JOURNALOF ENDOCONTICS
Copyright 0 2000 by The American Association of Endodontists
Apical Microleakage of Radiolabeled Lysozyme
Over Time in Three Techniques of Root
Canal Obturation
Youssef Haikel, DCD, DSO, PhD, Michel Freymann, DCD, DU, Virginie Fanti, DCD,
Anne Claisse, DCD, DSO, FranGoise Poumier, DCD, DU, and Morag Watson, BDS
The three methods of obturation assessed in this
study were lateral condensation (80 teeth), Ther-
mafil(40teeth), and McSpadden(40 teeth). All teeth
were prepared to the master apical file 30 and
widened coronally by Gates Glidden burs before
being randomly assigned to experimental groups
to be filled by each technique; they were then
sealed with nail polish, except for the apical 1 mm.
Quantitative evaluation of apical microleakage for
each technique was obtained after periods of 1
day, 7 days, 14 days, and 28 days of immersion in a
lysozyme solution labeled with radioactive iodine
by preparing horizontal sections of the teeth and
measuring the level of radioactivity in each section
using a gamma counter. Initial (1-day) leakage was
least in the Thermafil group and was significantly
different from the other techniques. Leakage was
greatest in the laterally condensed samples. By the
end of the study (28th day) values for lateral con-
densation were lowest, but were significantly dif-
ferent only for the McSpadden group. For all tech-
niques leakage was most significant in the first 3
mm from the apex and was very low below this
level so that all methods can be considered as
giving a hermetic seal below 3 mm.
Obturation is the final stage of endodontic treatment and has an
important role in preventing recontamination of the root canal and
encouraging healing of the penapical tissues (1). Classically canals
have been filled by cold lateral condensation, but limitations in this
technique have been identified (2). In an attempt to overcome these
problems and make obturation easier and less time-consuming (3),
other methods have been introduced, such as Thermafil (4, 5) and
the McSpadden technique (2, 6).
As techniques have been introduced, they have been assessed
using varying experimental designs. Most commonly dye penetra-
tion ( 2 ) has been used to evaluate the apical seal, but other
148
Printed in U.S.A.
VOL. 26, No. 3, MARCH 2000
techniques include scanning electron microscopy, bacterial pene-
tration studies (7), pressurized fluid measurements (8), electro-
chemical methods (9), radioactive isotopes detected by autoradiog-
raphy (6), scintillation spectrometry (lo), and gamma counters
(1 1). These procedures have given varying and often contradictory
results. Lateral condensation has often been used as a reference for
comparison with Thermafd (4) or with McSpadden techniques
(6, 12), but to date few experiments have compared all three of the
aforementioned techniques to each other.
The objective of this study was to compare the Thermafil and
McSpadden techniques with each other and with the more tradi-
tional lateral condensation technique. Apical leakage was analyzed
quantitatively using radiolabeled tracers that were detected and
measured by a gamma counter using a new method described in a
previous study (13). For each technique, samples were immersed
for varying periods of time in the radioactive solution so that
changes in the quality of the seal over time could be analyzed.
MATERIALS AND METHODS
The study involved 192 single-rooted human teeth without
marked apical curvature that had been stored in a 10% formalin
solution.
Endodontic Preparation
Teeth were prepared and obturated by a single operator. Access
cavities were cut with a diamond bur to gain entry to the root
canals. Apical patency was confirmed by inserting a #15 file
(Maillefer, Ballaigues, Switzerland) until it was just visible at the
apical foramen and the working length was taken as 1 mm short of
this. The canal was instrumented with frequent sodium hypochlo-
rite irrigation, alternating between K-files and H-files (Maillefer)
until a master apical file of 30 was reached and the coronal third
flared using Gates Glidden burs (Maillefer).
OBTURATION OF CANALS
The teeth were randomly divided into 1 group of 80 teeth and 2
groups of 40 teeth according to the method of obturation to be
used. To reduce variables between the groups, the same sealer AH
Vol. 26, No. 3, March 2000 Apical Microleakage 149

plus (Dentsply De Trey, Konstanz, Germany), which had been
shown to be the most effective in a previous study (13), was used
for each technique. The group of 80 teeth were filled by lateral
condensation; a medium-sized, nonstandardized master gutta-per-
cha point (Hygienic, Akron, OH) was inserted and adjusted until
apical tugback was achieved at the working length of 1 mm short
of the apex, then the canal walls were coated with sealer and the
master cone placed and condensed and accessory points added
until the canal was full. One of the groups of 40 teeth was obturated
by Thermafil (Thermafil Endodontic Obturators). The size was
chosen to correspond to the size of the verifier that best fit the
working length of the canal; sealer was placed in the entrance of
the canal and the Thermafil device heated in the oven supplied
(Thermaprep) for 4 min at 145°C and inserted to the working
length of the canal in one firm movement. The handle was removed
by cutting with a tapered diamond bur, and excess gutta-percha
was plugged round the carrier. The final group was filled by the
McSpadden technique: after coating the walls of the root canal
with sealer, a gutta-percha point was placed to the working length
and the compactor introduced running at 10 to 12,000 rpm for 1
min to compress the gutta-percha. The samples were then stored
for 48 h at 37°C in a humid environment to allow the sealer to set.
PREVENTION OF LATERAL LEAKAGE
Two coats of nail polish (L'Oreal, Clichy, France) were applied
to the surface of each root leaving 1 mm free at the apex so that
tracer could only penetrate the canal via the apical region.
CONTROL GROUPS
Two other groups of 16 teeth each were prepared as positive and
negative controls. The positive control group was obturated by
lateral condensation, but without sealer and the test group was
coated in nail polish except for the apical 1 mm. The negative
control group was left unobturated and was totally coated in nail
polish, including the apical region.
RADIOACTIVE SOLUTION
IMMERSION OF THE SAMPLES
The experiment was undertaken in a modified syringe (Fig. 1)
from which the end had been removed and the chamber was
enclosed in lead foil to limit ionization from the radioactive solu-
tion, as described in a previous study (13). Teeth were mounted by
their coronal aspects in transparent tubes attached to the pistons of'
the syringes. This equipment allowed each root to be immersed up
to 2 mm from the apex in radioactive solution. The three experi-
mental groups were each further subdivided into four equal groups,
depending on the duration of immersion-1 day, 7 days, 14 days,
or 28 days-and were stored in the solution for the allotted time at
a temperature of 23°C to limit evaporation.
PREPARATION OF SECTIONS
On removing the teeth from the solution, the root surfaces were
wiped with cellulose impregnated with a decontaminant (DCcon 90
diluted to 5%) until they no longer gave a reading when exposed
to a Geiger-Muller tube (Nardeux), indicating that all the radioac-
tive solution had been removed from the surface of the root. Each
root was then cut into six transverse sections using a fine diamond
wire (ESCIL diamented thread saw, Well 3032-4, Chassieu,
France) with lubrication to prevent overheating. The sections had
a thickness of 0.8 mm each, beginning 1 mm from the apical
foramen.
The first section from each tooth was discarded, because the
filling did not begin until 1 mm from the apex. Each subsequent
section was placed in a series of Pyrex tubes for measurement of
the radioactivity present by the gamma counter (Minaxi Auto
Gamma 5000 Series, United Technologies Packard, Downers
Grove, IL). This allowed the amount of apical infiltration and thus
the quality of seal of the filling to be evaluated.
STATISTICAL ANALYSIS
Results were analyzed using the Kruskal-Wallis one-way anal-
ysis of variance on ranks and the all painvise multiple comparison
procedure (Dunn) to determine the significance of the values
attained.

The radioactive tracer used was Iz5I combined with lysozyme at a

concentration of 20 mg/100 ml. This solution contains molecules
small enough to penetrate an inadequate apical seal due to the small
size (40 nm) and low molecular weight (14,600 Da) of the lysozyme,
while not reacting with the crystalline phase of the tooth substance.
The protein was made radioactive using the McFarlane technique (14,
15) in which '*'I attaches to the lysozyme molecule at its tyrosyl and
histidyl sites by an iodination reaction. The labeled protein solution
was then divided into 2 ml aliquots and frozen.
The concentration of this solution was determined using the
values of protein absorbance by spectrophotometry and radioac-
tivity using a gamma counter, allowing the samples to be thawed
and diluted with phosphate-buffered saline to a concentration of 20
FIG1. Diagram of apparatus for immersion of samples. System used
mg/100 ml for the experiment. The iodine is a gamma emitter with
to mount the samples during the study. 7, piston;2, sliding chamber;
a half-life of 60 days. The lysozyme used was extracted from egg 3, lead foil; 4 , syringe tip; 5, PVC tube; 6, sample; 7, sample support;
white and had a purity in excess of 96%. 8, sterile tube; and 9,radioactive solution.
150 Haikel et al. Journal of Endodontics

RESULTS

The positive control showed high levels of tracer penetration

present from day 1, but increasing as immersion time increased. -Mc Spadden
This verified that penetration of the tracer through the apical
foramen was possible when sealer was absent (Fig. 2).
The negative control gave very low readings that did not in-
crease with duration of immersion. This indicated that very little
penetration had occurred and confirmed that nail polish was ef-
fective in sealing the external root surface (Fig. 3). L

- -x--

1 Day (Fig. 4) I
1 2 3 4 5
CrOOS root section from the apex
Readings at this stage were fairly low; they were greatest in the
first (1 to 1.8 mm) section, especially with lateral condensation. In FIG4. Radioactiveintensity signal (cpm) as a function of the distance
the second (2 to 2.18 mm) section, levels of radioactivity seemed (mm) from the apex: 1-day period of immersion. Cond. Lat., lateral
condensation.
to settle at a lower level. There was a significant difference be-
tween Thermafil and lateral condensation and between Thermafil
and McSpadden at the level of the first section. Comparison
between all the techniques showed that, after 1 day, Thermafil
performed best with no significant differences between the two
others.
1200 ,+
-Ye Spadden

a-
7 Days (Fig. 5)

After 1 wk, radioactivity had not increased very much, but the
order of the leakage values had changed. Again readings were
highest in the first section and reduced in subsequent sections.
Statistical analysis of sections at the first level and overall illus-
trated no significant differences between the techniques.
1 2 3 4 5
Cross root sections from the apex

:,\
FIG5. Radioactiveintensity signal (cpm) as a function of the distance
(mm) from the apex: 7-day period of immersion. Cond. Lat., lateral
condensation.

30000 CPm
3000 -..
-1Mc Spadden

2 3 4 5 6
Cross root section from the apex
FIG2. Positive control group.

1 2 3 4 5
Cross root sections from the apex
FIG 6. Radioactiveintensity signal (cprn) as a function of the distance

I +D14
*m8l
(mm) from the apex: 14-day period of immersion. Cond. Lat., lateral
condensation.

14 Days (Fig. 6)
200
0 #- d
-*
After 2 wk higher levels of infiltration were evident. Values
1 2 3 4 5
were again higher at the level of the first section, reducing toward
Cross root sections from the apex the second section, and leveling out, with all techniques showing
FIG3. Negative control group. similar low levels of infiltration from the third section. Statistical
Vol. 26, No. 3, March 2000
analysis of results at the first level showed no significant difference
between the techniques, as was the case when all sections were
considered together.
28 Days (Fig. 7)
After 1 month, highest readings were attained in the first sec-
tion, with infiltration being much lower in the teeth filled by lateral
condensation than the other techniques. After the second level,
however, readings from the Thermafil and McSpadden groups had
greatly reduced to levels similar to those of lateral Condensation.
Statistical analysis of the results shows a significant difference,
with lateral condensation performing better than McSpadden at the
level of the first section and also when all sections are taken into
account.
Overall
Throughout the experiment, infiltration was mainly important in
the first 2 to 3 mm (i.e. sections 1 and 2 ) , where the infiltration
increased over the time of the experiment and varied between the
techniques. More coronal sections showed very little infiltration,
and levels of leakage did not appear to increase with time. It would
therefore appear that all techniques give a hermetic obturation from
the level of the third section; so all methods studied can be
considered to result in effective sealing of the root canals.
Comparison of the total infiltration of lysozyme at each time
period allowed the changes in permeability over time to be inves-
tigated. Initially, Thermafil seems the most effective technique;
but, over time, infiltration increased (Fig. 8). Lateral condensation
and McSpadden show similar levels of leakage at 1 day and 7 days,
and leakage began to rise after this time.
DISCUSSION
One of the major problems with studies of apical microleakage
is the lack of standardization of samples and techniques (5, 16). In
this study all canals were prepared to the same size and a single
operator conducted the preparation and obturation to try to over-
come variations. An attempt was made to make the samples of
cpm
-K
3500
3000
-Mc Spadden
-1hermaRI
2500
2000
1500
IOOa
500
0 Various isotopes have been used to study leakage, because they
I 2 3 4 6
Cross root section from the apex
FIG7. Radioactive intensity signal (cpm) as a function of the distance
(mm) from the apex: 28-day period of immersion. Cond. Lat., lateral
condensation.
Apical Microleakage 151
cpm
8000 -
-Cond. Lat
6000
-Mc
-ThennaRI
Spadden
P
4000 .-
0 0
Jt1 J+7 Jt14 Jt28
Days
FIG 8. Total radioactive intensity signal (cprn) as a function of the
immersion period in days. Cond. Lat., lateral condensation.
teeth comparable by using teeth with a single straight root, to avoid
the problems of multiple canals or differences between roots with
different curvatures. The teeth were also all of similar lengths so
that they would have the same volume available for infiltration.
Despite these attempts, natural anatomical variation among the
teeth was still a factor, but it was hoped that randomly assigning
the teeth to experimental groups and examining each group as a
whole rather than looking at individual teeth would give a fair
comparison between the groups.
The different obturation techniques also brought the potential
for variation. For example, Beatty et al. ( 2 ) noted that leakage was
reduced if the finger spreader tip was inserted to within 1 mm of
the working length. Uniformity of the depth reached by the finger
spreader is, however, difficult to standardize. In the same study
variations in the Thermafil technique were noted as a result of
nonuniform heating of the gutta-percha in a flame. This problem
was reduced by a newer Thermafil technique in which the carrier
is heated in an oven at a standard temperature for a uniform time.
Variations had also been shown with the McSpadden technique by
Harris et al. (6). who found that when used, sealer may become
incorporated in the gutta-percha giving the same effect as if no
sealer had been used, but this does not happen in every case. Again
the use of a single operator and analyzing the samples in groups
rather than as individual teeth attempts to reduce these problems.
Although it is important to minimize such variables, they will not
detract significantly from the results obtained because, clinically,
such anatomical and operator variations are also evident.
Subjective measurements in the previous studies have also in-
creased the margin of error (9, 13). In this study the use of the Iz5I
radioactive tracer detected by a gamma counter allows for quan-
titive measurements to be made at each root level, without affect-
ing the filling. The measurements were not subject to observer
variation and could be easily analyzed and compared. The gamma
counter gave accurate values for radioactivity in each sample while
taking into account the background radiation by measuring it
before beginning each sample.
are smaller than dye molecules and diffuse more rapidly than other
small molecules (7). The '2sI-labeled lysozyme used in this case
was chosen because of its low molecular weight. Although pene-
tration of bacteria into the root canal may be minimal due to their
large particle size and the fact that they are absorbed specifically
152 Haikel et al.
to the root surface by the action of immunoglobulins, penetration
of much smaller nutrients into the root canal system may encour-
age multiplication of bacteria that have not been removed during
canal preparation (8),and some antigenic substances released from
bacteria in the periapical region may easily enter the canal because
they may have a molecular weight as low as 1,000 D (10). Another
reason for choosing lysozyme as the tracer was that it does not
interact with tooth substance, whereas 45Ca that has been used in
the past undergoes ionic exchange with the apatite crystals and so
interferes with the true pattern of leakage (9).
To prevent isotope penetration apart from in the apical region,
nail polish was used to seal the external root surface. This tech-
nique is frequently used, but a study of surface sealing materials
(17) found high leakage levels with nail polish and concluded that
nail polish was not an adequate sealant for short-term microleakage
studies. Despite these findings, the negative control group showed
very low levels of isotope penetration, suggesting that nail polish
would seal the roots sufficiently for this study.
The high levels of leakage seen in the positive control group
indicated that lack of sealant cement will result in greatly increased
levels of apical microleakage and so confirmed the necessity for its
use in root canal obturation.
Another potential problem with the experimental technique that
had occurred in previous studies (12) was loss of tooth substance on
preparing the horizontal sections. The use of a fine diamond wire (0.17
mm diameter), which cut slowly and without pressure under lubrica-
tion, enabled the amount of tooth substance lost to be minimized.
One of the main findings of this study was that levels of apical
penetration varied over time. The results obtained support the
opinion of Delvanis and Chapman (9), who suggested that 2 days
(the period used in many apical leakage studies) is not long enough
for maximal leakage to be detected. Rhome et al. (1 8) undertook a
6-month study of leakage that demonstrated a rise and fall of
leakage values over the time for all methods of condensation
(lateral and vertical gutta-percha condensation and Hydron). They
found leakage to be greatest in the initial weeks (which is the time
period of this study).
Changes in permeability of the apical seal may be due to a
change in the properties of the filling over time. These changes
may be due to the material used, for example, the sealer cement is
likely to resorb over time resulting in increased leakage (19). The
obturation techniques involving heating of gutta-percha may also
cause an increase in permeability in the initial stages when shrink-
age occurs as the gutta-percha cools (2).It should, however, not be
forgotten that, as the samples are immersed for a longer time
period, readings will increase simply because infiltration is occur-
ring with the extended time. Throughout the experiment, the vari-
ations in permeability meant that different methods of obturation
appeared effective at different stages, so that no one method could
be said to be overall better than any other. In general the levels of
infiltration observed in all samples were low coronal to the third
section so that any of the techniques can be considered effective.
Journal of Endodontics
The authors thank B. Senger for his statistical assistance.
Drs. Haikel, Freymann, Fanti, and Poumier are affiliated with the Universite
Louis Pasteur, Faculte de Chirurgie Dentaire, Strasbourg, France. Drs. Hai’kel
and Poumier are affiliated with the Centre de Recherches Odontologiques.
National Institute of Health and Medical Research, Strasbourg, France. Dr.
Watson is affiliated with the ERASMUS Exchange Program, University of
Glasgow, Glasgow, Scotland. Dr. Claisse is affiliated with the Faculte de
Chirurgie Dentaire, Place de Verdun, Lille, France. Address requests for re-
prints to Dr. Y. Haikel, Universite Louis Pasteur, lnserm U-424, Faulte de
Chirurgie Dentaire, 1 Place de I’Hbpital, 67000 Strasbourg, France.
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