The Effect of Diode Laser on Planktonic Enterococcus faecalis in Infected Root Canals in an Ex Vivo Model

Abstract

Objective: This study examined the bactericidal effect of diode laser irradiation against intracanal Enterococcus faecalis. Methods and materials: m total of 128 extracted single-rooted and single-canal teeth were treated with ProTaper instruments (Dentsply Maillefer, Ballaigues, Switzerland). A total of 120 root canals were inoculated with E. faecalis for 21 days, and the samples were randomly divided into five groups: Group 1 (n = 24) samples were irrigated with only saline solution (positive controls); Group 2 (n = 24) was treated with only 5.25% sodium hypochlorite; Group 3 (n = 24) was irrigated with saline solutions activated by diode laser; Group 4 (n = 24) was treated with 5.25% sodium hypochlorite activated by diode laser; and Group 5 (n = 24) was irrigated with saline solution with methylene blue dye activated by the diode laser Fox (Sweden & Martina, Padova, Italy); additionally, eight teeth were not contaminated and their canals were irrigated with saline solution and used as a negative control. The Uro-Quick system was used to determine the microbial residual charge. The data were analyzed using Pearson's chi-square test (p < 0.001). Results: A statistically significant reduction in bacterial count was observed in Group 2 and Group 4 (p < 0.001). There were no statistically significant differences among the other groups (p > 0.001). Conclusions: Evidence indicates that the diode laser was not more effective than sodium hypochlorite in reducing free bacteria.

Introduction

The objective of endodontic therapy is to remove infection and eradicate intracanal bacterial populations.¹ The cleaning and shaping of root canals constitute the most important phases of endodontic therapy because they can greatly influence the final result. Root canal instruments are used for shaping and cleaning, and irrigants are used for cleaning and disinfecting, especially in areas that cannot be reached by instruments or that require further cleaning.

Studies have revealed that instrumentation and irrigation with antimicrobial irrigants, such as NaOCl, do not reliably render root canals free of bacteria, with ∼40–60% of the canals still containing cultivable bacteria after chemomechanical preparation.² Currently NaOCl is the irrigant of choice. It is recommended as an antiseptic for irrigation of the root canal system because of its effective antimicrobial and tissue-dissolving action and is applied during conventional root canal treatment.³ Needle irrigation, however, cannot be successful in the apical part of the root canal or in oval extensions, isthmuses, and anastomoses.⁴

The most common microorganism in root canal reintervention with or without periradicular lesions is the coccus, gram-positive Enterococcus faecalis, which is resistant to endodontic therapy procedures. E. faecalis is known for its ability to form intra- and extraradicular biofilms, which makes it difficult to control. In addition E. faecalis is also well known for to its resistance against chemical disinfectants, high temperatures, and desiccation.⁵

Several studies have also investigated the role of E. faecalis in primary endodontic infections, demonstrating that it is more often related to asymptomatic cases rather than symptomatic cases.⁶

The diode laser, which was introduced in dentistry in the mid-nineties, has been recommended as an adjunct to improve current disinfectant techniques.⁷ The spectrum of the diode laser light is absorbed by the water more than by the dental tissue, reducing its interaction with the dentin while also allowing greater laser light penetration. Several studies indicated some antimicrobial effectiveness of the diode laser.^8,9 Benedicenti et al. showed that concurrent use of the diode laser 810 nm with sodium hypochlorite is statistically more effective in root canal decontamination compared with only hypochlorite.¹⁰ Silbert et al. found 40% killing of E. faecalis using methylene blue and a 670 nm diode laser in infected root canals.¹¹ On the other hand, studies have also shown that the use of a diode laser does not produce the advantages of irrigation with NaOCl.¹²

The aim of this in vitro study was to evaluate the antibacterial effects of a diode laser against E. faecalis in the root canal.

Methods and Materials

Selection and preparation of samples

A total of 128 extracted human single-rooted and single-canal teeth were selected for the study.

The teeth were extracted for periodontal and orthodontic reasons. The external surface was cleaned ultrasonically. Access to the pulp chamber had been obtained for each tooth, and a working length was established. For the root canal instrumentation of all dental elements, a crown-down technique was performed with ProTaper Ni-Ti rotary instruments (Dentsply Maillefer, Ballaigues, Switzerland). Constant irrigation of the root canals was performed with 3 mL of 5.25% NaOCl. All samples were irrigated with 5 mL of 17% EDTA for 3 min (Hydroglide, Stomygen, Roma, Italia), followed by a rinse with 1 mL of 5.25% NaOCl for 3 min, and then a rinse with 1 mL of saline solution.

An apical seal of composite resin (Charisma, Heraeus Kulzer, Milano, Italia) was applied to each tooth. The canals were then dried with sterile ProTaper paper points. The teeth were singularly packed and sterilized by autoclaving at 130°C for 20 min. After the sterilization cycle, the teeth were rehydrated with saline solution under a sterile hood to restore physiological hydration (Steril-VBH Compact, Angelantoni, Mazzo di Rho, Italia). The teeth were stored in saline solution for 24 h and incubated in a thermostat at 37°C in an atmosphere of 5% CO₂.

Microbiological phase

The saline solution used to store the samples was sown on blood agar for 24 h to verify sterility.

One hundred twenty teeth were contaminated with E. faecalis ATCC 29212. Eight teeth were not contaminated, but were irrigated with saline solution.

Each tooth was dried with sterile paper cones and placed in a Falcon tube with 2 mL of culture liquid containing E. faecalis, prepared the same day at a turbidity of 1 MacFarland (3 × 10⁸ CFU/mL). This liquid was first inserted into the root canal to avoid the formation of air bubbles and was then inserted in the test tube until it completely covered the tooth. The 120 test tubes were incubated in a thermostat (Angelantoni, Mazzo di Rho, Italy) at 37°C in an atmosphere of 5% CO₂. The incubation period was 21 days with a change of the culture liquid every 48 h. At the end of the incubation period, each tooth was removed from the tube of inoculum, and steps were taken to determine the bacterial load reached in the root canal before switching to the laser treatment.

Laser-assisted treatment

The samples were randomly divided into five experimental groups:

Group 1 (n = 24): Root canals were irrigated with 5 mL of saline solution (positive control).

Group 2 (n = 24): Root canals were irrigated with 5 mL of 5.25% NaOCl for 3 min.

Group 3 (n = 24): Root canals were rinsed with sterile saline solution activated by diode laser.

Group 4 (n = 24): Samples were irrigated with 5.25% NaOCl activated by diode laser.

Group 5 (n = 24): Root canals were treated using saline solution colored with methylene blue and activated by diode laser.

Intracanal irradiation was performed using the high-power diode laser Fox (Sweden & Martina, Padova, Italy) with a wavelength of 810 nm, power output of 8 W, radiant energy of 75 J, and radiant power of 2.5 W. Three cycles were performed (30 s per cycle) for a total of 90 s of disbursement. For the first two cycles, an optical fiber of 200 μm in scope and 1 mm in working length was used, and helicoidal movements, from apical to cervical, were manually performed.⁹ The third cycle was carried out using a fiber of 300 μm to radiate the middle third and the coronal third of the root canal. Before starting treatment, both of the optical fibers were activated on a dark surface.

Determination of microbial residual charge

The root canal of each tooth was filled with saline solution using a sterile syringe, and a sterile paper cone was inserted inside the root canal for 10 min. Each completely soaked paper point was inserted into a Falcon test tube containing sterile culture liquid. After the Falcon tubes containing the microtubes were whirlpooled, the bacteria taken from the root canal of each sample were placed into the liquid medium, and the bacterial load of each sample was counted by colony-forming units (CFUs). The count was carried out with the Uro-Quick system (Alifax, Padova, Italy), an automatic dynamic system that combines the technique of light scattering (LS, light scattering) with measurements of growth kinetics. The count of E. faecalis (CFU/mL) before and after disinfection was scored according to a three-point scale: 0 (<2.5 × 10⁴ CFU/mL); 1 (2.5–7 × 10⁴ CFU/mL); and 2 (>7 × 10⁴ CFU/mL). Pearson's chi-square test was used to analyze the CFU values among the five experimental groups and within each group before and after disinfection. The significance level was set at p < 0.001.

Results

The counts of E. faecalis (CFU/mL) before and after disinfection are shown in Table 1.

Table 1.

The Percentage of Samples with Scores 0, 1, and 2 (CFU/ml) Pre- and Post-Treatment for Each Group

	0	1	2
	<2,5 × 10⁴ CFU/mL (%)	2,5–7 × 10⁴ CFU/mL (%)	>7 × 10⁴ CFU/mL (%)
Group 1
Pre	0	0	100
Post	0	0	100
Group 2
Pre	0	0	100
Post	100	0	0
Group 3
Pre	0	0	100
Post	0	100	0
Group 4
Pre	0	0	100
Post	100	0	0
Group 5
Pre	0	0	100
Post	0	100	0

In all experimental groups, there was a reduction in the number of CFUs after disinfection, and this reduction was significantly superior to the positive control (Group 1). However, the difference was statistically significant (p < 0.001) for the samples treated with 5.25% NaOCl (Group 2) and the samples treated with sodium hypochlorite activated by laser (Group 4), which were completely decontaminated with a total absence of charge-free bacteria; for Groups 3 and 5, a nonsignificant reduction of bacterial growth was obtained.

Discussion

E. faecalis is associated with persistent endodontic infections and becomes resistant to common intracanal medication by forming biofilms.^13
–15 The use of E. faecalis as a microbiological marker in several studies is well documented; the bacterial colonization and invasion of root dentinal tubules, in addition to important factors such as adherence capacity to dentin collagen,¹⁶ nutrient supply, and time for colonization, determine the pathogenic potential of E. faecalis.¹⁷

The high-power diode laser in some conditions reduced dentine permeability, although it did not provoke melting of the dentine.¹⁸ Laser-induced killing of bacteria can be attributed to thermal heating of the environment above lethal values and local heating inside the bacteria.¹⁹ The survival of E. faecalis and lower reduction rates can be attributed to its cell wall structure, which allows for high resistance to heat.²⁰ Laser irradiation with its inherent properties of light scattering, local intensity enhancement, and attenuation allowed an increase in the effectiveness and success rate of root canal treatment due to the laser's ability to penetrate into deeper parts of the dentinal tissues.^21,22

The reduction of bacterial load obtained in Group 3 (sterile saline solution activated by diode laser) is likely attributed to bactericidal effects of the laser. Mehrvarzfar et al. showed that activating saline solution with the diode laser was able to only disinfect root canals up to 80% at 2 W;²³ the partial eradication of bacteria was achieved when teeth were treated using only high-power irradiation that produced higher temperatures on the root surface.²⁴ da Costa Ribeiro et al. analyzed the increase in temperature during the use of 810 nm diode laser in a root canal at different radiant powers: at 2.5 W, the same radiant power of our study, the thermal rise in the apical part of the canal varied from 1.6°C to 8.6°C, remaining within safety limits for periodontal tissue.²⁵ On the other hand, de Moura-Netto et al. showed a partial debris and smear layer removal and area of alterations (fusion and recrystallization) using a diode laser (810 nm) at 2.5 W.²⁶

In Group 5 (saline solution colored with methylene blue activated by diode laser), the bactericidal action of the laser was enhanced by the dye. In fact, methylene blue, like other exogenous dyes, favors the absorption of laser radiation. The bacterial load was not eliminated in both Group 3 and Group 5 (p > 0.001).

Several studies have revealed a complete disinfection of 100% of bacteria when using high-power diode laser irradiation, followed by canal irrigation with 0.5% NaOCl and 17% EDTA, compared with ∼98.39% disinfection with the use of only hypochlorite.^18,27 In our study, the conventional NaOCl syringe irrigation (Group 2) and high-power diode laser (Group 4) provided complete decontamination of root canals with a total absence of charge-free bacteria, and the groups did not differ significantly from each other. Our findings are in disagreement with previous studies where the effect of high-power diode laser in association with sodium hypochlorite seemed to increase antimicrobial efficacy, especially in the coronal and medium regions of the root canal.^12,28
–30

However, some studies have indicated that a diode laser is unable to completely eliminate E. faecalis.^8,9,31 These previous studies also demonstrated greater difficulty in eliminating gram-positive E. faecalis using diode and Nd:YAG lasers.^19,20 In other studies, the high-power diode laser and conventional NaOCl syringe irrigation were demonstrated to have equal or lower antibacterial effects.^28,32,33

However, our results could be explained by the limitations of this system of measurement: a distinguishing feature of E. faecalis is the creation of a biofilm and, in this case, only the bacterial load was measured. While these results may be useful in a preventive strategy, further parallel studies would be needed to evaluate the effectiveness of laser alone and in combination with various irrigants in the disruption and elimination of biofilms associated with chronic infections.

Numerous alternative irrigation methods have been proposed³⁴; several studies affirm that sonic activation of the irrigant resulted in significantly more bacteria and smear layer removal than conventional syringe irrigation in straight root canals.^35,36 In addition, the EndoActivator and PAD (photoactivated disinfection) therapy succeeded in reducing root canal infection and could eradicate E. faecalis.

Conclusions and Summary

The use of a high-power diode laser has been proposed not as an alternative to the conventional approach of cleaning, disinfecting, and shaping of the root canal, but as an adjuvant to conventional endodontic cleaning procedures. The diode laser in combination with sodium hypochlorite was not more effective than sodium hypochlorite in reducing free E. faecalis. Due to limitations of the evaluation technique and complications during treatment procedures in a clinical situation, further studies are required to investigate the clinical effectiveness of this approach.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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