Evaluation of sonic, ultrasonic, and laser irrigation activation systems to eliminate bacteria from the dentinal tubules of the root canal system

Abstract Aiming to kill bacteria in dentin tubules of infected dental pulp cavities, we evaluated the effects of sodium hypochlorite (NaOCl) solution agitated by different irrigation protocols, i.e., conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), the EDDY tip, and the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser. The EDDY achieved good antibacterial effects as passive ultrasonic irrigation in the coronal and middle thirds. Nd:YAP laser irradiation and PUI were effective in the apical third of the root canal. Objectives: To evaluate the ability of NaOCl agitated by high-frequency sonic irrigation–EDDY, PUI, and Nd:YAP laser–to kill bacteria in infected root canal walls and if the associated temperature increases at the root surface during application. Methodology: Infected root canal models were established, and roots were randomly divided into six groups: negative control, positive control, CNI, PUI, sonic agitation with EDDY, and Nd:YAP laser groups. After irrigation, the teeth were split and stained using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacteria depth was evaluated by a confocal laser scanning microscopy and the temperature at the root surface was assessed using a thermal imaging camera during the irrigation process. Results: In the coronal and middle thirds of the root canal, PUI and EDDY had stronger antibacterial effects than CNI (p<0.05); in the apical third, the antibacterial effects of PUI and Nd:YAP laser-activated irrigation were better than CNI (p<0.05). The maximum change in temperature was significantly greater during continuous Nd:YAP laser application compared with the other methods, but intermittent irrigation helped lessening this trend. Conclusions: NaOCl agitated by EDDY tip and PUI exhibited a similar bacteria elimination effect in the coronal and middle root canal. Nd:YAP laser was effective in the apical third and intermittent irrigation reduced its thermal impact.


Introduction
Chemo-mechanical cleaning and shaping of the root canal system to remove or reduce bacterial populations are essential for infection control, which is the main goal of root canal treatment. 1 However, the complex anatomy of the root canal system limits the mechanical preparation, particularly in the apical third of the root. 2 In infected root canals, bacteria can grow 400 μm or more into the dentinal tubules. 3 Due to the strength and resistance of the remaining root canal wall, classic mechanical preparation generally only cuts the dentin at a depth of 150 μm, which is impossible to remove deep infection. 4 Chemical irrigation is an effective supplemental method as it can reach more areas of the root canal surface. 5 Techniques such as passive ultrasonic irrigation (PUI), sonic irrigation, and laseractivated irrigation enhance the disinfection effects of chemical irrigation and improve clinical outcomes. 6 PUI activates irrigation via acoustic streaming and cavitation and its disinfecting effect is better than the conventional needle irrigation (CNI). 7,8 The EDDY system (VDW, Munich, Germany) provides high-frequency sonic irrigation at 6000 Hz. 9 Although previous studies have compared the antibacterial efficacies of EDDY, PUI, and CNI, the results have been inconsistent, presumably due to the different evaluation indicators employed. [10][11][12] Most studies have quantified cultured residual bacteria, but some anaerobic bacteria are difficult to cultivate under normal conditions. 13 Additionally, most studies 12,14,15 used sterile paper points for sampling the root canals, however, this method only evaluates infection clearance for the overall root canal space, and not specifically for the dentinal tubules of the root canal wall.
Studies on the application of lasers to kill bacteria in root canals have been conducted since Fegan and Steiman 16 (1995) evaluated the antibacterial effects of intracanal Nd:YAG laser irradiation, which may improve disinfection through a photothermal effect and by enhancing the chemical effects of irrigants. 17 Commonly used lasers for root canal disinfection include the erbium-doped chromium-yttriumscandium-gallium garnet (Er,Cr:YSGG) laser, the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser, and the photon-induced photoacoustic streaming laser. 18 Wang, et al. 17   Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol. 26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During

Establishment of E. faecalis infection
To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection.
The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan).
Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.

Root canal irrigation protocols
After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.

Group 1: Negative control (n=2)
No root canal irrigation was performed in this group after bacterial incubation.

Group 2: Positive control (n=2)
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.

Group 3: CNI (n=5)
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure.
Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval.
This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation.

Group 5: High-frequency sonic irrigation (n = 5)
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.

Heat production evaluation
Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots.
Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.

Statistical analysis
The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games-Howell test applied for pairwise comparisons.

Evaluation of heat production
All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.
The mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly  Several studies showed that the cleaning effects of dynamic irrigation are limited in the apical region. 12,27,28 In our study, we found that the effects of NaOCl agitated by the PUI and EDDY showed a similar trend in the apical third. This finding probably relates to the small diameter of the root canal in the apical area and the short distance between the working tip and the root canal wall, which limits the effects of acoustic flow. 6 However, we failed to find significant differences in the effects of the Nd:YAP laser in the coronal, middle, or apical thirds. The laser irradiation mainly inhibits bacterial growth via photothermal action. Its fiber head heat output can directly destroy the cell wall and the irrigants can absorb it, transferring heat into the dentinal tubules to kill bacteria. 18 In this study, we moved the Nd:YAP laser fiber up and down 2 mm short of the WL, so that the irrigants in the root canal space could evenly absorb the heat. The uniform distribution of heat probably led to the relatively even depth of red fluorescence observed throughout the root canal.
In coronal and middle thirds, the antibacterial effect of the EDDY was equivalent to PUI. This finding indicates that the ultrasonic oscillation and sonic working tips could potentially achieve greater acoustic flow due to the unlimited space in middle and coronal parts of root canal. 29 Although the EDDY frequency is lower than PUI, its vibration amplitude is larger. The irrigants velocity positively relates to   (2021) found that the penetration depth of 2% chlorhexidine irrigated by EDDY or PUI was similar in the cervical and middle region of the root canal. Meanwhile, the laser-activated irrigation was not significantly weaker than PUI or EDDY, nor significantly better than CNI.  biofilm on the root canal wall or in the dentinal tubules.
It also requires bacterial sampling, inoculation, and incubation increasing the chances of introducing microorganisms. 39 In our study, we evaluated the depth of dead bacteria via CLSM. This step can be performed directly after staining the samples, which may reduce the potential of bacterial contamination. 40 We focused on killing the bacteria in dentinal tubules, as we think that the depth of dead bacteria better reflects the antibacterial effect.
This study also showed that intermittent laser irradiation significantly reduced its heat-generation effect. The main limitation of laser is its thermal impact during application, which may damage the periodontal ligament 41 and may cause postoperative pain. 42 In this study, the maximum increases in temperature in the sonic irrigation, PUI, and intermittent Nd:YAP groups were all <10°C during the 30 s irrigation interval, which agrees with previous results. 20 However, the increase in temperature in the Nd:YAP continuous irrigation group exceeded 10°C within 6-8 s; and its highest temperature during irrigation reached approximately 55°C. These results indicate that the heat generated by the laser is absorbed by water molecules in the dentin and then transmitted to the root outer surface. Therefore, to avoid damage to periodontal tissue when irrigating root canals, the Nd:YAP laser should be limited to intermittent use with short pulses.
This study had some limitations. First, the sample size was relatively small; and to avoid anatomical differences between groups and validate the results more strongly, further research with larger samples is needed. Second, the roots of teeth were exposed directly to air, whereas in another study the teeth were in 37°C water bath condition to simulate human body temperature. 20 Under water bath conditions, the temperature at the roots outer surface could only be measured at specific points using a thermocouple, thus it may not represent its highest temperature reached.

Conclusions
The NaOCl solution agitated by the EDDY system showed a potent bacterial killing effect in the dentinal tubules of coronal and middle thirds of root canal wall, but this effect was limited in the apical third.
The NaOCl solution agitated by the Nd:YAP laser showed no advantage in terms of killing bacteria in dentinal tubules of coronal and middle thirds of the root canal compared to the PUI or EDDY but achieved similar antibacterial effect to the PUI in the apical third. Under the conditions used in this study, the increase in temperature at the root surface caused by the intermittent irrigation protocol is safe for clinical application.

Conflict of interest
The authors declare no conflict of interest.

Data availability statement
All data generated or analyzed during this study are included in this published article.