Evaluation of Antibacterial Efficiency of Different Root Canal Disinfection Techniques in Primary Teeth

Abstract

Background: A successful primary root canal treatment depends on effective shaping and cleaning the root canal system and finally filling it with a hermetic sealer. Clinically, roots of primary teeth are difficult to shape and the irrigation/disinfection protocol has great importance on prognosis. Objective: The present study evaluated the antibacterial efficiency of Endosafe (Orangedental GmbH & Co. KG), photo-activated disinfection (PAD; Orangedental GmbH & Co. KG), diode laser (Epic 10; Biolase, Inc.), ozone (O₃, Ozonytron; Biozonix, München, Germany), and sodium hypochloride applications in primary root canals that were infected with Enterococcus faecalis after standard mechanical instrumentation. Methods: The study was conducted on roots of 100 human primary molar teeth, which were extracted due to excessive caries. The roots were divided in 5 groups with 15 roots in each root disinfection protocol. In addition, 15 samples and 10 samples served as positive and negative controls, respectively. The Shapiro–Wilk test, the Kruskal–Wallis test and then by post hoc group comparisons with the Bonferroni-adjusted Mann–Whitney U test (unpaired observations) was used. Results: Sodium hypochlorite (NaOCl) exhibited the highest antibacterial effect (0 colony-forming units per mL). Diode laser irradiation was statistically more effective than the ozone, PAD, and Endosafe groups (p < 0.001). Endosafe, PAD, and ozone groups showed similar antibacterial effect (p > 0.05). Although not statistically significant, the Endosafe was more effective in reducing the bacterial count when compared with ozone and PAD. Conclusions: The five tested irrigation systems were shown to be effective in disinfection of the E. faecalis-contaminated primary root canals and best results were obtained with 2.5% NaOCl and diode laser.

Introduction

Today, despite the progress in preventive interventions, dental infection and early loss of primary teeth are still among the major problems affecting the quality of life in children. As per maintainence of irreversible pulpitis in primary teeth, root canal treatment (RCT) is indicated. A successful primary RCT depends on effective shaping and cleaning of the root canal system followed by obturation with a hermetic root canal filling. However, proper shaping is difficult to achieve in primary teeth due to their internal complex nature and ramifications in the apical areas. Thus, optimal irrigation and root canal disinfection is gaining more importance with the current evidence in dental literature.^1

–4

The most widely accepted method in root canal irrigation is using a syringe with a needle or cannula with handheld. Sodium hypochlorite (NaOCl) is the most preferred irrigation solution due to its antibacterial properties, low surface tension, easy manipulation, and inexpensiveness. Although varying concentrations of NaOCl between 0.5% and 5.25% are used as root canal irrigation, yet, no consensus exists regarding its optimal concentration, contact time, and temperature for clinical use.^3,5
–7 The utilization of 2.5% NaOCl concentration in pediatric dentistry is recommended due to prevention of complications, which may occur depending on the pathological resorption areas on primary root. The major disadvantage of NaOCl irrigation, besides it foul smell and taste, is its cytotoxicity when injected into periradicular tissues. Thus, research is focusing on alternative approaches for an ideal root canal disinfection.^3,6,8

Recently, various technologically driven approaches are introduced to enhance effectiveness of irrigation protocol. One is Endosafe (Orangedental GmbH & Co. KG), which uses high-frequency electrical pulses for bacteria elimination. However, there is no literature about the antimicrobial effect of Endosafe in primary teeth to our knowledge.

Another approach is the “light-activated disinfection technique” named as PAD (photo-activated disinfection). In this technique, tolonium chloride solution, which is sensitive to light is sent into the root canal and after it surrounds the bacteria, solution is activated with low-energy laser light (635 nm).^1,3,9 Several researchers have tested the effectiveness of PAD in primary teeth endodonic procedure and reported favorable results.^1,2,4

Another interventional application in disinfection of infected root canal is lasers. Among different types of lasers, the diode laser is the most desirable type due to the properties, such as high penetration depth into the dentinal tubules and proper antibacterial effect.^10
–12 In pediatric dental clinic practice, lasers have been generally used in caries detection and diagnosis, pulpotomy, apexogenesis, and frenectomy of primary teeth.^13

–16 There is limited knowledge of their effectiveness in primary teeth RCT.^12,17

The last but not the least, ozone (O₃) is recently evaluated as a root canal disinfection agent.^3,18,19 In pediatric dentistry, there are several studies about ozone's effect on the retention of fissure sealant and dental caries treatment, however, very few reports have been published on the effectiveness of gaseous ozone in root canal disinfection in primary tooth.^20
–22 Kapdan et al.²² showed that KTP laser and gaseous ozone application provided a significant antibacterial effect in primary root canals.

The aim of this study was to evaluate and compare the antibacterial efficiency of Endosafe (Orangedental GmbH & Co. KG), photo-activated disinfection (PAD; Orangedental GmbH & Co. KG), diode laser (Epic 10, Biolase, Inc.), ozone (Ozonytron; Mymed), and sodium hypochloride applications in primary root canals, which were infected with Enterococcus faecalis with a standard protocol. The null hypothesis was that there would be no difference among the antibacterial effects of tested irrigation protocols.

Materials and Methods

The study sample comprised of 100 primary incisors extracted due to excessive caries. Teeth extracted did not have any radiographically visible physiological or pathological resorption and they were not subjected to any treatment before their extraction. All teeth were stored in sterile saline solution until the experiment. This study was approved by the Baskent University Institutional Review Board and Ethics Committee (process No. DKA16/17) and supported by the Baskent University Research Fund.

Tooth selection

Teeth were cleaned with a soft curettage and were kept in sterile saline solution. Clinically, teeth were included in the study, where 10 stainless steel Kerr file could reach the apical foramen. The crowns of the teeth were reduced to the cementoenamel junction by using 1000 Precision Sectioning Saw (Buehler, LakeBluff, IL). An access opening was prepared, the working length was determined, which was 1 mm shorter than the length of the specimen. The pulp was removed and the root canals were enlarged using stainless steel K-files (Kerr-files; Maillefer Instruments SA, Ballaigues, Switzerland) up to a size of ISO 40. Irrigation was done in the same protocol in each of the seven groups. Saline solution was used as irrigant and the root canals were dried using paper points. The teeth were randomly divided into seven groups and each group consisted of 15 primary incisors. These groups are Endosafe, PAD, diode laser, ozone, sodium hypochloride (n = 15), positive (n = 15), and negative (n = 10). The apical foramina were sealed using flowable composite resin and the teeth were embedded in acrylic resin blocks, which allowed handling of the teeth during the experiment. The samples were autoclaved for 15 min at 121°C. From this stage forward, all samples were processed using strict aseptic protocols.

Contamination of root canals

This part of the study was held at the Istanbul University Faculty of Dentistry Oral Microbiology Laboratory. The reference strain of E. faecalis was obtained from American Type Culture Collection. The bacterium was subcultured on trypticase soy agar (TSA; Merck, Darmstadt, Germany) and incubated aerobically at 37°C for 24 h. To generate a stock inoculum, a single colony was then harvested, placed in tryptic soy broth (TSB, Merck), following the same incubation conditions; the turbidity of E. faecalis culture was adjusted to number 0.5 Mc Farland Standard. Five microliters of bacterial suspension (final concentration of about 3 × 10⁸ colony-forming units per mL (CFU/ml) were applied into the mechanically enlarged root canals with a sterile micropipette. The suspension was worked into the canal using a sterile endodontic file size ISO 20 for the same period for each sample (Kerr-files; Maillefer Instruments SA). The opening of the canals was sealed with a temporary filling material (Coltosol^®; Coltene Whaledent). All samples were incubated at 37°C for 7 days under aerobic conditions. In the first, fourth, and sixth days, fresh TSB was added in root canals after removing the temporary filling material.

Disinfection procedures

Five different methods were used for the disinfection of bacteria-infected root canals. These were Endosafe, PAD, diode laser, ozone, and NaOCl groups. The root canals were irrigated with 1 mL of sterile saline solution before disinfection procedures and then started different methods.

Endosafe group

The root canal was irrigated with physiological serum solution and then after excess water was taken with 30 number paper point and provided the root canal slight moistening. High-frequency electrical pulses set by Endosafe system was applied inside the root canal. Endosafe probe was promoted to the root tip and three shots were done in auto mode, and in every shot the probe was pulled backward to each canal apical third (coronal, middle, apical) by using red needle (length 24 mm, diameter 0.12 mm). Final irrigation was performed with 2 mL saline solution.

PAD group

In this group, the disinfection was performed with a PAD system (Orangedental GmbH & Co. KG), which consists of the smart-pad and light-sensitive material (photosensitizing agent) and LED light device. The light-sensitive material in the PAD system was tolonium chloride solution (7.12 mg/mL), and between two distinct types as being low and high density, tolonium chloride solution 1.2 mL of low density was preferred and injected into the root canal. The excess solution was collected with sterile paper points and a 600 μm thin flexible endodontic fiber optic tip was placed in the apical third of the root canal and irradiated during 120 sec with a 670 nm light power.

Diode laser group

In this group, the roots were irradiated by using diode laser (Epic 10; Biolase, Inc.) and the endodontic tip (21 mm/300 μm) at a wavelength of 940 nm wavelength was transmitted in accordance to the manufacturer's recommendations in continuous mode with a 1.5 W power level with a repeated pulse mode using a pulse duration of 0.05 msec and a pulse interval of 0.2 msec. The optical fiber end was inserted up to 1 mm short of the working length and irradiated the root canal for 20 sec according to the recommendations of Gutknecht et al.²³ Then it is moved from apical to coronal in three consecutive cycles at intervals of 15 sec between each one.

Ozone group

Ozonytron-X (Ozonytron; Biozonix) was used for disinfection of this group. Before being ozonized, root canals were dried using sterile paper point (ProTaper F2; Maillefer Instruments SA). According to the manufacturer's recommendations, a special KP probe was applied for 5 sec at the speed of ≈0.4 mL/sec with a reservoir of 2.2 mL for each root canal. KP syringe is a specific type of syringe that contains a centrally positioned simple glass tube filled with noble gas surrounded with titanium mash. Ozone was generated from aspirated atmospheric air by dielectric barrier discharge in space between the glass tube and titanium mash.

NaOCl group

The roots were irrigated with 10 mL of 2.5% sodium hypochloride before shaping. Irrigation was continued with 10 mL of 2.5% NaOCl between increasing file diameters and final irrigation was done three consecutive times with 10 mL of 2.5% NaOCl. After final irrigation, there was a 5-min contact time. Irrigation procedures were performed with 30-gauge Perio/Endo irrigation needles (KerrHaweSa, Bioggio, Switzerland).

Positive control group

One group was left untreated for determining the amount of bacteria inside the root canal and named as positive controls.

Negative control group

One group was inoculated with sterile TSB and no disinfection was performed to control the sterilization of the working conditions.

Evaluation of antibacterial effect

After the disinfection procedures, the root canals in all groups were rinsed with 1 mL sterile saline solution. The saline solution was collected from canals with sterile number 20 paper points for a standard 15 sec contact for sample collection. The paper points were transferred to Eppendorf vials containing 1 mL of VMG II transport fluid. All collected samples were sent to the Istanbul University Dental Faculty Oral Microbiology Department within 12 h. The samples were vortexed for 10 sec and 10-fold dilutions were prepared in the laboratory. Aliquots of 0.1 mL suspensions were inoculated on TSA plates and incubated at 37°C for 24 h. CFU/mL were enumerated per sample.

Statistical analysis

Data were analyzed with statistical software SPSS (version 21.0; SPSS, Inc., Chicago, IL). The level of significance was set at 0.05. The power of study (1-β) was 0.8 with a sample size of 12. The Shapiro–Wilk test was used to determine whether data were normally distributed; the Kruskal–Wallis test and then by post hoc group comparisons with the Bonferroni-adjusted Mann–Whitney U test (unpaired observations) was used to compare the intergroup values when a normal distribution was not observed. The medians with 25th to 75th percentile values were calculated for each parameter using the bacterial counts and percent difference according to baseline bacterial count as the statistical units. A p value <0.005 was considered statistically significant.

Results

The distribution of remaining bacterial counts after disinfection procedures was shown in Table 1 and intergroup comparisons were shown in Table 2, respectively. No bacterial growth occurred in the negative control group. The positive control group revealed the highest number of bacteria (median 37 × 10⁸ CFU/mL) as expected. Among the tested irrigation procedures, NaOCl exhibited the highest antibacterial effect (0 CFU/mL) followed by the laser irradiation. Laser irradiation was statistically more effective than the ozone, PAD, and Endosafe groups (p < 0.005). Endosafe, PAD, and ozone groups showed similar antibacterial effect. Although not statistically significant, the Endosafe was more effective in reducing the bacterial count when compared with ozone and PAD. In intergroup comparisons, the decrease in bacterial count in the NaOCl group was significantly higher than diode laser (p < 0.005) (Table 2).

Table 1.

Distribution of Bacterial Counts Among Groups (Colony-Forming Units per mL)

	Bacteria count
Groups, n = 15	Median	25th Percentiles	75th Percentiles
Positive control	37 × 10⁸	32 × 10⁸	41 × 10⁸
NaOCl	0	0	0
Diode laser	2 × 10⁵	0.75 × 10⁵	5 × 10⁵
Ozone	192 × 10⁵	166 × 10⁵	260 × 10⁵
PAD	176 × 10⁵	93 × 10⁵	209 × 10⁵
Endosafe	128 × 10⁵	88 × 10⁵	174 × 10⁵

Values were given as median (25th–75th) percentiles.

NaOCl, sodium hypochlorite; PAD, photo-activated disinfection.

Table 2.

Intergroup Comparisons Between Groups

	p
Comparisons	Bacterial count	Percent difference according to baseline bacterial count
Positive control to NaOCl	0.000^*	0.000^*
Positive control to diode laser	0.000^*	0.000^*
Positive control to ozone	0.000^*	0.000^*
Positive control to PAD	0.000^*	0.000^*
Positive control to Endosafe	0.000^*	0.000^*
NaOCl to diode laser	0.000^*	0.000^*
NaOCl to ozone	0.000^*	0.000^*
NaOCl to PAD	0.000^*	0.000^*
NaOCl to Endosafe	0.000^*	0.000^*
Diode laser to ozone	0.000^*	0.000^*
Diode laser to PAD	0.000^*	0.000^*
Diode laser to Endosafe	0.000^*	0.000^*
Ozone to PAD	0.217	0.217
Endosafe to ozone	0.008	0.008
Endosafe to PAD	0.412	0.412

Kruskal–Wallis test, Mann–Whitney U test with Bonferroni adjustment were performed. p Value was adjusted to <0.005 for statistical significance.

Statistical difference between groups.

Discussion

An ideal intracanal irrigating solution should exhibit maximum antibacterial and minimum toxic effects. Today, NaOCl is the most commonly used antibacterial irrigation solution in endodontic practice. Moreover, it is clear that the antibacterial and tissue dissolution action of NaOCl increases with its concentration, but also accompanied by an increase in its toxicity. Around 2.5% NaOCl proved to be a better solution in primary teeth because it has greater effectiveness than 0.5% and 1% concentrations and lower cytotoxicity than 5.25% NaOCl concentration.^{5,6,8,12,17,24,25} Therefore, 2.5% NaOCl was used for 5 min in this study. Since a single concentration of NaOCl was tested in the present, further studies are needed evaluating different concentrations, contact time, and different setting parameters.

The working principle of Endosafe is based on emitting a calibrated electromagnetic impulse, which reduces bacteria inside through the electrode. To our knowledge, the present study is the first which evaluates Endosafe system. However, similar operating systems based on high-frequency alternating current (HFAC) can be found named as Endox Plus system.^26
–28 Aranda-Garcia et al.²⁶ compared ex vivo the antibacterial efficacy of Endox Plus system with 2.5% NaOCl/MTAD and showed that Endox Plus system had the weakest antimicrobial effect compared with study groups. In another in vitro study, Karale et al.²⁷ revealed that 3% NaOCl showed 100% reduction of E. faecalis after 24 and 48 h and HFAC showed 70% reduction of growth after 24 h, and 55% reduction of growth after 48 h. Herein, we demonstrated that Endosafe resulted in a 99% decrease in the bacterial count after 7 days. Although the incubation period in the present study was longer and the samples were collected 7 days after irrigation, the results were favorable. The reason for the difference between studies can be attributed to different devices as well as different incubation and sample collection periods. According to the data we obtained, NaOCl had the best results followed by the diode laser. In addition, although samples did not show significant differences between Endosafe, PAD, and ozone, Endosafe was more effective when compared with PAD and ozone. The results revealed significant difference in the irrigation systems used when compared with positive control groups (p < 0.005). Therefore, the null hypothesis was rejected.

The laser application with different wavelengths in endodontic practice is well documented for adults, but there are few studies on pediatric endodontics.^12,16,29 In a previous study, Kuvvetli et al.¹² compared the efficacy of a 810 nm diode laser irradiation and 5.25% NaOCl in primary molar root canals contaminated with E. faecalis. The results showed that NaOCl provided complete elimination of the bacteria in all root canals after 24 h. Additionally, diode laser was found to be as effective as 5.25% NaOCl application. Moreover, in some of the samples, diode laser revealed complete bacterial elimination. The results of the present study showed that 2.5% NaOCl irrigation resulted in 100% bacterial elimination, whereas a 980 nm diode laser resulted in 99.99% bacterial elimination. Although the concentration of NaOCl was lower from Kuvvetli et al.¹² the results showed similar effectiveness.

Diode laser has gained popularity for its potential antibacterial effect and affordable cost. For our study, we followed the manufacturer's recommendations using a 940 nm wavelength diode laser Epic 10 (Biolase, Inc.) having a 300 μm laser beam, which was transmitted in continuous mode with 1.5 W power level. Gutknecht et al.²³ supported that a 980 nm diode laser has a supportive mean in endodontics with an average of between 77% and 97% in root canals contaminated with E. faecalis with energy outputs of 1.7, 2.3, and 2.8 W. Castelo-Baz et al.³⁰ evaluated the bactericidal efficacy of a 940 nm diode laser alone or in combination with 5% NaOCl against mature biofilms of E. faecalis. After 7 days, they observed NaOCl alone and diode laser applications with NaOCl showed statistically significant good initial bactericidal effect.³⁰ The present results of this study are in agreement with previous studies demonstrating bacterial reduction with 99%; however, the limitations of this study need to be proved with diode laser-assisted techniques, such as laser-activated irrigation with concentrations of NaOCl (0.01%, 0.1%) or photodynamic therapy.

There is limited literature assessing the antibacterial effect of diode lasers in primary root canals considering the safety control to heat damage of the surrounding tissues and the effect of permanent tooth underneath the primary tooth. Recent in vitro studies showed the temperature rise stays in the range of 1–7°C. Since the threshold bone necrosis temperature is defined as a critical limit of 47°C for 1 min, this temperature rise is below the danger mark. This has cleared the way for the clinical use of lasers in root canal disinfection.^13,16,31 We used a 940 nm wavelength diode laser (Epic 10, Biolase, Inc.), which is known as a tissue-friendly laser showing lesser temperature rise compared with other lasers, such as Nd-Yag laser. The irradiation protocol followed for this study was as described by Gutknecht et al.²³ in continuous mode with a 1.5 W power level. In a systematic review, within a value of 1.5 W for the diode laser, thermal damage is excluded and bactericidal effects are acceptable.¹³ However, particular care must be taken within the application of laser energy into primary root canals for disinfection due to the characteristic anatomy of the apex and to the penetration depth of surrounding tissues.

As another alternative approach to overcome these problems, a new approach by using low power lasers was introduced, which is termed as PAD.^32

–35 Recently, Dickers et al.³⁴ showed that the temperature rise was 0.16°C ± 0.08°C after 150 sec of PAD application and considered the use of PAD safety for periodontal tissues. The recent researchs are interested with PAD application alone and its efficacy was found to be poor in bacterial inhibition.^30,33

Mohan et al.³³ compared the efficacy of PAD with conventional endodontic treatment (CET) and also their combination of CET and PAD. The researchers preferred 2.5% NaOCl as the irrigating solution. They found that a combination of CET with 2.5% NaOCl irrigation procedure followed by PAD reduced bacteria by about 99.5% and found it the most effective method.

In the present study, PAD irrigation could not achieve 100% bacterial reduction. Following PAD application, the count of bacteria was significantly higher compared with NaOCl and laser groups, whereas it was similar with Endosafe and Ozone. Thus, the use of PAD as an adjuvant to other irrigation systems may be suggested. Further clinical studies are needed to confirm the efficacy of PAD, especially on E. faecalis.

To date, ozone was used in root canal disinfection therapy for antimicrobial effect in several researches.^17,23,24 There are few reports on the use of gaseous ozone in the primary root canals for disinfection.^17,24 Kapdan et al.¹⁷ investigated the most effective disinfection method, conventional or gaseous ozone application (150 sec), in primary root canals. They showed that 2.5% NaOCl achieved complete sterilization in all the samples. Although gaseous ozone provided a significant reduction of the E. faecalis, it could not achieve total inhibition of bacteria. Our results are in accordance with Kapdan et al.'s¹⁷ study. Although 150 sec gaseous ozone allowed a significant bacterial reduction, its efficacy was lower as compared with diode laser. Presently ozone is an adjunct to other conventional treatment procedures and combination usage with other irrigation systems should be better than independent usage.

Conclusions

• The five tested irrigation systems were shown to be effective in disinfection of the E. faecalis-contaminated primary root canals and best results were obtained with 2.5% NaOCl and conventional needle irrigation as well as diode laser.

• Within the limitations of the current study, NaOCl and/or laser would be choice in achieving a “disinfected” root canal. On the other hand, due to the possible toxic effects of the NaOCl to the developing tooth germ, laser may gain more popularity in pediatric endodontics.

Footnotes

Acknowledgments

The authors would like to thank Emine Mutlu (Faculty of Dentistry, Department of Microbiology, Istanbul University) for her valuable technical asistance in the laboratory phases of the study.

Author Disclosure Statement

No competing financial interests exist.

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