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A micro-computed tomographic evaluation of apicalroot canal preparation using three instrumentationtechniques
J. Moore, P. Fitz-Walter & P. ParashosMelbourne Dental School, The University of Melbourne, Melbourne, Victoria, Australia
Abstract
Moore J, Fitz-Walter P, Parashos P. A micro-computed
tomographic evaluation of apical root canal preparation using
three instrumentation techniques. International Endodontic
Journal, 42, 1057–1064, 2009.
Aim To investigate the morphological changes in the
apical third of the root canal after preparation with
three techniques.
Methodology Forty molar teeth were scanned
using micro-computed tomography before and after
instrumentation with: Group 1 – stainless steel K-files
using the balanced force technique; Group 2 –
stainless steel K-files (balanced force) and then refining
the apical preparation with the equivalent size 0.04
taper FlexMaster instrument; Group 3 – a hybrid
ProTaper/FlexMaster (ProFile for sizes 45 and 60)
sequence. Eight canals were excluded because of
artefacts in the images or unnegotiable blockages
leaving 110 canals that could be analysed. Apical root
canal preparation was evaluated with respect to the
amount of dentine removed, canal roundness, trans-
portation and how the dimensions of the prepared
apical root canal correlated with those of the final
instrument used.
Results The median apical preparation sizes for the
three groups respectively were: 30, 30 and 40.
Despite the larger size and less experienced operator,
the volumetric change (the amount of dentine
removed) in canals prepared with a hybrid rotary
nickel–titanium instrumentation technique remained
small and, a more rounded preparation (P < 0.001)
that closely matched the final instrument dimensions
(P < 0.001) was produced. There was a trend for less
canal transportation using rotary nickel–titanium
instruments.
Conclusions Stainless steel hand preparation was
not conservative of apical dentine. When used cor-
rectly, even by less experienced operators, rotary
nickel–titanium instruments were able to precisely
‘machine’ a canal to larger apical sizes with minimal
risk of iatrogenic damage.
Keywords: apical preparation, micro-computed
tomography, rotary NiTi, transportation.
Received 15 November 2008; accepted 15 July 2009
Introduction
It is well established that intra-radicular microbial
infection is the primary cause of apical periodontitis
(Kakehashi et al. 1965, Sundqvist 1976, Moller et al.
1981). Instrumentation forms an integral part of the
process of eliminating or reducing the number of
microorganisms to a level that will allow healing of the
periapical tissues (Bystrom & Sundqvist 1981).
Technically, the goal of endodontic instrumentation
is to remove all necrotic and vital pulp tissue along
with heavily infected radicular dentine. Instrumenta-
tion also shapes the root canals for improved irrigation,
placement of intracanal medicaments and facilitates
obturation to a high technical standard (Haapasalo
et al. 2005). However, any instrumentation that
removes excessive dentine and substantially changes
the canal anatomy will not only lead to iatrogenic
Correspondence: Peter Parashos, Melbourne Dental School,
Faculty of Medicine Dentistry and Health Sciences, The
University of Melbourne, 720 Swanston Street, Melbourne,
Vic. 3010, Australia (Tel.: +613 9341 1472; fax: +613 9341
1599; e-mail: [email protected] ).
doi:10.1111/j.1365-2591.2009.01626.x
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1057–1064, 2009 1057
Page 2
preparation errors (Weine et al. 1975, 1976) but it
may adversely affect the strength of the tooth (Sathorn
et al. 2005, Versluis et al. 2006).
Micro-computed tomography (micro-CT) is a nonde-
structive analytical method that has enabled research-
ers to examine the effects of root canal instrumentation
in three-dimensions (Nielsen et al. 1995, Peters et al.
2000, Rhodes et al. 2000, Bergmans et al. 2001). A
number of investigations have now been carried out
using micro-CT to examine the effects of different hand
and rotary instrumentation techniques on root canal
anatomy (Peters et al. 2001a,b, 2003, Bergmans et al.
2002, 2003, Hubscher et al. 2003). However, none
has specifically focussed on the apical portion of the
root canal or how the preparation dimensions relate to
the final instrument used.
This is an area of interest, as there is evidence that
the apical portion of the root canal may harbour a
critical level of microorganisms that could maintain
apical periodontitis (Nair et al. 1990) and that
increased apical debridement improves reduction in
the microbial levels (Siqueira et al. 1999, Shuping et al.
2000, Card et al. 2002, Rollison et al. 2002). Larger
apical preparation sizes will decrease the amount of
infected dentine, pulpal debris and canal irregularities
(Tan & Messer 2002), as well as improving efficacy of
irrigant solutions (Shuping et al. 2000, Khademi et al.
2006) and potentially clinical outcomes. Furthermore,
considering that the average size of the apical canal is
approximately 0.30–0.35 mm (Kuttler 1955, Kerekes
& Tronstad 1977), preparation to larger apical sizes
appears logical (Spangberg 2001).
Despite these benefits and knowledge of the apical
canal anatomy, the concept of larger apical sizes has not
been widely adopted because of concern over iatrogenic
apical preparation errors. The aim of this study was to
investigate the morphological changes in the apical
third of root canals after preparation with three instru-
mentation techniques using micro-CT scanning.
Materials and methods
Ethics approval for this research project was obtained
from the Health Sciences Human Ethics Sub-Commit-
tee, The University of Melbourne, Melbourne, Victoria,
Australia (Ethics ID: 0714905).
Preparation of specimens
Twenty-one maxillary and nineteen mandibular first
molar teeth with no history of endodontic treatment
were used. All teeth were stored in 0.9% saline
solution. Caries and restorations were removed and
access cavities prepared with a high speed diamond
bur. The occlusal surface was reduced by 2 mm to
provide reproducible reference points and positioning in
the micro-CT scanners (SkyScan 1072 and SkyScan
1076, Kontich, Belgium).
Teeth were mounted in the scanners with the flat
occlusal surfaces against an SEM stub (SkyScan 1072,
Kontich, Belgium) or resin disc (SkyScan 1076, Kon-
tich, Belgium) to allow reproducible orientation in the
pre- and post-instrumentation micro-CT scans. All
teeth were scanned by micro-CT prior to negotiation
of canals. No attempt was made to instrument second
mesio-buccal canals because their anatomy was too
variable and it may have compromised the assessment
of the main mesio-buccal canal.
Canals were negotiated to patency with size 8 and 10
Hedstrom files (Dentsply Maillefer, Ballaigues, Switzer-
land) and working length was set 1 mm from the apical
foramen. Digital radiographs were taken from bucco-
lingual and mesio-distal directions with size 10
Hedstrom files (Dentsply Maillefer, Ballaigues, Switzer-
land) in the canals to allow calculation of canal angles
and radius of curvature using image processing
software (ImageTool v3.0; UT Health Science Centre,
San Antonio, TX, USA) for incorporation into the
statistical analysis (Schneider 1971, Pruett et al.
1997). Teeth were divided evenly, ensuring an equita-
ble distribution of canal numbers, canal curvatures and
radii between the two operators and the canals were
allocated into groups representing three instrumenta-
tion protocols.
Group 1 was prepared by one operator using Gates
Glidden burs 2 and 3 (Dentsply Maillefer) for coronal
flaring and gaining straight line access to the middle
third, and the balanced force technique (Roane et al.
1985) with stainless steel K-files (Dentsply Maillefer)
for the remaining preparation. All the mesio-buccal
canals of maxillary and mandibular teeth in Group 1
were refined with the corresponding FlexMaster 0.04
taper (VDW, Munich, Germany) rotary nickel–
titanium instrument (e.g. 30 K-file refined with size
30, 0.04 taper FlexMaster) used in a torque-
controlled handpiece (Endo IT Professional, VDW,
Munich, Germany) at manufacturer recommended
settings. Those canals were then considered a sepa-
rate group (Group 2) in the analysis of the results.
Group 3 was prepared by the second operator using
a hybrid rotary instrumentation technique similar to
that described by Walsch (2004). After negotiation
Apical root canal preparation assessed by micro-CT Moore et al.
International Endodontic Journal, 42, 1057–1064, 2009 ª 2009 International Endodontic Journal1058
Page 3
with a size 15 Hedstrom file (Dentsply Maillefer),
ProTaper S1, S2 and F1 (Dentsply Maillefer) were
used to working length. The apical preparation was
finished with a FlexMaster 0.04 taper nickel–titanium
instrument (VDW). In situations where 0.04 taper
instruments larger than size 40 were required but
not available in the FlexMaster range, ProFile 0.04
taper rotary nickel–titanium instruments (Dentsply
Maillefer) were used (i.e. sizes 45 and 60). All rotary
nickel–titanium instruments were used in a torque-
controlled handpiece (Endo IT Professional, VDW,
Munich, Germany) at manufacturer recommended
settings.
New instruments were used for each tooth and 1%
sodium hypochlorite was used as an irrigant. The
method used to determine the final apical preparation
size in Groups 1 and 2 (where the balanced force
technique was utilized) was based on the original
Grossman criterion of three sizes larger than the first
file that bound at working length (Grossman et al.
1988). In Group 3, the operator observed the apical
portion of the instrument for the presence of dentine
debris. Once the instrument flutes in the apical third
were loaded with dentine debris, the apical preparation
was considered to be complete. The minimum, median
and maximum master apical file sizes for each group
are presented in Table 1. The preparation technique
described for Group 1 was chosen because that
operator had successfully used it in private specialist
endodontic practice for over 20 years. The technique
described in Group 2 was a recent modification to it.
The canal preparation technique used for Group 3 was
what is taught in the graduate endodontic programme
at the Melbourne Dental School and as such the second
operator was familiar with it, having used it exclusively
for over 2 years.
Micro-CT measurements and evaluations
The micro-CT machines (SkyScan 1072 and 1076,
Kontich, Belgium) were used at 80 kV to scan the
specimens. Two machines were used in order to allow
scanning in the shortest period of time. Both scanners
produced the same resolution images and all the scans
were analysed with the same software. The same
machine was used for a specific tooth before and after
instrumentation to avoid inter-machine variability.
Typically 700-900 slices (voxel size 17.4 · 17.4
· 17.4lm) were scanned per tooth. Canals were
reconstructed using NRecon volumetric reconstruction
software (v1.4.4. SkyScan, Kontich, Belgium) and
analysed with CT Analyser image analysis software
(v1.6.1.1. SkyScan, Kontich, Belgium). Of the original
118 root canals, eight (two from Group 1, one from
Group 2 and five from Group 3) had to be excluded
because of artefacts in either the pre or post-instru-
mentation images or blockages that prevented negoti-
ation to the apical foramen. This left a total of 110 root
canals that could be analysed quantitatively.
The volume of interest was set using the technique
described by Peters et al. (2000). However, the vertical
range was limited to 7 mm from the apical foramen
allowing calculation of the amount of dentine removed
and the roundness of the apical 6 mm of canal
preparation. In contrast, canal transportation and the
difference between the canal dimensions and those of
the final instrument used were only calculated over the
apical 1 mm of canal preparation (located 1–2 mm
from the apical foramen).
The amount of dentine removed was represented by
the change in volume (D Volume) and was calculated
as the difference between the pre- and post-instrumen-
tation canal values. The post-instrumentation struc-
Table 1 Group information and morphometric scores evaluating apical canal preparation (mean ± SD)
Group n
Minimum, Median,
Maximum MAF D Volume (mm3)
Structural model
index D Centroid (mm) D Diameter (mm)
1 39 25, 30, 35 1.00 ± 0.66 2.63 ± 0.18 0.060 ± 0.047 0.026 ± 0.020
2 18 25, 30, 30 0.94 ± 0.58c 2.66 ± 0.15 0.076 ± 0.052 0.023 ± 0.018d
1/2 57 25, 30, 35 0.98 ± 0.63 2.64 ± 0.17 0.065 ± 0.049 0.025 ± 0.019
3 53 30, 40, 60 0.96 ± 0.47 2.83 ± 0.12a 0.052 ± 0.057 0.013 ± 0.012b
Master apical file (MAF) values refer to the minimum, median or maximum final instrument size used in any canal in that particular
group.aStatistically different from Group 1 (P < 0.001), Group 2 (P = 0.003) and combined Group 1/2 (P < 0.001).bStatistically different from Group 1 (P = 0.007) and combined Group 1/2 (P < 0.001).cMean is less than Group 1 because this group does not contain any P or D canals which had the highest mean D Volume scores (1.03
and 1.27 mm3 respectively).dMean is less than Group 1 because the average instrument diameter is larger, making the difference between the preparation and
instrument dimensions smaller.
Moore et al. Apical root canal preparation assessed by micro-CT
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1057–1064, 2009 1059
Page 4
tural model index (SMI) was recorded to quantify the
canal ‘roundness’ (Lorensen & Cline 1987, Hildebrand
& Ruegsegger 1997b, Peters et al. 2000). SMI values
will range between 0 and 4, where 0 corresponds to an
ideal plate, 3 an ideal cylinder and 4 a sphere
(Hildebrand & Ruegsegger 1997b). In an endodontic
application of this morphological parameter, the closer
a value is to 3, the closer the preparation represents a
cylinder and the ‘rounder’ the canal (Peters et al.
2000).
The average canal diameter, described by Hildebrand
& Ruegsegger (1997a), was calculated over the apical
1 mm of the preparation and compared to the theoret-
ical average diameter of the master apical file (MAF).
Any difference was recorded as D Diameter and
indicated how the preparation dimensions correlated
with those of the MAF. Transportation (D Centroid)
over the apical 1 mm of canal preparation was also
recorded. This involved calculating the three-dimen-
sional distance between the centre of the canal and the
centre of the external root surface using a Euclidean
distance calculator (Teknomo 2006). Any change
between the pre- and post-instrumentation distances
represented transportation.
Statistical analysis
Means and standard deviations were calculated for
each test group and analysed using Minitab� 15.1.1.0
(2007). anova tests were conducted to determine if
there was any statistical difference between the exper-
imental groups for a special outcome variable at
P = 0.05. Where a statistical difference between groups
was noted, pair-wise analysis was undertaken using
Fisher’s LSD (least significant difference) test.
To determine the impact of any pre- or intra-
operative variable (working length, canal angle, radius
of curvature, presence of an s-shaped canal and MAF),
the anova model was refit and pair-wise analysis using
Fisher’s LSD test was used when a statistical difference
was noted between groups. Analysis was conducted
without the pre-operative variables of S-shaped canals
because of the lack of a unique canal angle or radius.
The complete statistical analysis was repeated after
data from Groups 1 and 2 were combined.
Results
Scanning of the canals before and after instrumenta-
tion produced cross-sectional images that were exam-
ined for shape and the presence of any procedural
errors. The cross-sections were subsequently recon-
structed and analysed to quantify the three-dimen-
sional morphological changes in each canal. Generally,
as is demonstrated in Fig. 1, rotary nickel–titanium
instruments maintained the original canal position,
produced round, uniformly tapered preparations and
were free from procedural errors. Canals prepared with
stainless steel instruments had a more irregular cross-
sectional appearance and taper, with deficiencies noted
apically and where Gates Glidden instruments were
used to remove dentine irregularities and interferences.
A summary of the data is presented in Table 1. As
Group 2 was essentially the same preparation tech-
nique as Group 1, but with the final canal shape refined
using a rotary nickel–titanium instrument, those two
groups were combined for further statistical analysis
(Group 1/2). The mean (± SD) bucco-lingual angle and
radius of curvature for all the canals were
20.03 ± 13.23 degrees and 5.60 ± 1.55 mm respec-
tively; and the mean (± SD) mesio-distal canal angle
and radius of curvature were 13.65 ± 11.21 degrees
and 5.61 ± 1.64 mm respectively.
The only statistical differences observed between the
experimental groups were how ‘round’ the canals were
(SMI) and how close the prepared canal dimensions
were to the final instrument used (D Diameter). The
SMI in Group 3 (2.83 ± 0.12) was statistically different
from Group 1 and Group 2 as well as the combined
Group 1/2 (P < 0.001, P = 0.003 and P < 0.001
respectively). With respect to the D Diameter, Group
3 was statistically different from Group 1 and the
combined Group 1/2 (P = 0.007 and P < 0.001
respectively). The amount of dentine removed (DVolume) and the canal transportation (D Centroid)
were less in Group 3 compared to Group 1 and the
combined Group 1/2 however, the results were not
statistically different.
Structural model index and D Diameter were further
analysed using an anova model adjusting for pre- and
intra-operative variables. Group 1 always remained
statistically different from Group 3 with respect to both
SMI and D Diameter. However, increasing the MAF had
a detrimental effect on canals in Group 2 with the
Group 2–3 comparison of D Diameter becoming statis-
tically significant (P = 0.036). When examining the
effect of the canal angle, as it increased, the mean DDiameter of Groups 1 and 2 became significant
(P < 0.001). These results indicated that the two
influential factors on the results were the final instru-
ment size (MAF) and the canal angle. As either of those
was increased, the difference between the instrumen-
Apical root canal preparation assessed by micro-CT Moore et al.
International Endodontic Journal, 42, 1057–1064, 2009 ª 2009 International Endodontic Journal1060
Page 5
tation techniques became more noticeable. When
Groups 1 and 2 were combined and the analysis
repeated, no pre- or intra-operative variable was found
to be influential. As the preparation in each group was
only performed by one operator, it was not possible to
determine the effect of group over operator in the
statistical analysis.
Discussion
The principal limitations concerning the methodology
of this study were the fact that the final instrument size
(MAF) was not standardized and that the experience of
the operators varied greatly. The nonstandardized
apical preparation sizes do make a direct comparison
of the change in volume (D Volume) and the conclu-
sions drawn from it initially seem invalid. However,
when consideration is given to the fact that the median
MAF in Group 3 is the largest, yet the change in
volume remains small, it would appear that the results
do in fact support the hypothesis that rotary nickel–
titanium instruments are conservative in the total
amount of dentine they remove. A similar argument
could be proposed with respect to operator variability
and the effect on the results. Whilst it cannot be ruled
out statistically, as the less experienced operator
actually achieved better results, this would seem to
offer support to the notion that it was the technique
rather than the operator that had the greatest effect on
the results seen in this investigation.
The morphological parameters measured in this
study have been previously described and applied to
endodontic investigations in the literature (Peters et al.
2000, 2001a,b, 2003, Bergmans et al. 2002, 2003,
(a) (b) (c)
(d) (e) (f)
Figure 1 Examples of micro-computed tomography (micro-CT) cross-sectional images from the apical third of root canals and the
three-dimensional reconstructions. Pre-instrumentation (a) and post-instrumentation (b) micro-CT slices of a tooth prepared using
Gates Glidden drills and stainless steel K-files then refined with a rotary nickel–titanium instrument in the MB canal (final
preparation sizes: MB 30/0.04, DB 25/0.02, P 35/0.02); three-dimensional reconstruction of tooth presented in a–b (c); pre-
instrumentation (d) and post-instrumentation (e) micro-CT slices of a tooth prepared using rotary nickel–titanium instruments
(final preparation sizes: MB 40/0.04, DB 40/0.04, P 45/0.04); three-dimensional reconstruction of tooth presented in d–e (f). Note
the rounder, more centered preparations in canals prepared with rotary nickel–titanium instruments (e) and the excessive dentine
removal from Gates Glidden preparation steps (c).
Moore et al. Apical root canal preparation assessed by micro-CT
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1057–1064, 2009 1061
Page 6
Hubscher et al. 2003). However, the results of this
study are not directly comparable because of the
different area of interest. All of the previous studies
(Peters et al. 2001a,b, 2003, Bergmans et al. 2002,
2003, Hubscher et al. 2003) examined the entire
length of the canal (apical foramen to furcation)
whereas this study was only interested in the apical
portion. Consequently, D volume was not as large as
that seen in Peters et al. (2001a,b, 2003), Bergmans
et al. (2002, 2003) or Hubscher et al. (2003) and
postoperative SMI was smaller because of the more
complex apical morphology.
The results for transportation were similar to previ-
ous studies, though again, it is difficult to compare
them because Peters et al. (2001a,b, 2003) and Hub-
scher et al. (2003) measured changes in centroid over a
larger range and Bergmans et al. (2002, 2003) mea-
sured movement in eight different directions, not
stating an overall figure. A recent study (Cheung &
Cheung 2008) has compared the final canal dimen-
sions to that of the final instrument used and concluded
similarly to this study, that the final preparation taper
and dimensions closely match those of the rotary
nickel–titanium instruments used. However, whilst a
similar trend is observed, because they did not state the
actual values in their paper, a direct comparison of
results is not possible.
The results of this study indicated that a hybrid
rotary instrumentation technique (Group 3) (Walsch
2004), removed similar or less dentine despite the
largest median MAF size. The hybrid technique also
produced the least mean transportation although both
these findings were not statistically significant. Canals
prepared by rotary nickel–titanium instruments (Group
3) were statistically rounder and the dimensions more
closely matched that of the final instrument used. This
correlates well with the results showing less dentine
removal (D Volume) and less transportation (D Cen-
troid). Investigation into the effects of pre- and intra-
operative variables revealed that the canal angle and
MAF had an impact on the results. As the canal angle
or the MAF size was increased, the difference between
Group 3 and either Group 1 or 2 became more obvious,
reflecting the advantages of rotary nickel–titanium
instruments over that of stainless steel in curved root
canals. Clinically, this would equate to a more ‘con-
servative’ and ‘safer’ preparation despite the increased
apical debridement.
The results of this study agree with previous inves-
tigations into the advantages of rotary nickel–titanium
instruments over stainless steel instruments (Esposito &
Cunningham 1995, Glossen et al. 1995). However,
they are not as dramatic because the methodology in
this study did not specify an MAF size. Instead, each
was prepared to a size determined by the operator to be
appropriate for the specific canal which may be less
scientifically valuable but more clinically relevant.
The clinical implications of this study are that using
a predominantly stainless steel hand preparation tech-
nique may not be as conservative of apical root canal
dentine and that when used correctly, even by less
experienced practitioners, rotary nickel–titanium
instruments are able to precisely prepare a canal to
larger apical sizes with minimal risk of iatrogenic
damage.
Conclusion
The results of this study suggest that a hybrid rotary
nickel–titanium instrumentation technique produces
rounder canals (P < 0.001) whose dimensions more
closely match those of the final instrument used
(P < 0.001). They also highlight the trend that despite
a larger median apical preparation size, rotary nickel–
titanium instruments remove less total dentine and
result in less transportation. This was especially the
case in canals with more severe curvatures where
undesired apical dentine removal by stainless steel
instruments became more pronounced.
Based on this evidence and that of previous studies
comparing stainless steel hand preparation with rotary
nickel–titanium instrumentation, using rotary nickel–
titanium instruments to prepare root canals to larger
apical sizes (around 0.40 mm) is supported and carries
a minimal risk of iatrogenic damage even in the hands
of less experienced operators.
Acknowledgements
This study was supported by a grant from the Austra-
lian Society of Endodontology Incorporated. All end-
odontic instruments were donated by Dentsply
(Australia) Pty Ltd and Gunz Dental. Sandy Clarke,
from the Statistical Consulting Centre, University of
Melbourne provided statistical support.
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Distance from file tip to the major apical foramen inrelation to the numeric meter reading on the displayof three different electronic apex locators
R. A. Higa, C. G. Adorno, A. K. Ebrahim & H. SudaPulp Biology and Endodontics, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University,
Tokyo, Japan
Abstract
Higa RA, Adorno CG, Ebrahim AK, Suda H. Distance
from file tip to the major apical foramen in relation to
the numeric meter reading on the display of three
different electronic apex locators. International End-
odontic Journal, 42, 1065–1070, 2009.
Aim To establish and compare the relationship
between the distance from the file tip to the apical
foramen and the numeric meter reading on the display
of three different electronic apex locators (EALs).
Methodology A total of 12 extracted intact,
straight, single-rooted human teeth with complete roots
were used. The actual root canal length (AL) was
determined after access preparation. For the electronic
measurements with each EAL, silicon stops were fixed
with auto-polymerizing resin to size 15 K-files at AL and
0.5, 1, 2, 3, 4 mm short of AL. The data was analysed
by two-way anova and Tukey’s honestly significant
difference (HSD) test for multiple comparisons amongst
EALs. Additionally, one-way anova and Tukey’s HSD
test were carried out for multiple comparisons amongst
the measurements of each EAL.
Results There was a statistically significant difference
amongst all EALs in indicating the position of file tips in
relation to the major foramen (P < 0.05). The corre-
lation between the meter reading and the position of
the file tip from the apical foramen was statistically
significant in the three EALs. There were significant
differences amongst the measurements at distances
from 0 to 2 mm in Justy III. In Dentaport, significant
differences were found from 0 to 1 mm. However, the
E-Magic Finder showed significant differences from 0 to
0.5 mm.
Conclusions Justy III was more capable of display-
ing the intracanal position of the file tip from the major
foramen in mm whilst advancing through the root
canal during electronic measurements than the Den-
taport and E-Magic Finder Deluxe.
Keywords: distance to apical foramen, electronic
apex locator, meter reading display, root canal length
determination.
Received 21 April 2008; accepted 05 August 2009
Introduction
Working length determination is an essential step in
root canal treatment. The apical constriction is the
recommended end-point of instrumentation and obtu-
ration (Ricucci & Langeland 1998). The tooth pulp is
narrow at the apical constriction; therefore the wound
is minor, potentially providing optimal healing condi-
tions (Kuttler 1955). The location of the apical
constriction is considered to be 0.5–1 mm short of
the anatomical apex (Kuttler 1955, Tselnik et al.
2005). Over-instrumentation and over-filling has been
reported to cause tissue destruction, inflammation and
foreign body reaction in the periapical tissue area
(Kuttler 1955, Seltzer et al. 1968, 1969).
The development of electronic apex locators (EALs)
has helped to make the assessment of working length
Correspondence: Romina Andrea Higa, Fray Jose Leon Torres
1112, Alta Cordoba, CP 5001, Cordoba, Argentina (Tel.: 54
0351 4718387; fax: 54 0351 4718387; e-mail: romikawa@
hotmail.com).
doi:10.1111/j.1365-2591.2009.01629.x
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1065–1070, 2009 1065
Page 10
more accurate and predictable (Pratten & McDonald
1996, Fouad & Reid 2000, Hoer & Attin 2004, Plotino
et al. 2006). Modern EALs determine distance from
the end of the apex by comparing impedances, which
are measured by using different current frequencies
(Gordon & Chandler 2004). The difference in imped-
ance is calculated in order to determine a position of
the file in the canal (Kobayashi & Suda 1994, Azabal
et al. 2004).
The Justy III (Yoshida Co., Tokyo, Japan) and the
E-Magic Finder Deluxe (DESTI S-Denti Co., Ltd, Chung-
nam, Korea) are new EALs. The Justy III uses 500 Hz
and 2 kHz as measuring frequencies. It is presented as a
foldable LCD display, and when the meter value of the
scale becomes 2.5, a larger image is shown on the
screen. On the other hand, the E-Magic Finder Deluxe
uses 500 Hz and 5 kHz as measuring frequencies. Also
designed with a foldable LCD display, it can be
connected to a computer which allows a vivid graphic
display. Both the Justy III and the E-Magic Finder
Deluxe claim that their numeric meter reading display
show the distance in mm from the apical foramen
during their measurements.
Several studies have evaluated the accuracy of
different apex locators by calculating the distance from
the file tip to the apical foramen or apical constriction
using apex or 0.0, apical constriction or 0.5, and 1
reading marks (Martinez-Lozano et al. 2001, Tselnik
et al. 2005, D’Assuncao et al. 2007). However, few
studies have considered the display of all meter
readings on the display.
The aim of this laboratory study was to establish and
compare the relation between the distance from the file
tip to the major apical foramen and the numeric meter
reading on the display of three different apex locators:
Justy III, Dentaport and E-Magic Finder Deluxe.
Materials and methods
Extracted intact, straight, single-rooted human teeth
with complete root formation were selected randomly.
Teeth with resorption or fracture were excluded. Pre-
operative digital radiographic images in both buccolin-
gual and mesiodistal directions were taken to evaluate
root canal anatomy and teeth with accessory canals or
invisible main canals were excluded. Twelve teeth were
finally selected. All teeth were soaked in tap water for
2 h before use. Standard access preparation was carried
out using a high speed diamond fissure bur (Mani,
Tochigi, Japan) under water-cooling. The incisal or
occlusal edges were ground to create a flat surface to
facilitate length measurements. The actual root canal
length (AL) was determined by introducing a size 10 or
15 K-file (Zipperer, Munich, Germany) into the canal
until the tip of the file emerged through the major
apical foramen under a digital microscope (VH-S30;
Keyence, Osaka, Japan) at 20· magnification. The long
axis of the tooth was placed perpendicular to the line of
sight and the tip of the file was positioned tangential to
the major apical foramen (Fig. 1). A rubber stop was
carefully adjusted to the reference point and the
distance between the file tip and the rubber stop was
measured with a digital caliper (Sankin; Mitutoyo Co,
Kanagawa, Japan) to the nearest 0.5 mm. The mea-
surements were repeated three times and the mean was
taken as the definitive length.
Gates Glidden drills (size 1–4, Mani) were used to
prepare the coronal portion of the canals. Each canal
was irrigated using 2 mL of 6% sodium hypochlorite
solution (NaOCl) through a 27-gauge needle (Nipro,
Osaka, Japan) during cleaning. Patency was constantly
checked using a size 10 K-file.
The lid of a polystyrene specimen bottles (20 mL,
Iuchi, Osaka, Japan) was used to fix each tooth. The
bottles were filled with alginate (GC Corporation,
Tokyo, Japan) and, upon setting the root of the
corresponding tooth was embedded in it, leaving
Figure 1 Actual canal length determination. A size 15 K-file
was introduced into the canal until the tip of the file emerged
through the major apical foramen. The tip of the file was
positioned tangential to the major apical foramen.
Apex locators’ meter reading Higa et al.
International Endodontic Journal, 42, 1065–1070, 2009 ª 2009 International Endodontic Journal1066
Page 11
approximately 2 mm of the cervical root surface
exposed for stabilization using auto-polymerizing resin.
The tooth was kept in that position until the alginate
had completely set (Fig. 2).
The three EAL used to measure the twelve teeth in
this experiment: the Dentaport ZX (J. Morita Co.,
Kyoto, Japan), the Justy III and the E-Magic Finder
Deluxe. Each device was used according to the
manufacturers’ instructions. Size 15 K-files were used
with the EALs. Silicon stops were fixed with auto-
polymerizing resin to the files at the following
distances: AL and 0.5, 1, 2, 3, 4 mm short of the
AL. A file was gently inserted into the root canal until
the signal was emitted by the corresponding EAL. All
the electronic measurements were performed three
times and the mean was calculated.
Two-way analysis of variance (anova) and Tukey’s
HSD test were used to evaluate differences amongst
EALs. One-way anova and the Tukey’s HSD test were
used to evaluate differences amongst the measurements
of each EAL. In addition, the correlation between the
file tip-apical foramen distance and electronic measure-
ments, meter reading mean values, was analysed with
the Pearson Correlation Coefficient. The analysis was
carried out with JMP 7 software (SAS Institute, Cary,
NC, USA).
Results
Table 1 illustrates one-way anova and Tukey’s HSD
test results. The mean and standard deviation of the
meter readings with three EALs at different distances of
the file tip from the apex are shown. The indicated
mean meter reading of Justy III were significantly
different except at 3 and 4 mm (P < 0.05). For the
Dentaport, the mean readings at 0, 0.5 and 1 mm were
significantly different (P < 0.05). The mean reading of
the E-Magic Finder Deluxe at 0.5, 2, and 3 mm showed
no significant difference with 1, 3, and 4 mm respec-
tively (P < 0.05).
Two-way anova and Tukey’s HSD test showed
significant differences amongst the three EALs (P <
0.05). The correlation between distance of the file
tip from the major apical foramen and mean meter
readings was statistically significant (P < 0.001). The
Pearson Correlation Coefficient was 0.88 for Justy III,
0.83 for Dentaport and 0.74 for E-Magic Finder Deluxe.
Discussion
Many studies have reported the accuracy of EALs to
determine root canal length (Hoer & Attin 2004,
Lucena-Martin et al. 2004, ElAyouti & Lost 2006,
Plotino et al. 2006, Smadi 2006, Bernardes et al. 2007,
D’Assuncao et al. 2007, Wrbas et al. 2007). In addi-
tion, it is common knowledge that the numbers on the
display of the EALs do not correspond to the actual
distance in millimetres to the minor or major foramen.
Rather, they are arbitrary units indicating if the file tip
is moving closer or further from the foramen (Tselnik
et al. 2005). However, two new devices on the market,
Justy III and E-Magic Finder Deluxe, claim that the
readings on the display do show the distance in
millimetres from the apical foramen during measure-
ments. The purpose of this study was to evaluate the
capability of EALs to determine the distance in mm
from the apical foramen whilst the file tip is advancing
through the root canal.
Laboratory studies on EALs have made use of
different media in which the teeth are embedded to
simulate the clinical situation. The alginate model was
chosen for this experiment for its good electroconduc-
tive properties, ease of preparation, stability and firm
consistency (Baldi et al. 2007, Herrera et al. 2007).
The actual canal length was determined before the
flaring with Gates Glidden drills. Owing to the fact that
flaring with Gates Glidden files might alter the root
canal length, measurements were performed before and
after flaring and no difference was found largely
because teeth with straight roots were used.
The Dentaport ZX is comprised of two modules: the
Root ZX and the Tri Auto ZX, a rotary canal prepara-
tion handpiece with a nickel titanium instrument. The
Root ZX has become the benchmark to which other
EALs are compared (Plotino et al. 2006, Bernardes
et al. 2007). No data about the Justy III and E-Magic
Finder Deluxe could be found. Six percent NaOCl
was selected as the irrigant solution for this experi-
ment. Previous studies reported that NaOCl irrigation
Figure 2 Experimental set-up used in this study.
Higa et al. Apex locators’ meter reading
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1065–1070, 2009 1067
Page 12
improved the accuracy of measurements with Root ZX
(Meares & Steiman 2002, Ebrahim et al. 2006).
The present study showed that, as the distance of
the file tip from the apical foramen increased, the
differences amongst the mean of the numeric meter
readings became larger. The greatest differences were
noticed when the distance between the file tip and the
apical foramen was 4 mm. According to previous
reports, the accuracy of measurements increases as
the file tip approaches the foramen (Kobayashi & Suda
1994, Venturi & Breschi 2007).
ElAyouti & Lost (2006) suggested that accuracy and
repeatability should be considered in the evaluation of
EALs. The Dentaport provided the most stable elec-
tronic measurements when considering the mean
standard deviation (SD) of the meter readings of the
distance of the file tip from the apical foramen. The
maximum and minimum SD (max SD and min SD)
were 0.53 at 1 mm and 0.04 at 4 mm, respectively. On
the other hand, Justy III and E-Magic Finder Deluxe
showed min SD at 0 mm and 0.5 mm, respectively,
and max SDs were close to 1 at 4 mm. Although SDs at
0 mm are greater than the average at the same
distance, all the measurements were within the accept-
able clinical range of AL ± 0.5 mm. Similar results
were reported by Meares & Steiman (2002) and Venturi
& Breschi (2005).
If the estimated working length is considered to be
AL ± 0.5 mm, which is clinically acceptable, then the
measurements made with the three EALs at 0.5 mm
from apical foramen were acceptable. The results are in
agreement with the previous reports that EALs can
accurately determine root canal length within
±0.5 mm from the apical constriction (Fouad et al.
1989, Czerw et al. 1995, Vajrabhaya & Tepmongkol
1997, Plotino et al. 2006). When the position of the file
tip was at the major apical foramen, some of the
measurements by the three EALs were positive as the
file tip was beyond the major foramen. According to
Wrbas et al. (2007) and D’Assuncao et al. (2007), the
apical constriction should be used as a benchmark for
working length determination instead of the major
apical foramen to reduce overpreparation.
Clinically, the measurement of root canal length
with the use of EAL in conjunction with tactile
sensation has better results than radiographs (Pilot &
Pitts 1997). However, for inexperienced dental clini-
cians, the numeric meter reading values of EALs could
become a useful guide if they indicate the file tip
position within the root canal whilst developing tactile
sensitivity skills. In the present study, the relationship
between the numeric meter readings and the position of
the file was based on correlation analysis, and signif-
icant differences were found amongst the mean
numeric readings within each EAL. The Pearson
Correlation Coefficient indicated that the three EALs
revealed a statistically significant correlation between
the numeric meter reading and the distance of the file
tip from the major apical foramen. The Justy III
presented a higher level of correlation followed by the
Dentaport and the E-Magic Finder Deluxe. On the other
hand, the mean numeric meter readings by Justy III at
different distances from the apical foramen were
significantly different at 0, 0.5, 1 and 2 mm. The
Dentaport readings were significantly different at 0, 0.5
and 1 mm. This result shows discrepancy with the
previous study. Oishi et al. (2000) reported that the
Root ZX showed correlation between measurements
and file tip position whilst the file is up to 5 mm from
the apex. The E-Magic Finder Deluxe results showed
that mean meter readings at 0.5, 2, and 3 mm were
not significantly different from the 1, 3, and 4 mm
ones, respectively. According to the results obtained in
the present study, the Justy III correlates the distance
and the numeric meter reading display when the file
is within 2 mm from the apical foramen whilst the
Table 1 Mean ± SD of meter readings at
different intracanal positions of file tip
from the major apical foramen
Intracanal position of
file tip from major
apical foramen (mm) Justy III Dentaport E-Magic Finder
Meter readings
0 0.08 ± 0.12 A +0.09 ± 0.33 A 0.13 ± 0.33 A
0.5 0.72 ± 0.27 B 0.90 ± 0.49 B 0.59 ± 0.13 B
1 1.60 ± 0.61 C 2.13 ± 0.53 C 0.85 ± 0.38 B
2 3.32 ± 0.93 D 2.93 ± 0.13 D 1.50 ± 0.75 C
3 3.86 ± 0.86 E 2.93 ± 0.21 D 1.93 ± 0.96 C D
4 4.12 ± 0.94 E 3.01 ± 0.04 D 2.24 ± 1.02 D
Different alphabet letters (A, B, C, D and E) indicate statistically significant differences
(P < 0.05) amongst measurements within each EAL.
EAL, electronic apex locator; +, measurements are beyond the apical foramen.
Apex locators’ meter reading Higa et al.
International Endodontic Journal, 42, 1065–1070, 2009 ª 2009 International Endodontic Journal1068
Page 13
Dentaport shows correlation when the file is within
1 mm. These results disagree with the claims made in
Justy III and E-Magic Finder’s catalogue that state the
numeric reading on the display shows the distance in
millimetres from the apical foramen.
Conclusion
The accuracy of monitoring root canal length varies
amongst EALs. The Justy III was more capable of
displaying the intracanal position of the file tip to the
major foramen in mm whilst advancing through the
root canal during electronic measurements than the
Dentaport and E-Magic Finder Deluxe.
The relation between the distance from the major
apical foramen and the numeric meter reading display
was proved in Justy III when the file was within 2 mm
from the apical foramen whilst in the Dentaport when
the file was within 1 mm.
The readings ‘0.0’ or ‘apex’ and ‘0.5’ showing the
intracanal position of the file tip at the major and the
minor foramen was satisfactory by Justy III, Dentaport
and E-Magic Finder Deluxe.
Further studies are needed to evaluate the three EALs
clinically. The locators developed to date have their
own internal circuit and characteristics to process and
establish the file tip intracanal position from the major
apical foramen and to express this numerically on the
LCD display of the meter.
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Micro-computed tomography of tooth tissuevolume changes following endodontic proceduresand post space preparation
O. H. Ikram, S. Patel, S. Sauro & F. MannocciDepartment of Conservative Dentistry, King’s College London Dental Institute, London, UK
Abstract
Ikram OH, Patel S, Sauro S, Mannocci F. Micro-computed
tomography of tooth tissue volume changes following end-
odontic procedures and post space preparation. International
Endodontic Journal, 42, 1071–1076, 2009.
Aim To compare the volume of hard tooth tissue lost
after caries removal, access cavity preparation, root
canal preparation, fibre post space and cast post
preparation in carious premolar teeth. The null
hypothesis tested was that there is no difference
between the volumes of hard tooth tissue lost expressed
as a percentage of the preoperative hard tooth tissue
volume, after each operative procedure.
Methodology Twelve extracted human premolars
with mesial or distal carious cavities penetrating into
the pulp chamber were selected. Teeth were scanned
using a microCT scanner. After each operative proce-
dure the loss of hard tooth tissue volume was
measured. The data were statistically analysed using
one-way analysis of variance and Fisher’s PLSD test
with statistical significance set at a = 0.01.
Results The percentage of preoperative hard tooth
tissue volume lost after caries removal was 8.3 ± 5.83,
after access cavity preparation the loss of volume reached
12.7 ± 6.7% (increase of 4.4%). After root canal prepa-
ration, fibre post space and cast post preparation the hard
tissue volume lost reached, 13.7 ± 6.7 (increase of 1%),
15.1 ± 6.3 (increase of 1.4%) and 19.2 ± 7.4 (increase
of 4.1%) respectively. Each procedure performed after
caries removal significantly increased (P < 0.01) the
amount of hard tissue volume lost with the exception of
the root canal preparation.
Conclusions Access cavity and post space prepara-
tion are the procedures during root canal treatment
which result in the largest loss of hard tooth tissue
structure. Cast post space preparation causes a larger
loss of tooth structure than fibre post space preparation.
This should be taken into account when planning root
canal treatment and restoration of root filled teeth that
are to be restored with cuspal coverage restorations.
Keywords: cast posts, dentine, fibre posts.
Received 25 February 2009; accepted 5 August 2009
Introduction
Root filled teeth are more susceptible to fracture when
compared with teeth with vital pulps. There are several
reasons for the high incidence in fractures observed in
root filled teeth. First, the physical properties of the
dentine may be altered by the interaction of medica-
ments and irrigants (Grigoratos et al. 2001). A loss of
proprioception occurs when the pulp tissue is removed.
It has been shown that teeth with non vital pulps have
a higher load perception and take up to twice the
amount of loading compared with a vital pulp to
register discomfort (Randow & Glantz 1986). Finally,
loss of tooth structure, in particular loss of the marginal
ridge(s) results in increased cusp flexure ex vivo (Reeh
et al. 1989).
To assess how the loss of tooth tissue caused by
restorative procedures and root canal treatment may
Correspondence: Francesco Mannocci, Department of Conser-
vative Dentistry, King’s College London Dental Institute, Guy’s
Tower, Guy’s Hospital, St Thomas’ Street, London SE1 9RT,
UK (Tel.: +447515398390; fax +442071881583; e-mail:
[email protected] ).
doi:10.1111/j.1365-2591.2009.01632.x
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1071–1076, 2009 1071
Page 16
weaken the tooth it is important to measure the
amount of hard tooth tissue (dentine) removed at each
stage of root canal treatment and subsequent restora-
tion. This has not been assessed previously.
High resolution micro-computed tomography (micro
CT) has been extensively used to evaluate three
dimensional shapes and volumes of canals following
root canal instrumentation (Peters et al. 2000, 2003).
In clinical studies comparing the survival of root
filled treated teeth restored with different techniques,
attempts have been made to standardize the loss of
tooth structure before the start of the restorative
treatment (Bolla et al. 2007). In one recent randomized
clinical trial the loss of tooth structure was classified on
the basis of the number of dental walls left (Ferrari et al.
2007), other studies have limited their investigation to
Class 2 cavities (Mannocci et al. 2002). The use of posts
in premolar teeth with three coronal walls is supported
by the favourable results of a recent randomized clinical
trial (Ferrari et al. 2007).
The aim of this micro CT study was to compare the
loss of hard tooth tissue volume caused by various
operative stages (caries removal, access cavity, root
canal preparation with nickel titanium instruments,
fibre post and cast post space preparation) involved in
root canal treatment and subsequent restoration of the
tooth in extracted premolar teeth with mesial or distal
carious cavities penetrating into the pulp space.
The null hypothesis tested was that there is no
difference between the loss of volume of hard tooth
tissue expressed as a percentage of the preoperative
hard tooth tissue volume, after each operative
procedure.
Materials and methods
Twelve extracted human mature premolar teeth were
used. The relationship of the lesion and the pulp
chamber was assessed using periapical radiographs
taken in bucco-lingual and mesio-distal projections. All
teeth had mesial or distal lesions penetrating into the
chamber. Teeth with both mesial and distal lesions
and/or with previous restorations were excluded from
study.
The teeth were scanned using a GE Locus SP
microCT scanner (GE Pre-clinical Imaging, London,
ON, Canada). A custom sample holder was built to
position the specimens in the sample holder of the
microCT scanner. A 0.01 mm aluminium and
0.01 mm copper filter were used to reduce beam-
hardening artefacts and scattering. The geometrical
magnification was chosen according to the principle of
cone beam geometry. The reconstruction algorithm
was a half scan Feldkamp Parker algorithm less
weighting function. The settings for the Micro CT
scanner were 80 kvp and 80 lA. The distance between
each observed section was 21 lm.
The specimens were characterized further by making
three-dimensional isosurfaces, generated, segmented
and measured using Microview software (GE). Once
the scan was completed the operator assessed the
volume of hard tissue remaining. The setting for
surface quality used was 0.85 and the setting for
decimation factor was 26. To assess the hard tissue
volume, each tooth was selected as the region of
interest (ROI).
The automatic threshold tool was used with a
histogram plot to identify the mid point between the
tooth tissue and air. This value was then recorded
and kept consistent for each tooth at the beginning
and then used in subsequent scans to make the
isosurfaces.
Caries removal
All operative procedures (caries removal, access cavity
preparation, root canal preparation, fibre post and cast
post space preparation) were carried out by the same
operator (OI), the operator was unaware of the
objectives of the study.
Once the preoperative volume of the tooth and root
canals were recorded, caries was removed from each
tooth. The occlusal section and box of the cavity were
prepared using a diamond bur (REF 878–2800 Henry
Schein, Gillingham, UK), in a high speed handpiece
with water cooling and the caries was excavated with a
slow speed hand piece and steel rose head bur (size 7
REF 100–3223 Henry Schein Gillingham, UK). Caries
removal was verified using an explorer (04108 Dents-
ply; Ash Instruments, Dentsply, Gloucester, UK). If the
remaining dentine did not cause the probe to stick
caries removal was assumed to be completed. The teeth
were then scanned again and the new volume of hard
tissue volume was recorded. The scanning procedure
was repeated after access cavity, root canal, fibre post
and cast post space preparation.
Access cavity preparation
An oval access cavity was made in each tooth in the
occlusal aspect. Access was completed when the roof of
the pulp chamber was completely removed and a DG16
Micro-CT investigation Ikram et al.
International Endodontic Journal, 42, 1071–1076, 2009 ª 2009 International Endodontic Journal1072
Page 17
endodontic probe (DG-16 Endodontic Explorer, Ash UK)
could be placed in the pulp chamber and the canals
were visible to the naked eye.
Preparation of root canal
The root canals of the teeth were prepared initially
using size 2, 3, and 4 Gates Glidden drills at 600 rpm.
The working length was then measured using a size
10 K-file. The file was passed through the apical
foramen and then wound backwards when it was no
longer visible the length was recorded from a noted
landmark. The teeth were then prepared up to a size 20
file with hand instruments to the working length and
irrigated with sodium hypochlorite after each file.
Recapitulation was performed with a size 10 K-file
between instruments.
ProTaper� rotary instruments (Maillefer Dentsply,
Baillagues, Switzerland) were then used to prepare
the root canals. The Shaper 1 and Shaper 2 ProTaper
files were used to the working length and the
Finisher 1 and Finisher 2 files were used 1 mm short
of the working length. During the root canal prep-
aration a brushing technique was used on the
outward stroke to permit three-dimensional coronal
flaring of the canal. As before, between each file
recapitulation with a size 10 file was performed and a
1% sodium hypochlorite solution was used to irrigate
the canals.
Fibre post preparation
Post spaces were prepared using the Fibre White� post
kit (Coltene/Whaledent, NJ, USA). For this the blue post
drill (1.14 mm in diameter) was used. The post space
preparation was carried out leaving at least 4 mm of
the prepared apical root canal undisturbed. If the tooth
had two canals the widest canal was selected for post
preparation.
Cast post preparation
The preparations for the fibre posts were than modified
into preparations for Parapost� (Coltene/Whaledent)
cast post- cores of 1.14 mm in diameter by removing any
undercuts that would prevent the cementation of the cast
postcores. The Fibre White and Parapost are produced
with identical diameters, for this reason no adjustment
was made to the post space canal preparations.
The percentages of the preoperative hard tooth tissue
volume lost after each procedure were calculated and
statistically compared using one-way analysis of vari-
ance and Fisher’s PLSD test with statistical significance
set at a = 0.01.
Results
The values of hard tooth tissue lost in each tooth after
each procedure are reported in Table 1. The mean
Table 1 Tooth hard tissue volumes in mm3 and percentages of tooth hard tissue lost after caries removal, access cavity preparation,
root canal preparation, fibre post space preparation, and cast post space preparation
Tooth
1
Tooth
2
Tooth
3
Tooth
4
Tooth
5
Tooth
6
Tooth
7
Tooth
8
Tooth
9
Tooth
10
Tooth
11
Tooth
12
Initial
Volume 462.227 417.72 310.667 447.408 469.022 465.954 450.965 264.278 411.56 502.265 378.143 512.933
% volume lost 0 0 0 0 0 0 0 0 0 0 0 0
After caries removal
Volume 428.151 388.957 278.737 428.632 436.094 444.945 372.911 210.327 395.394 479.587 333.203 507.538
% volume lost 7.3 7 10.3 4.2 7.2 4.5 17.3 20.4 3.9 4.5 11.9 1.2
After access cavity
Volume 393.115 346.75 272.105 416.72 413.415 434.462 352.32 199.931 371.932 470.873 313.601 499.089
% volume lost 15.1 17.1 12.2 6.9 12 6.6 21.7 24.6 9.7 6.3 17.1 2.5
After root-canal preparation
Volume 389.969 340.961 265.301 409.991 398.337 433.615 351.024 197.07 371.646 469.189 308.237 494.14
% volume lost 15.8 18.4 14.5 8.5 15.1 6.9 22 25.3 9.7 6.5 18.5 3.5
After fibre post preparation
Volume 389.2 339.98 264.78 408.65 395.24 425.95 344.256 194.52 361.78 457.67 307.81 482.81
% volume lost 15.6 18.7 15.9 8.7 15.7 9.6 23.5 26.5 13.1 9 18.5 5.9
After cast post preparation
Volume 381.335 321.922 262.088 386.012 367.562 411.028 295.97 188.718 325.413 429.6 295.291 471.026
% volume lost 17.5 22.3 15.4 13.6 21.7 11.6 34.4 28.7 20.9 14.5 21.9 8
Ikram et al. Micro-CT investigation
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1071–1076, 2009 1073
Page 18
values of the percentage of preoperative hard tooth
tissue volume lost after each procedure are reported in
Table 2.
The percentages of the preoperative hard tooth
volume lost after caries removal, access cavity prepa-
ration, root canal preparation, fibre post space and cast
post preparation were 8.3 ± 5.83, 12.7 ± 6.7,
13.7 ± 6.7, 15.1 ± 6.3 and 19.2 ± 7.4 respectively.
With the exception of the root canal preparation
all procedures performed significantly increased
(P < 0.01) the amount of hard tissue volume lost.
Fig. 1 shows the tooth crown view and Fig. 2 the
mesio-distal view of a tooth after each operative
procedure.
Discussion
The micro CT technique used in this study produced
slice thicknesses of 21 lm. This allowed a precise three-
dimensional reconstruction of the teeth; however,
the precision of the mass measurement is limited by
the resolution of the machine (Peters et al. 2000). The
measurement of absolute values of hard tooth tissue
volumes was not the objective of the present study.
Instead the objective was to assess the change of hard
tooth tissue volume after each operative procedure. It is
therefore reasonable to assume that the imprecision of
volume measurement would be similar in scans con-
ducted after the completion of each step of the
endodontic treatment/postspace preparation.
In this study only premolars with three coronal walls
left were used, this meant the inclusion of teeth with a
similar amount of residual tooth structure. The largest
loss of hard tooth structure was caused by caries
removal (�8%). This confirms that, in a case of a tooth
with three remaining coronal walls (in all likelihood
the smallest possible loss of tooth tissue associated with
a ‘nonelective’ root canal treatment), caries removal is
the major cause of tooth tissue loss and potentially, the
major cause of tooth weakening. The loss of tooth
tissue because of caries removal varied from 1.2 to
20.4%, this suggested that teeth with three remaining
coronal walls may present with very different amounts
of loss of tooth structure and this may affect their long-
term prognosis. The preparation of the access cavity
caused the second largest loss of tooth structure
(�4.4%), followed by cast post (4.1%) and fibre post
(1.4%) space preparation. The only procedure that did
not result in a significant increase of tooth tissue loss
was the root canal preparation (1%). These results
(a) (b) (c)
(d) (e) (f)
Figure 1 Tooth crown view of a tooth
before caries removal (a), after caries
removal (b), after access cavity
preparation (c), after root canal pre-
paration (d), after fibre post preparation
(e), and after cast post preparation (f).
Table 2 Mean percentages* and standard deviations of hard
tissue volume loss after caries removal, access cavity preparation,
root canal preparation fibre post and cast post space prepa-
ration
Initial 0% % increase
After caries removal 8.3 ± 5.83a 8.3
After access cavity 12.7 ± 6.7b 4.4
After root canal preparation 13.7 ± 6.7b 1
After fibre post preparation 15.1 ± 6.3c 1.4
After cast post preparation 19.2 ± 7.4d 4.1
*Groups with the same letter showed no statistically significant
difference (P < 0.01).
Micro-CT investigation Ikram et al.
International Endodontic Journal, 42, 1071–1076, 2009 ª 2009 International Endodontic Journal1074
Page 19
suggest that the loss of tooth structure caused by root
canal instrumentation alone is small, especially taking
into account the relatively aggressive root canal
preparation technique used in this study which
included the use of Gates Glidden drills in the coronal
aspect and F2 ProTapers in the apical aspect of the root
canal. The loss of tooth tissue in the coronal and root
structure might well have a very different effect on the
fracture resistance of the teeth, indeed finite element
analysis studies have shown a high concentration of
stress caused by occlusal forces in the mid-root area
when posts are used (Lanza et al. 2005). The loss of
root structure caused by root canal and post space
preparation may result in a significant loss of fracture
resistance. However, this is relatively insignificant
when compared with the loss of coronal tooth structure
after access cavity preparation.
Lang et al. (2006) assessed the rigidity of the teeth
after access cavity preparation and post space prepa-
ration, they found a significant reduction of the root
rigidity after both clinical procedures. The results of the
present study strongly suggest that this loss of rigidity is
associated with significant loss of hard tooth tissue
structure. The loss of tooth structure caused by fibre
and cast post space preparation observed in this study
bears perhaps, the most relevant clinical implication.
(a) (b) (c)
(d) (e) (f)
Figure 2 Mesio distal view of the same
tooth shown in Figure 1 before caries
removal (a), after caries removal (b),
after access cavity preparation (c), after
root canal preparation (d), after fibre
post preparation (e), and after cast post
preparation (f).
Ikram et al. Micro-CT investigation
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1071–1076, 2009 1075
Page 20
A recent systematic review of the literature (Bolla et al.
2007) found only one randomized clinical trial
comparing fibre and cast posts (Ferrari et al. 2000)
providing evidence of a longer survival for fibre post
restored teeth, but the evidence was regarded as weak.
In 2 year (Ferrari et al. 2007) and 3 year (Cagidiaco
et al. 2008) randomized clinical trials on root filled
premolars restored with crowns, it was shown that the
cementation of a fibre post increased the survival
probability of teeth which initially presented with loss
of tooth tissue similar to that investigated in the present
study. There is no clinical study proving that the same
is true for cast posts. This study demonstrates that
modification of a preparation from a fibre post to a cast
post of the same shape and size by removing the
undercuts to facilitate the cementation of the cast post
and core placement, more than doubles the loss of hard
tooth tissue. This provides further support for the use of
direct fibre post/composite restorations of root filled
premolars with three remaining coronal walls which
are to be subsequently restored with cuspal coverage
restorations.
Conclusion
Access cavity and post space preparation are the
procedures during root canal treatment that cause
the largest loss of hard tissue structure. The loss of
coronal tooth structure caused by the cast post space
preparation is larger than that caused by the prepara-
tion of a fibre post of the same size. This needs to be
taken into account in planning root canal treatments
and restorations of root filled teeth that are to be
restored with cuspal coverage restorations.
Acknowledgements
The authors wish to thank Chris Healy (Department of
Craniofacial Development, King’s College London Den-
tal Institute, London, UK) for his technical support
The authors acknowledge support from the Depart-
ment of Health via the National Institute for Health
Research (NIHR) comprehensive Biomedical Research
Centre award to Guy’s & St Thomas’ NHS Foundation
Trust in partnership with King’s College London and
King’s College Hospital NHS Foundation Trust.
The findings of this paper were partially presented at
the AAE (American Association of Endodontists) meet-
ing in Orlando (FL) (Abstract OR 58)
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Peters OA, Laib A, Ruegsegger P, Barbakow F (2000) Three
dimensional analysis of root canal geometry by high
resolution computed tomography. Journal of Dental Research
79, 1405–9.
Peters OA, Peters CI, Schonberger K, Barbakow F (2003)
Protaper rotary root canal preparation: effects of canal
anatomy on final shape analysed by micro CT. International
Endodontic Journal 36, 86–92.
Randow K, Glantz PO (1986) On cantilever loading of vital
and non vital teeth. An experimental clinical study. Acta
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Micro-CT investigation Ikram et al.
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Page 21
Laser-activated irrigation within root canals:cleaning efficacy and flow visualization
S. D. de Groot1*, B. Verhaagen2,3*, M. Versluis2,3, M.-K. Wu1, P. R. Wesselink1 & L. W. M. vander Sluis1
1Department of Cariology, Endodontology & Pedodontology, Academic Center for Dentistry, Amsterdam; 2Physics of Fluids Group,
Faculty of Science and Technology, University of Twente, Enschede; and 3Research Institute for Biomedical Technology BMTI,
University of Twente, Enschede, The Netherlands
Abstract
de Groot SD, Verhaagen B, Versluis M, Wu M.-K,
Wesselink PR, van der Sluis LWM. Laser-activated irriga-
tion within root canals: cleaning efficacy and flow visualization.
International Endodontic Journal, 42, 1077–1083, 2009.
Aim To test ex vivo the efficiency of laser-activated
irrigation in removing dentine debris from the apical
part of the root canal and to visualize in vitro the fluid
dynamics during the activation of the irrigant by laser,
using high-speed imaging at a relevant timescale.
Methodology Root canals with a standardized
groove in one canal wall filled with dentine debris
were irrigated with syringe irrigation, ultrasonically or
laser-activated irrigation (LAI) using 2% sodium hypo-
chlorite as irrigant. The quantity of dentine debris after
irrigation was determined. Visualization of the fluid
dynamics during activation was achieved using a high-
speed camera and a glass model.
Results Laser-activated irrigation was significantly
more effective in removing dentine debris from the
apical part of the root canal than passive ultrasonic
irrigation or hand irrigation when the irrigant was
activated for 20 s.
Conclusions The in vitro recordings suggest that
streaming, caused by the collapse of the laser-induced
bubble, is the main cleaning mechanism of LAI.
Keywords: irrigation, laser, root canal, ultrasound,
visualization.
Received 16 April 2009; accepted 18 August 2009
Introduction
An important procedure during root canal treatment
is the irrigation of the root canal. Syringe irrigation
is the standard procedure but unfortunately, syringe
irrigation is not effective in the apical part of the root
canal (Ram 1977, Salzgeber & Brilliant 1977, Abou-
Rass & Patonai 1982, Druttman & Stock 1989) and
in isthmuses or oval extensions (Lee et al. 2004,
Burleson et al. 2007). Therefore, acoustic and hydro-
dynamic activation of the irrigant have been devel-
oped (Weller et al. 1980, Lumley et al. 1991, Lussi
et al. 1993), which have been shown to contribute
to the cleaning efficiency (Lumley et al. 1991, Lussi
et al. 1993, Roy et al. 1994). The physical mecha-
nisms underlying these cleaning procedures, how-
ever, are not well-understood (van der Sluis et al.
2007a).
Laser-activated irrigation (LAI) has been introduced
as a powerful method for root canal irrigation
(Blanken & Verdaasdonk 2007, George & Walsh
2008, George et al. 2008). The laser radiation pro-
duces transient cavitation in the liquid through
optical breakdown by strong absorption of the laser
energy (Blanken & Verdaasdonk 2007). LAI can
result in smear layer removal from the root canal
wall, but also cause extrusion of irrigant through the
apex (George & Walsh 2008, George et al. 2008).
However, the removal of dentine debris from the root
canal by LAI has not yet been studied. Furthermore,
Blanken & Verdaasdonk (2007) suggested repeating
Correspondence: Bram Verhaagen, MSc, University of Twente,
Meander 213, PO Box 217, 7500 AE Enschede, The Nether-
lands (Tel.: +31 53 489 3084; fax: +31 53 489 8068; e-mail:
[email protected] ).
*These two authors should both be listed as primary author, as
both contributed equally to this study.
doi:10.1111/j.1365-2591.2009.01634.x
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1077–1083, 2009 1077
Page 22
their visualization experiment with a single high-
speed camera recording, visualizing a single pulse, to
improve the understanding of the cavitation process.
The purpose of this study was to evaluate ex vivo the
removal of artificially placed dentine debris in stan-
dardized root canals by syringe irrigation, passive
ultrasonic irrigation (PUI) and LAI. LAI was also
visualized in vitro using high-speed imaging at a
timescale relevant to the cleaning process (ls). The
resulting flow is theoretically described using a fluid-
dynamical model.
Materials and methods
Dentine debris removal
Maxillary canines with straight root canals were
decoronated; the length of the remaining root was
15 mm for all teeth. The roots were then embedded in
self-curing acrylic resin (Ostron 100, GC Tokyo, Japan)
and then split longitudinally through the canal in
mesio-distal direction. To remove the imprint of the
root canal, both halves were ground with sandpaper
and fixed with four screws (see Fig. 1a). Then, the root
canals were prepared by K-files hand instruments
(Dentsply Maillefer, Ballaigues, Switzerland) and
mechanically driven Race NiTi instruments (FKG
Dentaire, La Chaux-de-Fonds, Switzerland), to a length
of 15 mm, size 35 and 0.06 taper resulting in a
standardized root canal. To verify the standardization
of the models, the canal diameter of six randomly
chosen models was measured at 2, 6 and 10 mm from
the apical end of the canal, using a KS100 Imaging
system 3.0 (Carl Zeiss Vision GmbH, Halbermoos,
Germany). At 2 mm, the average canal diameter was
found to be 0.47 ± 0.02 mm (diameter of the Race
NiTi instrument: 0.47 mm); at 6 mm the average
canal diameter was 0.71 ± 0.02 (0.71) and at 10 mm
the diameter was 0.94 ± 0.02 (0.95). These measured
values demonstrate that the root canals were indeed
uniform and standardized.
The coronal 3 mm of the canal was enlarged by a no.
23 round bur (Dentsply Maillefer) with a diameter of
2.3 mm, simulating a pulp chamber. A standard
(a)
(b)
(c)
Figure 1 Schematic representations of the
standardized root canal model (a), its groove
(b), and its cross section (c).
Laser-activated irrigation within root canals de Groot et al.
International Endodontic Journal, 42, 1077–1083, 2009 ª 2009 International Endodontic Journal1078
Page 23
groove of 4 mm in length, 0.5 mm deep and 0.2 mm
wide, situated at 2–6 mm from working length, was
cut in the wall of one-half of each root canal with an
ultrasonically driven tip (Fig. 1b,c) (P5 Booster, Sat-
elec, Acteongroup, Merignac-cedex, France). The
dimension of the groove was comparable with that of
an oval extension of a root canal. Each groove was
filled with dentine debris mixed with 2% NaOCl to
simulate a situation in which dentine debris accumu-
lates in uninstrumented canal extensions (Lee et al.
2004). This model was introduced to standardize the
root canal anatomy and the amount of dentine debris
present in the root canal before the irrigation proce-
dure, in order to increase the reliability of dentine
debris removal evaluation. The methodology is sensi-
tive and the data are reproducible (van der Sluis et al.
2007b).
Three irrigation protocols were tested. In all
groups, the needle, wire and fibre were inserted
1 mm short of the working length and were moved
slowly up and down 4 mm in the apical half of the
root canal; the activation time was 20 s, the total
irrigation time was 50 s and the total irrigant
volume was 4 mL. In group 1 (n = 20) syringe
irrigation with 4 mL of 2% NaOCl solution was
performed with a 10 mL syringe and a 30 gauge
needle (Navitip, Ultradent, South Jordan, UT, USA).
In group 2 (n = 20), the 2% NaOCl solution was
activated by ultrasound using PUI. A stainless steel
noncutting wire (size 20) (Irrisafe, Satelec, Acteon-
group) was used, driven by an ultrasonic device
(Suprasson Pmax Newtron, Satelec, Acteongroup) at
power setting ‘blue 4’ (frequency 30 KHz, displace-
ment amplitude ca. 30 lm according to the manu-
facturer). Subsequently the canal was flushed with
2 mL of 2% NaOCl solution using a 10 mL syringe
and a 30 gauge needle. In group 3 (n = 20), the 2%
NaOCl solution was activated by laser radiation
(KEY2 laser, KaVo Dental GmbH, Biberach, Germany)
from an optical fibre laser tip with outer diameter
280 lm and length 30 mm (type Gr. 30 · 28, Kavo
Dental GmbH). Calibration by the manufacturer
showed that the optical fibre has a reduction factor
of 0.36, which results in a fluence of 146 mJ mm)2
for a laser pulse energy setting of 100 mJ. The
Er:YAG laser emits at a wavelength of 2.94 lm
which coincides with the major absorption band of
water (Robertson & Williams 1971). A pilot study
demonstrated that the optimal settings for dentine
debris removal from the root canal are a low power
setting of 80 mJ per pulse and a pulse repetition
frequency of 15 Hz. Finally, the canal was flushed
with 2 mL of 2% NaOCl solution using a 10 mL
syringe and a 30 gauge needle.
After irrigation the root canals were dried with paper
points. Subsequently, the two halves were separated
and the amount of debris in the groove was evaluated.
Before and after the irrigation, a digital image was
taken of the groove, using a Photomakroskop M400
microscope with a digital camera (Wild, Heerbrugg,
Switzerland) at 40· magnification. The quantity of
dentine debris in the groove before and after irrigation
was scored double blind and independently by three
dentists using the following scores: score 0: the groove
is empty, score 1: less than half of the groove is filled
with dentine debris; score 2: more than half of the
groove is filled with dentine debris; score 3: the groove
is completely filled with dentine debris. The differences
in dentine debris scores between the different groups
were analysed by means of the Kruskal–Wallis and
Mann–Whitney tests (level of significance a = 0.05).
High-speed imaging experiments
An optical setup was constructed in order to visualize
the effect of the Er:YAG laser radiation in an artificial
root canal containing water or NaOCl. Optical record-
ings were made at a pulse repetition rate of the Er:YAG
laser of 1 Hz and a pulse energy between 80 and
250 mJ per pulse. The laser fibre tip was inserted up to
1 mm from the apical end of a glass root canal model.
The canal was 12 mm in length with an apical
diameter of 0.35 mm and taper 0.06. Imaging was
performed using a high-speed camera (FastCam APX-
RS, Photron, Tokyo, Japan), recording at a frame rate
of 14 000 frames per second, attached to a microscope
with 12· magnification (SZX12, Olympus, Tokyo,
Japan). The root canal model was illuminated in
bright-field by a continuous wave light source (ILP-1,
Olympus).
Results
Dentine debris removal
The debris scores before and after irrigation are
presented in Table 1. The difference between the
groups was statistically significant (Kruskal–Wallis test,
P < 0.0001). The debris score in group 3 was signif-
icantly lower than group 2 (P = 0.002) and group 1
(P < 0.0001), and the score in group 2 was signifi-
cantly lower than group 1 (P < 0.0001).
de Groot et al. Laser-activated irrigation within root canals
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1077–1083, 2009 1079
Page 24
High-speed imaging experiments
The high-speed recordings of the laser activity inside
the artificial (glass) canal showed that irrigant was
vapourized by the laser pulse energy and that a large
vapour bubble was created at the fibre tip, similar to
that observed previously (Lauterborn 1972). The
bubble grew with a velocity of the order of 1 m s)1
during the pulse duration (see Fig. 2 and video clips S1
and S2); a higher energy laser pulse corresponded to a
longer growth time of the bubble. When the laser pulse
ended, the bubble collapsed with a velocity of the order
of 1 m s)1. Upon collapse, a shockwave was generated
(Holzfuss et al. 1998), whose negative-pressure tail
caused secondary cavitation in the root canal with a
relatively large bubble near the collapse site (which was
usually at the apex). The cavitation bubble then
collapsed again and this cycle repeated for a number of
times, until it was damped out within a few milliseconds
(6 ms at 250 mJ per pulse). Smaller bubbles with a
typical diameter of 10 lm remain buoyant for a longer
time (even up to the next pulse), also at the apical end of
the root canal.
The laser-induced bubble grew predominantly in the
coronal direction, as there was a confinement at the
apex. The depth reached by this bubble depended on
the position of the fibre and the laser energy, but never
fully extended to the apex. It was observed that when a
small bubble was present at the apex, it grew during
the collapse phase of the laser-induced bubble and
Table 1 Dentine debris score in the groove after the irrigation
procedures per group (no. cases and percentage of total; 20
cases in total for each irrigation procedure)
Score:
0
n (%)
1
n (%)
2
n (%)
3
n (%)
Syringe irrigation 0 0 4 (20%) 16 (80%)
Ultrasonic irrigation 6 (30%) 8 (40%) 6 (30%) 0
Laser-activated
irrigation
16 (80%) 4 (20%) 0 0
Scoring system: 0: the groove is empty; 1: less than half of the
groove is filled with debris; 2: more than half of the groove is
filled with debris; 3: the complete groove is filled with debris.
a b c d e f g h i j k l m n o p
a b c d e f g h i j k l m n o p
(a)
(b)
Figure 2 (Video clips S1 and S2) Visualization of the laser-generated vapor bubble. The laser energy was 60 mJ per pulse in (a)
and 250 mJ per pulse in (b). Image sequence is from left to right. The interframe time is 140 ls. Panel p in (a) shows a sketch of the
setup, with 1) the root canal model, 2) the laser fiber tip (outer diameter 280 lm), 3) the laser-induced cavitation bubble, and 4) a
stable cavitation bubble at the apex.
Laser-activated irrigation within root canals de Groot et al.
International Endodontic Journal, 42, 1077–1083, 2009 ª 2009 International Endodontic Journal1080
Page 25
collapsed and renucleated in anti-phase with the laser-
induced bubble (Fig. 2a (indicated with the no. 4 in
panel p), whereas in Fig. 2b this bubble was not
present).
It was observed that the laser-induced bubble grew
larger when NaOCl was used as an irrigant solution.
Consequently, it had a longer collapse time as com-
pared with having water as an irrigant. It was also
found that a higher amount of smaller bubbles were
present after laser activation when using NaOCl as the
irrigant solution.
Because of the impulsive growth of the laser-
induced bubble the fluid was pushed outward at the
free surface at the coronal part (see Fig. 3 and Video
Clip S3). For a laser energy exceeding 120 mJ per
pulse it was observed that some fluid was ejected
from the root canal, leaving less irrigant in the root
canal.
Discussion
The results of the ex vivo experiments demonstrate that
within the time frame of 20 s, LAI is more effective in
removing dentine debris from an artificial groove in the
apical part of the root canal than ultrasonically
activated (PUI) or syringe-activated irrigation.
The high-speed recordings have shown that vapou-
rization of the irrigant causes a large bubble to grow,
which then collapses and renucleates a few times.
During this process, secondary cavitation bubbles are
formed. The fluid flow associated with such an inertial
collapse, combined with acoustic streaming resulting
from the oscillations of smaller bubbles, could explain
the cleaning efficacy of LAI; however, a more detailed
study is required to elucidate the principal cleaning
mechanism. The secondary cavitation bubbles can also
assist in the cleaning of the root canal wall, as they are
excited by the bubble collapse of the consecutive laser
pulse. As the flow does not penetrate all the way into
the apex, a trapped bubble in the apex (most likely a
remainder of previous laser pulses) could assist in the
cleaning of the apical part of the root canal.
The irrigant flow in the root canal due to the
collapsing laser-induced bubble can be modelled by a
flow in concentric annuli for heights above the
insertion depth of the fibre. For the typical flow velocity
of 1 m s)1 (value obtained from the high-speed record-
ings by measuring the bubble wall displacement
between consecutive frames), the Reynolds number
Re = Udq/l (with U the flow velocity, d the distance
between the cylinders, q the density of the liquid and lthe dynamic viscosity) for a flow in annuli is of the
order of 300. According to Rothfus et al. (1950), the
transition to turbulence occurs over the range 2100–
3700, therefore the flow in this problem is treated as
laminar flow.
Rothfus et al. (1950) also give the laminar flow
velocity distribution for flow in concentric annuli:
(a) (b) (c) (d) (e) (f)
Figure 3 (Video clip S3) Pinch-off at the free surface at the coronal part of the glass root canal model. Secondary cavitation
bubbles are formed (in e) upon passage of a shockwave generated by the vapor bubble inertial collapse at the laser fiber tip. The
frame rate is 14,000 frames/second.
de Groot et al. Laser-activated irrigation within root canals
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1077–1083, 2009 1081
Page 26
uðrÞ ¼ 2uav
r21 � r2 þ r2
2 � r21
lnr2
r1
ln rr1
264
375
r22 þ r2
1 � 2r2m
ð1Þ
where rm is the radius of maximum velocity, given
by:
rm ¼r2
2 � r21
2 ln r2
r1
" #12
ð2Þ
Using s ¼ �l @u@r the shear stress for laminar flow in
annuli is given by:
sðrÞ ¼ �2luav
r22�r2
1
r � 2r ln r2
r1
� �ðr2
2 þ r21Þ ln r2
r1� r2
2 þ r21
ð3Þ
Using standard values for density q = 1000 kg m)3
and dynamic viscosity l = 1 · 10)3 m2 s)1, and a
measured average velocity uav = 5 m s)1 and cylinder
radii r1 = 140 lm (inner) and r2 = 300 lm (outer),
the shear stress on the inner wall is 496 N m)2 and on
the outer wall 436 N m)2. These values are one order
of magnitude lower than the shear stress generated by
a laser-induced cavitation bubble of radius 0.75 mm
next to a single wall, which is reported to generate a
shear stress of up to 3.5 · 103 N m)2 (Dijkink & Ohl
2008). No quantitative data on the adhesion strength
of dental intracanal biofilms to dentine or its failure
shear stress is available in the literature.
Figure 4 shows the velocity profile calculated with
the theory described above in a tapered canal with a
cylinder inserted, assuming an average velocity of
5 m s)1 at the fibre tip (taken from experiment). The
profile on the left of the inner cylinder is the velocity
profile; the profile on the right is the shear stress
distribution. The plot clearly shows that on the inner
cylinder (the laser fibre) the shear stress is higher than
on the outer cylinder (the root canal wall).
The root canal diameter increases with height,
therefore the average velocity decreases with height.
This results in the shear stress being highest next to the
tip of the laser fibre. LAI is therefore expected to be most
effective in the region close to the fibre tip, with
decreasing efficiency away from the tip.
Using a 27G needle and a volume flow rate of
0.30 mL s)1 (Boutsioukis et al. 2007) it follows that
the typical fluid velocity in syringe irrigation is of the
order of 1 m s)1 at the needle orifice, which is the same
order of magnitude as the flow velocities developed
with LAI. Likewise for PUI with u = xe02/a (Ahmad
et al. 1988; x = oscillation frequency, e0 = oscillation
amplitude and a = file radius) a typical fluid velocity of
the order of 1 m s)1 was found. One possible explana-
tion for the improvement in cleaning efficacy with LAI
is the impulsive nature of the laser-generated bubble
dynamics. Because of the pulsations the fluid becomes
accelerated at every pulse and the acceleration gives
rise to inertial forces, whereas a steady streaming as in
syringe irrigation and PUI only exerts viscous stress.
This would also explain why the irrigation duration is
an important factor and why a high pulse repetition
rate of the laser is more efficient than a lower one, as
found in the pilot-study.
Previous studies have shown side-effects caused by
the use of these types of lasers in the root canal.
Carbonization of the root canal and cracks were
observed when laser tips were used in the root canal
(Matsuoka et al. 2005). Kimura et al. (2002) have
shown a temperature increase of the root canal wall of
3–6 �C. The current study did not monitor these side-
effects, because the aim of this study was clarification of
the fluid mechanical working mechanisms.
Conclusion
Laser-activated irrigation was more effective in remov-
ing the artificially placed dentine debris from the root
canal than syringe irrigation or PUI when the irrigant
was activated for 20 s.
250
300
350
400
450 N m–2u ττm s–1
200
150
100
50
00
1
2
3
4
5
6
7
Figure 4 Average velocity profile (left) and sheer stress
distribution (right) between two concentric cylinders of which
the outer cylinder represents the tapered root canal wall. The
average velocity at the laser fiber tip is set at 5 m s)1. The
region below the laser fiber tip is intentionally left blank, as
details of the streaming pattern in the apical part are missing
and are part of a future study.
Laser-activated irrigation within root canals de Groot et al.
International Endodontic Journal, 42, 1077–1083, 2009 ª 2009 International Endodontic Journal1082
Page 27
Acknowledgements
We thank Gert-Wim Bruggert for technical support and
Gerrit J. de Bruin for valuable discussions on the
theoretical approach of the fluid flow in tapered
canals.
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Supporting Information
Additional supporting information may be found in the
online version of this article.
Video Clip S1. Visualization at the apex of the root
canal, laser intensity 60 mJ/pulse (apex_energy60mj.
wmv).
Video Clip S2. Visualization at the apex of the root
canal, laser intensity 250 mJ/pulse (apex_energy250mj.
wmv).
Video Clip S3. Visualization at the corona of the
root canal, laser intensity 250 mJ/pulse (corona_
energy250mj.wmv)
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supporting infor-
mation supplied by the authors. Any queries (other
than missing material) should be directed to the
corresponding author for the article.
de Groot et al. Laser-activated irrigation within root canals
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1077–1083, 2009 1083
Page 28
Sealing properties of a new root canal sealer
U. Salz, D. Poppe, S. Sbicego & J.-F. RouletIvoclar Vivadent AG, Research & Development, Bendererstr. 2, FL-9494 Schaan, Liechtenstein
Abstract
Salz U, Poppe D, Sbicego S, Roulet J.-F. Sealing properties
of a new root canal sealer. International Endodontic Journal, 42,
1084–1089, 2009.
Aim To evaluate bacterial leakage of Apexit Plus, a
new root canal sealer, in comparison with AH Plus.
Methodology A total of 56 single-rooted human
teeth were randomly divided into two experimental
groups of 16 roots and two control groups. Roots were
filled by lateral condensation with Gutta-percha and
AH Plus or with Gutta-percha and Apexit Plus. A split
chamber microbial leakage model was used in which
Streptococcus mutans placed in the upper chamber could
reach the lower chamber only through the filled canal.
Positive controls were filled only with Gutta-percha and
tested with bacteria, whereas negative controls were
sealed with wax to test the seal between chambers.
Additionally, film thickness, solubility and dimensional
change were determined.
Results All positive controls leaked within 24 h,
whereas none of the negative controls leaked after
30 days. Apexit Plus had significant less bacterial
leakage (log-rank test, P < 0.0001) than AH Plus.
AH Plus (0.3% solubility) showed a slightly lower
solubility than Apexit Plus (0.5% solubility) but a
larger film thickness (28 vs. 11 lm) according to ISO
6876:2001.
Conclusion Apexit Plus had a better sealing ability in
comparison with AH Plus.
Keywords: AH Plus, Apexit Plus, bacterial leakage,
root canal sealer.
Received 18 September 2008; accepted 17 August 2009
Introduction
In general, a root filling is composed of two materials: a
solid core material and a sealer. The most commonly
used core material is Gutta-percha, which can be
placed into the root canal in a cold or a warm state.
The main purpose of the root canal sealer is to fill the
interface between the core material and the dentine
wall, the voids inside the core material and the
accessory canals, to serve as a lubricant and to obtain
a hermetic apical seal (Skinner & Himel 1987).
Although the most important property of a root canal
sealer is its sealing ability, there is no standardized
sealing test that is part of the ISO 6876:2001 (‘Dental
root canal sealing materials’). However, limits of water
solubility, film thickness and dimensional change
following setting are components of the standard
providing an indirect indication of sealing ability.
Dye penetration experiments have been performed
for the assessment of the sealing behaviour of end-
odontic materials. However, the frequent approach of
sectioning teeth vertically or horizontally to determine
tracer depth is not reproducible, and rarely yields
statistically significant differences between materials
(Schuurs et al. 1993, Wu & Wesselink 1993, Camps &
Pashley 2003). A quantitative measurement of pene-
trated dye should be possible by dissolving roots in
nitric acid and spectrophotometric determination of
extracted dye (Mandras et al. 1993, Camps & Pashley
2003). Unfortunately, methylene blue and other dyes
are not stable against nitric and hydrochloric acid
(Mandras et al. 1993). Hence, dyes are unsuitable for
such studies. Further applied test methods include,
amongst others (Al-Ghamdi & Wennberg 1994), fluid
filtration measurements (Wu et al. 1993, Pommel et al.
2003) and movement of glucose solution under low
hydrostatic pressure (Xu et al. 2005).
Correspondence: Ulrich Salz, Ivoclar Vivadent AG, Benderer-
str. 2, FL-9494 Schaan, Liechtenstein (Tel.: +423/235 34 21;
fax: +423/233 12 79; e-mail: ulrich.salz@ivoclarvivadent.
com).
doi:10.1111/j.1365-2591.2009.01635.x
International Endodontic Journal, 42, 1084–1089, 2009 ª 2009 International Endodontic Journal1084
Page 29
Kersten & Moorer (1989) suggested that penetration
experiments with particles the size of bacteria may be
more relevant than using small molecules. If bacteria
are used as a leakage tracer the experiment will be
more closely related to the clinical situation (Wolanek
et al. 2001). For this purpose, different types of bacteria
have been used, e.g. Enterococcus faecalis (Saleh et al.
2008, Fransen et al. 2008) as well as Streptococcus
mutans (Monticelli et al. 2007).
Therefore, the main purpose of this laboratory study
was to assess the penetration of S. mutans through
coronally unsealed root canals to compare the effec-
tiveness of AH Plus and Apexit Plus to resist bacterial
leakage.
Materials and methods
Tests according to ISO 6876
Sample preparation and measurement of solubility, film
thickness and dimensional change following setting
were performed in accordance with ISO 6876:2001.
Solubility means the amount of material dissolved by
water out of a test specimen of 20 mm diameter and
1.5 mm height after 24 h at 37 �C. Film thickness was
determined by displacement of mixed sealer under a
load of 150 N for 7 min. Dimensional change is the
longitudinal change of a test specimen having a
diameter of 6 mm and a height of 12 mm after
30 days storage in water at 37 �C. The dimensional
change was determined by an independent institute
(NIOM; test-report T051/03). Following sealers were
used: Apexit Plus (Ivoclar Vivadent AG, Schaan,
Liechtenstein) and AH Plus (Dentsply de Trey GmbH,
Konstanz, Germany).
Bacterial leakage test
A total of 56 extracted, single-rooted human teeth were
divided into two experimental (n = 16) and two control
groups (n = 12). To standardize the length of root
canals involved in each experimental group, the length
of all roots was measured and root segments ranging
11–16 mm were equally distributed to the groups.
Each root canal was coronally enlarged with Largo
Peeso Reamers (Dentsply Maillefer, Ballaigues, Switzer-
land)) to size 90 or 110 to obtain standardized round
root canal shapes. Hand K-files were also used to finish
the enlargement and achieve better adaptation of core
materials to the root segments. In both experimental
groups the canals were enlarged to the same size.
The root canals were irrigated with 1.25% NaOCl
during instrumentation and then sterilized by auto-
claving for 20 min at 121 ± 2 �C. The smear layer was
removed with 17% EDTA and the canals rinsed with
sterile water.
The canals were filled with Gutta-percha and sealer
(except positive control) using cold lateral condensation
technique as follows: a size 50 master Gutta-percha
cone (Kerr, Orange, CA, USA) was coated with the
sealer and placed into the root canal to working length.
A size 30 finger spreader (Dentsply Maillefer) was then
inserted into the canal to a level about 1 mm short of
working length. Lateral condensation with fine acces-
sory Gutta-percha cones (Kerr) was performed.
Group 1
Cold lateral condensation of Gutta-percha with AH Plus
(Dentsply) according to the instructions for use.
Group 2
Cold lateral condensation of Gutta-percha with Apexit
Plus (Ivoclar Vivadent) according to the instructions for
use.
Positive control
Cold lateral condensation of Gutta-percha without
sealer.
Negative control
Cold lateral condensation of Gutta-percha with AH
Plus, sticky wax was applied to completely cover the
root and coronal orifice of canal.
All root canal treatment and filling procedures were
completed by one endodontist at the Scandinavian
Institute of Dental Materials (NIOM; test-report T074/
04). Teeth were then placed in an incubator at 37 �C
for 14 days to allow the sealer to set.
The microbial leakage test was performed in a two-
chamber set-up as described by Shipper et al. (2004).
The upper chamber consisted of a 15-mL polycarbonate
centrifuge tube (Corning Inc, Corning, NY, USA) with a
small hole prepared at the bottom to receive the root-
end (see Fig. 1). The tooth was inserted into the tube
and gently pushed through the opening until approx-
imately one-half of it protruded through the tube. The
space between the tube and the tooth was then sealed
with sticky wax (Kerr Cooperation). The tube was
introduced into and sealed to the neck of a flat-
bottomed scintillation vial. The tip of the root was
mounted to reach approximately 3 mm into the sterile
TSB (Trypticase Soy Broth) with streptomycin in the
Salz et al. Sealing properties of new sealer
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1084–1089, 2009 1085
Page 30
lower chamber. The upper chamber was filled with
medium without bacteria on day )1, and checked for
leakage until day 0. Streptococcus mutans (VA 159) was
added to the upper chamber and the time for S. mutans
to eventually penetrate into the lower chamber was
noted. Bacteria penetrating along the root filling were
detected by turbidity observed in the lower chamber.
Maximum observation time was 30 days. Bacterial
growth in teeth with turbidity was checked by seeding
the liquid on agar plates, followed by incubation and
microscopy.
Statistical analysis
The Kaplan–Meier method was used to estimate the
survival curves. Specimens that did not leak until the
end of the observation time were computed with an
event time of 30 days as censored variables. The non-
parametric log-rank test was used to compare the
survival curves using a significance level of 0.05. The
results at the end of the observation time (30 days) was
further analysed by chi-square testing.
Results
Results of measurements of solubility, film thickness
and dimensional change following setting are summa-
rized in Table 1. AH Plus had a slightly lower solubility
(0.3% solubility) than Apexit Plus (0.5% solubility).
The film thickness of AH Plus was higher (28 lm) than
Apexit Plus (11 lm).
In the bacterial leakage test, all positive controls
leaked within 24 h and no penetration of bacteria was
observed in the negative controls during the observa-
tion time of 30 days. The Kaplan–Meier survival
probabilities for the experimental groups are shown in
Fig. 2. Significant differences were observed amongst
the experimental groups (P < 0.0001, log-rank test)
and also at the end of the observation time after 30 day
a significant difference was observed (P < 0.0005, chi-
squared test). The mean time for the bacteria to
penetrate the root canal in the AH Plus group was
5.3 days (CI: 3.7–7.0 days). For the Apexit Plus group,
the mean penetration time was not calculated because
at the end of the observation time more than 63% (10
of 16) of the specimens had not allowed passage of
bacteria.
Discussion
To ensure a permanent seal of the root canal and
prevent the penetration of bacteria into the apical
periodontium, the root canal sealer must be insoluble.
A low solubility of endodontic sealers is a requirement
of the ISO 6876 standard. To comply with this
standard, the solubility of the sealer after 24-h immer-
sion in water must not exceed 3% (w/w). For Apexit
Plus and AH Plus, low solubility values were observed
(0.5% solubility and 0.3% solubility respectively).
These values are comparable with AH 26 (Dentsply
DeTrey, 0.4%) (Schafer & Zandbiglari 2003) and
RoekoSeal (Coltene/Whaledent, 0.5%) (Schafer & Zan-
dbiglari 2003). According to the literature, Ketac Endo
(3 M Espe AG, 6.1%) (Schafer & Zandbiglari 2003),
Epiphany (Pentron, 3.4%) (Versiani et al. 2006) and
both Sybron Endo sealers Sealapex (4.2%) (Schafer &
Zandbiglari 2003) and Pulp Canal Sealer (3.6%)
Root canal sealer
Gutta-percha
S. mutans
Sticky wax
Sterile broth Contaminatedbroth
Δ t
Figure 1 Experimental setup of the bacterial leakage model
used: A culture of Streptococcus mutans was placed in the upper
chamber of the setup and sterile broth in the lower chamber.
Growth of bacteria in the lower chamber as indicator of
leakage was observed visually by appearance of turbidity.
Table 1 Solubility after 24 h, film
thickness and dimensional change
following setting of Apexit Plus and
AH Plus according to ISO 6876:2001
ISO 6876 limits AH Plus Apexit Plus
Solubility/% £3.0 0.3 0.5
Film thickness/lm £50 28 11
Dimensional change
following settinga/%
£0.1 (expansion)
£1 (shrinkage)
0.2 (expansion) 0.4 (expansion)
aNIOM; test-report T051/03.
Sealing properties of new sealer Salz et al.
International Endodontic Journal, 42, 1084–1089, 2009 ª 2009 International Endodontic Journal1086
Page 31
(Ørstavik 1983) fail to fulfil the requirements of ISO
6876. The high solubility of Epiphany may explain
why the apical sealing ability of Resilon/Epiphany is
reduced after 16 months of water storage (Paque &
Sirtes 2007). A matter of particular interest is the
significant difference in solubility between Apexit Plus
and Sealapex, because both materials are calcium
salicylate based sealers. This may indicate that the
setting reaction of Apexit Plus is more consistent than
that of Sealapex.
The film thickness describes the ability of the
material to adapt to the geometry of the canal wall.
The lower the film thickness, the better is the adapta-
tion of the material. ISO 6876 requires a maximum of
50 lm. The film thickness of Apexit Plus was 11 lm
and of AH Plus 28 lm. AH 26 (36 lm) and Sealapex
(36 lm) exhibit a higher film thickness; however, they
do fulfil the ISO requisite (Oguntebi & Shen 1992).
Dimensional change following setting may lead to
gap and channel formation along the interface between
sealer and dentine or Gutta-percha. These gaps and
channels may be large enough for microorganisms to
pass (Ørstavik et al. 2001). Therefore, the ISO require-
ment of linear shrinkage was set to not more than 1%.
An expansion after setting has a two-edged effect: On
the one hand, a slight expansion favours a better seal.
On the other hand, expansion of root canal sealers
increases the risk of root fracture caused by radial
pressure on the pulpal aspect of dentine. This risk is
highly material dependent, the higher the bulk mod-
ulus of the sealer the higher the pressure. The limit
within ISO 6876 is expansion of 0.1%. Therefore,
neither Apexit Plus nor AH Plus complied with this
requirement. However, because of its low bulk modu-
lus, at least for Apexit Plus a risk of root fracture caused
by expansion can be neglected. Ørstavik et al. (2001)
observed for RoekoSeal a slight (0.2%) and for AH 26 a
distinct expansion (4%), whilst both Pulp Canal Sealer
and Ketac Endo shrank around 1%. For Sealapex,
determination of dimensional change was not possible
because the test specimens expanded and disintegrated
during the experiment. Versiani et al. (2006) reported a
pronounced expansion of 8% for the resin-based sealer
Epiphany.
All three tested parameters imply a good sealing
ability of Apexit Plus. However, these parameters are
only indirect indicators for sealing performance. There-
fore, a laboratory bacterial leakage test was conducted.
For the bacterial leakage model, S. mutans was used
as bacterial marker. It is a nonmotile facultative aerobic
bacteria that often is found in endodontic infections
(Baumgartner & Falkler 1991). It penetrates easily
along root fillings (Saleh et al. 2008) and is easy to
handle in the laboratory setting (Weinberger & Wright
1989). The number of bacteria penetrating through
the root canal filling was not determined because the
purpose of the experiment was only to test if the root
filling can prevent penetration of bacteria into the
lower chamber.
The comparator product AH Plus had a mean
leakage time of 5.3 days. This result is consistent with
the findings in other studies investigating coronal
bacterial leakage in teeth obturated employing cold
lateral condensation techniques (Yucel et al. 2006,
Eldeniz & Ørstavik 2007). However, in studies with
alternative microbial markers, different mean break-
through-times were observed (Timpawat et al. 2001,
Miletic et al. 2002, De-Deus et al. 2006). For Apexit
Plus, more than 50% of the specimens did not leak at
the end of the experiment. One explanation for the
observed difference between the two products could be
the more optimal film thickness of Apexit Plus. Another
explanation could be that Apexit Plus exhibits some
antimicrobial activity. Calcium hydroxide-based root
canal sealers are often suspected to have a high pH
value that kills the bacteria (Heling & Chandler 1996,
Fuss et al. 1997). At least for Apexit Plus, this is a
misunderstanding, as during the setting reaction the
calcium hydroxide forms a stable complex with the
Figure 2 Kaplan–Meier survival curves of the sealed root
canals for the two different sealers in the bacterial leakage test
(NIOM; test-report T074/04).
Salz et al. Sealing properties of new sealer
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1084–1089, 2009 1087
Page 32
salicylat derivative resulting in a product with a pH<8.
To insure complete setting of the sealer, the bacterial
penetration experiment commenced after a pre-incu-
bation period of 14 days. At this time-point no antimi-
crobial activity could be detected for Apexit Plus
(Slutzky-Goldberg et al. 2008). This is in agreement
with the results for Apexit, were no antibacterial
activity was detected (Evcil & Colak 2004, Kayaoglu
et al. 2005, Eldeniz et al. 2006).
Conclusion
The better sealing ability of Apexit Plus compared with
AH Plus may be explained by the physical-chemical
properties and not by a potential antimicrobial effect of
the material.
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Page 34
A tactile method for canal length determinationin teeth with open apices
A. ElAyouti, E. Dima & C. LostDepartment of Conservative Dentistry and Endodontology, University of Tubingen, Tubingen, Germany
Abstract
ElAyouti A, Dima E, Lost C. A tactile method for canal length
determination in teeth with open apices. International End-
odontic Journal, 42, 1090–1095, 2009.
Aim To present a tactile method for working length
determination in teeth with open apices and to deter-
mine its accuracy and repeatability.
Methodology Ninety teeth with 129 root canals
were prepared to create open apices. The correct
working length (CWL) for each canal was determined
by introducing a file into the root canal until it was
visible at the apex. Consequently, the tactile working
length (TWL) was determined by the ‘Tactile Method’
using a K-file that was bent at the tip. Two operators
repeated the measurement once in each root canal. The
accuracy of the TWL was determined by comparing the
TWL with the CWL. The mean of the absolute
differences and the corresponding 99% confidence
interval (CI) were calculated. Both the repeatability
and inter-operator agreement of the tactile method
were determined by performing paired analysis of the
differences between repeated measurements and the
two operators.
Results Overall, 97% (CI: 91–99) of the TWL were
within 0.5 mm from the CWL, the mean of absolute
differences was 0.1 mm (CI: 0.1–0.2). The maximum
difference between repeated measurements was
0.2 mm and between the two operators was 0.6 mm.
Conclusions The tactile method may provide an
accurate determination of canal length in teeth with
open apices.
Keywords: accuracy and repeatability, inter operator
agreement limits, open apex in immature teeth,
simulated root resorption, tactile methods, working
length determination.
Received 14 April 2009; accepted 17 August 2009
Introduction
The term ‘open apex’ is used to indicate the presence of
an exceptionally wide root canal at the apex. Open
apices typically occur in immature teeth when root
development ceases as a sequel of pulp necrosis. Whilst
trauma is regarded as the main cause of open apices in
immature anterior teeth, caries may also lead to open
apices in both anterior and posterior immature teeth. In
fully developed teeth causes of open apices include
extensive apical resorption, root-end resection and
overinstrumentation.
There are many problems associated with the treat-
ment of teeth with open apices; the short thin-walled
roots increase the risk of fracture and have an
unfavourable crown-root ratio; the extensive apical
resorption, facilitated by the thin-walled dentine and
long-standing infection, impedes accurate canal length
determination; the wide and often apically divergent
canals necessitate tailored canal filling techniques to
achieve an optimal seal (Gutmann & Heaton 1981,
Morse et al. 1990, Kerezoudis et al. 1999, Mackie & Hill
1999, Allen & Mackie 2003, Dominguez et al. 2005,
Bogen & Kuttler 2009).
Successful root canal treatment occurs when over-
instrumentation and overfilling are avoided and filling
materials confined to the limits of the canal (Ricucci
1988, Ricucci & Langeland 1988, Shabahang et al.
1999, Holland et al. 2007). Accordingly, accurate
working length determination is essential in achieving
Correspondence: Ashraf ElAyouti, Department of Conservative
Dentistry and Endodontology, University of Tubingen. Osia-
nderstraße 2–8, 72076 Tubingen, Germany (Tel.: 0049 7071
29 83498; fax: 0049 7071 29 5656; e-mail: ashraf.elayouti
@med.uni-tuebingen.de).
doi:10.1111/j.1365-2591.2009.01636.x
International Endodontic Journal, 42, 1090–1095, 2009 ª 2009 International Endodontic Journal1090
Page 35
optimal healing. Unfortunately, open apices pose many
difficulties to contemporary methods of canal length
determination.
Radiographic methods known for their inherent
interpretation difficulties are even more challenging
in open apices where dentinal walls frequently end at
different levels and have irregular margins. Conse-
quently, the apical end of the canal that is circumfer-
entially surrounded by dentine is located a few
millimetres short of the radiographic apex, which
results in overestimation of the radiographic working
length (Baggett et al. 1996).
Apex locators have been shown to be highly accurate
in locating the apical foramen and constriction (Gordon
& Chandler 2004, Kim & Lee 2004). Unfortunately, in
open apices they give incorrect measurements (Huls-
mann & Pieper 1989, Ebrahim et al. 2006, Herrera
et al. 2007, Tosun et al. 2008) because wide root
canals (e.g. >size 60), associated with open apices,
adversely influence the function of apex locators. In
wide canals, the electronic working length is shorter
than the actual canal length (Wu et al. 1992, ElAyouti
et al. 2005).
Paper point techniques may be used to determine
canal length in open apices (Baggett et al. 1996) and to
check or adjust the electronic working length (Rosen-
berg 2003). These techniques require the canal to be
completely dry and the periapical tissues to be relatively
moist (i.e. not excessively dry or moist). In open apices,
the control of moisture is difficult because the contact
area to the inflamed periapical tissues is large, and
excess moisture is common, which results in measure-
ment error. Moreover, to obtain accurate measure-
ments when using tactile techniques the periapical
tissues must be located at the same level of the apical
terminus, a condition that may not be fulfilled in open
apices, because the periapical tissues may grow down
the canal up to a distance of 3 mm (Baggett et al.
1996) and result in short measurements.
The aim of the present paper was to present a
consistent tactile method for working length determi-
nation in teeth with open apices, and to determine the
accuracy, repeatability and inter-operator agreement of
the Tactile Method under simulated clinical conditions.
Materials and methods
The Tactile Method implements a hand instrument to
probe the dentinal walls of the root canal. A stainless
steel hand file, plugger or spreader can be used. In this
study a size 25 K-File was used. The file was bent at the
tip (0.5–1 mm) to a 90� angle using an endodontic
gauge (Dentsply Maillefer, Ballaigues, Switzerland). The
tip of the file was placed in the gauge hole correspond-
ing to the size of the file and bent to be parallel to the
gauge surface. The angle of the bent tip was checked to
be right angle using a square gauge. Instead of
conventional rubber stoppers a small silicon ring was
attached to the shaft of the file. The marking line on
the silicon ring was used to indicate the direction of the
bent tip. The file was slightly curved to facilitate the
engagement of the bent tip on the apical edge of
dentinal walls (Fig. 1).
Ninety teeth (30 anterior teeth, 30 premolars and 30
molars) with 129 root canals were selected after
excluding curved roots (>10 degrees). To simulate
immature open apices, the apical 3–4 mm of the roots
were removed and the canal was widened with large
files and Gates Glidden burs to obtain 0.5–1.5 mm
dentinal walls thickness at the apex. Subsequently,
apical resorption was simulated by rendering the
dentinal walls at the apex irregular using fine diamond
round burs and SONICflex ultrasonic tips (Kavo, Bibe-
rach, Germany). The differences between dentinal-wall
lengths in the same root ranged from 2 to 5 mm.
The correct working length (CWL) was defined to be
at the level of the shortest dentinal wall as at this level
Figure 1 Schematic presentation of the Tactile Method and
the measuring file.
ElAyouti et al. Tactile working length
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1090–1095, 2009 1091
Page 36
the root canal is surrounded by dentine. The CWL for
each root canal was determined by inserting a file into
the canal to the level of the shortest dentinal wall. A
silicon stopper was adjusted to a coronal reference
point. The length of the file corresponding to the CWL
was measured using a digital micrometer under a
stereomicroscope; Stemi (Carl Zeiss, Jena, Germany) at
16· magnification. The roots were then embedded in a
low viscosity Impression material (President, Coltene/
Whaledent AG, Altstatten, Switzerland) using a 15 mm
brass ring. To prevent the impression material from
flowing into the canal the apices of teeth were covered
with a piece of wax that was removed after the setting
of the material. The teeth with the embedded roots
were fixed according to their anatomical position in
either the madibular or maxillary tooth model (G50;
Kavo) of a dental simulation unit (DSEplus; Kavo). The
face mask and the antagonist jaw tooth-model of the
dental mannequin allowed for simulated clinical con-
ditions by limiting the accessibility of the teeth.
The Tactile Method
The aim of the Tactile Method is to circumferentially
probe the dentinal walls with the bent tip of the file
to determine the length of the shortest dentinal wall.
A K-File size 25 curved and bent at the tip, as described,
was used (Fig. 1). The bent tip was placed against a
dentinal wall in the root canal and displaced apically
until it engaged the edge of dentinal wall at the apex
(Fig. 1). The silicon ring was adjusted to a coronal
reference point and the file was then rotated to
disengage the bent tip. The same procedure was
repeated to circumferentially probe all dentinal walls.
When a shorter length was detected the silicon ring
was readjusted, the shortest adjusted length of the file
represented the tactile working length (TWL). The file
length (from the bent tip to the silicon ring) was
measured using a digital micrometer under magnifica-
tion (·16). The length of each root canal was measured
by two operators and each operator repeated the
measurement once. All measurements were recorded
and performed successively on each tooth. The first
operator had 1-year experience with the Tactile Method
and the second operator had a practical demonstration
and practised the method on extracted teeth 1 week
prior to the study.
The accuracy of the Tactile Method was determined
by comparing the CWL with the TWL of the first
operator. The mean of absolute differences (positive
values) and the 99% confidence interval (CI) were
calculated. A regression analysis was performed to
evaluate the influence of tooth type and canal length
on the accuracy of the Tactile Method.
The repeatability of the Tactile Method was deter-
mined by performing paired analysis of the repeated
measurements in each tooth. The coefficient of repeat-
ability that includes 95% of the differences was
calculated (Bland & Altman 1986).
The inter-operator agreement was determined by
comparing the average of the repeated measurements
per tooth. The limits of agreement, which are twice the
standard deviations around the mean, were calculated
(Bland & Altman 1986).
Results
The accuracy of the Tactile Method within a range of
0.5 mm was 97.7% (126/129 canals). The mean of
absolute distances between TWL and CWL was 0.1 mm
(99% CI: 0.1–0.2). Box and whiskers plots (Fig. 2)
present the distances to the CWL in each root canal.
Statistically, there were no differences between anterior
teeth, premolars or molars. The length of the canal did
not influence the measurements (Fig. 3).
The coefficient of repeatability of the Tactile Method
was 0.12 mm, the maximum difference between
repeated TWL was 0.2 mm.
The inter-operator agreement upper and lower limits
were )0.2 and 0.5 mm, the maximum difference
between the two operators using the Tactile Method
was 0.6 mm. The readings of the second operator were
shorter than those of the first operator in most of the
canals (Fig. 4).
Discussion
The accuracy of the Tactile Method, calculated in teeth
with simulated open apices, was high (97.7%). It seems
that the wide, short and straight root canals used in the
present study facilitated the measuring procedure.
Nevertheless, the Tactile Method is not feasible in
curved canals or in teeth with an apical size smaller
than 80, but these clinical situations are uncommon
for teeth with open apices.
When Goldberg et al. (2002) evaluated 50 teeth with
simulated apical resorption they found that the accu-
racy of Root ZX apex locator was 62.7% (with a
tolerance of ±0.5 mm). But, Mente et al. (2002)
concluded by inspecting 24 cleared teeth that the
presence of apical resorption did not affect the accuracy
of apex locators. They found that the mean distance to
Tactile working length ElAyouti et al.
International Endodontic Journal, 42, 1090–1095, 2009 ª 2009 International Endodontic Journal1092
Page 37
the acceptable working length in teeth without resorp-
tion (0.26 mm) was similar to that with resorption
(0.29 mm). Apical resorption is one factor that may
affect the electronic working length in open apices, but
the associated wide root canals (size 60 and more) is
another factor. Although, different studies have showed
that wide canals may not affect the accuracy of apex
locators (Nguyen et al. 1996, Lee et al. 2002), it must
be emphasized that the maximum size of the examined
canals was 60, which is not comparable with the large
sized canals associated with open apices. Other studies
examining apex locators in canals with larger sizes (>
60) showed that wide canals do result in short
electronic measurements (Wu et al. 1992, ElAyouti
et al. 2005). Hulsmann & Pieper (1989) found that
apex locators did not function in teeth with open
apices, but after apexification apex locators determined
the canal length correctly.
Radiographic methods may lead to overestimation of
the canal length (Stein & Corcoran 1992, ElAyouti
et al. 2001, Williams et al. 2006). The main reason is
the fact that the apical foramen is frequently (92%)
located short of the apex (Burch & Hulen1972) and the
length of measuring file appears radiographically
shorter than its actual length (Stein & Corcoran
1992). In teeth with open apices the radiographic
interpretation of canal length is even more difficult due
to the altered apical anatomy and the missing peri-
odontal ligament space at the apex.
The paper point techniques (Baggett et al. 1996,
Rosenberg 2003) may deliver accurate measurements
provided that the periapical tissues exist at the same
Figure 2 Box and whiskers plot of the
differences between tactile working
length (TWL) and correct working
length (CWL) in each tooth group.
Figure 3 Paired analysis of the differ-
ences between correct working length
(CWL) and tactile working length (TWL)
in relation to root canal length.
ElAyouti et al. Tactile working length
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1090–1095, 2009 1093
Page 38
level of canal terminus and that moisture control is
possible within the canal as well as from the periapical
tissues. Baggett et al. (1996) calculated an accuracy of
95% for the paper point technique when all measure-
ments within ±1 mm from the radiographic apex were
considered accurate. Nevertheless, the accuracy of the
paper point techniques remains to be determined in
relation to the actual canal length, which is a more
valid reference than the radiographic length.
Root canal treatment of teeth with open apices is
more common in anterior rather than posterior teeth.
Even so, molars and premolars were included in this
study because there are clinical situations that neces-
sitate the treatment of a posterior tooth with an open
apex, for example, treatment of infected resected teeth
(Bogen & Kuttler 2009), necrotic immature teeth
(Gutmann & Heaton 1981) or teeth with extensive
apical resorption (Kerezoudis et al. 1999).
Whilst carbide burs have been used to simulate apical
resorption (Goldberg et al. 2002), in the present study
ultrasonic tips were also used to render the irregularities
of the dentine walls smooth. Indeed, the apical anatomy
of open apices may deviate from the simulated form, and
therefore clinical studies are still necessary to validate
the accuracy of the Tactile Method.
The file used for the Tactile Method was curved to
allow an easy and reproducible engagement of the bent
file tip on dentinal wall margins. Also, the use of a small
silicon ring instead of conventional stoppers facilitated
the manoeuvring of the instrument without interfering
with the coronal reference point. The size of the file used
was 25; this provided enough instrument stiffness to
probe the dentinal walls. However, instruments with
larger sizes may also be used in wider root canals.
The minor differences between repeated measure-
ments (0.2 mm) showed that repeating the measure-
ment in the same canal was not necessary. Clinically,
this high repeatability may not be attainable because it
is impractical to measure the length of the file with a
digital micrometer under magnification, and therefore
clinically, repeated measurements may still yield more
accuracy. Notably, the high repeatability was also
observed in the measurements of the second operator
who learned the Tactile Method 1 week prior to the
study, this demonstrated the consistency of the Tactile
Method and the reproducibility of the apical and
coronal reference points.
The inter-operator differences were at a maximum of
0.6 mm, whereas the second operator delivered shorter
measurements in most of canals. This could be explained
by different interpretation of the distance between the
bent tip and stopper. This was in agreement with an
earlier study that reported the inter-operator agreement
limits to be around 0.7 mm when the stopper of a file
was adapted to a reference point and the length of the file
was measured (ElAyouti & Lost 2006).
Operators who used the Tactile Method for the first
time, as the second operator in the present study,
experienced difficulties in disengaging of the file tip
from dentinal wall. This difficulty can be overcome by
curving the file and slightly rotating it on removal out
of the canal. Also, a helpful orientation may be
provided by adjusting the marking line on the silicon
ring to indicate the direction of the bent tip.
Figure 4 Plot of inter-operator agreement showing the limits of agreement and the differences between the first and second
operator in each canal.
Tactile working length ElAyouti et al.
International Endodontic Journal, 42, 1090–1095, 2009 ª 2009 International Endodontic Journal1094
Page 39
Conclusion
In teeth with open apices, the presented Tactile Method
may offer an accurate alternative to contemporary
methods of working length determination.
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ElAyouti et al. Tactile working length
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1090–1095, 2009 1095
Page 40
Preliminary study of the presence and associationof bacteria and archaea in teeth with apicalperiodontitis
Y. T. Jiang1, W. W. Xia1, C. L. Li2, W. Jiang1 & J. P. Liang1
1Department of Endodontics and Operative Dentistry, School of Stomatology, Ninth People’s Hospital, Shanghai Jiao Tong
University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai; and 2Department of Periodontology, School of
Stomatology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of
Stomatology, Shanghai, China
Abstract
Jiang YT, Xia WW, Li CL, Jiang W, Liang JP. Preliminary
study of the presence and association of bacteria and archaea in
teeth with apical periodontitis. International Endodontic
Journal, 42, 1096–1103, 2009
Aim To investigate, by reverse transcription polymer-
ase chain reaction (RT-PCR), the presence and associ-
ation of bacteria and archaea in primary and secondary
root canal infections.
Methodology A total of 77 root canal samples from
77 Chinese patients, 42 with necrotic pulp tissues
(primary infection) and 35 with failed prior conven-
tional root canal treatment (secondary infection),
aseptically exposed at the first patient visit, were
studied. Total RNA was isolated directly from each
sample, and 16S rRNA gene-based RT-PCR assays were
used to determine the presence of bacteria and archaea,
respectively.
Results Bacteria were detected in 39/42 (93%) of
root canal samples from teeth with primary infections,
and archaea in 16/42 (38%). In the cases diagnosed as
secondary root-infected canals, bacteria were detected
in 30/35 (86%), whilst archaea were detected in 6/35
(17%) of cases. Amongst the canals, which were
positive for bacteria, archaea were always found in
combination with bacteria. The incidence of symptom-
atic cases positive for both bacteria and archaea (16/
22, 73%) were significantly higher than those positive
for bacteria alone (21/47, 45%) (P < 0.05).
Conclusions This study confirms the presence of
archaea in root canal infections and further implicates
them in an association with clinical symptoms. The
nature of this association requires further study.
Keywords: archaea, bacteria, root canal infections,
RT-PCR.
Received 24 August 2008; accepted 1 September 2009
Introduction
Chronic apical periodontitis is a condition describing
a group of inflammatory diseases with a multitude of
clinical features that afflicts humans (Baumgartner
et al. 2006). Contemporary knowledge of the patho-
genesis of apical periodontitis shows that primary
infections are polymicrobial in character and
dominated by anaerobic gram-negative bacteria
(Sundqvist 1992, Gomes et al. 2004). Secondary
infections may be caused by microorganisms that
gain entry into the canal system after professional
intervention and or as a result of coronal leakage
before or after root filling (Siqueira 2002, Sakamoto
et al. 2008). Besides bacteria, other infective factors
such as Candida spp. (Sundqvist et al. 1998, Peciuli-
ene et al. 2001), human cytomegalovirus and
Epstein-Barr virus (Sabeti et al. 2003) have also been
detected in infected root canals. More than 150
microbial species have been isolated and cultured
from root canals (Sundqvist 1976, Molander et al.
Correspondence: Prof. Jing Ping Liang, Department of End-
odontics and Operative Dentistry, Ninth People’s Hospital,
Shanghai Jiao Tong University School of Medicine, Shanghai
Key Laboratory of Stomatology, Shanghai, China (Tel.: +86
21 6313 5412; fax: +86 21 6313 5412; e-mail: lian-
[email protected] ).
doi:10.1111/j.1365-2591.2009.01639.x
International Endodontic Journal, 42, 1096–1103, 2009 ª 2009 International Endodontic Journal1096
Page 41
1998, Baumgartner et al. 2004). These various
microbes form a complex community of organisms
that interact with each other and play an important
role in the aetiology of apical periodontitis (Sedgley
et al. 2008). Therefore, it seems unreasonable to
reduce the suspected causative agents to a simple and
specific (e.g. single species) aetiology. In order to have
a more complete understanding of the role of
microorganisms in root canal infections, the patho-
genic theory should be evaluated from a microbial
community perspective.
Archaea, one of the three domains of life (Woese
et al. 1990), have been isolated from the human oral
cavity (Kulik et al. 2001), as well as the human gut
(Miller & Wolin 1982) and vagina (Belay et al. 1990).
Although they are now recognized as a component of
human microbiota, none of the archaea domain has
been established as a causative agent in human disease.
However, they do share some characteristics with
known pathogens that may reflect the potential to
cause disease. Such characteristics include ample
access to a host (i.e. opportunity) and capabilities for
long-term colonization and coexistence with endoge-
nous microbiota in a host (Miller & Wolin 1982, Belay
et al. 1988, 1990, Kulik et al. 2001). Recently, there
has been increasing interest in the relationship between
archaea and periapical disease (Siqueira et al. 2005,
Vianna et al. 2006, Vickerman et al. 2007), whereas
there is little information on the association between
bacteria and archaea in primary and secondary root
canal infections.
The emergence of a variety of cultivation-indepen-
dent molecular methods, based mainly on 16S rDNA
sequences, has widened the scope of detectable
microorganisms to include uncultivable organisms
that might play significant roles, as yet undefined, in
pathogenesis (Munson et al. 2002, Saito et al. 2006,
Siqueira et al. 2007). Most of these studies focus on
the detection of DNA, which may originate from dead
cells, or even from free DNA, giving an erroneous
account of current viable infection. As the ribosome-
per-cell ratio is roughly proportional to the growth
rate of bacteria (Wagner 1994), rRNA is regarded as
an indicator of total bacterial activity. Hence, the
purpose of this study was to detect the presence of
metabolically active bacteria and archaea in untreated
and treated root canals using 16S rRNA derived from
isolated ribosomes by reverse transcription polymerase
chain reaction (RT-PCR) (Williams et al. 2006), and to
compare their presence with the incidence of clinical
symptoms.
Materials and methods
Patient selection and clinical features
Seventy-seven teeth (one tooth per patient) were
selected from patients who sought root canal treatment
or retreatment at the Shanghai Ninth People’s Hospital.
Forty-two teeth presented with necrotic pulp tissues
and 35 had been root filled >4 years previously and
showed radiographic evidence of apical periodontitis.
A detailed medical and dental history was obtained
from each patient. Patients having received antibiotic
treatment in the previous 3 months or having a
systemic disease were excluded from the study. The
Ethics Committee of Shanghai Jiao Tong University
School of Medicine approved a protocol describing
the specimen collection for this investigation, and all
patients signed an informed consent form to participate
in this study.
Patients were classified as symptomatic if they had a
history of spontaneous pain, pain on percussion or pain
upon palpation immediately prior to the consultation.
The presence of swelling, lymphadenopathy or evi-
dence of a sinus tract was considered symptomatic
whether or not pain was present. Patients without the
above criteria were considered asymptomatic. No teeth
showed significant gingival recession or any of peri-
odontal pockets deeper than 4 mm.
Sampling procedure
Samples from infected root canals were collected as
previously described (Ng et al. 2003). After a two-stage
access cavity preparation, which was made without the
use of water spray but under manual irrigation with
sterile saline solution and employing sterile burs, the
teeth involved were individually isolated from the oral
cavity with a previously disinfected rubber dam.
Disinfection of the rubber dam and teeth was carried
out using first 30% hydrogen peroxide and then 2.5%
sodium hypochlorite. The solution was inactivated with
5% sodium thiosulphate to avoid interference with the
bacteriological sampling. Aseptic techniques were used
throughout endodontic therapy and sample acquisi-
tion. After initial entry into the pulp space, the patency
of the root canal was established with minimal instru-
mentation and without the use of any chemically active
irrigant. Pre-existing root filling material was removed
using Gates Glidden drills and endodontic files without
the use of chemical solvents. Irrigation with sterile
saline solution was performed to remove any remaining
Jiang et al. Presence and association of bacteria and archaea
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1096–1103, 2009 1097
Page 42
treatment materials prior to sample collection. In
multi-rooted teeth, the criterion used to choose the
canal to be microbiologically investigated was the
presence of exudate or, in its absence, the canal
associated with the periapical radiolucency. In each
case, a single root canal associated with the criterion
above was sampled in order to confine the microbial
evaluation to a single ecological environment.
After minimal canal enlargement with sterile saline
irrigant to allow access to the working length, dry,
autoclaved paper points were placed in the canal space
for 60 s. The samples were collected with as many
paper points necessary to absorb all the fluid inside the
canal and inserted to the full length of the canal as
calculated from the preoperative radiograph. After-
wards, the paper points per root canal were pooled in
a sterile tube containing 1 mL)1 Sample Protector
(Takara, Dalian, China) and transported to the micro-
biology laboratory in dry ice, then stored at )80 �C for
4 weeks or less before extraction of total genomic
RNA.
Nucleic acid isolation
The frozen paper point samples were thawed and
dispersed by vortexing for 60 s. The Sample Protector
contained glass beads 3 mm in diameter to facilitate
mixing and homogenization of the sample prior to
extraction. Then, the samples were centrifuged for
5 min at 12 000g, with the supernatant discarded and
the pellet resuspended in 1 mL)1 TRIzol Reagent
(Invitrogen, Carlsbad, CA, USA). The microbial RNA
was extracted from the samples according to the
manufacturer’s protocol and reference method (Chom-
czynski & Sacchi 1987, 2006). Briefly, after incubating
the homogenized samples for 5 min at room tempera-
ture to obtain complete dissociation of nucleoprotein
complexes, 0.2 mL)1 of chloroform was added per
0.75 mL)1 of TRIzol Reagent in capped sample tubes.
The tubes were shaken vigorously by hand for 15 s,
incubated at room temperature for 15 min and centri-
fuged at 12 000g for 15 min at 5 �C. Following
centrifugation, the mixture separated into a lower
red, phenol–chloroform phase, an interphase and a
colourless upper aqueous phase containing RNA. After
transferring the aqueous phase to a clean tube, RNA
was precipitated by adding 0.5 mL)1 isopropyl alcohol
per 0.75 mL)1 of TRIzol Reagent used for the initial
homogenization. Samples were then incubated at room
temperature for 10 min and centrifuged at 12 000g for
10 min at 5 �C. The RNA precipitate, often invisible
before centrifugation, formed a gel-like pellet on the
side and bottom of the tube. After removing the
supernatant, the RNA pellet was washed once by
adding 1 mL)1 of 75% ethanol per 0.75 mL)1 of TRIzol
Reagent used for the initial homogenization. Samples
were mixed on a vortex and centrifuged at 7500g for
5 min at 5 �C. The RNA pellet was briefly dried and
then reconstituted in RNAse free water.
DNAse treatments
Extracted crude RNA was treated enzymatically with
DNAse to remove contaminant genomic DNA. For each
reaction, 8 lL of extract was incubated for 30 min at
37 �C with DNAse (RQ1 RNAse free DNAse; Promega,
Shanghai, China) in buffer plus inhibitors of RNAse
(Recombinant RNasins Ribonuclease Inhibitor, Pro-
mega, Shanghai, China). After incubation, 1 lL of
STOP DNAse was added to each tube and samples were
incubated for 15 min at 70 �C to inactivate DNAse and
to denature RNA. Samples were chilled on ice for
10 min. The absence of genomic DNA was confirmed
by PCR performed with universal bacterial and archa-
eal primers (Yu & Morrison 2001, Lepp et al. 2004).
The integrity and quantity of the purified RNA were
examined by absorbance ratio A260/A280 and RNA gel
electrophoresis (Cury et al. 2008).
Reverse transcription of total RNAs
Complementary DNA (cDNA) synthesis was carried out
with the Reverse Transcription System (Promega,
Madison, WI, USA), and cDNA were quantified based
on absorbance at 260 nm. The purified cDNA were
checked on a 1.5% agarose gel and stored at )20 �C
prior to amplification.
Universal bacterial primers and PCR conditions
The variable V3–V5 region of 16S rRNA was enzy-
matically amplified with primers located on conserved
ends of the V3 and V5 region (Yu & Morrison 2001).
The primers were as follows: primer 341f, 5¢-CCTACG-
GGAGGCAGCAG-3¢; primer 926r, 5¢-CCGTCAATTCCT-
TTGAGTTT-3¢. A combination of primer 341f and 926r
was used to amplify the V3–V5 region of 16S rRNA in
the different bacterial species, which correspond to
positions 341–926 in E. coli. Each reaction mixture
contained 2.5 lL of 10· PCR buffer [100 mmol L)1
Tris–HCI (pH 9), 15 mmol L)1 MgCl2, 500 mmol L)1
KCI, 0.1% (w/v) gelatin, 1% (v/v) Triton X-100],
Presence and association of bacteria and archaea Jiang et al.
International Endodontic Journal, 42, 1096–1103, 2009 ª 2009 International Endodontic Journal1098
Page 43
0.2 mmol deoxynucleotide triphosphate, 1 U of Hot-
StarTaq DNA polymerase (Qiagen, Hamburg, Ger-
many), 0.25 mmol of each forward and reverse
primer, 50 ng template cDNA and enough sterile
MilliQ water to bring the final volume to 50 lL. PCR
amplification was performed using the Techne thermo-
cycler (Biometra, Gottingen, Germany). Amplification
consisted of 30 cycles of denaturation for 30 s at 94 �C,
annealing for 30 s at 58 �C, and extension for 1 min at
72 �C. The first cycle was preceded by an initial
template denaturation step of 4 min at 94 �C, and
the last cycle was followed by a final extension step
of 7 min at 72 �C. PCR products were separated by
electrophoresis in 1.5% agarose gels in 1· TAE buffer
(40 mmol L)1 Tris acetate, 20 mmol L)1 sodium acet-
ate, 1 mmol L)1 EDTA, pH 8.0) and visualized under
UV light, following an ethidium bromide staining, the
positive samples were recorded.
Universal archaeal primers and PCR conditions
Fragments of 16S rRNA from samples were PCR
amplified by using broad-range archaeal primers
SDArch0333aS15 (5¢-TCCAGGCCCTACGGG-3¢) and
SDArch0958aA19 (5¢-YCCGGCGTTGAMTCCAATT-3¢)(Lepp et al. 2004). Each reaction mixture contained
2.5 lL of 10· PCR buffer [100 mmol L)1 Tris–HCI (pH
9), 15 mmol L)1 MgCl2, 500 mmol L)1 KCI, 0.1% (w/v)
gelatin, 1% (v/v) Triton X-100], 0.2 mmol deoxy-
nucleotide triphosphate, 1 U of HotStarTaq DNA
polymerase (Qiagen), 0.25 mmol of each forward and
reverse primer, 50 ng template cDNA and enough
sterile MilliQ water to bring the final volume to 50 lL.
Archaeal 16S rRNA genes were amplified under the
following cycle conditions: 35 cycles of 94 �C (30 s),
58 �C (30 s) and 72 �C (30 s) followed by a 3-min
extension at 72 �C. PCR products were separated by
electrophoresis in 1.5% agarose gels in 1· TAE buffer
and visualized under UV light, following an ethidium
bromide staining, the positive samples were recorded.
Statistical analysis
Chi-squared analysis was used to determine a statisti-
cally significant difference between the prevalence of
bacteria and archaea, and statistical correlation of
clinical symptoms with the prevalence of bacteria alone
or both bacteria and archaea. Statistical analysis was
performed with SAS Software (version 6.12, SAS
Institute, Cary, NC, USA). Significance level was set at
P < 0.05.
Results
A total of 77 samples, 42 teeth with primary endodon-
tic infection and 35 with secondary endodontic
infection (i.e. failed treatment), were subjected to RT-
PCR with universal bacterial primers and archaeal
primers. Table 1 shows the distribution of bacteria and
archaea in different root canal infections. In all
subjects, the prevalence was 88.14% and 28.5%,
respectively. The positive rate of bacteria was 92.9%
in primary apical periodontitis (39/42) and 85.7% in
secondary apical periodontitis (30/35). Archaea were
detected in 38.1% (16/42) of canals with necrotic pulps
and 17.1% (6/35) in treated canals.
Of the 69 root canals positive for bacteria, 37
(53.6%) were from patients with symptoms. Archaea
were always found in combination with bacteria, and
the symptomatic cases positive for both bacteria and
archaea were significantly higher than those positive
for bacteria alone (Table 2) (P < 0.05).
Discussion
Historically, conventional culture methods have been
used to detect bacteria in infected root canals, thus only
allowing detection of bacteria capable of dividing
(Sundqvist 1994, Le Goff et al. 1997). However, over
Table 1 Prevalence of bacteria and archaea found in 77
root canalsa
Group Bacteria (%) Archaea (%) P*
Primary root canal
infection (n = 42)
39 (92.9) 16 (38.1)
Secondary root canal
infection (n = 35)
30 (85.7) 6 (17.1)
Total (n = 77) 69 (88.1) 22 (28.5) 0.001
aData are number and (percentage) of subjects.
*Percentage of archaea is obviously lower than that of bacteria
(P < 0.01).
Table 2 Correlation of clinical symptoms with prevalence of
bacteria alone or in combination with archaeaa
Symptomatic
(%)
Asymptomatic
(%) P*
Bacteria + archaea
(n = 22)
16 (72.7) 6 0.03
Bacteria alone
(n = 47)
21 (44.7) 26
aData are number and (percentage) of subjects.
*Symptomatic cases positive for both bacteria and archaea
were significantly higher than those positive for bacteria alone
(P < 0.05).
Jiang et al. Presence and association of bacteria and archaea
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1096–1103, 2009 1099
Page 44
the past few years it has been demonstrated that
nonculturable bacteria make up an undetermined
proportion of the microbial population in the infected
root canal system (Siqueira & Rocas 2003, 2005, Saito
et al. 2006). Moreover, several reports have found that
bacteria become less culturable under starvation con-
ditions, and these viable but nonculturable (VBNC)
bacteria demonstrate metabolic activity (Mason et al.
1986, Kaprelyants et al. 1993, Oliver 1995), maintain
their pathogenic features and resume division when
favourable environmental conditions are restored (Lleo
et al. 2001). The VBNC state might be a survival
strategy that persists in the root canal. Because the
nonculturable microorganisms could play a part in the
perpetuation of periapical disease, it becomes manda-
tory, for proper study of infected root canals, to develop
and apply methods capable of detecting such bacterial
forms (Siqueira & Rocas 2003, 2005).
Amongst the various molecular methods, PCR has
proven useful for detecting target microorganisms in
endodontic samples (Siqueira & Rocas 2003). Conven-
tional PCR assays, however, detect only the presence or
absence of genomic DNA of microorganisms present in
the root canal space and cannot distinguish between
viable and nonviable microorganisms. Recent research
demonstrates that PCR-detectable DNA from dead
bacteria might persist after cell death (Young et al.
2007). Ribosomes can be used as markers for bacterial
activity because the number of ribosomes (and their
rRNA) per cell maybe roughly proportional to the
growth activity of bacteria in pure culture (Wagner
1994). For successful isolation of intact RNA, it is
important to avoid the death of bacteria and enzymatic
degradation of RNA during the handling and process-
ing of samples. In this study, Sample Protector (an
aqueous tissue storage reagent) was used to overcome
these problems by simply adding the reagent directly to
the root canal samples and providing immediate RNA
stabilization prior to RNA isolation. Isolation of high-
quality RNA is another important step for the down-
stream processes. Any extracted RNA must be devoid of
contaminants such as salt, protein, solvents and
genomic DNA. The extracted RNA was ‘quality con-
trolled’ using gel electrophoresis, PCR and optical
density measurements. Gel electrophoresis and ‘no-RT’
control during RT-PCR were used to check for genomic
DNA contamination. Optical density was used to assay
the RNA yield and to check for contamination by salt,
solvent, protein, etc.
Although the total number of viable cells present in a
population can be determined by using 4¢,6-diamidino-
2-phenylindole (DAPI) or acridine orange staining
or by establishing the presence of an intact cyto-
plasmic membrane [(BacLight�, Molecular Probes, Inc,
Eugene, OR, USA) or propidium iodide] (Oliver 2005),
detection of mRNA by RT-PCR is regarded as the most
appropriate method of evaluating the specific RNA
against a large background of procaryotic and eucary-
otic cells present in root canal samples. This study
reports the application of ribosome isolation and
subsequent RT-PCR, leading to the identification of
the metabolic portion of root canal microbial commu-
nities. Such data should provide a more realistic basis
for discussion about the correlation between clinical
symptoms and viable microbial species.
Archaea are microorganisms distinct from bacteria
and eukaryotes (Woese et al. 1990). They can be found
in most ecosystems and are often prevalent in extreme
environments. RT-PCR of the present study indicated
that both bacteria and archaea can be detected in
primary and secondary root canal infections, support-
ing the notion of the poly microbial nature of infected
root canal systems. The prevalence of archaea in
infected root canals in a Chinese population sample was
28.5%, which is in agreement with other surveys of
endodontic infections (Vianna et al. 2006). Despite
their abundant and ubiquitous association with
humans, animals and plants, no pathogenic archaea
have so far been described.
However, amongst 700 different bacterial species that
have been identified from dental plaque and oral cavity
(Paster et al. 2001, Aas et al. 2005), only a relatively
small and select group of bacteria are detected in the root
canal, and appear to have the properties necessary to
invade tubules and survive within the intratubular
environment (Love & Jenkinson 2002). Furthermore,
the infected root canal is a unique environment, unlike
other infectious oral diseases. Apical periodontitis is
caused by infection of the root canal space, normally
devoid of microbes in a healthy state (Nair 2004). Hence,
there is good reason to assume that archaea share some
characteristics with known pathogens that may reflect
the potential to cause apical disease. Such characteris-
tics include ability to colonize the human host and
evasion of host defenses. These virulence factors have
recently been demonstrated in other medical fields
(Cavicchioli et al. 2003, Eckburg et al. 2003).
In the present study, in which the percentage of
archaea in patients with apical periodontitis was
obviously lower than that of bacteria (88.1%;
P < 0.01), it is noteworthy that archaea were always
found in combination with bacteria, and there was a
Presence and association of bacteria and archaea Jiang et al.
International Endodontic Journal, 42, 1096–1103, 2009 ª 2009 International Endodontic Journal1100
Page 45
statistically significant difference between the percent-
age of symptomatic cases positive for both archaea and
bacteria and bacteria alone (P < 0.05). More recently,
it has been demonstrated that bacteria may co-operate
for invasion of dentinal tubules (Love & Jenkinson
2002). With this in mind, the potential symbiotic
relationship between archaea and bacteria may fulfil a
similar role in endodontic infections.
Methanogens might be the only archaea in the
human body (Vianna et al. 2006, Vickerman et al.
2007). They are strict anaerobes characterized by the
ability to produce methane from H2/CO2 and, in some
cases, from formate, acetate or methanol. Hydrogen is
the waste end product of the metabolism of microor-
ganisms in anoxic environments. Maintaining a low
hydrogen concentration is important because the
anaerobic fermentative process becomes increasingly
unfavourable as the partial pressure of hydrogen
increases, which affects microbial growth. The meth-
anogens depend on the hydrogen and carbon dioxide
produced by other species; in return some of these other
species grow better in the presence of the methanogens
because of the altered patterns of redox balance
associated with reduced partial pressure of hydrogen
due to interspecies hydrogen transfer (Lovley 1985,
Bonch-Osmolovskaya & Stetter 1991, Conrad 1999). It
can be deduced from the mechanism of ‘interspecies
hydrogen transfer’ that methanogens may play an
important role in increasing activity of some species of
microorganisms in the root canal system and contrib-
ute to local apical tissue damage.
The microbial community in the root canal system is
thought to undergo ecological succession as different
species combinations emerge at different levels (Sundq-
vist & Figdor 2003). It has been suggested that
metabolic competition for hydrogen with sulphate-
reducing bacteria (Vianna et al. 2006), such as the
Desulfovibrio or treponemal species (Lepp et al. 2004),
might inhibit the coexistence of these bacteria with
methanogenic archaea. This might explain in part the
presence of archaea in some, but not all, cases of
endodontic infections. An understanding of the inter-
actions between archaea, bacteria and other members
of the root canal microbiota may help elucidate the
bacterial physiological and pathological functions
underlying periapical disease activity.
Conclusion
This study showed archaea to be present in root canals
but always with bacteria. Their combined presence was
associated with a significantly higher prevalence
of clinical symptoms compared with the sole presence
of bacteria.
Acknowledgements
This work was supported by the Science and Technol-
ogy Commission of Shanghai (08DZ2271100), Shang-
hai Leading Academic Discipline Project (S30206) and
the National Natural Science Foundation of China
(30801291).
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Sonic extracts from a bacterium related toperiapical disease activate gelatinase A andinactivate tissue inhibitor of metalloproteinasesTIMP-1 and TIMP-2
Y. Sato1, J. Kishi2, K. Suzuki1, H. Nakamura1 & T. Hayakawa2
1Department of Endodontics, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan; and 2Department of Biochemistry,
School of Dentistry, Aichi-Gakuin University, Nagoya, Japan
Abstract
Sato Y, Kishi J, Suzuki K, Nakamura H, Hayakawa T.
Sonic extracts from a bacterium related to periapical disease
activate gelatinase A and inactivate tissue inhibitor of metallo-
proteinases TIMP-1 and TIMP-2. International Endodontic
Journal, 42, 1104–1111, 2009.
Aim To examine the effects of sonicated bacterial
extracts (SBEs) from three related to periapical
disease bacteria (Porphyromonas gingivalis, P. endodon-
talis and F. nucleatum) on the activation of matrix
metalloproteinase (MMP-2) and the inactivation of
tissue inhibitors of metalloproteinase (TIMP-1 and
TIMP-2).
Methodology Each SBE was added to cultures of
human periodontal ligament (PL) cells or HT1080
cells and their supernatants were analysed by zymo-
graphy for MMP-2. Each SBE was added to PL cell
cultures, and the amount of TIMP-1 was determined
by ELISA. P. gingivalis SBE was incubated with
HT1080 cell culture supernatants, and the amounts
of TIMP-1 and TIMP-2 were determined by ELISA.
Statistical analysis was performed with the paired
Student’s t-test.
Results In extracts of PL cells that had been
incubated in the presence of P. gingivalis SBE, one
representing pro-MMP-2 (72 kDa) and a band
corresponding to the active MMP-2 (66 kDa) were
observed; but in the other extracts it was not detected.
When HT1080 cells were treated with P. gingivalis SBE,
the pro-MMPs was processed into 86- and 66-kDa
fragments, but in the other extracts, the processing did
not occur when the other SBEs were used. When PL
cells were incubated with the same SBEs, the amount of
TIMP-1 was markedly decreased (P < 0.01), but in the
other extracts, it was not. The amounts of both TIMP-1
and TIMP-2 were decreased in a dose-dependent
manner when HT1080 cell culture supernatant was
incubated with P. gingivalis SBE.
Conclusions These findings suggest that P. gingiva-
lis SBE may cause connective tissue to be destroyed,
contributing to the process of periapical disease, by
activating pro-MMP-2 as well as by inactivating
TIMP-1 and TIMP-2.
Keywords: ECM, MMP-2, P. gingivalis, SBE, TIMP-1,
TIMP-2.
Received 23 September 2008; accepted 01 September 2009
Introduction
Inflammatory changes in major pathological lesions in
oral tissues could progress via the destruction of the
extracellular matrix (ECM) in the periodontal ligament
and alveolar bone. It has been reported that the
degradation of the ECM is associated, in large part,
with matrix metalloproteinases (MMPs), including
interstitial collagenase (MMP-1), gelatinase A (72-kDa
gelatinase/IV type collagenase, MMP-2), gelatinase B
(92-kDa gelatinase/type IV collagenase, MMP-9) and
stromelysin-1 (MMP-3). Twenty-three members of this
MMP family have been detected in humans (Visse &
Nagase 2003). These MMPs are also produced and
Correspondence: Hiroshi Nakamura, Department of Endodon-
tics, School of Dentistry, Aichi-Gakuin University, 2-11
Suemori-dori, Chikusa-ku, Nagoya 464-8651, Japan (Tel.:
+81 52 759 2147; fax: +81 52 764 2299; e-mail: nakaendo
@dpc.aichi-gakuin.ac.jp).
doi:10.1111/j.1365-2591.2009.01640.x
International Endodontic Journal, 42, 1104–1111, 2009 ª 2009 International Endodontic Journal1104
Page 49
secreted by inflammatory cells, including neutrophils
and macrophages that migrate into inflamed sites, as
well as by noninflammatory cells, including fibroblasts
(Nagase & Woessner 1999). Four tissue inhibitors of
metalloproteinases (TIMPs), the common endogenous
inhibitors of these MMPs have been reported: TIMP-1,
TIMP-2, TIMP-3, and TIMP-4 (Declerk et al. 1989,
Goldberg et al. 1989, Stetler-Stevenson et al. 1989,
Kishi et al. 1991, Ward et al. 1991, Apte et al. 1994,
Uria et al. 1994, Green et al. 1996). These TIMPs play
roles in controlling the degradation of ECM components
by inhibiting MMP activity; the imbalance between
levels of MMPs and TIMPs during inflammation is
thought to be of importance (Nagase & Woessner
1999). Study of infected root canal systems using
modern anaerobic bacterial culture techniques has
revealed many obligate anaerobic bacteria (Bergenholtz
1974, Kantz & Henry 1974, Wittgow & Sabiston 1975,
Brook et al. 1981, Oguntebi et al. 1982, Williams et al.
1983, Haapasalo 1989) including Porphyromonas,
Bacteroides, Prevotella, Fusobacterium, Eubacterium, and
Veillonella species (Sundqvist 1992). ECM-degrading
enzymes, including collagenase, hyaluronidase, and
LPS (endotoxic activity) produced by such bacteria
have been reportedly involved in the development and
progress of periapical diseases (Higerd et al. 1978,
Singer & Dutton 1979, Nair et al. 1983, Berit & Klaus
1986).
In this study, sonicated bacterial extracts (SBEs) from
three obligate anaerobic bacteria (P. endodontalis, Por-
phyromonas gingivalis, and F. nucleatum) were used to
examine their effects on gelatinase A (MMP-2), TIMP-1,
and TIMP-2, which are known to be present in
periapical tissue, by using human periodontal ligament
(PL) cell cultures and human fibrosarcoma (HT1080)
cell culture supernatants.
Materials and methods
Preparation of human periodontal ligament cell
cultures
Three freshly extracted human teeth for orthodontic
reasons were immediately soaked and washed with
sterilized physiological saline containing penicillin
(1000 U mL)1; Meiji Seika Kaisha, Ltd, Tokyo, Japan)
and Fungizone (30 lg mL)1; Nippon Squib, Tokyo,
Japan). Periodontal ligament tissue was then isolated
from the surfaces of the roots and washed thoroughly
with RPMI1640 (Nisui Pharmaceuticals, Tokyo, Japan)
containing penicillin (100 U mL)1), streptomycin
(100 lg mL)1; Meiji Seika Kaisha) and Fungizone
(3 lg mL)1). It was then minced into small pieces of
approximately 1.5 mm · 1.5 mm; and the pieces were
placed in a 35-mm cell culture dishes (Corning, NY,
USA), containing RPMI1640 medium supplemented
with 10% foetal bovine serum (FBS; Immuno-Biological
Laboratories Co. Ltd, Saitama, Japan), penicillin
(100 U mL)1), kanamycin (100 lg mL)1), and strep-
tomycin (100 lg mL)1), prior to being cultured at
37 �C in a 5% CO2 atmosphere. When the cells
migrating from the pieces of tissue became confluent,
they were subcultured and used for experiments at
subculture levels 4–8.
Bacteria used and preparation of sonicated bacterial
extracts
Three obligate anaerobic gram-negative bacteria,
P. endodontalis ATCC 35406, P. gingivalis 381, and
F. nucleatum ATCC 10953, were cultured in an
anaerobic glove box (Sanyo, Tokyo, Japan) at 37 �C
and harvested at the late log phase of growth. The cells
were harvested by centrifugation at 5000 g for 15 min,
washed twice with PBS, suspended in PBS again, and
then sonicated twice for 9 min each time in an icebox
by using a Sonifer 250 (Taikex Co., Saitama, Japan).
Thereafter, the sonicates were centrifuged at 10 000g
for 30 min, the intact cells were removed, and the
supernatants were collected. After the supernatants
had been sterilized by passing through a 0.45-lm filter
(Corning), the protein content of each SBE (unfraction-
ated) was determined with a protein assay kit (Bio-Rad,
Tokyo, Japan; data is not shown). Then all superna-
tants were diluted to a concentration of 1 mg of protein
per millilitre and used for further experiments as
sonicated bacterial extracts.
Preparation of human fibrosarcoma (HT1080) cell
culture supernatants
HT1080 cells were cultured with RPMI1640 medium
containing 10% Fetal Bovine Serum (FBS) in 100-mm
cell culture dishes at 37 �C in a 5% CO2 atmosphere.
When they had become confluent, they were washed
twice with PBS, which was then replaced with serum-
free RPMI1640 medium, and cultured for a further
48 h. Then, their supernatants were collected and
centrifuged at 3000 g for 5 min to remove cell com-
ponents, before being centrifuged further at 10 000 g
for 20 min. The supernatants produced were used as
samples for experiments.
Sato et al. SBE from bacteria on activation of MMP-2
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1104–1111, 2009 1105
Page 50
Preparation of TIMP-1 and TIMP-2
TIMP-1 was purified from human gingival cell culture
medium according to the previous method (Kodama
et al. 1981); and TIMP-2, as reported earlier (Sakamoto
et al. 1996).
Cell culture and SBE response experiment
The present study was conducted to examine the MMP-
2-activating ability of and the amounts of TIMP-1 and
TIMP-2 produced by PL cells and HT1080 cells in the
presence of P. gingivalis, F. nucleatum, and P. endodon-
talis SBEs. PL cells (adjusted to 1.5 · 105 cells mL)1 in
RPMI1640 medium containing 10% FBS) were divided
into 24-well culture plates (Funakoshi, Tokyo, Japan),
each well containing 1 mL, and allowed to grow to
confluence at 37 �C in a 5% CO2 atmosphere. They
were then washed twice with PBS followed by replace-
ment with 1 mL of serum-free RPMI1640 medium
containing 10 lg mL)1 of SBE. The cells were subse-
quently cultured for 48 h and supernatants were
collected and centrifuged at 3000 g for 5 min to
remove cell components, before being centrifuged
further at 10 000 g for 20 min. The supernatants
produced were used as samples for experiments and
were used to determine MMP-2 activity and the
amounts of TIMP-1 and TIMP-2.
Treatment of cell culture supernatants with SBEs
One millilitre of HT1080 cell culture supernatant was
added to 1 mL of each SBE, and the mixture was
incubated at 37 �C for 18 h.
Determination of MMP-2 activity
Gelatin zymography was used to determine MMP-2
activity. Electrophoresis was conducted according to
the previous method (Laemmli 1970). The acrylamide
concentrations of the gel were 3% (condensing gel) and
10% (separating gel). The separating gel had gelatin
added to it (DIFCO, Lakes, NJ, USA) to a final concen-
tration of 0.3 mg mL)1. Tris–glycine buffer containing
0.1% sodium dodecyl sulphate (SDS) was used as the
running buffer. After electrophoresis, the gel was soaked
in a 2.5% TritonX-100 solution, and shaken twice at
room temperature for 30 min to remove the SDS. The
gel was then soaked in a 30 mmol L)1 Tris sodium
chloride buffer containing 5 mmol L)1 CaCl2 and
0.2 mol L)1 NaCl (pH 7.5; refer buffer A below) before
being shaken twice at room temperature for 30 min.
Then the TritonX-100 was removed, and the gel was
incubated at 37 �C for 24 h. Finally, the gel was stained
with Coomassie brilliant blue R (Tokyo Chemical
Industry Co., Ltd, Tokyo, Japan) at room temperature
for 1 h before being decolorized with 5% methanol–
7.5% acetic acid. This procedure allowed the MMP-2
activity to be observed as clear bands on a blue
background. For the experiments, Pre-stained Stan-
dards Low Range for SDS-PAGE (Bio-Rad) was used as
molecular weight markers, and HT1080 cell culture
supernatants were used as the positive control.
Determination of amounts of TIMP-1 and TIMP-2
The amounts of TIMP-1and TIMP-2 were determined
by using a previously reported Enzyme Immunoassays
(EIA) method (Kodama et al. 1990, Fujimoto et al.
1993).
Determination of TIMP-1 inhibition of MMP-1 and
MMP-2
The TIMP-1 inhibition of MMP-1 and MMP-2 was by the
solution method (Terato et al. 1976) with [14C]glycine-
labelled collagen used as substrate.
The TIMP-1 inhibition of MMP-2 was also deter-
mined by reverse zymography. After electrophoresis
and gel treatment in the same way as for gelatin
zymography, active gelatinase was added to buffer A
(1 U mL)1); and the resultant gel was incubated at
37 �C for 24 h. Then the gel was stained with
Coomassie brilliant blue R at room temperature for
1 h and decolorized with 5% methanol–7.5% acetic
acid. In this way, TIMP-1 was observed as a blue band.
Treatment of MMP-2 or TIMP-1 with SBEs
Purified MMP-2 (0.5 lg mL)1) or purified TIMP-1
(0.4 lg mL)1) was incubated with P. gingivalis SBE
(0.1 lg mL)1) at 37 �C for 18 h.
Statistical analysis
All measurements were made in triplicate, and the
average values were calculated for each group. Data
were expressed as mean ± standard deviation of the
means (SD; n = 3 for each group). Differences between
control and experimental treatment groups were
determined by using the paired Student’s t-test. Differ-
ences were considered significant if P < 0.01.
SBE from bacteria on activation of MMP-2 Sato et al.
International Endodontic Journal, 42, 1104–1111, 2009 ª 2009 International Endodontic Journal1106
Page 51
Results
Processing by sonicated bacterial extracts
of progelatinase A into a 66-kDa fragment
Progelatinase A in periodontal ligament cell culture medium
Periodontal ligament cell culture medium was treated
with each SBE and subjected to gelatin zymography. As
shown in Fig. 1, neither F. nucleatum nor P. endodontalis
SBE at 10 lg mL)1 showed any effect on the 72-kDa
single band corresponding to progelatinase A (pro-
MMP-2), the density of which was no different from that
of the control. In the presence of 10 lg mL)1 P. gingivalis
SBE; however, the band corresponding to pro-MMP-2
completely disappeared. By reducing the concentration
of P. gingivalis SBE down to 1 lg mL)1, two bands with
molecular masses of 72- and 66-kDa were detected.
Progelatinase A in HT1080 cell culture medium
Electrophoresis of HT1080 cell culture medium pro-
duced 92- and 72-kDa bands, which corresponded to
gelatinase B (pro-MMP-9) and pro-MMP-2, respectively
(Fig. 2, lane 2). Only P. gingivalis SBE processed both
pro-MMPs, pro-MMP-9 into an 86-kDa fragment and
pro-MMP-2 into a 66-kDa fragment (lane 3). Neither
F. nucleatum nor P. endodontalis SBE had any effect on
either pro-MMP (lanes 5 and 7). None of these SBEs
alone possessed MMP-processing activities for either
MMP (lanes 4, 6, and 8).
Purified progelatinase A
Purified pro-MMP-2 was processed by P. gingivalis SBE.
As shown in Fig. 3, two bands, corresponding to 72-
kDa pro-MMP-2 and its 66-kDa fragment, were
detected by gelatin zymography.
Amount of TIMP-1 in PL cell culture medium
Periodontal ligament cell culture medium was first
incubated with a given SBE, and the medium was then
subjected to a sandwich EIA for TIMP-1. TIMP-1 was
not detected in the culture medium of cells treated with
P. gingivalis SBE, suggesting that TIMP-1 was degraded
by P. gingivalis SBE (Fig. 4). Neither the SBE from F.
nucleatum nor that from P. endodontalis affected the
amount of TIMP-1 in the culture medium.
Dose-dependent degradation of TIMP-1 and TIMP-2
P. gingivalis SBE at different concentrations degraded
both TIMPs dose-dependently, as shown in Fig. 5.
Figure 1 Gelatin zymograph of periodontal ligament (PL) cell
culture medium treated with sonicated bacterial extracts
(SBEs) at 37 �C for 48 h. 1, molecular weight markers; 2,
PL cell culture medium alone; 3, 2 + Porphyromonas gingivalis
SBE (10 lg mL)1); 4, 2 + P. gingivalis SBE (1 lg mL)1); 5,
2 + F. nucleatum SBE (10 lg mL)1); 6, 2 + P. endodontalis SBE
(10 lg mL)1).
Figure 2 Gelatin zymograph of HT1080 cell culture medium
treated with SBEs at 37 �C for 18 h. 1, molecular weight
markers; 2, HT1080 cell culture medium alone; 3, 2 +
Porphyromonas gingivalis SBE(1 lg mL)1); 4, P. gingivalis
SBE(1 lg mL)1) alone; 5, 2 + F. nucleatum SBE(10 lg mL)1);
6, F. nucleatum SBE(10 lg mL)1) alone; 7, 2 + P. endodontalis
SBE (10 lg mL)1); 8, P. endodontalis SBE (10 lg mL)1) alone.
Figure 3 Gelatin zymograph of purified progelatinase A (pro-
MMP-2) treated with Porphyromonas gingivalis SBE at 37 �C for
48 h. 1, molecular weight markers; 2, pro-MMP-2 alone; 3,
2 + P. gingivalis SBE (1 lg mL)1); 4, P. gingivalis SBE
(1 lg mL)1) alone.
Sato et al. SBE from bacteria on activation of MMP-2
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1104–1111, 2009 1107
Page 52
TIMP-1 was more susceptible to P. gingivalis SBE than
TIMP-2.
Inhibitory activity of TIMP-1
The effect of each SBE on the inhibitory activity of
TIMP-1 against interstitial collagenase (MMP-1) was
Figure 4 Effects of SBEs on the amount of TIMP-1 in PL cell
culture medium. PL cell culture medium was first treated with
10 lg mL)1 of each SBE at 37 �C for 48 h, and then was
subjected to the sandwich EIA for TIMP-1. 1, PL cell culture
medium alone; 2, 1 + Porphyromonas gingivalis SBE; 3, 1 + F.
nucleatum SBE; 4, 1 + P. endodontalis SBE.
Figure 5 Dose-dependent degradation by Porphyromonas gin-
givalis SBE of TIMP-1 and TIMP-2 in HT1080 cell culture
medium. HT1080 cell culture medium was first treated with
1 lg mL)1 of P. gingivalis SBE at 37 �C for 48 h, and then EIAs
for TIMP-1 and TIMP-2 were performed. Amounts of TIMPs
are expressed as a percentage of the amount in the culture
medium without treatment.
Figure 6 Effects of SBEs on the inhibitory activity of purified
TIMP-1 against interstitial collagenase (MMP-1). Purified
TIMP-1 (0.4 lg) was first with 0.1 lg of each SBE at 37 �C
for 48 h, and then was subjected to the inhibition assay for
MMP-1. Porphyromonas gingivalis SBE alone significantly
reduced the inhibitory activity of TIMP-1 toward MMP-1 to
about the 100% of the control (P < 0.01). 1, TIMP-1 alone; 2,
1 + P. gingivalis SBE; 3, 1 + F. nucleatum SBE; 4, 1 + P.
endodontalis SBE. *P < 0.01.
Figure 7 Reverse gelatin zymograph of purified TIMP-1
treated with Porphyromonas gingivalis SBE. The same sample
prepared for the inhibition assay for MMP-1 shown in Fig. 6
was subjected to reverse zymography. 1, molecular weight
markers; 2, 3 + P. gingivalis SBE (0.1 lg mL)1); 3, TIMP-1
(0.4 lg mL)1) alone.
SBE from bacteria on activation of MMP-2 Sato et al.
International Endodontic Journal, 42, 1104–1111, 2009 ª 2009 International Endodontic Journal1108
Page 53
examined. P. gingivalis SBE alone significantly reduced
the inhibitory activity of TIMP-1 toward MMP-1 to
about the 100% of the control (Fig. 6; P < 0.01). The
effect of P. gingivalis SBE on the inhibitory activity of
purified TIMP-1 was further confirmed by reverse
zymography of TIMP-1. As shown in Fig. 7, essentially
no inhibition against MMP-2 was observed with
purified TIMP-1 pre-treated with P. gingivalis SBE.
Discussion
One of the types of enzyme that could be of importance in
the degradation of ECM is the MMP family. The collapse
of the balance between MMPs and their common
endogenous inhibitors, TIMPs, is an important point in
understanding the progress of connective tissue destruc-
tion. It is known, however, that most MMPs are secreted
from cells as inactive pro-enzymes; therefore, MMPs are
subject to two different processes: activation by an
activator(s) and inactivation via TIMPs.
For activation of pro-MMP in vivo, proteases could be
major factors, and trypsin, plasmin, cathepsin G,
elastase, and MMP-3 have been shown to act as
activators of MMPs. However, pro-MMP-2 is not
activated by any of these proteases (Collier et al.
1988), and for a long time it was not known which
activators could activate pro-MMP-2 in vivo. Recently,
membrane-type MMP (MT-MMP) has been found to be
activating substance that is specific for pro-MMP-2, and
four subclasses of it have been reported. It has been
elucidated that MT-MMP has a transmembrane
domain, which other MMPs do not have, at its
C-terminus and that MT-MMP specifically activates
pro-MMP-2 on cell surfaces (Sato et al. 1994). The only
pro-MMP-2 activator that has been reported is MT1-
MMP, one of the MT-MMPs.
At the outset, it was believed that MMP-2 might be
activated by bacteria related to periapical diseases.
Studying pro-MMP-2 activation by exogenous factors
including bacterial components could be useful for
increasing understanding of the mechanisms of tissue
destruction during inflammation, and could be also of
help to clarify the development, progress, and mecha-
nism underpinning the progression of periapical
diseases.
In the current study, the MMP-2-activating ability of
PL cells and HT1080 cells in the presence of
P. gingivalis, F. nucleatum, and P. endodontalis SBEs
was examined for the first time, although similar
studies have been conducted with human periodontal
ligament or human gingival fibroblasts in the presence
of P. gingivalis supernatant (Pattamapun et al. 2003,
Tiranathanagul et al. 2004, Zhou & Windsor 2006).
Also, it is unclear whether TIMP-1 and TIMP-2 can be
inactivated by P. gingivalis, although similar studies
have been conducted (Grenier & Mayrand 2001,
Tiranathanagul et al. 2004). In this study, MMP-2 in
PL cell culture medium treated with P. gingivalis SBE
was analyzed by gelatin zymography, and a band of
pro-MMP-2 and a band that could be its active form
were observed. In the samples with P. gingivalis SBE
alone, no similar bands of gelatin-degrading activity
were observed. The new band, which disappeared
when Ca2+ was removed by Ethylen Diamine Tetra
Acetic Acid (EDTA) (data not shown), could be a band
derived from MMPs; and this suggests that it could be
an active band produced from pro-MMP-2 being
converted to be a smaller molecule by P. gingivalis
SBE. The reason for the emergence of this band
(molecular weight of 66 kDa) may be that P. gingivalis
SBE first activates pro-MT1-MMP, and then the active
MT1-MMP activates the pro-MMP-2 produced by PL
cells. Therefore, similar experiments using a system
without cells were planned. However, PL cell in
primary culture only produce low amounts of pro-
MMP-2 and TIMP-1 and have slow growth. To
overcome these problems, HT1080 cell culture super-
natants were used, because these cells have been
studied in detail with regard to their production of pro-
MMP-2, TIMP-1, and TIMP-2, and also because they
are derived from fibroblasts, which are the same as PL
cells. HT1080 cell culture supernatants were directly
incubated with each SBE, and a band corresponding to
a smaller molecule (66 kDa), which could be the active
form of MMP-2, emerged along with a band represent-
ing pro-MMP-2 (72 kDa). In addition to that, treatment
of purified pro-MMP-2 with P. gingivalis SBE resulted in
a similar outcome. These results strongly suggest that
pro-MMP-2 activation would not be associated with
MT1-MMP, but with factors derived from P. gingivalis
SBE. No 66-kDa bands were observed in the experi-
ments using either F. nucleatum SBE or P. endodontalis
SBE, therefore, the activating factors could be consid-
ered to be specific to P. gingivalis. When P. gingivalis SBE
(10 lg mL)1) was added to the PL cell culture system,
TIMP-1 was not detected in the medium of PL cultures,
whereas in the media of cultures treated with either
P. endodontalis SBE or F. nucleatum SBE, the same
amount of TIMP-1 as in the control was detected. These
results suggest the following four possibilities: (i)
P. gingivalis SBE could inhibit the production of TIMP-
1 by PL cells, (ii) P. gingivalis SBE could inhibit and
Sato et al. SBE from bacteria on activation of MMP-2
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1104–1111, 2009 1109
Page 54
suppress the secretion of TIMP-1 by PL cells, (iii) P.
gingivalis SBE could degrade the TIMP-1 secreted from
the PL cells, (iv) P. gingivalis SBE could kill PL cells.
Amongst these, four was rejected because there was no
cytotoxity against PL cells at the concentration of SBE
used (data not shown). Regarding 1 and 2, when a cell
culture medium was incubated with P. gingivalis SBE,
the amount of TIMP-1 in the medium decreased in a
dose-dependent manner; therefore, it is unlikely that
the TIMP-1 production and secretion capabilities of the
cells were impaired by P. gingivalis SBE. Therefore, the
third possibility was suggested. To clarify this, purified
TIMP-1 was reacted with P. gingivalis SBE, and the
TIMP-1 inhibition of MMPs (for MMP-1 and MMP-2)
was found to be decreased, which suggested that TIMP-
1 could be degraded by P. gingivalis SBE (Grenier &
Mayrand 2001).
For TIMP-2, the same experiments were conducted.
When each SBE was added to PL cell cultures, the
amount of TIMP-2 produced by PL cells was extremely
small; therefore, the effects of P. gingivalis SBE could not
be confirmed. However, in the experiments using
HT1080 cell culture medium, the amount of TIMP-2
protein in the medium decreased in a P. gingivalis SBE
dose-dependent manner, the same as the findings for
TIMP-1, which suggests that TIMP-2 could also be
degraded by P. gingivalis SBE.
It is well known that P. gingivalis possesses fimbriae
and LPS and that it produces hemagglutinin and
various proteases. It has recently been shown that a
trypsin-like protease, the main type of these proteases,
is one of the cysteine proteases termed gingipain and
that there are two strains of gingipain (Arg-gingipain,
Lys-gingipain), each having a different substrate spec-
ificity (Pike et al. 1994). It was also shown that
gingipain has collagenolytic activity (Yamamoto
1995), and can activate pro-MMP-1 and pro-MMP-9
(DeCarlo et al. 1997). It was also confirmed that pro-
MMP-9 was activated by P. gingivalis SBE (Fig. 2). Both
pro-MMP-2 activation and TIMP-1, TIMP-2 inactiva-
tion were only confirmed by using the media treated
with P. gingivalis SBE; however, it can not be concluded
that these results were due to the same components of
P. gingivalis. It is also unclear whether the results of
experiments such as these conducted in cell cultures or
in vitro will be the same in vivo. However, because
obligate anaerobic bacteria are isolated in great num-
bers from infected root canals with progressing peri-
apical lesions (Sundqvist 1992), the results obtained
from these present experiments should be helpful for
clarifying the development and progression of periapi-
cal disease. It would be worthwhile to determine the
mechanisms of enzyme synthesis and activation of
pro-MMP-2 and others, as well as the identity of
the enzymes by Matrix Assisted Laser Desorbtion
Ionization-Time of Flight-MS (MALDI-TOF MS) peptide
fingerprinting in the future.
Conclusions
P. gingivalis SBE may contribute to the destruction of
connective tissue in a developing periapical lesion by
activating pro-MMP-2 as well as by inactivating TIMP-
1 and TIMP-2, which may change the balance between
MMPs and TIMPs in the ECM.
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Antibiotic prescribing in dental practice in Belgium
A. Mainjot1, W. D’Hoore2, A. Vanheusden1 & J.-P. Van Nieuwenhuysen3
1Department of Fixed Prosthodontics, Institute of Dentistry, University Hospital (CHU) of Liege, University of Liege, Liege, Belgium;2School of Public Health, Health Systems Research, Universite Catholique de Louvain, Louvain, Belgium; and 3Department of
Restorative Dentistry and Endodontics, Universite Catholique de Louvain, Louvain, Belgium
Abstract
Mainjot A, D’Hoore W, Vanheusden A, Van Nieuwen-
huysen J-P. Antibiotic prescribing in dental practice in
Belgium. International Endodontic Journal, 42, 1112–1117, 2009.
Aim To assess the types and frequency of antibiotic
prescriptions by Belgian dentists, the indications for
antibiotic prescription, and dentists’ knowledge about
recommended practice in antibiotic use.
Methodology In this cross-sectional survey, dental
practitioners were asked to record information about all
antibiotics prescribed to their patients during a 2-week
period. The dental practitioners were also asked to
complete a self-administered questionnaire regarding
demographic data, prescribing practices, and knowl-
edge about antibiotic use. A random sample of 268
Belgian dentists participated in the survey.
Results During the 2-week period, 24 421 patient
encounters were recorded; 1033 patients were pre-
scribed an antibiotic (4.2%). The median number of
prescriptions per dentist for the 2 weeks was 3. Broad
spectrum antibiotics were most commonly prescribed:
82% of all prescriptions were for amoxycillin, amoxy-
cillin-clavulanic acid and clindamycin. Antibiotics were
often prescribed in the absence of fever (92.2%) and
without any local treatment (54.2%). The most
frequent diagnosis for which antibiotics were prescribed
was periapical abscess (51.9%). Antibiotics were pre-
scribed to 63.3% of patients with periapical abscess and
4.3% of patients with pulpitis. Patterns of prescriptions
were confirmed by the data from the self-reported
practice.
Conclusions Discrepancies between observed and
recommended practice support the need for educational
initiatives to promote rational use of antibiotics in
dentistry in Belgium.
Keywords: antibiotics, audit, dentistry, drug use
review, prescription.
Received 31 March 2009; accepted 2 September 2009
Introduction
Overuse and misuse of antibiotics are well-known
problems with a negative impact on the general
population (American Dental Association Council on
Scientific Affairs 2004). Adverse reactions, emergence
and dissemination of resistance of some species through
genetic routes, increase in the prevalence of drug-
resistant bacterial infections, and economic waste have
drawn the attention of health professionals, scientists
and policymakers to the problems of antibiotic mis/
overuse (Hawkey 2008). Dentists prescribe a consider-
able amount of antibiotics: It was estimated that
antibiotic prescriptions amounted to 1.1 Defined Daily
Doses/1000 inhabitants/day, for a total antibiotic
expenditure of 7.4 million EUR in Belgium in 2004
(URL http://www.inami.fgov.be/drug/fr/statistics-
scientific-information/pharmanet/Statistics-group/2004/
pdf/t13101311.pdf). Surveys of dentists’ prescribing
habits have raised awareness of the quality of prescrip-
tions in developed countries. For example, question-
naire surveys showed that 12.5% of UK general dental
practitioners and 16.8% of US endodontists prescribed
antibiotics for the treatment of acute pulpitis (Palmer
et al. 2000, Yingling et al. 2002). However, a clinical
study warned against this practice (Nagle et al. 2000).
Whilst, some surveys have concluded that dental
prescriptions do not follow clinical guidelines (Palmer
& Batchelor 2004) other authors have emphasized that
Correspondence: Amelie Mainjot, Institut de Dentisterie
Universite de Liege, Quai G.Kurth, 45, B-4020 Liege, Belgium
(Tel.: +32 4 270 31 00; fax: +32 4 270 31 10; e-mail:
[email protected] ).
doi:10.1111/j.1365-2591.2009.01642.x
International Endodontic Journal, 42, 1112–1117, 2009 ª 2009 International Endodontic Journal1112
Page 57
there is a lack of scientific information regarding
appropriate and efficient prescription (Keenan et al.
2005).
In Belgium, little is known about the antibiotic
prescribing patterns of dentists. A questionnaire survey
was conducted to assess the types and frequency of
antibiotic prescriptions by dentists, the indications for
antibiotics, and dentists’ knowledge about recom-
mended practice in antibiotic use.
Material and methods
This cross-sectional survey was performed in September
2004 and consisted of two parts. In part 1 (prescrip-
tions), dental practitioners were asked to record infor-
mation about all the antibiotics they prescribed to their
patients during a 2-week period. In part 2 (self-reported
practice), dental practitioners completed a self-adminis-
tered questionnaire about demographic data, prescrib-
ing practices, and knowledge about antibiotic use.
Questionnaires were sent in August 2004 and the
survey ended on October 7th, 2004.
Participants
The study sample was drawn from the Belgian popu-
lation of dentists accreditated in 2004 by the Belgian
social security office (Institut National d’Assurance
Maladie Invalidite – Rijksinstituut voor Ziekte- en
Invaliditeitsverzekering). The Belgian accreditation is
a premium based system focusing on continuing
education and participating to epidemiologic data
collection. In 2004, there were 3917 accredited
dentists, i.e. 45.6% of all dentists. A random sample
of 150 Dutch-speaking and 150 French-speaking
dentists was drawn.
Prescriptions
Informed consent was required from every patient. Den-
tists were asked to complete a form for every patient who
was prescribed antibiotics, including type, dose, and
duration of antibiotic, patient history (allergies and
pregnancy), patient-related factors which may influence
prescription, concomitant prescription of nonsteroidal
anti-inflammatory drugs (NSAIDs), analgesics, and
mouthwashes. A difference was made between thera-
peutic and prophylactic (e.g. prevention of endocarditis)
prescriptions. In cases of antibiotherapy, dentists were
also asked to supply details about diagnosis and dental
treatment.
Self-reported practice
Practitioners were invited to complete a self-adminis-
tered questionnaire including demographic data (age,
gender), speciality, average activity (patients per week,
emergencies). The numbers of cases of pulpitis, periapical
abscess and periodontal abscess diagnosed during the 2-
week period were also recorded. Dentists were asked to
describe their usual prescription patterns (type and
frequency of antibiotic prescriptions; type, dosage and
duration of antibiotics prescribed for selected diseases
and prophylaxis), and factors influencing prescription
(patient demand and health status, antibiotic cost).
Dentists were also asked to rate their satisfaction with the
information that they receive about antibiotics from
various channels (social security authorities, university
training sessions, scientific or professional societies,
peers and pharmaceutical companies). Finally, the
dentists were asked to estimate their own role in and
information level about antimicrobial resistance, and its
possible influence on their prescribing behaviour. Space
was allowed for additional comments. Dentists received
either the Dutch or French version of the questionnaire.
Translations were supervised by professional translators
and native Dutch- and French-speaking dentists. Ques-
tionnaires were formatted for optical reading.
Statistical analysis
Chi-square and Fisher’s exact tests were used to test the
significance of associations in cross Tables. To compare
means, Student’s t-test and anova were used.
All statistical analysis was performed with the sas
system software (9.1 release; SAS Institute Inc., Cary,
NC, USA).
Results
A total of 268 practitioners participated in the survey
(response rate = 89.3%); 56.3% were male. The sam-
ple represented 3.1% of the Belgian dentist population
and 6.8% of accredited dentists. Only 3.7% of the
dentists were qualified in periodontics or orthodontics,
the two official dental specializations in Belgium.
During the 2-week period, 24 421 patient encoun-
ters were recorded; 1033 were prescribed an antibiotic
(4.2% of all patient encounters). In 936 patient
encounters (90.6%), antibiotics were prescribed for
therapeutic reasons, in 46 patient encounters for
prophylactic reasons, and the reasons for the remain-
ing 51 were undetermined.
Mainjot et al. Antibiotic prescribing
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1112–1117, 2009 1113
Page 58
Amongst the respondents, 11.2% did not prescribe
any antibiotics during the 2-week period. The median
number of prescriptions for the 2 weeks was 3 (min-
imum = 0, maximum = 21).
As seen in Table 1, the most frequently prescribed
antibiotics for therapeutic reasons were amoxycillin
and the combination of amoxycillin and clavulanic acid
(75.1%). The orders in which antibiotics were ranked
were similar in the prescription and the self-reported
data.
In penicillin-allergic patients, 49 prescriptions were
recorded. As expected, the most frequently prescribed
antibiotics in these patients were macrolides (57.1%),
followed by clindamycin (16.3%).
There was no difference in antibiotic choice accord-
ing to the diagnosis: Amoxycillin followed by the
combination of amoxycillin and clavulanic acid were
the most frequently prescribed antibiotics, except for
rapidly progressive periodontitis for which doxycycline
was second on the list.
The antibiotics most commonly prescribed for pro-
phylaxis were amoxycillin and the combination of
amoxycillin and clavulanic acid (73.9%).
In Table 2, the number of actual patient encounters
and associated diagnoses for which antibiotherapy was
ordered is compared with self-reported prescription
patterns. The most common indications for antibio-
therapy were periapical abscess (51.9%) and periodon-
tal abscess (14.2%). Pulpitis accounted for 4.4% of all
prescriptions. In more than 90% of antibiotherapy for
periapical or periodontal abscess and pulpitis, fever was
absent. Prescription rates, i.e. the proportion of diag-
noses leading to antibiotic prescriptions, were very high
for periapical abscess (63.3%) and high for periodontal
abscess (28.8%); they were much lower for pulpitis
(4.3%). Antibiotics were prescribed without any local
treatment in 59.0% of periapical abscesses, in 46.4% of
periodontal abscesses, and in 31.7% of cases of pulpitis.
The data for antibioprophylaxis were not analysed
because of the small number of cases.
Several items were analysed to describe patterns of
antibiotherapy. First, in 40.7% of prescriptions, dentists
reported that there was patient demand for antibiotics,
meaning that prescribing does not only depend on the
oral health status. Secondly, in 33.4% of prescriptions,
dentists recommended that antibiotics not be taken
unless symptoms become more severe, meaning that
Table 1 Distribution of actual antibiotics prescribed for ther-
apeutic reasons versus self-reported prescribing patterns
Antibiotic
Prescriptions
n (%)
Self-reported
prescribing
patterns (%)
Amoxycillin 478 (51.1) 33.6
Amoxycillin + clavulanic acid 225 (24.0) 22.1
Clindamycin 62 (6.6) 13.9
Azythromycin 50 (5.3) 4.8
Clarithromycin 41 (4.4) 5.1
Doxycycline 34 (3.6) 9.0
Spiramycin 21 (2.2) 1.6
Erythromycin 11 (1.2) 7.4
Other 10 (1.1) 0.9
Ciprofloxacin 2 (0.2) 0
Cefadroxil 1 (0.1) 0.3
Minocycline 1 (0.1) 0.3
Cefuroxime 0 (0) 0.15
Never prescribe antibiotic – 1
Total 936 (100) 100
Table 2 Indications for antibiotherapy: comparison between actual prescriptions and self-reported prescribing patterns
Diagnosis
Indications for
antibiotherapy
(1), n (%)
Frequency of
diagnosis (2)
Prescription
rate
(1)/(2) %
Self-reported
prescription
patterns (3) %
Periapical abscess 476 (51.9) 752 63.3 82.7
Periodontal abscess 130 (14.2) 452 28.8 63.2
Others 75 (8.2) – – –
Pericoronitis 70 (7.6) – – 52.2
Rapidly progressive periodontitis 43 (4.7) – – 23.5
Pulpitis 40 (4.4) 933 4.3 5.9
Alveolar osteitis 37 (4.0) – – 41.9
Chronic adult periodontitis 25 (2.7) – – –
Cellulitis 22 (2.4) – – 44.1
Total 918 (100) – – –
(1): Total number and % of diagnoses for which antibiotics were prescribed, during the 2-week period.
(2): Total number of three selected diagnoses recorded during the 2-week period.
(1)/(2): Proportion of diagnoses leading to antibiotic prescription.
(3): Proportion of dentists who reported antibiotic prescription for each diagnosis.
Antibiotic prescribing Mainjot et al.
International Endodontic Journal, 42, 1112–1117, 2009 ª 2009 International Endodontic Journal1114
Page 59
the link between antibiotic prescriptions and antibiotic
consumption is not linear. Thirdly, there was wide
variability in antibiotic courses and regimens. For
example, a higher initial dose was prescribed in 17.2%
of cases of antibiotherapy (with unequal distribution
amongst antibiotics: 17.9% for amoxycillin versus
13.8% for the combination of amoxycillin and clavul-
anic acid, P < 0.01). Treatment duration varied
around an average of 4.8 days (SD = 2.1) with a
lower mean of 3.0 days for azithromycin and a higher
mean of 7.9 days for doxycycline (P < 0.01).
Companion treatments of antibiotherapy included
NSAIDs (38.7%), analgesics (22.8%) and mouthwashes
(45.0%). The NSAIDs and analgesics most frequently
prescribed were ibuprofen (82.5%) and paracetamol
(78.7%). Surprisingly, a majority of patients with pain
did not receive analgesics (75.2%) or NSAIDs (54.6%).
The satisfaction rate of practitioners with informa-
tion about antibiotic use was high (81%). Colleagues
were considered as the best source of information
(87%) whereas university continuing education ses-
sions were less satisfying (73.5%). However, satisfac-
tion with information contrasted with knowledge. For
example, in self-reported data, it was observed that
American Heart Association and American Academy of
Orthopaedic Surgeons guidelines (Tong & Rothwell
2000) were followed in 44.8% and 35.8% of cases,
respectively. Finally, amongst practitioners who felt
responsible for development of resistant strains
(64.6%), the majority (61.8%) did not change their
prescribing practices.
Discussion
The findings of this cross-sectional survey show that a
minority of patient encounters (4.2%) lead to antibiotic
prescription. Periapical abscess was the most frequent
diagnosis associated with prescription (51.9%) and the
prescription rate for this diagnosis was 63.3%. In a
majority of patients, antibiotics were prescribed in the
absence of general symptoms, indicating defensive
practice. Moreover, a substantial percentage of cases
(54.2%) were treated without any local treatment.
If continuing education and the Belgian system of
accreditation improve quality of care, the sample
studied here should reflect best practice amongst
Belgian practitioners. The combination of data about
actual prescriptions and from the self-administered
questionnaire allows reported and perceived practices
to be compared. Collection of data about diagnosis,
local treatment, and patient-related factors that can
influence prescription provided information about the
qualitative aspects of the prescriptions. In particular,
records of the numbers of cases of pulpitis, periapical
abscess, and periodontal abscess diagnosed during the
2-week period highlighted the prescription rate for
these diagnoses. The self-administered questionnaire
enabled evaluation of practitioners’ knowledge about
antibiotic use and about the dissemination of knowl-
edge in Belgium.
Some quantitative estimates of the frequency of
antibiotic prescriptions by dentists were found in the
literature. Two studies conducted in England, on 175
and 212 dental practitioners, respectively, over a 6-
week period (Palmer et al. 2001, Chate et al. 2006)
reported similar prescription rates (2.2 and 2.31
prescriptions/dentist/week), from a quantitative point
of view, to the present study (1.9 prescriptions/dentist/
week). In contrast, in a study conducted in Saudi
Arabia, the mean number of declared prescriptions per
week was 5–10 (Al-Mubarak et al. 2004). However,
comparisons between regions or countries are mean-
ingless as case-mix, professional standards, and local
regulations are unknown. In Belgium, no economic
incentives favour prescribing antibiotics.
Qualitative analysis of prescription practices allows
evaluation of the quality of treatment. It highlights the
misuse and abuse of antibiotics, which can increase the
risk of toxicity and may also result in development of
antibiotic-resistant bacteria. Indeed, the development of
resistance to antibiotics by many important human
pathogens has been linked to exposure to antibiotics
over time (Hawkey 2008). In this study, broad spec-
trum antibiotics were most commonly prescribed: Amox-
ycillin, amoxycillin-clavulanic acid and clindamycin
accounted for 82% of all prescriptions. However, use of
broad-spectrum antibiotics, like amoxycillin-clavulanic
acid (24.0% of prescriptions for therapeutic reasons in
our study), is questionable. Selected antibiotics should
possess a spectrum of action as narrow as possible
(Handal & Olsen 2000), based on the susceptibility of
pathogens (Sweeney et al. 2004). Empirical and inap-
propriate prescription leads to selection of resistant
strains which is potentially damaging to the community
(Sweeney et al. 2004). In contrast with Belgian
practitioners, in 2004, dentists in Norway showed a
conservative antibiotic practice and prescribed the
narrow-spectrum, phenoxymethylpenicillin, as their first
choice (75% of their total prescriptions) (Al-Haroni &
Skaug 2007).
Regarding the indications for antibiotherapy, the
prescription data showed that antibiotics were pre-
Mainjot et al. Antibiotic prescribing
ª 2009 International Endodontic Journal International Endodontic Journal, 42, 1112–1117, 2009 1115
Page 60
scribed independently of local treatments, as shown in
previous audits in the United Kingdom (Choudhury
et al. 2001, Dailey & Martin 2001). For periapical
abscesses, the prescription rate was 63.3%, although
drainage of the purulent collection and suppression of
the cause of infection by disinfection of the root canal
system alone are recognized as adequate treatment
(Matthews et al. 2003, Kuriyama et al. 2005, Lopez-
Piriz et al. 2007). The prescription rates for periodontal
abscesses and pulpitis also seemed rather high (28.8
and 4.3%, respectively) as it is known that local
treatment constitutes effective treatment (Keenan et al.
2005). Moreover, in patients with periapical abscess
who were prescribed antibiotics, 59.0% did not receive
any local treatment; the percentages for periodontal
abscess and pulpitis were 46.4% and 31.7%, respec-
tively. Our study did not provide information about the
organization of patient care, but it is likely that lack of
time, working schedules, and technical difficulties
prevent dentists from performing timely local treat-
ments. It was also striking that antibiotics were used in
the absence of general symptoms and fever (92.2%).
Antibiotic use should be saved for patients with general
symptoms of disseminating infection, such as the
presence of fever, extended swelling (cellulitis), or for
cases where local treatment is impossible.
On the qualitative side of this study, other findings
suggested poor antibiotic use. First, in 33.4% of
registered prescriptions, patients were told not to take
the prescribed antibiotics unless symptoms worsening.
This defensive attitude results in waste for the social
security, and may also promote risky self-prescription,
leaving patients with the opportunity to misuse the
same drug later. Another consequence of this practice
is that the amount of antibiotics prescribed does not
reflect the amount of antibiotics used by patients. This
apparently positive situation may hamper the collec-
tion of accurate data about antibiotic consumption and
the evaluation of antibiotic effectiveness due to the
uncertainty regarding antibiotic consumption. Finally,
this could generate environmental problems. Secondly,
there was wide variability in antibiotic courses and
regimens, as indicated also in a study by Roy & Bagg in
Scotland (Roy & Bagg 2000). One reason for this
finding may be the lack of relevant recommendations
about antibiotic use.
The results of the analysis of prescriptions contrast
with the respondents’ high level of satisfaction about
information on antibiotic use, indicating a lack of
awareness of good clinical practices. This is confirmed
by responses to simulated cases about prophylaxis of
endocarditis and artificial joint infections: results
show that a majority of practitioners (55.2 and
64.2%, respectively) do not follow international
guidelines (Tong & Rothwell 2000). The importance
of colleagues as information source highlights the
poor efficacy, visibility, and/or legitimacy of official
sources, such as university continuing education
sessions.
The findings of this study support the need for
interventions to promote rational use of antibiotics in
dentistry. Experience with medical practitioners shows
that various interventions may improve antibiotic
prescribing practices in ambulatory care, as demon-
strated in the Cochrane review by Arnold & Straus
(2005). Selection of the most effective intervention
appears to be condition and situation specific. In
particular, as patient demand for antibiotics is still
problematic (in 40.7% of registered prescriptions
patients expected antibiotics), patient education
should form part of multi-faceted interventions,
which appear to play an important role in reducing
the inappropriate use of antibiotics in community
settings. Educational components and setting stan-
dards for antibiotic prescribing by dental practitioners
were successfully tested in England (Palmer et al.
2001, Palmer & Dailey 2002, Chate et al. 2006).
Pre- and post-audit measurements should provide
feedback on practitioners’ practices. Repeated surveys,
measuring the impact and durability of interventions,
can be used in this perspective. In this context, the
present study constitutes the first step in a drug
utilization review concerning antibiotic prescribing in
dental practice in Belgium. It should be followed by
national consensus meetings to elaborate guidelines
in this area. Guidelines about antibiotic choice should
be dynamic and take into consideration local factors,
such as local resistant bacteria status and profes-
sional realities. Post- and re-audit should be planned
after introduction interventions designed to alter
prescribing practices.
Conclusions
Discrepancies between observed and recommended
practice support the need for educational initiatives to
promote rational use of antibiotics in dentistry.
Acknowledgements
The Belgian «Institut National d’Assurance Maladie-
Invalidite» supported this research.
Antibiotic prescribing Mainjot et al.
International Endodontic Journal, 42, 1112–1117, 2009 ª 2009 International Endodontic Journal1116
Page 61
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CASE REPORT
One step pulp revascularizationtreatment of an immature permanenttooth with chronic apical abscess:a case report
S. Y. Shin, J. S. Albert & R. E. MortmanEndodontic Division, Atlantic Coast Dental Research Clinic, West Palm Beach, FL, USA
Abstract
Shin SY, Albert JS, Mortman RE. One step pulp revascularization treatment of an immature
permanent tooth with chronic apical abscess: a case report. International Endodontic Journal, 42,
1118–1126, 2009.
Aim To describe a case in which a mandibular right second premolar with a necrotic pulp,
sinus tract, periradicular radiolucency and an immature apex underwent revascularization
via a single treatment approach.
Summary Revascularization procedures have the potential to heal a partially necrotic
pulp, which can be beneficial for the continued root development of immature teeth.
However, it is not clear which revascularization protocols are the most effective. This case
report details the outcome of a successful revascularization procedure on tooth 45 (FDI) in
a 12-year-old patient, eliminating the associated periapical pathosis within 19 months. The
tooth was treated using coronal root irrigation with 6% NaOCl and 2% chlorhexidine
without instrumentation in a single visit. The successful outcome of this case report
suggests that this conservative revascularization treatment approach can preserve the
vitality of the dental pulp stem cells and create a suitable environment for pulp
regeneration, resulting in the completion of root maturation.
Key learning points
• The noninstrumentation procedure using 6% NaOCl and 2% chlorhexidine coronal
irrigation may help preserve the remaining vital dental pulp stem cells believed to be critical
for pulp revascularization.
• A single visit pulp revascularization protocol can be a favourable treatment option for an
immature permanent tooth with a partially necrotic pulp.
Keywords: dental pulp stem cells, immature apex, pulp regeneration, revascular-
ization, stem cells of the apical papilla.
Received 21 June 2009; accepted 21 August 2009
doi:10.1111/j.1365-2591.2009.01633.x
Correspondence: Dr Sang Shin, 1501 Presidential Way, Suite 7, West Palm Beach, FL 33401,
USA (e-mail: [email protected] ).
International Endodontic Journal, 42, 1118–1126, 2009 ª 2009 International Endodontic Journal1118
Page 63
Introduction
Recently there has been evidence indicating that a better alternative to conventional
calcium hydroxide apexification exists in immature permanent teeth exhibiting periapical
pathology (Shah et al. 2008). Procedures attempting to preserve the potentially remaining
dental pulp stem cells and mesenchymal stem cells of the apical papilla can result in canal
revascularization and the completion of root maturation (Sonoyama et al. 2006, Huang
et al. 2008). Revascularization of a partially necrotic pulp in an immature root is based on
the concept that vital stem cells located in the apical papilla can survive pulpal necrosis,
even in the presence of a periradicular infection (Huang et al. 2008). These stem cells are
believed to differentiate into secondary odontoblasts, ultimately allowing for dentinal
deposition (Huang et al. 2008). Survival of the stem cells is aided by an abundant blood
supply to the apical papilla, contributing to pulp revascularization. In addition, it has been
speculated that some vital dental pulp stem cells in the apical canal may survive partial
pulpal necrosis, even in cases with associated periapical pathology (Lin et al. 1984, Iwaya
et al. 2001, Huang et al. 2008). Previous studies involving tooth reimplantation indicate
that apically survived pulp tissue can proliferate and replace the remnant coronal necrotic
tissue (Ohman 1965, Barrett & Reade 1981, Skoglund & Tronstad 1981). Furthermore,
some of these dental pulp stem cells may have the capacity to differentiate into
odontoblast-like cells, contributing to root maturation (Yousef 1988, Shah et al. 2008).
Maintaining the viability of the remaining survived pulp tissue and the stem cells of the
apical papilla are considered critical for revascularization to succeed. Therefore, most
recent case reports follow a protocol of no canal instrumentation throughout the
revascularization procedure in order preserve these essential enduring stem cells (Iwaya
et al. 2001, Chueh & Huang 2006, Jung et al. 2008).
The literature indicates several advantages of promoting apexogenesis in immature teeth
with open apices (Murray et al. 2007). Contrary to apexification, apexogenesis encourages
a longer and thicker dentinal composed root to develop (Rafter 2005). These beneficial
anatomic properties may decrease the propensity of long-term root fracture, a significant
risk associated with apexification procedures (Andreasen et al. 2002, Reynolds et al.
2009). Revascularization procedures attempt to obtain a longer and thicker root, whilst
restoring vital pulpal conditions. A successfully revascularized tooth would require no
additional treatment. Conversely, apexification involves supplementary treatment visits to
replenish the calcium hydroxide and ultimately requires an apical plug of mineral trioxide
aggregate (MTA) or final Gutta-percha canal filling (Rafter 2005).
Although it has been demonstrated clinically that revascularization procedures can be
successful, it is not completely understood to what extent the preservation of the apical
papilla is involved in final root maturation (Huang et al. 2008, Shah et al. 2008). Continued
research is needed to determine if the stem cells of the apical papilla are irrefutably
responsible for differentiation into odontoblasts and subsequently accountable for the
characteristic dentinal deposition involved in typical root maturation (Chueh et al. 2009).
Finally, research experiments investigating the outcome of intentionally removing the
apical papilla in minipigs has failed to determine whether terminated root maturation is
related to the destruction of the apical papilla stem cells or damage to Hertwig’s epithelial
root sheath (Huang et al. 2008).
Drawbacks of the revascularization process include a lack of long term follow up data on
root canal morphology and pulpal cellular composition following the procedure on patients.
This refers to the possibility of accelerated canal calcification, rendering the tooth more
difficult to treat endodontically in the future (Shah et al. 2008). Furthermore, it has not
been determined the stage and duration of pathosis that will ultimately lead to the
complete destruction of the resistant apical mesenchymal cells and surviving dental pulp
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stem cells. Under the circumstances of total pulpal and apical papilla necrosis,
revascularization treatment may not be possible. As a result, it is difficult to case select
appropriate teeth that clinically test nonvital, but maintain vital apical cells believed to be
necessary to successfully perform the procedure. Additional complications such as
various systemic health conditions and immunologic problems may offer other obstacles
in achieving adequate root maturation in the presence of a periradicular infection.
The current case report examines the concept of pulp revascularization of a mandibular
right second premolar via a single visit treatment approach. The objective was to
determine if the presented protocol would result in the formation of a longer and thicker
root in a tooth believed to exhibit a partial loss of vital pulp tissue. The resolution of
periradicular pathology and related symptoms was considered essential for a successful
outcome. Previous case reports illustrate a multi-visit treatment method to achieve
satisfactory revascularization results (Chueh & Huang 2006, Jung et al. 2008, Shah et al.
2008). Banchs & Trope (2004) reported the successful revascularization of an immature
mandibular right second premolar diagnosed with chronic apical abscess. The canal was
disinfected with NaOCl and peridex to 1 mm of the apex without mechanical instrumen-
tation. A tri-antibiotic paste, composed of metronidazole, ciprofloxacine and minocycline
was placed for 2 weeks. At the second visit, a blood clot was produced to the level of the
cementoenamel junction to provide a scaffold for the in-growth of new tissue,
subsequently using MTA to provide an effective seal (Hoshino et al. 1996, Banchs &
Trope 2004). In a similar clinical report, Chueh & Huang (2006) followed a more
conservative revascularization technique to achieve analogous results. A periradicularly
involved immature tooth was treated without instrumentation and irrigation with NaOCl
was confined to the pulp chamber. A calcium hydroxide paste was then placed. Two
additional visits were required to replenish the calcium hydroxide at coronal portion of the
root to achieve comparable root maturation results (Chueh & Huang 2006). The current
case report attempts to provide an utmost conservative single visit, modified technique to
revascularize a partially necrotic pulp with associated chronic apical periodontitis.
Case report
A 12-year-old girl of Hispanic descent was referred by her general dentist for evaluation
and root canal treatment of the mandibular right second premolar. The medical history
was unremarkable. The patient was scheduled as an emergency visit with her general
dentist 3 days prior, complaining of pain in the mandibular right premolar region. The
dentist prescribed amoxicillin 500 mg PO tid. The intra-oral exam revealed an asymp-
tomatic tooth 45 with an associated draining sinus tract located distal to the root (Fig. 1a).
Vitality, percussion and palpation exams were performed on the tooth and adjacent teeth.
Tooth 45 exhibited occlusal caries (Fig. 2a) with slight palpation and percussion sensitivity.
It did not respond to 1, 1, 1, 2-tetrafluoroethane (Endo-Ice; Hygenic Corp., Akron, OH,
USA) or the electric pulp test (Analytic Technology, Redmond, WA, USA). The adjacent
teeth were caries free, asymptomatic and tested vital. The periodontal exam presented
probings and physiologic mobility within normal limits.
Radiographic evaluation showed an immature open apex, measuring 2 mm in diameter
with a large periradicular rarefaction approximately 9 · 9 mm in size, extending from the
apex of tooth 45 to the distal crestal bone area. The periapical radiograph demonstrated a
carious lesion associated with a pre-existing Oehlers type I dens invaginatus, where the
developmental anomaly presents an enamel lined invagination terminating in a blind sac
located within the crown. There was visible external inflammatory resorption on the mid
distal portion of the root. Condensing osteitis was apparent at the periapical area of the
tooth (Fig. 3a).
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The initial diagnosis of pulpal necrosis with suppurative chronic periapical abscess was
determined for tooth 45. Following local anaesthesia administration, rubber dam isolation
and occlusal access preparation using the dental operating microscope, all remaining
caries and hypomineralized enamel were removed. Upon entering the coronal aspect of
(a) (b)
(c) (d)
Figure 1 Clinical observations. (a) Pre-op clinical photograph illustrates a draining sinus tract distal to
the mandibular right second premolar. Hypomineralized enamel is visible on the distal occlusal pit. (b)
Two-week post-op photograph depicts a reduction in size of the sinus tract. (c) Six-week follow-up
photograph demonstrates complete healing of the sinus tract. (d) At the seven-month post-op visit,
re–establishment of normal gingival contour was observed.
(a) (b)
(c) (d)
Figure 2 Root canal revascularization procedure. (a) Clinically, the mandibular right second premolar
presented with distal pit caries. (b) After controlling the haemorrhage, viable tissue was observed in
the canal. (c) The placement of white MTA in the canal. (d) Final composite restoration.
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the root canal, haemorrhage into the pulp chamber was observed (Fig. 2b). A size 10 K-file
(Kerr, Romulus, MI, USA) was inserted into the canal and the patient reported discomfort,
potentially indicating the survival of residual vital pulp tissue. The clinical diagnosis was
revised from total pulpal necrosis to partial necrosis. After evaluating the treatment
options, it was established that the patient would benefit greatest from a revascularization
procedure. A thorough explanation of the potential risks, complications and benefits of the
suggested treatment was carried out. The alternative option of conventional calcium
hydroxide apexification was discussed. Based on the increased long-term risk of root
fracture attributed to traditional calcium hydroxide apexification and the potential rewards
of revascularization, maternal consent was obtained to initiate revascularization treatment.
The proposed, most conservative treatment protocol is a modification of the Banchs &
Trope (2004) and Chueh et al. (2009) clinical case reports. It was explained to the mother
that they would be given additional options of the triple antibiotic paste technique or
calcium hydroxide apexification if the current revascularization procedure did not succeed.
The haemorrhaging coronal portion of the canal was irrigated with 10 mL of 6% NaOCl and
then rinsed with sterile saline solution. The coronal canal was then irrigated with 10 mL of
2% chlorhexidine gluconate (Vista Dental, Racine, WI, USA) and left there for 5 min. No
instrumentation was performed. The coronal canal was dried with paper points and white
MTA (Dentsply Tulsa Dental, Tulsa, OK, USA) was gently packed into the coronal canal
(Fig. 2a). A thin layer of thermoplastic Gutta-percha (Calamus system, Dentsply Tulsa
Dental, Johnson City, TN, USA) was temporarily placed over the MTA to prevent washing out
and the chamber walls were etched with 37% phosphoric acid, rinsed with water and dried.
Prior to restoring the access cavity with a final resin-bonded composite (ESPE Filtek, 3M, St
Paul, MN, USA) restoration (Fig. 2d), the Gutta-percha was removed from the pulp chamber.
The draining sinus tract was rinsed with 3 mL of 0.12% chlorhexidine gluconate (Peridex,
Zila Pharmaceuticals, Inc, Cincinnati, OH, USA). The patient was instructed to complete the
amoxicillin provided by her dentist and was prescribed ibuprofen 800 mg for pain. The
mother was informed to call if there were any complications.
(a) (b)
(c) (d)
Figure 3 (a, b) Pre-treatment radiographs of tooth 45 (FDI). Radiographic examination demonstrates
type I dens invaginatus with associated caries and incomplete root formation with diffuse periapical
radiolucency measuring 9 · 9 mm in size. (c) Post-op radiographs show coronal canal MTA placement
with a composite restoration. (d) Two-week follow-up radiograph shows a wide open apex.
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The patient returned for the 2-week follow-up visit, asymptomatic with no sensitivity to
palpation or biting. The tooth exhibited minimal sensitivity to percussion. No significant
radiographic changes were noted. (Fig. 3d) The smaller sinus tract (Fig. 1b) was irrigated
with 3 mL of 0.12% chlorhexidine gluconate.
At the 6-week recall appointment, the patient returned asymptomatic. Tests for percussion,
mobility, palpation and biting sensitivity were all within normal limits. The sinus tract had
completely healed (Fig. 1c) and the periapical radiolucency became less radiolucent. The
diameter of the open apex had decreased and thickening of the radicular walls were evident.
Periodontal probing depths were normal. No additional treatment was administered.
The patient returned for the 7-month post-op visit completely asymptomatic. Radiograph-
ically, the lamina dura could be traced around the entire root surface and the periodontal
ligament space was re-established. The alveolar crestal bone around the tooth had healed
and condensing osteitis became less radiopaque (Fig. 4b). Clinically, there was a complete
re-establishment of gingival contour (Fig. 1d). The Endo Ice test and electric pulp test did
not elicit a response.
At the 13- and 19-month follow-up appointments, the patient remained asymptomatic. No
tenderness to percussion or palpation was noted. Periodontal pocket depths and
physiologic mobility were within normal limits. The Endo ice test and electric pulp test
were negative. The radiographs demonstrated evidence of complete periradicular bone
healing and root maturation (Fig. 4c,d). The 19-month follow-up radiograph showed
complete resolution of condensing osteitis (Fig. 4d).
Discussion
The conventional calcium hydroxide apexification procedure has been extensively studied
and appears to be a reliable treatment option. However, the technique has several
disadvantages. These include a lengthy treatment period, complications relating to poor
(a) (b)
(c) (d)
Figure 4 Post-treatment radiographs of tooth 45. (a) Six-week post-op radiograph shows thickening
of dentinal walls with no evidence of the lamina dura apically. (b) At the seven-month recall, a re-
establishment of the periodontal ligament space and lamina dura was observed. Root maturation is
visible. (c) Thirteen-month follow-up radiograph shows further thickening of the lamina dura and
maturation of the root. (d) At the 19-month post-op visit, a complete maturation of the apex and
resolution of condensing osteitis are evident.
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patient compliance and resulting thin dentinal walls with a high risk of long-term root
fracture (Cvek & Sundstrom 1974, Andreasen et al. 2002). Contemporary research articles
examine alternative treatment options to encourage a root maturation process emulating
natural root formation, even in the presence of extensive periapical pathology (Banchs &
Trope 2004, Chueh & Huang 2006, Jung et al. 2008). Revascularization treatment has
been suggested to be a favourable alternative, yielding the development of a longer and
thicker root, less susceptible to fracture. Using a modified technique, originally outlined by
Banchs & Trope (2004), the current investigation offers a more conservative single visit
approach, avoiding apical irritation and focuses on preserving the remaining vital pulp
tissue and mesenchymal stem cells of the apical papilla. Preservation of these cells is
believed to be critical for successful revascularization (Huang et al. 2008).
An Oehlers type I dens invaginatus is a developmental anomaly characterized by a
hypomineralized, enamel-lined invagination appearing as a radiolucent blind sac in the
crown (Canger et al. 2009). The deep invagination is susceptible to carious progression
because of the hypomineralized quality of the enamel and the exposure of the invagination
to the oral environment (Canger et al. 2009). If left untreated it often results in necrosis of
the pulp and periradicular infection (Cengiz et al. 2006, Canger et al. 2009). Depending on
the stage of pathogenesis, treatment options can vary from preventive and restorative
options to nonsurgical root canal treatment (Er et al. 2007). When conventional root canal
treatment fails, surgical treatment may be necessary (Canger et al. 2009). In the present
case, tooth 45 appeared radiographically as an Oehlers type I dens invaginatus, with the
formation of an associated carious lesion and periapical abscess. The developmental
anomaly and pathology was successfully treated through the revascularization procedure
and final composite restoration.
The presented case report used NaOCl and chlorhexidine irrigation of the coronal necrotic
tissue and systemic antibiotics to provide a favourable environment for pulpal revascu-
larization to proceed. The amoxicillin, prescribed by the general dentist, may have aided
the bactericidal activity in the periapical area. The irrigant, 2% chlorhexidine was selected
based on its extended residual anti-microbial properties and a relative absence of toxicity
(Greenstein et al. 1986, Jeansonne & White 1994). An in vitro study has reported that root
canals treated with 2% chlorhexidine had 72 h of residual antimicrobial activity against
Streptococcus mutans (White et al. 1997). Recent questions concerning the use of 2%
chlorhexidine relates to the potential cytotoxicity on cultured dental pulp stem cells. In
addition, it has been reported that interactions between NaOCl and chlorhexidine forms
para-chloroaniline, which is known to be a carcinogen (Basrani et al. 2007). Basrani et al.
(2007) suggested that prior to irrigating with chlorhexidine, it is recommended to wash
away the existing NaOCl to diminish the formation of para-chloroaniline. The current report
used copious irrigation of saline solution to reduce the interaction between NaOCl and
chlorhexidine. Further research is needed to weigh the benefits of the residual
antimicrobial activity of chlorhexidine versus the cytotoxicity and carcinogenicity of para-
chloroaniline.
Mineral trioxide aggregate was used in the study to provide an effective pulpal seal.
Contrary to calcium hydroxide, MTA exhibits biocompatibility with adjacent pulp tissue,
even capable of inducing pulpal cell proliferation (Kettering & Torabinejad 1995, Fridland &
Rosado 2005). In addition, MTA sustains a high pH for extended periods of time and has
exceptional marginal adaptation (Torabinejad et al. 1995, Moghaddame-Jafari et al. 2005).
MTA was used as a coronal plug based on its known beneficial properties demonstrated
during vital pulp therapy (Torabinejad & Chivian 1999).
There are several advantages of the single visit revascularization protocol. Eliminating
subsequent access appointments to the root canal environment may reduce the
possibility of further bacterial contamination of the canal. Single visit procedures also
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act to diminish the detrimental consequences of poor patient compliance for regular
follow-up evaluation. Decreased successive trauma to the tooth and increased patient
comfort are other potential benefits of completing the treatment in one visit.
A variety of revascularization techniques have demonstrated that periapically involved
immature teeth that initially appear to have necrotic pulps can undergo the procedure and
respond positive to vitality testing over extended periods of evaluation (Iwaya et al. 2001,
Banchs & Trope 2004, Reynolds et al. 2009). These reports indicate that the revascularized
teeth regained vitality between 15 months to 2 years. The current case did not achieve a
conclusive positive vitality response at the 19-month follow-up appointment. It is possible
that over longer periods of evaluation, the tooth may generate a positive response.
In conclusion, the presented case report demonstrates a conservative, single visit
revascularization approach, resulting in the elimination of periapical pathology and a stronger
mature root. Whilst the discussion advocates following a more conservative technique, it is
possible that this single visit approach may not be applicable to all revascularization cases. It
is believed that with teeth exhibiting complete pulpal necrosis, the presented protocol would
not have succeeded. A more aggressive technique may be required to eradicate the bacteria
in the canal system and periapical tissues. A multi-visit, tri-antibiotic paste sequence could be
a better treatment choice for teeth potentially presenting with total pulpal necrosis. As a
result, case selection is critical when deciding which revascularization protocol is ideal for a
particular pulpal condition. Patients that report discomfort to an advancing file within the
canal may indicate the presence of viable canal tissue. It is suggested that in these cases, the
current technique can be beneficial prior to attempting the less conservative tri-antibiotic
sequence or calcium hydroxide apexification. Further investigation is needed to properly
diagnose the correct pulpal status of a tooth and design treatment guidelines depending on
the stage of pulpal necrosis to obtain a predictable outcome.
Acknowledgement
We would like to give special thanks to Dr Peter Murray for reviewing and advising for this
case report.
Disclaimer
Whilst this article has been subjected to Editorial review, the opinions expressed, unless
specifically indicated, are those of the author. The views expressed do not necessarily
represent best practice, or the views of the IEJ Editorial Board, or of its affiliated Specialist
Societies.
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