Vol. 99 No.

ENDODONTOLOGY

January 2005

Editor: Larz S. W. Spangberg

A new quantitative method using glucose for analysis of endodontic leakage

Qiong Xu, DDS, PhD,a Ming-wen Fan, DDS,b Bing Fan, DDS, PhD,c
Gary S. P. Cheung, BDS, MSc, MDS,d and Han-lin Hu, MS,e Wuhan and Hong Kong, China
WUHAN UNIVERSITY AND UNIVERSITY OF HONG KONG

Objective. The purpose of this study was to introduce a new method for quantitative testing of endodontic leakage.
Study design. Eighty straight maxillary anterior teeth were divided randomly into 3 experimental groups of 20 samples each and
2 control groups. The experimental groups were prepared using the modified double-flared technique and obturated by lateral
compaction of cold gutta-percha with Pulp Canal Sealer EWT, Sealapex, or AH Plus sealer. With the leakage test device, coronal
1 mol/L glucose solution was forced under a hydrostatic pressure of 1.5 kPa toward the apical part of the root. Leakage was
measured by the concentration of leaked glucose in apical reservoir at 1, 2, 4, 7, 10, 15, 20, and 30 days with the enzymatic
glucose oxidase method.
Results. No significant difference of sealing ability was found among 3 test groups at 1, 2, 4, and 7 days. From the tenth day, Pulp
Canal Sealer EWT showed the highest leakage, and the leakage was not significantly different between Sealapex and AH Plus.
Conclusions. The quantitative method is sensitive, nondestructive, and clinically relevant. Pulp Canal Sealer EWT showed more
leakage than Sealapex and AH Plus in most observation time.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:107-11)

Adequate obturation of the root canal system following

intracanal preparation is a major objective of endodontic
treatment. A great deal of attention has been given
to the evaluation of sealing ability of root canal filling
materials and associated obturation techniques. Various
test methods have been described to evaluate the quality
of seal by such methods as dye penetration,1 radioactive
isotopes test,2 bacteria or bacterial metabolites leakage
a

Formerly, Assistant Professor, School of Stomatology, Wuhan

University; currently, Assistant Professor, Department of Endodontics, Guanghua School of Stomatology, Sun Yat-sen University,
Guangzhou, China.
b
Professor, Department of Endodontics, School of Stomatology,
Wuhan University.
c
Professor, Department of Endodontics, School of Stomatology,
Wuhan University.
d
Associate Professor, Department of Conservative Dentistry and
Endodontics, Faculty of Dentistry, University of Hong Kong.
e
Associate Professor, Department of Laboratory, Zhongnan Hospital,
Wuhan University.
Received for publication Mar 29, 2004; returned for revision May 2,
2004; accepted for publication Jun 1, 2004.
Available online 1 September 2004.
1079-2104/$ - see front matter
2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.tripleo.2004.06.006

test,3,4 electrochemical technique,5 and fluid filtration.6

However, the published reports often reach different or
even conflicting conclusions. As pointed out by Wu and
Wesselink, there was a high level of variation in these
results and it was difficult to draw firm conclusions as to
which filling technique or material was the best in
sealing the root canal system.7 It was suggested that
more studies should be devoted to perfecting microleakage methodology.
When filling the root canal system, the sealer plays an
important role in reducing microleakage.8 Many types
and brands of sealers are available, which may be
broadly classified into zinc oxideeeugenol based,
calcium hydroxide based, epoxy resin, and glass
ionomer cement. Numerous studies have been carried
out to compare sealing property of various sealers, but
there is hardly any common consensus. Some studies
showed that the resin-based sealer provided a better seal
than other sealers,9,10 and others indicated that there was
no significant difference in leakage of different types of
sealers.3,11 The variety of evaluation methodologies
and test parameters might account for the varying and
sometimes contradictory results.
The purpose of the present study was to introduce
a new method for analysis of endodontic microleakage,
107

108 Xu et al

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January 2005

MATERIAL AND METHODS

Eighty recently extracted human maxillary anterior
teeth with a single, straight root canal were selected for
this study. Roots with cracks, open apices, resorptive
defects, or large carious lesions approaching the pulp
had been excluded. After removal of bony debris,
calculus and soft tissues on the root surface, the teeth
were stored in deionized water at 48C until use. A
trained operator performed the root canal instrumentation and obturation described below.

confirmed by inserting a size 15 file through the apical

foramen before and after the root canal preparation. The
coronal portion of each canal was preflared with GatesGlidden drills (Dentsply Maillefer) of sizes 3 to 6 in
a step-back manner. The apical portion of the canal was
instrumented to a size 50 master file using the balanced
force technique12 and Flexofile. A step-back flaring was
then performed at 1 mm increments to size 80. The canal
was irrigated with 2 mL freshly prepared 2.5% sodium
hypochlorite solution with a 27-gauge needle after every
instrument. A final rinse with 10 mL 17% EDTA
(pH = 7.7) and 10 mL 2.5% sodium hypochlorite was
given after completion of preparation to remove the
smear layer.13 The canal was then dried with paper
points.
The prepared roots were randomly divided into 3
experimental groups of 20 roots each and 2 control
groups of 10 each. The sealers used in 3 test groups
were Pulp Canal Sealer EWT (Kerr, Romulus, Mich),
Sealapex (Kerr), and AH Plus (Dentsply DeTrey,
Konztanz, Germany). The root canals were filled using
lateral compaction technique. The sealers were
mixed according to the manufactures instructions and
introduced into the canal using a size 45 file with
counterclockwise rotation. A size 50 master guttapercha (GP) cone (Dentsply Maillefer) lightly coated
with sealer was then placed to the full working length.
Lateral compaction was achieved using size 25
accessory gutta-percha cones and a size B finger
spreader (Dentsply Maillefer) that initially reached
to within 1 mm of the working length. The tip of
each accessory GP cone was lightly coated with sealer,
placed, and compacted laterally. The process was
repeated until the cone could not be inserted more than
3 mm into the canal. The 10 positive control teeth were
filled in a manner identical to that of the experimental
teeth but without sealer. The 10 negative control teeth
were obturated with GP cones and AH Plus.
The gutta-percha at the root canal orifice was
removed with a hot plugger leaving 10 mm of the filling
material in the canal. All teeth were stored in aqueous
solution containing 0.1% sodium azide (NaN3) for
1 week.

Instrumentation and obturation of root canals

The coronal portion of all teeth was removed with
diamond disks so that each specimen was 15 mm long.
A diamond bur was used to gain a straight-line entry to
the root canal. After removal of the pulp tissue using
a barbed broach, a size 15 K-Flexofile (Dentsply
Maillefer, Ballaigues, Switzerland) was introduced into
the root canal until the tip was just visible at the major
apical foramen. The working length was determined by
subtracting 1 mm from this length. Apical patency was

Preparation of specimens
The entire specimen of the negative control group,
including the root canal orifice and apical foramen, was
completely coated with sticky wax (Kerr). The roots of
the experimental groups and the positive controls were
covered with sticky wax, except for the coronal access
cavities, root canal orifices, and apical apices.
The coronal part of each root was glued to the end of
an Eppendorf vial using cyanoacrylate. Leakage at this
connection was eliminated by the generous use of sticky

Fig 1. The device for microleakage test.

based on the filtration rate of glucose along the root

canal filling. The amount of leakage was quantified with
spectrophotometry. Three commonly used root canal
sealers, Pulp Canal Sealer EWT, Sealapex, and AH Plus,
were used to evaluate this method.

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Volume 99, Number 1

Xu et al 109

Table I. Microleakage of glucose in three experimental groups (mM/L)

Time
(days)

Fig 2. Leakage of positive and negative control groups. The

positive control group showed high values of glucose leakage
that increased rapidly. The negative control group showed no
leakage of glucose.

wax. A hole was created in the cap of the Eppendorf vial,

through which a plastic tube of at least 15 mm long was
connected. A seal was obtained using cyanoacrylate
glue and sticky wax. The assembly was then placed in
a sterile 5 mL glass bottle with a screw cap and sealed
with sticky wax (Fig 1).
The tracer used in the present study was a 1 mol/L
glucose solution (pH = 7.0), which density was 1.09 3
103 g/L and viscosity 1.18 3 10
3 Pas at 378. Glucose
has a low molecular weight of 180 Da, and is
hydrophilic and chemically stable. About 5 mL of the
glucose solution, containing 0.2% NaN3, was injected
into the Eppendorf vial from the plastic tube until the top
of the solution was 14 cm higher than the top of guttapercha in the canal, which created a hydrostatic pressure
of 1.5 kPa (15 cm H2O). The glass bottle contained 1 mL
0.2% solution of NaN3, in which glucose that passed
through the obturated canal would be collected. The
NaN3 was used here to inhibit the proliferation of
microorganisms that might decompose glucose. The
seal at all junctions of the system was checked by
connecting the open end of the plastic tube to
compressed air. Any bubbles would indicate leakage
of the assembly.
The model was then transferred to an incubator that
provided 100% humidity and 378C temperature for the
duration of observation periods. To examine if there
might be any evaporation of solution in the tube/vial,
another such assembly containing 1 mL 0.2% NaN3 was
placed in the incubator under the same condition and
weighted every day during test time.
Measurement of microleakage
A 10 mL aliquot of solution was drawn from the glass
bottle using a micropipette at 1, 2, 4, 7, 10, 15, 20, and 30
days. After drawing the sample, 10 mL of fresh 0.2%
NaN3 was added to the glass bottle reservoir to maintain
a constant volume of 1 mL. If there was any decrease
in volume in the control bottle due to evaporation,

1
2
4
7
10
15
20
30

AH plus
0.06
0.12
0.22
0.34
0.52
1.27
3.09
5.39

6
6
6
6
6
6
6
6

0.08
0.10
0.14
0.13
0.19
0.51
1.77
3.07

Sealaplex
0.05
0.12
0.20
0.32
0.49
1.72
4.01
7.28

6
6
6
6
6
6
6
6

0.08
0.09
0.10
0.10
0.11
0.74
1.59
2.84

Pulp canal
sealer EWT
0.07
0.15
0.32
0.61
2.04
7.34
12.88
20.76

6
6
6
6
6
6
6
6

0.11
0.16
0.27
0.50
1.31
3.49
6.25
9.21

corresponding amount of sterile deionized water was

added to the glass bottle. The sample was then analyzed with a Glucose kit (Diasys, Shanghai, China)
in a UV-Vis recording spectrophotometer (Shimadzu;
Kyoto, Japan) at 500 nm wavelength. Two blinded
independent evaluators conducted the spectrophotometric determination of glucose concentration.
The results of leakage in all groups were calculated as
mmol/L at that particular time after obturation. Since
the data were not normally distributed, analysis by
means of the Kruskal-Wallis test was adopted. When the
difference of leakage in terms of glucose concentration
among groups was significant, further multiple comparisons by means of the Nemenyi test were performed.
RESULTS
The positive control group showed high values of
glucose leakage from the first day and it increased
rapidly over time. In the negative control group, no
glucose was detected in all apical reservoirs throughout
the experiment (Fig 2). This indicated that the seal of the
system was effective and reliable.
There was a tendency of increase in leakage in all
experimental groups from the first day to the end of
experimental period. The mean values of leakage are
given in Table I. No significant difference was found
among the 3 experimental groups at 1, 2, 4, and 7 days
(P [.05). From the tenth day onward, there was
a significant difference among the groups (P \ .001).
The results of Nemenyi test indicated that Pulp Canal
Sealer EWT showed the greatest amount of leakage,
whereas the difference between Sealapex and AH Plus
groups was not significant at 10, 15, 20, and 30 days
(P [.05) (Fig 3).
DISCUSSION
Various methods have been developed to assess
sealing ability of root canal filling materials, usually
based on the same principle, ie, to evaluate the
penetration of a tracer along the obturated canal of an
extracted tooth.4,14 Several tracers, such as dye,

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January 2005

110 Xu et al

Fig 3. The microleakage of 3 types of sealers. Pulp Canal

Sealer EWT sealer showed the highest leakage from the tenth
day. Sealapex and AH Plus showed similar behavior of
leakage.

radioisotope, and bacteria and their products, had been

used for evaluation of microleakage. The dye penetration test is the most popular, probably because it is
a simple and inexpensive method. However, this method
often yielded a large variation of the result, and could
hardly be reproducible and comparable.7 Assessment of
bacterial leakage might be more biologically relevant
than that of dye or radioisotope penetration, but the
conclusions might vary with the bacterial species used.
Maintaining aseptic conditions throughout all steps of
the experiment can be difficult. Radioisotope labeling
and electrochemical technique were less frequently
employed because they pose a radiation hazard and
require sophisticated materials and apparatus. The fluid
filtration method, which was developed by Derkson et al
for measuring dentin permeability,15 and later modified
by Wu et al to evaluate endodontic leakage,16 is gaining
popularity because it is sensitive and nondestructive and
permits repeated observation of the same specimen over
times. However, there was no standardization of the
methods, such as the measurement time, the applied
pressure, the diameter of the tube containing the bubble,
and the length of the bubble, which might influence the
results.17
The choice of tracer should be carefully chosen
because its size and physicochemical properties may
influence the result. The use of tracer of a small
molecular size was favored by some authors.7 The
smaller molecular size, and the stricter test, may be seen
as more relevant to clinical outcome.18 In the present
study, glucose was selected as the tracer because it is of
small molecular size (MW = 180 Da) and is a nutrient
for bacteria. If glucose could enter the canal from the
oral cavity, bacteria that might survive root canal
preparation and obturation could multiply and potentially lead to periapical inflammation. Glucose, therefore, was thought to be more clinically relevant than
other tracers used in microleakage tests. Quantitative
analysis of leakage was possible by determining the
concentration of glucose in the apical reservoir that
leaked through the filled root canal.

To determine the concentration of glucose, the

enzymatic glucose oxidase method was chosen because
it provided the ultimate degree of specificity and high
sensitivity when compared with other methods, such as
copper or ferricyanide methods.19 With this method,
glucose is oxidized by the enzyme glucose oxidase in
the presence of oxygen to gluconic acid with formation
of hydrogen peroxide as shown in reaction (1) below.
Then in the presence of a peroxidase enzyme, a
chromogenic oxygen acceptor (4-aminoantipyrine and
phenol) is oxidized by the hydrogen peroxide, resulting
in the formation of a red product (oxidized chromogen).
The quantity of this oxidized chromogen is proportional
to the glucose present initially in the first reaction, which
quantity is determined by spectrophotometry.
Glucose oxidase

Glucose1H2 O1O2

!

Gluconic acid1H2 O2

H2 O2 14-Aminoantipyrine
Peroxidase

1 phenol

! Oxidized chromogen1H2 O

With this model, it was possible to quantify the

endodontic microleakage continuously over time. The
amount of microleakage was the cumulative value of
leaked glucose. In addition, the coronal low pressure
used in the study could help rule out entrapped air or
fluid and seemed to be sufficient for a device with high
sensitivity.17
The results of the present study indicated that the 3
types of sealer all showed similar level of leakage in the
first 10 days during the observation time. Thereafter, the
greatest amount of leakage was observed with Pulp
Canal Sealer EWT. That might be owing to the
dissolution of the sealer in the solution. Sealapex and
AH Plus showed similar behavior of long-term seal;
although AH Plus seemed to perform better, the
difference was not significant. With our leakage testing
device, most canals showed low degree of leakage at day
2 and all samples showed leakage to a small extent at
day 7. This suggested the need to reconsider those
studies that reported on short-term leakage patterns of
different obturation materials or techniques. The authors
note the need, however, to test the validity and reproducibility of the currrent method.
In summary, under the conditions of this study, this
new in vitro method allowed a nondestructive, longterm, quantitative evaluation of endodontic microleakage. It appeared that Pulp Canal Sealer EWT
showed a greater amount of leakage than Sealapex and
AH Plus after an extended period of observation.
REFERENCES
1. Starkey DL, Anderson RW, Pashly DH. An evaluation of the
effects of methylene blue dye pH on apical leakage. J Endod
1993;19:435-9.

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2. Hakel Y, Wittenmeyer W, Bateman G, Bentaleb A, Allemann C.
A new method for the quantitative analysis of endodontic
microleakage. J Endod 1999;25:172-7.
3. Chailertvanikul P, Saunders WP, Mackenzie D. An assessment
of microbial coronal leakage in teeth root filled with gutta-percha
and three different sealers. Int Endod J 1996;29:387-92.
4. Kersten HW, Moorer WR. Particles and molecules in endodontic
leakage. Int Endod J 1989;22:118-24.
5. Jacquot B, Panighi M, Steinmetz P, GSell C. Evaluation of
temporary restorations by means of electrochemical impedance
measurements. J Endod 1996;22:586-9.
6. Pommel L, Camps J. In vitro apical leakage of system B
compared with other filling techniques. J Endod 2001;27:449-51.
7. Wu M-K, Wesselink PR. Endodontic leakage studies reconsidered. Part I. Methodology, application and relevance. Int Endod J
1993;26:37-43.
8. Canalda-Sahli C, Brau-Aguade E, Sentis-Vilalta J, AguadeBruix S. The apical seal of root canal sealing cements using
a radionuclide detection technique. Int Endod J 1992;25:250-6.
9. Timpawat S, Amornchat C, Trisuwan WR. Bacterial coronal
leakage after obturation with three root canal sealers. J Endod
2001;27:36-9.
10. Kataoka H, Yoshioka T, Suda H, Imai Y. Dentin bonding and
sealing ability of a new root canal resin sealer. J Endod 2000;26:
230-5.
11. Miletic I, Anic I, Pezelj-Ribaric S, Jukic S. Leakage of five root
canal sealers. Int Endod J 1999;32:415-8.
12. Roane JB, Sabala CL. The balanced force concept for
instrumentation of curved canals. J Endod 1985;11:203-11.

Xu et al 111
13. Taylor JK, Jeansonne BG, Lemon RR. Coronal leakage: effects
of smear layer, obturation technique, and sealer. J Endod 1997;
23:505-12.
14. Al-Ghamdi A, Wennberg A. Testing of sealing ability of
endodontic filling materials. Endod Dent Traumatol 1994;10:
249-55.
15. Derkson GD, Pashley DH, Derkson ME. Microleakage measurement of selected restorative material: a new in vitro method.
J Prosthet Dent 1986;56:435-40.
16. Wu M-K, De Gee AJ , Wesselink PR, Moorer WR. Fluid
transport and bacterial penetration along root canal fillings. Int
Endod J 1993;26:203-8.
17. Pommel L, Camps J. Effects of pressure and measurement time
on the fluid filtration method in endodontics. J Endod 2001;27:
256-8.
18. Friedman . Retrograde approaches in endodontic therapy. Endod
Dent Traumatol 1991;7:97-107.
19. Bishop ML, Duben-Engelkirk JL, Fody EP. Clinical chemistry
principles, procedures, correlations. 2nd ed. Philadelphia:
Lippincott; 1985. pp. 307-9.
Reprint requests:
Professor Ming-wen Fan
Department of Endodontics
School of Stomatology
Wuhan University
65 Luoyu Road
Wuhan 430079
China
kqyywjtx@public.wh.hb.cn