape of the periradicular lesion and its location (6).
Lately, alternative diagnostic imaging systems with computed tomography were
suggested (7, 8). Although these systems could visualize VRF, they all use harmful
ionized radiation and present relatively low sensitivity in detection of VRF (7).
Bahcall and Barss (9) suggested a fiberoptic endoscope to image the canal inter-
nally, but images of a VRF were never shown by using this system. Furthermore, the
minimal thickness of the imaging probe used, 0.7 mm, means that the canal should be
enlarged to ISO size 70 – 80. Such a preparation is seldom performed in adult teeth.
Another requirement for the endoscope is a dry canal, which might prove difficult to
achieve in teeth with VRF.
Optical coherence tomography (OCT) is a high-resolution imaging technique that
allows micrometer scale imaging of biologic tissues over small distances (10). It was
introduced in 1991 (11) and uses infrared light waves that reflect off the internal
microstructure within the biologic tissues. It achieves a depth resolution of the order of
10 ␮m and an in-plane resolution similar to the optical microscope. OCT has previously
been shown to be a valuable tool in assessing intracanal anatomy, cleanliness of the
canal after preparation, and even perforations (12).
The purpose of this experiment was to evaluate the ability of an OCT system to
diagnose VRF under different conditions.
Materials and Methods
Twenty-five lower premolars were chosen and accessed coronally with a diamond
bur (FG 173; Horico, Berlin, Germany). The canal opening was enlarged with Gates-
Glidden drills #3 and #4, which were inserted 4 and 3 mm into the canal, respectively.
The canal was instrumented to a size 50 stainless steel K-file (Dentsply Maillefer, Bal-
laigues, Switzerland). Patency with file size 30 was verified. Irrigation with 2% sodium
hypochlorite (NaOCl) by using a 26-gauge needle followed after every instrument, so
that a total of 15 mL solution was used per tooth. The teeth were then divided into 3
groups: In the control group (n ⫽ 5) and group 2 (n ⫽ 10), teeth were flushed with
5 mL of water. In group 1 (n ⫽ 10), passive ultrasonic irrigation (PUI) was performed
(13), and 10 mL of ethylenediaminetetraacetic acid (EDTA) 17% was used to flush the
canals for 1 minute (14). A final flush of tap water followed. After preparation, the
external root surface was inspected under a microscope (Zeiss Stemi SV6; Carl Zeiss,
Gottingen, Germany) to exclude any external defects or cracks. During these proce-
dures, roots were handled with a wet swab around them to keep them in 100% humidity
and to avoid drying. VRF was artificially made immediately after in the 2 experimental
groups; a size D stainless steel finger spreader (Hu-friedy, Leimen, Germany) was cut at
the tip so its length was 12 mm, and it was inserted as far possible into the canal. Vertical
pressure was applied to the spreader with a screw-table until a vertical line appeared on
the outside surface of the root. No forces were applied on the control group teeth. A
Diagnosis of Vertical Root Fractures with OCT 739

Basic Research—Technology
Figure 1. Appearance of VRF through an OCT scan. (A) VRF extends through the root 2.5 mm from the apex on 2 opposing sides of the canal; it appears as a bright
line. The OCT catheter (C) is inside the canal. (B) VRF that has separated the dentin, 8 mm from the apex; the crack can be traced to the external root surface (S),
and dentinal tubules can also be identified (T).
stereomicroscope (Zeiss Stemi SV6; Carl Zeiss) with a cold light source
(KL 2500 LCD; Carl Zeiss) was used to inspect the roots. Pictures were
taken with a camera (Axio cam; Carl Zeiss) at a magnification of ⫻12 to
determine the location of the fracture line along the external surface of
the root. All teeth were pooled and scanned with an M2-CV OCT system
(LightLab Imaging Inc, Westford, MA) in combination with an Im-
ageWire 2 catheter that has a diameter of 0.3 mm at its thinnest part,
with a “pullback” technique (12) starting at the apex. The pullback
speed was 1 mm/second, and video files were generated at a rate of 10
frames per second. Each frame consisted of 312 lines, with a scan depth
of 3.3 mm in water. Distances were measured from the apex tip.
The presence of VRF in the OCT scan was noted at 3, 6, and 9 mm
from the apex by 2 observers. The same observers reviewed the same
scans again after an interval of 4 weeks. Interobserver and intraobserver
agreement was assessed on the basis of the proportion of agreement and
the values of the kappa coefficient. The sensitivity and specificity of the
OCT scan in diagnosing VRF were calculated as well as the 95% confi-
dence intervals for both groups.
Results
Several features were observed and illustrated in Figs. 1–3. A
bright line extending from the canal (Fig. 1A) and 2 bright lines with a
void between them separating the dentin (Fig. 1B) were both consid-
ered a VRF. Fig. 2 compares a microscope photograph with registered
OCT imagery, whereas Fig. 3 demonstrates how cracks can propagate
inside the dentin.
The specificity and sensitivity are shown in Table 1 and demon-
strate high to very high values of the OCT system in diagnosing VRF.
For interobserver agreement, the mean kappa values were 0.9 for
group 1 and 0.7 for group 2, showing very good to excellent reliability.
For intraobserver agreement, the mean kappa value of 0.9 for both
observers shows very high reproducibility. Because the values of sensi-
tivity and specificity of one group fell inside the 95% confidence interval
of the other group, we concluded that the groups did not differ in regard
to these parameters.
740 Shemesh et al.
63
Discussion
According to the current results, more information on the pres-
ence of VRF could become available with the use of OCT. No statistical
significance for the difference between sensitivity and specificity was
demonstrated between the 2 groups. The higher agreement between the
observers after the ultrasonic cleaning and use of EDTA could be a result
of better light propagation through dentin when the smear layer is
absent or cleaner canal devoid of debris. The penetration of light
through the dentin tubules allows imaging of structures beyond the
canal walls and in some cases up to the cementum layer (12). If the
tubules are blocked, less light could penetrate, giving rise to lower-
quality imaging beyond the canal walls or in areas where the dentin is
thicker.
Two factors might contribute to the development of VRF during the
endodontic treatment: First, excessive canal shaping especially in teeth
with curved roots or oval canals (15, 16) and excessive removal of tooth
structure contribute to the overall weakening of the tooth, which pro-
motes a higher incidence of VRF (17). Second, excessive hand pressure
during lateral and vertical compaction of filling material can result in
development of VRF (18).The detection of these fractures is usually not
made during treatment and is diagnosed only years later (4, 19), lead-
ing to bone loss, malfunction of the involved tooth or area, and pain. The
immediate identification of a fracture during treatment thus has great
clinical value.
There is still confusion in the literature regarding the definition of
various terms used to describe different fracture types. Split teeth, ver-
tical fracture, complete, incomplete, primary, secondary fractures,
crack, craze line, and other terms are sometimes used interchangeably.
Furthermore, a craze line might propagate into a fracture in certain
conditions or along different levels of the same root (20, 21) (Fig.3).
Although some authors try to define the different terms (4, 22), many
others avoid the definitions (16, 20, 23) or only refer to the clinical or
diagnostic aspects (24). Still, a VRF is usually referred to as a “through
and through” crack with a connection between the canal and the outer
root surface or periodontal ligament (21, 25). Because the current
study investigated teeth in which a clear fracture line was visible on the
JOE — Volume 34, Number 6, June 2008
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Basic Research—Technology

Figure 2. Root sample (A) where the fracture is marked by an arrow, and the corresponding OCT images at 9 mm (B), 6 mm (C), and 3 mm (D) from the apex. OCT
visualization of the VRF is marked by arrows.

outer surface of the root and the force applied originated from within
the canal, a “through and through” defect was assumed. Nevertheless,
because OCT is an in situ imaging technique, cracks are always imaged
at the site where they originate, and even if they do not propagate all the
way to the exterior root wall, they could, in principle, still be imaged
with OCT. Although this class of defects was not included in this study,
they might be important for future in vitro work.
It is worth noting that there is not yet a commercially available OCT
system for dental use. The OCT unit used in this study is designed for intra-
coronary imaging in atherosclerotic plaque diagnosis and is commercially
available for clinical use in cardiac catheterization laboratories. Lantis Laser
Inc (Denville, NJ) is currently developing an OCT dental system for lateral
scanning, which will allow caries detection and periodontal examination
(26). We believe that a specially designed endodontic probe will improve
visualization for conventional endodontic treatment and could lead to better
decision making and elevated standard of care.
In conclusion, OCT represents a powerful tool for evaluating VRF
and has the potential to both identify VRF and detect its specific location

Figure 3. Sequence of video frames from a pullback, recorded approximately 4.5 mm from the apex, spaced 0.1 mm apart. A crack (white arrow) that originates in
the canal (top left frame) can be followed as it propagates in the dentin.

JOE — Volume 34, Number 6, June 2008 Diagnosis of Vertical Root Fractures with OCT 741
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TABLE 1. Sensitivity and Specificity of Detecting VRF with OCT (%) 10. Brezinski ME. Optical coherence tomography-principles and applications. Burling-
ton, MA: Academy Press-Elsevier, 2006.
Group 1 Group 2 11. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science
Observer A 1991;254:1178 – 81.
Sensitivity 95 95 12. Shemesh H, van Soest G, Wu MK, van der Sluis LW, Wesselink PR. The ability of optical
Specificity 100 100 coherence tomography to characterize the root canal walls. J Endod
Observer B 2007;33:1369 –73.
Sensitivity 91 74 13. van der Sluis LW, Wu MK, Wesselink PR. The efficacy of ultrasonic irrigation to
Specificity 89 91 remove artificially placed dentine debris from human root canals prepared using
Overall instruments of varying taper. Int Endod J. 2005;38:764 – 8.
Sensitivity 93 85 14. Çalt S, Serper A. Time dependent effects of EDTA on dentin structures. J Endod
Specificity 95 96 2002;28:17–9.
15. Frank RJ. Endodontic mishaps. In: Ingle, JI Backland, eds. LK Endodontics. 4th ed.
VRF, vertical root fracture; OCT, optical coherence tomography. Hamilton, Ontario, Canada: BC Decker, 2002.
16. Wu MK, van der Sluis LW, Wesselink PR. Comparison of mandibular premolars and
along the root. In vivo studies are needed to substantiate the clinical canines with respect to their resistance to vertical root fracture. J Dent
usage of OCT during endodontic treatment. 2004;32:265– 8.
17. Nissan J, Zukerman O, Rosenfelder S, Barnea E, Shifman A. Effect of endodontic
access type on the resistance to fracture of maxillary incisors. Quintessence Int
References 2007;38:e364 –7.
1. Tamse A, Fuss Z, Lustig J, Kaplavi J. An evaluation of endodontically treated vertically 18. Obermayr G, Walton RE, Leary JM, Krell KV. Vertical root fracture and relative defor-
fractured teeth. J Endod 1999;25:506 – 8. mation during obturation and post cementation. J Prosthet Dent 1991;66:181–7.
2. Morfis AS. Vertical root fractures. Oral Surg Oral Med Oral Pathol 1990;69:631–5. 19. Fuss Z, Lustig J, Katz A, Tamse A. An evaluation of endodontically treated vertical root
3. Fuss Z, Lustig J, Tamse A. Prevalence of vertical root fractures in extracted endodon- fractured teeth: impact of operative procedures. J Endod 2001;27:46 – 8.
tically treated teeth. Int Endod J 1999;32:283– 6. 20. Lertchirakarn V, Palamara JE, Messer HH. Load and strain during lateral condensa-
4. Paul RA, Tamse A, Rosenberg E. Cracked and broken teeth-definitions, differential tion and vertical root fracture. J Endod 1999;25:99 –104.
diagnosis and treatment. Refuat Hapeh Vehashinayim 2007;24:7–12. 21. Wilcox LR, Roskelley C, Sutton T. The relationship of root canal enlargement to
5. Rud J, Omnell KA. Root fractures due to corrosion: diagnostic aspects. Scand J Dent finger-spreader induced vertical root fracture. J Endod 1997;23:533– 4.
Res 1970;78:397– 403. 22. Sutherland JK, Teplitsky PE, Moulding MB. Endodontic access of all-ceramic crowns.
6. Tamse A, Fuss Z, Lustig J, Ganor Y, Kaffe I. Radiographic features of vertically frac- J Prosthet Dent 1989;61:146 –9.
tured, endodontically treated maxillary premolars. Oral Surg Oral Med Oral Pathol 23. Nair MK, Grondahl HG, Webber RL, Nair UP, Wallace JA. Effect of iterative restoration
Oral Radiol Endod 1999;88:348 –52. on the detection of artificially induced vertical radicular fractures by Tuned Aperture
7. Nair MK, Nair UDP, Grondahl HG, Webber RL, Wallace JA. Detection of artificially Computed Tomography. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
induced vertical radicular fractures using tuned aperture computed tomography. Eur 2003;96:118 –25.
J Oral Sci 2001;109:375–9. 24. Meister F Jr, Lommel TJ, Gerstein H. Diagnosis and possible causes of vertical root
8. Hannig C, Dullin C, Hulsmann M, Heidrich G. Three-dimensional, non-destructive fractures. Oral Surg Oral Med Oral Pathol 1980;49:243–53.
visualization of vertical root fractures using flat panel volume detector computer 25. Walton RE, Michelich RJ, Smith GN. The histopathogenesis of vertical root fractures.
tomography: an ex vivo in vitro case report. Int Endod J 2005; 38:904 –13. J Endod 1984;10:48 –56.
9. Bahcall J, Barss J. Orascopic visualization technique for conventional and surgical 26. Otis LL, Everett MJ, Sathyam US, Colston BW Jr. Optical coherence tomography: a new
endodontics. Int Endod J 2003;36:441–7. imaging technology for dentistry. J Am Dent Assoc. 2000;131:511– 4.

742 Shemesh et al. JOE — Volume 34, Number 6, June 2008

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Chapter IV
Inspection of dentinal defects in the root canal wall

This chapter is enhanced by additional high-resolution images available on

www.shemesh.nl

1. The effects of canal preparation and filling on the incidence of dentinal defects. Shemesh H,
Bier CA. Wu MK, Tanomaru-Filho M, Wesselink PR. Int Endod J 2009 Mar;42(3);208-13.
2. The ability of different Ni-Ti rotary instruments to induce dentinal damage during canal
preparation . Bier CA. Shemesh H, Tanomaru-Filho M, Wesselink PR, Wu MK, J Endod. 2009
Feb;35 (2):236-8.
68

The effects of canal preparation and filling on the
incidence of dentinal defects
H. Shemesh1, C. A. S. Bier2, M.-K. Wu1, M. Tanomaru-Filho2 & P. R. Wesselink1
1
Department of Cariology, Endodontology, Pedodontology, Academic Centre of Dentistry Amsterdam, Amsterdam, The
Netherlands; and 2Department of Restorative Dentistry, Araraquara Dental School, São Paulo State University, UNESP,
Araraquara, SP, Brazil
Abstract
Shemesh H, Bier CAS, Wu M-K, Tanomaru-Filho M,
Wesselink PR. The effects of canal preparation and filling on
the incidence of dentinal defects. International Endodontic
Journal, 42, 208–213, 2009.
Aim To evaluate ex vivo the incidence of defects in
root dentine before and after root canal preparation
and filling.
Methodology Eighty extracted mandibular premo-
lars were divided equally in four groups. Group 1 was
left unprepared. All other root canals were prepared
using Gates Glidden drills and System GT files up to size-
40, 0.06 taper at the working length. Group 2 was not
filled while the canals of the other groups were filled
with gutta-percha and AH26, either with a master cone
and passive insertion of secondary gutta percha points
(group 3) or lateral compaction (group 4). Roots were
then sectioned horizontally 3, 6, and 9 mm from the
Introduction
Vertical root fracture (VRF) is a clinical complication
that may be associated with root canal treatment and
lead to extraction (Tamse et al. 1999). Endodontic
procedures might contribute to the development of VRF
as well as other localized defects such as craze lines or
incomplete cracks in root dentine (Onnink et al. 1994,
Sathorn et al. 2005). These localized defects may have
the potential to develop into fractures (Wilcox et al.
Correspondence: H. Shemesh, Department of Cariology, En-
dodontology, Pedodontology, Academic Centre of Dentistry
Amsterdam (ACTA), Louwesweg 1 1066 EA Amsterdam, The
Netherlands (Tel.: +31 20 5188651; fax: +31 20 6692881;
e-mail:hshemesh@acta.nl).
208 International Endodontic Journal, 42, 208–213, 2009
69
doi:10.1111/j.1365-2591.2008.01502.x
apex and observed under a microscope. The presence of
dentinal defects (fractures, craze lines or incomplete
cracks) was noted and the differences between the
groups were analysed with the Fisher’s exact test.
Results No defects were observed in the roots with
unprepared canals. The overall difference between the
groups was significant (P < 0.05). Canal preparation
alone created significantly more defects than unpre-
pared canals (P < 0.05). The total number of defects
after lateral compaction was significantly larger than
after noncompaction canal filling.
Conclusion Root canal preparation and filling of
extracted teeth created dentine defects such as frac-
tures, craze lines and incomplete cracks.
Keywords: craze lines, dentine, obturation, prepara-
tion, vertical root fracture.
Received 12 August 2008; accepted 14 October 2008
1997, Shemesh et al. 2008) and should therefore be
prevented. Several factors may be responsible for the
formation of dentinal defects: instrumentation and root
filling (Onnink et al. 1994), high concentration of
hypochlorite (Sim et al. 2001), tooth anatomy (Wu
et al. 2004) and post-placement (Kishen 2006).
Lateral compaction of gutta-percha is widely used to
fill the root canal system and was reported previously to
be associated with an increased risk of VRF (Meister
et al. 1980, Wilcox et al. 1997). Spreader design and
applied forces were suggested as contributing factors to
the appearance of VRF during lateral compaction (Pitts
et al. 1983, Dang & Walton 1989). However, labora-
tory stress distribution studies consistently conclude
that the pressure applied during lateral compaction is
insufficient to cause VRF (Dalat & Spångberg 1994,
ª 2009 International Endodontic Journal

Lertchirakarn et al. 1999). Thus, it remains unclear
whether lateral compaction can cause VRF. As an
alternative, several techniques where no compaction
forces are used have been proposed and shown to
produce an apical seal similar to that of lateral
compaction when used in conjunction with dimen-
sionally stable sealers (Dalat & Spångberg 1994,
Tidswell et al. 1994, Ozok et al. 2008).
The purpose of this study was to compare the
incidence of fractures and other dentinal defects before
and after canal preparation and filling either with
lateral compaction of gutta-percha or a technique
where no compaction forces were used.
Materials and methods
Eighty extracted mandibular premolar teeth were
selected and stored in purified filtered water. Proximal
radiographs were taken to verify the presence of a
single canal. The clinical crowns of all teeth were
removed using an Isomet 11-1180 low-speed saw
(Buehler Ltd, Evanston, IL, USA), leaving roots approx-
imately 16 mm in length. All roots were observed
under 8· magnifications in a stereomicroscope (Zeiss
Stemi SV6; Carl Zeiss, Jena, Germany) to exclude
cracks. Twenty root canals were left unprepared (group
1) and the remaining 60 teeth (groups 2, 3 and 4) were
subjected to the procedures described below. All roots
were kept moist in purified filtered water throughout
the experimental procedures in order to prevent dehy-
dration.
Cleaning and shaping
Canal patency was established with a size-20 K-Flexo
File (Dentsply Maillefer, Ballaigues, Switzerland). The
canal opening was enlarged with Gates Glidden drills 3
(size-90) and 4 (size-110) to a depth of 4 and 3 mm
from the coronal orifice respectively. Canal preparation
followed with System GT rotary files (Dentsply Maille-
fer) and a torque-control motor (Technika, Pistoia,
Italy) at 300 rpm using the crown-down technique
with series 40 and 30 files, ending with a size-40, 0.06
taper instrument to 1 mm short of the apical foramen.
Each canal was irrigated with a freshly prepared 2%
solution of sodium hypochlorite (NaOCl) between each
instrument, using a syringe and a 27-gauge needle.
Twelve millilitres of NaOCl solution was used for each
root canal. After completion of instrumentation, pas-
sive ultrasonic irrigation was performed using an
ultrasonic file size-15 (Satelec, Merignac Cedex, France)
ª 2009 International Endodontic Journal
70
Shemesh et al. Canal preparation and filling
in order to activate the irrigation solution and clean the
canals (van der Sluis et al. 2007). After completion of
the procedure, canals were rinsed with 2 mL distilled
water. The prepared roots were divided into three
experimental groups of 20 roots each (groups 2, 3, and
4). Group 2 remained without root filling. Group 3 and
4 were filled with gutta-percha and AH26, using two
different techniques according to the protocol described
below.
Canal filling
Canals were dried using paper point size-40. AH 26
(Dentsply De trey, Konstanz, Germany) was mixed
according to the manufacturer’s instructions and
introduced into the canal on two occasions, using a
lentulo spiral, for 5 s each (Hall et al. 1996) rotating at
400 rpm to 1 mm short of the working length.
Standardized size-40 gutta-percha cones (Henry
Schein, Mexico City, Mexico), with a 0.02 taper, were
coated with sealer, and placed into the root canal to the
working length. In group 3, two additional size-25
cones were placed to a depth where resistance was met
without use of a spreader (Souza et al. 2008). Group 4
was sealed with the lateral compaction technique using
a size C spreader (D1 diameter 0.3 mm, 0.04 taper)
(Dentsply Maillefer) and size-25 standardized gutta-
percha cones. The force applied to the spreader was
controlled using a digital scale and kept at a maximum
of 2 kg. A polyether impression material (President,
Coltene, Altstätten, Switzerland) was used around the
tooth during the filling procedures in order to mimic
the mechanisms of stress distribution. The coronal
gutta-percha was removed with a flame-heated plugger
(0.5-mm diameter, Dentsply Maillefer), and the impres-
sion material was removed. Roots were stored for
1 week at 37 C and 100% humidity to allow the
sealers to set.
Examination of roots
All roots were sectioned horizontally at 3, 6 and 9 mm
from the apex with a low-speed saw under water
cooling (Leica SP1600, Wetzlar, Germany). Slices were
then viewed through a stereomicroscope (Zeiss Stemi
SV6; Carl Zeiss) using a cold light source (KL 2500
LCD; Carl Zeiss). Pictures were taken with a camera
(Axio cam; Carl Zeiss) at a magnification of X12. The
dentine was inspected and defects were noted. Defects
were categorized as: ‘no defect’, ‘fracture’ and ‘all other
defects’ (Fig. 1). ‘No defect’ was defined as root dentine
International Endodontic Journal, 42, 208–213, 2009 209

Canal preparation and filling Shemesh et al.
(a)
(c1)
Figure 1 Classification of the different dentinal defects. A – ‘no defects’, B – ‘VRF’, C – ‘other defects’ (C1 = ‘craze line’,
C2 = ‘incomplete crack’).
devoid of any lines or cracks where both the external
surface of the root and the internal root canal wall had
no defects (Fig. 1a). ‘Fracture’ was defined as a line
extending from the root canal space to the outer
surface of the root (Wilcox et al. 1997) (Fig. 1b). ‘Other
defects’ were defined as all other lines observed that did
not extend from the root canal to the outer root
surface. For example, a craze line – line extending from
the outer surface into the dentine that did not reach the
canal lumen (Wilcox et al. 1997) (Fig. 1.C1), or a
partial crack extending from the canal wall into the
dentine without reaching the outer surface of the root
(Fig. 1.C2).
Statistical analysis
Fisher’s exact test was performed to compare the
incidence of fractures and other defects between the
four groups using the SPSS/PC version 15 (SPSS Inc.,
210 International Endodontic Journal, 42, 208–213, 2009
71
(b)
(c2)
Chicago, IL, USA). The level of significance was set at
a = 0.05.
Results
Figures 2 and 3 summarize the results. The groups
were significantly different from each other (P < 0.05).
The unprepared canals (group 1) had no defects. When
considering the overall appearance of defects, the
lateral compaction group (group 4) demonstrated
significantly more defects than all other groups
(P < 0.05), while roots with prepared canals (group
2) had significantly more defects than teeth with
unprepared canals (group 1) (P < 0.05). When con-
sidering only fractures, the lateral compaction (group
4) had significantly more defects than the unprepared
group (group 1) (P < 0.05) but not significantly more
than the canal preparation-only group (group 2)
(P > 0.05).
ª 2009 International Endodontic Journal
 72

Shemesh et al. Canal preparation and filling

Fracture present (with/without other defects) 1999) that ex vivo methods often could not clarify. It
Other defect (fracture not present)
may be hypothesized that craze lines and incomplete
12
cracks might propagate and develop into fractures after
10 completion of endodontic procedures, following addi-
tional treatments such as post-space preparation or re-
Number of teeth

8
treatment (Wilcox et al. 1997), or simply by forces
6
transferred to the root during masticatory function and
occlusal loading (Assif et al. 2003).
4 Previous ex vivo experiments studying the influence
of endodontic procedures on root dentine mainly used
2
one or other of the following methodologies: resistance
0 to fracture, stress distribution measurements or obser-
No Only No Lateral vations of the presence of defects in tooth sections
preparation preparation compaction compaction
(Obermayr et al. 1991, Onnink et al. 1994, Saw &
Figure 2 Number of teeth presenting fractures or other Messer 1995, Wilcox et al. 1997, Lertchirakarn et al.
defects. Groups with the same letter denote no statistical 1999, Mayhew et al. 2000, Ribeiro et al. 2008).
significance between them (P = 0.05). Resistance to fracture is frequently measured to
assess the weakening of the root after different proce-
dures. This method differs from the one used in the
All defects
18 current study in that it applies an external force until
16 the root fractures (Ribeiro et al. 2008), while in the
14 current experiment, the influence of various procedures
on the root canal walls was directly observed and no
No. of teeth

12
10
8 fracture provides no information on the incidence of
6 dentinal defects other than VRF.
4 Stress distribution studies record stress transmission
2
0
No Only No
preparation preparation compaction
Figure 3 Number of teeth presenting a defect (either VRF or
other defects or both). Letters denote statistical resemblance.
Groups with the same letter have no statistical difference
between them (P = 0.05).
Discussion
The sectioning method used in the current study
allowed the evaluation of the effect of root canal
treatment procedures on the root dentine by direct
inspection of the root canal wall, observing not only
VRF but also dentinal defects such as craze lines and
incomplete cracks. When considering all the defects,
the lateral compaction group had significantly more
defects than all other groups (P < 0.05). Although no
definitive conclusion can be made of the clinical
consequences of the dentinal defects developed under
these ex vivo experimental conditions, these findings
may contribute to the understanding of long-term
clinical observations (Meister et al. 1980, Tamse et al.
external forces were applied. Furthermore, resistance to
to dentine during different procedures using strain
Lateral gauges (Obermayr et al. 1991, Saw & Messer 1995,
compaction Lertchirakarn et al. 1999), or a photoelastic material
(Mayhew et al. 2000). These studies have shown that
the force needed to fracture a root is much higher than
that formed during lateral compaction. The current
study found relatively few VRF in all groups, confirm-
ing this conclusion.
The observation that many of the defects did not
connect with the pulp space, and were located in places
away from direct contact with intra-canal instruments
is baffling. Wilcox et al. (1997) speculated that the
stresses generated from inside the root canal are
transmitted through the root to the surface where they
overcome the bonds holding the dentine together. In
the current study, craze lines were not seen in
unprepared teeth proving that they were caused by
the preparation and filling procedures. Onnink et al.
(1994) claimed that a fracture contained within the
dentine in one section could communicate with the
canal space in an adjacent section. This supposition
was recently supported by nondestructive observations
of VRF induced in extracted teeth and viewed with
optical coherence tomography (Shemesh et al. 2008).

ª 2009 International Endodontic Journal International Endodontic Journal, 42, 208–213, 2009 211

Canal preparation and filling Shemesh et al.
In a series of scans depicting a dentinal defect in one
root, an incomplete crack is shown to originate at the
root canal wall but later propagates into dentine,
demonstrating no communication with the root canal
lumen at subsequent sections. In another recent
publication, Soros et al. (2008) claimed that VRF is
mainly a matter of crack propagation and should not
be considered as an instant phenomenon.
In the current experiment, the roots were sur-
rounded with an impression material during filling in
an attempt to mimic the bony socket that may change
the force distribution around the tooth when external
forces are used, namely lateral compaction (Lertchira-
karn et al. 1999). However, the clinical situation is
more complex because of the presence of the periodon-
tal ligament that could further influence the distribu-
tion of forces. While some studies did not attempt to
imitate bone or periodontal ligament (Onnink et al.
1994, Ribeiro et al. 2008), others have made various
attempts to do so. For example, Wilcox et al. (1997)
used a single layer of aluminium foil and then
embedded the teeth in acrylic resin, while Lertchira-
karn et al. (1999) covered the roots with silicone paste.
However, it seems that these attempts are insufficient to
mimic the anatomical and biological aspects of tooth
structure (Saw & Messer 1995) and could contribute to
the introduction of artificial changes in force distribu-
tion themselves. Soros et al. (2008) stated that elasto-
meric materials are incapable of withstanding
compaction forces in the way that the natural ligament
does and that they may collapse under pressure. In
agreement with the current findings, they highlight the
significance of crack initiation and propagation in the
evaluation of VRF formation rather than the actual
appearance of a VRF as a result of a specific stress
application.
The forces of extraction may also contribute to the
observation of incomplete fractures. However, since
most of the premolars selected had calculus and
staining but no deep carious lesions, an assumption
could be made that they were extracted for periodontal
reasons with minimal trauma (Onnink et al. 1994).
This is further confirmed by the finding that unpre-
pared root canals had no defects. The Storage medium
used to keep the teeth was purified filtered water. This
medium was previously recommended for investiga-
tions of human dentine (Strawn et al. 1996) as it
causes the smallest changes in dentine over time.
System GT instruments have lands, a U-shaped
cross section and a noncutting tip. The lands make
the instruments passive and prevent canal
212 International Endodontic Journal, 42, 208–213, 2009
73
transportation (Schirrmeister et al. 2006). This design
increases the contact area with the canal wall as
compared to cutting instruments devoid of lands such
as the ProTaper system, and might increase friction
and torque and thereby fracture risk (Blum et al.
1999). This may explain the significantly larger
number of defects in the preparation-only group
(Fig. 2). It will be interesting to study whether similar
defects will be present after preparation with instru-
ments having different designs. Furthermore, Gates
Glidden drills were used during the preparation
procedure, as they were shown to improve working-
safety, avoiding apical transportation and reducing
working time (Bergmans et al. 2002). They, too,
might be a contributing factor to the formation of
root defects during preparation, through the action of
the burs on dentine, and the excess removal of root
structure resulting in weakening of the root (Pilo
et al. 1998, Kuttler et al. 2004).
Little is known on the effects of ultrasonic irrigation
on the root canal walls. Further studies should be
conducted on the effect of these instruments on root
dentine.
Conclusion
Under the conditions of this ex vivo study, the use of
System GT rotary files and Gates Glidden drills to
prepare canals resulted in dentinal defects. The use of a
passive compaction technique to fill the canals of
extracted teeth significantly reduced the incidence of
defects compared to lateral compaction. Clinicians
should be aware of the potential damage risks during
canal enlargement, preparation and some root filling
procedures.
Acknowledgements
Carlos Alexandre Souza Bier was supported by
the Brazilian Agency Capes, Grants No. 2094/07.
The authors wish to thank Irene H.A. Aartman from
the Department of Social Dentistry and Behavioral
Sciences, ACTA, Amsterdam, the Netherlands for her
help with the statistical interpretation of the results.
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International Endodontic Journal, 42, 208–213, 2009 213

Basic Research—Technology
The Ability of Different Nickel-Titanium Rotary Instruments
To Induce Dentinal Damage During Canal Preparation
Carlos Alexandre Souza Bier, DMD,* Hagay Shemesh, DMD,†
Mário Tanomaru-Filho, DDS, MSc, PhD,* Paul R. Wesselink, DDS, PhD,† and
Min-Kai Wu, MD, MSD, PhD†
Abstract
The purpose of this study was to compare the incidence
of dentinal defects (fractures and craze lines) after
canal preparation with different nickel-titanium rotary
files. Two hundred sixty mandibular premolars were
selected. Forty teeth were left unprepared (n ⫽ 40). The
other teeth were prepared either with manual Flexofiles
(n ⫽ 20) or with different rotary files systems: ProTaper
(Dentsply-Maillefer, Ballaigues, Switzerland), ProFile
(Dentsply-Maillefer), SystemGT (Dentsply-Maillefer), or
S-ApeX (FKG Dentaire, La Chaux-de-Fonds, Switzer-
land) (n ⫽ 50 each). Roots were then sectioned 3, 6,
and 9 mm from the apex and observed under a micro-
scope. The presence of dentinal defects was noted.
There was a significant difference in the appearance of
defects between the groups (p ⬍ 0.05). No defects
were found in the unprepared roots and those prepared
with hand files and S-ApeX. ProTaper, ProFile, and GT
preparations resulted in dentinal defects in 16%, 8%,
and 4% of teeth, respectively. Some endodontic prep-
aration methods might damage the root and induce
dentinal defects. (J Endod 2009;35:236 –238)
Key Words
Craze lines, dentinal defects, nickel-titanium instru-
ments, root canal preparation
From the *Department of Restorative Dentistry, Arara-
quara Dental School, São Paulo State University, UNESP,
Araraquara, SP, Brazil; and †Department of Cariology, Endod-
ontology, Pedodontology Academic Centre of Dentistry Am-
sterdam, the Netherlands.
Carlos Alexandre Souza Bier was supported by the Brazil-
ian agency Capes, grant no. 2094/07.
Address requests for reprints to Dr Hagay Shemesh, ACTA,
CEP, Louwesweg 1, 1066 EA Amsterdam, The Netherlands.
E-mail address: hshemesh@acta.nl.
0099-2399/$0 - see front matter
Copyright © 2008 American Association of Endodontists.
doi:10.1016/j.joen.2008.10.021
236 Souza Bier et al.
75
T he goals of endodontic instrumentation are to completely remove microorganisms,
debris, and tissue by enlarging the canal diameter and create a canal form that allows
a proper seal. Complications such as transportation, ledge formation, and perforation
are well documented (1, 2). However, preparation procedures could also damage the
root dentin resulting in fractures or craze lines (3). Wilcox et al. (3) examined teeth
after hand preparation and lateral compaction of gutta-percha and sealer. Teeth not
presenting vertical root fractures (VRFs) were retreated by removing the root canal
filling and further enlarging the canal. No VRFs were observed after the initial prepara-
tion, whereas numerous fractures had occurred after further enlargement. All roots that
eventually fractured had previously shown craze lines. They concluded that the more
dentin removed the more chance for a fracture. The observation that all teeth presented
craze lines led the authors to speculate that these could later propagate into VRF if the
tooth is subjected to repeated stresses from endodontic or restorative procedures.
Indeed, evidence of recent years concentrate on the findings that VRFs are probably
caused by a propagation of smaller, less pronounced defects and not by the force
practiced during preparation or obturation (4, 5).
An increasing number of rotary nickel-titanium (NiTi) file systems have been
marketed by various manufacturers. These systems differ from one another in the
design of the cutting blades, body taper, and tip configuration. Despite the obvious
clinical advantages of these techniques over hand instrumentation, the influence of the
design of the cutting blades is still controversial (6, 7) and could generate increased
friction and stresses within the root canal (8). Rotary instrumentation requires less time
to prepare canals as compared with hand instrumentation but result in significantly
more rotations of the instruments inside the canal (9). This may cause more friction
between the files and the canal walls.
The goal of this ex vivo study was to compare the damage observed in the root
dentin after endodontic preparations with different NiTi rotary files systems.
Materials and Methods
Two hundred sixty extracted lower premolars were selected and stored in purified
filtered water. This storage medium causes the smallest changes in dentin over time and
was previously recommended for investigations of human dentin (10). The coronal
portion of all teeth was removed by using an Isomet 11–1180 low-speed saw (Buehler
Ltd, Evanston, IL) with water cooling, leaving roots approximately 16 mm in length. All
roots were observed with a stereomicroscope under 12⫻ magnification (Zeiss Stemi
SV6, Jena, Germany) to exclude cracks. Forty teeth were left unprepared and served as
control group A. Twenty teeth were prepared with hand files (K-Flexofiles; Dentsply-
Maillefer, Ballaigues, Switzerland) using balance force crown down to file 40 and
step-back 1-mm increments with Flexofiles #45 to 80 (Dentsply-Maillefer), resulting in
a preparation with a taper of about 0.05. These teeth formed control group B. The
remaining 200 teeth were randomly divided into four groups of 50 teeth each. Canal
patency was established with a #20 K-Flexofile (Dentsply Maillefer). Thereafter, canal
preparation followed with rotary files according to the relevant group (1– 4) using a
torque-control motor (ATR; Technika, Pistoia, Italy) and at the torque and speed
recommended by the manufacturer for the specific system used.
In group 1, the following sequence of ProTaper rotary files (Dentsply Maillefer)
was used at 300 rpm to prepare the canals; an SX file was used to enlarge the coronal
JOE — Volume 35, Number 2, February 2009

portion of the canal, and then all files were used to working length: S1¡
S2¡ F1¡ F2¡ F3. The last file used was F4, which corresponds to file
40 with a taper 0.06 at the apical area.
In group 2, canal preparation with SystemGT files (Dentsply
Maillefer) at 300 rpm using a crown-down technique. Files 50/0.12 and
35/0.12 were used to enlarge the canal opening, and GT series 30 and
40 taper 0.1, 0.08, 0.06, 0.04 were used consequently to prepare the
canal till GT file 40/0.06 reached 1 mm short of the apical foramen.
In group 3, ProFile rotary NiTi files (Dentsply Maillefer) at 300
rpm were used in a crown-down sequence. ProFile Orifice shapers 2
and 3 were used to preflare the canal opening, and the preparation
ended when a 40/0.06 file reached 1 mm short of the apex.
In group 4, S-ApeX rotary files (FKG Dentaire, La Chaux-de-Fonds,
Switzerland) sizes 15 to 60 were used at a speed of 600 rpm. The
manufacturer’s instructions were followed using the full sequence of 6
files available at the working length. This system has an inverted taper,
and the preparation may result in a nontapered form.
In all experimental and control groups, each canal was irrigated
with a freshly prepared 2% solution of sodium hypochlorite (NaOCl)
between each instrument during the preparation procedure using a
syringe and a 27-G needle. Twelve milliliters of NaOCl solution was used
for each root. After the completion of instrumentation, passive ultra-
sonic irrigation was performed with an Irrisafe 20/21 file (Satelec,
Merinac Cedex, France) in order to efficiently clean the canals and
remove any debris still present (11). After completion of the procedure,
canals were rinsed with 2 mL distilled water. All roots were kept moist
in distilled water throughout the experimental procedures.
Sectioning and Microscopic Observations
All roots were cut horizontally at 3, 6, and 9 mm from the apex with
a low-speed saw under water cooling (Leica SP1600, Wetzlar, Ger-
many). Slices were then viewed through a stereomicroscope (Zeiss
Stemi SV6, Carl Zeiss, Jena, Germany) using a cold light source (KL
2500 LCD, Carl Zeiss). The appearance of dentinal defects was regis-
tered by the author (CASB), after which pictures were taken with a
camera (Axio Cam, Carl Zeiss) at a magnification of ⫻12. The pictures
were then blindly inspected by the second author (HS). In four cases out
of a total of 780 slices (0.5%), there was a discrepancy in the observa-
tions, and a consensus was reached after inspecting the slices again. In
order to avoid confusing definitions of root fractures, three distin-
guished categories were made: “no defect,” “fracture,” and “other de-
fects” (12). “No defect” was defined as root dentin devoid of any lines
or cracks where both the external surface of the root and the internal
root canal wall did not present any evident defects. “Fracture” was
defined as a line extending from the root canal space all the way to the
outer surface of the root (3). “Other defects” were defined as all other
lines observed that did not seem to extend from the root canal to the
outer root surface (eg, a craze line, a line extending from the outer
surface into the dentin but does not reach the canal lumen (3), or a
partial crack, a line extending from the canal walls into the dentin
without reaching the outer surface).
Statistical Analysis
Roots were classified as “defected” if at least one of three sections
showed either a craze line, partial crack, or a fracture. Results were
expressed as the number and percentage of defected roots in each
group. A chi-square test was performed to compare the appearance
of defected roots between the experimental groups by using the
SPSS/PC version 15 (SPSS Inc, Chicago, IL).The level of significance
was set at ␣ ⫽ 0.05.
JOE — Volume 35, Number 2, February 2009
76
Basic Research—Technology
Figure 1. The number of teeth and percentage showing defect.
Results
No complete fractures were observed in any of the samples. Con-
trol groups A (unprepared canals) and B (hand preparation) showed
no defected roots. Craze lines and partial cracks (“other defects”) were
found in the SystemGT, ProFile, and ProTaper groups, whereas no de-
fects were observed in canals prepared with S-Apex files (Fig. 1). The
difference between the experimental groups in the appearance of de-
fects was significant (chi-square test, p ⫽ 0.001).
Discussion
Recent ex vivo studies suggest that VRF is probably not an instant
phenomenon but rather a result of gradual diminution of root structure
(4, 5) .The current results could confirm that fractures did not occur
immediately after canal preparation. However, craze lines occurred in
4% to 16%, and these may develop into fractures during retreatment or
after long-term functional stresses like chewing (3). This is in agree-
ment with Onnink et al. (13), who were the first to report dentinal
defects as a consequence of canal preparation but only found small
defects entirely within dentin that did not communicate with the canal
wall. Less pronounced dentinal defects like craze lines were observed in
the current study. Considering the crucial clinical importance of VRF
and its determinant effect on tooth survival, even a small percentage of
damaged teeth could be of clinical importance.
Ultrasonic irrigation was used in order to effectively clean the
canal walls (11). However, ultrasonic action can also result in rough
canal walls (14) and cause fractures after root-end preparations (15).
In the current experiment, ultrasonic irrigation was performed in all
groups (11). Because all teeth were irrigated following the same pro-
tocol and roots prepared with hand files and S-ApeX files did not show
any dentinal defects, the conclusion that the ultrasonic irrigation per-
formed in this study did not contribute to the appearance of dentinal
defects seems justified.
Sawing action could also result in dentinal defects. However, be-
cause both the controls and the S-ApeX groups did not show any defect,
we may conclude that the defects seen were a result of preparation
procedures.
The only rotary file system among those used in this study that did
not show any dentin damage is S-ApeX. A literature search revealed no
previous studies regarding this file system. It is the only system available
with an inverted taper (Fig. 2) and may result in a parallel preparation
similar to the previously described LightSpeed system (LightSpeed
Technology, Inc, San Antonio, TX) (16).
The taper of the preparation and the files could be a contributing
factor in the generation of dentinal defects. Wilcox et al. (3) concluded
that the more root dentin that is removed the more likely a root is to
fracture. However, in the present study, a uniformed tapered prepara-
tion (0.05– 0.06) was attempted in all groups except for the S-ApeX
Inducing Dentinal Damage during Canal Preparation with NiTi Instruments 237

Basic Research—Technology
Figure 2. The S-Apex file has an inverted taper.
preparation, which is not tapered at all. The observation that all groups
prepared with rotary NiTi files showed various degrees of damage ex-
cept for the nontapered group supports the idea that tapered files may
generate an increased stress on the dentin wall. This observation is
supported by Sathorn et al. (17) who concluded that by maintaining the
canal size as small as practical, a reduction in fracture susceptibility
could be expected.
The amount of material removed from the root canal depends on
the shape of the rotary instrument and the penetration depth in the
canal. Because the roots prepared with rotary files that had a taper of at
least 0.06 in this study showed defects, it should be realized that these
instruments remove more apical dentin as compared with hand files
that have a taper of 0.02. It is noteworthy that ProTaper file F3 has a large
apical taper of 0.09 (18), which could explain the higher incidence of
damage observed in the ProTaper group as compared with the other
tapered rotary files. Furthermore, significantly more rotations in the
canal are necessary to complete a preparation with rotary NiTi files as
compared with hand files (9). This, in itself, may contribute to the
formation of dentinal defects.
Some of the defects seen did not connect with the pulp space. Wilcox
et al. (3) speculated that the stresses generated from inside the root canal
are transmitted through the root to the surface where they overcome the
bonds holding the dentin together. Onnink et al. (13) speculated that a
fracture contained within the dentin in one section could communicate with
the canal space in an adjacent section. This was recently supported by
nondestructive observations of dentinal defects induced in extracted teeth
and viewed with optical coherence tomography (5).
Under the tested conditions and within the limitations of this ex
vivo investigation, it may be concluded that the use of some rotary NiTi
instruments could result in an increased chance for dentinal defects.
Acknowledgments
The authors thank Wim van der Borden, DDS, Department of
Cariology Endodontology Pedodontology, Academic Centre of Den-
238 Souza Bier et al.
77
tistry Amsterdam, The Netherlands, for his clinical expertise with
S-ApeX files.
References
1. Schilder H. Cleaning and shaping the root canal. Dent Clin North Am
1974;18:269 –96.
2. Pettiette MT, Metzger Z, Phillips C, Trope M. Endodontic complications of root canal
therapy performed by dental students with stainless-steel K-files and nickel-titanium
hand files. J Endod 1999;25:230 – 4.
3. Wilcox LR, Roskelley C, Sutton T. The relationship of root canal enlargement to
finger-spreader induced vertical root fracture. J Endod 1997;23:533– 4.
4. Soros C, Zinelis S, Lambrianidis T, Palaghias G. Spreader load required for vertical
root fracture during lateral compaction ex vivo: evaluation of periodontal simulation
and fracture load information. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2008;106:e64 –70.
5. Shemesh H, van Soest G, Wu MK, Wesselink PR. Diagnosis of vertical root fractures
with optical coherence tomography. J Endod 2008;34:739 – 42.
6. Peters OA.Current challenges and concepts in the preparation of root canal systems:
a review. J Endod 2004;30:559 – 67.
7. Bergmans L, Van Cleynenbreugel J, Beullens M, Wevers M, Van Meerbeek B,
Lambrechts P. Smooth flexible versus active tapered shaft design using NiTi rotary
instruments. Int Endod J 2002;35:820 – 8.
8. Blum JY, Machtou P, Ruddle C, Micallef JP.Analysis of mechanical preparations in
extracted teeth using ProTaper rotary instruments: value of the safety quotient. J
Endod 2003;29:567–75.
9. Pasqualini D, Scotti N, Tamagnone L, Ellena F, Berutti E. Hand-operated and rotary
ProTaper instruments: a comparison of working time and number of rotations in
simulated root canals. J Endod 2008;34:314 –7.
10. Strawn SE, White JM, Marshall GW, Gee L, Goodis HE, Marshall SJ. Spectroscopic
changes in human dentine exposed to various storage solutions-short term. J Dent
1996;24:417–23.
11. van der Sluis LW, Versluis M, Wu MK, Wesselink PR. Passive ultrasonic irrigation
of the root canal: a review of the literature. Int Endod J 2007;40:415–26.
12. Shemesh H, Bier CA, Wu MK, Tanomaru Filho M, Wesselink PR. The effects of root
preparation and obturation on the incidence of dentinal defects. Int Endod J (in
press).
13. Onnink PA, Davis RD, Wayman BE. An in vitro comparison of incomplete root frac-
tures associated with three obturation techniques. J Endod 1994;20:32–7.
14. Stock CJ. Current status of the use of ultrasound in endodontics. Int Dent J
1991;41:175– 82.
15. Navarre SW, Steiman HR. Root-end fracture during retropreparation: a comparison
between zirconium nitride-coated and stainless steel microsurgical ultrasonic instru-
ments. J Endod 2002;28:330 –2.
16. Thompson SA, Dummer PM. Shaping ability of Lightspeed rotary nickel-titanium
instruments in simulated root canals. Part 1. J Endod 1997;23:698 –702.
17. Sathorn C, Palamara, Messer HH. A comparison of the effects of two canal prepara-
tion techniques on root fracture susceptibility and fracture pattern. J Endod
2005;31:283–7.
18. West JD. Introduction of a new rotary endodontic system: progressively tapering files.
Dent Today 2001;20:50 –2, 54 –7.
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 79

Chapter V
Summary and conclusions
Samenvatting en conclusies
80

Summary and Conclusions
New in vitro and ex vivo methodologies were
presented in this thesis that measure, image and
explore the root canal walls and related interfaces.
Numerous experiments were conducted and could
lead to a better understanding of the influence of
endodontic procedures on the root canal wall and
vice versa. This will help to improve current practices
that seem to fail in providing a reliable, predictable
outcome.
In chapter II Different experiments were presented
using the glucose penetration model. The ability of
this model to detect leakage patterns through various
materials, set ups and conditions was tested. The
following could be concluded:
- The glucose penetration model was more sensitive
in detecting leakage along the root canal filling as
compared to the fluid transport model.
- Removing the smear layer before filling did not
improve the sealing of the apical 4 mm of filling.
- Resilon, a recently introduced resin type filling
material, allowed more glucose penetration but the
same amount of fluid transport as gutta percha root
fillings in the apical 4 mm of the root, while no
difference in leakage was detected with both
models when the coronal 9 mm of the filling was
tested.
- When glucose penetration and fluid transport were
used to measure the leakage through coronal root
structures no leakage was detected. This confirms
the assumption that root structures do not allow
penetration of water or glucose through them.
- Root fillings with gutta percha and AH26 using the
warm vertical compaction technique sealed the root
canal better when passive ultrasonic irrigation had
been used.
- Portland cement, MTA, Ca(OH)2 and Sealer 26
react with glucose solution. This may result in less
detectable glucose in the penetration test.
Therefore, these materials should not be evaluated
for sealing ability with the glucose leakage model.
In chapter III New endodontic applications for
novel imaging techniques like ultrasound scan and
optical coherence tomography were suggested.
- Ultrasound scan could generate three dimensional
structural imaging of streptococcus mutans
biofilms of various thicknesses and on different
substrates without damaging the biofilm layer in a
quick and easy manner and can therefore be used to
evaluate biofilms longitudinally as a function of
time.
81
- Optical coherence tomography proved to be a
reliable method to image root canals and root
dentin in a nondestructive way and without using
ionized radiation. This technique holds promise for
full in vivo endodontic imaging. Furthermore, it is a
promising non destructive imaging method for the
diagnosis of vertical root fractures. Both the
sensitivity and specificity of this technique to
diagnose vertical root fractures are high and a
tendency for even higher specificity was
demonstrated in canals where the smear layer was
first removed.
In chapter IV the formation and incidence of
dentinal defects in the root canal wall after different
preparation and filling procedures was evaluated. It
was concluded that some procedures cause more
damage than others and that caution should be
exercised with the use of rotary Ni-Ti files and lateral
compaction of gutta percha.
- Root canal preparation and filling of extracted teeth
created dentin defects such as fractures, craze lines
and incomplete cracks. Canals filled with the lateral
compaction technique showed more defects than
those filled with non compaction methods.
- Some endodontic preparation methods might
damage the root and induce dentinal defects.
Rotary Ni-Ti instruments damaged the teeth more
than hand-filing. The new S-Apex system was the
only rotary system that did not result in dentinal
defects probably due to the inverted taper design.
The techniques described in this thesis could serve as
a promising tool for future research:
New knowledge on the different aspects of the root
canal wall will hopefully help to improve the clinical
outcomes and predictability of the endodontic
treatment.

Samenvatting en conclusies
In dit proefschrift werden nieuwe in vitro en ex vivo
onderzoekmethodes gepresenteerd die de wanden van
het wortelkanaal en de eraan gerelateerde
raakvlakken kunnen meten, visualiseren en
verkennen. Talrijke experimenten werden verricht die
zouden kunnen leiden naar een beter begrip van de
invloed van endodontische behandelingen op de
wortelkanaalwand en vice versa. Mogelijk kunnen
hierdoor bestaande procedures die nog geen
betrouwbaar en voorspelbaar resultaat bieden,
verbeteren.
In hoofdstuk II werden verschillende experimenten
gepresenteerd waarbij het glucose penetratie model
werd gebruikt. De capaciteit van het model om
lekkagepatronen te herkennen bij verschillende
materialen en opstellingen onder verschillende
omstandigheden, werd getest. De conclusies waren
als volgt:
- Het glucose penetratiemodel had een grotere
sensitiviteit voor het opsporen van lekkage langs
wortelkanaalvullingen dan het vloeistoftransport
model.
- Het verwijderen van de smeerlaag voorafgaand aan
het vullen, leidde niet tot verbetering van de
afsluiting op 4 mm afstand van de wortelpunt.
- Resilon, een kunsthars dat recent geïntroduceerd is
als vulmateriaal, vertoonde tussen 0 en 4 mm
gemeten vanaf de apex, meer glucose penetratie dan
Gutta Percha. Bij het vloeistoftransport model werd
bij zowel Resilon als gutta percha een gelijke
hoeveelheid lekkage waargenomen. Bij 0-9 mm
gemeten vanaf coronaal werd er echter geen verschil
waargenomen tussen beide testopstellingen.
- Bij het testen van lekkage door coronale
wortelstructuren met zowel het glucose penetratie
model als het vloeistoftransport model werd geen
lekkage waargenomen. Dit bevestigt de aanname dat
er geen water of glucose door wortelstructuren kan
penetreren.
- Wortelkanaal vullingen van gutta percha met
AH26, aangebracht door middel van warme verticale
compactie, sluiten het kanaal beter af wanneer eerst
passieve ultrasone irrigatie is toegepast.
- Portlandcement, MTA, Ca(OH)2 en Sealer 26
reageren met glucose oplossingen. Dit kan ertoe
leiden dat er minder glucose wordt waargenomen in
de penetratie test. Evaluatie van het afsluitend
vermogen van deze materialen kan daarom niet
worden onderzocht met het glucose lekkage model.
In hoofdstuk III werden nieuwe endodontische
toepassingen geïntroduceerd voor moderne
82
beeldvormende technieken zoals ultrageluidscans en
optical coherence tomografie.
- Met ultrageluidscans bleek het mogelijk om snel en
gemakkelijk een driedimensionaal structureel beeld
streptococcus mutans biofilms van verschillende
diktes en op verschillende substraten te maken,
zonder de driedimensionale structuur en vitaliteit van
de biofilm te beschadigen. Dit maakt deze methode
geschikt om biofilms longitudinaal te vervolgen.
- Optical coherence tomografie bleek een
betrouwbare methode om wortelkanalen en
worteldentine weer te geven zonder deze te
beschadigen, en zonder ioniserende straling te
gebruiken. De techniek heeft het vermogen in zich
om een volledig endodontisch beeld te geven in vivo.
Ook kan deze vorm van tomografie gebruikt worden
om verticale wortelfracturen te diagnosticeren zonder
het element of omringende structuren te beschadigen.
De sensitiviteit en specificiteit hierbij is hoog, en als
eerst de smeerlaag wordt verwijderd is er een tendens
naar een nog grotere specificiteit.
In hoofdstuk IV werd het ontstaan en de incidentie
van dentinedefecten op de wortelkanaal wand ten
gevolge van verschillende preparatie en
vultechnieken, geëvalueerd. Er werd geconcludeerd
dat de ene techniek meer schade veroorzaakt dan de
andere, zo is voorzichtigheid geboden bij het gebruik
van roterende Ni-Ti vijlen en laterale compactie van
gutta percha.
- Het prepareren en vullen van wortelkanalen bij
geëxtraheerde elementen veroorzaakten dentine
defecten zoals fracturen, crazelijnen en onvolledige
barsten. Kanalen gevuld door middel de laterale
compactie techniek vertoonden meer defecten dan
kanalen die met de non-compactie techniek werden
gevuld.
-Sommige endodontische preparatietechnieken
kunnen mogelijk de wortel beschadigen en dentine
defecten veroorzaken. Roterende Ni-Ti instrumenten
beschadigen het element meer dan handvijlen. Het
nieuwe S-apex systeem is het enige roterende
systeem dat geen dentine defecten liet zien,
waarschijnlijk ten gevolge van het omgekeerde taper
ontwerp.
De beschreven onderzoektechnieken bieden een
enorme kans voor de toekomst:
Nieuwe kennis over de verschillende aspecten van de
wand van het wortelkanaal kan er hopelijk toe
bijdragen dat de klinische resultaten en de
voorspelbaarheid van de endodontische behandeling
zullen verbeteren.
 83

Acknowledgements
Min Kai Wu, Paul Wesselink and Luc van der Sluis taught me a lot during the last years. They
showed me how to read an article and how to write one, how to design an experiment and how to
abandon one, how to present my ideas and how to conceal them, and especially, how to ask a question
and how to try and answer one.

Rifat Ozok For his help with almost every
question, dilemma or demand, and for his
friendship and patience.
The laboratory staff and especially Marc Buijs,
Rob Exterkate and Michel Hoogenkamp - what a
professional, serious and reliable team you are.
Thanks for your instructions, remarks and
criticism- I learned a lot from all of you.
extending my horizons to unknown territories
which will end up with a big breakthrough.
Erick Souza and Alexandre Bier from Brazil for
their excellent work and enthusiasm.
Special thanks to the students I worked with and to
the post-graduate students (TEO’s) for their
critical questions and input.

Irene Aartman for helping me with the statistics.
Annemarie Verhoef for het advice on teaching,
and for the Dutch translation…
Marijke Voorn - for helping me with my Dutch,
from the very beginning, with the ordering of
materials and instruments and for her wise (and
often controversial) remarks during lunch…
To all the other members of the department, and
especially Linda Peters for their advise and
support.
David Goertz and Gijs van Soest from the
Erasmus MC in Rotterdam (David is now back to
his native Canada) and Paul Zaslansky from the
Max Planck institute in Potsdam, Germany for
The staff of the Verwijspraktijk in Amsterdam for
being much more than my main source of income,
and especially to Nicolien, Abe, Erwin, Rian, Jan,
Tjebbe, Peter, Gordon. And Els and Andreas –also
for agreeing to be my Paranymphs.
Tineke Looman: for the valuable advice,
friendship and honest critic.
Anne Mieke Eggenkamp for the enthusiasm,
professionalism and time. What a boost to my
thesis!
Piet Dinkla- for the website and for your patience.
And Wijb- you already know I don't like to get
sentimental, and you often heard what I think
about these (too) long “thank-you” sections, so I
am NOT going to thank you on these pages, but in
a more personal way.

For more acknowledgements, links and contacts please go to

www. shemesh.nl
HAGAY SHEMESH was born in Israel and immigrated to
the Netherlands in 2002.
He lives in Amsterdam and works at the Academic Centre for
Dentistry Amsterdam (ACTA) where he conducted this PhD thesis
research. He also works as an endodontist in a private practice.

For more on this thesis, complete up-to-date list of publications,

abstracts and presentations, curriculum vitae and additional
information, please go to: www.shemesh.nl