Disinfection of gutta-percha cones using three reagents and their residual effects.

AIMS AND
OBJECTIVES: The purpose of this study was to compare the efficacy of 5.25% sodium hypochlorite, 2% chlorehexidine and MTAD in disinfecting gutta-percha cones and to analyze the surface topography of gutta-percha cones after the rapid chemical disinfection procedure.
MATERIALS AND METHODS: Gutta-percha cones were immersed in suspensions of Enterococcus faecalis and Staphylococcus aureus separately. The cones were then immersed in 5.25% NaOCl, 2% CHX and MTAD for 30 seconds, 1 minute and 5 minutes separately. The disinfected cones were then incubated in thioglycollate media for 7 days. The thioglycolate media was sub-cultured and colony forming units were counted. For topographical examination of gutta-percha cones, the cones were immersed in respective solutions for one minute and allowed to air dry for 30 minutes after rinsing or without rinsing the cones with distilled water. The topography of the cones were then analyzed under SEM. Data was statistically analyzed using one way anova.
RESULTS: MTAD was found to be the most effective disinfecting solution. The topographical examination of GP cones found some deposits after the disinfection procedure in every group. These deposits were removed when the GP cones were rinsed with distilled water.
CONCLUSIONS: MTAD possesses superior bactericidal activity when compared with NaOCl and CHX and a final rinse with distilled water is essential after the disinfection procedure.

INTRODUCTION

Success of endodontic treatment depends on thorough biomechanical preparation and the use of aseptic techniques during the treatment.[1] The practitioner must be concerned not only with endogenous oral microbial flora, but with exogenous bacterial contamination as well.[1] For optimum infection control, every instrument and material placed within root canals should be sterile. Gutta-percha is the most commonly used obturating material. Although gutta-percha cones are manufactured under aseptic conditions they can get contaminated by aerosols, improper storage and physical handling of cones.[12]

It is occasionally seen that even in endodontic procedures performed with utmost care, reinfection may occur.[7] One possible explanation for this may be the use of contaminated gutta-percha cones in the root canals.[7] Studies have revealed the presence of microorganisms in freshly opened boxes.[12] Even though the number of these organisms was quite low at the time of packaging, clinical usage increased the number of microorganisms contaminating the gutta-percha cones.[12] According to various studies, Staphylococcus genus is found to be the most common microorganism contaminating gutta-percha cones in their boxes and after handling with gloves.[23] According to Guimarães et al, the recovery rate of Staphylococcus genus from infected root canal is about 15.7%; this result justifies the need of gutta-percha disinfection.[21]Enterococcus faecalis is another organism that is found to be the most resistant intra-canal pathogen in failed root canals that serves as a gold standard bacterium in endodontic research.[4] Due to its superior virulence, it was selected in this study to represent the other possible organisms that may get contaminated with gutta-percha cones. However due to the thermoplastic nature of gutta-percha, the conventional methods of moist or dry heat sterilization cannot be used. A rapid chair side chemical disinfection is indicated.

A 5.25% sodium hypochlorite solution is a broad spectrum antimicrobial agent and it is recognized as efficient, inexpensive and reliable. Chlorhexidine gluconate is a broad spectrum antimicrobial agent with substantively and relatively low toxicity. MTAD, a mixture of doxycycline, citric acid and tween-80 has been introduced by Torabinejad et al., as a final irrigant for disinfection of the root canal system.

During gutta-percha disinfection, sodium hypochlorite and MTAD have been found to leave some deposits on the surface of gutta-percha cones. It is advisable to remove these layers or crystals before using the cones for obturation of root canals, since they may compromise the obturation seal.

Therefore this study was undertaken to compare the efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine and MTAD in disinfecting gutta-percha cones and to analyze the surface topography of gutta-percha cones after the rapid chemical disinfection procedure.

For topographical examination, SEM at 1000× magnification was used since it allows a detailed examination of topography of any material at different magnifications.[11]

MATERIALS AND METHODS

In all, 280 gutta-percha cones (Size 80, Dentsply) were taken from freshly opened boxes and arranged in 8 groups.

Groups I, II, III, IV and V were used to evaluate the efficacy of the disinfecting solutions. Groups I, II and III were further divided into a, b and c according to the disinfecting time.

Group I: Ten contaminated gutta-percha cones immersed in 5.25% sodium hypochlorite for 30 seconds (Group I a), 1 minute (Group I b) and 5 minutes (Group I b), respectively.

Group II: Ten contaminated gutta-percha cones immersed in 2% chlorhexidine for 30 seconds (Group II a), 1 minute (Group II b) and 5 minutes (Group II c), respectively.

Group III: Ten contaminated gutta-percha cones immersed in MTAD for 30 seconds (Group III a), 1 minute (Group III b) and 5 minutes (Group III c), respectively.

Group IV: Ten gutta-percha cones contaminated with E. faecalis/S. aureus cultivated in thioglycollate media as a positive control.

Group V: Ten gutta-percha cones from freshly opened boxes and individually cultivated in thioglycollate media as a negative control.

Gutta-percha cones from groups VI, VII and VIII were used to check the topography of gutta-percha cones after the disinfection procedure. Groups VI and VII were subdivided to a, b, c according to the disinfecting solutions used.

Group VI: Ten gutta-percha cones were immersed in 5.25% sodium hypochlorite (Group VIa), 2% chlorhexidine (Group VI b) or MTAD (Group VI c) for 1 minute and dried for 30 minutes without rinsing in distilled water.

Group VII: Ten gutta-percha cones were immersed in 5.25% sodium hypochlorite (Group VII a), 2% chlorhexidine (Group VII b) or MTAD (Group VII c) for 1 minute and dried for 30 minutes after rinsing in distilled water with a gentle agitation.

Group VIII: Ten gutta-percha cones were directly taken from the box and examined under scanning electron microscope.

Artificial contamination of gutta-percha cones

Microbial suspension of E. faecalis (ATCC29212) and S. aureus (ATCC6538) of approximately 10⁸ CFU/ml in trypticase soy broth (HiMedia Laboratories) was used for this study.

Gutta-percha cones from Groups I, II, III and IV were immersed in 20 ml of either of the above microbial suspensions for 30 minutes. The cones were then transferred to sterile paper pads and allowed to air dry for 10 minutes.

Disinfection of gutta-percha cones

After artificial contamination, 10 gutta-percha cones from Groups I, II and III were arranged on a stainless steel mesh separately and immersed in the respective disinfectant solutions contained in a petridish for 30 seconds, 1 minute and 5 minutes separately.

The cones were then individually transferred to sterile test tubes containing 10 ml of thioglycollate media (HiMedia Laboratories) and incubated at 37°C for 7 days. After 7 days, a micropipette (Precica) was used to transfer the thioglycollate media to a petridish containing brain heart infusion agar and a sterile cotton tip was used to spread the thioglycollate media in a thin layer over BHI agar. The plates were then incubated for 48 hours aerobically at 37°C and the colony forming units were counted with a digital colony counter (Labmate).

Topographical examination of gutta-percha cones

Scanning electron microscope (FEI Quanta 200 ESEM FEG) at 1000× magnification was used for the topographical examination of gutta-percha cones. Gutta-percha cones from Groups VI and VII were subjected to topographical examination after immersing in 5.25% sodium hypochlorite, 2% chlorhexidine and MTAD for 1 minute. In Group VI, the gutta-percha cones were arranged in the stainless steel mesh and immersed in the respective disinfecting solutions for 1 minute. The cones were then transferred to sterile paper pads and allowed to air dry for 30 minutes without rinsing the cones in distilled water.

In Group VII, the gutta-percha cones were arranged in the stainless steel mesh and immersed in the respective disinfecting solutions for 1 minute. The cones were then individually rinsed with 10 ml of distilled water in a petridish with a gentle agitation. The cones were then transferred to sterile paper pads and allowed to air dry for 30 minutes.

The topography of cones was then analyzed under scanning electron microscope at 1000× magnification.

Statistical analysis

The data were analyzed by one-way ANOVA (analysis of variance) using SPSS 17.0 software.

RESULTS

The mean bacterial count of E. faecalis or S. aureus was found to be lesser after treating the cones with MTAD when compared with other disinfecting solutions for all time intervals tested. 5.25% NaOCl was found to be the second most effective disinfecting solution while 2% CHX was the least effective among the solutions tested [Table 1]. Topographical examination of gutta-percha cones revealed the presence of some residues over the cones after the disinfection process. The deposits were removed after rinsing the cones with distilled water [Figure 1].

Table 1

Comparison of mean number of colonies (X10-1) among disinfecting solutions against contaminated organisms at different time intervals

Figure 1

SEM image of gutta-percha cones before and after immersing in disinfecting solutions for 1 minute, (a) Control group (b) 5.25% NaOCl — Before rinse (c) 5.25% NaOCl — After rinse (d) 2% CHX — Before rinse (e) 2% CHX — After rinse (f) MTAD — Before rinse (g) MTAD — After rinse

DISCUSSION

Even though gutta-percha cones are produced under aseptic conditions, they may get contaminated by aerosols, improper storage and physical handling.[8] Studies have revealed the presence of microorganisms in 5-19% of freshly opened gutta-percha packs.[1345] Though the number of these organisms was quite low at the time of packaging, clinically it is routine practice for dentists to use gutta-percha points ‘straight out of box’ without a thought about its sterility. It has been demonstrated that the clinical usage of the package increased the number of microorganisms contaminating the gutta-percha cones.[1]

S. aureus is a facultative anaerobic coccus and is found in the saliva and skin.[5]S. aureus was selected as one of the test organisms, as it is the most common microorganism contaminating gutta-percha cones in their boxes and after handling with gloves.[359] Incorrect handling of gutta-percha cones during the retreatment or checking the fit of the master cone before final rinse may lead to the contamination of the cones with E. faecalis, which is known for its superior virulence factors and considered as a gold standard to represent the other possible microorganisms that may contaminate gutta-percha cones. Therefore, E. faecalis was selected as the second test organism. For the evaluation of efficacy of disinfecting solutions, the disinfected cones were cultured in thioglycollate media for 7 days and it was sub-cultured in BHI agar using culture plate method as described in previous studies.[10]

Earlier studies by Senia et al showed that 1-minute immersion of gutta-percha cones in 5.25% NaOCl eliminated microorganisms, whereas CHX was efficient only at 5 minutes.[5912] The minimum contact time required for MTAD to eliminate the microorganisms is unknown. Therefore, immersion time intervals of 30 seconds, 1 minute and 5 minutes were selected for this study.

In the current study, the mean bacterial count of E. faecalis or S. aureus was found to be lesser after treating the cones with MTAD when compared with other disinfecting solutions in all time intervals tested. This may be due to the synergistic effect of an antibiotic, acid and a detergent, which increases its bactericidal activity. The results of the current study were not in concurrence with the results of Senia et al who found that 1-minute immersion in 5.25% NaOCl was sufficient for the disinfection of gutta-percha cones. 5.25% NaOCl and 2% CHX completely eliminated S. aureus only in 5 minutes and they were unable to completely eliminate E. faecalis even after 5 minutes. The current study was in concurrence with Giardino et al., and Davis et al., who demonstrated that, 5.25% of NaOCl showed high antibacterial activity against anaerobic bacteria but was ineffective in eliminating facultative anaerobic bacteria.[1314] According to their study MTAD was equally effective against anaerobic bacteria and facultative anaerobic bacteria. The test organisms used in the current study were facultative anaerobic bacteria. This may be the reason why 5.25% of NaOCl was less effective in eliminating microorganisms in the current study.

The topographical examination of gutta-percha cones found some deposits after 1-minute disinfection procedure with all disinfecting solutions used. MTAD and CHX were found to leave lesser deposits compared to NaOCl. These deposits were removed when the gutta-percha cones were rinsed in distilled water with gentle agitation after the disinfection procedure.

CONCLUSIONS

Within the limitations of this study, MTAD was found to be the most efficient disinfecting agent followed by 5.25% of NaOCl in eliminating E. faecalis and S. aureus at different time intervals tested. A 5-minute immersion is required for the complete disinfection and a final rinse of gutta-percha cones with distilled water is essential to eliminate the surface deposits.