Bacterial Coronal Leakage after Obturation with Three Root Canal Sealers

Bacterial Coronal Leakage after Obturation with Three Root Canal Sealers

JOURNAL OF ENDODONTICS Copyright © 2001 by The American Association of Endodontists Printed in U.S.A. VOL. 27, NO. 1, JANUARY 2001 Bacterial Coronal...

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JOURNAL OF ENDODONTICS Copyright © 2001 by The American Association of Endodontists

Printed in U.S.A. VOL. 27, NO. 1, JANUARY 2001

Bacterial Coronal Leakage after Obturation with Three Root Canal Sealers Siriporn Timpawat, DDS, MS, Cholticha Amornchat, DDS, MS, and Wirong-rong Trisuwan, DDS, Grad. Dip. Clin. Sc.

and with various assays such as dyes, radioisotopes, or bacteria. Using bacteria as a leakage tracer will provide more biologically significant and clinically relevant information (4). In 1987 Madison et al. (5) found that microleakage in endodontically treated teeth by using artificial saliva would cause coronal leakage in 7 days, depending on type of sealer used. Later Torabinejad et al. (6) used Staphylococcus epidermidis and Proteus vulgaris, and found that root canals were completely contaminated after 19 days, whereas Khayat et al. (7) determined coronal leakage with natural human saliva would thoroughly penetrate the entire length of the root canal in ⬍30 days. Chailertvanitkul et al. (8) found that coronal leakage of an obligated anaerobe and its products into root canals obturated with gutta-percha and two sealers (AH26, DeTrey, Zurich, Switzerland; Tubliseal EWT, Kerr, Romulus, MI) was apparent within 12 wk. They reported later that 30% and 75% of Apexit (Vivadent, Schann, Liechteinstein) and Tubliseal EWT groups, respectively, showed bacterial (Anaerobic streptococcus and Prevotella intermedia) leakage at 90 days (9). To minimize the seal of the root canal system, sealers are used to eliminate the interface between gutta-percha and dentinal walls. Currently endodontic sealers containing glass ionomer have become commercially available. It has been shown that the glass ionomer sealer presents physiochemically properties comparable with other sealers commonly used in endodontics (10). Some studies showed that the glass-ionomer sealer provided a better seal than other sealers because it has the property to bond chemically to the hydroxyapatite of the root canal wall. (10, 11). Recent studies (12, 13) have shown that Ketac-Endo provides an effective apical seal when used with a single cone gutta-percha obturation method. The purpose of this study was to clarify the coronal seal of the root canal using Enterococcus faecalis as a bacterial marker and the effectiveness of coronal seals of different types of root canal sealer when used with gutta-percha filling material. The three different types of root canal sealers were epoxy root canal sealer (AH-Plus, Detrey, Zurich, Switzerland), calcium hydroxide root canal sealer (Apexit), and GIC root canal sealer (Ketac-Endo, Espe, Seefield, Germany).

The purpose of this study was to compare the bacterial leakage of root canals obturated with three root canal sealers, using Endodontalis faecalis as a microbial tracer to determine the length of time for bacteria to penetrate through the obturated root canal to the root apex. Seventy-five, single-rooted teeth with straight root canals had the crown cut off at the cementoenamel junction. Root canals were instrumented by a step-back technique. The prepared teeth were randomly divided into 3 groups of 19 teeth each and another 2 groups as positive and negative controls (9 teeth each). The experimental groups were dependent on the sealer used: AH-Plus, Apexit, and Ketac-Endo. The root canals were obturated using a lateral condensation technique. After 24 h the teeth were attached to microcentrifuge tubes with 2 mm of the root apex submerged in Brain Heart Infusion broth in glass test tubes. The coronal portions of the root canal filling materials were placed in contact with E. faecalis. The teeth were observed for bacterial leakage daily for 30 and 60 days. With the ␹2 test for comparing pairs of groups at the 0.05 level (p < 0.05), there was no statistical difference between Ketac-Endo and AH-Plus (p > 0.05), but Apexit had significantly higher leakage (p < 0.05) at 30 days. After 60 days there was no statistical difference between Ketac-Endo and Apexit (p > 0.05), but Apexit leaked more than AH-Plus. The conclusion drawn from this experiment was that epoxy resin root canal sealer was found to be more adaptable to the root canal wall and filling material than a calcium hydroxide sealer when bacterial coronal leakage was studied.

Obturation of the root canal in three dimension is an important factor for root canal success, although several factors are related to treatment failures, including inadequate apical and coronal seal, over- or underinstrumentation, and procedural errors (1). It has been indicated that both apical and coronal leakage may lead to loss of more endodontically treated teeth than other failures of endodontic therapy (2, 3). Many studies have been performed to evaluate coronal leakage

MATERIALS AND METHODS Seventy-five caries free, human maxillary incisors and canines with straight roots were used in this study. The root surfaces were scraped with a scalpel to remove calculus or soft tissue. The teeth were kept in 2.5% NaOCl solution overnight for surface disin36

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TABLE 1. No. of teeth and percentage of total leakage of obturated root canal after exposure to E. faecalis for 30 days Type of Cement

n

Leakage (%)

No Leakage (%)

Range (days)

AH-Plus Apexit Ketac-Endo Negative control Positive control

16 17 15 9 8

25.0 (4†) 58.8 (10) 13.3 (2) 0 (0) 100 (8)

75.0 (12) 41.2 (7) 86.7 (13) 100 (9) 0 (0)

21–30 21–30 16–30 — —

]*

]*

* Significant difference, p ⬍ 0.05. † Number of teeth are in parentheses.

fection and later stored in phosphoric buffer saline solution before the study. The crowns were removed at the cementoenamel junction, with a tapered fissure carbide bur in a highspeed handpiece under water spray. The root lengths were standardized at 13 mm, facilitating instrumentation and evaluation. Instrumentation of the canals was performed serially to a size 40 K-file (Kerr/Sybron, Romulus, MI) as a master apical file (MAF) and then using a step-back technique for the subsequent three file sizes larger than the MAF. An apical stop was designated 1 mm short of the point at which a #15 file exited the apical foramen. All instrumentation was accompanied by copious irrigation with 2.5% NaOCl delivered in a glass syringe fitted with a 27-gauge needle with a blunt endodontic tip. After instrumentation was completed a #15 file was passed 1 mm through the apex to the foramen. Final irrigation of all root canals was performed with 2 ml of 2.5% NaOCl, and the canals were dried with paper points. The prepared teeth were randomly divided into 3 experimental groups of 19 teeth each and another 2 groups as the positive and negative controls (9 teeth each). The root canals were obturated by using a lateral condensation technique and one of three sealers, experimental group 1 (AH-Plus), group 2 (Apexit), or group 3 (Ketac-Endo). Group 4 and group 5 each consisted of nine control teeth and were obturated with lateral condensation and no sealer, and used as positive and negative controls. The master gutta-percha cone (Hygenic, Hygenic Corporation, Akron, OH) corresponding to the last file size at working length was selected. A standardized gutta-percha cone the same size as the latest file used to MI the canal was put in the root canal until tug-back at working length was obtained. The main gutta-percha cone was coated with root canal sealer that was mixed to a thickness according to the manufacturer’s direction. Sealer was placed with the MAF. After placing the master cone, lateral condensation was accomplished with fine accessory cones (P D., Products Dentaire, Vevey, Switzerland) and a D11T spreader (Hufriedy, Chicago, IL). The medium-fine accessory cones were added until the plugger would not penetrate more than 3 mm from the coronal surface. Gutta-percha was removed with a heated instrument 1 mm below the coronal surface, and vertical force was applied with a cold instrument to compact the remaining mass. The obturated teeth were checked buccolingually and mesiodistally using a roentgenogram to check the homogeneity of the filling material. If there were some voids in the fill further condensation was performed until good obturation was obtained. The teeth were kept moist in a humidor at 37°C for 48 h to allow the sealer to set. Leakage Apparatus Preparation The roots of 66 teeth (groups 1 to 3 and positive control) were sealed with two coats of colored nail polish (Revlon, Inc., New

York, NY), except for the apical 3 mm around the apical foramen. The nine teeth used as negative controls had the entire root surface sealed. The tapered end of 1.5-ml microcentrifuge tubes (Treff Lab, Schweiz, Switzerland) 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. These prepared microcentrifuge tubes could be put in 10-ml glass tubes so that 2 mm of the root apex was submerged in the Brain Heart Infusion (BHI) broth (Difco Laboratory, Detroit, MI). The junction between the microcentrifuge tube and glass tube was sealed tightly with Whatman Laboratory Sealing Film (Whatman International Ltd., Maidstone, UK). The test apparatus was put in a box with a lid and then sterilized with ethylene oxide gas for 12 h. To ensure sterilization the whole apparatus was incubated at 37°C for 3 days. Any test apparatus that showed signs of turbidity in the BHI broth was discarded.

Bacterial Leakage Test The upper chamber of the apparatus (microcentrifuge tubes) was filled with 1 ml of 24 h growth of E. faecalis in BHI broth and the whole box incubated at 37°C. Bacterial leakage to BHI broth in the lower chambers was checked daily for 30 and 60 days. A new 24-h growth of E. faecalis was added every week to ensure the viability of the bacterial marker. The turbidity of BHI broth in the lower chambers indicated bacterial leakage. The bacterial growth in the lower chambers was identified as E. faecalis by colonial morphology, cell morphology, growth in 6.5% NaCl BHI broth, and a positive bile esculin test (Difco Laboratory). The upper chamber was also tested for the presence of viable E. faecalis every week. Samples were excluded if there were signs of contamination with other bacteria. Data were statistically analyzed using the ␹2 test. A value of p ⬍ 0.05 was considered statistically significant.

RESULTS In the test apparatus that was put in an incubator for 3 days before starting the experiment, there were signs of turbidity in 10 samples. Three of these were from the group of AH-Plus, two from the Apexit group, four from the Ketac-Endo group, and one from the positive control group. These 10 samples were discarded. Table 1 shows the time for leakage of E. faecalis through 13 mm root canal filling at the apex in 30 days. All specimens in the negative control group showed no turbidity throughout the experiment. There was evidence of broth turbidity in all specimens in the positive control group, except for one specimen that was discarded

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Timpawat et al.

Journal of Endodontics

TABLE 2. No. of teeth and percentage of total leakage of obturated root canal after exposure to E. faecalis for 60 days Type of Cement

n

Leakage (%)

No Leakage (%)

Range (days)

AH-Plus Apexit Ketac-Endo Negative control Positive control

16 17 15 9 8

31.3 (5†) 76.5 (13) 53.3 (8) 0 (0) 100 (8)

68.7 (11) 23.5 (7) 46.7 (13) 100 (9) 0 (0)

21–60 21–60 16–60 — —

]*

* Significant difference, p ⬍ 0.05. † Number of teeth are in parentheses.

because the test apparatus was contaminated. In Table 1, 2 of the 15 root canals (13.3%) of Ketac-Endo showed entire leakage at 30 days. The first specimen in which leakage was observed was at 16 days from the Ketac-Endo group and the first specimen in which leakage was observed from AH-Plus and Apexit was at 21 days. Four of 16 specimens (25.0%) of AH-Plus and 10 of 17 specimens (58.8%) of Apexit showed complete leakage in 30 days. There was a statistically significant difference between AH-Plus and Apexit, and also Ketac-Endo and Apexit (p ⬍ 0.05), but no statistical difference between Ketac-Endo and AH-Plus. Apexit showed greater leakage. Data in Table 2 reveal a statistical difference between AH-Plus and Apexit at 60 days (p ⬍ 0.05). Apexit leaked more than AH-Plus. There was no statistically significant difference between Apexit and Ketac-Endo, or between AH-Plus and Ketac-Endo. Leakage in the experimental teeth in the AH-Plus group was 31.1% at 60 days, whereas Ketac-Endo and Apexit showed 53.3% and 76.5% of leakage, respectively, at 60 days. Bacteriological testing for turbidity of the lower chamber showed E. faecalis in every case. DISCUSSION This study showed that leakage occurred after loss of the coronal seal, as had previously been shown by other studies (6, 8, 11). Bacteria were used to evaluate the coronal leakage, because this method is considered to have more biological and clinical relevance than the dye leakage test (8, 9, 12, 13). Kersten and Moorer (4) demonstrated that molecules of dyes have a low molecular weight and can penetrate in sites where bacterial cells cannot, whereas bacteria-sized particles (latex beads) and large-sized protein molecules (endotoxin) could be prevented only when both sealer and vertical pressure were used in obturation techniques. 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 (14). E. faecalis is also one of the most commonly isolated microbes from the root canal. A study by Torabinejad and co-workers (6) showed that highly motile bacteria (P. vulgaris) penetrated completely in 66 days, whereas nonmotile bacteria (S. epidermidis) penetrated in 30 days. The use of human saliva is advantageous to some degree because it is closely approximates the real clinical situation, but it does not simulate some conditions such as temperature changes, influence of diet, and salivary flow (13). The model in this study attempted to simulate conditions in the clinic, and E. faecalis was chosen because of ease of arrangement and interpretation of the data. Most sealers have antibacterial effects and these properties may limit the ingress of bacteria. The pH of calcium hydroxide-con-

taining root canal sealer is known to inhibit growth of bacteria. The effect of a sealer such as Apexit is seemingly high at 2 and 40 days (15). It was demonstrated by previous studies that at 20 days calcium hydroxide that is released by Apexit might have been neutralized by another compound in the formula so that Apexit has less effect on the bacteria than other types of root canal formula (15, 16). This finding is also supported by Mickel and Wright (17). Regarding the effect of Ketac-Endo, it seems that the antimicrobial activity is based on fluoride-ion release rather than a subsequent variation in pH. AH-Plus is basically the same material as AH26, except that there is no formaldehyde in its formula. A previous study (15) has shown that AH-Plus had no inhibitory effect on Candida albicans, Staphylococcus aureus, or Streptococcus mutans. This study found that bacterial penetration occurred 58.8% in the group-obturated root canal with Apexit, whereas the AH-Plus group and the Ketac-Endo group showed 25.5% and 13.3% coronal leakage in 30 days (Table 1). The bacterial coronal penetration in 60 days was slightly different in the Ketac-Endo group, because there was a higher percentage of penetration than AH-Plus (53.3% and 31.3%, respectively), but statistical analysis showed that there was no significant difference (p ⬎ 0.05). The Apexit group presented more leakage than AH-Plus (p ⬍ 0.05, Table 2). This may be due to the antibacterial effects of Ketac-Endo and Apexit being less than AH-Plus. Not only antibacterial activities of the sealers, but also the physical properties—such as adhesion, adaptability, or solubility—are important. From a previous study (18), AH-Plus demonstrated better sealing ability than a calcium hydroxide-based root canal sealer that showed a large increase of leakage after 14 days. This evidence was presumably a result of cement dissolution. There was a similar result in Siqueira et al.’s (13) study, which found that 80% of root canals obturated with calcium hydroxide sealer (Sealapex) showed complete leakage in 60 days. Our study found 76.5% of the Apexit group had complete leakage in 60 days. This may be due to adaptability and solubility of calcium hydroxide sealer being poorer than the other two types of root canal sealer. This study shows that an inadequate coronal seal can cause bacterial coronal leakage. Different types of root canal sealers may affect the seal differently. AH-Plus, which is an epoxy resin root canal sealer, was found to be more adaptable and involve less leakage than Apexit, which is the calcium hydroxide sealer.

Dr. Timpawat is affiliated with the Department of Operative Dentistry; and Dr. Amornchat is affiliated with the Department of Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand. Dr. Trisuwan is affiliated with the Dental Section, Rajavithi Hospital, Bangkok, Thailand. Address requests for reprints to Dr. Siriporn Timpawat, Department of Operative Dentistry, Faculty of Dentistry, Mahidol University, Yotee Street, Phayathai, Bangkok 10400, Thailand.

Vol. 27, No. 1, January 2001 References 1. Ray HA, Trope M. Periapical status of endodontically treated teeth in relation to the technical quality of root filling and coronal restoration. Int Endod J 1995;28:12– 8. 2. Ingle JI. Endodontics. 3rd ed. Philadelphia: Lea Febiger, 1985:27. 3. Saunders W, Saunders E. Coronal leakage as a cause of failure in root canal therapy. A review. Endod Dent Traumatol 1994;10:105– 8. 4. Kersten HW, Moorer WR. Particles and molecules in endodontic leakage. Int Endod J 1989;22:118 –24. 5. Madison S, Swanson K, Chiles SA. An evaluation of coronal microleakage in endodontically treated teeth. Part II. J Endodon 1987;13:109 –12. 6. Torabinejad M, Borasmy U, Kettering JD. In vitro bacterial penetration of coronally unsealed endodontically treated teeth. J Endodon 1990; 16:556 –9. 7. Khayat A, Lee S-J, Torabinejad M. Human saliva penetration of coronally unsealed obturated root canals. J Endodon 1993;19:458 – 61. 8. Chailertvanitkul P, Saunders WP, MacKenzie D, Weeman DA. An in vitro study of the coronal leakage of two root canal sealers using an obligate anaerobe in microbial marker. Int Endod J 1996;29:249 –55. 9. Chailertvanitkul P, Saunders WP, MacKenzie D. Coronal leakage in teeth root-filled with gutta-percha and two different sealers after long-term storage. Endod Dent Traumatol 1997;13:82–7. 10. Ray H, Seltzer S. A new glass ionomer root canal sealer. J Endodon

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1991;17:598 – 603. 11. Swanson K, Madison S. An evaluation of coronal microleakage in endodontically treated teeth. Part 1. J Endodon 1987;13:56 –9. 12. 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. 13. Siqueira J, Rocas I, Loper H, Uzeda M. Coronal leakage of two root canal sealers containing calcium hydroxide after exposure to human saliva. J Endodon 1999;25:14 – 6. 14. Baumgartner JC, Falkler WA. Bacteria in the apical 5 mm of infected root canals. J Endodon 1991;17:380 –3. 15. Kaplan AE, Picca M, Gonzalez MI, Macchi RL, Molgatini SL. Antibacterial effect of six endodontic sealers: an in vitro evaluation. Endod Dent Traumatol 1999;15:42–5. 16. Georgopoulou M, Kontakiotis E, Nakon M. In vitro evaluation of the effectiveness of calcium hydroxide and paramonochlorophenol on anaerobic bacteria from the root canal. Endod Dent Traumatol 1993;9:259 –53. 17. Mickel A, Wright R. Growth inhibition of Streptococcus anginosus (mitleri) by three calcium hydroxide sealers and one zinc oxide eugenol sealer. J Endodon 1999;25:34 –7. 18. Haikel Y, Wittenmeyer W, Bateman G, Bentaleb A, Alconan C. A new method for the quantitative analysis of endodontic microleakage. J Endodon 1999;25:172–7.

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