- Email: [email protected]
Bacterial Penetration After Obturation With Four Different Root Canal Sealers
Basic Research—Technology
Bacterial Penetration After Obturation With Four Different Root Canal Sealers Ali Çag˘ın Yücel, DDS, PhD,* Eda Güler, DDS,* Ahmet Umut Güler, DDS, PhD,† and Ertan Ertas¸, DDS, PhD* Abstract The aim of this study was to compare bacterial penetration after obturation with lateral compaction technique using four different root canal sealers. This study was performed on 100 teeth including negative control (n ⫽ 10), positive control (n ⫽ 10), and experimental groups (n ⫽ 80). 80 teeth were randomly divided into five groups of 20 teeth each and obturated with AH 26 (A), AH Plus (AP), Sealapex (S), Ketac-Endo (K) root canal sealers. Evaluation was carried out for 60 days. After 30 days of comparing the bacterial penetration values, total penetration was observed in 85% of the Group AP, and group K, 80% of the group S, and 75% of the group A. According to the results of 2 test, there was no statistically significant difference observed between any groups (p ⬎ 0.05). After 60 days of comparing the bacterial penetration values, total penetration was observed in 100% of the group AP, group K, and group S and 95% of the group A. It may be concluded that under the conditions of this study, there was no difference in the bacterial penetration of the four root canal sealers tested at 30 and 60 days. (J Endod 2006;32:890 – 893)
Key Words AH Plus, AH26, Bacterial penetration, Ketac-Endo, root canal sealer, Sealapex
From the *Department of Restorative Dentistry, and the Department of Prosthodontics, Ondokuz Mayıs University, Faculty of Dentistry, Samsun, Turkey. Address requests for reprint Dr. Ali Çag˘ın Yücel, Ondokuz Mayıs University, Faculty of Dentistry, Department of Restorative Dentistry, 55139 Kurupelit, Samsun, Turkey. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2006.04.002
C
omplete obturation of the pulp space with a biocompatible and inert material is considered to be an important part of root canal treatment. Although, there are a large number of filling materials and obturation techniques, the combination of guttapercha and a sealer is the most widely used in clinical practice (1). The aim of obturating the root canal system is to prevent penetration of bacteria and their products into the periradicular tissues and to seal within the root canal any irritants that cannot be fully removed. Many studies show that root canal fillings leak coronally in a relatively short time when exposed to the oral environment. Swanson and Madison (2) demonstrated extensive coronal leakage of dye along the canal wall and within the root canal filling material in coronally unsealed but obturated canals exposed to artificial saliva. This was later confirmed in vivo when Madison and Wilcox (3) demonstrated dye penetration along the entire length of obturated root canals exposed to the oral environment. Torabinejad et al. (4) stated that when the coronal part of the filled root canal system is exposed to oral flora, it may allow ingress of bacteria to the periapical tissues. More recently, Trope et al. (5) reported the penetration of bacterial endotoxin through the obturated root canal in as few as 21 days. Recently the significance of the integrity of the coronal seal has become more evident in the long-term success of root canal treatment (5). Some of the causes for a loss of the coronal seal after endodontic therapy are delay of placing a restoration, fracture of restoration, or post space preparation when the remaining apical section of root filling is inadequate (6). It has been suggested that leakage occurs between the root filling and the root canal wall (7), and the sealer plays an important role in preventing leakage. A wide variety of root canal sealers are available and these include cement based upon organic resin, calcium hydroxide, or glass ionomer. The purpose of this study was to compare coronal bacterial penetration after obturation with lateral compaction technique using four different sealers at 30 and 60 days.
†
890
Yücel et al.
Materials and Methods The sealer tested in this study were AH26 silver free (Dentsply DeTrey GmbH, Konstanz, Germany), AH Plus (Dentsply DeTrey), Sealapex (Kerr Italia S.p.A., Salerno, Italy) and Ketac-Endo (3M ESPE AG, Seefeld, Germany). There were 100 extracted single-rooted teeth selected, cleaned of extraneous tissue and calculus, and then rinsed and stored in distilled water. To ensure that all specimens were of the same length, they were resected 16 mm from the apex using a water-cooled diamond bur and then stored in deionized water. The canals were accessed and the working length (WL) was determined by inserting a #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) into the canal until it was just visible at the apical foramen, then subtracting 1 mm. Apical patency was maintained throughout instrumentation using a #15 K-file (Dentsply Maillefer). The canals were instrumented using a crown-down technique with rotary ProTaper nickel-titanium files (Dentsply Maillefer) to a master apical file size of finishing file No 3. and were irrigated between files with 2 ml of a freshly prepared solution of 5.25% sodium hypochlorite. After instrumentation, the smear layer was removed with 17% EDTA, followed by 5.25% NaOCl (8 –11). The canal was then dried with sterile paper points.
JOE — Volume 32, Number 9, September 2006
Basic Research—Technology These prepared Eppendorf tubes could be put in 30 ml glass tubes so that 2 mm of the root apex was submerged in the Brain Hearth Infusion (BHI) broth. The junction between the Eppendorf tube and glass tube was sealed tightly with cyanoacrylate glue and acrylic resin (Fig. 1). The system was sterilized using ethylene oxide gas for 12 h. To ensure sterilization the whole system was incubated at 37°C for 3 days. Any test apparatus that showed signs of turbidity in the BHI Broth was discarded. Enterococcus faecalis organism (OG1X) was cultured at 37°C for 24 h in BHI Broth. The bacterial suspension was adjusted to ensure that the amount of bacteria was 108 colony forming units (CFU)/ml by spectrophotometer (Biotech UV1101, Biochrom, Cambridge, UK) and colony counting. An inoculum of E. faecalis (0.1 ml, 107 CFU) was injected into the tubes using a micropipette. The tubes were incubated aerobically at 37°C for 60 days. An inoculum of E. faecalis in BHI was changed with fresh inoculum every 5 days. In this period, the color of BHI with phenol red in tubes changed red to yellow (Fig. 1), which was the indicator of bacterial penetration, was checked every 24 h. The tubes, which the yellow color was seen, were taken out of experiment and qualified as E. faecalis penetrate and growth. The days of the bacterial penetration was noted for evaluation. To confirm the purity of E. faecalis in the inoculum a sample was taken in these tubes and was cultivated on D-coccosel agar plates and incubated for 24 h at 37°C. The data were statistically analyzed using the 2 test. A value of p ⬍ 0.05 was statistically significant.
Figure 1. Test apparatus (A, no bacterial penetration; B, bacterial penetration).
Teeth were then randomly divided into four groups of 20 teeth. Specimens of all groups were obturated using lateral compaction which was described Gutmann and Witherspoon (12) by using standardized (usually size 35) and non-standardized (fine-medium size) gutta-percha cones (Dentsply Maillefer) as master and accessory cones, respectively. Accessory gutta-percha cones were added until the spreader penetrated into the coronal one-third of the root canal space. In the group A, AH26 was used as a sealer. AH Plus was used as a sealer for obturation of the group AP. In the group K, Ketac-Endo was used as a sealer. Sealapex was used as a sealer for obturation of the group S. After obturation, excess gutta-percha 1 mm below the coronal surface was removed. The 10 teeth that were instrumented but not obturated were used as positive controls to demonstrate bacterial leakage through the entire length of the canal. The negative control teeth (n ⫽ 10) were instrumented but not obturated and sealed externally with two layers of nail polish except coronal access. After obturation, radiographs of all specimens were taken in buccolingual and mesiodistal directions to evaluate the quality of the root canal filling with regard to homogeneity and apical extension. Specimens used this experiment had radiographically well-compacted fillings that extended to 1 mm short of the apical foramen. And then the all specimens were stored in 100% humidity for 7 days to ensure the sealer was set. The roots of 90 teeth (group A, AP, K, S, and positive control) were sealed with two coats of colored nail polish, except for the apical 3 mm around the apical foramen. The 10 teeth used as negative controls had the entire root surface sealed. The tapered end of 2 ml Eppendorf plastic tube (Eppendorf-Elkay, Shrewsbury, MA) were cut, and the obturated roots were inserted into the tubes until the roots protruded through the end. The junction between each tube and root was sealed with sticky wax to prevent leakage of the connection.
Results The positive controls demonstrated penetration after 1 day, where as the negative controls did not penetrate for the entire observation period. The bacterial penetration data are showed in Table 1. After 30 days of comparing the bacterial penetration values, total penetration was observed in 85% of the group AP, and group K, 80% of the group S, and 75% of the group A. The highest ratio of penetration in the all experimental groups was observed in group AP, and group S. According to the results of 2 test, there was no statistically significant difference observed between any groups (p ⬎ 0.05). The 2 test results are listed in Table 2. After 60 days of comparing the bacterial penetration values, total penetration was observed in 100% of the group AP, group K, and group S and 95% of the group A. However, there was no statistically significant difference observed between any groups (p ⬎ 0.05). (Table 2)
Discussion Numerous studies have attempted to assess in vitro leakage (4, 11, 13–23). However, there is a great deal of variability in the results and minimal consensus as to the most appropriate method to assess leakage (13, 24, 25). The bacterial leakage model is commonly used and ap-
TABLE 1. Day of Bacterial Penetration According to Sealers Number of penetration specimens Groups
n
Days 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
30
60
Positive Control Negative Control Group AP Group A Group S Group K
10 10
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
20 20 20 20
— — — —
— — — —
1 — — —
2 — 1 —
2 — 2 —
3 — 2 —
3 1 3 2
4 3 4 2
5 3 6 3
5 5 6 4
7 5 9 5
8 9 10 7
12 11 11 10
13 12 13 11
17 15 14 13
17 15 16 17
20 19 20 20
JOE — Volume 32, Number 9, September 2006
Bacterial Penetration After Obturation
891
Basic Research—Technology TABLE 2. Differences Between Groups According to the 2 Test in 30 Days and 60 Days Groups
n
Group AP Group K Group S Group A
20 20 20 20
30 days
60 days
Penetration
No Penetration
Penetration
No Penetration
17 (85%) 17 (85%) 16 (80%) 15 (75%)
3 (15%) 3 (15%) 4 (20%) 5 (25%)
20 (100%) 20 (100%) 20 (100%) 19 (95%)
0 (0%) 0 (0%) 0 (0%) 1 (5%)
Vertical lines connect groups that are not significantly different at p ⬍ 0.05.
pears most clinically relevant. The model described by Malone & Donnelly (26), Barthel et al. (27) was chosen in the present study. Facultative bacteria are predominant found in failed previously treated root canals (28), with the most frequently isolated bacteria being enterococci (29). Furthermore, E. faecalis are part of normal flora in humans and are frequently found in mixed infections with other aerobes and facultative anaerobes (30, 31). Therefore; E. faecalis was used as the test bacteria for evaluation of leakage in this study. In the present study, teeth with single straight canals were chosen as they may be prepared in a standardized manner with rotary instruments. AH Plus initially exhibited the fastest bacterial penetration of all experimental groups. Bacterial penetrations were firstly determined at the third days for AH Plus, fourth days for Sealapex, and seventh days for AH 26 and Ketac-Endo. Although, Sealapex exhibited slower bacterial penetration than AH Plus, faster than AH 26 and Ketac-Endo groups (Table 1). According to the results at 30 and 60 days, there were no statistically differences among root canal sealers tested from the view of bacterial penetration. Zmener et al. (32) evaluated that sealing properties of AH Plus and AH 26 by dye penetration techniques. They stated that AH Plus more leaked than AH 26 at 10 days period. Orucoglu et al. (33) demonstrated that AH Plus allowed greater leakage than Diaket using a computerized fluid filtration meter technique at seven days period. Cobankara et al. (34) evaluated that the apical sealing of different root canal sealers and showed better apical sealing in Sealapex group when compared with AH Plus group at 21 days. These studies are in contrast with the present results because there was no statistical difference at 30 and 60 days. This contrast may probably stem from the fact that the experiment periods of the above mentioned studies are shorter than that of present study. Williamson et al. (35) evaluated that the effect of root canal filling technique and sealer type on the penetration of endotoxin from a mixed anaerobic bacterial community through the apical foramen of root canal treated teeth. Their results showed that endotoxin leaks more readily through the root canal system in root canal treated teeth using AH 26 as the sealer. A number of factors could explain the differences in the result such as instrumentation and obturation techniques, the type of microorganisms used, experimental design and the nature of the irrigant to which the canal was exposed (36). On the other hand, the result of this study showed the slowest bacterial penetration was observed in samples sealed with the AH26 and Ketac-Endo cements, the differences among them were not statistically significant. AH 26 might be explained by the sealer is an epoxy-resin based material and showed good sealing ability even when used as the sole filling in a root canal (37). The long setting time and fluidity of materials result in no cracking or rapid separation from dentinal walls (38). It also has the ability to solidify in a wet medium. It is bioinert, and during penetration into lateral canals it showed a contraction of less than 0.5%. The findings of present study are similar to results of Miletic et al. (39). They evaluated the sealing ability in vitro and found that AH 26 had the least leakage and there were no statistically difference between AH 26 and AH Plus. Regarding the 892
Yücel et al.
effect of Ketac-Endo, it seems that the antimicrobial activity is based on fluoride-ion release rather than a subsequent variation in pH. Timpawat et al. (40) evaluated bacterial leakage in vitro and found that no significant difference was observed between Ketac-Endo and AH Plus at 60 days. Our results are similar. All samples were bacterial penetrating after 60 days of observation. Clinically, these findings point to the need to provide a suitable coronal restoration after the root canal treatment has been completed. This may enhance the protection from contamination of the root canal system by microorganisms. From a clinical standpoint, coronal exposure of the root canal obturation for a relatively short time might be considered an indication for endodontic retreatment. It may be concluded that under the conditions of this study; although, bacterial penetrations were firstly determined at different time intervals for different sealers, there was no difference in the bacterial penetration of the four root canal sealers tested at 30 and 60 days.
References 1. Nguyen NT. Obturation of the root canal system. In: Cohen S, Burns RC, eds. Pathways of the pulp, 5th ed. St Louis, MO: CV Mosby, 1991:199 –210. 2. Swanson K, Madison S. An evaluation of coronal microleakage in endodontically treated teeth. Part I. Time periods. J Endod 1987;13:56 –9. 3. Madison S, Wilcox LR. An evaluation of coronal microleakage in endodontically treated teeth. Part III. In vivo study. J Endod 1988;14:455– 8. 4. Torabinejad M, Ung B, Kettering JD. In vitro bacterial penetration of coronally unsealed endodontically treated teeth. J Endod 1990;16:566 –9. 5. Trope M, Chow E, Nissan R. In vitro endotoxin penetration of coronally unsealed endodontically treated teeth. Endod Dent Traumatol 1995;11:90 – 4. 6. Saunders WP, Saunders EM. Coronal leakage as a cause of failure in root canal treatment: a review. Endod Dent Traumatol 1994;10:105– 8. 7. Hovland EJ, Dumsha TJ. Leakage evaluation in vitro of the root canal sealer Sealapex. Int Endod J 1985;18:179 – 82. 8. Yamada RS, Armas A, Goldman M, Sun Lin P. A scanning electron microscopic comparison of a high volume flush with several irrigating solutions. Part 3. J Endod 1983;9:137– 42. 9. Ciucchi B, Khettabi M, Holtz J. The effectiveness of different endodontic irrigation procedures on the removal of the smear layer: a scanning electron microscope study. Int Endod J 1986;22:21– 8. 10. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 11. Gutmann JL. Adaptation of injected thermoplasticized gutta-percha in the absence of the dentinal smear layer. Int Endod J 1993;26:87–92. 12. Gutmann JL, Witherspoon DE. Obturation of the cleaned and shaped root canal system. In: Cohen S, Burns RC, eds. Pathways of the pulp, 8th ed. St Louis: Mosby, 2002;293–364. 13. Wu MK, Wesselink PR. Endodontic leakage studies reconsidered. Part I. Methodology, application and relevancy. Int Endod J 1993;26:37– 43. 14. Peters LB, Harrison JW. A comparison of leakage of filling materials in demineralized and non demineralized resected root ends under vacuum and non-vacuum conditions. Int Endod J 1992;25:273– 8. 15. Scott AC, Vire DE, Swanson R. An evaluation of the Thermafil endodontic obturation technique. J Endod 1992;18:340 –3. 16. Dummer PMH, Kelly T, Meghji A, Sheikh I, Vanitchai JT. An in vitro study of the quality of root fillings in teeth obturated by lateral condensation of gutta-percha or Thermafil obturators. Int Endod J 1993;26:99 –105. 17. Baumgardner KR, Taylor J, Walton R. Canal adaptation and coronal leakage: lateral condensation compared to Thermafil. JADA 1995;126:351– 6.
JOE — Volume 32, Number 9, September 2006
Basic Research—Technology 18. Lloyd A, Thompson J, Gutmann JL, Dummer PMH. Sealability of the Trifecta technique in the presence or absence of a smear layer. Int Endod J 1995;28:35– 41. 19. Hovland EJ, Dumsha TC. Leakage evaluation in vitro of the root canal sealer Sealapex. Int Endod J 1985;18:179 – 82. 20. Rhome BH, Solomon EA, Rabinowitz JL. Isotopic evaluation of the sealing properties of lateral condensation, vertical condensation, and Hydron. J Endod 1981;7:458 – 61. 21. Wu MK, Degee AJ, Wesselink PR, Moorer WR. Fluid transport and bacterial penetration along root canal fillings. Int Endod J 1993;26:203– 8. 22. Khayat A, Lee SJ, Torabinejad M. Human saliva penetration of coronally unsealed obturated root canals. J Endod 1993;19:458 – 61. 23. Behrend GD, Cutler CW, Gutmann JL. An in-vitro study of smear layer removal and microbial leakage along root canal fillings. Int Endod J 1996;29:99 –107. 24. Goldman M, Simmonds S, Rush R. The usefulness of dye-penetration studies reexamined. Oral Surg Oral Med Oral Pathol 1989;67:327–32. 25. Kersten HW, Moorer WR. Particles and molecules in endodontic leakage. Int Endod J 1989;22:118 –24. 26. Malone KH, Donnelly JC. An in vitro evaluation of coronal microleakage in obturated root canals without coronal restoration. J Endod 1997;23:35– 8. 27. Barthel CR, Moshonov J, Shuping G, Orstavik D. Bacterial leakage versus dye leakage in obturated root canals. Int Endod J 1999;32:370 –5. 28. Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1–7. 29. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257– 62.
JOE — Volume 32, Number 9, September 2006
30. Baumgartner JC, Falkler WA. Bacteria in the apical 5 mm of infected root canals. J Endod 1991;17:380 –3. 31. Timpavat S, Amornchat C, Trisuwan W. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36 –9. 32. Zmener O, Spielberg C, Lamberghini F, Rucci M. Sealing properties of a new epoxybased root canal sealer. Int Endod J 1997;30:332– 4. 33. Orucoglu H, Sengun A, Yilmaz N. Apical leakage of resin based root canal sealers with a new computerized fluid filtration meter. J Endod 2005;31:886 –90. 34. Cobankara FK, Orucoglu H, Sengun A, Belli S. The quantitative evaluation of apical sealing of four endodontic sealers. J Endod 2006;32:66 – 8. 35. 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. 36. Chailertvanitkul P, Saunders WP, Mackenzie D. An assessment of microbial coronal leakage in teeth root filled with gutta-percha and three different sealers. Int Endod J 1996;29:387–92. 37. Wu M-K, Dee Gee AJ, Wesselink PR. Leakage of four root canal sealers at different thicknesses. Int Endod J 1994;27:304 – 8. 38. Dee Gee AJ, Wu M-K, Wesselink PR. Sealing Properties of Ketac-Endo glass ionomer cement and AH26 root canal sealers. Int Endod J 1999;32:415– 8. 39. Miletic I, Prpic-Mehicic G, Marsan T, et al. Bacterial and fungal microleakage of AH 26 and AH Plus root canal sealers. Int Endod J 2002;35:428 –32. 40. Timpawat S, Amornchat C, Trisuwan W. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36 –9.
Bacterial Penetration After Obturation
893
Bacterial Penetration After Obturation With Four Different Root Canal Sealers Ali Çag˘ın Yücel, DDS, PhD,* Eda Güler, DDS,* Ahmet Umut Güler, DDS, PhD,† and Ertan Ertas¸, DDS, PhD* Abstract The aim of this study was to compare bacterial penetration after obturation with lateral compaction technique using four different root canal sealers. This study was performed on 100 teeth including negative control (n ⫽ 10), positive control (n ⫽ 10), and experimental groups (n ⫽ 80). 80 teeth were randomly divided into five groups of 20 teeth each and obturated with AH 26 (A), AH Plus (AP), Sealapex (S), Ketac-Endo (K) root canal sealers. Evaluation was carried out for 60 days. After 30 days of comparing the bacterial penetration values, total penetration was observed in 85% of the Group AP, and group K, 80% of the group S, and 75% of the group A. According to the results of 2 test, there was no statistically significant difference observed between any groups (p ⬎ 0.05). After 60 days of comparing the bacterial penetration values, total penetration was observed in 100% of the group AP, group K, and group S and 95% of the group A. It may be concluded that under the conditions of this study, there was no difference in the bacterial penetration of the four root canal sealers tested at 30 and 60 days. (J Endod 2006;32:890 – 893)
Key Words AH Plus, AH26, Bacterial penetration, Ketac-Endo, root canal sealer, Sealapex
From the *Department of Restorative Dentistry, and the Department of Prosthodontics, Ondokuz Mayıs University, Faculty of Dentistry, Samsun, Turkey. Address requests for reprint Dr. Ali Çag˘ın Yücel, Ondokuz Mayıs University, Faculty of Dentistry, Department of Restorative Dentistry, 55139 Kurupelit, Samsun, Turkey. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2006.04.002
C
omplete obturation of the pulp space with a biocompatible and inert material is considered to be an important part of root canal treatment. Although, there are a large number of filling materials and obturation techniques, the combination of guttapercha and a sealer is the most widely used in clinical practice (1). The aim of obturating the root canal system is to prevent penetration of bacteria and their products into the periradicular tissues and to seal within the root canal any irritants that cannot be fully removed. Many studies show that root canal fillings leak coronally in a relatively short time when exposed to the oral environment. Swanson and Madison (2) demonstrated extensive coronal leakage of dye along the canal wall and within the root canal filling material in coronally unsealed but obturated canals exposed to artificial saliva. This was later confirmed in vivo when Madison and Wilcox (3) demonstrated dye penetration along the entire length of obturated root canals exposed to the oral environment. Torabinejad et al. (4) stated that when the coronal part of the filled root canal system is exposed to oral flora, it may allow ingress of bacteria to the periapical tissues. More recently, Trope et al. (5) reported the penetration of bacterial endotoxin through the obturated root canal in as few as 21 days. Recently the significance of the integrity of the coronal seal has become more evident in the long-term success of root canal treatment (5). Some of the causes for a loss of the coronal seal after endodontic therapy are delay of placing a restoration, fracture of restoration, or post space preparation when the remaining apical section of root filling is inadequate (6). It has been suggested that leakage occurs between the root filling and the root canal wall (7), and the sealer plays an important role in preventing leakage. A wide variety of root canal sealers are available and these include cement based upon organic resin, calcium hydroxide, or glass ionomer. The purpose of this study was to compare coronal bacterial penetration after obturation with lateral compaction technique using four different sealers at 30 and 60 days.
†
890
Yücel et al.
Materials and Methods The sealer tested in this study were AH26 silver free (Dentsply DeTrey GmbH, Konstanz, Germany), AH Plus (Dentsply DeTrey), Sealapex (Kerr Italia S.p.A., Salerno, Italy) and Ketac-Endo (3M ESPE AG, Seefeld, Germany). There were 100 extracted single-rooted teeth selected, cleaned of extraneous tissue and calculus, and then rinsed and stored in distilled water. To ensure that all specimens were of the same length, they were resected 16 mm from the apex using a water-cooled diamond bur and then stored in deionized water. The canals were accessed and the working length (WL) was determined by inserting a #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) into the canal until it was just visible at the apical foramen, then subtracting 1 mm. Apical patency was maintained throughout instrumentation using a #15 K-file (Dentsply Maillefer). The canals were instrumented using a crown-down technique with rotary ProTaper nickel-titanium files (Dentsply Maillefer) to a master apical file size of finishing file No 3. and were irrigated between files with 2 ml of a freshly prepared solution of 5.25% sodium hypochlorite. After instrumentation, the smear layer was removed with 17% EDTA, followed by 5.25% NaOCl (8 –11). The canal was then dried with sterile paper points.
JOE — Volume 32, Number 9, September 2006
Basic Research—Technology These prepared Eppendorf tubes could be put in 30 ml glass tubes so that 2 mm of the root apex was submerged in the Brain Hearth Infusion (BHI) broth. The junction between the Eppendorf tube and glass tube was sealed tightly with cyanoacrylate glue and acrylic resin (Fig. 1). The system was sterilized using ethylene oxide gas for 12 h. To ensure sterilization the whole system was incubated at 37°C for 3 days. Any test apparatus that showed signs of turbidity in the BHI Broth was discarded. Enterococcus faecalis organism (OG1X) was cultured at 37°C for 24 h in BHI Broth. The bacterial suspension was adjusted to ensure that the amount of bacteria was 108 colony forming units (CFU)/ml by spectrophotometer (Biotech UV1101, Biochrom, Cambridge, UK) and colony counting. An inoculum of E. faecalis (0.1 ml, 107 CFU) was injected into the tubes using a micropipette. The tubes were incubated aerobically at 37°C for 60 days. An inoculum of E. faecalis in BHI was changed with fresh inoculum every 5 days. In this period, the color of BHI with phenol red in tubes changed red to yellow (Fig. 1), which was the indicator of bacterial penetration, was checked every 24 h. The tubes, which the yellow color was seen, were taken out of experiment and qualified as E. faecalis penetrate and growth. The days of the bacterial penetration was noted for evaluation. To confirm the purity of E. faecalis in the inoculum a sample was taken in these tubes and was cultivated on D-coccosel agar plates and incubated for 24 h at 37°C. The data were statistically analyzed using the 2 test. A value of p ⬍ 0.05 was statistically significant.
Figure 1. Test apparatus (A, no bacterial penetration; B, bacterial penetration).
Teeth were then randomly divided into four groups of 20 teeth. Specimens of all groups were obturated using lateral compaction which was described Gutmann and Witherspoon (12) by using standardized (usually size 35) and non-standardized (fine-medium size) gutta-percha cones (Dentsply Maillefer) as master and accessory cones, respectively. Accessory gutta-percha cones were added until the spreader penetrated into the coronal one-third of the root canal space. In the group A, AH26 was used as a sealer. AH Plus was used as a sealer for obturation of the group AP. In the group K, Ketac-Endo was used as a sealer. Sealapex was used as a sealer for obturation of the group S. After obturation, excess gutta-percha 1 mm below the coronal surface was removed. The 10 teeth that were instrumented but not obturated were used as positive controls to demonstrate bacterial leakage through the entire length of the canal. The negative control teeth (n ⫽ 10) were instrumented but not obturated and sealed externally with two layers of nail polish except coronal access. After obturation, radiographs of all specimens were taken in buccolingual and mesiodistal directions to evaluate the quality of the root canal filling with regard to homogeneity and apical extension. Specimens used this experiment had radiographically well-compacted fillings that extended to 1 mm short of the apical foramen. And then the all specimens were stored in 100% humidity for 7 days to ensure the sealer was set. The roots of 90 teeth (group A, AP, K, S, and positive control) were sealed with two coats of colored nail polish, except for the apical 3 mm around the apical foramen. The 10 teeth used as negative controls had the entire root surface sealed. The tapered end of 2 ml Eppendorf plastic tube (Eppendorf-Elkay, Shrewsbury, MA) were cut, and the obturated roots were inserted into the tubes until the roots protruded through the end. The junction between each tube and root was sealed with sticky wax to prevent leakage of the connection.
Results The positive controls demonstrated penetration after 1 day, where as the negative controls did not penetrate for the entire observation period. The bacterial penetration data are showed in Table 1. After 30 days of comparing the bacterial penetration values, total penetration was observed in 85% of the group AP, and group K, 80% of the group S, and 75% of the group A. The highest ratio of penetration in the all experimental groups was observed in group AP, and group S. According to the results of 2 test, there was no statistically significant difference observed between any groups (p ⬎ 0.05). The 2 test results are listed in Table 2. After 60 days of comparing the bacterial penetration values, total penetration was observed in 100% of the group AP, group K, and group S and 95% of the group A. However, there was no statistically significant difference observed between any groups (p ⬎ 0.05). (Table 2)
Discussion Numerous studies have attempted to assess in vitro leakage (4, 11, 13–23). However, there is a great deal of variability in the results and minimal consensus as to the most appropriate method to assess leakage (13, 24, 25). The bacterial leakage model is commonly used and ap-
TABLE 1. Day of Bacterial Penetration According to Sealers Number of penetration specimens Groups
n
Days 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
30
60
Positive Control Negative Control Group AP Group A Group S Group K
10 10
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
10 —
20 20 20 20
— — — —
— — — —
1 — — —
2 — 1 —
2 — 2 —
3 — 2 —
3 1 3 2
4 3 4 2
5 3 6 3
5 5 6 4
7 5 9 5
8 9 10 7
12 11 11 10
13 12 13 11
17 15 14 13
17 15 16 17
20 19 20 20
JOE — Volume 32, Number 9, September 2006
Bacterial Penetration After Obturation
891
Basic Research—Technology TABLE 2. Differences Between Groups According to the 2 Test in 30 Days and 60 Days Groups
n
Group AP Group K Group S Group A
20 20 20 20
30 days
60 days
Penetration
No Penetration
Penetration
No Penetration
17 (85%) 17 (85%) 16 (80%) 15 (75%)
3 (15%) 3 (15%) 4 (20%) 5 (25%)
20 (100%) 20 (100%) 20 (100%) 19 (95%)
0 (0%) 0 (0%) 0 (0%) 1 (5%)
Vertical lines connect groups that are not significantly different at p ⬍ 0.05.
pears most clinically relevant. The model described by Malone & Donnelly (26), Barthel et al. (27) was chosen in the present study. Facultative bacteria are predominant found in failed previously treated root canals (28), with the most frequently isolated bacteria being enterococci (29). Furthermore, E. faecalis are part of normal flora in humans and are frequently found in mixed infections with other aerobes and facultative anaerobes (30, 31). Therefore; E. faecalis was used as the test bacteria for evaluation of leakage in this study. In the present study, teeth with single straight canals were chosen as they may be prepared in a standardized manner with rotary instruments. AH Plus initially exhibited the fastest bacterial penetration of all experimental groups. Bacterial penetrations were firstly determined at the third days for AH Plus, fourth days for Sealapex, and seventh days for AH 26 and Ketac-Endo. Although, Sealapex exhibited slower bacterial penetration than AH Plus, faster than AH 26 and Ketac-Endo groups (Table 1). According to the results at 30 and 60 days, there were no statistically differences among root canal sealers tested from the view of bacterial penetration. Zmener et al. (32) evaluated that sealing properties of AH Plus and AH 26 by dye penetration techniques. They stated that AH Plus more leaked than AH 26 at 10 days period. Orucoglu et al. (33) demonstrated that AH Plus allowed greater leakage than Diaket using a computerized fluid filtration meter technique at seven days period. Cobankara et al. (34) evaluated that the apical sealing of different root canal sealers and showed better apical sealing in Sealapex group when compared with AH Plus group at 21 days. These studies are in contrast with the present results because there was no statistical difference at 30 and 60 days. This contrast may probably stem from the fact that the experiment periods of the above mentioned studies are shorter than that of present study. Williamson et al. (35) evaluated that the effect of root canal filling technique and sealer type on the penetration of endotoxin from a mixed anaerobic bacterial community through the apical foramen of root canal treated teeth. Their results showed that endotoxin leaks more readily through the root canal system in root canal treated teeth using AH 26 as the sealer. A number of factors could explain the differences in the result such as instrumentation and obturation techniques, the type of microorganisms used, experimental design and the nature of the irrigant to which the canal was exposed (36). On the other hand, the result of this study showed the slowest bacterial penetration was observed in samples sealed with the AH26 and Ketac-Endo cements, the differences among them were not statistically significant. AH 26 might be explained by the sealer is an epoxy-resin based material and showed good sealing ability even when used as the sole filling in a root canal (37). The long setting time and fluidity of materials result in no cracking or rapid separation from dentinal walls (38). It also has the ability to solidify in a wet medium. It is bioinert, and during penetration into lateral canals it showed a contraction of less than 0.5%. The findings of present study are similar to results of Miletic et al. (39). They evaluated the sealing ability in vitro and found that AH 26 had the least leakage and there were no statistically difference between AH 26 and AH Plus. Regarding the 892
Yücel et al.
effect of Ketac-Endo, it seems that the antimicrobial activity is based on fluoride-ion release rather than a subsequent variation in pH. Timpawat et al. (40) evaluated bacterial leakage in vitro and found that no significant difference was observed between Ketac-Endo and AH Plus at 60 days. Our results are similar. All samples were bacterial penetrating after 60 days of observation. Clinically, these findings point to the need to provide a suitable coronal restoration after the root canal treatment has been completed. This may enhance the protection from contamination of the root canal system by microorganisms. From a clinical standpoint, coronal exposure of the root canal obturation for a relatively short time might be considered an indication for endodontic retreatment. It may be concluded that under the conditions of this study; although, bacterial penetrations were firstly determined at different time intervals for different sealers, there was no difference in the bacterial penetration of the four root canal sealers tested at 30 and 60 days.
References 1. Nguyen NT. Obturation of the root canal system. In: Cohen S, Burns RC, eds. Pathways of the pulp, 5th ed. St Louis, MO: CV Mosby, 1991:199 –210. 2. Swanson K, Madison S. An evaluation of coronal microleakage in endodontically treated teeth. Part I. Time periods. J Endod 1987;13:56 –9. 3. Madison S, Wilcox LR. An evaluation of coronal microleakage in endodontically treated teeth. Part III. In vivo study. J Endod 1988;14:455– 8. 4. Torabinejad M, Ung B, Kettering JD. In vitro bacterial penetration of coronally unsealed endodontically treated teeth. J Endod 1990;16:566 –9. 5. Trope M, Chow E, Nissan R. In vitro endotoxin penetration of coronally unsealed endodontically treated teeth. Endod Dent Traumatol 1995;11:90 – 4. 6. Saunders WP, Saunders EM. Coronal leakage as a cause of failure in root canal treatment: a review. Endod Dent Traumatol 1994;10:105– 8. 7. Hovland EJ, Dumsha TJ. Leakage evaluation in vitro of the root canal sealer Sealapex. Int Endod J 1985;18:179 – 82. 8. Yamada RS, Armas A, Goldman M, Sun Lin P. A scanning electron microscopic comparison of a high volume flush with several irrigating solutions. Part 3. J Endod 1983;9:137– 42. 9. Ciucchi B, Khettabi M, Holtz J. The effectiveness of different endodontic irrigation procedures on the removal of the smear layer: a scanning electron microscope study. Int Endod J 1986;22:21– 8. 10. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 11. Gutmann JL. Adaptation of injected thermoplasticized gutta-percha in the absence of the dentinal smear layer. Int Endod J 1993;26:87–92. 12. Gutmann JL, Witherspoon DE. Obturation of the cleaned and shaped root canal system. In: Cohen S, Burns RC, eds. Pathways of the pulp, 8th ed. St Louis: Mosby, 2002;293–364. 13. Wu MK, Wesselink PR. Endodontic leakage studies reconsidered. Part I. Methodology, application and relevancy. Int Endod J 1993;26:37– 43. 14. Peters LB, Harrison JW. A comparison of leakage of filling materials in demineralized and non demineralized resected root ends under vacuum and non-vacuum conditions. Int Endod J 1992;25:273– 8. 15. Scott AC, Vire DE, Swanson R. An evaluation of the Thermafil endodontic obturation technique. J Endod 1992;18:340 –3. 16. Dummer PMH, Kelly T, Meghji A, Sheikh I, Vanitchai JT. An in vitro study of the quality of root fillings in teeth obturated by lateral condensation of gutta-percha or Thermafil obturators. Int Endod J 1993;26:99 –105. 17. Baumgardner KR, Taylor J, Walton R. Canal adaptation and coronal leakage: lateral condensation compared to Thermafil. JADA 1995;126:351– 6.
JOE — Volume 32, Number 9, September 2006
Basic Research—Technology 18. Lloyd A, Thompson J, Gutmann JL, Dummer PMH. Sealability of the Trifecta technique in the presence or absence of a smear layer. Int Endod J 1995;28:35– 41. 19. Hovland EJ, Dumsha TC. Leakage evaluation in vitro of the root canal sealer Sealapex. Int Endod J 1985;18:179 – 82. 20. Rhome BH, Solomon EA, Rabinowitz JL. Isotopic evaluation of the sealing properties of lateral condensation, vertical condensation, and Hydron. J Endod 1981;7:458 – 61. 21. Wu MK, Degee AJ, Wesselink PR, Moorer WR. Fluid transport and bacterial penetration along root canal fillings. Int Endod J 1993;26:203– 8. 22. Khayat A, Lee SJ, Torabinejad M. Human saliva penetration of coronally unsealed obturated root canals. J Endod 1993;19:458 – 61. 23. Behrend GD, Cutler CW, Gutmann JL. An in-vitro study of smear layer removal and microbial leakage along root canal fillings. Int Endod J 1996;29:99 –107. 24. Goldman M, Simmonds S, Rush R. The usefulness of dye-penetration studies reexamined. Oral Surg Oral Med Oral Pathol 1989;67:327–32. 25. Kersten HW, Moorer WR. Particles and molecules in endodontic leakage. Int Endod J 1989;22:118 –24. 26. Malone KH, Donnelly JC. An in vitro evaluation of coronal microleakage in obturated root canals without coronal restoration. J Endod 1997;23:35– 8. 27. Barthel CR, Moshonov J, Shuping G, Orstavik D. Bacterial leakage versus dye leakage in obturated root canals. Int Endod J 1999;32:370 –5. 28. Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1–7. 29. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257– 62.
JOE — Volume 32, Number 9, September 2006
30. Baumgartner JC, Falkler WA. Bacteria in the apical 5 mm of infected root canals. J Endod 1991;17:380 –3. 31. Timpavat S, Amornchat C, Trisuwan W. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36 –9. 32. Zmener O, Spielberg C, Lamberghini F, Rucci M. Sealing properties of a new epoxybased root canal sealer. Int Endod J 1997;30:332– 4. 33. Orucoglu H, Sengun A, Yilmaz N. Apical leakage of resin based root canal sealers with a new computerized fluid filtration meter. J Endod 2005;31:886 –90. 34. Cobankara FK, Orucoglu H, Sengun A, Belli S. The quantitative evaluation of apical sealing of four endodontic sealers. J Endod 2006;32:66 – 8. 35. 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. 36. Chailertvanitkul P, Saunders WP, Mackenzie D. An assessment of microbial coronal leakage in teeth root filled with gutta-percha and three different sealers. Int Endod J 1996;29:387–92. 37. Wu M-K, Dee Gee AJ, Wesselink PR. Leakage of four root canal sealers at different thicknesses. Int Endod J 1994;27:304 – 8. 38. Dee Gee AJ, Wu M-K, Wesselink PR. Sealing Properties of Ketac-Endo glass ionomer cement and AH26 root canal sealers. Int Endod J 1999;32:415– 8. 39. Miletic I, Prpic-Mehicic G, Marsan T, et al. Bacterial and fungal microleakage of AH 26 and AH Plus root canal sealers. Int Endod J 2002;35:428 –32. 40. Timpawat S, Amornchat C, Trisuwan W. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36 –9.
Bacterial Penetration After Obturation
893