An in vivo comparison of the antibacterial efficacy of photoactivated disinfection, diode laser, and 5% sodium hypochlorite in root canal disinfection.

BACKGROUND: Pulpal infections are caused by varied number of organisms and are located in various sites of root canal system. Thus, the success of root canal treatment depends on complete eradication of microbiota.
MATERIALS AND METHODS: Thirty-two patients were selected and divided into four groups (photoactivated disinfection [PAD], diode laser, 5% sodium hypochlorite [NaOCl], and normal saline). The treatment was done according to groups, following manufacturer's instructions. The efficacies of groups were evaluated using microbial culturing technique. Three samples were taken for each group and were incubated separately for aerobic and anaerobic culture. Microbial counting was done for each sample in the form of colony-forming units.
RESULTS: It was evaluated using two-way analysis of variance and Friedman test. All the groups showed reduction in number of bacteria. However, for group A P < 0.0001 when analyzed for aerobic and anaerobic microorganisms, whereas, with Group B and C, P < 0.0001 for aerobic microorganisms.
CONCLUSION: PAD, diode laser, and 5% NaOCl showed antibacterial action against aerobic and anaerobic bacteria.

INTRODUCTION

The success of endodontic therapy depends mainly on elimination of bacteria and irritants from root canal before obturation.[1] The chemical debridement helps in removal of residual tissue and bacterial biofilm, mainly from the noninstrumented areas of root canal system.[2] The mechanical objective involves flushing out debris, lubricating canal, and dissolving organic and inorganic tissue whereas biological function adds to the antimicrobial effect.[3] The irrigating solutions used for chemical debridement are sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid, chlorhexidine, citric acid, MTAD [mixture of doxycycline, citric acid, and a detergent (Tween80)], tetraclean, and other herbal irrigants.[23]

Coolidge introduced hypochlorite in endodontics and is the most commonly used endodontic irrigant.[4] It has unique properties of organic tissue dissolution, saponification, transformation of amines, and antibacterial activity.[5] However, the recent literature has focused on LASER and photoactivated disinfection (PAD) for root canal disinfection. The word “LASER” is an acronym for light amplification by stimulated emission of radiation. In 1960, Theodore Maiman developed the first working laser device, which emitted a deep red-colored beam from ruby crystal.[6] In endodontics, it was first used for sealing apical foramen of freshly extracted teeth. Further, in 2000, diode laser was introduced for root canal disinfection.[7] PAD is an antimicrobial strategy in which low laser energy is used to activate a nontoxic photosensitizer. The singlet oxygen released from these dyes causes damage to the membrane and DNA of microorganisms. It was found that the PAD technique was successful in eliminating all culturable bacteria, with combination of photosensitizer and correct energy doses.[8]

Various researches had been done on PAD, diode laser, and 5% NaOCl, but none have compared them. The aim of this randomized clinical trial was to evaluate and compare antibacterial efficacy of PAD, diode laser, and 5% NaOCl in root canal disinfection. The null hypothesis to be tested was that there was no difference in the antibacterial efficacy among the groups.

MATERIALS AND METHODS

The protocol for the present study was approved by institutional ethical committee. All the patients who presented for treatment of nonvital permanent maxillary central incisors with mature apices were prospective candidates for the study. Both males and females of age group 18–55 years were included. The teeth with vital pulp, periapical cyst, calcified canals, and immature apices were excluded. These teeth were analyzed by preoperative radiographs and electric pulp test for monitoring their pulpal and periapical status. A detailed medical and dental history was taken. Patients were explained about the procedure, and written consent was taken before treatment. Thirty-two permanent maxillary central incisors were randomly divided into groups corresponding to the disinfection protocol. The groups were PAD (n = 10), diode laser (n = 10), 5% NaOCl (n = 10), and saline (n = 2).

Local anesthesia (Lox 2%, Neon Lab Ltd, India) was administered, and isolation was done with rubber dam. The tooth and surrounding area were disinfected by swabbing with 5% iodine tincture.[9] A high-speed handpiece and sterilized round bur (BR40:Mani, Inc., Tochigi, Japan) were used for root canal access opening, and walls were modified using safe end cutting diamond point (EX24:Mani, Inc., Tochigi, Japan). The root canal was accessed with size 10K-file (Mani, Inc., Tochigi, Japan). Sterile water was deposited with hypodermic syringe, and a 27-gauge needle and the contents were debrided from canal walls by push-pull motion. Then, initial pretreatment root canal culture sample was taken with presterilized paper points. Two paper points were placed in the canal for 60 s and then transferred into two separate presterilized tubes of 2 ml brain heart infusion broth (BHI broth) (marked as aerobic and anaerobic sample). This was designated as the first sample (Sample 1). These tubes were transferred in 10 min for culturing under aerobic and anaerobic conditions. The working length was determined using electronic apex locator (Dentaport ZX, JMorita Corp.) which was kept 0.5 mm short of the apex. This was confirmed by radiograph. Cleaning and shaping was done using conventional step-back technique. Master apical size was kept three sizes larger than the initial binding instrument. Five ml of sterile water was used for irrigation during cleaning and shaping. Depending on the disinfection protocol to be tested, the canals were treated according to the groups.

Group A: Photoactivated disinfection

PAD plus device (Denfotex Ltd, Inverkeithing, UK) was used as per the manufacturer's instructions. The canal was inoculated with PAD solution (tolonium chloride) till the pulp chamber and was agitated for 60 s. The emitter tip was set 4 mm short of working length and inserted in canal. It was activated for 120 s in endodontic mode. The canal was then flushed with 2 ml of sterile water and dried with paper points.

Sterile water was again inserted in the canal with a hypodermic syringe and 27-gauge needle. It was kept in the canal for 5 min without agitating. Two paper points were placed in the canal for 60 s and were transferred into two separate test tubes as mentioned for Sample 1 and was designated as second sample (Sample A2). These tubes were transferred in 10 min for microbial culturing under aerobic and anaerobic conditions. A similar technique for acquiring samples from the canal was followed for all the test groups.

Group B: diode laser

Intracanal irradiation was done with Diode laser (PicassoLite, AMD, LASERS® LLC, USA) having wavelength of 810 nm. The settings used were, noninitiated tip with power of 0.8 Watts, interval and duration of 20 ms in a repeat mode. The fiber optic tip was kept 1 mm short of working length and was activated.[7] This tip was removed from the canal in helicoidal movements (speed of 2 mm/s) and was repeated 4 times at intervals of 20 s. After completion of irradiation, sterile water was placed in the canal and second sample was taken as described for Group A (Sample B2).

Group C: 5% sodium hypochlorite

Irrigation was done with 5 ml of 5% NaOCl (Prime Dental Products, Mumbai, India) for 5 min[10] with hypodermic syringe and 27-gauge, beveled needle, which was kept 1 mm short of the working length. The canal was irrigated with 5 ml of 5% sodium thiosulfate to neutralize the effect of NaOCl.[11] This was followed by flushing the canal with sterile water and canals were dried with paper points. An immediate postoperative sample was taken as described for Group A (Sample C2).

Group D: normal saline

Irrigation was done with 5 ml of normal saline with hypodermic syringe and 27-gauge, beveled needle, which was kept 1 mm short of the working length. Sterile water was used to rinse and the canals were dried with paper points. An immediate postoperative sample was taken as described for Group A (Sample D2).

After obtaining the second sample for each group, the canals were dried and Cavit™ G (3M ESPE, Germany) was used for coronal seal. The patients were recalled after 7 days. In the second appointment, root canal was reopened under rubber dam isolation and was subjected to one more application of disinfection protocol as per groups. The third sample was taken as described before (Sample A3, B3, C3, and D3).

Microbial analysis

The microbial analysis was done by counting the colony-forming units (CFUs).

Aerobic culturing technique

Ten μl of BHI broth was inoculated using micropipette on 5% Columbia sheep blood agar plates (HiMedia Laboratories Pvt Ltd, Mumbai, India). The inoculation loop was heated on the blue flame of Bunsen burner, till it became red hot and was allowed to cool down at room temperature. Using loop, streaking was done on agar plates and was placed in incubator at 37°C for 24 h. Then, the bacterial growth was counted as CFUs using manual counting technique.

Anaerobic culturing technique

The lids of test tubes were opened slightly, and paper points were inserted immediately in BHI broth. Ten μl of BHI broth was inoculated using micropipette on agar plates, and streaking was done. The culture plates were placed in an anaerobox chamber with accessories (Anaerogas pouch and indicator tablet) and were incubated for 24 h at 37°C, that maintains anaerobic environment. After 24 h, the bacterial growth was counted as CFUs.

Calculation formula for manual counting technique

Statistical analysis

Microbial counting was done for both aerobic and anaerobic culture in the form of CFUs. The mean was tabulated and analyzed using statistical analysis software MedCalc (MedCalc software for windows, Version 12.7.5, Ostend, Belgium). The results were evaluated statistically using ANOVA and Friedman test. Individual comparison within the group was done with post hoc individual comparison using Mann–Whitney U-test.

RESULTS

Aerobic condition

Intragroup comparison of mean CFUs at baseline and after treatments for each group showed significant reduction for Group A, B, and C (P < 0.0001) and nonsignificant for Group D (P = 0.156) [Table 1]. Intergroup comparison of antibacterial efficacy after each application of various disinfection protocols showed significant reduction in CFUs after first, second, and between first and second application of disinfection protocols (P < 0.05) [Table 2]. A significantly higher reduction in CFUs was seen after second disinfection protocol when comparison between PAD and Diode laser was done [Table 3]. The reduction in CFUs after first and second application of disinfection protocols was significantly higher when comparison was done between diode laser and NaOCl [Table 3].

Table 1

Mean colony-forming unit (×106) for aerobic bacteria at baseline and after treatments

Table 2

Reduction in colony-forming unit of aerobic bacteria (×106) after each application of various disinfection protocols

Table 3

Individual comparisons for change in colony-forming unit (×106) for aerobic bacteria

Anaerobic condition

Intragroup comparison of mean CFUs at baseline and after treatments for each group showed significant reduction for Group A and C (P < 0.0001 and P = 0.025, respectively), and nonsignificant for Group B and D [Table 4]. Intergroup comparison of antibacterial efficacy after each application of disinfection protocols showed a nonsignificant reduction in CFUs [Table 5]. Comparison in between the individual groups also showed nonsignificant results [Table 6].

Table 4

Mean colony-forming unit (×106) for anaerobic bacteria at baseline and after treatments

Table 5

Reduction in colony-forming unit of anaerobic bacteria (×106) after each application of various disinfection protocols

Table 6

Individual comparisons for change in colony-forming unit count (×102) for anaerobic bacteria

DISCUSSION

Microorganisms and their end products are considered as the main cause of pulpal and periapical pathosis.[12] Root canal disinfectant destroys microorganisms and neutralizes their products without damaging host tissue. In the current study, maxillary central incisors were chosen as they have single large, wide, and straight canal in which disinfection protocol can be evaluated without procedural errors.[13]

PAD was found to be an effective antimicrobial agent for both aerobic and anaerobic bacteria. Its antimicrobial activity is due to the photosensitizer solution (tolonium chloride penetrates up to300 μm), mediated by singlet oxygen because of its high chemical reactivity that has direct effect on extracellular molecules. Thus, the polysaccharides present in extracellular matrix proteins of bacterial biofilm are susceptible to photodamage. The breakdown of biofilm inhibits plasmid exchange involved in the transfer of antibiotic resistance and disrupts colonization.[14] In addition, PAD has shown significantly more reduction in CFUs after second application of disinfection protocol when compared with the first application. These findings were in accordance with the observations of other studies.[1516] The reasons for this findings are 2-fold, first being recolonization of microorganisms in a less complex biofilm as compared to the initial biofilm and[17] secondly the phototoxicity of phenothiazine dyes like tolonium chloride to both DNA and the outer membrane of target species.[18]

Laser irradiation has inherent properties of light scattering, local intensity enhancement, and attenuation that allow light penetration deeper in the dentinal tubules contributing to a superior antimicrobial efficacy.[19] The bactericidal effect of diode laser could be attributed to its greater depth of penetration (1000 μm into dentinal tubules) when compared to NaOCl which penetrates to 100 μm.[20] In addition, diode laser causes thermal photodisruptive action in unreachable parts of dentin, resulting in an enhanced bactericidal effect in the root canal dentin.[21]

In the present study, antimicrobial efficacy of diode laser was significantly high for aerobic bacteria (P < 0.0001) and was nonsignificant for anaerobic bacteria (P = 0.318). These findings were in accordance with Moritz et al.[22] The diode laser was less effective against anaerobic bacteria, this could be attributed to low output power (0.8W) of laser and secondly the cell-wall structure of bacteria.[20]

In the present study, 5% NaOCl has shown significant reduction in CFUs for both aerobic (P < 0.0001) and anaerobic conditions (P = 0.025). These findings were in consonance with other studies.[2324] The effects of reduction in CFUs are promoted by mechanical debriding action of instruments and irrigation hydrodynamics. This effect is further enhanced by the concentration and volume of irrigant.[20]

In a clinical study, diode laser radiation and 5% NaOCl were equally effective for disinfecting the root canals containing aerobic and anaerobic bacteria.[25] Our findings differ; diode laser was effective for reduction of aerobic bacteria only whereas the percentage reduction for anaerobic bacteria was not significant. The reason for this could be attributed to the use of low output power laser used in the current disinfection protocol.

Thus, We Can Conclude That for Aerobic Bacteria, Pad Was More Effective Than 5% Naocl Whereas Diode Laser Was Least Effective. Pad and 5% Naocl Were More Antibacterial Than Diode Laser for Anaerobic Bacteria. the Null Hypothesis Was Rejected as There Was Difference in the Antibacterial Efficacy of Pad, Diode Laser, and 5% Naocl.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.