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ORIGINAL ARTICLE
Year : 2012  |  Volume : 6  |  Issue : 1  |  Page : 2-6

An assessment of bactericidal effect of two different types of lasers on enterococcus faecalis: An in vitro study


1 Department of Conservative Dentistry and Endodontics, Meghna Institute of Dental Sciences, Nizamabad, India
2 Department of Conservative Dentistry and Endodontics, SIBAR Institute of Dental Sciences, Guntur, India

Date of Web Publication15-Sep-2012

Correspondence Address:
Mohammed Ahmeduddin
Department Of Conservative Dentistry and Endodontics, MEGHNA Institute of Dental Sciences. 503003, Nizambad. AP
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-2868.100975

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  Abstract 

Aim and Objective: To evaluate the bactericidal efficacy of the Nd:YAG laser and the Diode laser at different parameters to that of calcium hydroxide, and to know the effect of increase in lasing time on degree of disinfection. Materials and Methods: Fifty human single-rooted teeth were taken and were kept in 3% NaOCl for 15 mins and access cavities were prepared, the apical third of these roots was gradually enlarged until reaching the ISO 40- K file. Later the teeth were autoclaved and mounted on 2.5 ml eppendorf tubes filled with BHI medium which was autoclaved again. The teeth were inoculated with a standard strain of Enterococcus faecalis and incubated for one week. They were randomly divided into five groups and treated as follows, G-I: - Negative control. G-II:- 980 nm diode laser irradiation for five seconds and five cycles at 2W power, 10 Hz frequency and pulsed mode. G-III:- 980 nm diode laser irradiation for 10 seconds and five cycles at 2W power, 10 Hz frequency and pulsed mode. G-IV:- Nd:YAG laser irradiation for five seconds and five cycles at 2 W power, 15 Hz frequency and pulsed mode. G-V:- Nd:YAG laser irradiation for 10 seconds and five cycles at 2 W power, 15 Hz frequency and pulsed mode. The dentin dust was collected from superficial and deep dentin of coronal and apical halves of all the samples and CFU/ ml were evaluated after three days. Results: The results were calculated and statistical analysis was done using Kruskal Wallis ANOVA test by ranks and Mann Whitney's U test. Comparisons were done between the superficial and deep dentin of different groups and between the groups. Conclusion: Under the parameters of the samples tested Nd:YAG laser provided the best results for disinfection compared to Diode laser. However, both the Nd:YAG and Diode lasers provided clinically acceptable disinfection.

Keywords: Colony forming units, diode laser, enterococcus faecalis, Nd:YAG laser


How to cite this article:
Ahmeduddin M, Nagesh B, Reddy K N, Raj K S. An assessment of bactericidal effect of two different types of lasers on enterococcus faecalis: An in vitro study. J Dent Lasers 2012;6:2-6

How to cite this URL:
Ahmeduddin M, Nagesh B, Reddy K N, Raj K S. An assessment of bactericidal effect of two different types of lasers on enterococcus faecalis: An in vitro study. J Dent Lasers [serial online] 2012 [cited 2022 Dec 3];6:2-6. Available from: http://www.jdentlasers.org/text.asp?2012/6/1/2/100975


  Introduction Top


The main objective of endodontic treatment is to completely eliminate bacteria from the root canal system and obtain three dimensional obturation of the tubular network. Endodontic procedures rely on mechanical instrumentation, intracanal irrigants, and medicaments to disinfect the root canal system. Mechanical cleansing of the root canal removes the bulk of infected tissue, but chemical and other means of disinfection are necessary for complete elimination of microorganisms, which may cause persistent inflammation and treatment failure. [1] Although current instrumentation techniques involving hand or rotary instruments as well as ultrasonic and sonic devices can greatly reduce the bacterial load in the infected canal, they fall short of the goal of total disinfection of the root canal system. [2]

Irrigants such as sodium hypochlorite and chlorhexidine have demonstrated useful antimicrobial effects, but infection of the root canal and adjacent dentin may still persist owing to the inability of these agents to reach all the infectious microorganisms. [3] Conventional irrigants used for disinfection have disadvantage the of reduced penetration into dentinal tubules due to the complexity of the root canal system and presence of smear layer. [4],[5] The introduction of lasers in endodontics has dramatically improved the effectiveness and success rate of root canal treatment. In general, dental lasers provide greater accessibility of formerly unreachable parts of the tubular network due to their better penetration into dentinal tissues. The use of lasers for sterilization of root canals has shown promising results in previous studies. [1],[4],[6],[7] Diode lasers can be used for a multitude of dental procedures which are predominantly soft tissue procedures including soft tissue surgery, periodontal pocket therapy and peri-implantitis, but can also be used for certain applications root canal disinfection. [8]

The efficacy of Nd:YAG lasers for photo-thermal disinfection has been investigated and laser application was found to be safe and having potential advantage of using for various purposes like cleaning and shaping and sealing of dentin walls and root canal sterilization. [1],[4],[9] Although studies have been done to evaluate the antibacterial efficacy of the diode laser and Nd:YAG laser individually, the comparative evaluation of these lasers has not been less reported. Taking this into account, this in vitro study has been done to evaluate and compare the degree of disinfection and penetrating ability of both the Diode and the Nd:YAG laser irradiation of infected root canals at two different time intervals.


  Materials and Methods Top


Specimen collection

Fifty human single-rooted teeth were taken and were kept in 3 % NaOCl for 15 minutes, access cavities were prepared, the canals were cleaned and pulp remnants were removed with the help of ISO 15 K- file (Mani inc. Japan.). All the teeth were cut to a standard size of 15 mm from the apex. Following this, the apical third of these roots was gradually enlarged until reaching the ISO 40- K file, 1 mm short of anatomical apex. During cleaning and shaping 2.5 ml of each 3% sodium hypochlorite (Prime dental products. India) and 17 % EDTA (Pulpdent.USA) were used. The teeth were mounted on the eppendorf tubes after autoclaving 134°C for seven minutes. Color coding was done accordingly on the tubes [Table 1].
Table 1: Colour coding of specimens

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Later the eppendorf tubes were filled with brain heart infusion medium. The set was again sterilized in an autoclave at 134°C for seven minutes.

Inoculation of specimen

The standard strain of Enterococcus faecalis ATCC 29212 (Himedia, India), and incubated in 50 ml of brain heart infusion culture medium at 37°C for 24 hrs, it was then vortexes (Vortex meter Hind medical equipment, India) to get a cell concentration of approximately 10 8 cells/ml. The prepared canals of the sterilized specimens were inoculated with the suspension and later incubated for one week in a 37°C. All the procedures were carried out in a laminar flow chamber.

Group design

The specimens were randomly divided into five groups. After one week of incubation the samples were taken out of the incubator, the BHI medium in the tubes was removed, the canals of all the samples were irrigated with saline and cleaned and later treated as follows:

  • G-I- Negative control Group
  • G-II- 980 nm diode laser irradiation for five seconds and five cycles at 2 W power, 10 Hz frequency and pulsed mode.
  • G-III - 980 nm diode laser irradiation for 10 seconds and five cycles at 2 W power, 10 Hz frequency and pulsed mode.
  • G-IV - Nd:YAG laser irradiation for five seconds and five cycles at 2 W power, 15 Hz frequency and pulsed mode.
  • G-V - Nd:YAG laser irradiation for 10 seconds and five cycles at 2 W power, 15 Hz frequency and pulsed mode.
The irradiation of the specimens with the lasers was done according the technique given by Gutknecht N. and Moritz A. Utilizing a 200 μm optical fiber, A gap period of 10 seconds was given between each radiation cycle performed.

Sample collection

The apical 1 mm of the samples were removed with diamond disc and the specimens were sliced into two halves transversely. Each coronal and apical half was mounted over a 0.2 ml eppendorf tube and was drilled with a sterile Gates glidden drills. The dentinal dust was collected, from lumen of the main canal 0.5 mm in dentin for circumpulpal dentin, and 0.5 mm further deep for deep dentin.

Each group provided four samples sub - groups namely

Subgroup A - superficial dentin in coronal half,

Subgroup B - deep dentin in coronal half,

Subgroup C - superficial dentin in apical half, and

Subgroup D - deep dentin in apical half.

The BHI medium was added to the collected dentinal dust and vortexed after which 0.1 ml of the suspension was spread over the agar in petridishes (Applied biosystems, Singapore) and incubated.


  Results Top


The viable colony forming units were counted as (CFU's/ ml) after three days. The means and standard deviations were calculated for all the groups. The statistical analysis was done by using Kruskhal wallis test as the data obtained did not have normal distribution. The minimum Colony forming units were obtained sub groups of Gr: V (Nd:YAG laser irradiation for 10 secs and five cycles) A- 9x10, B-1x10, C-0 and D- 0 and hence the overall disinfection was also high amongst all the groups. The degree of disinfection was not consistant in the sub groups A, B, C and D [Graph 1].




  Discussion Top


Several protocols have been proposed for the eradication of bacteria including use of highly concentrated Sodium hypochlorite (6 %), chlorohexidine 2% and a combination of chlorohexidine and calcium hydroxide. [10] Use of concentrated antibacterial rinsing solutions may have cytotoxic effects over the periapical tissues. More over the formation of precipitates during combined irrigating regimen may reduce the penetration of this irrigating solutions. [11] The use of Diode and Nd:YAG lasers for root canal disinfection has been well researched and have been found promising in reducing the number of bacteria from the root canal dentin. [1],[9] In the present study the Diode and Nd:YAG lasers have been used at different parameters to evaluate the comparative disinfection and the effect of increase in time on degree of disinfection.

In this study, the maximum concentration of bacterial cells during inoculation was 10 8 cells/ml and the bacterial concentration after the irradiation was evaluated. The laser irradiation was done for 10 and five seconds with a gap period of 10 seconds given between successive cycles of irradiations. The degree of disinfection which was achieved in superficial dentine (A and B sub groups) was statistically highly significant, i.e. P<0.001 [Table 2] and [Table 3]. Disinfection was comparatively more in superficial dentin of Group IV and V (Nd:YAG laser irradiated groups) than Group II and III (Diode laser irradiated groups) and the Group I [Figure 1] (Negative control group) [Table 4]. Both Laser irradiated groups showed better disinfection than the control group. But the degree of disinfection was not consistent throughout the degree of disinfection was more in the deep dentin of the coronal and apical half than in the superficial dentin.
Figure 1: Group I

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Table 2: Comparison of three groups (I, II+III, IV+V) with respect to CFU counts in sub group CD by Kruskal Wallis test

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Table 3: Summary statistics of counts according groups (I, II, III, IV and V)

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Table 4: Comparison of three groups (I, II+III, IV+V) with respect to CFU counts in sub group AB by Kruskal Wallis test

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Overall disinfection was better in Group V [Figure 2] where Nd:YAG laser was used for 10 seconds in five successive cycles, which was followed by Group II > Group III > Group II > Group I [Table 5]. The results obtained here show that the Nd:YAG laser when used at 2 W, 15 Hz in a pulsed mode for 10 seconds and five successive cycles provides optimum results for disinfection. However there was no complete eradication of bacteria in any of the five groups. This may be attributed to straight nature of the laser beam during irradiation. The power and frequency parameters used in this study were considered to be safe in the previous studies. [6],[12] During the irradiation the laser beam to a great extent absorbed by blood, hemoglobin, melanin and other pigmented tissue components which contribute to its thermal effects hence there is reduction in temperature in a very short period of time.
Figure 2: Group V

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Table 5: Comparison of five groups (I, II, III, IV and V) with respect to CFU counts of total samples by Kruskal Wallis test by ranks

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The possible mechanisms regarding the antibacterial effect of laser are thermal and photodisruptive effects. [6] These effects are considered the principal reasons for the laser to eliminate the bacteria. Immediate cell death might not occur during laser irradiation, but sublethal damage inhibits the cell growth after exposure to laser irradiation. The sub lethal damage includes destruction of cell wall integrity and possibly the accumulation of denatured protein. Integrity of cell wall is intimately related to the mechanical stability of gram-positive bacteria. The damage of cell wall will cease the cell growth and successive cell lysis. On the other hand, the cellular protein is highly sensitive to thermal changes. The laser irradiation might produce denatured protein and induce the cell to create new proteins to compensate the denaturation of the proteins. In a Bacterial cell some proteins such as IDG-60 immunodominant glycoprotein is indispensable for maintaining the integrity of the cell wall and the structure uniformity of cell shape. The stress on the cells to prevent the accumulation of denatured protein debris could also cause cell death. [13]

Regardless the type of laser is used for disinfection the principle effect is through heat, which destroys the contents of microbes. With the coherent nature of laser beam it is not possible to irradiate laterally on the root canal dentin and the focus is straight. This hinders the penetration of laser light laterally into the canals and ramifications and hence becoming less effective. The Nd:YAG and the diode laser have more affinity towards the pigmented tissue. Further research could be done taking the above concerns into account by modifying the lasing fiber tip and using dyes for bacterial staining before irradiation.


  Conclusion Top


Within confines of this study it can be concluded that both the Nd:YAG and diode lasers provide clinically acceptable disinfection. The diode laser when used for 10 seconds and five successive cycles provides disinfection similar to that of the Nd:YAG laser. The increase in intracanal lasing time increases disinfection. Nd:YAG laser provides better disinfection than diode laser, and the use of lasers as adjunct to intracanal disinfection procedures is indispensible in modern endodontic practice.

 
  References Top

1.Moshonov J, Orstavik D, Yamauchi S, Trope M. Nd:YAG laser irradiation in root canal disinfection. Endod Dent Traumatol 1995;11:220-4.  Back to cited text no. 1
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2.Plotino G, Pameijer CH, Grande NM, Somma F. Ultrasonics in endodontics: A Review of the Literature. J Endod 2007;33:81-95.  Back to cited text no. 2
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3.Gordon W, Atabakhsh VA, Meza F, Doms A. The antimicrobial efficacy of the ErCr:YSGG laser with radial emitting tips on root canal dentin walls infected with enterococcus faecalis. J Am Dent Assoc 2007;138:992-1002.  Back to cited text no. 3
    
4.Gutknecht N, Franzen R, Schippers M, Lampert F. Bactericidal effect of a 980-nm diode laser in the root canal wall dentin of bovine teeth. J Clin Laser Med Surg 2004;22:9-13.  Back to cited text no. 4
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5.Gutknecht N. Lasers in endodontics. J Laser Health Acad 2008;4:1-4.  Back to cited text no. 5
    
6.Hardee MW, Miserendino LJ, Kos W, Walia H. Evaluation of the antibacterial effects of intracanal Nd: YAG laser irradiation. J Endod 1994;20:377-80.  Back to cited text no. 6
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7.Rooney J, Midda M, Lleeming J. A laboratory investigation of the bactericidal effect of a Nd:YAG laser. Br Dent J 1994;176:61-4.  Back to cited text no. 7
    
8.Pirnat S. Versatility of an 810 nm diode laser in dentistry: An overview. J Laser Health Acad 2007;4:1-9.  Back to cited text no. 8
    
9.Bergmans L, Moisiadis P, Teughels W, Van Meerbeek B. Bactericidal effect of Nd:YAG laser irradiation on some endodontic pathogens ex vivo. Int Endod J 2006;39:547-57.  Back to cited text no. 9
    
10.Stuart CH, Schwartz SA. Enterococcus Faecalis; Its role In root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.  Back to cited text no. 10
    
11.Lessa FC, Aranha AM, Nogueira I, Giro EM, Hebling J, Costa CA. Toxicity of chlorhexidine on odontoblast-like cells. J Appl Oral Sci 2010;18:50-8.  Back to cited text no. 11
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12.de Souza EB, Cai S, Simionato MR, Lage-Marques JL. High-power diode laser in the disinfection in depth of the root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:e68-72.  Back to cited text no. 12
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13.Lee BS, Lin YW, Chia JS, Hsieh TT, Chen MH, Lin CP, et al. Bactericidal effects of diode laser on streptococcus mutans after irradiation through different thickness of dentin. Lasers Surg Med 2006;38:62-6.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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