|Year : 2014 | Volume
| Issue : 1 | Page : 71-74
Lack of correlation between microbial penetration method and electro chemical technique for assessment of leakage through the root canal fillings
Maryam Kazemipoor1, Jalil Modaresi1, Hengameh Zandi2, Narges Vasee1, Vaziri Farzaneh3
1 Department of Endodontics, Shahid Sadooghi University of Medical Sciences, Yazd, Iran
2 Department of Microbiology, School of Medicine, Shahid Sadooghi University of Medical Sciences, Yazd, Iran
3 Department of Periodontics, School of Dentistry, Shahid Sadooghi University of Medical Sciences, Yazd, Iran
|Date of Web Publication||13-Mar-2014|
Department of Endodontics, School of Dentistry, Shahid Sadooghi University of Medical Sciences, Emam Street, Dahe Fajr Blv, Yazd
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims: The purpose of this study was to compare microbial penetration method and electrochemical technique for evaluation of the apical seal. Materials and Methods: A total of 28 teeth were prepared using the step-back technique to ISO size 40 master apical files. The specimens were randomly divided into an experimental group, two positive and two negative controls. Root canals in the experimental and negative control group were filled with gutta-percha (GP) and sealer, using lateral condensation technique. In the positive control group, canals were filled with GP without any sealer. The external surface of each tooth was coated with two layers of the nail varnish, except for the access opening and the apical foramen. In the negative control group, the teeth were completely covered with nail varnish. The apical seal was recorded with two methods, each successively used on the same teeth: An electrochemical method and a bacterial penetration method. Statistical Analysis Used: The correlation of the electro chemical readings with the results obtained from bacterial micro leakage test was evaluated by Pearson's correlation coefficient. Results: Correlation coefficient of the measurements obtained from the two evaluation methods, was 0.23 (r = 0.23), so the correlation was not statistically significant (P = 0.275). Conclusions: This study shows that several studies by different methods are necessary before evaluation of the marginal leakage.
Keywords: Apical leakage, electrochemical technique, microbial penetration, root canal fillings
|How to cite this article:|
Kazemipoor M, Modaresi J, Zandi H, Vasee N, Farzaneh V. Lack of correlation between microbial penetration method and electro chemical technique for assessment of leakage through the root canal fillings. Contemp Clin Dent 2014;5:71-4
|How to cite this URL:|
Kazemipoor M, Modaresi J, Zandi H, Vasee N, Farzaneh V. Lack of correlation between microbial penetration method and electro chemical technique for assessment of leakage through the root canal fillings. Contemp Clin Dent [serial online] 2014 [cited 2020 Feb 18];5:71-4. Available from: http://www.contempclindent.org/text.asp?2014/5/1/71/128670
| Introduction|| |
Cleansing and shaping of the root canal system, followed by adequate obturation are the major objectives of endodontic treatment.  Root canal obturation provides a seal that prevents microleakage and subsequent reinfection of the canal and the periradicular tissues.  For this reason, different endodontic materials and obturation techniques have been applied for decreasing microleakage and improvement of seal. , Along with these improvements, various test methods have been described to assess the sealing ability of these materials and techniques. 
In vitro methods are used, generally can be divided in to two categories: Methods that used a tracer agent penetrating the filled canal and those that rendered without a tracer. Tracers such as, dyes, radioisotopes, bacteria and their products (i.e. endotoxins) are commonly used in microleakage studies. Other methods include fluid filtration technique, electro chemical technique and etc.
Assessment of bacterial leakage might be more biologically relevant than that of dye or radioisotope penetration, but this method has two limitations: Depending on the bacterial species has been used, conclusions might vary and maintaining aseptic condition throughout all steps of the experiment can be difficult. ,
Jacobsen and von Fraunhofer depicted the electrochemical micro leakage method for the first time.  In this technique the tooth is immersed in an ionic solution (i.e. NaCl solution). A stainless steel wire (working electrode) is placed into the coronal access cavity, which was filled with the same ionic solution and another stainless steel wire (counter electrode) is submerged in to the solution. The two electrodes are attached to a constant power supply with a multimeter. As the leakage occurs, the solution penetrates through the apical seal. It is assumed that the magnitude of the current detected will indicate the degree of the penetration. 
The aim of this study was to compare the results of bacterial and electro chemical micro leakage tests and evaluate any correlation between these tests.
| Materials and Methods|| |
Preparation of samples
A total of 28 freshly extracted human maxillary and mandibular anterior teeth with a single, straight root canal were selected for this study. The exclusion criteria include: Existing cracks, large carious lesions, open apices and resorptive defects. After removal of bony debris, calculus and soft-tissues on the root surface, the teeth were stored in saline solution. All preparation and obturation procedures were carried out by a trained operator as described below.
The coronal fragments of all teeth were removed with a diamond disks and left roots with 15 mm length.  A diamond bur was used to gain a straight-line access to the root canal. Following pulp extirpation, a size 15 K-file (Dentsply, Maillefer, Ballaigues) inserted in to the canal until it was seen at the apical foramen. The working length was determined by subtracting 1 mm from this length. A size 15 file was percolated through the apical foramen before and after the root canal preparation to attain the apical patency. The root canals were instrumented using step-back technique to ISO size 40 master apical file within 1 mm of the apex.
A volume of 2 ml of saline solution were used for irrigation between each file size. After the completion of the preparation procedure, the teeth were randomly divided into one experimental group consisting of 24 samples, two positive and two negative control groups. The canals were dried using paper point (Sina Dent, Iran). The root canals were filled using lateral compaction technique as described below.
The ZOE sealer (Gholchai, Iran) was mixed according to the manufacturer instructions and introduced into the canal using a size 30 file with counter clock wise rotation. Size 40 master Gutta-percha (GP) cones (Diadent, Korea), lightly coated with sealer and was then placed to the full working length. Lateral compaction was achieved using size 25 accessory GP cones and a size B finger spreader (Dentsply Maillefer) that initially reached to within 1 mm of the working length.
The two positive control teeth were obturated as the same manner as experimental teeth but without any sealer. The two negative control teeth were obturated with GP cones and ZOE sealer. After the obturation procedures were completed, all roots were stored at 100% humidity for 24 h in order to allow complete setting of the sealer cement. Before the evaluation of the microleakage, the excess coronal GP was removed with a Peeso Reamer ISO size 3 (approximately 5 mm GP was remained in each root canal.). Apical sealing ability of the obturated canals was then assessed, using electro-chemical and bacterial micro leakage tests.
Electro-chemical micro leakage test
The exterior surfaces of the teeth were completely covered with two coats of nail varnish except for the access opening and the apical foramen. The root surfaces in the negative controls were entirely covered with two coats of nail varnish.
The roots were mounted with silicon through the bottom of plastic cylinders leaving the open access opening inside the cylinder. The cylinders were filled with saline as electrolyte. The cylinders with teeth were mounted in Petri dish More Detailses filled with saline electrolyte. Only 2 mm of the root endings were immersed in the solution.
For measurement, a #70 k file (Dentsply, Maillefer, Ballaigues) was placed in each upper chamber and a stainless steel wire was inserted into the Petri dish. The electrode in each upper chamber was separately connected to the electrode in the lower chamber through an electric circuit with an 8-v, DC power supply (Z-IC, 8V1A. Siehe ECA). The electrical conductivity in this circuit was measured in μA with a multimeter (Case, Japan) for each root.
Bacterial microleakage test
The teeth were inserted in to the plastic Eppendorf test tubes with screw caps and then fixed through it. A volume of 2 ml of sterile culture medium (Tripticase Soy Broth (TSB); Merck, Darmstadt, Germany) were added to sterile glass test tubes. The Eppendorf root assembly was mounted inside the test tube with the root tip contacting the culture medium.
A standard Enterococcus faecalis bacterium was cultured in Tripticase Soy Agar and then microbial suspension, with 0/5 McFarland score for turbidity, was prepared in TSB.
Using a sterile micropipette, the microbial suspension was placed into the Eppendorf test tubes in contact with the coronal access opening of the filled roots. The test assemblies were incubated at 37°C. The turbidity of the culture medium in the lower chamber was monitored daily for 20 days. As the turbidity was observed, microbial samples were cultured again to confirm the presence of E. faecalis bacterium.
The data were analyzed using SPSS software (SPSS ver. 11, Statistical Package for Social Science, IBM Corporation, NY-USA). The statistical significance was set as a 0.05 level. The correlation of the electro chemical readings with the results obtained from bacterial micro leakage test was evaluated by Pearson's correlation coefficient.
| Results|| |
Electro chemical method
The teeth in the negative control group showed no flow of electrical current. In the positive control group maximum current flow was recorded (90 μA). The results in the experimental group ranged from 10 μA to 60 μA.
Bacterial infiltration method
As expected; the negative controls showed no significant infiltration during the experimental period. Samples in positive control group showed infiltration after 1 day of contamination. In the experimental group, infiltration occurred between day 4 and day 17.
The results of leakage, obtained by electro chemical method and bacterial infiltration method were compared using Pearson's correlation coefficient [Figure 1]. Since, the correlation coefficient of the measurements obtained from the two aforementioned methods, was 0/23 (r = 0/23), the correlation was not statistically significant (P = 0.275).
|Figure 1: Scattering diagram of microbial penetration results and electrochemical leakage evaluation (r = 0.23)|
Click here to view
| Discussion|| |
Various methods have been developed to assess the sealing ability of root canal filling materials. ,,,, The previous studies that measured and compared different methods for evaluation of the leakage, mostly, failed to show any correlation. , Barthel et al.  applied the dye leakage test after the bacterial test on the same teeth and found no correlation between these two tests. Pommel et al.  also compared fluid filtration, electro chemical and dye leakage tests for assessing the sealing ability of two obturation techniques, using the same teeth. They found no correlation among the tests. They described that this result was not surprising because the leakage phenomena are dependent to different factors.
In Modaresi et al.  study, electrochemical method was compared with dye penetration method. No correlation was found between two techniques. Delivanis and Chapman  compared the electrochemical method to the dye penetration or the radioisotope method. They found a correlation, but only at the two ends of the electric score range. Martell and Chandler  compared three root end restorative materials using the electrochemical and dye penetration methods and found a correlation between two methods. A study by Wu et al.  compared bacterial penetration to the fluid transport along root canal fillings. They found no correlation between two methods.
In the present study, the quantitative measurements recorded by electrochemical method and bacterial penetration method gave contradictory results. This may be due to the differences in working principles of various tests methods.
The electrochemical method is based on the diffusion of ions through very narrow spaces and the outcome of this method likely depends on electrical laws.  It is assumed that the magnitude of the electrical current that produced by ions diffusion, between two electrodes is directly proportional to the degree of leakage. Any change in ion concentration can affect the results.
Seidler  has emphasized that all sealers undergo dimensional changes, these changes occur upon setting and dissolution in fluids. Dissolution of inorganic salts that used in sealers formulation may affect the ionic concentration.
Another parameter that could be measured in the electrochemical test is electrical resistance. Resistance and leakage related to each other reversely. As the leakage increases, the electrical resistance value declines.
Jacobson and von Fraunhofer  applied two different types of metal as electrode (stainless steel and copper). This procedure may lead to an electrical potential that creates between two electrodes and effects on our measurements. The results of electrochemical microleakage tests varied considerably. This may be partly because of differences in the composition of the electrolyte, electrode type and distance between the two electrodes, electrode thickness and electrical conductivity of the ionic solution.
We have used saline solution, as electrolyte, in our study because; osmotic pressure and ionic composition of saline solution are relatively similar to the interstitial fluid and may not interfere with the results obtained by electrochemical tests, as opposed to NaOCL solution. The ionic concentration in hypochlorite solutions is very high especially in thick ones. This may be impact on the electrical conductivity that measured by electrochemical test. Since the electrochemical method did not destroy the tooth structure, we could assess leakage in one sample, repeatedly.
According to Timpawat et al.,  use of bacteria for assessing the leakage (mainly coronal) is considered to be of greater clinical and biological relevance than the dye penetration method. Many different strains of bacteria have been used to detect marginal leakage and this has led to contradictory results, because the methods depend on the type of bacteria used. A reference E. faecalis strain was chosen because they are part of normal oral flora in humans and are frequently found in mixed infections with other aerobes and facultative anaerobes. ,,, E. faecalis is also one of the most commonly isolated microbes from the root canal.  In this study, E. faecalis was selected due to ease of arrangement and interpretation of the data.
According to the results of this study there was not a significant correlation between electrochemical and bacterial penetration tests for evaluation of the leakage. Thus, the clinical relevance of leakage evaluation in vitro may be questioned. Moreover, lack of the correlation between the two methods, which were applied in this study, is likely related to the differences in criteria. So that it is proposed that several methods of evaluation should be used, to have several sets of data before drawing any conclusion.
| Acknowledgments|| |
Special thanks are given to Dr. Fatemeh Mokhtari for her contributions to the manuscript.
| References|| |
|1.||Moogi PP, Rao RN. Cleaning and shaping the root canal with an Nd: YAG laser beam: A comparative study. J Conserv Dent 2010;13:84-8. |
|2.||Saatchi M, Barekatain B, Behzadian M. Comparing the apical microleakage of lateral condensation and chloroform dip techniques with a new obturation method. Dent Res J (Isfahan) 2011;8:22-7. |
|3.||Brosco VH, Bernardineli N, Torres SA, Consolaro A, Bramante CM, de Moraes IG, et al. Bacterial leakage in root canals obturated by different techniques. Part 1: Microbiologic evaluation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e48-53. |
|4.||Williamson AE, Marker KL, Drake DR, Dawson DV, Walton RE. Resin-based versus gutta-percha-based root canal obturation: Influence on bacterial leakage in an in vitro model system. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:292-6. |
|5.||Veríssimo DM, do Vale MS. Methodologies for assessment of apical and coronal leakage of endodontic filling materials: A critical review. J Oral Sci 2006;48:93-8. |
|6.||Xu Q, Fan MW, Fan B, Cheung GS, Hu HL. A new quantitative method using glucose for analysis of endodontic leakage. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:107-11. |
|7.||Timpawat S, Amornchat C, Trisuwan WR. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36-9. |
|8.||Jacobson SM, von Fraunhofer JA. The investigation of microleakage in root canal therapy. An electrochemical technique. Oral Surg Oral Med Oral Pathol 1976;42:817-23. |
|9.||Delivanis PD, Chapman KA. Comparison and reliability of techniques for measuring leakage and marginal penetration. Oral Surg Oral Med Oral Pathol 1982;53:410-6. |
|10.||Inan U, Aydemir H, Taºdemir T. Leakage evaluation of three different root canal obturation techniques using electrochemical evaluation and dye penetration evaluation methods. Aust Endod J 2007;33:18-22. |
|11.||Karagenç B, Gençoglu N, Ersoy M, Cansever G, Külekçi G. A comparison of four different microleakage tests for assessment of leakage of root canal fillings. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:110-3. |
|12.||Tasdemir T, Yesilyurt C, Ceyhanli KT, Celik D, Er K. Evaluation of apical filling after root canal filling by 2 different techniques. J Can Dent Assoc 2009;75:201a-201d. |
|13.||Williamson AE, Dawson DV, Drake DR, Walton RE, Rivera EM. Effect of root canal filling/sealer systems on apical endotoxin penetration: A coronal leakage evaluation. J Endod 2005;31:599-604. |
|14.||Pommel L, Jacquot B, Camps J. Lack of correlation among three methods for evaluation of apical leakage. J Endod 2001;27:347-50. |
|15.||Barthel CR, Moshonov J, Shuping G, Orstavik D. Bacterial leakage versus dye leakage in obturated root canals. Int Endod J 1999;32:370-5. |
|16.||Modaresi J, Davari AR, Daneshkazemi AR. Comparison of apical leakage patterns shown by two different methods. Artigo Internacional Original 2007;7:169-72. |
|17.||Martell B, Chandler NP. Electrical and dye leakage comparison of three root-end restorative materials. Quintessence Int 2002;33:30-4. |
|18.||Wu MK, De Gee AJ, Wesselink PR, Moorer WR. Fluid transport and bacterial penetration along root canal fillings. Int Endod J 1993;26:203-8. |
|19.||Seidler B. The technique and rational of filling root canals. N Y J Dent 1954;24:276-85. |
|20.||Baumgartner JC, Falkler WA Jr. Bacteria in the apical 5 mm of infected root canals. J Endod 1991;17:380-3. |
|21.||Brosco VH, Bernardineli N, Torres SA, Consolaro A, Bramante CM, de Moraes IG, et al. Bacterial leakage in obturated root canals-part 2: A comparative histologic and microbiologic analyses. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:788-94. |
|22.||Fransen JN, He J, Glickman GN, Rios A, Shulman JD, Honeyman A. Comparative assessment of ActiV GP/glass ionomer sealer, Resilon/Epiphany, and gutta-percha/AH plus obturation: A bacterial leakage study. J Endod 2008;34:725-7. |
|23.||Siqueira JF Jr, Rôças IN, Favieri A, Abad EC, Castro AJ, Gahyva SM. Bacterial leakage in coronally unsealed root canals obturated with 3 different techniques. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90:647-50. |