iej.12.2009

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

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

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


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-



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).



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.

References

Bergmans L, Van Cleynenbreugel J, Wevers M, Lambrechts P

(2001) A methodology for quantitative evaluation of root

canal instrumentation using microcomputed tomography.

International Endodontic Journal 34, 390–8.



Bergmans L, Van Cleynenbreugel J, Beullens M, Wevers M,

Van Meerbeek B, Lambrechts P (2002) Smooth flexible

versus active tapered shaft design using NiTi rotary instru-

ments. International Endodontic Journal 35, 820–8.

Bergmans L, Van Cleynenbreugel J, Beullens M, Wevers M,

Van Meerbeek B, Lambrechts P (2003) Progressive versus

constant tapered shaft design using NiTi rotary instruments.


Bystrom A, Sundqvist G (1981) Bacteriologic evaluation of the

efficacy of mechanical root canal instrumentation in end-

odontic therapy. Scandinavian Journal of Dental Research 89,

321–8.

Card SJ, Sigurdsson A, Ørstavik D, Trope M (2002) The

effectiveness of increased apical enlargement in reducing

intracanal bacteria. Journal of Endodontics 28, 779–83.

Cheung LHM, Cheung GSP (2008) Evaluation of a rotary

instrumentation method for c-shaped canals with

micro-computed tomography. Journal of Endodontics 34,

1233–8.

Esposito PT, Cunningham CJ (1995) A comparison of canal

preparation with nickel–titanium and stainless steel instru-

ments. Journal of Endodontics 21, 173–6.

Glossen CR, Haller RH, Dove SB, del Rio CE (1995) A

comparison of root canal preparations using Ni–Ti hand,

Ni–Ti engine-driven, and K-Flex endodontic instruments.

Journal of Endodontics 21, 146–51.

Grossman LI, Oliet S, del Rio CE (1988) Preparation of the root

canal: equipment and technique for cleaning, shaping and

irrigation. In: Grossman LI, Oliet S, del Rio CE, eds.

Endodontic practice, 11th edn. Philadelphia: Lea & Febiger,

pp. 179–227.

Haapasalo M, Endal U, Zandi H, Coil JM (2005) Eradication of

endodontic infection by instrumentation and irrigation

solutions. Endodontic Topics 10, 77–102.

Hildebrand T, Ruegsegger P (1997a) A new method for the

model-independent assessment of thickness in three-dimen-

sional images. Journal of Microscopy 185, 67–75.

Hildebrand T, Ruegsegger P (1997b) Quantification of bone

microarchitecture with the structural model index. Com-

puter Methods in Biomechanics and Biomedical Engineering 1,

15–23.

Hubscher W, Barbakow F, Peters OA (2003) Root-canal

preparation with FlexMaster: canal shapes analysed by

micro-computed tomography. International Endodontic Jour-

nal 36, 740–7.

Kakehashi S, Stanley HR, Fitzgerald RJ (1965) The effect

of surgical exposures of dental pulps in germ-free and

conventional laboratory rats. Oral Surgery Oral Medicine

Oral Pathology Oral Radiology and Endodontology 20,

340–9.

Kerekes K, Tronstad L (1977) Morphometric observations on

the root canals of human molars. Journal of Endodontics 3,

114–8.

Khademi A, Yazdizadeh M, Feizianfard M (2006) Determina-

tion of the minimum instrumentation size for penetration of

irrigants to the apical third of root canal systems. Journal of

Endodontics 32, 417–20.

Kuttler Y (1955) Microscopic investigation of root apexes.

Journal of the American Dental Association 50, 544–52.

Lorensen WE, Cline HE (1987) Marching cubes: a high

resolution 3D surface construction algorithm. Computer

Graphics 21, 163–9.

Moller AJ, Fabricius L, Dahlen G, Ohman AE, Heyden G (1981)

Influence on periradicular tissues of indigenous oral bacteria

and necrotic pulp tissue in monkeys. Scandinavian Journal of

Dental Research 89, 475–84.

Nair PRN, Sjogren U, Krey G, Kahnberg KE, Sundqvist G

(1990) Intraradicular bacteria and fungi in root-filled,

asymptomatic human teeth with therapy-resistant periapi-

cal lesions: a long-term light and electron microscopic

follow-up study. Journal of Endodontics 16, 580–8.

Nielsen RB, Alyassin AM, Peters DD, Carnes DL, Lancaster J

(1995) Microcomputed tomography: an advanced system

for detailed endodontic research. Journal of Endodontics 21,

561–8.

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, Laib A, Gohring TN, Barbakow F (2001a) Changes

in root canal geometry after preparation assessed by high-

resolution computed tomography. Journal of Endodontics 27,

1–6.

Peters OA, Schonenberger K, Laib A (2001b) Effects of four

Ni–Ti preparation techniques on root canal geometry

assessed by micro computed tomography. International

Endodontic Journal 34, 221–30.

Peters OA, Peters CI, Schonenberger K, Barbakow F (2003)

ProTaper rotary root canal preparation: effects of canal

anatomy on final shape analysed by micro CT. International


Pruett JP, Clement DJ, Carnes DL Jr (1997) Cyclic fatigue

testing of nickel–titanium endodontic instruments. Journal of


Rhodes JS, Ford TR, Lynch JA, Liepins PJ, Curtis RV (2000) A

comparison of two nickel–titanium instrumentation tech-

niques in teeth using microcomputed tomography. Interna-

tional Endodontic Journal 33, 279–85.

Roane JB, Sabala CL, Duncanson MG (1985) The ‘‘balanced

force’’ concept for instrumentation of curved canals. Journal

of Endodontics 11, 203–11.

Rollison S, Barnett F, Stevens RH (2002) Efficacy of bacterial

removal from instrumented root canals in vitro related to

instrumentation technique and size. Oral Surgery Oral

Medicine Oral Pathology Oral Radiology and Endodontology

94, 366–71.

Sathorn C, Palamara JEA, Messer HH (2005) A comparison of

the effects of two canal preparation techniques on root

fracture susceptibility and fracture pattern. Journal of




Schneider SW (1971) A comparison of canal preparations in

straight and curved root canals. Oral Surgery Oral Medicine

Oral Pathology Oral Radiology and Endodontology 32, 271–5.

Shuping GB, Ørstavik D, Sigurdsson A, Trope M (2000)

Reduction of intracanal bacteria using nickel–titanium

rotary instrumentation and various medications. Journal of


Siqueira JF, Lima KC, Magalhaes FA, Lopes HP, de Uzeda M

(1999) Mechanical reduction of the bacterial population in

the root canal by three instrumentation techniques. Journal


Spangberg L (2001) The wonderful world of rotary root canal

preparation. Oral Surgery Oral Medicine Oral Pathology Oral

Radiology and Endodontology 92, 479.

Sundqvist G (1976) Bacteriological studies of necrotic dental pulps

(Dissertation). Umea, Sweden: Umea University.

Tan BT, Messer HH (2002) The quality of apical canal

preparation using hand and rotary instruments with specific

criteria for enlargement based on initial apical file size.


Teknomo K (2006) Similarity measurement. [Website, up-

dated 2006]. URL http://people.revoledu.com/kardi/

tutorial/Similarity/EuclideanDistance.html [accessed on 14

December 2007].

Versluis A, Messer HH, Pintado MR (2006) Changes in

compaction stress distributions in roots resulting from canal

preparation. International Endodontic Journal 39, 931–9.

Walsch H (2004) The hybrid concept of nickel–titanium

rotary instrumentation. Dental Clinics of North America 48,

183–202.

Weine FS, Kelly RF, Lio PJ (1975) The effect of preparation

procedures on original canal shape and on apical foramen

shape. Journal of Endodontics 1, 255–62.

Weine FS, Kelly RF, Bray KE (1976) Effect of preparation with

endodontic handpieces on original canal shape. Journal of




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


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.


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.


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


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.



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.

References

Azabal M, Garcia-Otero D, De la Macorra JC (2004) Accuracy

of Justy II Apex locator in determining working length in

simulated horizontal and vertical fractures. International


Baldi JV, Victoriano FR, Bernardes RA et al. (2007) Influence

of embedding media on the assessment of electronic apex

locators. Journal of Endodontics 33, 476–9.

Bernardes RA, Duarte MAH, Vasconcelos BC et al. (2007)

Evaluation of precision of length determination with 3

electronic apex locators: Root ZX, Elements Diagnostic Unit

and Apex Locator and RomiAPEX D-30. Oral Surgery, Oral

Medicine, Oral Pathology, Oral Radiology and Endodontics 104,

e91–4.

Czerw RJ, Fulkerson MS, Donnelly JC, Walmann JO (1995)

In vitro evaluation of the accuracy of several electronic apex

locators. Journal of Endodontics 21, 572–5.

D’Assuncao FLC, Santana de Albuquerque D, Salazar-Silva JR,

Correia de Queiroz Ferreira L, Medeiros Bezerra P (2007)

The accuracy of root canal measurements using the Mini

Apex Locator and Root ZX-II: an evaluation in vivo. Oral

Surgery, Oral Medicine, Oral Pathology, Oral Radiology and

Endodontics 104, e50–3.

Ebrahim AK, Yoshioka T, Kobayashi C, Suda H (2006) The

effects of file size, sodium hypochlorite solution and blood on

the accuracy of Root ZX apex locator in enlarged root canals:

an in vitro study. Australian Dental Journal 51, 153–7.

ElAyouti A, Lost C (2006) A simply mounting model for

consistent determination of the accuracy and repeatability

of apex locators. International Endodontic Journal 39, 108–

12.

Fouad AF, Reid LC (2000) Effect of using electronic apex

locators on selected endodontic treatment parameters.


Fouad AF, Krell KV, McKendry DJ, Koorbusch GF, Olson RA

(1989) A clinical evaluation of five electronic root-canal

length measuring instruments. Journal of Endodontics 16,

446–9.

Gordon MPJ, Chandler NP (2004) Electronic apex locators.


Herrera M, Abalos C, Jimenez Planas A, Llamas R (2007)

Influence of apical constriction diameter on Root ZX apex

locator precision. Journal of Endodontics 33, 995–7.

Hoer D, Attin T (2004) The accuracy of electronic working

length determination. International Endodontic Journal 37,

125–31.

Kobayashi C, Suda H (1994) New electronic canal measuring

device based on the ratio method. Journal of Endodontics 20,

111–4.

Kuttler Y (1955) Microscopic investigation of root apexes.

Journal of the American Dental Association 50, 544–52.

Lucena-Martin C, Robles-Guijon V, Ferrer-Luque CM, de

Mondelo JM (2004) In vitro evaluation of the accuracy of

three electronic apex locators. Journal of Endodontics 30,

231–3.

Martinez-Lozano MA, Forner-Navarro L, Sanchez-Cortes JL,

Llena-Puy C (2001) Methodological considerations in the

determination of working length. International Endodontic

Journal 34, 371–6.

Meares WA, Steiman HR (2002) The influence of sodium

hypochlorite irrigation on the accuracy of the Root ZX

electronic apex locator. Journal of Endodontics 28, 595–8.

Oishi A, Yoshioka T, Kobayashi C, Suda H (2000) Location of

the file tip and the meter value of apex locators. The Japanese

Journal of Conservative Dentistry 6, 1145–53.

Pilot TF, Pitts DL (1997) Determination of impedances

changes at varying frequencies in relation to root canal

file position and irrigant. Journal of Endodontics 23, 719–

24.

Plotino G, Grande NM, Brigante L, Lesti B, Somma F (2006)

Ex vivo accuracy of three electronic apex locators: Root ZX,

Elements Diagnostic Unit and Apex Locator and Propex.

International Endondontic Journal 39, 408–14.

Pratten DH, McDonald NJ (1996) Comparison of radiographic

and electronic working lengths. Journal of Endodontics 22,

173–6.

Higa et al. Apex locators’ meter reading


Ricucci D, Langeland K (1998) Apical limit of root canal

instrumentation and obturation. Part 2. A histological

study. International Endodontic Journal 31, 394–409.

Seltzer S, Soltanoff W, Sinai I, Goldenberg A, Bender IB (1968)

Biologic aspects of endodontics. Part III. Periapical tissue

reactions to root canal instrumentation. Oral Surgery, Oral

Medicine and Oral Pathology 26, 534–46.

Seltzer S, Soltanoff W, Sinai I, Smith J (1969) Biologic aspects

of endodontics. Part IV. Periapical tissue reactions to root-

filled teeth whose canals had been instrumented short of

their apices. Oral Surgery, Oral Medicine and Oral Pathology

28, 724–38.

Smadi L (2006) Comparison between two methods of working

length determination and its effect on radiographic extent of

root canal filling: a clinical study. BMC Oral Health 6, 4.

Tselnik M, Baumgartner JC, Marshall JG (2005) An evaluation

of Root ZX and Elements Diagnostic Apex locators. Journal of


Vajrabhaya L, Tepmongkol P (1997) Accuracy of apex

locator. Endodontics and Dental Traumatology 13, 180–2.

Venturi M, Breschi L (2005) A comparison between two

electonic apex locators: an in vivo investigation. International


Venturi M, Breschi L (2007) A comparison between two

electonic apex locators: an ex vivo investigation. Interna-


Wrbas KT, Ziegler AA, Altenburger MJ, Schirrmeister JF

(2007) In vivo comparison of working length determination

with two electronic apex locators. International Endodontic

Journal 40, 133–8.



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


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.


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.


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 postcores 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


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).



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 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)

References

Bolla M, Muller-Bolla M, Borg C, Lupi-Pegurier L, Laplanche O,

Leforestier E (2007) Root canal posts for the restoration of

root filled teeth. Cochrane Database of Systematic Reviews 1,

CD004623.

Cagidiaco MC, Garcıa-Godoy F, Vichi A, Grandini S, Goracci C,

Ferrari M (2008) Placement of fiber prefabricated or custom

made posts affects the 3-year survival of endodontically

treated premolars. American Journal of Dentistry 21, 179–84.

Ferrari M, Vichi A, Garcia-Godoy F (2000) Clinical evaluation

of fiber reinforced epoxy resin posts and cast post and cores.

American Journal of Dentistry 13, (Spec No): 15B–8B.

Ferrari M, Cagidiaco MC, Grandini S, De Sanctis M, Goracci C

(2007) Post placement affects survival of endodontically

treated premolars. Journal of Dental Research 86, 729–34.

Grigoratos D, Knowles J, Ng YL, Gulabivala K (2001) Effect of

exposing dentine to sodium hypochlorite and calcium

hydroxide on its flexural strength and elastic modulus.


Lang H, Korkmaz Y, Schneider K, Raab WHM (2006) Impact

of endodontic treatments on the rigidity of the root. Journal

of Dental Research 85, 364–8.

Lanza A, Aversa R, Rengo S, Apicella D, Apicella A (2005) 3D

FEA of cemented steel, glass and carbon posts in a maxillary

incisor. Dental Materials 21, 709–15.

Mannocci F, Bertelli E, Sherriff M, Watson TF, Pitt Ford TR

(2002) Three year clinical comparison of survival of

endodontically treated teeth restored with either full cast

coverage or with direct composite restoration. Journal of

Prosthetic Dentistry 88, 297–301.

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


Randow K, Glantz PO (1986) On cantilever loading of vital

and non vital teeth. An experimental clinical study. Acta

Odontologica Scandinavia 44, 271–7.

Reeh ES, Messer HH, Douglas WH (1989) Reduction in tooth

stiffness as a result of endodontic and restorative procedures.




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.


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


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.


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.


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


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.



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.



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.



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.

References

Abou-Rass M, Patonai FJ (1982) The effects of decreasing

surface tension on the flow of irrigating solutions in narrow

root canals. Oral Surgery, Oral Medicine, Oral Pathology 53,

524–6.

Ahmad M, Pitt Ford TR, Crum LA, Walton AJ (1988)

Ultrasonic debridement of root canals: acoustic streaming

and its relevance. Journal of Endodontics 14, 486–93.

Blanken JW, Verdaasdonk RM (2007) Cavitation as a working

mechanism of the Er,Cr:YSGG Laser in endodontics: a

visualization study. Journal of Oral Laser Applications 7, 97–

106.

Boutsioukis C, Lambrianidis T, Kastrinakis E, Bekiaroglou P

(2007) Measurement of pressure and flow rates during

irrigation of a root canal ex vivo with three endodontic

needles. International Endodontic Journal 40, 504–13.

Burleson A, Nusstein J, Reader A, Beck M (2007) The in vivo

evaluation of hand/rotary/ultrasound instrumentation in

necrotic, human mandibular molars. Journal of Endodontics

33, 782–7.

Dijkink R, Ohl C-D (2008) Measurement of cavitation

induced wall shear stress. Applied Physics Letters 93,

2541071–3.

Druttman AC, Stock CJ (1989) An in vitro comparison of

ultrasonic and conventional methods of irrigant replace-

ment. International Endodontic Journal 22, 174–8.

George R, Walsh LJ (2008) Apical extrusion of root canal

irrigants when using Er:YAG and Er,Cr:YSGG lasers with

optical fibers: an in vitro dye study. Journal of Endodontics 34,

706–8.

George R, Meyers IA, Walsh LJ (2008) Laser activation of

endodontic irrigants with improved conical laser fiber tips

for removing smear layer in the apical third of the root

canal. Journal of Endodontics 34, 1524–7.

Holzfuss J, Ruggeberg M, Billo A (1998) Shock wave emissions

of a sonoluminescing bubble. Physical Review Letters 81,

5434–7.

Kimura Y, Yonaga K, Yokoyama K, Kinoshita J, Ogata Y,

Matsumoto K (2002) Root surface temperature increase

during Er:YAG laser irradiation of root canals. Journal of


Lauterborn W (1972) High-speed photography of laser-

induced breakdown in liquids. Applied Physics 21, 27–9.

Lee S-J, Wu M-K, Wesselink PR (2004) The efficacy of

ultrasonic irrigation to remove artificially placed dentin

debris from different sized simulated plastic root canals.

International Endododontic Journal 37, 607–12.

Lumley PJ, Walmsley AD, Laird WRE (1991) Streaming

patterns around endosonic files. International Endodontic

Journal 24, 290–7.

Lussi A, Nussbacher U, Grosrey J (1993) A novel noninstru-

mented technique for cleansing the root canal system.


Matsuoka E, Jayawardena JA, Matsumoto K (2005) A

morphological study on root canal preparation using

Erbium, Chromium:YSGG laser. Journal of Oral Laser Appli-

cations 5, 17–21.

Ram Z (1977) Effectiviness of root canal irrigation. Oral

Surgery, Oral Medicine, Oral Pathology 44, 306–11.

Robertson CW, Williams D (1971) Lambert absorption coef-

ficients of water in the infrared. Journal of the Optical Society

of America 61, 1316–20.

Rothfus RR, Monrad CC, Senecal VE (1950) Velocity distribu-

tion and fluid friction in smooth concentric annuli. Industrial

& Engineering Chemistry 42, 2511–20.

Roy RA, Ahmad M, Crum LA (1994) Physical mechanisms

governing the hydrodynamic response of an oscillating

ultrasonic file. International Endodontic Journal 27, 197–207.

Salzgeber RM, Brilliant JD (1977) An in vitro evaluation of the

penetration of an irrigating solution in root canals. Journal of


van der Sluis LMW, Wu M-K, Versluis M, Wesselink PR

(2007a) Passive ultrasonic irrigation of the root canal: a

review of the literature. International Endodontic Journal 40,

415–26.

van der Sluis LWM, Wu MK, Wesselink PR (2007b) The

evaluation of removal of Ca(OH)2 from an artificial standard-

ized groove in the apical root canal using different irrigation

methodologies. Internaional Endodontic Journal 40, 52–7.

Weller RN, Brady JN, Bernier WE (1980) Efficacy of ultrasonic

cleaning. Journal of Endodontics 6, 740–3.

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.



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


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.


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


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.


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.


(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).



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.

References

Al-Ghamdi A, Wennberg A (1994) Testing of sealing ability of

endodontic filling materials. Endodontics & Dental Trauma-

tology 10, 249–55.

Baumgartner JC, Falkler WA Jr (1991) Bacteria in the apical

5 mm of infected root canals. Journal of Endodontics 17,

380–3.

Camps J, Pashley D (2003) Reliability of the dye penetration

studies. Journal of Endodontics 29, 592–4.

De-Deus G, Coutinho-Filho T, Reis C, Murad C, Paciornik S

(2006) Polymicrobial leakage of four root canal sealers at

two different thicknesses. Journal of Endodontics 32, 998–

1001.

Eldeniz AU, Ørstavik D (2007) Five new endodontic sealers’ in

vitro resistance to bacterial penetration. Journal of Dental

Research (abstract) 86, 2654.

Eldeniz AU, Erdemir A, Hadimli HH, Belli S, Erganis O (2006)

Assessment of antibacterial activity of EndoREZ. Oral

Surgery, Oral Medicine, Oral Pathology, Oral Radiology &


Evcil MS, Colak M (2004) The pH changes of four different root

canal sealers after mixing at various time intervals in vitro.

Journal of Contemporary Dental Practice 15, 71–8.

Fransen JN, He J, Glickman GN, Rios A, Shulman JD,

Honeyman A (2008) Comparative assessment of ActiV

GP/glass ionomer sealer, resilon/epiphany, and gutta-

percha/AH plus obturation: a bacterial leakage study.


Fuss Z, Weiss EI, Shalhay M (1997) Antibacterial activity of

calcium hydroxide-containing endodontic sealers on Entero-

coccus faecalis in vitro. International Endodontic Journal 30,

397–402.

Heling I, Chandler NP (1996) The antimicrobial effect within

dentinal tubules of four root canal sealers. Journal of


Kayaoglu G, Erten H, Alacam T, Ørstavik D (2005) Short-term

antibacterial activity of root canal sealers towards Entero-

coccus faecalis. International Endodontic Journal 38, 483–8.

Kersten HW, Moorer WR (1989) Particles and molecules in

endodontic leakage. International Endodontic Journal 22,

118–24.

Mandras RS, Retief DH, Russell CM (1993) Quantitative

microleakage of six dentin bonding systems. American

Journal of Dentistry 6, 119–22.

Miletic I, Prpic-Mehicic G, Marsan T et al. (2002) Bacterial and

fungal microleakage of AH26 and AH Plus root canal

sealers. International Endodontic Journal 35, 428–32.

Monticelli F, Sadek FT, Schuster GS et al. (2007) Efficacy of

two contemporary single-cone filling techniques in prevent-

ing bacterial leakage. Journal of Endodontics 33, 310–3.

Oguntebi BR, Shen C (1992) Effect of different sealers on

thermoplasticized gutta-percha root canal obturations.


Ørstavik D (1983) Weight loss of endodontic sealers, cements

and pastes in water. Scandinavian Journal of Dental Research

91, 316–9.

Ørstavik D, Nordahl I, Tibballs JE (2001) Dimensional change

following setting of root canal sealer materials. Dental

Materials 17, 512–9.

Paque F, Sirtes G (2007) Apical sealing ability of resilon/

epiphany versus gutta-percha/AH Plus: immediate and 16-

months leakage. International Endodontic Journal 40, 722–9.

Pommel L, About I, Pashley D, Camps J (2003) Apical leakage

of four endodontic sealers. Journal of Endodontics 29, 208–

10.

Saleh IM, Ruyter IE, Haapasalo M, Ørstavik D (2008) Bacterial

penetration along different root canal filling materials in the

presence or absence of smear layer. International Endodontic

Journal 41, 32–40.

Schafer E, Zandbiglari T (2003) Solubility of root-canal sealers

in water and artificial saliva. International Endodontic Journal

36, 660–9.

Schuurs AH, Wu MK, Wesselink PR, Duivenvoorden HJ

(1993) Endodontic leakage studies reconsidered. Part II.

Statistical aspects. International Endodontic Journal 26, 44–

52.

Shipper G, Ørstavik D, Teixeira FB, Trope M (2004) An

evaluation of microbial leakage in roots filled with a

thermoplastic synthetic polymer-based root canal filling

material (Resilon). Journal of Endodontics 30, 342–7.

Skinner RL, Himel VT (1987) The sealing ability of injection-

molded thermoplasticized gutta-percha with and without

the use of sealers. Journal of Endodontics 13, 315–7.

Slutzky-Goldberg I, Slutzky H, Solomonov M, Moshonov J,

Weiss EI, Matalon S (2008) Antibacterial properties of four

endodontic sealers. Journal of Endodontics 34, 735–8.

Timpawat S, Amornchat C, Trisuwan WR (2001) Bacterial

coronal leakage after obturation with three root canal

sealers. Journal of Endodontics 27, 36–9.

Sealing properties of new sealer Salz et al.


Versiani MA, Carvalho-Junior JR, Padilha MI, Lacey S,

Pascon EA, Sousa-Neto MD (2006) A comparative study

of physicochemical properties of AH Plus and epiphany

root canal sealants. International Endodontic Journal 39,

464–71.

Weinberger SJ, Wright GZ (1989) Correlating Streptococcus

mutans with dental caries in young children using a

clinically applicable microbiological method. Caries Research

23, 385–8.

Wolanek GA, Loushine RJ, Weller RN, Kimbrough WF,

Volkmann KR (2001) In vitro bacterial penetration of

endodontically treated teeth coronally sealed with a dentin

bonding agent. Journal of Endodontics 27, 354–7.

Wu MK, Wesselink PR (1993) Endodontic leakage studies

reconsidered. Part I. Methodology, application and rele-

vance. International Endodontic Journal 26, 37–43.

Wu MK, De Gee AJ, Wesselink PR, Moorer WR (1993) Fluid

transport and bacterial penetration along root canal fillings.


Xu Q, Fan MW, Fan B, Cheung GS, Hu HL (2005) A new

quantitative method using glucose for analysis of endodon-

tic leakage. Oral Surgery, Oral Medicine, Oral Pathology, Oral

Radiology & Endodontics 99, 107–11.

Yucel AC, Guler E, Guler AU, Ertas E (2006) Bacterial

penetration after obturation with four different root canal

sealers. Journal of Endodontics 32, 890–3.



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.


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


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.


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


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.


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.



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.


Conclusion

In teeth with open apices, the presented Tactile Method

may offer an accurate alternative to contemporary

methods of working length determination.

References

Allen R, Mackie IC (2003) Management of the immature apex-

a clinical guide. Dentistry Update 30, 437–41.

Baggett FJ, Mackie IC, Worthington HV (1996) An investiga-

tion into the measurement of the working length of

immature incisor teeth requiring endodontic treatment in

children. British Dental Journal 10, 96–8.

Bland JM, Altman DG (1986) Statistical methods for assessing

agreement between two methods of clinical measurement.

Lancet 1, 307–10.

Bogen G, Kuttler S (2009) Mineral trioxide aggregate obtura-

tion: a review and case series. Journal of Endodontics 35,

777–90.

Burch JG, Hulen S (1972) The relationship of the apical

foramen to the anatomic apex of the tooth. Oral Surgery,

Oral Medicine, and Oral Pathology 34, 262–8.

Dominguez RA, Munoz ML, Aznar MT (2005) Study of

calcium hydroxide apexification in 26 young permanent

incisors. Dental Traumatology 21, 141–5.

Ebrahim AK, Wadachi R, Suda H (2006) Ex vivo evaluation of

the ability of four different electronic apex locators to

determine the working length in teeth with various foramen

diameters. Australian Dental Journal 51, 258–62.

ElAyouti A, Lost C (2006) A simple mounting model for

consistent determination of the accuracy and repeatability of

apex locators. International Endodontic Journal 39, 108–12.

ElAyouti A, Weiger R, Lost C (2001) Frequency of overin-

strumentation with an acceptable radiographic working

length. Journal of Endodontics 27, 49–52.

ElAyouti A, Kimionis I, Chu AL, Lost C (2005) Determining

the apical terminus of root-end resected teeth using three

modern apex locators: a comparative ex vivo study. Inter-

national Endodontic Journal 38, 827–33.

Goldberg F, De Silvio AC, Manfre S, Nastri N (2002) In vitro

measurement accuracy of an electronic apex locator in teeth

with simulated apical root resorption. Journal of Endodontics

28, 461–3.

Gordon MP, Chandler NP (2004) Electronic apex locators.


Gutmann JL, Heaton JF (1981) Management of the open

(immature) apex. 2. Non-vital teeth. International Endodontic

Journal 14, 173–8.

Herrera M, Abalos C, Planas AJ, Llamas R (2007) Influence of

apical constriction diameter on root ZX apex locator

precision. Journal of Endodontics 33, 995–8.

Holland R, Mazuqueli L, de Souza V, Murata SS, Dezan Junior

E, Suzuki P (2007) Influence of the type of vehicle and limit

of obturation on apical and periapical tissue response in

dogs’ teeth after root canal filling with mineral trioxide

aggregate. Journal of Endodontics 33, 693–7.

Hulsmann M, Pieper K (1989) Use of an electronic apex

locator in the treatment of teeth with incomplete root

formation. Endodontics and Dental Traumatology 5, 238–41.

Kerezoudis NP, Valavanis D, Prountzos F (1999) A method of

adapting gutta-percha master cones for obturation of open

apex cases using heat. International Endodontic Journal 32,

53–60.

Kim E, Lee SJ (2004) Electronic apex locator. Dental Clinics of

North America 48, 35–54.

Lee SJ, Nam KC, Kim YJ, Kim DW (2002) Clinical accuracy of

a new apex locator with an automatic compensation circuit.


Mackie IC, Hill FJ (1999) A clinical guide to the endodontic

treatment of non-vital immature permanent teeth. British

Dental Journal 186, 54–8.

Mente J, Seidel J, Buchalla W, Koch MJ (2002) Electronic

determination of root canal length in primary teeth with

and without root resorption. International Endodontic Journal

35, 447–52.

Morse DR, O’Larnic J, Yesilsoy C (1990) Apexification: review

of the literature. Quintessence International 21, 589–98.

Nguyen HQ, Kaufman AY, Komorowski RC, Friedman S

(1996) Electronic length measurement using small and

large files in enlarged canals. International Endodontic Journal

29, 359–64.

Ricucci D (1988) Apical limit of root canal instrumentation

and obturation, part 1. Literature review. International


Ricucci D, Langeland K (1988) Apical limit of root canal

instrumentation and obturation, part 2. A histological

study. International Endodontic Journal 6, 394–409.

Rosenberg DB (2003) The paper point technique, part 1.

Dentistry Today 22, 80–6.

Shabahang S, Torabinejad M, Boyne PP, Abedi H, McMillan P

(1999) A comparative study of root-end induction using

osteogenic protein-1, calcium hydroxide, and mineral

trioxide aggregate in dogs. Journal of Endodontics 25, 1–5.

Stein TJ, Corcoran JF (1992) Radiographic ‘‘working length’’

revisited. Oral Surgery, Oral Medicine, and Oral Pathology 74,

796–800.

Tosun G, Erdemir A, Eldeniz AU, Sermet U, Sener Y (2008)

Accuracy of two electronic apex locators in primary teeth

with and without apical resorption: a laboratory study.


Williams CB, Joyce AP, Roberts S (2006) A comparison

between in vivo radiographic working length determination

and measurement after extraction. Journal of Endodontics 32,

624–7.

Wu YN, Shi JN, Huang LZ, Xu YY (1992) Variables affecting

electronic root canal measurement. International Endodontic

Journal 25, 88–92.



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


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.


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


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.


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.


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).



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



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).

References

Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005)

Defining the normal bacterial flora of the oral cavity. Journal

of Clinical Microbiology 43, 5721–32.

Baumgartner JC, Siqueira JF Jr, Xia T, Rocas IN (2004)

Geographical differences in bacteria detected in endodontic

infections using polymerase chain reaction. Journal of


Baumgartner JC, Hutter JW, Siqueira JF Jr (2006) Endodontic

microbiology and treatment of infections. In: Cohen S,

Hargreaves KM eds. Pathways of the Pulp, 9th edn. St Louis,

MO: CV Mosby/Elsevier, pp. 580–607.

Belay N, Johnson R, Rajagopal BS, de Macario EC, Daniels L

(1988) Methanogenic bacteria from human dental plaque.

Applied and Environmental Microbiology 54, 600–3.

Belay N, Mukhopadhyay B, Conway de Macario E, Galask R,

Daniels L (1990) Methanogenic bacteria in human vaginal

samples. Journal of Clinical Microbiology 28, 1666–8.

Bonch-Osmolovskaya EA, Stetter KO (1991) Interspecies

hydrogen transfer in cocultures of thermophilic archaea.

Systematic and Applied Microbiology 14, 205–8.

Cavicchioli R, Curmi PM, Saunders N, Thomas T (2003)

Pathogenic archaea: do they exist? BioEssays 25, 1119–

28.

Chomczynski P, Sacchi N (1987) Single-step method of RNA

isolation by acid guanidinium thiocyanate–phenol–chloro-

form extraction. Analytical Biochemistry 162, 156–9.

Chomczynski P, Sacchi N (2006) The single-step method of

RNA isolation by acid guanidinium thiocyanate–phenol–

chloroform extraction: twenty-something years on. Nature

Protocols 1, 581–5.

Conrad R (1999) Contribution of hydrogen to methane

production and control of hydrogen concentrations in

methanogenic soils and sediments. FEMS Microbiology

Ecology 28, 193–202.

Cury JA, Seils J, Koo H (2008) Isolation and purification of

total RNA from Streptococcus mutans in suspension cultures

and biofilms. Brazilian Oral Research 22, 216–22.

Eckburg PB, Lepp PW, Relman DA (2003) Archaea and their

potential role in human disease. Infection and Immunity 71,

591–6.



Gomes BP, Pinheiro ET, Gade-Neto CR et al. (2004) Microbi-

ological examination of infected dental root canals. Oral

Microbiology and Immunology 19, 71–6.

Kaprelyants AS, Gottschal JC, Kell DB (1993) Dormancy in

non-sporulating bacteria. FEMS Microbiology Reviews 104,

271–86.

Kulik EM, Sandmeier H, Hinni K, Meyer J (2001) Identification

of archaeal rDNA from subgingival dental plaque by PCR

amplification and sequence analysis. FEMS Microbiology

Letters 196, 129–33.

Le Goff A, Bunetel L, Mouton C, Bonnaure-Mallet M (1997)

Evaluation of root canal bacteria and their antimicrobial

susceptibility in teeth with necrotic pulp. Oral Microbiology

and Immunology 12, 318–22.

Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC,

Relman DA (2004) Methanogenic Archaea and human

periodontal disease. Proceedings of the National Academy of

Sciences of the United States of America 101, 6176–81.

Lleo MM, Bonato B, Tafi MC, Signoretto C, Boaretti M,

Canepari P (2001) Resuscitation rate in different entero-

coccal species in the viable but non-culturable state. Journal

of Applied Microbiology 91, 1095–102.

Love RM, Jenkinson HF (2002) Invasion of dentinal tubules by

oral bacteria. Critical Reviews in Oral Biology and Medicine

13, 171–83.

Lovley DR (1985) Minimum threshold for hydrogen metabo-

lism in methanogenic bacteria. Applied and Environmental

Microbiology 49, 1530–1.

Mason CA, Hamer G, Bryers JD (1986) The death and lysis of

microorganisms in environmental processes. FEMS Micro-

biology Reviews 39, 373–401.

Miller TL, Wolin MJ (1982) Enumeration of Methanobrevibacter

smithii in human feces. Archives of Microbiology 131, 14–8.

Molander A, Reit C, Dahlen G, Kvist T (1998) Microbiological

status of root-filled teeth with apical periodontitis. Interna-


Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG

(2002) Molecular and cultural analysis of the microflora

associated with endodontic infections. Journal of Dental

Research 81, 761–6.

Nair PN (2004) Pathogenesis of apical periodontitis and the

causes of endodontic failures. Critical Reviews in Oral Biology

and Medicine 15, 348–81.

Ng YL, Spratt D, Sriskantharajah S, Gulabivala K (2003)

Evaluation of protocols for field decontamination before

bacterial sampling of root canals for contemporary micro-

biology techniques. Journal of Endodontics 29, 317–20.

Oliver JD (1995) The viable but non-culturable state in the

human pathogen, Vibrio vulnificus. FEMS Microbiology

Letters 133, 203–8.

Oliver JD (2005) The viable but nonculturable state in

bacteria. The Journal of Microbiology 43, 93–100. Review.

Paster BJ, Boches SK, Galvin JL et al. (2001) Bacterial diversity

in human subgingival plaque. Journal of Bacteriology 183,

3770–83.

Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M (2001)

Isolation of yeasts and enteric bacteria in root-filled teeth

with chronic apical periodontitis. International Endodontic

Journal 34, 429–34.

Sabeti M, Simon JH, Slots J (2003) Cytomegalovirus and

Epstein-Barr virus are associated with symptomatic peri-

apical pathosis. Oral Microbiology and Immunology 18,

327–8.

Saito D, Leonardo Rde T, Rodrigues JL, Tsai SM, Hofling JF,

Goncalves RB (2006) Identification of bacteria in endodon-

tic infections by sequence analysis of 16S rDNA clone

libraries. Journal of Medical Microbiology 55, 101–7.

Sakamoto M, Siqueira JF Jr, Rocas IN, Benno Y (2008)

Molecular analysis of the root canal microbiota associated

with endodontic treatment failures. Oral Microbiology and

Immunology 23, 275–81.

Sedgley CM, Lee EH, Martin MJ, Flannagan SE (2008)

Antibiotic resistance gene transfer between Streptococcus

gordonii and Enterococcus faecalis in root canals of teeth ex

vivo. Journal of Endodontics 34, 570–4.

Siqueira JF Jr (2002) Endodontic infections: concepts, para-

digms and perspectives. Oral Surgery, Oral Medicine, Oral

Pathology, Oral Radiology, and Endodontics 94, 281–93.

Siqueira JF Jr, Rocas IN (2003) PCR methodology as a

valuable tool for identification of endodontic pathogens.

Journal of Dentistry 31, 333–9.

Siqueira JF Jr, Rocas IN (2005) Exploiting molecular methods

to explore endodontic infections. Part 2. Redefining the

endodontic microbiota. Journal of Endodontics 31, 488–98.

Siqueira JF Jr, Rocas IN, Baumgartner JC, Xia T (2005)

Searching for Archaea in infections of endodontic origin.


Siqueira JF Jr, Rocas IN, Paiva SS, Magalhaes KM, Guimaraes-

Pinto T (2007) Cultivable bacteria in infected root canals as

identified by 16S rRNA gene sequencing. Oral Microbiology

and Immunology 22, 266–71.

Sundqvist G (1976) Bacteriological Studies of Necrotic Dental

Pulps. (PhD Dissertation). Sweden: University of Umea.

Sundqvist G (1992) Associations between microbial species in

dental root canal infections. Oral Microbiology and Immunol-

ogy 7, 257–62.

Sundqvist G (1994) Taxonomy, ecology, and pathogenicity of

the root canal flora. Oral Surgery, Oral Medicine, and Oral

Pathology 78, 522–30.

Sundqvist G, Figdor D (2003) Life as an endodontic pathogen:

ecological differences between the untreated and root filled

root canals. Endodontic Topics 6, 3–28.

Sundqvist G, Fidgor D, Persson S, Sjogren U (1998) Microb-

iologic analysis of teeth with failed endodontic treatment

and the outcome of conservative retreatment. Oral Surgery,

Oral Medicine, and Oral Pathology 85, 86–93.

Vianna ME, Conrads G, Gomes BP, Horz HP (2006) Identifi-

cation and quantification of Archaea involved in primary

endodontic infections. Journal of Clinical Microbiology 44,

1274–82.



Vickerman MM, Brossard KA, Funk DB, Jesionowski AM, Gill

SR (2007) Phylogenetic analysis of bacterial and archaeal

species in symptomatic and asymptomatic endodontic

infections. Journal of Medical Microbiology 56, 110–8.

Wagner R (1994) The regulation of ribosomal RNA synthesis

and bacterial cell growth. Archives of Microbiology 161,

100–6.

Williams JM, Trope M, Caplan DJ, Shugars DC (2006)

Detection and quantitation of E. faecalis by real-time PCR

(qPCR), reverse transcription-PCR (RT-PCR), and cultiva-

tion during endodontic treatment. Journal of Endodontics 32,

715–21.

Woese CR, Kandler O, Wheelis ML (1990) Towards a natural

system of organisms: proposal for the domains Archaea,

Bacteria, and Eucarya. Proceedings of the National Academy of

Sciences of the United States of America 87, 4576–9.

Young G, Turner S, Davies JK, Sundqvist G, Figdor D (2007)

Bacterial DNA persists for extended periods after cell death.


Yu Z, Morrison M (2001) Comparisons of different hypervari-

able regions of rrs genes for use in fingerprinting of

microbial communities by PCR-denaturing gradient gel

electrophoresis. Applied and Environmental Microbiology 70,

4800–6.



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


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.


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


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.


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.


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.



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.



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



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.

References

Apte SS, Mattei MG, Olsen BR (1994) Cloning of the cDNA

encoding human tissue inhibitor of metalloproteinases-3

(TIMP-3) and mapping of the TIMP-3 gene to chromosome

22. Genomix 19, 86–90.

Bergenholtz G (1974) Micro-organisms from necrotic pulp of

traumatized teeth. Odontologisk Revy 25, 347–58.

Berit J, Klaus B (1986) Chemical composition and biological

properties of a lipopolysaccharide from Bacteroides interme-

dius. Acta Pathologica et Microbiologica Scandinavia. Section B:

Microbiology and Immunology 94, 265–71.

Brook I, Grimm S, Kielich RB (1981) Bacteriology of acute

periapical abscess in children. Journal of Endodontics 7, 378–

80.

Collier IE, Wilhelm SM, Eisen AZ et al. (1988) H-ras oncogene-

transformed human bronchial epithelial cells (TBE-1)

secrete a single metalloprotease capable of degrading

basement membrane collagen. The Journal of Biological

Chemistry 263, 6579–87.

DeCarlo AA Jr, Windsor LJ, Bodden MK, Harber GJ, Birkedal-

Hansen B, Birkedal-Hansen H (1997) Activation and novel

processing of matrix metalloproteinases by a thiol-protein-

ase from the oral anaerobe Porphyromonas gingivalis. Journal

of Dental Research 76, 1260–70.

Declerk YA, Yean TD, Ratzkin BJ, Lu HS, Langley KE (1989)

Purification and characterization of two related but distinct

metalloproteinase inhibitors secreted by bovine aortic

endothelial cells. The Journal of Biological Chemistry 264,

17445–53.

Fujimoto N, Zhang J, Iwata K, Shinya T, Okada Y, Hayakawa

T (1993) A one-step sandwich enzyme immunoassay for

tissue inhibitor of metalloproteinases-2 using monoclonal

antibodies. Clinica Chimica Acta 220, 31–45.

Goldberg GI, Marmer BL, Grant GA, Eisen AZ, Wilhelm S, He C

(1989) Human 72-kilodalton type-collagenase forms a

complex with a tissue inhibitor of metalloproteases desig-

nated TIMP-2. Proceeding of the National Academy of Science of

the United States America 86, 8207–11.



Green J, Wang M, Liu YE, Raymond LA, Rosen C, Shi YE

(1996) Molecular cloning and characterization of human

tissue inhibitor of metalloproteinase 4. The Journal of

Biological Chemistry 271, 30375–80.

Grenier D, Mayrand D (2001) Inactivation of tissue inhibitor of

metalloproteinases-1(TIMP-1) by Porphyrmonas gingivalis.

FEMS Microbiology Letters 203, 161–4.

Haapasalo M (1989) Bacteroides spp. in dental root canal

infections. Endodontic Dental Traumatology 5, 1–10.

Higerd TB, Vesole DH, Goust JM (1978) Inhibitory effects of

extracellular products from oral bacteria on human fibro-

blasts and stimulated lymphocytes. Infection and Immunity

21, 567–74.

Kantz WE, Henry CA (1974) Isolation and classification of

anaerobic bacteria from intact pulp chambers of non-vital

teeth in man. Archives Oral Biology 19, 91–6.

Kishi J, Ogawa K, Yamamoto S, Hayakawa T (1991) Purifi-

cation and characterization of a new tissue inhibitor of

metalloproteinases (TIMP-2) from mouse colon 26 tumor

cell. Matrix 11, 10–6.

Kodama S, Kishi J, Okada K, Iwata K, Hayakawa T (1981)

Monoclonal antibodies to bovine collagenase inhibitor.

Collagen Related Research 7, 341–50.

Kodama S, Iwata K, Iwata H, Yamashita K, Hayakawa T

(1990) Rapid one-step sandwich enzyme immunoassay for

tissue inhibitor of metalloproteinases. An application for

rheumatoid arthritis serum and plasma. Journal Immunolog-

ical Methods 127, 103–8.

Laemmli UK (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227,

680–5.

Nagase H, Woessner JF Jr (1999) Matrix metalloproteinases.

The Journal of Biological Chemistry 274, 21491–4.

Nair BC, Mayberry WR, Dziak R, Chen PB, Levine MJ,

Hausmann E (1983) Biological effects of a purified lipopoly-

saccharide from Bacteroides gingivalis. Journal of Periodontal


Oguntebi B, Slee AM, Tanzer JM, Langeland K (1982)

Predominant microflora associated with human dental

periapical abscesses. Journal of Clinical Microbiology 15,

964–6.

Pattamapun K, Tiranathangui S, Yongchaitrakul T, Kuwata-

nasuchat J, Pavasant P (2003) Activation of MMP-2 by

Porphyromonas gingivalis in human periodontal ligament

cells. Journal of Periodontal Research 38, 115–21.

Pike R, McGran W, Potempa J, Travis J (1994) Lysine- and

arginine-specific proteinases from Porphyromonas gingivalis.

The Journal of Biological Chemistry 269, 406–11.

Sakamoto W, Fujie K, Kaga M et al. (1996) Degradation of T-

kininogen by cathepsin D and matrix metalloproteinases.

Immunopharmacology 32, 73–5.

Sato H, Takino T, Okada Y et al. (1994) A matrix metallo-

proteinase expressed on the surface of invasive tumour cells.

Nature 370, 61.

Singer RE, Dutton WG (1979) A comparison of the effects of

endotoxin upon fibroblast proliferation and macromolecular

synthesis. Journal of Dental Research 58, 1634–9.

Stetler-Stevenson WG, Krutzsch HC, Liotta LA (1989) Tissue

inhibitor of 12-metalloproteinase (TIMP-2). A new member

of the metalloproteinase inhibitor family. The Journal of

Biological Chemistry 264, 17374–8.

Sundqvist G (1992) Associations between microbial species in

dental root canal infections. Oral Microbiology and Immunol-

ogy 7, 257–62.

Terato K, Nagai Y, Kawanishi K, Yamamoto S (1976) A rapid

assay method of collagenase activity using 14C-labeled

soluble collagen as substrate. Biochimica et Biophysica Acta

445, 753–62.

Tiranathanagul K, Pattamapun T, Yongchaitrakul T, Pava-

sant P (2004) MMP-2 activation by Actinomycetemcomitans

supernatant in human PDL cells was corresponded with

reduction of TIMP-2. Oral Biology 10, 383–8.

Uria JA, Ferrando AA, Velasco G et al. (1994) Structure and

expression in breast tumors of human TIMP-3, a new

member of the metalloproteinases inhibitor family. Cancer


Visse R, Nagase H (2003) Matrix metalloproteinases and tissue

inhibitors of metalloproteinases: structure, function, and

biochemistry. Circulation Research 92, 827–39.

Ward RV, Hembry RM, Reynolds JJ, Murphy G (1991) The

purification of tissue inhibitor of metalloproteinases-2 from

its 72 kDa progelatinase complex. Biochemical Journal 278,

179–87.

Williams BL, McCann GF, Schoenknecht FD (1983) Bacteri-

ology of dental abscesses of endodontic origin. Journal of

Clinical Microbiology 18, 770–4.

Wittgow WCJ, Sabiston CBJ (1975) Microorganisms from

pulpal chambers of intact teeth with necrotic pulps. Journal


Yamamoto K (1995) Studies on periodontal pathogenic

proteinase from Porphyromonas gingivalis and host cell. Folia

Phormacologica Japonica 105, 345–55.

Zhou J, Windsor LJ (2006) Porphyromonas gingivalis affects

host collagen degradation by affecting expression, activa-

tion, and inhibition of matrix metalloproteinases. Journal of

Periodontal Research 41, 47–54.



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


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.


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


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.


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-



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.


References

Al-Haroni M, Skaug N (2007) Incidence of antibiotic pre-

scribing in dental practice in Norway and its contribution to

national consumption. Journal of Antimicrobial Chemotherapy

59, 1161–6.

Al-Mubarak S, Al-Nowaiser A, Abou Rass M et al. (2004)

Antibiotic prescription and dental practice within Saudi

Arabia; the need to reinforce guidelines and implement

specialty needs. Journal of the International Academy of

Periodontology 6, 47–55.

American Dental Association Council on Scientific Affairs

(2004) Combating antibiotic resistance. Journal of the

American Dental Association 135, 484–7.

Arnold SR, Straus SE (2005) Interventions to improve

antibiotic prescribing practices in ambulatory care. Cochrane

Database of Systematic Reviews issue 4, Art. No. CD003539.

pub.2. DOI:10.1002/14651858.

Chate RA, White S, Hale LR et al. (2006) The impact of clinical

audit on antibiotic prescribing in general dental practice.

British Dental Journal 201, 635–41.

Choudhury M, Needleman I, Gillam D, Moles DR (2001)

Systemic and local antimicrobial use in periodontal therapy

in England and Wales. Journal of Clinical Periodontology 28,

833–9.

Dailey YM, Martin MV (2001) Are antibiotics being used

appropriately for emergency dental treatment? British Dental

Journal 191, 391–3.

Handal T, Olsen I (2000) Antimicrobial resistance with focus

on oral beta-lactamases. European Journal of Oral Sciences

108, 163–74.

Hawkey PM (2008) The growing burden of antimicro-

bial resistance. Journal of Antimicrobial Chemotherapy 62,

i1–9.

Keenan JV, Farman AG, Fedorowicz Z, Newton JT (2005)

Antibiotic use for irreversible pulpitis. Cochrane Database of

Systematic Reviews issue 2, Art. No.: CD004969.pub2.

DOI:10.1002/14651858.

Kuriyama T, Absi EG, Williams DW, Lewis MAO (2005) An

outcome audit of the treatment of acute dentoalveolar

infection: impact of penicillin resistance. British Dental

Journal 198, 759–63.

Lopez-Piriz R, Aguilar L, Gimenez M (2007) Management of

odontogenic infection of pulpal and periodontal origin.

Medicina Oral, Patologıa Oral y Cirugıa Bucal 12, E154–9.

Matthews DC, Sutherland S, Basrani B (2003) Emergency

management of acute apical abscesses in the permanent

dentition: a systematic review of the literature. Journal of the

Canadian Dental Association 69, 660.

Nagle D, Reader A, Beck M, Weaver J (2000) Effect of systemic

penicillin on pain in untreated irreversible pulpitis. Oral

Surgery, Oral Medicine, Oral Pathology, Oral radiology End-

odontics 90, 636–40.

Palmer NA, Batchelor PA (2004) An audit of antibiotic

prescribing by vocational dental practitioners. Primary

Dental Care 1, 77–80.

Palmer NA, Dailey YM (2002) General dental practitioners’

experiences of a collaborative clinical audit on antibiotic

prescribing: a qualitative study. British Dental Journal 193,

46–9.

Palmer NA, Pealing R, Ireland RS, Martin MV (2000) A study

of therapeutic antibiotic prescribing in National Health

Service general dental practice in England. British Dental

Journal 10, 554–8.

Palmer NA, Dailey YM, Martin MV (2001) Can audit improve

antibiotic prescribing in general dental practice? British

Dental Journal 191, 253–5.

Roy KM, Bagg J (2000) Antibiotic prescribing by general

dental practitioners in the Greater Glasgow Health Board,

Scotland. British Dental Journal 188, 674–6.

Sweeney LC, Dave J, Chambers PA, Heritage J (2004) Antibiotic

resistance in general dental practice – a cause for concern?

Journal of Antimicrobial Chemotherapy 53, 567–76.

Tong DC, Rothwell BR (2000) Antibiotic prophylaxis in

dentistry: a review and practice recommendations. Journal

of the American Dental Association 131, 366–74.

Yingling NM, Byrne EB, Hartwell GR (2002) Antibiotic use by

members of the American association of endodontists in the

year 2000: report of a national survey. Journal of Endodontics

28, 396–404.



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]).


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

CA

SE

RE

PO

RT


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).

CA

SE

RE

PO

RT


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.

CA

SE

RE

PO

RT


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.

CA

SE

RE

PO

RT


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.

CA

SE

RE

PO

RT


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

CA

SE

RE

PO

RT


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.

References

Andreasen JO, Farik B, Munksgaard EC (2002) Long-term calcium hydroxide as a root canal dressing

may increase the risk of root fracture. Dental Traumatology 18, 134–7.

Banchs F, Trope M (2004) Revascularization of immature permanent teeth with apical periodontitis:

new treatment protocol? Journal of Endodontics 30, 196–200.

Barrett AP, Reade PC (1981) Revascularization of mouse tooth isografts and allografts using

autoradiography and carbon-profusion. Archives in Oral Biology 26, 541–5.

Basrani B, Manek S, Sodhi R, Fillery E, Manzur A (2007) Interaction between sodium hypochlorite and

chlorhexidine gluconate. Journal of Endodontics 33, 966–9.

Canger EM, Kayipmaz S, Celenk P (2009) Bilateral dens invaginatus in the mandibular premolar region.

Indian Journal of Dental Research 20, 238–40.

Cengiz SB, Korasli D, Ziraman F, Orhan K (2006) Non-surgical root canal treatment of dens invaginatus:

reports of three cases. International Dental Journal 56, 17–21.

CA

SE

RE

PO

RT


Chueh LH, Huang GT (2006) Immature teeth with periradicular periodontitis or abscess undergoing

apexogenesis: a paradigm shift. Journal of Endodontics 32, 1205–13.

Chueh LH, Ho YC, Kuo TC, Lai WH, Chen YH, Chiang CP (2009) Regenerative endodontic treatment

for necrotic immature permanent teeth. Journal of Endodontics 35, 160–4.

Cvek M, Sundstrom B (1974) Treatment of non-vital permanent incisors with calcium hydroxide, V.

Histologic appearance of roentgenologically demonstrable apical closure of immature roots.

Odontologisk Revy 25, 379–92.

Er K, Kustraci A, Ozan U, Tasdemir T (2007) Nonsurgical endodontic treatment of dens invaginatus in a

mandibular premolar with large periradicular lesion: a case report. Journal of Endodontics 33, 322–4.

Fridland M, Rosado R (2005) MTA solubility (a long term study). Journal of Endodontics 31, 376–9.

Greenstein G, Berman C, Jaffin R (1986) Chlorhexidine: an adjunct to periodontal therapy. Journal of

Periodontology 57, 370–6.

Hoshino E, Kurihara-Ando N, Sato I et al. (1996) In-vitro antibacterial susceptibility of bacteria taken

from infected root dentine to a mixture of ciprofloxacin, metronidazole and minocycline.


Huang G, Sonoyama W, Liu Y (2008) The hidden treasure in apical papilla: the potential role in pulp/

dentin regeneration and bioroot engineering. Journal of Endodontics 34, 645–51.

Iwaya S, Ikawa M, Kubota M (2001) Revascularization of an immature permanent tooth with apical

periodontitis and sinus tract. Dental Traumatology 17, 185–7.

Jeansonne JJ, White R (1994) A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium

hypochlorite as antimicrobial endodontic irrigants. Journal of Endodontics 20, 276–8.

Jung IY, Lee SJ, Hargreaves KM (2008) Biologically based treatment of immature permanent teeth

with pulpal necrosis: a case series. Journal of Endodontics 34, 876–87.

Kettering JD, Torabinejad M (1995) Investigation of mutagenicity of mineral trioxide aggregate and

other commonly used root end filling materials. Journal of Endodontics 21, 537–9.

Lin L, Shovlin F, Skribner J, Langeland K (1984) Pulp biopsies from the teeth associated with periapical

radiolucency. Journal of Endodontics 10, 436–48.

Moghaddame-Jafari S, Mantellini MG, Botero TM, McDonald NJ, Nor JE (2005) Effect of proroot MTA

on pulp cell apoptosis and proliferation in vitro. Journal of Endodontics 31, 387–91.

Murray PE, Garcia-Godoy F, Hargreaves KM (2007) Regenerative endodontics: a review of current

status and a call for action. Journal of Endodontics 33, 377–90.

Ohman A (1965) Healing and sensitivity to pain in young replanted human teeth: an experimental,

clinical and histological study. Odontologisk Tidskrift 73, 168–227.

Rafter M (2005) Apexification: a review. Dental Traumatology 21, 1–8.

Reynolds K, Johnson D, Cohenca N (2009) Pulp revascularization of necrotic bilateral bicuspids using a

modified novel technique to eliminate potential coronal discolouration: a case report. International


Shah N, Logani A, Bhaskar U (2008) Efficacy of revascularization to induce apexification/apexogenesis

in infected, nonvital, immature teeth: a pilot clinical study. Journal of Endodontics 34, 919–25.

Skoglund A, Tronstad L (1981) Pulpal changes in replanted and autotransplanted immature teeth of

dogs. Journal of Endodontics 7, 309–16.

Sonoyama W, Lin Y, Fang D et al. (2006) Mesenchymal stem cell-mediated functional tooth

regeneration in swine. PLoS ONE 1, e79.

Torabinejad M, Chivian N (1999) Clinical applications of mineral trioxide aggregate. Journal of


Torabinejad M, Wilder Smith P, Pitt Ford TR (1995) Comparative investigation of marginal adaptation of

mineral trioxide aggregate and other commonly used root-end filling materials. Journal of


White R, Hays G, Janer L (1997) Residual antimicrobial activity after canal irrigation with chlorhexidine.


Yousef SaadA (1988) Calcium hydroxide and apexogenesis. Oral Surgery, Oral Medicine & Oral

Pathology 66, 499–501.

CA

SE

RE

PO

RT


iej.12.2009

Documents

refiningthe apical preparation

apical rootcanal preparation

larger apical sizes

canal roundness

rounded preparation

preparedapical root

root canals

changesthe canal anatomy