Iej 12 2009

doi:10.1111/j.1365-2591.2009.01626.
A micro-computed tomographic evaluation of apical root canal preparation using three instrumentation techniques
J. Moore, P. Fitz-Walter & P. Parashos

Melbourne 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, 10571064, 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-les using the balanced force technique; Group 2 stainless steel K-les (balanced force) and then rening 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, transportation and how the dimensions of the prepared
apical root canal correlated with those of the nal 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 nickeltitanium instrumentation technique remained small and, a more rounded preparation (P < 0.001) that closely matched the nal instrument dimensions (P < 0.001) was produced. There was a trend for less canal transportation using rotary nickeltitanium instruments. Conclusions Stainless steel hand preparation was not conservative of apical dentine. When used correctly, even by less experienced operators, rotary nickeltitanium 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, Mo ller et al. 1981). Instrumentation forms an integral part of the
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: parashos@unimelb.edu.au).
process of eliminating or reducing the number of microorganisms to a level that will allow healing of the periapical tissues (Bystro m & Sundqvist 1981). Technically, the goal of endodontic instrumentation is to remove all necrotic and vital pulp tissue along with heavily infected radicular dentine. Instrumentation 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
2009 International Endodontic Journal
International Endodontic Journal, 42, 10571064, 2009
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Apical root canal preparation assessed by micro-CT Moore et al.
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 nondestructive analytical method that has enabled researchers 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, Hu bscher et al. 2003). However, none has specically focussed on the apical portion of the root canal or how the preparation dimensions relate to the nal 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 efcacy 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 0.300.35 mm (Kuttler 1955, Kerekes & Tronstad 1977), preparation to larger apical sizes ngberg 2001). appears logical (Spa Despite these benets 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 instrumentation techniques using micro-CT scanning.
Materials and methods

Ethics approval for this research project was obtained from the Health Sciences Human Ethics Sub-Committee, The University of Melbourne, Melbourne, Victoria, Australia (Ethics ID: 0714905).
Preparation of specimens
Twenty-one maxillary and nineteen mandibular rst 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 at occlusal surfaces against an SEM stub (SkyScan 1072, Kontich, Belgium) or resin disc (SkyScan 1076, Kontich, 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 Hedstro m les (Dentsply Maillefer, Ballaigues, Switzerland) and working length was set 1 mm from the apical foramen. Digital radiographs were taken from buccolingual and mesio-distal directions with size 10 Hedstro m les (Dentsply Maillefer, Ballaigues, Switzerland) 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 equitable distribution of canal numbers, canal curvatures and radii between the two operators and the canals were allocated into groups representing three instrumentation protocols. Group 1 was prepared by one operator using Gates Glidden burs 2 and 3 (Dentsply Maillefer) for coronal aring and gaining straight line access to the middle third, and the balanced force technique (Roane et al. 1985) with stainless steel K-les (Dentsply Maillefer) for the remaining preparation. All the mesio-buccal canals of maxillary and mandibular teeth in Group 1 were rened with the corresponding FlexMaster 0.04 taper (VDW, Munich, Germany) rotary nickel titanium instrument (e.g. 30 K-le rened with size 30, 0.04 taper FlexMaster) used in a torquecontrolled handpiece (Endo IT Professional, VDW, Munich, Germany) at manufacturer recommended settings. Those canals were then considered a separate 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
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with a size 15 Hedstro m le (Dentsply Maillefer), ProTaper S1, S2 and F1 (Dentsply Maillefer) were used to working length. The apical preparation was nished with a FlexMaster 0.04 taper nickeltitanium 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 nickeltitanium instruments (Dentsply Maillefer) were used (i.e. sizes 45 and 60). All rotary nickeltitanium instruments were used in a torquecontrolled 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 nal 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 rst le 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 utes in the apical third were loaded with dentine debris, the apical preparation was considered to be complete. The minimum, median and maximum master apical le 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 modication 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 specic 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 ve from Group 3) had to be excluded because of artefacts in either the pre or post-instrumentation images or blockages that prevented negotiation 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 nal instrument used were only calculated over the apical 1 mm of canal preparation (located 12 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-instrumentation canal values. The post-instrumentation struc-
Table 1 Group information and morphometric scores evaluating apical canal preparation (mean SD)
Group 1 2 1/2 3 n 39 18 57 53 Minimum, Median, Maximum MAF 25, 25, 25, 30, 30, 30, 30, 40, 35 30 35 60 D Volume (mm3) 1.00 0.94 0.98 0.96 0.66 0.58c 0.63 0.47 Structural model index 2.63 2.66 2.64 2.83 0.18 0.15 0.17 0.12a D Centroid (mm) 0.060 0.076 0.065 0.052 0.047 0.052 0.049 0.057 D Diameter (mm) 0.026 0.023 0.025 0.013 0.020 0.018d 0.019 0.012b
Master apical le (MAF) values refer to the minimum, median or maximum nal instrument size used in any canal in that particular group. a Statistically different from Group 1 (P < 0.001), Group 2 (P = 0.003) and combined Group 1/2 (P < 0.001). b Statistically different from Group 1 (P = 0.007) and combined Group 1/2 (P < 0.001). c Mean 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). d Mean is less than Group 1 because the average instrument diameter is larger, making the difference between the preparation and instrument dimensions smaller.
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tural model index (SMI) was recorded to quantify the canal roundness (Lorensen & Cline 1987, Hildebrand & Ru egsegger 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 & Ru egsegger 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 & Ru egsegger (1997a), was calculated over the apical 1 mm of the preparation and compared to the theoretical average diameter of the master apical le (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-dimensional 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 experimental groups for a special outcome variable at P = 0.05. Where a statistical difference between groups was noted, pair-wise analysis was undertaken using Fishers LSD (least signicant difference) test. To determine the impact of any pre- or intraoperative variable (working length, canal angle, radius of curvature, presence of an s-shaped canal and MAF), the anova model was ret and pair-wise analysis using Fishers 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 instrumentation produced cross-sectional images that were examined for shape and the presence of any procedural
errors. The cross-sections were subsequently reconstructed and analysed to quantify the three-dimensional morphological changes in each canal. Generally, as is demonstrated in Fig. 1, rotary nickeltitanium 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 crosssectional appearance and taper, with deciencies 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 technique as Group 1, but with the nal canal shape rened using a rotary nickeltitanium 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 respectively; 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 nal 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 (D Volume) 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 23 comparison of D Diameter becoming statistically signicant (P = 0.036). When examining the effect of the canal angle, as it increased, the mean D Diameter of Groups 1 and 2 became signicant (P < 0.001). These results indicated that the two inuential factors on the results were the nal instrument size (MAF) and the canal angle. As either of those was increased, the difference between the instrumen-
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(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-les then rened with a rotary nickeltitanium instrument in the MB canal (nal preparation sizes: MB 30/0.04, DB 25/0.02, P 35/0.02); three-dimensional reconstruction of tooth presented in ab (c); preinstrumentation (d) and post-instrumentation (e) micro-CT slices of a tooth prepared using rotary nickeltitanium instruments (nal preparation sizes: MB 40/0.04, DB 40/0.04, P 45/0.04); three-dimensional reconstruction of tooth presented in de (f). Note the rounder, more centered preparations in canals prepared with rotary nickeltitanium instruments (e) and the excessive dentine removal from Gates Glidden preparation steps (c).
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 inuential. 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 nal 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 conclusions 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,
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Hu bscher 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, Hu bscher 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 Hu bscher et al. (2003) and postoperative SMI was smaller because of the more complex apical morphology. The results for transportation were similar to previous studies, though again, it is difcult to compare them because Peters et al. (2001a,b, 2003) and Hu bscher et al. (2003) measured changes in centroid over a larger range and Bergmans et al. (2002, 2003) measured movement in eight different directions, not stating an overall gure. A recent study (Cheung & Cheung 2008) has compared the nal canal dimensions to that of the nal instrument used and concluded similarly to this study, that the nal preparation taper and dimensions closely match those of the rotary nickeltitanium 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 ndings were not statistically signicant. Canals prepared by rotary nickeltitanium instruments (Group 3) were statistically rounder and the dimensions more closely matched that of the nal instrument used. This correlates well with the results showing less dentine removal (D Volume) and less transportation (D Centroid). Investigation into the effects of pre- and intraoperative 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, reecting the advantages of rotary nickeltitanium instruments over that of stainless steel in curved root canals. Clinically, this would equate to a more conservative and safer preparation despite the increased apical debridement. The results of this study agree with previous investigations into the advantages of rotary nickeltitanium 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 specic canal which may be less scientically valuable but more clinically relevant. The clinical implications of this study are that using a predominantly stainless steel hand preparation technique may not be as conservative of apical root canal dentine and that when used correctly, even by less experienced practitioners, rotary nickeltitanium 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 nickeltitanium instrumentation technique produces rounder canals (P < 0.001) whose dimensions more closely match those of the nal 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 nickeltitanium 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 Australian Society of Endodontology Incorporated. All endodontic 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, 3908.
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Bergmans L, Van Cleynenbreugel J, Beullens M, Wevers M, Van Meerbeek B, Lambrechts P (2002) Smooth exible versus active tapered shaft design using NiTi rotary instruments. International Endodontic Journal 35, 8208. 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. International Endodontic Journal 36, 28895. Bystro m A, Sundqvist G (1981) Bacteriologic evaluation of the efcacy of mechanical root canal instrumentation in endodontic therapy. Scandinavian Journal of Dental Research 89, 3218. Card SJ, Sigurdsson A, rstavik D, Trope M (2002) The effectiveness of increased apical enlargement in reducing intracanal bacteria. Journal of Endodontics 28, 77983. Cheung LHM, Cheung GSP (2008) Evaluation of a rotary instrumentation method for c-shaped canals with micro-computed tomography. Journal of Endodontics 34, 12338. Esposito PT, Cunningham CJ (1995) A comparison of canal preparation with nickeltitanium and stainless steel instruments. Journal of Endodontics 21, 1736. Glossen CR, Haller RH, Dove SB, del Rio CE (1995) A comparison of root canal preparations using NiTi hand, NiTi engine-driven, and K-Flex endodontic instruments. Journal of Endodontics 21, 14651. 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. 179227. Haapasalo M, Endal U, Zandi H, Coil JM (2005) Eradication of endodontic infection by instrumentation and irrigation solutions. Endodontic Topics 10, 77102. Hildebrand T, Ru egsegger P (1997a) A new method for the model-independent assessment of thickness in three-dimensional images. Journal of Microscopy 185, 6775. Hildebrand T, Ru egsegger P (1997b) Quantication of bone microarchitecture with the structural model index. Computer Methods in Biomechanics and Biomedical Engineering 1, 1523. Hu bscher W, Barbakow F, Peters OA (2003) Root-canal preparation with FlexMaster: canal shapes analysed by micro-computed tomography. International Endodontic Journal 36, 7407. 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, 3409. Kerekes K, Tronstad L (1977) Morphometric observations on the root canals of human molars. Journal of Endodontics 3, 1148. Khademi A, Yazdizadeh M, Feizianfard M (2006) Determination of the minimum instrumentation size for penetration of
irrigants to the apical third of root canal systems. Journal of Endodontics 32, 41720. Kuttler Y (1955) Microscopic investigation of root apexes. Journal of the American Dental Association 50, 54452. Lorensen WE, Cline HE (1987) Marching cubes: a high resolution 3D surface construction algorithm. Computer Graphics 21, 1639. hman AE, Heyden G (1981) n G, O Mo ller AJ, Fabricius L, Dahle Inuence on periradicular tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scandinavian Journal of Dental Research 89, 47584. Nair PRN, Sjo gren U, Krey G, Kahnberg KE, Sundqvist G (1990) Intraradicular bacteria and fungi in root-lled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study. Journal of Endodontics 16, 5808. Nielsen RB, Alyassin AM, Peters DD, Carnes DL, Lancaster J (1995) Microcomputed tomography: an advanced system for detailed endodontic research. Journal of Endodontics 21, 5618. Peters OA, Laib A, Ru egsegger P, Barbakow F (2000) Threedimensional analysis of root canal geometry by highresolution computed tomography. Journal of Dental Research 79, 14059. Peters OA, Laib A, Go hring TN, Barbakow F (2001a) Changes in root canal geometry after preparation assessed by highresolution computed tomography. Journal of Endodontics 27, 16. Peters OA, Scho nenberger K, Laib A (2001b) Effects of four NiTi preparation techniques on root canal geometry assessed by micro computed tomography. International Endodontic Journal 34, 22130. Peters OA, Peters CI, Scho nenberger K, Barbakow F (2003) ProTaper rotary root canal preparation: effects of canal anatomy on nal shape analysed by micro CT. International Endodontic Journal 36, 8692. Pruett JP, Clement DJ, Carnes DL Jr (1997) Cyclic fatigue testing of nickeltitanium endodontic instruments. Journal of Endodontics 23, 7785. Rhodes JS, Ford TR, Lynch JA, Liepins PJ, Curtis RV (2000) A comparison of two nickeltitanium instrumentation techniques in teeth using microcomputed tomography. International Endodontic Journal 33, 27985. Roane JB, Sabala CL, Duncanson MG (1985) The balanced force concept for instrumentation of curved canals. Journal of Endodontics 11, 20311. Rollison S, Barnett F, Stevens RH (2002) Efcacy 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, 36671. 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 Endodontics 31, 2837.
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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, 2715. Shuping GB, rstavik D, Sigurdsson A, Trope M (2000) Reduction of intracanal bacteria using nickeltitanium rotary instrumentation and various medications. Journal of Endodontics 26, 7515. Siqueira JF, Lima KC, Magalha es FA, Lopes HP, de Uzeda M (1999) Mechanical reduction of the bacterial population in the root canal by three instrumentation techniques. Journal of Endodontics 25, 3325. ngberg L (2001) The wonderful world of rotary root canal Spa preparation. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 92, 479. Sundqvist G (1976) Bacteriological studies of necrotic dental pulps , Sweden: Umea University. (Dissertation). Umea Tan BT, Messer HH (2002) The quality of apical canal preparation using hand and rotary instruments with specic
criteria for enlargement based on initial apical le size. Journal of Endodontics 28, 65864. Teknomo K (2006) Similarity measurement. [Website, updated 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, 9319. Walsch H (2004) The hybrid concept of nickeltitanium rotary instrumentation. Dental Clinics of North America 48, 183202. 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, 25562. Weine FS, Kelly RF, Bray KE (1976) Effect of preparation with endodontic handpieces on original canal shape. Journal of Endodontics 2, 298303.
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doi:10.1111/j.1365-2591.2009.01629.x
Distance from le tip to the major apical foramen in relation to the numeric meter reading on the display of three different electronic apex locators
R. A. Higa, C. G. Adorno, A. K. Ebrahim & H. Suda

Pulp 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 le tip to the major apical foramen in relation to the numeric meter reading on the display of three different electronic apex locators. International Endodontic Journal, 42, 10651070, 2009.
Aim To establish and compare the relationship between the distance from the le 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 xed with auto-polymerizing resin to size 15 K-les at AL and 0.5, 1, 2, 3, 4 mm short of AL. The data was analysed by two-way anova and Tukeys honestly signicant difference (HSD) test for multiple comparisons amongst EALs. Additionally, one-way anova and Tukeys HSD test were carried out for multiple comparisons amongst the measurements of each EAL.
Results There was a statistically signicant difference amongst all EALs in indicating the position of le tips in relation to the major foramen (P < 0.05). The correlation between the meter reading and the position of the le tip from the apical foramen was statistically signicant in the three EALs. There were signicant differences amongst the measurements at distances from 0 to 2 mm in Justy III. In Dentaport, signicant differences were found from 0 to 1 mm. However, the E-Magic Finder showed signicant differences from 0 to 0.5 mm. Conclusions Justy III was more capable of displaying the intracanal position of the le tip from the major foramen in mm whilst advancing through the root canal during electronic measurements than the Dentaport 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 obturation (Ricucci & Langeland 1998). The tooth pulp is
Leo n Torres Correspondence: Romina Andrea Higa, Fray Jose rdoba, CP 5001, Co rdoba, Argentina (Tel.: 54 1112, Alta Co 0351 4718387; fax: 54 0351 4718387; e-mail: romikawa@ hotmail.com).
narrow at the apical constriction; therefore the wound is minor, potentially providing optimal healing conditions (Kuttler 1955). The location of the apical constriction is considered to be 0.51 mm short of the anatomical apex (Kuttler 1955, Tselnik et al. 2005). Over-instrumentation and over-lling has been reported to cause tissue destruction, inammation 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
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Apex locators meter reading Higa et al.
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 impedance is calculated in order to determine a position of the le 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, Chungnam, 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 le 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, DAssunc a o 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 le 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.
facilitate length measurements. The actual root canal length (AL) was determined by introducing a size 10 or 15 K-le (Zipperer, Munich, Germany) into the canal until the tip of the le emerged through the major apical foramen under a digital microscope (VH-S30; Keyence, Osaka, Japan) at 20 magnication. The long axis of the tooth was placed perpendicular to the line of sight and the tip of the le 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 le tip and the rubber stop was measured with a digital caliper (Sankin; Mitutoyo Co, Kanagawa, Japan) to the nearest 0.5 mm. The measurements were repeated three times and the mean was taken as the denitive length. Gates Glidden drills (size 14, 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-le. The lid of a polystyrene specimen bottles (20 mL, Iuchi, Osaka, Japan) was used to x each tooth. The bottles were lled with alginate (GC Corporation, Tokyo, Japan) and, upon setting the root of the corresponding tooth was embedded in it, leaving

Extracted intact, straight, single-rooted human teeth with complete root formation were selected randomly. Teeth with resorption or fracture were excluded. Preoperative digital radiographic images in both buccolingual and mesiodistal directions were taken to evaluate root canal anatomy and teeth with accessory canals or invisible main canals were excluded. Twelve teeth were nally selected. All teeth were soaked in tap water for 2 h before use. Standard access preparation was carried out using a high speed diamond ssure bur (Mani, Tochigi, Japan) under water-cooling. The incisal or occlusal edges were ground to create a at surface to
Figure 1 Actual canal length determination. A size 15 K-le
was introduced into the canal until the tip of the le emerged through the major apical foramen. The tip of the le was positioned tangential to the major apical foramen.
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no signicant difference with 1, 3, and 4 mm respectively (P < 0.05). Two-way anova and Tukeys HSD test showed signicant differences amongst the three EALs (P < 0.05). The correlation between distance of the le tip from the major apical foramen and mean meter readings was statistically signicant (P < 0.001). The Pearson Correlation Coefcient was 0.88 for Justy III, 0.83 for Dentaport and 0.74 for E-Magic Finder Deluxe.
Discussion
Figure 2 Experimental set-up used in this study.
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-les were used with the EALs. Silicon stops were xed with autopolymerizing resin to the les at the following distances: AL and 0.5, 1, 2, 3, 4 mm short of the AL. A le 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 Tukeys HSD test were used to evaluate differences amongst EALs. One-way anova and the Tukeys HSD test were used to evaluate differences amongst the measurements of each EAL. In addition, the correlation between the le tip-apical foramen distance and electronic measurements, meter reading mean values, was analysed with the Pearson Correlation Coefcient. The analysis was carried out with JMP 7 software (SAS Institute, Cary, NC, USA).
Results
Table 1 illustrates one-way anova and Tukeys HSD test results. The mean and standard deviation of the meter readings with three EALs at different distances of the le tip from the apex are shown. The indicated mean meter reading of Justy III were signicantly different except at 3 and 4 mm (P < 0.05). For the Dentaport, the mean readings at 0, 0.5 and 1 mm were signicantly different (P < 0.05). The mean reading of the E-Magic Finder Deluxe at 0.5, 2, and 3 mm showed
Many studies have reported the accuracy of EALs to determine root canal length (Hoer & Attin 2004, Lucena-Martin et al. 2004, ElAyouti & Lo st 2006, Plotino et al. 2006, Smadi 2006, Bernardes et al. 2007, DAssunc a o et al. 2007, Wrbas et al. 2007). In addition, 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 le 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 measurements. The purpose of this study was to evaluate the capability of EALs to determine the distance in mm from the apical foramen whilst the le 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 electroconductive properties, ease of preparation, stability and rm consistency (Baldi et al. 2007, Herrera et al. 2007). The actual canal length was determined before the aring with Gates Glidden drills. Owing to the fact that aring with Gates Glidden les might alter the root canal length, measurements were performed before and after aring 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 preparation 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 experiment. Previous studies reported that NaOCl irrigation
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Intracanal position of le tip from major apical foramen (mm) Meter readings 0 0.5 1 2 3 4
Justy III
Dentaport
E-Magic Finder
Table 1 Mean SD of meter readings at different intracanal positions of le tip from the major apical foramen
0.08 0.72 1.60 3.32 3.86 4.12
0.12 0.27 0.61 0.93 0.86 0.94
A B C D E E
+0.09 0.90 2.13 2.93 2.93 3.01
0.33 0.49 0.53 0.13 0.21 0.04
A B C D D D
0.13 0.59 0.85 1.50 1.93 2.24
0.33 0.13 0.38 0.75 0.96 1.02
A B B C CD D
Different alphabet letters (A, B, C, D and E) indicate statistically signicant differences (P < 0.05) amongst measurements within each EAL. EAL, electronic apex locator; +, measurements are beyond the apical foramen.
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 le 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 le tip and the apical foramen was 4 mm. According to previous reports, the accuracy of measurements increases as the le tip approaches the foramen (Kobayashi & Suda 1994, Venturi & Breschi 2007). ElAyouti & Lo st (2006) suggested that accuracy and repeatability should be considered in the evaluation of EALs. The Dentaport provided the most stable electronic measurements when considering the mean standard deviation (SD) of the meter readings of the distance of the le 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 acceptable 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 le tip was at the major apical foramen, some of the measurements by the three EALs were positive as the
le tip was beyond the major foramen. According to Wrbas et al. (2007) and DAssunc a o 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 clinicians, the numeric meter reading values of EALs could become a useful guide if they indicate the le 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 le was based on correlation analysis, and significant differences were found amongst the mean numeric readings within each EAL. The Pearson Correlation Coefcient indicated that the three EALs revealed a statistically signicant correlation between the numeric meter reading and the distance of the le 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 signicantly different at 0, 0.5, 1 and 2 mm. The Dentaport readings were signicantly 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 le tip position whilst the le 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 signicantly 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 le is within 2 mm from the apical foramen whilst the
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Dentaport shows correlation when the le is within 1 mm. These results disagree with the claims made in Justy III and E-Magic Finders 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 le 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 le was within 2 mm from the apical foramen whilst in the Dentaport when the le was within 1 mm. The readings 0.0 or apex and 0.5 showing the intracanal position of the le 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 le 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 Endodontic Journal 37, 1747. Baldi JV, Victoriano FR, Bernardes RA et al. (2007) Inuence of embedding media on the assessment of electronic apex locators. Journal of Endodontics 33, 4769. 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, e914. Czerw RJ, Fulkerson MS, Donnelly JC, Walmann JO (1995) In vitro evaluation of the accuracy of several electronic apex locators. Journal of Endodontics 21, 5725. DAssunc a o 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, e503. Ebrahim AK, Yoshioka T, Kobayashi C, Suda H (2006) The effects of le 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, 1537. ElAyouti A, Lo st 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. Journal of Endodontics 26, 3647. Fouad AF, Krell KV, McKendry DJ, Koorbusch GF, Olson RA (1989) A clinical evaluation of ve electronic root-canal length measuring instruments. Journal of Endodontics 16, 4469. Gordon MPJ, Chandler NP (2004) Electronic apex locators. International Endodontic Journal 37, 42537. Herrera M, Abalos C, Jimenez Planas A, Llamas R (2007) Inuence of apical constriction diameter on Root ZX apex locator precision. Journal of Endodontics 33, 9957. Hoer D, Attin T (2004) The accuracy of electronic working length determination. International Endodontic Journal 37, 12531. Kobayashi C, Suda H (1994) New electronic canal measuring device based on the ratio method. Journal of Endodontics 20, 1114. Kuttler Y (1955) Microscopic investigation of root apexes. Journal of the American Dental Association 50, 54452. 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, 2313. 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, 3716. Meares WA, Steiman HR (2002) The inuence of sodium hypochlorite irrigation on the accuracy of the Root ZX electronic apex locator. Journal of Endodontics 28, 5958. Oishi A, Yoshioka T, Kobayashi C, Suda H (2000) Location of the le tip and the meter value of apex locators. The Japanese Journal of Conservative Dentistry 6, 114553. Pilot TF, Pitts DL (1997) Determination of impedances changes at varying frequencies in relation to root canal le 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, 40814. Pratten DH, McDonald NJ (1996) Comparison of radiographic and electronic working lengths. Journal of Endodontics 22, 1736.
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Ricucci D, Langeland K (1998) Apical limit of root canal instrumentation and obturation. Part 2. A histological study. International Endodontic Journal 31, 394409. 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, 53446. Seltzer S, Soltanoff W, Sinai I, Smith J (1969) Biologic aspects of endodontics. Part IV. Periapical tissue reactions to rootlled teeth whose canals had been instrumented short of their apices. Oral Surgery, Oral Medicine and Oral Pathology 28, 72438. Smadi L (2006) Comparison between two methods of working length determination and its effect on radiographic extent of root canal lling: 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 Endodontics 31, 5079. Vajrabhaya L, Tepmongkol P (1997) Accuracy of apex locator. Endodontics and Dental Traumatology 13, 1802. Venturi M, Breschi L (2005) A comparison between two electonic apex locators: an in vivo investigation. International Endodontic Journal 38, 3645. Venturi M, Breschi L (2007) A comparison between two electonic apex locators: an ex vivo investigation. International Endodontic Journal 40, 36273. 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, 1338.
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doi:10.1111/j.1365-2591.2009.01632.x
Micro-computed tomography of tooth tissue volume changes following endodontic procedures and post space preparation
O. H. Ikram, S. Patel, S. Sauro & F. Mannocci

Department of Conservative Dentistry, Kings College London Dental Institute, London, UK
Abstract
Ikram OH, Patel S, Sauro S, Mannocci F. Micro-computed
tomography of tooth tissue volume changes following endodontic procedures and post space preparation. International Endodontic Journal, 42, 10711076, 2009.
Aim To compare the volume of hard tooth tissue lost after caries removal, access cavity preparation, root canal preparation, bre 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 procedure the loss of hard tooth tissue volume was measured. The data were statistically analysed using one-way analysis of variance and Fishers PLSD test with statistical signicance 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 preparation, bre 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 signicantly 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 preparation 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 bre post space preparation. This should be taken into account when planning root canal treatment and restoration of root lled teeth that are to be restored with cuspal coverage restorations. Keywords: cast posts, dentine, bre posts.
Received 25 February 2009; accepted 5 August 2009
Introduction
Root lled 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 lled teeth. First, the physical properties of the
Correspondence: Francesco Mannocci, Department of Conservative Dentistry, Kings College London Dental Institute, Guys Tower, Guys Hospital, St Thomas Street, London SE1 9RT, UK (Tel.: +447515398390; fax +442071881583; e-mail: francesco.mannocci@kcl.ac.uk).
dentine may be altered by the interaction of medicaments 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 exure ex vivo (Reeh et al. 1989). To assess how the loss of tooth tissue caused by restorative procedures and root canal treatment may
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Micro-CT investigation Ikram et al.
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 restoration. 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 lled 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 classied 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, bre 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.
magnication 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, bre 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 8782800 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 1003223 Henry Schein Gillingham, UK). Caries removal was veried using an explorer (04108 Dentsply; 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, bre post and cast post space preparation.

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 lter were used to reduce beamhardening artefacts and scattering. The geometrical
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
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Ikram et al. Micro-CT investigation
endodontic probe (DG-16 Endodontic Explorer, Ash UK) could be placed in the pulp chamber and the canals were visible to the naked eye.
Fibre post preparation

Post spaces were prepared using the Fibre White post ` ne/Whaledent, NJ, USA). For this the blue post kit (Colte 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.
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-le. The le 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 le with hand instruments to the working length and irrigated with sodium hypochlorite after each le. Recapitulation was performed with a size 10 K-le between instruments. ProTaper rotary instruments (Maillefer Dentsply, Baillagues, Switzerland) were then used to prepare the root canals. The Shaper 1 and Shaper 2 ProTaper les were used to the working length and the Finisher 1 and Finisher 2 les were used 1 mm short of the working length. During the root canal preparation a brushing technique was used on the outward stroke to permit three-dimensional coronal aring of the canal. As before, between each le recapitulation with a size 10 le was performed and a 1% sodium hypochlorite solution was used to irrigate the canals.
Cast post preparation

The preparations for the bre posts were than modied ` ne/Whaledent) into preparations for Parapost (Colte 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 variance and Fishers PLSD test with statistical signicance 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, bre 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 % volume lost 0 0 After caries removal Volume 428.151 388.957 % volume lost 7.3 7 After access cavity Volume 393.115 346.75 % volume lost 15.1 17.1 After root-canal preparation Volume 389.969 340.961 % volume lost 15.8 18.4 After bre post preparation Volume 389.2 339.98 % volume lost 15.6 18.7 After cast post preparation Volume 381.335 321.922 % volume lost 17.5 22.3
310.667 447.408 469.022 465.954 450.965 264.278 411.56 0 0 0 0 0 0 0
502.265 378.143 512.933 0 0 0
278.737 428.632 436.094 444.945 372.911 210.327 395.394 479.587 333.203 507.538 10.3 4.2 7.2 4.5 17.3 20.4 3.9 4.5 11.9 1.2 272.105 416.72 12.2 6.9 413.415 434.462 352.32 12 6.6 21.7 199.931 371.932 470.873 313.601 499.089 24.6 9.7 6.3 17.1 2.5 371.646 469.189 308.237 494.14 9.7 6.5 18.5 3.5 361.78 13.1 457.67 9 307.81 18.5 482.81 5.9
265.301 409.991 398.337 433.615 351.024 197.07 14.5 8.5 15.1 6.9 22 25.3 264.78 15.9 408.65 8.7 395.24 15.7 425.95 9.6 344.256 194.52 23.5 26.5
262.088 386.012 367.562 411.028 295.97 15.4 13.6 21.7 11.6 34.4
188.718 325.413 429.6 28.7 20.9 14.5
295.291 471.026 21.9 8
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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 preparation, root canal preparation, bre 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 signicantly 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 threeTable 2 Mean percentages* and standard deviations of hard tissue volume loss after caries removal, access cavity preparation, root canal preparation bre post and cast post space preparation
Initial After After After After After caries removal access cavity root canal preparation bre post preparation cast post preparation 0% 8.3 12.7 13.7 15.1 19.2 5.83a 6.7b 6.7b 6.3c 7.4d % increase 8.3 4.4 1 1.4 4.1
*Groups with the same letter showed no statistically signicant difference (P < 0.01).
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 conducted 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 conrms 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 longterm prognosis. The preparation of the access cavity caused the second largest loss of tooth structure ($4.4%), followed by cast post (4.1%) and bre post (1.4%) space preparation. The only procedure that did not result in a signicant 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 preparation (d), after bre post preparation (e), and after cast post preparation (f).
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Ikram et al. Micro-CT investigation
(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 bre post preparation (e), and after cast post preparation (f).
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 nite 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 signicant loss of fracture resistance. However, this is relatively insignicant 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 preparation, they found a signicant 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 signicant loss of hard tooth tissue structure. The loss of tooth structure caused by bre and cast post space preparation observed in this study bears perhaps, the most relevant clinical implication.
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A recent systematic review of the literature (Bolla et al. 2007) found only one randomized clinical trial comparing bre and cast posts (Ferrari et al. 2000) providing evidence of a longer survival for bre 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 lled premolars restored with crowns, it was shown that the cementation of a bre 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 modication of a preparation from a bre 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 bre post/composite restorations of root lled premolars with three remaining coronal walls which are to be subsequently restored with cuspal coverage restorations.
The ndings of this paper were partially presented at the AAE (American Association of Endodontists) meeting 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 lled teeth. Cochrane Database of Systematic Reviews 1, CD004623. Cagidiaco MC, Garc a-Godoy F, Vichi A, Grandini S, Goracci C, Ferrari M (2008) Placement of ber prefabricated or custom made posts affects the 3-year survival of endodontically treated premolars. American Journal of Dentistry 21, 17984. Ferrari M, Vichi A, Garcia-Godoy F (2000) Clinical evaluation of ber reinforced epoxy resin posts and cast post and cores. American Journal of Dentistry 13, (Spec No): 15B8B. 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, 72934. Grigoratos D, Knowles J, Ng YL, Gulabivala K (2001) Effect of exposing dentine to sodium hypochlorite and calcium hydroxide on its exural strength and elastic modulus. International Endodontic Journal 34, 1139. Lang H, Korkmaz Y, Schneider K, Raab WHM (2006) Impact of endodontic treatments on the rigidity of the root. Journal of Dental Research 85, 3648. 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, 70915. 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, 297301. 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, 14059. Peters OA, Peters CI, Schonberger K, Barbakow F (2003) Protaper rotary root canal preparation: effects of canal anatomy on nal shape analysed by micro CT. International Endodontic Journal 36, 8692. Randow K, Glantz PO (1986) On cantilever loading of vital and non vital teeth. An experimental clinical study. Acta Odontologica Scandinavia 44, 2717. Reeh ES, Messer HH, Douglas WH (1989) Reduction in tooth stiffness as a result of endodontic and restorative procedures. Journal of Endodontics 15, 5126.
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 preparation of a bre post of the same size. This needs to be taken into account in planning root canal treatments and restorations of root lled teeth that are to be restored with cuspal coverage restorations.
Acknowledgements
The authors wish to thank Chris Healy (Department of Craniofacial Development, Kings College London Dental Institute, London, UK) for his technical support The authors acknowledge support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guys & St Thomas NHS Foundation Trust in partnership with Kings College London and Kings College Hospital NHS Foundation Trust.
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doi:10.1111/j.1365-2591.2009.01634.x
Laser-activated irrigation within root canals: cleaning efcacy and ow visualization
S. D. de Groot1*, B. Verhaagen2,3*, M. Versluis2,3, M.-K. Wu1, P. R. Wesselink1 & L. W. M. van der Sluis1
Department 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
1
Abstract
de Groot SD, Verhaagen B, Versluis M, Wu M.-K, Wesselink PR, van der Sluis LWM. Laser-activated irrigation within root canals: cleaning efcacy and ow visualization. International Endodontic Journal, 42, 10771083, 2009.
Aim To test ex vivo the efciency of laser-activated irrigation in removing dentine debris from the apical part of the root canal and to visualize in vitro the uid 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 lled with dentine debris were irrigated with syringe irrigation, ultrasonically or laser-activated irrigation (LAI) using 2% sodium hypochlorite as irrigant. The quantity of dentine debris after
irrigation was determined. Visualization of the uid dynamics during activation was achieved using a highspeed camera and a glass model. Results Laser-activated irrigation was signicantly 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, AbouRass & Patonai 1982, Druttman & Stock 1989) and in isthmuses or oval extensions (Lee et al. 2004, Burleson et al. 2007). Therefore, acoustic and hydrodynamic activation of the irrigant have been developed (Weller et al. 1980, Lumley et al. 1991, Lussi
Correspondence: Bram Verhaagen, MSc, University of Twente, Meander 213, PO Box 217, 7500 AE Enschede, The Netherlands (Tel.: +31 53 489 3084; fax: +31 53 489 8068; e-mail: b.verhaagen@tnw.utwente.nl). *These two authors should both be listed as primary author, as both contributed equally to this study.
et al. 1993), which have been shown to contribute to the cleaning efciency (Lumley et al. 1991, Lussi et al. 1993, Roy et al. 1994). The physical mechanisms underlying these cleaning procedures, however, 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 produces 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
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Laser-activated irrigation within root canals de Groot et al.
their visualization experiment with a single highspeed 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 articially placed dentine debris in standardized 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 ow is theoretically described using a uiddynamical model.
Materials and methods Dentine debris removal

Maxillary canines with straight root canals were decoronated; the length of the remaining root was 15 mm for all teeth. The roots were then embedded in self-curing acrylic resin (Ostron 100, GC Tokyo, Japan) and then split longitudinally through the canal in mesio-distal direction. To remove the imprint of the
root canal, both halves were ground with sandpaper and xed with four screws (see Fig. 1a). Then, the root canals were prepared by K-les 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).
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de Groot et al. Laser-activated irrigation within root canals
groove of 4 mm in length, 0.5 mm deep and 0.2 mm wide, situated at 26 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 rignac-cedex, France). The elec, Acteongroup, Me dimension of the groove was comparable with that of an oval extension of a root canal. Each groove was lled with dentine debris mixed with 2% NaOCl to simulate a situation in which dentine debris accumulates 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 procedure, in order to increase the reliability of dentine debris removal evaluation. The methodology is sensitive and the data are reproducible (van der Sluis et al. 2007b). Three irrigation protocols were tested. In all groups, the needle, wire and bre 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, Acteongroup) was used, driven by an ultrasonic device (Suprasson Pmax Newtron, Satelec, Acteongroup) at power setting blue 4 (frequency 30 KHz, displacement amplitude ca. 30 lm according to the manufacturer). Subsequently the canal was ushed 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 bre 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 bre has a reduction factor of 0.36, which results in a uence 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 ushed 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 magnication. 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 lled with dentine debris; score 2: more than half of the groove is lled with dentine debris; score 3: the groove is completely lled with dentine debris. The differences in dentine debris scores between the different groups were analysed by means of the KruskalWallis and MannWhitney tests (level of signicance 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 articial root canal containing water or NaOCl. Optical recordings 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 bre 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 APXRS, Photron, Tokyo, Japan), recording at a frame rate of 14 000 frames per second, attached to a microscope with 12 magnication (SZX12, Olympus, Tokyo, Japan). The root canal model was illuminated in bright-eld 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 signicant (KruskalWallis test, P < 0.0001). The debris score in group 3 was significantly lower than group 2 (P = 0.002) and group 1 (P < 0.0001), and the score in group 2 was signicantly lower than group 1 (P < 0.0001).
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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: Syringe irrigation Ultrasonic irrigation Laser-activated irrigation 0 n (%) 0 6 (30%) 16 (80%) 1 n (%) 0 8 (40%) 4 (20%) 2 n (%) 4 (20%) 6 (30%) 0 3 n (%) 16 (80%) 0 0
Scoring system: 0: the groove is empty; 1: less than half of the groove is lled with debris; 2: more than half of the groove is lled with debris; 3: the complete groove is lled with debris.
High-speed imaging experiments

The high-speed recordings of the laser activity inside the articial (glass) canal showed that irrigant was vapourized by the laser pulse energy and that a large vapour bubble was created at the bre 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
(a)
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 connement at the apex. The depth reached by this bubble depended on the position of the bre 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
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 ber tip (outer diameter 280 lm), 3) the laser-induced cavitation bubble, and 4) a stable cavitation bubble at the apex.
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(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 ber tip. The frame rate is 14,000 frames/second.
collapsed and renucleated in anti-phase with the laserinduced 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 compared 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 laserinduced bubble the uid 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 uid 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 articial groove in the apical part of the root canal than ultrasonically activated (PUI) or syringe-activated irrigation. The high-speed recordings have shown that vapourization 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 uid ow associated with such an inertial collapse, combined with acoustic streaming resulting from the oscillations of smaller bubbles, could explain the cleaning efcacy 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 ow 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 ow in the root canal due to the collapsing laser-induced bubble can be modelled by a ow in concentric annuli for heights above the insertion depth of the bre. For the typical ow velocity of 1 m s)1 (value obtained from the high-speed recordings by measuring the bubble wall displacement between consecutive frames), the Reynolds number Re = Udq/l (with U the ow velocity, d the distance between the cylinders, q the density of the liquid and l the dynamic viscosity) for a ow 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 ow in this problem is treated as laminar ow. Rothfus et al. (1950) also give the laminar ow velocity distribution for ow in concentric annuli:
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2
ln
3
7 m s1 6 5
r2 r2 6 2 7 4r1 r2 2 r2 1 ln rr1 5 ur 2uav

2 r2
450 N m2 400 350 300
2 r1
r1 2 2rm
where rm is the radius of maximum velocity, given by: " #1 2 r 2 r2 rm 2 r21 2 2 ln r1

u Using s l @ @ r the shear stress for laminar ow in annuli is given by: 2 2 r2 r1 r2 r 2r ln r1 sr 2luav 2 3 2 ln r2 r 2 r2 r2 r1 2 1 r1
4 3 2 1 0
250 200 150 100 50 0
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 biolms to dentine or its failure shear stress is available in the literature. Figure 4 shows the velocity prole 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 bre tip (taken from experiment). The prole on the left of the inner cylinder is the velocity prole; the prole on the right is the shear stress distribution. The plot clearly shows that on the inner cylinder (the laser bre) 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 bre. LAI is therefore expected to be most effective in the region close to the bre tip, with decreasing efciency away from the tip. Using a 27G needle and a volume ow rate of 0.30 mL s)1 (Boutsioukis et al. 2007) it follows that the typical uid velocity in syringe irrigation is of the order of 1 m s)1 at the needle orice, which is the same order of magnitude as the ow velocities developed with LAI. Likewise for PUI with u = xe02/a (Ahmad et al. 1988; x = oscillation frequency, e0 = oscillation
Figure 4 Average velocity prole (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 ber tip is set at 5 m s)1. The region below the laser ber tip is intentionally left blank, as details of the streaming pattern in the apical part are missing and are part of a future study.
amplitude and a = le radius) a typical uid velocity of the order of 1 m s)1 was found. One possible explanation for the improvement in cleaning efcacy with LAI is the impulsive nature of the laser-generated bubble dynamics. Because of the pulsations the uid 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 efcient 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 36 C. The current study did not monitor these sideeffects, because the aim of this study was clarication of the uid mechanical working mechanisms.
Conclusion
Laser-activated irrigation was more effective in removing the articially placed dentine debris from the root canal than syringe irrigation or PUI when the irrigant was activated for 20 s.
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Acknowledgements
We thank Gert-Wim Bruggert for technical support and Gerrit J. de Bruin for valuable discussions on the theoretical approach of the uid ow in tapered canals.
References
Abou-Rass M, Patonai FJ (1982) The effects of decreasing surface tension on the ow of irrigating solutions in narrow root canals. Oral Surgery, Oral Medicine, Oral Pathology 53, 5246. Ahmad M, Pitt Ford TR, Crum LA, Walton AJ (1988) Ultrasonic debridement of root canals: acoustic streaming and its relevance. Journal of Endodontics 14, 48693. 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 ow rates during irrigation of a root canal ex vivo with three endodontic needles. International Endodontic Journal 40, 50413. 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, 7827. Dijkink R, Ohl C-D (2008) Measurement of cavitation induced wall shear stress. Applied Physics Letters 93, 25410713. Druttman AC, Stock CJ (1989) An in vitro comparison of ultrasonic and conventional methods of irrigant replacement. International Endodontic Journal 22, 1748. George R, Walsh LJ (2008) Apical extrusion of root canal irrigants when using Er:YAG and Er,Cr:YSGG lasers with optical bers: an in vitro dye study. Journal of Endodontics 34, 7068. George R, Meyers IA, Walsh LJ (2008) Laser activation of endodontic irrigants with improved conical laser ber tips for removing smear layer in the apical third of the root canal. Journal of Endodontics 34, 15247. Holzfuss J, Ru ggeberg M, Billo A (1998) Shock wave emissions of a sonoluminescing bubble. Physical Review Letters 81, 54347. 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 Endodontics 28, 768. Lauterborn W (1972) High-speed photography of laserinduced breakdown in liquids. Applied Physics 21, 279. Lee S-J, Wu M-K, Wesselink PR (2004) The efcacy of ultrasonic irrigation to remove articially placed dentin debris from different sized simulated plastic root canals. International Endododontic Journal 37, 60712.
Lumley PJ, Walmsley AD, Laird WRE (1991) Streaming patterns around endosonic les. International Endodontic Journal 24, 2907. Lussi A, Nussba cher U, Grosrey J (1993) A novel noninstrumented technique for cleansing the root canal system. Journal of Endodontics 19, 54953. Matsuoka E, Jayawardena JA, Matsumoto K (2005) A morphological study on root canal preparation using Erbium, Chromium:YSGG laser. Journal of Oral Laser Applications 5, 1721. Ram Z (1977) Effectiviness of root canal irrigation. Oral Surgery, Oral Medicine, Oral Pathology 44, 30611. Robertson CW, Williams D (1971) Lambert absorption coefcients of water in the infrared. Journal of the Optical Society of America 61, 131620. Rothfus RR, Monrad CC, Senecal VE (1950) Velocity distribution and uid friction in smooth concentric annuli. Industrial & Engineering Chemistry 42, 251120. Roy RA, Ahmad M, Crum LA (1994) Physical mechanisms governing the hydrodynamic response of an oscillating ultrasonic le. International Endodontic Journal 27, 197207. Salzgeber RM, Brilliant JD (1977) An in vitro evaluation of the penetration of an irrigating solution in root canals. Journal of Endodontics 3, 3948. 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, 41526. van der Sluis LWM, Wu MK, Wesselink PR (2007b) The evaluation of removal of Ca(OH)2 from an articial standardized groove in the apical root canal using different irrigation methodologies. Internaional Endodontic Journal 40, 527. Weller RN, Brady JN, Bernier WE (1980) Efcacy of ultrasonic cleaning. Journal of Endodontics 6, 7403.
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 information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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Sealing properties of a new root canal sealer
U. Salz, D. Poppe, S. Sbicego & J.-F. Roulet

Ivoclar 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, 10841089, 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 lled 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 lled canal. Positive controls were lled only with Gutta-percha and tested with bacteria, whereas negative controls were
sealed with wax to test the seal between chambers. Additionally, lm 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 signicant 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 lm 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 lling 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 ll 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, lm thickness and dimensional change
Correspondence: Ulrich Salz, Ivoclar Vivadent AG, Bendererstr. 2, FL-9494 Schaan, Liechtenstein (Tel.: +423/235 34 21; fax: +423/233 12 79; e-mail: ulrich.salz@ivoclarvivadent. com).
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 endodontic materials. However, the frequent approach of sectioning teeth vertically or horizontally to determine tracer depth is not reproducible, and rarely yields statistically signicant differences between materials (Schuurs et al. 1993, Wu & Wesselink 1993, Camps & Pashley 2003). A quantitative measurement of penetrated 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), uid ltration measurements (Wu et al. 1993, Pommel et al. 2003) and movement of glucose solution under low hydrostatic pressure (Xu et al. 2005).
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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 effectiveness of AH Plus and Apexit Plus to resist bacterial leakage.
The root canals were irrigated with 1.25% NaOCl during instrumentation and then sterilized by autoclaving 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 lled 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 nger spreader (Dentsply Maillefer) was then inserted into the canal to a level about 1 mm short of working length. Lateral condensation with ne accessory 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 orice of canal. All root canal treatment and lling 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 twochamber 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 rootend (see Fig. 1). The tooth was inserted into the tube and gently pushed through the opening until approximately 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 atbottomed 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
Materials and methods Tests according to ISO 6876

Sample preparation and measurement of solubility, lm 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 1116 mm were equally distributed to the groups. Each root canal was coronally enlarged with Largo Peeso Reamers (Dentsply Maillefer, Ballaigues, Switzerland)) to size 90 or 110 to obtain standardized round root canal shapes. Hand K-les were also used to nish 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.
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Sealing properties of new sealer Salz et al.
Results
Results of measurements of solubility, lm thickness and dimensional change following setting are summarized in Table 1. AH Plus had a slightly lower solubility (0.3% solubility) than Apexit Plus (0.5% solubility). The lm 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 observation time of 30 days. The KaplanMeier survival probabilities for the experimental groups are shown in Fig. 2. Signicant 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 signicant difference was observed (P < 0.0005, chisquared test). The mean time for the bacteria to penetrate the root canal in the AH Plus group was 5.3 days (CI: 3.77.0 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.
S. mutans
Sticky wax Gutta-percha Root canal sealer
Sterile broth
Contaminated broth
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.
lower chamber. The upper chamber was lled 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 lling 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.
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 immersion 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 ` ne/Whaledent, 0.5%) (Schafer & ZanRoekoSeal (Colte 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%)
Table 1 Solubility after 24 h, lm thickness and dimensional change following setting of Apexit Plus and AH Plus according to ISO 6876:2001
The KaplanMeier 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 nonparametric log-rank test was used to compare the survival curves using a signicance level of 0.05. The results at the end of the observation time (30 days) was further analysed by chi-square testing.
ISO 6876 limits Solubility/% Film thickness/lm Dimensional change following settinga/%
a
AH Plus 0.3 28 0.2 (expansion)
Apexit Plus 0.5 11 0.4 (expansion)
3.0 50 0.1 (expansion) 1 (shrinkage)
NIOM; test-report T051/03.
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Figure 2 KaplanMeier survival curves of the sealed root
canals for the two different sealers in the bacterial leakage test (NIOM; test-report T074/04).
(rstavik 1983) fail to full 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 signicant 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 lm thickness describes the ability of the material to adapt to the geometry of the canal wall. The lower the lm thickness, the better is the adaptation of the material. ISO 6876 requires a maximum of 50 lm. The lm thickness of Apexit Plus was 11 lm and of AH Plus 28 lm. AH 26 (36 lm) and Sealapex (36 lm) exhibit a higher lm thickness; however, they do full 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 (rstavik et al. 2001). Therefore, the ISO requirement 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 modulus 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 modulus, at least for Apexit Plus a risk of root fracture caused by expansion can be neglected. rstavik 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. Therefore, 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 llings (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 lling was not determined because the purpose of the experiment was only to test if the root lling 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 ndings in other studies investigating coronal bacterial leakage in teeth obturated employing cold lateral condensation techniques (Yucel et al. 2006, Eldeniz & rstavik 2007). However, in studies with alternative microbial markers, different mean breakthrough-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 lm 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
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Sealing properties of new sealer Salz et al.
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-incubation period of 14 days. At this time-point no antimicrobial 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 lling materials. Endodontics & Dental Traumatology 10, 24955. Baumgartner JC, Falkler WA Jr (1991) Bacteria in the apical 5 mm of infected root canals. Journal of Endodontics 17, 3803. Camps J, Pashley D (2003) Reliability of the dye penetration studies. Journal of Endodontics 29, 5924. 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, rstavik 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 & Endodontics 102, 11926. 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, 718. Fransen JN, He J, Glickman GN, Rios A, Shulman JD, Honeyman A (2008) Comparative assessment of ActiV GP/glass ionomer sealer, resilon/epiphany, and guttapercha/AH plus obturation: a bacterial leakage study. Journal of Endodontics 34, 7257. Fuss Z, Weiss EI, Shalhay M (1997) Antibacterial activity of calcium hydroxide-containing endodontic sealers on Enterococcus faecalis in vitro. International Endodontic Journal 30, 397402. Heling I, Chandler NP (1996) The antimicrobial effect within dentinal tubules of four root canal sealers. Journal of Endodontics 22, 2579.
Kayaoglu G, Erten H, Alacam T, rstavik D (2005) Short-term antibacterial activity of root canal sealers towards Enterococcus faecalis. International Endodontic Journal 38, 4838. Kersten HW, Moorer WR (1989) Particles and molecules in endodontic leakage. International Endodontic Journal 22, 11824. Mandras RS, Retief DH, Russell CM (1993) Quantitative microleakage of six dentin bonding systems. American Journal of Dentistry 6, 11922. 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, 42832. Monticelli F, Sadek FT, Schuster GS et al. (2007) Efcacy of two contemporary single-cone lling techniques in preventing bacterial leakage. Journal of Endodontics 33, 3103. Oguntebi BR, Shen C (1992) Effect of different sealers on thermoplasticized gutta-percha root canal obturations. Journal of Endodontics 18, 3636. rstavik D (1983) Weight loss of endodontic sealers, cements and pastes in water. Scandinavian Journal of Dental Research 91, 3169. rstavik D, Nordahl I, Tibballs JE (2001) Dimensional change following setting of root canal sealer materials. Dental Materials 17, 5129. F, Sirtes G (2007) Apical sealing ability of resilon/ Paque epiphany versus gutta-percha/AH Plus: immediate and 16months leakage. International Endodontic Journal 40, 7229. 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, rstavik D (2008) Bacterial penetration along different root canal lling materials in the presence or absence of smear layer. International Endodontic Journal 41, 3240. Schafer E, Zandbiglari T (2003) Solubility of root-canal sealers in water and articial saliva. International Endodontic Journal 36, 6609. 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, rstavik D, Teixeira FB, Trope M (2004) An evaluation of microbial leakage in roots lled with a thermoplastic synthetic polymer-based root canal lling material (Resilon). Journal of Endodontics 30, 3427. Skinner RL, Himel VT (1987) The sealing ability of injectionmolded thermoplasticized gutta-percha with and without the use of sealers. Journal of Endodontics 13, 3157. Slutzky-Goldberg I, Slutzky H, Solomonov M, Moshonov J, Weiss EI, Matalon S (2008) Antibacterial properties of four endodontic sealers. Journal of Endodontics 34, 7358. Timpawat S, Amornchat C, Trisuwan WR (2001) Bacterial coronal leakage after obturation with three root canal sealers. Journal of Endodontics 27, 369.
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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, 46471. Weinberger SJ, Wright GZ (1989) Correlating Streptococcus mutans with dental caries in young children using a clinically applicable microbiological method. Caries Research 23, 3858. 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, 3547.
Wu MK, Wesselink PR (1993) Endodontic leakage studies reconsidered. Part I. Methodology, application and relevance. International Endodontic Journal 26, 3743. Wu MK, De Gee AJ, Wesselink PR, Moorer WR (1993) Fluid transport and bacterial penetration along root canal llings. International Endodontic Journal 26, 2038. Xu Q, Fan MW, Fan B, Cheung GS, Hu HL (2005) A new quantitative method using glucose for analysis of endodontic leakage. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology & Endodontics 99, 10711. Yucel AC, Guler E, Guler AU, Ertas E (2006) Bacterial penetration after obturation with four different root canal sealers. Journal of Endodontics 32, 8903.
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doi:10.1111/j.1365-2591.2009.01636.x
A tactile method for canal length determination in teeth with open apices
A. ElAyouti, E. Dima & C. Lo st

Department of Conservative Dentistry and Endodontology, University of Tu bingen, Tu bingen, Germany
Abstract
ElAyouti A, Dima E, Lo st C. A tactile method for canal length
determination in teeth with open apices. International Endodontic Journal, 42, 10901095, 2009.
Aim To present a tactile method for working length determination in teeth with open apices and to determine 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 le 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-le 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% condence
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: 9199) of the TWL were within 0.5 mm from the CWL, the mean of absolute differences was 0.1 mm (CI: 0.10.2). 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.
Correspondence: Ashraf ElAyouti, Department of Conservative Dentistry and Endodontology, University of Tu bingen. Osianderstrae 28, 72076 Tu bingen, Germany (Tel.: 0049 7071 29 83498; fax: 0049 7071 29 5656; e-mail: ashraf.elayouti @med.uni-tuebingen.de).
There are many problems associated with the treatment 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 lling 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 overinstrumentation and overlling are avoided and lling materials conned 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
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ElAyouti et al. Tactile working length
optimal healing. Unfortunately, open apices pose many difculties to contemporary methods of canal length determination. Radiographic methods known for their inherent interpretation difculties are even more challenging in open apices where dentinal walls frequently end at different levels and have irregular margins. Consequently, the apical end of the canal that is circumferentially 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 (Hu lsmann & 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 inuence 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 (Rosenberg 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 difcult because the contact area to the inamed periapical tissues is large, and excess moisture is common, which results in measurement error. Moreover, to obtain accurate measurements when using tactile techniques the periapical tissues must be located at the same level of the apical terminus, a condition that may not be fullled 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 determination in teeth with open apices, and to determine the accuracy, repeatability and inter-operator agreement of the Tactile Method under simulated clinical conditions.
tip (0.51 mm) to a 90 angle using an endodontic gauge (Dentsply Maillefer, Ballaigues, Switzerland). The tip of the le was placed in the gauge hole corresponding to the size of the le 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 le. The marking line on the silicon ring was used to indicate the direction of the bent tip. The le 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 34 mm of the roots were removed and the canal was widened with large les and Gates Glidden burs to obtain 0.51.5 mm dentinal walls thickness at the apex. Subsequently, apical resorption was simulated by rendering the dentinal walls at the apex irregular using ne diamond round burs and SONICex ultrasonic tips (Kavo, Biberach, Germany). The differences between dentinal-wall lengths in the same root ranged from 2 to 5 mm. The correct working length (CWL) was dened to be at the level of the shortest dentinal wall as at this level

The Tactile Method implements a hand instrument to probe the dentinal walls of the root canal. A stainless steel hand le, plugger or spreader can be used. In this study a size 25 K-File was used. The le was bent at the
Figure 1 Schematic presentation of the Tactile Method and
the measuring le.
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Tactile working length ElAyouti et al.
the root canal is surrounded by dentine. The CWL for each root canal was determined by inserting a le 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 le corresponding to the CWL was measured using a digital micrometer under a stereomicroscope; Stemi (Carl Zeiss, Jena, Germany) at 16 magnication. The roots were then embedded in a ` ne/ low viscosity Impression material (President, Colte Whaledent AG, Altsta tten, Switzerland) using a 15 mm brass ring. To prevent the impression material from owing 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 xed 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 conditions by limiting the accessibility of the teeth.
calculated. A regression analysis was performed to evaluate the inuence of tooth type and canal length on the accuracy of the Tactile Method. The repeatability of the Tactile Method was determined by performing paired analysis of the repeated measurements in each tooth. The coefcient of repeatability 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: 0.10.2). 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 inuence the measurements (Fig. 3). The coefcient 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 rst operator in most of the canals (Fig. 4).
The Tactile Method

The aim of the Tactile Method is to circumferentially probe the dentinal walls with the bent tip of the le 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 le 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 le represented the tactile working length (TWL). The le length (from the bent tip to the silicon ring) was measured using a digital micrometer under magnication (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 rst 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 rst operator. The mean of absolute differences (positive values) and the 99% condence interval (CI) were
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 accuracy 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
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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.
the acceptable working length in teeth without resorption (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). Hu lsmann & Pieper (1989) found that apex locators did not function in teeth with open
apices, but after apexication 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 le appears radiographically shorter than its actual length (Stein & Corcoran 1992). In teeth with open apices the radiographic interpretation of canal length is even more difcult due to the altered apical anatomy and the missing periodontal 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
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Tactile working length ElAyouti et al.
Figure 4 Plot of inter-operator agreement showing the limits of agreement and the differences between the rst and second
operator in each canal.
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 measurements 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 necessitate 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 le used for the Tactile Method was curved to allow an easy and reproducible engagement of the bent le 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 le 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 measurements (0.2 mm) showed that repeating the measurement 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 le with a digital micrometer under magnication, 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 le was adapted to a reference point and the length of the le was measured (ElAyouti & Lo st 2006). Operators who used the Tactile Method for the rst time, as the second operator in the present study, experienced difculties in disengaging of the le tip from dentinal wall. This difculty can be overcome by curving the le 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.
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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 apexa clinical guide. Dentistry Update 30, 43741. Baggett FJ, Mackie IC, Worthington HV (1996) An investigation into the measurement of the working length of immature incisor teeth requiring endodontic treatment in children. British Dental Journal 10, 968. Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 30710. Bogen G, Kuttler S (2009) Mineral trioxide aggregate obturation: a review and case series. Journal of Endodontics 35, 77790. 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, 2628. Dominguez RA, Munoz ML, Aznar MT (2005) Study of calcium hydroxide apexication in 26 young permanent incisors. Dental Traumatology 21, 1415. 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, 25862. ElAyouti A, Lo st C (2006) A simple mounting model for consistent determination of the accuracy and repeatability of apex locators. International Endodontic Journal 39, 10812. ElAyouti A, Weiger R, Lo st C (2001) Frequency of overinstrumentation with an acceptable radiographic working length. Journal of Endodontics 27, 4952. ElAyouti A, Kimionis I, Chu AL, Lo st C (2005) Determining the apical terminus of root-end resected teeth using three modern apex locators: a comparative ex vivo study. International Endodontic Journal 38, 82733. 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, 4613. Gordon MP, Chandler NP (2004) Electronic apex locators. International Endodontic Journal 37, 42537. Gutmann JL, Heaton JF (1981) Management of the open (immature) apex. 2. Non-vital teeth. International Endodontic Journal 14, 1738. Herrera M, Abalos C, Planas AJ, Llamas R (2007) Inuence of apical constriction diameter on root ZX apex locator precision. Journal of Endodontics 33, 9958. Holland R, Mazuqueli L, de Souza V, Murata SS, Dezan Junior E, Suzuki P (2007) Inuence of the type of vehicle and limit
of obturation on apical and periapical tissue response in dogs teeth after root canal lling with mineral trioxide aggregate. Journal of Endodontics 33, 6937. Hu lsmann M, Pieper K (1989) Use of an electronic apex locator in the treatment of teeth with incomplete root formation. Endodontics and Dental Traumatology 5, 23841. 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, 5360. Kim E, Lee SJ (2004) Electronic apex locator. Dental Clinics of North America 48, 3554. Lee SJ, Nam KC, Kim YJ, Kim DW (2002) Clinical accuracy of a new apex locator with an automatic compensation circuit. Journal of Endodontics 28, 7069. Mackie IC, Hill FJ (1999) A clinical guide to the endodontic treatment of non-vital immature permanent teeth. British Dental Journal 186, 548. 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, 44752. Morse DR, OLarnic J, Yesilsoy C (1990) Apexication: review of the literature. Quintessence International 21, 58998. Nguyen HQ, Kaufman AY, Komorowski RC, Friedman S (1996) Electronic length measurement using small and large les in enlarged canals. International Endodontic Journal 29, 35964. Ricucci D (1988) Apical limit of root canal instrumentation and obturation, part 1. Literature review. International Endodontic Journal 6, 38493. Ricucci D, Langeland K (1988) Apical limit of root canal instrumentation and obturation, part 2. A histological study. International Endodontic Journal 6, 394409. Rosenberg DB (2003) The paper point technique, part 1. Dentistry Today 22, 806. 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, 15. Stein TJ, Corcoran JF (1992) Radiographic working length revisited. Oral Surgery, Oral Medicine, and Oral Pathology 74, 796800. 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. International Endodontic Journal 41, 43641. Williams CB, Joyce AP, Roberts S (2006) A comparison between in vivo radiographic working length determination and measurement after extraction. Journal of Endodontics 32, 6247. Wu YN, Shi JN, Huang LZ, Xu YY (1992) Variables affecting electronic root canal measurement. International Endodontic Journal 25, 8892.
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doi:10.1111/j.1365-2591.2009.01639.x
Preliminary study of the presence and association of bacteria and archaea in teeth with apical periodontitis
Y. T. Jiang1, W. W. Xia1, C. L. Li2, W. Jiang1 & J. P. Liang1

Department of Endodontics and Operative Dentistry, School of Stomatology, Ninth Peoples Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai; and 2Department of Periodontology, School of Stomatology, Ninth Peoples Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China
1
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, 10961103, 2009
Aim To investigate, by reverse transcription polymerase chain reaction (RT-PCR), the presence and association 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 conventional root canal treatment (secondary infection), aseptically exposed at the rst 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 symptomatic cases positive for both bacteria and archaea (16/ 22, 73%) were signicantly higher than those positive for bacteria alone (21/47, 45%) (P < 0.05). Conclusions This study conrms 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 inammatory diseases with a multitude of clinical features that aficts humans (Baumgartner et al. 2006). Contemporary knowledge of the pathogenesis of apical periodontitis shows that primary
Correspondence: Prof. Jing Ping Liang, Department of Endodontics and Operative Dentistry, Ninth Peoples 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: liangjp123@yahoo.com.cn).
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 lling (Siqueira 2002, Sakamoto et al. 2008). Besides bacteria, other infective factors such as Candida spp. (Sundqvist et al. 1998, Peciuliene 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.
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Jiang et al. Presence and association of bacteria and archaea
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 specic (e.g. single species) aetiology. In order to have a more complete understanding of the role of microorganisms in root canal infections, the pathogenic 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 reect 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 endogenous 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-independent molecular methods, based mainly on 16S rDNA sequences, has widened the scope of detectable microorganisms to include uncultivable organisms that might play signicant roles, as yet undened, 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 ribosomeper-cell ratio is roughly proportional to the growth rate of bacteria (Wagner 1994), rRNA is regarded as an indicator of total bacterial activity. Hence, the purpose of this study was to detect the presence of metabolically active bacteria and archaea in untreated and treated root canals using 16S rRNA derived from isolated ribosomes by reverse transcription polymerase chain reaction (RT-PCR) (Williams et al. 2006), and to compare their presence with the incidence of clinical symptoms.
Materials and methods Patient selection and clinical features

Seventy-seven teeth (one tooth per patient) were selected from patients who sought root canal treatment or retreatment at the Shanghai Ninth Peoples Hospital. Forty-two teeth presented with necrotic pulp tissues and 35 had been root lled >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 classied 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 evidence of a sinus tract was considered symptomatic whether or not pain was present. Patients without the above criteria were considered asymptomatic. No teeth showed signicant gingival recession or any of periodontal 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 rst 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 acquisition. After initial entry into the pulp space, the patency of the root canal was established with minimal instrumentation and without the use of any chemically active irrigant. Pre-existing root lling material was removed using Gates Glidden drills and endodontic les without the use of chemical solvents. Irrigation with sterile saline solution was performed to remove any remaining
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Presence and association of bacteria and archaea Jiang et al.
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 conne 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 uid inside the canal and inserted to the full length of the canal as calculated from the preoperative radiograph. Afterwards, 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 microbiology laboratory in dry ice, then stored at )80 C for 4 weeks or less before extraction of total genomic RNA.
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 briey 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, Promega, 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 conrmed by PCR performed with universal bacterial and archaeal primers (Yu & Morrison 2001, Lepp et al. 2004). The integrity and quantity of the puried RNA were examined by absorbance ratio A260/A280 and RNA gel electrophoresis (Cury et al. 2008).
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 manufacturers protocol and reference method (Chomczynski & Sacchi 1987, 2006). Briey, after incubating the homogenized samples for 5 min at room temperature 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 centrifuged at 12 000g for 15 min at 5 C. Following centrifugation, the mixture separated into a lower red, phenolchloroform 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
Reverse transcription of total RNAs

Complementary DNA (cDNA) synthesis was carried out with the Reverse Transcription System (Promega, Madison, WI, USA), and cDNA were quantied based on absorbance at 260 nm. The puried cDNA were checked on a 1.5% agarose gel and stored at )20 C prior to amplication.
Universal bacterial primers and PCR conditions

The variable V3V5 region of 16S rRNA was enzymatically amplied with primers located on conserved ends of the V3 and V5 region (Yu & Morrison 2001). The primers were as follows: primer 341f, 5-CCTACGGGAGGCAGCAG-3; primer 926r, 5-CCGTCAATTCCTTTGAGTTT-3. A combination of primer 341f and 926r was used to amplify the V3V5 region of 16S rRNA in the different bacterial species, which correspond to positions 341926 in E. coli. Each reaction mixture contained 2.5 lL of 10 PCR buffer [100 mmol L)1 TrisHCI (pH 9), 15 mmol L)1 MgCl2, 500 mmol L)1 KCI, 0.1% (w/v) gelatin, 1% (v/v) Triton X-100],
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0.2 mmol deoxynucleotide triphosphate, 1 U of HotStarTaq DNA polymerase (Qiagen, Hamburg, Germany), 0.25 mmol of each forward and reverse primer, 50 ng template cDNA and enough sterile MilliQ water to bring the nal volume to 50 lL. PCR amplication was performed using the Techne thermocycler (Biometra, Go ttingen, Germany). Amplication 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 rst cycle was preceded by an initial template denaturation step of 4 min at 94 C, and the last cycle was followed by a nal 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 acetate, 1 mmol L)1 EDTA, pH 8.0) and visualized under UV light, following an ethidium bromide staining, the positive samples were recorded.
Results
A total of 77 samples, 42 teeth with primary endodontic infection and 35 with secondary endodontic infection (i.e. failed treatment), were subjected to RTPCR 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 signicantly higher than those positive for bacteria alone (Table 2) (P < 0.05).
Universal archaeal primers and PCR conditions

Fragments of 16S rRNA from samples were PCR amplied 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 TrisHCI (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 deoxynucleotide 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 nal volume to 50 lL. Archaeal 16S rRNA genes were amplied 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.
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 Primary root canal infection (n = 42) Secondary root canal infection (n = 35) Total (n = 77)
a
Bacteria (%) 39 (92.9) 30 (85.7) 69 (88.1)
Archaea (%) 16 (38.1) 6 (17.1) 22 (28.5)
P*
0.001
Data 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
Chi-squared analysis was used to determine a statistically signicant 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). Signicance level was set at P < 0.05.
Bacteria + archaea (n = 22) Bacteria alone (n = 47)
Symptomatic (%) 16 (72.7) 21 (44.7)
Asymptomatic (%) 6 26
P* 0.03
a Data are number and (percentage) of subjects. *Symptomatic cases positive for both bacteria and archaea were signicantly higher than those positive for bacteria alone (P < 0.05).
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the past few years it has been demonstrated that nonculturable bacteria make up an undetermined proportion of the microbial population in the infected c root canal system (Siqueira & Ro as 2003, 2005, Saito et al. 2006). Moreover, several reports have found that bacteria become less culturable under starvation conditions, 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 mandatory, for proper study of infected root canals, to develop and apply methods capable of detecting such bacterial c forms (Siqueira & Ro as 2003, 2005). Amongst the various molecular methods, PCR has proven useful for detecting target microorganisms in c endodontic samples (Siqueira & Ro as 2003). Conventional 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 processing 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 highquality RNA is another important step for the downstream processes. Any extracted RNA must be devoid of contaminants such as salt, protein, solvents and genomic DNA. The extracted RNA was quality controlled 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 cytoplasmic 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 specic RNA against a large background of procaryotic and eucaryotic cells present in root canal samples. This study reports the application of ribosome isolation and subsequent RT-PCR, leading to the identication of the metabolic portion of root canal microbial communities. 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, supporting 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 identied 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 reect the potential to cause apical disease. Such characteristics include ability to colonize the human host and evasion of host defenses. These virulence factors have recently been demonstrated in other medical elds (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
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statistically signicant difference between the percentage 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 full 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 microorganisms 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 methanogens 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 contribute 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 (Sundqvist & Figdor 2003). It has been suggested that metabolic competition for hydrogen with sulphatereducing 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 interactions between archaea, bacteria and other members of the root canal microbiota may help elucidate the bacterial physiological and pathological functions underlying periapical disease activity.
associated with a signicantly higher prevalence of clinical symptoms compared with the sole presence of bacteria.
Acknowledgements
This work was supported by the Science and Technology Commission of Shanghai (08DZ2271100), Shanghai 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) Dening the normal bacterial ora of the oral cavity. Journal of Clinical Microbiology 43, 572132. c Baumgartner JC, Siqueira JF Jr, Xia T, Ro as IN (2004) Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction. Journal of Endodontics 30, 1414. 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. 580607. Belay N, Johnson R, Rajagopal BS, de Macario EC, Daniels L (1988) Methanogenic bacteria from human dental plaque. Applied and Environmental Microbiology 54, 6003. Belay N, Mukhopadhyay B, Conway de Macario E, Galask R, Daniels L (1990) Methanogenic bacteria in human vaginal samples. Journal of Clinical Microbiology 28, 16668. Bonch-Osmolovskaya EA, Stetter KO (1991) Interspecies hydrogen transfer in cocultures of thermophilic archaea. Systematic and Applied Microbiology 14, 2058. 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 thiocyanatephenolchloroform extraction. Analytical Biochemistry 162, 1569. Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanatephenol chloroform extraction: twenty-something years on. Nature Protocols 1, 5815. Conrad R (1999) Contribution of hydrogen to methane production and control of hydrogen concentrations in methanogenic soils and sediments. FEMS Microbiology Ecology 28, 193202. Cury JA, Seils J, Koo H (2008) Isolation and purication of total RNA from Streptococcus mutans in suspension cultures and biolms. Brazilian Oral Research 22, 21622. Eckburg PB, Lepp PW, Relman DA (2003) Archaea and their potential role in human disease. Infection and Immunity 71, 5916.
Conclusion
This study showed archaea to be present in root canals but always with bacteria. Their combined presence was
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-Neto CR et al. (2004) MicrobiGomes BP, Pinheiro ET, Gade ological examination of infected dental root canals. Oral Microbiology and Immunology 19, 716. Kaprelyants AS, Gottschal JC, Kell DB (1993) Dormancy in non-sporulating bacteria. FEMS Microbiology Reviews 104, 27186. Kulik EM, Sandmeier H, Hinni K, Meyer J (2001) Identication of archaeal rDNA from subgingival dental plaque by PCR amplication and sequence analysis. FEMS Microbiology Letters 196, 12933. 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, 31822. 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, 617681. ` MM, Bonato B, Ta MC, Signoretto C, Boaretti M, Lleo Canepari P (2001) Resuscitation rate in different enterococcal species in the viable but non-culturable state. Journal of Applied Microbiology 91, 1095102. Love RM, Jenkinson HF (2002) Invasion of dentinal tubules by oral bacteria. Critical Reviews in Oral Biology and Medicine 13, 17183. Lovley DR (1985) Minimum threshold for hydrogen metabolism in methanogenic bacteria. Applied and Environmental Microbiology 49, 15301. Mason CA, Hamer G, Bryers JD (1986) The death and lysis of microorganisms in environmental processes. FEMS Microbiology Reviews 39, 373401. Miller TL, Wolin MJ (1982) Enumeration of Methanobrevibacter smithii in human feces. Archives of Microbiology 131, 148. n G, Kvist T (1998) Microbiological Molander A, Reit C, Dahle status of root-lled teeth with apical periodontitis. International Endodontic Journal 31, 17. Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG (2002) Molecular and cultural analysis of the microora associated with endodontic infections. Journal of Dental Research 81, 7616. Nair PN (2004) Pathogenesis of apical periodontitis and the causes of endodontic failures. Critical Reviews in Oral Biology and Medicine 15, 34881. Ng YL, Spratt D, Sriskantharajah S, Gulabivala K (2003) Evaluation of protocols for eld decontamination before bacterial sampling of root canals for contemporary microbiology techniques. Journal of Endodontics 29, 31720. Oliver JD (1995) The viable but non-culturable state in the human pathogen, Vibrio vulnicus. FEMS Microbiology Letters 133, 2038. Oliver JD (2005) The viable but nonculturable state in bacteria. The Journal of Microbiology 43, 93100. Review. Paster BJ, Boches SK, Galvin JL et al. (2001) Bacterial diversity in human subgingival plaque. Journal of Bacteriology 183, 377083.
Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M (2001) Isolation of yeasts and enteric bacteria in root-lled teeth with chronic apical periodontitis. International Endodontic Journal 34, 42934. Sabeti M, Simon JH, Slots J (2003) Cytomegalovirus and Epstein-Barr virus are associated with symptomatic periapical pathosis. Oral Microbiology and Immunology 18, 3278. Saito D, Leonardo Rde T, Rodrigues JL, Tsai SM, Ho ing JF, Gonc alves RB (2006) Identication of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries. Journal of Medical Microbiology 55, 1017. c Sakamoto M, Siqueira JF Jr, Ro as IN, Benno Y (2008) Molecular analysis of the root canal microbiota associated with endodontic treatment failures. Oral Microbiology and Immunology 23, 27581. 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, 5704. Siqueira JF Jr (2002) Endodontic infections: concepts, paradigms and perspectives. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 94, 28193. c Siqueira JF Jr, Ro as IN (2003) PCR methodology as a valuable tool for identication of endodontic pathogens. Journal of Dentistry 31, 3339. c Siqueira JF Jr, Ro as IN (2005) Exploiting molecular methods to explore endodontic infections. Part 2. Redening the endodontic microbiota. Journal of Endodontics 31, 48898. c Siqueira JF Jr, Ro as IN, Baumgartner JC, Xia T (2005) Searching for Archaea in infections of endodontic origin. Journal of Endodontics 31, 71922. c Siqueira JF Jr, Ro as IN, Paiva SS, Magalha es KM, Guimara esPinto T (2007) Cultivable bacteria in infected root canals as identied by 16S rRNA gene sequencing. Oral Microbiology and Immunology 22, 26671. 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 Immunology 7, 25762. Sundqvist G (1994) Taxonomy, ecology, and pathogenicity of the root canal ora. Oral Surgery, Oral Medicine, and Oral Pathology 78, 52230. Sundqvist G, Figdor D (2003) Life as an endodontic pathogen: ecological differences between the untreated and root lled root canals. Endodontic Topics 6, 328. Sundqvist G, Fidgor D, Persson S, Sjo gren U (1998) Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative retreatment. Oral Surgery, Oral Medicine, and Oral Pathology 85, 8693. Vianna ME, Conrads G, Gomes BP, Horz HP (2006) Identication and quantication of Archaea involved in primary endodontic infections. Journal of Clinical Microbiology 44, 127482.
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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, 1108. Wagner R (1994) The regulation of ribosomal RNA synthesis and bacterial cell growth. Archives of Microbiology 161, 1006. 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 cultivation during endodontic treatment. Journal of Endodontics 32, 71521.
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, 45769. Young G, Turner S, Davies JK, Sundqvist G, Figdor D (2007) Bacterial DNA persists for extended periods after cell death. Journal of Endodontics 33, 141720. Yu Z, Morrison M (2001) Comparisons of different hypervariable regions of rrs genes for use in ngerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 70, 48006.
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doi:10.1111/j.1365-2591.2009.01640.x
Sonic extracts from a bacterium related to periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases TIMP-1 and TIMP-2
Y. Sato1, J. Kishi2, K. Suzuki1, H. Nakamura1 & T. Hayakawa2

Department of Endodontics, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan; and 2Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan
1
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 metalloproteinases TIMP-1 and TIMP-2. International Endodontic Journal, 42, 11041111, 2009.
Aim To examine the effects of sonicated bacterial extracts (SBEs) from three related to periapical disease bacteria (Porphyromonas gingivalis, P. endodontalis 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 zymography 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 Students 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 ndings suggest that P. gingivalis 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
Inammatory changes in major pathological lesions in oral tissues could progress via the destruction of the
Correspondence: Hiroshi Nakamura, Department of Endodontics, 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).
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
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Sato et al. SBE from bacteria on activation of MMP-2
secreted by inammatory cells, including neutrophils and macrophages that migrate into inamed sites, as well as by noninammatory cells, including broblasts (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 inammation 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, Porphyromonas 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 brosarcoma (HT1080) cell culture supernatants.
(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 streptomycin (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 conuent, they were subcultured and used for experiments at subculture levels 48.
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 lter (Corning), the protein content of each SBE (unfractionated) was determined with a protein assay kit (Bio-Rad, Tokyo, Japan; data is not shown). Then all supernatants were diluted to a concentration of 1 mg of protein per millilitre and used for further experiments as sonicated bacterial extracts.
Materials and methods Preparation of human periodontal ligament cell cultures

Three freshly extracted human teeth for orthodontic reasons were immediately soaked and washed with sterilized physiological saline containing penicillin (1000 U mL)1; Meiji Seika Kaisha, Ltd, Tokyo, Japan) and Fungizone (30 lg mL)1; Nippon Squib, Tokyo, Japan). Periodontal ligament tissue was then isolated from the surfaces of the roots and washed thoroughly with RPMI1640 (Nisui Pharmaceuticals, Tokyo, Japan) containing penicillin (100 U mL)1), streptomycin
Preparation of human brosarcoma (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 conuent, they were washed twice with PBS, which was then replaced with serumfree 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 components, before being centrifuged further at 10 000 g for 20 min. The supernatants produced were used as samples for experiments.
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SBE from bacteria on activation of MMP-2 Sato et al.
Preparation of TIMP-1 and TIMP-2

TIMP-1 was puried 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 MMP2-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. endodontalis 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 conuence at 37 C in a 5% CO2 atmosphere. They were then washed twice with PBS followed by replacement with 1 mL of serum-free RPMI1640 medium containing 10 lg mL)1 of SBE. The cells were subsequently 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.
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 Standards 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]glycinelabelled collagen used as substrate. The TIMP-1 inhibition of MMP-2 was also determined 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% methanol7.5% acetic acid. In this way, TIMP-1 was observed as a blue band.
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 nal concentration of 0.3 mg mL)1. Trisglycine 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
Treatment of MMP-2 or TIMP-1 with SBEs

Puried MMP-2 (0.5 lg mL)1) or puried 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 Students t-test. Differences were considered signicant if P < 0.01.
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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 (proMMP-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 produced 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).
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.
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 rst 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.
Puried progelatinase A
Puried pro-MMP-2 was processed by P. gingivalis SBE. As shown in Fig. 3, two bands, corresponding to 72-
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 3 Gelatin zymograph of puried 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.
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Figure 4 Effects of SBEs on the amount of TIMP-1 in PL cell
Figure 6 Effects of SBEs on the inhibitory activity of puried
culture medium. PL cell culture medium was rst 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.
TIMP-1 against interstitial collagenase (MMP-1). Puried TIMP-1 (0.4 lg) was rst 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 signicantly 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 puried TIMP-1 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 rst 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.
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.
Inhibitory activity of TIMP-1

TIMP-1 was more susceptible to P. gingivalis SBE than TIMP-2. The effect of each SBE on the inhibitory activity of TIMP-1 against interstitial collagenase (MMP-1) was
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examined. P. gingivalis SBE alone signicantly 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 puried TIMP-1 was further conrmed by reverse zymography of TIMP-1. As shown in Fig. 7, essentially no inhibition against MMP-2 was observed with puried 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 destruction. 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 specic 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 specically activates pro-MMP-2 on cell surfaces (Sato et al. 1994). The only pro-MMP-2 activator that has been reported is MT1MMP, 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 inammation, and could be also of help to clarify the development, progress, and mechanism 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 rst time, although similar studies have been conducted with human periodontal ligament or human gingival broblasts 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 rst 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 proMMP-2 and TIMP-1 and have slow growth. To overcome these problems, HT1080 cell culture supernatants were used, because these cells have been studied in detail with regard to their production of proMMP-2, TIMP-1, and TIMP-2, and also because they are derived from broblasts, 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 representing pro-MMP-2 (72 kDa). In addition to that, treatment of puried 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 experiments using either F. nucleatum SBE or P. endodontalis SBE, therefore, the activating factors could be considered to be specic 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 TIMP1 by PL cells, (ii) P. gingivalis SBE could inhibit and
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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, puried 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 TIMP1 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 conrmed. 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 ndings for TIMP-1, which suggests that TIMP-2 could also be degraded by P. gingivalis SBE. It is well known that P. gingivalis possesses mbriae 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 specicity (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 conrmed that proMMP-9 was activated by P. gingivalis SBE (Fig. 2). Both pro-MMP-2 activation and TIMP-1, TIMP-2 inactivation were only conrmed 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 numbers from infected root canals with progressing periapical 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 ngerprinting 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 TIMP1 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, 8690. Bergenholtz G (1974) Micro-organisms from necrotic pulp of traumatized teeth. Odontologisk Revy 25, 34758. Berit J, Klaus B (1986) Chemical composition and biological properties of a lipopolysaccharide from Bacteroides intermedius. Acta Pathologica et Microbiologica Scandinavia. Section B: Microbiology and Immunology 94, 26571. 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 oncogenetransformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. The Journal of Biological Chemistry 263, 657987. DeCarlo AA Jr, Windsor LJ, Bodden MK, Harber GJ, BirkedalHansen B, Birkedal-Hansen H (1997) Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis. Journal of Dental Research 76, 126070. Declerk YA, Yean TD, Ratzkin BJ, Lu HS, Langley KE (1989) Purication and characterization of two related but distinct metalloproteinase inhibitors secreted by bovine aortic endothelial cells. The Journal of Biological Chemistry 264, 1744553. 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, 3145. 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 designated TIMP-2. Proceeding of the National Academy of Science of the United States America 86, 820711.
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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, 3037580. Grenier D, Mayrand D (2001) Inactivation of tissue inhibitor of metalloproteinases-1(TIMP-1) by Porphyrmonas gingivalis. FEMS Microbiology Letters 203, 1614. Haapasalo M (1989) Bacteroides spp. in dental root canal infections. Endodontic Dental Traumatology 5, 110. Higerd TB, Vesole DH, Goust JM (1978) Inhibitory effects of extracellular products from oral bacteria on human broblasts and stimulated lymphocytes. Infection and Immunity 21, 56774. Kantz WE, Henry CA (1974) Isolation and classication of anaerobic bacteria from intact pulp chambers of non-vital teeth in man. Archives Oral Biology 19, 916. Kishi J, Ogawa K, Yamamoto S, Hayakawa T (1991) Purication and characterization of a new tissue inhibitor of metalloproteinases (TIMP-2) from mouse colon 26 tumor cell. Matrix 11, 106. Kodama S, Kishi J, Okada K, Iwata K, Hayakawa T (1981) Monoclonal antibodies to bovine collagenase inhibitor. Collagen Related Research 7, 34150. 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 Immunological Methods 127, 1038. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 6805. Nagase H, Woessner JF Jr (1999) Matrix metalloproteinases. The Journal of Biological Chemistry 274, 214914. Nair BC, Mayberry WR, Dziak R, Chen PB, Levine MJ, Hausmann E (1983) Biological effects of a puried lipopolysaccharide from Bacteroides gingivalis. Journal of Periodontal Research 18, 409. Oguntebi B, Slee AM, Tanzer JM, Langeland K (1982) Predominant microora associated with human dental periapical abscesses. Journal of Clinical Microbiology 15, 9646. Pattamapun K, Tiranathangui S, Yongchaitrakul T, Kuwatanasuchat J, Pavasant P (2003) Activation of MMP-2 by Porphyromonas gingivalis in human periodontal ligament cells. Journal of Periodontal Research 38, 11521. Pike R, McGran W, Potempa J, Travis J (1994) Lysine- and arginine-specic proteinases from Porphyromonas gingivalis. The Journal of Biological Chemistry 269, 40611.
Sakamoto W, Fujie K, Kaga M et al. (1996) Degradation of Tkininogen by cathepsin D and matrix metalloproteinases. Immunopharmacology 32, 735. Sato H, Takino T, Okada Y et al. (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370, 61. Singer RE, Dutton WG (1979) A comparison of the effects of endotoxin upon broblast proliferation and macromolecular synthesis. Journal of Dental Research 58, 16349. 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, 173748. Sundqvist G (1992) Associations between microbial species in dental root canal infections. Oral Microbiology and Immunology 7, 25762. 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, 75362. Tiranathanagul K, Pattamapun T, Yongchaitrakul T, Pavasant P (2004) MMP-2 activation by Actinomycetemcomitans supernatant in human PDL cells was corresponded with reduction of TIMP-2. Oral Biology 10, 3838. 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 Research 54, 20914. Visse R, Nagase H (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circulation Research 92, 82739. Ward RV, Hembry RM, Reynolds JJ, Murphy G (1991) The purication of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Biochemical Journal 278, 17987. Williams BL, McCann GF, Schoenknecht FD (1983) Bacteriology of dental abscesses of endodontic origin. Journal of Clinical Microbiology 18, 7704. Wittgow WCJ, Sabiston CBJ (1975) Microorganisms from pulpal chambers of intact teeth with necrotic pulps. Journal of Endodontics 1, 16871. Yamamoto K (1995) Studies on periodontal pathogenic proteinase from Porphyromonas gingivalis and host cell. Folia Phormacologica Japonica 105, 34555. Zhou J, Windsor LJ (2006) Porphyromonas gingivalis affects host collagen degradation by affecting expression, activation, and inhibition of matrix metalloproteinases. Journal of Periodontal Research 41, 4754.
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doi:10.1111/j.1365-2591.2009.01642.x
Antibiotic prescribing in dental practice in Belgium
A. Mainjot1, W. DHoore2, A. Vanheusden1 & J.-P. Van Nieuwenhuysen3

Department of Fixed Prosthodontics, Institute of Dentistry, University Hospital (CHU) of Liege, University of Liege, Liege, Belgium; Catholique de Louvain, Louvain, Belgium; and 3Department of School of Public Health, Health Systems Research, Universite Catholique de Louvain, Louvain, Belgium Restorative Dentistry and Endodontics, Universite
2 1
Abstract
Mainjot A, DHoore W, Vanheusden A, Van Nieuwenhuysen J-P. Antibiotic prescribing in dental practice in
Belgium. International Endodontic Journal, 42, 11121117, 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 knowledge 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 prescribed 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, amoxycillin-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 prescribed to 63.3% of patients with periapical abscess and 4.3% of patients with pulpitis. Patterns of prescriptions were conrmed 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 Scientic Affairs 2004). Adverse reactions, emergence and dissemination of resistance of some species through genetic routes, increase in the prevalence of drugresistant 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-
lie Mainjot, Institut de Dentisterie Correspondence: Ame ` ge, Quai G.Kurth, 45, B-4020 Lie ` ge, Belgium de Lie Universite (Tel.: +32 4 270 31 00; fax: +32 4 270 31 10; e-mail: a.mainjot@chu.ulg.ac.be).
able amount of antibiotics: It was estimated that antibiotic prescriptions amounted to 1.1 Dened 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/statisticsscientic-information/pharmanet/Statistics-group/2004/ pdf/t13101311.pdf). Surveys of dentists prescribing habits have raised awareness of the quality of prescriptions in developed countries. For example, questionnaire 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
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Mainjot et al. Antibiotic prescribing
there is a lack of scientic information regarding appropriate and efcient 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 recommended practice in antibiotic use.
Self-reported practice
Practitioners were invited to complete a self-administered 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 2week 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 inuencing 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, scientic 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 inuence 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. Questionnaires were formatted for optical reading.
Material and methods

This cross-sectional survey was performed in September 2004 and consisted of two parts. In part 1 (prescriptions), dental practitioners were asked to record information 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-administered questionnaire about demographic data, prescribing 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 population of dentists accreditated in 2004 by the Belgian social security ofce (Institut National dAssurance Rijksinstituut voor Ziekte- en Maladie Invalidite 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.
Chi-square and Fishers exact tests were used to test the signicance of associations in cross Tables. To compare means, Students 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 Prescriptions
Informed consent was required from every patient. Dentists 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 inuence prescription, concomitant prescription of nonsteroidal anti-inammatory drugs (NSAIDs), analgesics, and mouthwashes. A difference was made between therapeutic and prophylactic (e.g. prevention of endocarditis) prescriptions. In cases of antibiotherapy, dentists were also asked to supply details about diagnosis and dental treatment. A total of 268 practitioners participated in the survey (response rate = 89.3%); 56.3% were male. The sample represented 3.1% of the Belgian dentist population and 6.8% of accredited dentists. Only 3.7% of the dentists were qualied in periodontics or orthodontics, the two ofcial dental specializations in Belgium. During the 2-week period, 24 421 patient encounters 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 remaining 51 were undetermined.
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Antibiotic prescribing Mainjot et al.
Table 1 Distribution of actual antibiotics prescribed for therapeutic reasons versus self-reported prescribing patterns
Prescriptions n (%) 478 (51.1) 225 (24.0) 62 (6.6) 50 (5.3) 41 (4.4) 34 (3.6) 21 (2.2) 11 (1.2) 10 (1.1) 2 (0.2) 1 (0.1) 1 (0.1) 0 (0) 936 (100) Self-reported prescribing patterns (%) 33.6 22.1 13.9 4.8 5.1 9.0 1.6 7.4 0.9 0 0.3 0.3 0.15 1 100
Antibiotic Amoxycillin Amoxycillin + clavulanic acid Clindamycin Azythromycin Clarithromycin Doxycycline Spiramycin Erythromycin Other Ciprooxacin Cefadroxil Minocycline Cefuroxime Never prescribe antibiotic Total
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 (minimum = 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 according 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 prophylaxis 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 antibiotherapy were periapical abscess (51.9%) and periodontal 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 diagnoses 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 2 Indications for antibiotherapy: comparison between actual prescriptions and self-reported prescribing patterns
Indications for antibiotherapy (1), n (%) 476 (51.9) 130 (14.2) 75 (8.2) 70 (7.6) 43 (4.7) 40 (4.4) 37 (4.0) 25 (2.7) 22 (2.4) 918 (100) Frequency of diagnosis (2) 752 452 933 Prescription rate (1)/(2) % 63.3 28.8 4.3 Self-reported prescription patterns (3) % 82.7 63.2 52.2 23.5 5.9 41.9 44.1
Diagnosis Periapical abscess Periodontal abscess Others Pericoronitis Rapidly progressive periodontitis Pulpitis Alveolar osteitis Chronic adult periodontitis Cellulitis Total
(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.
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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 clavulanic 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 information about antibiotic use was high (81%). Colleagues were considered as the best source of information (87%) whereas university continuing education sessions were less satisfying (73.5%). However, satisfaction 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 ndings 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 reect 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
inuence 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 knowledge 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 6week 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 510 (Al-Mubarak et al. 2004). However, comparisons between regions or countries are meaningless 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 spectrum antibiotics were most commonly prescribed: Amoxycillin, 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 inappropriate 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 rst choice (75% of their total prescriptions) (Al-Haroni & Skaug 2007). Regarding the indications for antibiotherapy, the prescription data showed that antibiotics were pre-
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Antibiotic prescribing Mainjot et al.
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, LopezPiriz 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%, respectively. Our study did not provide information about the organization of patient care, but it is likely that lack of time, working schedules, and technical difculties prevent dentists from performing timely local treatments. 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 ndings 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 reect the amount of antibiotics used by patients. This apparently positive situation may hamper the collection 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 nding 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 conrmed by responses to simulated cases about prophylaxis of
endocarditis and articial 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 efcacy, visibility, and/or legitimacy of ofcial sources, such as university continuing education sessions. The ndings 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 demonstrated in the Cochrane review by Arnold & Straus (2005). Selection of the most effective intervention appears to be condition and situation specic. 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 standards 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 rst 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 professional 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 dAssurance Maladie supported this research. Invalidite
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References
Al-Haroni M, Skaug N (2007) Incidence of antibiotic prescribing in dental practice in Norway and its contribution to national consumption. Journal of Antimicrobial Chemotherapy 59, 11616. 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, 4755. American Dental Association Council on Scientic Affairs (2004) Combating antibiotic resistance. Journal of the American Dental Association 135, 4847. 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, 63541. 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, 8339. Dailey YM, Martin MV (2001) Are antibiotics being used appropriately for emergency dental treatment? British Dental Journal 191, 3913. Handal T, Olsen I (2000) Antimicrobial resistance with focus on oral beta-lactamases. European Journal of Oral Sciences 108, 16374. Hawkey PM (2008) The growing burden of antimicrobial resistance. Journal of Antimicrobial Chemotherapy 62, i19. 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, 75963. Lopez-Piriz R, Aguilar L, Gimenez M (2007) Management of odontogenic infection of pulpal and periodontal origin. a Oral y Cirug a Bucal 12, E1549. Medicina Oral, Patolog 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 Endodontics 90, 63640. Palmer NA, Batchelor PA (2004) An audit of antibiotic prescribing by vocational dental practitioners. Primary Dental Care 1, 7780. Palmer NA, Dailey YM (2002) General dental practitioners experiences of a collaborative clinical audit on antibiotic prescribing: a qualitative study. British Dental Journal 193, 469. 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, 5548. Palmer NA, Dailey YM, Martin MV (2001) Can audit improve antibiotic prescribing in general dental practice? British Dental Journal 191, 2535. Roy KM, Bagg J (2000) Antibiotic prescribing by general dental practitioners in the Greater Glasgow Health Board, Scotland. British Dental Journal 188, 6746. Sweeney LC, Dave J, Chambers PA, Heritage J (2004) Antibiotic resistance in general dental practice a cause for concern? Journal of Antimicrobial Chemotherapy 53, 56776. Tong DC, Rothwell BR (2000) Antibiotic prophylaxis in dentistry: a review and practice recommendations. Journal of the American Dental Association 131, 36674. 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, 396404.
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doi:10.1111/j.1365-2591.2009.01633.x
CASE REPORT
One step pulp revascularization treatment of an immature permanent tooth with chronic apical abscess: a case report
S. Y. Shin, J. S. Albert & R. E. Mortman
Endodontic 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, 11181126, 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 benecial 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, revascularization, stem cells of the apical papilla.
Received 21 June 2009; accepted 21 August 2009
Correspondence: Dr Sang Shin, 1501 Presidential Way, Suite 7, West Palm Beach, FL 33401, USA (e-mail: shin_dmd@hotmail.com).
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CASE REPORT
Introduction Recently there has been evidence indicating that a better alternative to conventional calcium hydroxide apexication 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 apexication, apexogenesis encourages a longer and thicker dentinal composed root to develop (Rafter 2005). These benecial anatomic properties may decrease the propensity of long-term root fracture, a signicant risk associated with apexication 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, apexication involves supplementary treatment visits to replenish the calcium hydroxide and ultimately requires an apical plug of mineral trioxide aggregate (MTA) or nal Gutta-percha canal lling (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 nal 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 Hertwigs 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 calcication, rendering the tooth more difcult to treat endodontically in the future (Shah et al. 2008). Furthermore, it has not been determined the stage and duration of pathosis that will ultimately lead to the complete destruction of the resistant apical mesenchymal cells and surviving dental pulp
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stem cells. Under the circumstances of total pulpal and apical papilla necrosis, revascularization treatment may not be possible. As a result, it is difcult 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 instrumentation. A tri-antibiotic paste, composed of metronidazole, ciprooxacine 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 conned 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, modied technique to revascularize a partially necrotic pulp with associated chronic apical periodontitis.
CASE REPORT
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 asymptomatic 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-tetrauoroethane (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 inammatory resorption on the mid distal portion of the root. Condensing osteitis was apparent at the periapical area of the tooth (Fig. 3a).
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CASE REPORT
(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, reestablishment of normal gingival contour was observed.
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 2 Root canal revascularization procedure. (a) Clinically, the mandibular right second premolar presented with distal pit caries. (b) After controlling the haemorrhage, viable tissue was observed in the canal. (c) The placement of white MTA in the canal. (d) Final composite restoration.
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CASE REPORT
(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.
the root canal, haemorrhage into the pulp chamber was observed (Fig. 2b). A size 10 K-le (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 benet greatest from a revascularization procedure. A thorough explanation of the potential risks, complications and benets of the suggested treatment was carried out. The alternative option of conventional calcium hydroxide apexication was discussed. Based on the increased long-term risk of root fracture attributed to traditional calcium hydroxide apexication and the potential rewards of revascularization, maternal consent was obtained to initiate revascularization treatment. The proposed, most conservative treatment protocol is a modication 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 apexication 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 nal 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.
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CASE REPORT
(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 reestablishment 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.
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 signicant 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. Radiographically, 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 apexication 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
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patient compliance and resulting thin dentinal walls with a high risk of long-term root fracture (Cvek & Sundstrom 1974, Andreasen et al. 2002). Contemporary research articles examine alternative treatment options to encourage a root maturation process emulating natural root formation, even in the presence of extensive periapical pathology (Banchs & Trope 2004, Chueh & Huang 2006, Jung et al. 2008). Revascularization treatment has been suggested to be a favourable alternative, yielding the development of a longer and thicker root, less susceptible to fracture. Using a modied 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 nal 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 revascularization 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 benets of the residual antimicrobial activity of chlorhexidine versus the cytotoxicity and carcinogenicity of parachloroaniline. 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 benecial 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
CASE REPORT
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CASE REPORT
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 benets 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 le within the canal may indicate the presence of viable canal tissue. It is suggested that in these cases, the current technique can be benecial prior to attempting the less conservative tri-antibiotic sequence or calcium hydroxide apexication. 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 specically 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 afliated 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, 1347. Banchs F, Trope M (2004) Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? Journal of Endodontics 30, 196200. Barrett AP, Reade PC (1981) Revascularization of mouse tooth isografts and allografts using autoradiography and carbon-profusion. Archives in Oral Biology 26, 5415. Basrani B, Manek S, Sodhi R, Fillery E, Manzur A (2007) Interaction between sodium hypochlorite and chlorhexidine gluconate. Journal of Endodontics 33, 9669. Canger EM, Kayipmaz S, Celenk P (2009) Bilateral dens invaginatus in the mandibular premolar region. Indian Journal of Dental Research 20, 23840. 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, 1721.
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Chueh LH, Huang GT (2006) Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a paradigm shift. Journal of Endodontics 32, 120513. 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, 1604. 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, 37992. 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, 3224. Fridland M, Rosado R (2005) MTA solubility (a long term study). Journal of Endodontics 31, 3769. Greenstein G, Berman C, Jafn R (1986) Chlorhexidine: an adjunct to periodontal therapy. Journal of Periodontology 57, 3706. 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 ciprooxacin, metronidazole and minocycline. International Endodontic Journal 29, 12530. 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, 64551. Iwaya S, Ikawa M, Kubota M (2001) Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dental Traumatology 17, 1857. 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, 2768. Jung IY, Lee SJ, Hargreaves KM (2008) Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series. Journal of Endodontics 34, 87687. Kettering JD, Torabinejad M (1995) Investigation of mutagenicity of mineral trioxide aggregate and other commonly used root end lling materials. Journal of Endodontics 21, 5379. Lin L, Shovlin F, Skribner J, Langeland K (1984) Pulp biopsies from the teeth associated with periapical radiolucency. Journal of Endodontics 10, 43648. 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, 38791. Murray PE, Garcia-Godoy F, Hargreaves KM (2007) Regenerative endodontics: a review of current status and a call for action. Journal of Endodontics 33, 37790. Ohman A (1965) Healing and sensitivity to pain in young replanted human teeth: an experimental, clinical and histological study. Odontologisk Tidskrift 73, 168227. Rafter M (2005) Apexication: a review. Dental Traumatology 21, 18. Reynolds K, Johnson D, Cohenca N (2009) Pulp revascularization of necrotic bilateral bicuspids using a modied novel technique to eliminate potential coronal discolouration: a case report. International Endodontic Journal 42, 8492. Shah N, Logani A, Bhaskar U (2008) Efcacy of revascularization to induce apexication/apexogenesis in infected, nonvital, immature teeth: a pilot clinical study. Journal of Endodontics 34, 91925. Skoglund A, Tronstad L (1981) Pulpal changes in replanted and autotransplanted immature teeth of dogs. Journal of Endodontics 7, 30916. 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 Endodontics 25, 197205. Torabinejad M, Wilder Smith P, Pitt Ford TR (1995) Comparative investigation of marginal adaptation of mineral trioxide aggregate and other commonly used root-end lling materials. Journal of Endodontics 21, 2959. White R, Hays G, Janer L (1997) Residual antimicrobial activity after canal irrigation with chlorhexidine. Journal of Endodontics 23, 22931. Yousef SaadA (1988) Calcium hydroxide and apexogenesis. Oral Surgery, Oral Medicine & Oral Pathology 66, 499501.
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doi:10.1111/j.1365-2591.2009.01626.

J. Moore, P. Fitz-Walter & P. Parashos

2009 International Endodontic Journal

International Endodontic Journal, 42, 10571064, 2009

Apical root canal preparation assessed by micro-CT Moore et al.

Materials and methods

International Endodontic Journal, 42, 10571064, 2009

2009 International Endodontic Journal

Moore et al. Apical root canal preparation assessed by micro-CT

Micro-CT measurements and evaluations

2009 International Endodontic Journal

International Endodontic Journal, 42, 10571064, 2009

Apical root canal preparation assessed by micro-CT Moore et al.

International Endodontic Journal, 42, 10571064, 2009

2009 International Endodontic Journal

Moore et al. Apical root canal preparation assessed by micro-CT

2009 International Endodontic Journal

International Endodontic Journal, 42, 10571064, 2009

Apical root canal preparation assessed by micro-CT Moore et al.

International Endodontic Journal, 42, 10571064, 2009

2009 International Endodontic Journal

Moore et al. Apical root canal preparation assessed by micro-CT

2009 International Endodontic Journal

International Endodontic Journal, 42, 10571064, 2009

Apical root canal preparation assessed by micro-CT Moore et al.

International Endodontic Journal, 42, 10571064, 2009

2009 International Endodontic Journal

R. A. Higa, C. G. Adorno, A. K. Ebrahim & H. Suda

2009 International Endodontic Journal

International Endodontic Journal, 42, 10651070, 2009

Apex locators meter reading Higa et al.

Materials and methods

Figure 1 Actual canal length determination. A size 15 K-le

International Endodontic Journal, 42, 10651070, 2009

2009 International Endodontic Journal

Higa et al. Apex locators meter reading

2009 International Endodontic Journal

International Endodontic Journal, 42, 10651070, 2009

Apex locators meter reading Higa et al.

0.08 0.72 1.60 3.32 3.86 4.12

0.12 0.27 0.61 0.93 0.86 0.94

+0.09 0.90 2.13 2.93 2.93 3.01

0.33 0.49 0.53 0.13 0.21 0.04

0.13 0.59 0.85 1.50 1.93 2.24

0.33 0.13 0.38 0.75 0.96 1.02

International Endodontic Journal, 42, 10651070, 2009

2009 International Endodontic Journal

Higa et al. Apex locators meter reading

2009 International Endodontic Journal

International Endodontic Journal, 42, 10651070, 2009

Apex locators meter reading Higa et al.

International Endodontic Journal, 42, 10651070, 2009

2009 International Endodontic Journal

O. H. Ikram, S. Patel, S. Sauro & F. Mannocci

2009 International Endodontic Journal

International Endodontic Journal, 42, 10711076, 2009

Micro-CT investigation Ikram et al.

Materials and methods

Access cavity preparation

International Endodontic Journal, 42, 10711076, 2009

2009 International Endodontic Journal

Ikram et al. Micro-CT investigation