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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 10  |  Issue : 2  |  Page : 138-144

Determination of minimum instrumentation size for penetration of diode laser against Enterococcus faecalis in root canals using scanning electron microscope: An in vitro study


Department of Conservative Dentistry and Endodontics, KLE V. K. Institute of Dental Sciences, Belagavi, Karnataka, India

Date of Web Publication30-May-2017

Correspondence Address:
Niraj Jayant Godbole
807, Angol “C” Scheme, M. G. Road, Hindu Nagar, Tilakwadi, Belagavi - 590 006, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/kleuhsj.ijhs_429_16

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  Abstract 

Aim: The aim of this study was to determine minimum instrumentation size for penetration of diode laser against Enterococcus faecalis in root canals using scanning electron microscope (SEM).
Methodology: Fifty-five single-rooted human permanent maxillary anterior teeth were selected. The sterilized specimens were inoculated with 5 μl E. faecalis culture grown in diluted brain–heart infusion broth and incubated at 37°C for 4 weeks. After 4 weeks, 5 samples were selected as baseline group to evaluate the depth of penetration of E. faecalis. Remaining 50 samples were divided into 5 groups based on the apical enlargement size using ProTaper file system: Group 1 – canal preparation done till #F1 size file; Group 2 – canal preparation done till #F2 size file; Group 3– canal preparation done till #F3 size file; Group 4 – canal preparation done till #F4 size file; Group 5 – canal preparation done till #F5 size file. The canals were prepared and then irradiated with 940 nm diode laser at a power setting of 1.5W for 5 s. The procedure was repeated 4 times with intervals of 10 s between each one. All the samples were then transversely sectioned at 2 mm and 5 mm from the apex. Each section was then examined under a SEM to evaluate the depth of penetration of E. faecalis in the baseline group and depth of penetration of diode laser in the experimental groups. The results were analyzed using the two-way ANOVA and Tukey's multiple post hoc test.
Results: Depth of penetration of laser was highest in Group 3 in apical third region and was statistically significant when compared to Group 1 and Group 2 in the middle and apical third regions. However, no statistically significant difference was noted between Group 3, Group 4, and Group 5.
Conclusion: It can be concluded that the apical instrumentation to a size F3 using ProTaper file system along with irradiation using a diode laser showed a greater depth of penetration into dentin and thus reducing residual microorganisms. Furthermore, conserving as much tooth structure as possible, thereby increasing the predictability of success.

Keywords: Depth of penetration, diode laser, Enterococcus faecalis, scanning electron microscope


How to cite this article:
Godbole NJ, Dodwad PK, Bhat K, Patil A. Determination of minimum instrumentation size for penetration of diode laser against Enterococcus faecalis in root canals using scanning electron microscope: An in vitro study. Indian J Health Sci Biomed Res 2017;10:138-44

How to cite this URL:
Godbole NJ, Dodwad PK, Bhat K, Patil A. Determination of minimum instrumentation size for penetration of diode laser against Enterococcus faecalis in root canals using scanning electron microscope: An in vitro study. Indian J Health Sci Biomed Res [serial online] 2017 [cited 2022 Sep 29];10:138-44. Available from: https://www.ijournalhs.org/text.asp?2017/10/2/138/207259


  Introduction Top


The main goals of root canal therapy are disinfection and complete sealing of the root canal space and prevention of reinfection. The root canal system is very complex and makes it difficult for the irrigating solution to completely disinfect the root canal.[1]

Various studies have shown that microorganisms in failed root canal treatment cases are of distinct group which include Gram-positive cocci and facultative anaerobes such as Enterococcus faecalis (E. faecalis). They have the ability to grow in the presence or absence of oxygen. They are associated with various periradicular diseases such as primary endodontic infections and persistent infections. It has been shown that the chance of containing E. faecalis in failed root canals is up to nine times of primary infections. E. faecalis is completely resistant to intracanal medications such as calcium hydroxide and wide variations in pH. Various irrigants are used to eliminate these microorganisms, among which is sodium hypochlorite (NaOCl) which is the most widely used irrigant.[2]

However, these irrigants have a limited penetration into dentinal tubules in the apical 1/3rd when compared to middle and coronal 1/3rd; thus, lasers have become the latest choice to eliminate microorganisms in deeper layers of dentin.[3]

Another important aspect of root canal preparation is the minimum enlargement of the root canals during cleaning and shaping which would enable maximum amount of disinfection of the canals. The penetration of irrigation solution into the dentinal tubules is dependent on the removal of debris and the final size of the instrument used in the canals.[4]

Use of lasers in dentistry has been introduced in the late 1980's, with the production of the American dental laser.[5] The high-power diode laser has been used in several areas of dentistry and has shown promising results in relation to disinfection. Different wavelengths of diode lasers have been available for the use in endodontics: 810 nm, 830 nm, 940 nm, 980 nm. Recently, diode laser with a wavelength of 940 nm has been studied widely as it has been demonstrated that this wavelength has a greater penetration depth in the dentinal tubules. Lasers demonstrate some amount of cavitation that is generated in aqueous irrigation fluids around the top of the fiber tip.

The effect of laser beam is based on thermal changes, i.e., it generates heat which is bactericidal and thus kills the microorganisms in the root canal.[3] The antibacterial effect of diode lasers has been observed to be more than 1 mm deep into dentine, surpassing that of NaOCl.[6]

Combination therapy using various irrigation solutions and diode lasers has been shown to be more effective against different microorganism species when used in conjunction or in succession.[7]

Considering this, the present study was conducted to determine minimum instrumentation size for penetration of diode laser against E. faecalis in root canals.

Aim

The aim of this study was to determine minimum instrumentation size for penetration of diode laser against E. faecalis in root canals using scanning electron microscope (SEM).

Objectives

  • To determine the minimum enlargement of root canals using ProTaper Universal file system
  • To evaluate the depth of penetration of diode laser against E. faecalis in the dentinal tubules of prepared teeth.



  Methodology Top


Fifty-five extracted human single-rooted permanent maxillary anterior teeth were collected. Teeth were decoronated at length of 17 mm from the apex using a rotary diamond disc. Nail varnish was applied to each tooth to block any orifice present on the root surface.

Access to the root canals was gained using a contra-angle airotor handpiece and a round diamond point. Canal patency was checked using a #10 K-file, and working length of 16 mm and working width up to maximum of #20 K-file size (i.e., 0.2 mm) were standardized. The canal was enlarged till #20 K-file using 17% ethylenediaminetetraacetic acid (EDTA) as a lubricant under copious irrigation with 3% NaOCl and a final flush of sterile saline. The samples were then autoclaved at 121°C under 15 lbs. pressure for 15 min. Pure culture of E. faecalis (ATCC 35550) in brain–heart infusion broth at a concentration of 1.5 × 108 colony forming unit (CFU)/ml was used for inoculation.

A total of 50 μl of this suspension was inoculated into each canal using a micropipette. Then, the samples were incubated for 4 weeks at 37°C in an incubator. The inoculum in the canal was replaced with a fresh bacterial suspension thrice a week. After the incubation period of 4 weeks, 5 samples were randomly selected and stored separately, and no preparation was done in these samples.

The remaining 50 samples were divided into 5 groups (n = 10) depending on the file size used and the root canals were prepared using ProTaper Universal file system.

  • GROUP 1: Canal preparation done till #F1 size file
  • GROUP 2: Canal preparation done till #F2 size file
  • GROUP 3: Canal preparation done till #F3 size file
  • GROUP 4: Canal preparation done till #F4 size file
  • GROUP 5: Canal preparation done till #F5 size file.


The canals were prepared using 17% EDTA as a lubricant and intermittent irrigation with 2.5 ml of 3% NaOCl and normal saline. Later, the canals were dried using sterile paper points. Each canal was then irradiated using a 940 nm diode laser using 300 μm fiberoptic tips.

The power output was set at 1.5 W in a continuous mode. Irradiation was done in an oscillatory motion. The fiber was introduced 1 mm short of the apex and was retrieved in a helicoidal movement at a speed approximately 2 mm/s for 5 s. The procedure was repeated 4 times with intervals of 10 s between each one. All the 55 samples were transversely sectioned at 2 mm and 5 mm from the apex to analyze the apical and the middle regions, respectively.

The samples were embedded is a clear, cold-cure acrylic resin. The acrylic resin block along with the samples was mounted on the holder of the hard tissue microtome. The sections were prepared and were stored in a reduced transport fluid. Each sample was then mounted on an aluminum stub and was examined under SEM. Photomicrographs of apical and middle thirds of each sample at a magnification of ×100 and ×10,000 were taken for final evaluation.

The samples from the baseline group were examined under SEM in a clockwise direction, and the maximum depth of penetration of E. faecalis into the dentinal tubules from the root canal wall was analyzed. Remaining groups were examined in a similar manner to determine the absence of E. faecalis, i.e., the depth to which E. faecalis was eliminated from the root canal wall.

Statistical analysis

Statistical analysis was done by Tukey's multiple post hoc test and two-way ANOVA test.


  Results Top


The depth of penetration of diode laser in each of the experimental groups was analyzed [Table 1] and [Graph 1]. Group 1 showed statistically significant difference (P < 0.05) when compared to Group 3, Group 4, and Group 5 in both middle and apical third regions, respectively. There was a statistically significant difference between Group 1 in the apical third region and Group 2 in the middle third region. However, Group 1 middle third region showed no statistically significant difference when compared to Group 2 in the middle third region.
Table 1: Mean, standard deviation, standard error, and coefficient of variation of size for penetration in five groups and two region

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Group 2 showed statistically significant difference when compared to Group 3, Group 4, and Group 5 in both middle and apical third regions, respectively. However, there was no statistically significant difference in Group 3, Group 4, and Group 5 [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10].
Figure 1: Sample representing depth of penetration of diode laser in apical third region of Group 1

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Figure 2: Sample representing depth of penetration of diode laser in middle third region of Group 1

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Figure 3: Sample representing depth of penetration of diode laser in apical third region of Group 2

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Figure 4: Sample representing depth of penetration of diode laser in middle third region of Group 2

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Figure 5: Sample representing depth of penetration of diode laser in apical third region of Group 3

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Figure 6: Sample representing depth of penetration of diode laser in middle third region of Group 3

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Figure 7: Sample representing depth of penetration of diode laser in apical third region of Group 4

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Figure 8: Sample representing depth of penetration of diode laser in middle third region of Group 4

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Figure 9: Sample representing depth of penetration of diode laser in apical third region of Group 5

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Figure 10: Sample representing depth of penetration of diode laser in middle third region of Group 5

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  Discussion Top


Complete elimination of bacteria from the root canals is a challenging task and only reduction in microbial count can be achieved through multiple steps such as use of different irrigants and irrigating systems and different intracanal medicaments. However, the tubule penetration capacity of these irrigants and medicaments is very limited.[8]

NaOCl as a root canal irrigant is the most widely used irrigant.[9] However, NaOCl has few drawbacks among which is the limited depth of penetration of the solution into the dentinal tubules. Giardino et al.[10] evaluated the depth of penetration of NaOCl with or without surfactant and reported a maximum depth of penetration of 74 μm to 131 μm which is significantly less than the depth of penetration of E. faecalis which is about 800 μm – 1000 μm as reported by Haapasalo and Orstavik [Figure 11] and [Figure 12].[6]
Figure 11: Sample representing depth of penetration of Enterococcus faecalis in apical third region of baseline group

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Figure 12: Group sample representing depth of penetration of Enterococcus faecalis in middle third region of baseline group

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Diode lasers have a solid active medium; it is a solid-state semiconductor laser that combines with aluminum, gallium, and arsenide to change electric energy into light energy. The available wavelengths for use in dentistry range from about 810 to 980 nm, placing them at the beginning of the near-infrared invisible nonionizing part of the spectrum.[11],[12]

The sterilization effect of the diode laser resembles that of neodymium-doped yttrium aluminum garnet laser. Diode laser shows inhibiting effect on inflammation propagating enzymes.[3] The antibacterial effect of a diode laser is based on the heat generating property after the interaction of the tissue with the laser beam. Diode lasers have broad application spectrum. In addition to these qualities, they have a reasonable price, thus increasing their use.[3],[13] Hence, diode laser was used in the present study.

E. faecalis was used in the present study as it is a normal inhabitant of the oral cavity. E. faecalis is a Gram-positive facultative anerobic cocci, which has the ability to grow in the presence or absence of oxygen. E. faecalis is associated with both primary and secondary endodontic infection. Primary endodontic infections can be associated with asymptomatic chronic periradicular lesions, more often being acute periradicular abscesses. E. faecalis can be found in 4%–40% of primary infections. Hence, effective elimination of microorganisms including E. faecalis is very important for successful endodontic treatment [Figure 13] and [Figure 14].[14]
Figure 13: Representative image of dentinal tubules containing Enterococcus faecalis

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Figure 14: Representative image of dentinal tubules devoid of Enterococcus faecalis

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The concept of working width plays an important role in the chemomechanical preparation of the root canals.[15],[16] Rollison et al. noted that larger file sizes to #50 showed greater reduction in remaining microorganisms when compared to those prepared with #35 file.[17] Khademi et al. in a study concluded that the minimum instrumentation size needed for irrigant penetration in the apical third of the root canal was #30 file size. Enlarging the canals to a greater size increases the risk of ledge formation, perforation, apical transportation, and removal of excessive dentin.[4]

In this ever changing era of rotary instruments, the question of how large should be the enlargement, still remains. Akhlaghi et al. in one study suggested that master apical file of size #35 with 0.06 taper showed 100% acceptable debridement.[18]

With the advent of newer technologies such as ultrasonic irrigation and lasers, combining different approaches can have a synergistic effect.[19] de Souza et al. stated that high-power laser irradiation along with 0.5% NaOCl and 17% EDTA-T showed increased reduction of bacteria in deep radicular dentin.[20] Preethee et al. evaluated the bactericidal effects of 908 nm diode laser in conjunction with NaOCl, EDTA, and MTAD and concluded that 908 nm diode laser in conjunction with NaOCl demonstrated significant elimination in E. faecalis.[21]

No previous study was done evaluating the effect of instrumentation size on the disinfection efficacy of diode laser combined with NaOCl. With these considerations, the present study evaluated the depth of penetration of diode laser against E. faecalis in the dentinal tubules of prepared teeth and to determine the minimum enlargement of root canals using ProTaper Universal file system.

Irradiation was performed based on the criteria suggested by de Souza et al.[20] In all the studies evaluating the disinfection efficacy of diode laser, various researchers have evaluated the CFUs/mL using centrifugal drilling as stated by Haapasalo and Orstavik.[10]

In Group 1 and Group 2, it can be observed that the depth of penetration of diode laser was insufficient to reduce significant amount of E. faecalis. The result can be attributed to the presence of smear layer and debris due to insufficient preparation of the root canals as stated in a study done by Khademi et al.[4] who observed that the groups prepared till size #20 with 0.06 taper and #25 with 0.06 taper, which are equivalent to ProTaper file size F1 and F2, respectively, had the highest amount of smear layer and debris covering the canal wall. Srikanth et al.[22] found similar result in their study and observed that teeth prepared till file size #30 with 0.06 taper showed the highest removal of smear layer.

In Group 3 (samples prepared till size F3), the depth of penetration of diode laser in the middle region showed a mean ± standard deviation value of 899.01 ± 29.81 μm, and in the apical region, it was 920.61 ± 35.16 μm. These values are significantly higher than the mean depth of penetration of E. faecalis as observed in the present study. Statistical analysis revealed that the mean depth of penetration of diode laser in Group 3 in both middle and apical third regions is significantly greater than the mean values of Group 1 (samples prepared till size F1) and Group 2 (samples prepared till size F2), respectively. This difference can be attributed to the amount of smear layer and debris eliminated using ProTaper file size F3, i.e., tip size equivalent to #30 with 0.09 taper as suggested by Khademi et al.[4] and Akhlaghi et al.[18] Group 4 (samples prepared till size F4) and Group 5 (samples prepared till size F5) showed similar values. There was no statistically significant difference between Group 3, Group 4, and Group 5 in middle and apical third regions, respectively. The reason for these finding is same as that explained for Group 3, i.e., the removal of smear layer from the root canal wall allowing an increased penetration of diode laser irradiation.

Within the limitations of the present study, diode laser irradiation seems to be a viable adjunct in combination therapy using irrigants to effectively eliminate microorganisms present deep into the dentinal tubules. However, further in vitro and in vivo evaluation of diode laser irradiation as a combination therapy is recommended.


  Conclusion Top


Under the parameters of this study, it can be concluded that apical enlargement in maxillary anteriors to size F3 (Group 3) using the ProTaper system has shown promising results in reducing E. faecalis in deeper dentinal tubules when used in conjunction with 940 nm diode laser. Since the apical enlargement can be restricted to size F3, there is increased amount of remaining dentin after mechanical preparation, which increases the strength of the tooth and reduces the chance of fracture posttreatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Wu D, Fan W, Kishen A, Gutmann JL, Fan B. Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus faecalis biofilm. J Endod 2014;40:285-90.  Back to cited text no. 1
    
2.
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. 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. 2
    
3.
Kaiwar A, Usha HL, 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.  Back to cited text no. 3
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4.
Khademi A, Yazdizadeh M, Feizianfard M. Determination of the minimum instrumentation size for penetration of irrigants to the apical third of root canal systems. J Endod 2006;32:417-20.  Back to cited text no. 4
    
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Haapasalo M, Orstavik D.In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375-9.  Back to cited text no. 6
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Castelo-Baz P, Martín-Biedma B, Ruíz-Piñón M, Rivas-Mundiña B, Bahillo J, Perez-Estévez A, et al. Combined sodium hypochlorite and 940 nm diode laser treatment against mature E. Faecalis biofilms in vitro. J Lasers Med Sci 2012;3:116-21.  Back to cited text no. 7
    
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Mohammadi Z. Sodium hypochlorite in endodontics: an update review. Int Dent J 2008;58:329-41.  Back to cited text no. 9
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Giardino L, Cavani F, Generali L. Sodium hypochlorite solution penetration into human dentine: A histochemical evaluation. Int Endod J 2016. [Epub ahead of print].  Back to cited text no. 10
    
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Moritz A, Schoop U. Lasers in endodontics. In: Mortiz A, editor. Oral Laser Application. Ch. 6. Germany: Quintessenz Verlags-GmbH; 2006. p. 241-313.  Back to cited text no. 13
    
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Rôças IN, Siqueira JF Jr., Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315-20.  Back to cited text no. 14
    
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Jou YT, Karabucak B, Levin J, Liu D. Endodontic working width: Current concepts and techniques. Dent Clin North Am 2004;48:323-35.  Back to cited text no. 15
    
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Wu MK, Wesselink PR. Efficacy of three techniques in cleaning the apical portion of curved root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:492-6.  Back to cited text no. 16
    
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Rollison S, Barnett F, Stevens RH. Efficacy of bacterial removal from instrumented root canals in vitro related to instrumentation technique and size. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:366-71.  Back to cited text no. 17
    
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Akhlaghi NM, Dadresanfar B, Darmiani S, Moshari A. Effect of master apical file size and taper on irrigation and cleaning of the apical third of curved canals. J Dent (Tehran) 2014;11:188-95.  Back to cited text no. 18
    
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Jyotsna SV, Raju RV, Patil JP, Singh TV, Bhutani N, Kamishetty S, et al. Effect of diode laser on bacteria beyond the apex in relation to the size of the apical preparation – An in-vitro study. J Clin Diagn Res 2016;10:ZC63-5.  Back to cited text no. 19
    
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Preethee T, Kandaswamy D, Arathi G, Hannah R. Bactericidal effect of the 908 nm diode laser on Enterococcus faecalis in infected root canals. J Conserv Dent 2012;15:46-50.  Back to cited text no. 21
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Srikanth P, Krishna AG, Srinivas S, Reddy ES, Battu S, Aravelli S. Minimal apical enlargement for penetration of irrigants to the apical third of root canal system: A scanning electron microscope study. J Int Oral Health 2015;7:92-6.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14]
 
 
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