I. Miletie, G. Prpie-Mehifie, T. Maruan, A. Tambie-Andrauevie, S. Pleuko, Z. Karlovie & I. Anie
Department of Dental Pathology, School of Dentistry, University of Zagreb, Croatia.
University Hospital of Infectious Diseases ‘Dr Fran Mihaljevib, Zagreb, Croatia.

Introduction.
Complete 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, the combination of guttapercha and a sealer is the most widely used in clinical practice (Nguyen 1991). Previous studies have demonstrated good obturation and adhesion of AH26 (DeTrey Dentsply, Konstanz, Germany) on the root-canal walls (Limkangwalmonkol et al. 1991, DeGee et al. 1994, Wu et al. 1995, Miletib et al. 1999). AH Plus (DeTrey Dentsply) is said to have improved properties, including long-term dimensional stability (Leonardo et al. 1999). Because of the chemistry of the epoxy amines, the material no longer releases toxic substances like formaldehyde (Leonardo et al. 1999) compared to AH26 (Koch 1999).
One of the factors related to failure of root canal treatment is coronal leakage of the root filling (Saunders & Saunders 1994). Studies (Torabinejad et al. 1990) have demonstrated 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. Although bacteria have a significant role in endodontic treatment, it is known that in some cases fungi have also been isolated. Nair et al. (1990) and Sen et al. (1997) have both observed bacteria and fungi in infected root canals and dentinal tubules associated with periapical lesions. The presence of fungi in persistent infection may be the result of contamination during treatment or of differences in susceptibility to local root canal medicaments between fungi and bacteria (Waltimo et al. 1999).
The aim of this study was to evaluate the penetration of Candida albicans alone and combination of bacteria through root canals filled with gutta-percha and either AH26 or AH Plus sealer.

Materials and methods.

Preparation of teeth.
A sample of 100 single-rooted teeth with fully developed apices was used for the experiment. All of the teeth were extracted for periodontic and orthodontic reasons. After mechanical cleaning, the teeth were stored in deionized water with a few thymol crystals at 100% humidity and 37C (Chailertvanitkul et al. 1996a).
Prior to the study, the crowns of teeth were removed at the cemento–enamel junction using a water-cooled diamond bur. To ensure that all specimens were of the same length, they were than resected 8 mm from the apex.
A size 15 K-file (Dentsply Maillefer, Ballaigues, Switzerland) was introduced into each canal until it was just visible at the apical foramen and the working length determined by withdrawing the instrument 1 mm. The root canals were than prepared using a step-back technique with K-files (Dentsply Maillefer). The apical portion of the canals was instrumented to a size 40 K-file, and the middle portion to a size 80 K-file. After the use of each instrument, 5 mL of a freshly prepared solution of 2.5% sodium hypochlorite (NaOCl) was delivered by means of a syringe and 27-gauge needle. The coronal part of the root canals was shaped using size 2 and size 3 Gates-Glidden drills (Dentsply Maillefer). After instrumentation, the smear layer was removed with 17% EDTA for 3 min.
Final irrigation of all samples was carried out with 2.5% NaOCl, and the canals were dried with air and sterile paper points ( Johnson & Johnson, Slough, UK).

Obturation.
The specimens were randomly assigned to two groups of 40 teeth each, and obturated by lateral condensation of gutta-percha (Kerr, Romulus, MI, USA) and sealer. In the first group, AH26 silver free (DeTrey Dentsply) was used as the sealer, and AH Plus (DeTrey Dentsply) was used in the second group. The sealers were mixed according to the manufacturer’s instructions, and applied to the walls of the canal with a size 40 K-file (Dentsply Maillefer). The master cone corresponding to the last master apical file was lightly coated with the sealer and placed in the canal. A size B finger spreader (Dentsply Maillefer), and size 25 gutta-percha (Kerr), were used for lateral condensation. Gutta-percha cones were added until the spreader penetrated into the coronal one-third of the root canal space. After obturation, a hot plugger was used to remove excess gutta-percha 1 mm from the cemento–enamel junction.
Of the 20 teeth used as controls, 10 positive controls were laterally condensed with gutta-percha only, whilst for the 10 negative controls, five were filled with gutta-percha and AH Plus and five with gutta-percha and AH26. All 10 negative controls were completely covered with nail varnish, including the apex of the root and coronal access.
After obturation, all specimens were stored in saline solution at 37C for 3 weeks to allow full setting of the sealer. After that, they were thermocycled between 5and 55C for 300 cycles, for 10 s at each temperature (Rossomando & Wendt 1995).

Preparation of samples.
The external surfaces of specimens were covered with two layers of nail varnish. The teeth were inserted individually into an Eppendorf plastic tube (Eppendorf-Elkay, Shrewsbury, MA, USA) with the root apex protruding through the end. The coronal and middle portion of specimens were sealed with cyanoacrylate glue and acrylic resin. The system was sterilized using ethylene oxide gas and placed in a 5-mL glass bottle containing 3 mL sterile Schaedler broth (Schaedler anaerobe broth; Oxoid, Unipath Ltd, Basingstoke, UK). Around the entrance to the flask, a layer of cyanoacrylate glue and acrylic resin was applied. The model used in this study was refined from a system described by Imura et al. (1997) and shown in Figure 1.

Figure 1. The experimental model - a tooth attached in the Eppendorf tube and placed in a glass bottle with Schaedler broth.

Bacterial and fungal leakage.
The coronal chambers of 20 teeth of the 40 obturated with AH26, and 20 teeth of the 40 with AH Plus were inoculated with a suspension of bacteria containing approximately 108 organisms: Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Prevotella melaninogenica (ATCC 25845), and Lactobacillus acidophilus (ATCC 832) using a sterile syringe and 27-gauge needle. Another group of 20 specimens for each sealer was inoculated with Candida albicans (ATCC 10231) in the same concentration. The medium with microorganisms was changed every 7 days. The system was stored in an anaerobic incubator at 37C, and any changes in opacity of the broth in the apical chamber were checked every 72 h for 3 months. All samples with evidence of broth turbidity were checked by Gram staining and culture. Broth from the apical chamber was streaked onto blood agar culture plates and incubated under aerobic and anaerobic conditions. Microorganisms were identified by colony morphology, Gram stain and, in case of streptococci, by biochemical tests (api 20 Strep, BioMerieux, France). The specimens with turbid broth were removed from the experiment and replaced with new specimens.

Data analysis.
The data obtained for all experimental groups were analysed using survival analysis methodology, as the experimental variable (leakage day) was censored by the 90-day experiment duration. As ‘time before leakage’ is undefined for samples that did not show leakage during the experimental period and cannot be declared as missing cases, a score is assigned to each survival time, using Mantel’s procedure (Mantel 1967). To compare times before leakage, i.e. scores in the four groups (inoculated with bacteria or fungi, sealed with AH26 or AH Plus), a chi-squared value was computed based on the sums (for each group) of this score. All analyses were performed using Statistica for Windows, version 5.0 (Statsoft inc., Tulsa, OK, USA).

Results.
Leakage through the samples varied from 14 to 87 days. All positive controls exhibited bacterial and fungal leakage within 72 h, whilst the broth in lower chambers used for negative controls remained clear throughout the test period. There was no statistically significant difference in leakage amongst the four experimental groups (2 – 1.06, P = 0.787).
Descriptive results for Mantel scores in the four experimental groups are shown in Table 1.
The leakage rate is shown in Figure 2: the percentage of specimens without leakage was uniform for all four groups and no particular intervals of risk were detected.
Biochemical analysis of turbid broth from the apical chambers showed presence of either Streptococcus mutans and Streptococcus mitis, or a mixture of Streptococcus spp. with Lactobacillus acidophilus and/or Prevotella melaninogenica.

Table 1. Descriptive statistic for Mantel scores for four experimental groups.



Figure 2. Percentage of specimens without leakage during the experimental period in the four experimental groups.

Discussion.
Both sealers allowed leakage of bacteria and C. albicans during the experimental period. Forty-five per cent of the samples sealed with AH26 leaked bacteria, and 60% leaked C. albicans, whilst amongst the samples sealed with AH Plus, 50% leaked bacteria and 55% leaked C. albicans. There were no statistically significant differences amongst the groups regarding the leakage times and percentages.
Moshonov et al. (1995), in their study of leakage with Staphyloccocus epidermidis in samples sealed with AH26, Roth 801 and Ketac Endo, showed that only two samples with AH26 leaked. Chailertvanitkul et al. (1996a), who used Streptococcus sanguis, found leakage in 16 of 20 samples, whilst the results of our study show leakage in nine of 20 samples sealed with AH26. A number of factors could explain the differences in the results, such as instrumentation technique, obturation technique, the type of microorganisms used, experimental design, and the nature of the irrigant to which the canal was exposed (Chailertvanitkul et al. 1996b).
The number of microorganisms that had caused turbidity was not measured as the purpose was only to prove that some bacteria and yeasts were capable of penetrating root canal fillings. The fact that in some specimens only one microorganism could leak and cause turbidity and in another case more microrganisms could penetrate through the root filling was not considered. However, partial simulation of the complex and dynamic nature of the oral flora was possible by using a mixture of bacterial species, which were changed in the upper chambers every 7 days.
The bacteria most often isolated from the turbid broth were Streptococcus mutans and Streptococcus mitis, whilst Lactobacillus acidophilus and Prevotella melaninogenica were less common and only in combination with Streptococcus spp. Also, Chailertvanitkul et al. (1997) reported that no single Prevotella intermedia sp. alone penetrated through the experimental teeth into the apical chamber. They attributed higher penetration of anaerobic streptococci in comparison with Prevotella intermedia to their smaller size.
The similarity between the leakage of bacteria and fungi in samples sealed with both sealers leads to the conclusion that C. albicans has at least the same capability to penetrate through the sealed root canal as the bacteria. Sen et al. (1997) found that growing hyphae have a tendency to follow cracks and penetrate into dental tubules and this could explain the leakage of C. albicans. Further investigation, both in vitro and in vivo, is needed to establish possible clinical risks of fungal infection.

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