| Abstract|| |
Aims: The aim of this study is to verify the disinfection of diode laser, following chemo-mechanical procedures against Enterococcus fecalis.
Materials and Methods: Crowns of 30 extracted premolar teeth were sectioned at the cemento- enamel junction. The canals were shaped using step-back technique to K-file #40. The teeth were randomly assigned to three groups and placed into nutrient broth containing bacterial suspension of Enterococcus fecalis. Group A received no laser radiation. Specimens of group B and C were treated with diode laser (Sirona) with energy set at 1.5 and 3 W, respectively. After laser irradiation, the teeth were placed in vials, which contained 2 mL of the nutrient broth. The vials were incubated at 37°C for 24 h. Grown colonies were identified by standard methods.
Statistical Analysis Used: Statistical analysis used was the nonparametric Kruskal-Wallis test, with comparison using the Bonferroni methods of means.
Results: Higher mean CFU/mL is recorded in Group A (without laser disinfection) followed by Group B (with 1.5 W laser disinfection) and Group C (with 3 W laser disinfection), respectively. The difference in CFU/mL between the three groups is found to be statistically significant ( P < 0.001).
Conclusions: The results of this research show that the 980 nm diode laser can eliminate bacteria that has immigrated into dentin, thus being able to increase the success rate in endodontic therapy.
Keywords: Colony-forming unit, Enterococcus fecalis, 980 nm diode lasing techniques
|How to cite this article:|
Kaiwar A, Usha H L, Meena N, Ashwini P, Murthy CS. The efficiency of root canal disinfection using a diode laser: In vitro study. Indian J Dent Res 2013;24:14-8
In Endodontics, disinfection of the root canal system is essential step for ensuring successful root canal therapy. The contamination of the root canals with microorganisms and its propagation in remnants of necrotic soft tissue are considered one of the main reasons for failure in endodontic treatment.  The eradication of persisting microorganisms in distant areas of the tubular system is a major challenge in today's treatment regimens and is crucial for the long-term preservation of the endodontically treated tooth.
|How to cite this URL:|
Kaiwar A, Usha H L, Meena N, Ashwini P, Murthy CS. The efficiency of root canal disinfection using a diode laser: In vitro study. Indian J Dent Res [serial online] 2013 [cited 2018 Dec 15];24:14-8. Available from: http://www.ijdr.in/text.asp?2013/24/1/14/114916
Studies have shown that microorganisms present in failed root canal cases are distinct from those present in infected root canals before endodontic treatment. , Enterococcus fecalis is a facultative anaerobic Gram positive coccus, which is present in oral flora and is identified in persistent root canal infections and is also related to the failure of endodontic treatment.  They form intra and extra radicular biofilms, which are difficult to remove and they cause persistent or reinfection. ,
Irrigating solutions used during root canal treatment act through direct contact with the bacteria targeted. However irrigants that have insufficient penetration depth as a result of these microorganisms in the deeper layers are not destroyed.  Lasers have become latest choice to eradicate microorganisms in the root channel, especially in the lateral dentinal tubuli. This has been achieved by the development of a fiber delivery system. It has been proved in numerous studies that an emission of laser light directly in the root canal does have bactericidal effect.  The antibacterial effect of a laser beam is based on thermal properties of laser tissue interaction.  The high-power diode laser has been used in several areas of dentistry, with promising results in relation to dentinal disinfection. ,, Diode laser has been proved to be resource worth testing. The aim of this study is to verify the disinfection of diode laser with a wavelength of 980 nm, following chemo-mechanical procedures against E. fecalis.
| Materials and Methods|| |
Thirty extracted uniradicular premolar teeth were stored in 5.2% NaOCl solution for 30 min to remove organic residues and left in saline solution until the procedure began. The crowns were sectioned at the cemento-enamel junction using high-speed diamond disk to obtain root canal length of 15 mm [Figure 1]. Access opening was done. #10 K-file (Dentsply-Maillefer) was introduced into each canal until it appeared at the apical foramen. The working length was established by subtracting 0.5 mm from this length. The canals were shaped using step-back technique to #40 K-file (Denstply Maillefer). Irrigating solution used after each instrument was 5 mL of 2.5% sodium hypochlorite. The coronal third of the root canals was flared with #2, #3 and #4 Gates Glidden drills. The teeth were then allowed to air-dry overnight at room temperature and apical foramen was sealed externally and waterproofed with coats of clear nail polish. The teeth underwent sterilization in 121 o C autoclave for 15 min at 15 lbs pressure.
Preparation of the medium for E. faecalis
The strains of microorganisms used for the study were standard strains of E. fecalis ATCC29212, which were sub-cultured in UTI Hichrome agar plate (selective medium) and was incubated at 37°C for 24 h.
A pure, single E. fecalis colony was isolated from the same cultured plate and Gram's staining was done to confirm its growth, which was observed under microscope and then inoculated with a brain heart infusion (BHI) broth. The BHI broth was incubated at 37°C for a 24-h period and checked for bacterial growth by changes in turbidity. A drop of BHI containing E. fecalis was placed into saline solution and checked for correct bacterial concentration with a spectrophotometer. The density of the bacterial suspension is standardized by comparing the broth at a density equivalent to the barium sulphate standard of 0.5 McFarland units, which is equivalent to 1.5 × 10 8 colony forming units per milliliter (CFU/mL).
Before irradiation, a micropipette with a sterile needle was used to inoculate the canals with 10 mL of the bacterial suspension in nutrient broth. The teeth were randomly assigned to three groups and placed into nutrient broth containing bacterial suspension.
The specimens were randomly divided into three groups where two groups B, C received laser treatment. Group A received no laser radiation. Specimens of Groups B and C were treated with diode laser (Sirona) with energy set at 1.5 and 3 W, respectively. Irradiation followed oscillatory technique developed by Gutknecht et al.  The optical fiber is introduced 1 mm short of the apex and is recessed in helicoidal movements at a speed of approximately 2 mm/s for 5 s, repeated 4 times at intervals of 10 s, between each one. This rest period between irradiation avoided temperature change. After laser irradiation, the teeth were placed in vials, which contained 2 mL of the nutrient broth.
The vials were incubated at 37°C for 24 h. The vials were checked for turbidity after 24 h incubation. Dilution of the sample was maintained at 10 3 . Overall 6 mL of broth, in duplicate, from all the samples together was collected and seeded on a Petri dish More Details containing UTI Hichrome agar in order to count the CFUs. Incubation lasted 48 h at 37°C. Grown colonies were seen in all groups and were identified by standard methods  [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6] and [Figure 7].
| Results|| |
Colony counts were done with the aid of a magnifying lens. Statistical analysis used was the non-parametric Kruskal-Wallis test, with comparison using the Bonferroni methods of means. The mean numbers for CFUs/mL of groups were: Group A: 126/10 3 , Group B: 42/10 3 and Group C: 8/10 3 Statistical analysis demonstrated significant differences among the groups. Level of significance: α = 0.05.
The tables below give us the various computations and the P value [Table 1].
Higher mean CFU/mL is recorded in Group A followed by Group B and Group C, respectively. The difference in CFU/mL between the three groups is found to be statistically significant (P < 0.001) [Table 2].
In order to find out among which pair of groups there exist a significant difference, we carry out multiple comparisons using the Bonferroni method.
The difference in mean CFU/mL between Group A and Group B is found to be statistically significant (P < 0.001). The difference in mean CFU/mL between Group A and Group C is also found to be statistically significant (P < 0.001). Statistically significant difference is observed between Group B and Group C with respect to the mean CFU/mL (P < 0.001) [Figure 8].
| Discussion|| |
Infections of the root canal system consist of polymicrobial flora with approximately equal proportions of gram-positive and gram-negative bacteria. , Studies demonstrated that microorganisms are capable of invading the peri-luminal dentin up to a depth of 1100 micro meter, whereas the chemical irrigants penetrate no more than 130 micrometer into the dentin. ,
In recent years, different laser systems are used in endodontic field, which are effective for root canal disinfection. Initially Nd: YAG laser was first used for root canal disinfection, introduced by Hardee  and Myers and McDaniel.  The diode laser is a compact device and now used in diverse areas of dentistry. , Moritz et al. introduced use of diode laser for root canal disinfection. 
E. fecalis is a non-sporiferous vegetative microorganism and is resistant to high temperatures,  frequently found in cases of therapy resistant infection. Because of its resistant to heat, E. fecalis was preferred in this study in order to investigate the effect of the laser heat. Single rooted premolar teeth were instrumented up to #40 K-file to obtain the adequate size and easy access for the fiber tip.
In the present study, the cell concentration at the time of initial inoculation was 10 cells/mL  before the laser treatment. The methodology used for counting the CFUs in the present study showed that the diode laser irradiation provoked a higher level of disinfection than other group without laser disinfection with significant statistical difference.
The high-power diode laser reduces dentine permeability, although it does not provoke the dentine melting unlike neodymium laser. , The diode laser device is composed of two layers of semiconductor material interlaced with a non-conductive layer. Its light presents a spectrum that allows for greater absorption by water than dental tissues when compared with Nd: YAG laser. This characteristic means greater laser light penetration through the dentin with little interaction on the dentin, making it possible to act on the microorganisms present in the dentinal tubules.
The fine diameters of optic fibers (200-320 μm) enable effective delivery of laser light to the root canal to help with reduction of bacterial contamination. The antibacterial effect observed reaches over 1 mm deep into the dentin,  surpassing the effective range of chemical disinfectants, such as NaOCl and displaying moderate effectiveness against E. fecalis even in the deeper layers of dentin. Diode laser effectiveness in relation to diverse microorganisms has been demonstrated by many authors. ,
Moritz et al.,  determined that irradiation with a diode laser in two subsequent sessions resulted in almost complete elimination of bacteria and suggested that the diode laser could be considered equal to the Nd: YAG laser in endodontic treatment. Schoop et al.,  reported that Nd: YAG and diode lasers at 1 W are efficacious for E. fecalis; while the power increased to 1.5 W; only the diode laser was effective against the microorganisms. In the present study, the diode laser was used in both 1.5 W and 3 W. Although complete sterilization cannot be achieved, a significant bacterial reduction was seen.
The parameters used in this study were considered safe in accordance with Radaelli et al.  However the choice of the essential precautions and the correct laser parameters is crucial for a safe and efficient way of therapy. Studies should be done in vivo to check whether the radiation can cause cellular damage to periodontal ligament.  The diode laser used can be applied by all means as a support in endodontic treatment, thus increasing the success rate through its antibacterial effect in endodontic treatment, which should be confirmed by performing clinical studies.
| Conclusion|| |
The results of this research show that the 980 nm diode laser can eliminate bacteria that has immigrated into dentin, thus being able to increase the success rate in endodontic therapy.
| Acknowledgment|| |
We wish to extend our sincere gratitude to Sourav Bhattacharya, Department of Microbiology, Geniohelix Lab, Chamarajpet; Bangalore, India for guiding us in microbiology field for this research work. We also wish to thank all the faculties and their supporting staff members for their invaluable help for successful completion of the present study.
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Department of Conservative Dentistry and Endodontics, V.S. Dental College and Hospital, Bangalore, Karnataka
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2]