Antimicrobial effect of three disinfecting agents on Resilon cones and their effect on surface topography: An in vitro study.

AIM: The objective of this study was to evaluate the effectiveness of 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and 2% peracetic acid (PAA) in disinfecting Resilon cones and to evaluate topographical changes microscopically under scanning electron microscope (SEM) after rapid chemical disinfection.
MATERIALS AND METHODS: Resilon cones were disinfected in an ultraviolet (UV) light chamber for 20 min and contaminated by immersing in a microbial suspension of Enterococcus faecalis for 30 min. The contaminated cones were then immersed in the 5.25% NaOCl, 2% CHX, and 2% PAA for 1 min, 5 min, and 10 min, separately. The cones were then incubated at 37°C in thioglycollate broth for 7 days and examined for turbidity. The samples showing turbidity were subcultured on blood agar and incubated at 37°C for 48 h. Gram staining was done to confirm that the cultured bacteria were E. faecalis. Surface changes of disinfected Resilon cones were evaluated under SEM. STATISTICAL ANALYSIS USED: The data were analyzed statistically using Kruskal-Wallis, analysis of variance (ANOVA), and Mann-Whitney U-test. RESULT: In eliminating E. faecalis, 5.25% NaOCl was most effective followed by 2% PAA and 2% CHX. Topographic examination of tested Resilon cones revealed some surface deposits after disinfection with 5.25% NaOCl and 2% CHX, whereas 2% PAA caused surface erosion.
CONCLUSION: In disinfecting Resilon cones, 5.25% NaOCl is most effective followed by 2% PAA and 2% CHX.

INTRODUCTION

Thorough biomechanical preparation and intracanal medication is essential for the proper disinfection of the root canal system during endodontic treatment. At the same time it is also important to ensure that no exogenous bacteria enter the disinfected root canal subsequently. Such cross contamination can be avoided by ensuring the use of sterile obturating materials and instruments. Gutta-percha which is the most commonly used obturating material can be successfully disinfected by immersing in 5.25% NaOCl or MTAD for 5 minutes just before use.[1]

Obturating materials such as gutta-percha and Resilon cones are manufactured and packed under sterile conditions. They remain sterile till their first use when they are opened from their packages. Once handled by the operator and exposed to the dental operatory environment, they get contaminated with exogenous microorganisms.[2] The use of such contaminated obturating materials in endodontic treatment may result in reinfection of treated root canals. Since gutta-percha and Resilon are thermoplastic materials, sterilization by means of heat is not possible. Hence chemicals are used for rapid chair side disinfection procedures.

Several antibacterial root canal irrigants like NaOCl, CHX, hydrogen peroxide, MTAD and surgical disinfectants like, glutaraldehyde, ethyl alcohol, povidone-iodine, have been used to sterilize the gutta-percha cones. The efficacy of these agents is time and concentration dependent. Lower concentrations take longer time for disinfection. NaOCl and CHX are two commonly used solutions for disinfection of gutta-percha cones.[34567] These agents may take anywhere between 1 to 5 minutes to accomplish complete disinfection

NaOCl has bactericidal and sporicidal property owing to release of hypochlorite ion and hypochlorous acid.[8] Chlorine combines with protein amino groups and forms chloramines. Chlorine also inhibits bacterial enzymes leading to irreversible oxidation of the sulfydryl group of bacterial enzymes.[8] CHX is bacteriostatic at low concentration and bactericidal at high concentration.[9] It is a cationic biguanide that interacts with the negatively charged phospholipids and lipopolysaccharides in the cell membrane of bacteria.[10] Peracetic acid (PAA) has been used as a disinfectant in food industry and for water and sewage treatment.[11] PAA is a strong oxidizing agent and has stronger oxidation potential than chlorine. It oxidizes the outer cell membrane of vegetative bacterial cells, spores, yeast and virus.

Resilon is a thermoplastic polycaprolactone based material with difunctional methacrylate resin.[12] The thermoplastic nature of Resilon is provided by polycaprolactone, an biodegradable polyester with a moderately low melting point.[12] Therefore chemicals are used for rapid chair side disinfection of Resilon cones. In the present study, 5.25% NaOCl, 2% CHX and 2% PAA were used. These have broad spectrum of antibacterial activity.[6810] However, these solutions may cause changes on the surface of Resilon cones which may compromise the obturation seal.[1] Hence this study further evaluated effect of these solutions on the surface topography of Resilon cones examined under Scanning Electron Microscopy.

MATERIALS AND METHODS

A total of 114, size 80 Resilon (SybronEndo), cones were used in this study. The Resilon cones were disinfected in an ultraviolet (UV) light chamber for 20 min and divided into three experimental groups of 30 cones each and 2 control groups of 10 cones each. The remaining four cones were used for topographical evaluation. Each of the experimental groups were further subdivided into 3 subgroups as “a, b, and c” containing 10 cones as follows:

Group I: Ten Resilon cones each immersed in 5.25% NaOCl for 1 min (group I a) and 5 min (group I b) and 10 min (group I c), respectively.

Group II: Ten Resilon cones each immersed in 2% CHX for 1 min (group II a) and 5 min (group II b) and 10 min (group II c), respectively.

Group III: Ten Resilon cones each immersed in 2% PAA for 1 min (group III a) and 5 min (group III b), and 10 min (group III c), respectively.

Group IV: Ten Resilon cones after disinfection in a UV light chamber were individually cultivated in thioglycollate broth as a negative control

Group V: Ten Resilon cones contaminated with Enterococcus faecalis cultivated in thioglycollate broth as a positive control.

After disinfection, ten Resilon cones of group IV (negative control) were individually transferred to thioglycollate broth and incubated at 37°C for 7 days to evaluate the primary sterility of the Resilon cones.

Artificial contamination of Resilon cones

Microbial suspension of E. faecalis (ATCC2912) in trypticase soy broth (Himedia Laboratories, Mumbai, India) was used in this study. Resilon cones from groups I, II, III, and V were immersed in 20 mL of microbial suspension for 30 min. They were then allowed to dry for 10 min at room temperature.

Disinfection of contaminated Resilon cones

After artificial contamination, 10 Resilon cones from each of the subgroups of groups I, II, and III were immersed in 20 mL of respective disinfection solution contained in a Petri dish for 1 min, 5 min, and 10 min. The Resilon cones from experimental groups were immersed in detergent (3% Tween) for 5 min and washed in 10 mL of sterile distilled water. Each of these Resilon cones was individually transferred to separate test tubes containing 10 mL of thioglycollate broth and incubated at 37°C for 7 days. The appearance of turbidity indicated bacterial growth in the test tube.

At the end of 7 days, the test tubes showing turbidity were subcultured by inoculating thioglycollate broth in blood agar plate and incubated at 37°C for 48 h. The microbial growth on blood agar plate was used to make smears for Gram staining and observed under light microscope (Olympus BX, Haryana, India). Gram-positive staining of smears confirms the growth of E. faecalis.

Topographical examination of Resilon cones

After analyzing the results of the disinfection procedure one Resilon cone was immersed in respective disinfectants. The immersion time evaluated for topographical changes was the time at which respective disinfectants were 100% effective. Therefore, one Resilon cone was immersed in 5.25% NaOCl for 1 min, 2% PAA for 5 min, and 2% CHX for 10 min, separately. One unaltered Resilon cone was used that simulated as negative control. Following this, all four Resilon cones were gold sputter coated and examined under SEM at 500× magnification.

Statistical analysis

The data were analyzed using Kruskal–Wallis, analysis of variance (ANOVA), and Mann–Whitney U-test.

RESULTS

All the three disinfectants eliminated E. faecalis at different time intervals. In eliminating E. faecalis 5.25% NaOCl was most effective followed by 2% PAA and 2% CHX. E. faecalis was eliminated by 5.25% of NaOCl in 1 min itself. E. faecalis was eliminated by 2% PAA in 5 min, whereas 2% CHX took 10 min. Topographic examination showed deposits on the surface of Resilon cones in NaOCl and CHX group [Figure 1b and d]. In the PAA group, surface destruction was seen by the time disinfection was complete in 5 min [Figure 1c].

Figure 1

SEM images of Resilon cones. (a) Negative control, (b) NaOCl 1 min, (c) PAA 5 min, and (d) CHX 10 min

DISCUSSION

For success of endodontic therapy, it is important to maintain an aseptic chain during the procedure.[5] Since obturation is the final step of root canal treatment, the use of contaminated obturating material is a potential source of microorganisms for secondary infections.[7] Therefore, it is important to disinfect obturating materials before placing them in the root canal system.

E. faecalis is a nonspore-forming, facultatively anaerobic gram-positive coccus.[13] It was chosen as test microorganism because it has shown resistance to irrigants and medicaments commonly used in endodontics.[13] The disinfection time intervals evaluated in this study are based on the findings of Senia et al., Dumani et al., and Tonomaru et al.[71415] The microbiological study carried out was turbidity test. It is a quick and efficient method of estimating the presence of bacteria in a liquid medium. This method of estimation is fast and is preferred when large numbers of cultures are to be counted. In the present study, the disinfected Resilon cones were transferred to test tubes containing thioglycollate broth and cultured at 37°C for 7 days. Every 24 h, the test tubes were checked for turbidity. Samples that showed turbidity were subcultured in blood agar plate for 48 h as described in a previous study by Zand et al.[5]

The results of this study are in accordance with the previous study by Senia et al., which concluded that gutta-percha cones immersed in 5.25% NaOCl for 1 min will be successfully disinfected.[14] This has been confirmed in other studies.[3457] This study is also in accordance with the study by Shuba et al. who showed that immersion in 2% PAA for 5 min will eliminate E. faecalis.[16] With respect to 2% CHX, the results of the present study do not agree with the results of Cardoso et al., Royal et al., Gomes et al., and Dumani et al.[34717] They found 2% CHX to be effective at a time range of 15 s to 5 min.

Topographical examination of Resilon cones revealed some deposits after disinfection with 5.25% NaOCl or 2% CHX and erosion of the surface after disinfection with 2% PAA [Figure 1]. However, 2% CHX produced fewer deposits than 5.25% NaOCl. These deposits can be removed by gentle agitation in distilled water.[1] In a study by Mahima et al., both 5.25% NaOCl and 2% CHX did not cause alteration on the surface of Resilon cones evaluated at different time period. In their study, surface topography was evaluated under atomic force microscope and roughness was as criteria to compare root mean square values.[18] Gomes et al., also showed in their study, that 2% CHX did not cause alteration on the surface of Resilon cones.[19]

CONCLUSION

Within the limited scope of this study, Resilon cones can be disinfected completely by immersing either in 5.25% NaOCl for 1 min, or in 2% PAA for 5 min, or in 2% CHX for 10 min. Only a few surface deposits were seen on Resilon cones that were completely disinfected using 5.25% NaOCl or 2% CHX. For the disinfection of Resilon cones, 2% PAA cannot be recommended as it causes alteration to the surface in 5 min.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors deny any conflicts of interests related to this study.