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  Table of Contents 
Year : 2022  |  Volume : 25  |  Issue : 6  |  Page : 895-902

Effect of different irrigation activation methods on non-infected dentinal tubule penetration of medicaments: A CLSM study

1 Department of Endodontics, Faculty of Dentistry, Sivas Cumhuriyet University, Sivas, Turkey
2 Department of Endodontics, Faculty of Dentistry, Erciyes University, Kayseri, Turkey
3 Department of Restorative Dentistry, Faculty of Dentistry, Sivas Cumhuriyet University, Sivas, Turkey
4 Department of Prosthodontics, Faculty of Dentistry, Dokuz Eylül University, İzmir, Turkey

Date of Submission09-Nov-2021
Date of Acceptance11-Mar-2022
Date of Web Publication16-Jun-2022

Correspondence Address:
Dr. R Zan
Department of Endodontics, Faculty of Dentistry, Sivas Cumhuriyet University, 58140 Sivas
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njcp.njcp_1932_21

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Background: The whole root canal disinfection is crucial in terms of long-term endodontic success. In this context, applying medicaments following effective irrigation activation procedures becomes an important point. Aims: The purpose of this study was to evaluate and compare the efficacy of various irrigation activation techniques on non-infected dentinal tubule penetration of calcium hydroxide (CH), double (DAP), and triple (TAP) antibiotic pastes. Materials and Methods: A total of 180 extracted human permanent mandibular premolar single-rooted teeth were selected and randomly divided into a control group and four main groups according to the irrigation activation procedures (n = 36) as KTP laser irradiation, conventional needle, NaviTip FX, sonic (SI) and ultrasonic activation (UI) procedures. Each group was randomly subdivided into three subgroups according to the medicament type (n = 12); CH, DAP, and TAP. After the activation procedures, the root canals were filled with CH, TAP, and DAP mixed with 0.1% fluorescent rhodamine B isothiocyanate. Specimens were sectioned at 2, 5, and 8 mm from the apex, and all the sections were examined under a confocal laser scanning microscope to calculate the dentinal tubule penetration. Data were analyzed using a three-way analysis of variance and Tukey's post hoc tests (P = 0.05). Results: TAP provided a statistically significant greater penetration than the other groups (P < 0.05). UI provided a statistically significant higher dentinal tubules penetration area than other activation procedures (P < 0.05). Conclusion: Medicament penetration depends on the type of medicament, activation procedures, and root canal level. The TAP may be preferred following the ultrasonic activation in terms of long term root canal treatment success.

Keywords: Confocal microscope, endodontics, intracanal medicament, root canal irrigants

How to cite this article:
Zan R, Topcuoglu H S, Hubbezoglu I, Gorler O, Altunbas D, Ayhan M. Effect of different irrigation activation methods on non-infected dentinal tubule penetration of medicaments: A CLSM study. Niger J Clin Pract 2022;25:895-902

How to cite this URL:
Zan R, Topcuoglu H S, Hubbezoglu I, Gorler O, Altunbas D, Ayhan M. Effect of different irrigation activation methods on non-infected dentinal tubule penetration of medicaments: A CLSM study. Niger J Clin Pract [serial online] 2022 [cited 2022 Jul 5];25:895-902. Available from:

   Introduction Top

Some of the most important aims of root canal treatments are the elimination of the microorganisms, controlling infections and preventing reinfections, and achieving the hermetic obturation of root canals to prevent recontamination.[1] However, the irregular anatomical formations of root canal systems might make disinfection difficult in specific ramifications and dentinal tubules. Even though the chemomechanical preparation may perform inadequate elimination of microorganisms because of these complexities of the root canal system. Therefore, the effective irrigation and antibacterial efficacy of medicaments are playing a crucial role in terms of the success of endodontic treatments, especially for inter-appointment flare-ups.[2] Some intracanal medicaments are proposed for the aim of eliminating the bacteria and preventing re-growth of bacteria between the appointments. The dentinal tubule penetration of root canal medicaments may be impressed negatively by debris and microorganisms that may remain within untouched areas along the root canal.[3]

Penetration analysis of endodontic sealers was achieved with a confocal laser scanning microscope (CLSM), which enables the visualization of deep structures of the optical sections. The application of fluorescent dyes in addition to confocal microscopy has advantages in terms of imaging contribution, especially for penetration studies and also with cementation of endodontic fiber posts in endodontics. For instance, it provides crucial information about the presence and distribution of root canal sealers within the dentinal tubules.[4] Moreover, it has been used to assess the topographic properties of the root canal filling since it provides a detailed evaluation of the samples in-depth, even in a wet environment.[5]

Researchers have investigated the effect of different medicaments based on this critical importance role of medicaments in root canals against microorganisms. Calcium hydroxide (CH) is the highly preferred effective medicament used as an intracanal medicament.[6] On the other hand, it is not successful enough to eliminate E. faecalis because of its insufficient ability in terms of penetration directly into the dentinal tubules.[7] The use of triple antibiotic paste (TAP) consisting of ciprofloxacin, metronidazole, and minocycline in endodontics is widespread due to its antimicrobial and biocompatible features, but crown coloration caused by minocycline is one of its undesirable effects.[8] Therefore, double antibiotic paste (DAP) was developed to eliminate the coloration problem.[9] Ciprofloxacin and metronidazole were used, excluding minocycline from DAP content.[10] There are many studies[11],[12] on these antibiotic mixtures, but the number of studies emphasizing their effectiveness on dentinal tubules is limited.[9]

Root canal instrumentation is insufficient to achieve complete root canal debridement because of the complex nature of root canal anatomy. Therefore, various machine-assisted agitation instruments and manual agitation techniques have been suggested to increase the efficiency of chemomechanical preparation.[13] Conventional needle irrigation (CI) alone is not an effective and sufficient chemomechanical preparation.[14] Hence different irrigation activation techniques have been used for an effective and sufficient chemomechanical preparation, involving syringe irrigation with needles/cannulas, sonic and ultrasonic systems, and laser systems.[14],[15]

Ultrasonic irrigation has been used to facilitate the cleaning and disinfection of root canals in endodontics.[16] It was stated that it is more successful than conventional needle irrigation in terms of root canal cleaning efficiency. Ultrasonic irrigation has shown a high cleaning efficacy of the root canal system. Studies have shown that irrigation solutions used with ultrasonic and sonic devices can be effectively delivered to the apical third.[17] It has been stated that sonic and ultrasonic activations are effective in increasing the penetration of root canal sealer into the lateral canals. Sonic devices operate at low frequency (< 20 kHz) using flexible tips and appear to be more effective than conventional needle irrigation.[18] However, ultrasonic activation has been reported to be superior regarding penetration to working length and into lateral canals.[18] Another alternative irrigation activation method is laser activation. Recently, the use of lasers for irrigation activation has increased. Intracanal agitation by laser energy has been reported to be effective at smear layer removal or sealer penetration into dentinal tubules. The KTP laser has a wavelength of 532 nm, and it is a frequency-doubled Nd: YAG laser. It was mainly introduced for whitening applications in dentistry. However, the KTP laser has also been used for other dental procedures (such as root canal and cavity disinfection, treatment of dentin tenderness, vital pulp treatments, and soft tissue surgery) similar to those using the Nd: YAG laser. Also, ultrasonics and lasers render the irrigants to reach the apical third of the root canal system.[19]

In the light of all this information, the purpose of the present study was to evaluate and compare the effect of KTP laser irradiation, conventional needle, NaviTip FX, sonic (SI), and ultrasonic activation (UI) procedures on dentinal tubules penetration of CH, DAP, and TAP antibiotic pastes by using a laser scanning confocal microscope. The null hypothesis tested was that the different irrigation activation techniques do not affect medicament penetration.

   Subjects and Methods Top

Approval was obtained from the Non-Interventional Clinical Research Ethics Committee of Sivas Cumhuriyet University, Sivas, Turkey (ethics committee code: 2021-01/28). In the present study, 180 single-rooted and single-canal mandibular premolars were extracted for periodontal disease, orthodontic, or prosthetic treatment planning, without root canal calcification, and root surface fractures, cracks, and caries were used. All tissue and debris residues on the root surfaces of the teeth were removed, and teeth were stored in physiological saline solution (Polifarma, Tekirdağ, Turkey) at + 4°C until usage. Endodontic access cavities were prepared under water cooling using a diamond bur (Distech; Coltene Whaledent, Altstätten, Switzerland) with a highspeed handpiece (Sirona T3 Racer, Dentsply, Germany). Size 10 K-file (Dentsply, Maillefer, Ballaigues, Switzerland) was forward until it appeared apical foramen, and the working length was determined to be 1 mm shorter than this length. To ensure standardization, the lengths of all teeth were standardized to 19 mm by flattening the crowns of the teeth with a diamond fissure bur. Root canals were shaped with ProTaper Next (Dentsply, Tulsa Endodontics, Oklahoma, USA) Ni-Ti rotary instruments using an endomotor (Sendoline, Perfect Endo, Australia). Rotary files X1 (size 17.,04 taper), X2 (size 26.,06 taper), X3 (size 30.,07 taper) and X4 (size 40.,06 taper) were respectively used at the WL for shaping the root canals. After each instrumentation, the root canals were irrigated with 2 ml of 5.25% NaOCl. 17% EDTA, 5.25% NaOCl, and distilled water were applied for 5 minutes, respectively, to remove the smear layer that may affect the tubule penetration of the medicaments to be used. Then teeth were dried with paper points and sterilized using ethylene oxide. Teeth were randomly divided into a control group (conventional needle) and four main groups according to the irrigation activation procedures (n = 36) as conventional needle, KTP laser irradiation, NaviTip FX, sonic (SI), and ultrasonic activation (UI) procedures and three subgroups.

Main groups

In all groups, each root canal was flushed with 10 mL of 2% NaOCl during activation procedures.

Conventional needle irrigation: Conventional needle (Berika Teknoloji Medikal, Konya, Turkey) was inserted into the root canal 2 mm shorter than the working length and used in up and down motion in the canal, which facilitated the removal of debris (total time: 3 min).

NaviTip FX: NaviTip FX needle (Ultradent, South Jordan, UT) was inserted into the root canal 2 mm shorter than the working length and used in up and down motion in the canal, which facilitated the removal of debris (total time: 3 min).

KTP laser: KTP laser (SMARTLITE D, DEKA, Calenzano, Firenze, Italy) at 532 nm wavelength was applied with a fiber tip (Preciso, DEKA) of 200 μm diameter with 2.0 W, (100 mJ, 20 Hz) parameters during irradiation, the fiber tip was positioned 2 mm shorter than the working length and then applied in 15 s recovery intervals for each irradiation, motions at a speed of 2 mm/s towards the canal mouth. This process was repeated nine times (total time: 3 min).

Sonic Activation (SI): Irrigation solution was activated by a sonic Endoactivator system (Dentsply, Tulsa, OK) without touching the canal walls (total time: 3 min).

UltraSonic Activation (UI): The ultrasonic activation was performed with a stainless steel #20/.00 file (IrriSafe; Satelec Acteon, Merignac, France) energized by a piezoelectric unit (Suprasson PMax; Satelec Acteon) at power setting ''blue'' (total time: 3 min).


Sub-group A (CH): CH powder (Kalsin, Aktu Tic, Izmir, Turkey) was mixed with distilled water (2:1 powder/liquid ratio).

Sub-group B (DAP): The mixture of medicament was made using equal amounts (250 mg tablets) of metronidazole (Eczacıbaşı, Istanbul, Turkey) and ciprofloxacin (Biofarma, Istanbul, Turkey) with distilled water.

Sub-group C (TAP): The mixture of medicament was made using equal amounts (167 mg tablets) of metronidazole, ciprofloxacin, and minocycline (Ratiopharm, Ulm, Germany) with distilled water (0.50 mL) (1 g/mL).

Root canal medicaments were mixed with 0.1% Rhodamine B (İnterlab Laboratuar Ürünleri San. ve Tic. A.Ş., Istanbul, Turkey) to give fluorescent images for CLSM analysis. After preparation, root canal medicaments were placed in the root canals using 40# lentulo (Shenzhen Denco Medical Co., Ltd., Shenzhen, China) until they appeared from the apex. Then access cavities were sealed (Cavit W; 3M ESPE, Seefeld, Germany), and teeth were incubated at 37°C for 24 hours before analysis. The application procedures of sub-groups:

Confocal laser scanning microscope analysis

After the root canal medicament had been placed, each specimen was sectioned perpendicular to its long axis using a precision saw (IsoMet 1000; Buehler, Lake Bluff, IL) at a slow speed under water cooling. The tooth was sliced into three slices with approximately 1 mm thickness at 2, 5, and 8 mm (apical, middle, and coronal) depths.

Silicon carbide abrasive paper was used for polished the sections. The samples were fixed by placing them on glass slides properly after polishing, and it was examined with a Leica TCS-SPE confocal laser scanning microscope (Leica, Mannheim, Germany) set at 10× at wavelengths of 560–600 nm. If a single image of the root canal could not be obtained, different images of the canal were taken, and then these images were combined into a single image using Photoshop (Adobe Systems, Inc., San Jose, CA). Digital images [Figure 1] obtained to measure and evaluate the total area of dentin tubule penetration were transferred to the ImageJ program (ImageJ software, NIH). The dentinal tubule penetration area was measured as micrometers (μm) and converted to square millimeters (mm2) for the statistical analysis.
Figure 1: Representative CLSM images of dentinal tubule penetration at coronal (8 mm), middle (5 mm), and apical (2 mm) sections

Click here to view

Statistical analysis

The data were analyzed with SPSS software (v22.0, IBM Corp., New York, USA) using the three-way analysis of variance (ANOVA) and Tukey's post hoc tests to detect the effects of the independent variables and their interactions on the dentinal tubule penetration into the root canal dentin.

   Results Top

Mean and statistical data obtained from dentin tubule penetration area values by root canal medicament type, irrigation activation procedure, and root canal region are shown in [Table 1]. TAP provided a statistically significant greater penetration than the other all medicament groups (P = 0.000). UI provided a statistically significant higher dentinal tubules penetration area than other activation procedures (P = 0.000). When the root canal regions were compared among themselves, the same irrigation activation techniques and medicaments showed statistically higher tubule penetration in the coronal part compared to the middle third and the middle part compared to the apical third (P = .000).
Table 1: Mean and statistical data obtained from dentin tubule penetration area values by root canal medicament type, irrigation activation procedure, and root canal region

Click here to view

   Discussion Top

Elimination of the root canal bacterial micro organisms plays a crucial role in the long-term success of endodontic treatment. Different irrigation activation methods and medicaments have been preferred to achieve the whole disinfection of the root canal system.[20] It is important to clean the main canals of the tooth deeply from all the microorganisms, but sometimes it can be challenging because of the complexity of root canals.They may have accessory and lateral canals, isthmuses, and fins which might not be apparent in radiographs. These areas are very difficult to clean because they are too small for mechanical instrumentation. Also, these areas are eligible for microorganisms as bacteria can colonize and start an infection. It has been proved that the irrigating needle delivering technique only is not adequate for proper cleaning of the root canals.[21]

Therefore, different activation techniques are recommended, such as sonic, ultrasonic, and laser; they have been used to increase the effectiveness of irrigation solutions and to reach the places where mechanical preparation cannot reach within the root canal anatomy.[13] On the other hand, different studies have shown that irrigation activation has more effective results in removing the smear layer,[22] debridement ability, and postoperative pain. Irrigation systems also increase the tubule penetration of irrigants.[23]

The use of laser irrigation activation procedures has recently attracted the attention of researchers. Especially beneficial effects of Er:YAG laser activation were detected in many researches.[24],[25],[26] Example, Bitter et al.,[24] demonstrated the efficacy of Er:YAG laser on bond strength to root canal dentin, and this type of laser showed significantly higher results after laser treatment. Additionally, the effect of a few activation techniques such as conventional irrigation, passive ultrasonic agitation (PUA), and Nd: YAG laser-activated irrigation (LAI) were evaluated on the penetration of sodium hypochlorite into dentin tubules.[25],[27] As a result, they demonstrated that the standard protocol caused deeper penetration of irrigation solutions than PUA and LAI groups. Furthermore,[26] in another study, the effect of various irrigation agitation methods that include Er:YAG, photon-induced photoacoustic streaming (PIPS), sonic, and passive ultrasonic activation on the final irrigation solution penetration was examined into dentinal tubules by using a laser scanning confocal microscope.[28] They demonstrated Er:YAG laser activation put forth the highest penetration value than the other groups. In addition, passive ultrasonic activation exhibited significantly higher penetration than the sonic activation and the control group. When the laser energy is absorbed by the irrigant, explosive vapor bubbles form and implode, acting as a fluid pump in the root canal and causing a cavitation effect.[29] This effect may remove the smear layer and enable medicaments to penetrate dentinal tubules. As a result, laser activation is an effective technique for irrigation activation during root canal treatment, because it acts as a liquid pump by causing acoustic cavitation characterized by the formation of large oval vapor bubbles that will expand and explode following the use of ablative lasers in an aqueous environment.[30] However, according to the present study's result, ultrasonic activation indicated a stronger effect than laser irradiation. Therefore, the null hypothesis that the different irrigation activation techniques do not affect medicament penetration is rejected. The results of upper mentioned studies show contradiction when compared with the present study; these differences can be explained with different materials and methods such as tooth type, analysis type, laser type (This may be due to the fact that the KTP laser is not absorbed by water), activation time, Hz and mj, laser mode, activation application times, amount of solution used.

The other effective current approaches in modern endodontics are sonic and ultrasonic activation procedures. Sonic irrigation differs from ultrasonic irrigation in that its frequency (1–6 kHz) is lower, and it creates smaller shear stresses.[31] Ultrasonic agitation technique causes at –25,000 vibrations/s in the endodontic file. According to Martin and Cunningham, this superior effect with the acoustic flow of irrigation solutions that causes cavitation on root canal dentin surfaces leads to better penetration in the dentinal tubules. They suggested that the irrigation solution showed better active biological-chemical properties during ultrasonic application. In other studies, it was observed that ultrasonic activation gave more successful results than conventional syringe and sonic activation, which supports the present study's results.[32] We also put forth the superior results in terms of the effect of ultrasonic activation on dentinal tubules penetration compared with sonic and laser activation techniques.

These superiorities of ultrasonic activation that are widely used in endodontic treatments may depend on a few specific properties. For example, ultrasonically activated irrigation solutions reach at least 3 mm beyond the tip of the oscillating.[33] Furthermore, during ultrasonically activated irrigation, the irrigant is activated corresponding to a characteristic pattern of nodes and antinodes along with the oscillating file. Induced acoustic flow causes irrigation jets directed towards the root canal wall.[34] These jets provide better cleaning of the root canal system with the lateral cleaning effect of the oscillating ultrasonic file. This cleaning effect is observable from the coronal to the apical of the root canal. With the increase of intensity of the ultrasonic power, a cleaning effect that can reach 0.5 mm beyond the file tip was reported.[34] In our opinion, the present and upper mentioned studies' results that show similarity and dissimilarities demonstrated that although either laser and sonic activation or NaviTip FX may prefer, ultrasonic techniques stand out significantly as a remarkably applicable technique in terms of long term endodontic success.

Although instrumentation procedures have improved over the years, current techniques are insufficient to achieve complete cleaning of the root canal system. Therefore, the use of medicaments is recommended for better root canal disinfection that increases the degree of endodontic success. CH has been accepted as a commonly used intra-canal medicament due to its properties, such as its antimicrobial effect and biocompatibility against the majority of endodontic pathogens.[35] Various other medicaments can be preferred as an alternative to CH, such as TAP in root canal disinfection. The TAP is consists of metronidazole, ciprofloxacin, and minocycline mixture. It is also preferred by clinicians in regenerative endodontics due to its antimicrobial effect, biocompatibility, and effective tubule penetration ability.[36]

In a study carried out,[34] the penetration efficacy of the various types of medicaments after different agitating methods were researched in root canals.[37] Consequently, no statistically significant difference was observed in terms of tubule penetration in the apical third. On the other hand, in the present study, we found a statistically significant difference in terms of tubule penetration among CH and other groups following the irrigation activation groups in the apical third. This different result may be caused by differences in irrigation activation procedures and the use of vehiclesA few studies evaluated the degree of the penetration of dentinal tubules between TAP and CH as intracanal medicaments. Consequently, the researchers put forward that deeper tubule penetration was observed in TAP compared to CH regardless of the surface tension reducers used.[35],[36] In a study by Sabrah et al.[12] TAP and DAP were observed to be more effective than CH against E. faecalis and  P.gingivalis Scientific Name Search . In addition, in that study, it was stated that DAP is an effective antibacterial agent comparable to TAP.

In the present study, the lowest tubule penetration was observed in the CH group, while the highest value was obtained in the TAP group. The results mentioned above indicated that the use of CH for routine endodontic treatments has become useful especially to prevent flare-ups between sessions. Furthermore, the use of medicaments with higher tubule penetration and antibiotic effects such as TAP and DAP is recommended for more specific treatments (disinfection of immature necrotic teeth during regenerative endodontic procedures).[8]

The superior properties of TAP in terms of dentinal tubule penetration depend on the penetration ability of TAP up to 200 μm of the dentin tubules. The tubule penetration of intracanal medicament depends on its surface tension; low surface tension provides more penetration into inaccessible areas.[38]

Penetration into dentinal tubules has been evaluated using several techniques, including scanning electron microscopy, stereomicroscopy, confocal laser scanning microscopy, microcomputed tomography, and spiral computed tomography. The present study used confocal microscopy as it is an easy and commonly used approach to visualize and quantify medicament penetration based on fluorescence. Confocal microscopic approaches are less demanding in terms of the time required for imaging, reconstruction, and analysis.[39] In the confocal laser technique, there is no need for additional sample preparation that may cause artifacts, and it also allows the same samples to be used for the placement of CH. This aspect eliminates concerns such as inability to solve anatomical issues that may affect the results. Also, it has been reported that in penetration studies, root canal sealers and CH mixed with fluorescein green and rhodamine B have been shown to put forth very successful images.[40]

Untouched areas create risky areas for the storage of microorganisms, their by-products, and residues. These areas can also prevent tubule penetration of root canal medications. Therefore, the inaccessible areas of the canals can create a new source of infection by causing the formation of a suitable environment for the reproduction and proliferation of microorganisms. As a result, undesirable pulpal and periapical tissue diseases may occur in the post-treatment period, and the treatment will be unsuccessful. Accordingly, complete disinfection of dentinal tubules plays a critical role in endodontic success.[41]

Penetration of materials into dentinal tubules is affected by the smear layer formed during root canal preparation, as well as its chemical and physical properties and anatomical structure of the root canal system can affect the depth of penetration. Various permeability levels in different regions of the roots can be explained by important factors. The fact that the apical part of the root canal is less permeable than the coronal and middle third parts of the root canal can be attributed to factors such as the apical parts containing fewer dentin tubules, smaller tubules diameters, and tubular sclerosis.[42] The results of this study were similar to other studies, and when the penetration degrees of the same medicament type used in the same activation technique were evaluated, lower penetration of the root canal was observed at the apical 2 mm level compared to 5 mm, while lower penetration degrees were obtained at the 5 mm level compared to 8 mm.[28],[43] In addition, the preference for straight canal teeth in our study is due to the possibility that curved canals may adversely affect the penetration depth of the medicament to be used.[44] In the present study conducted, it was found that the tubule penetration of TAP was higher than that of CH regardless of the vehicles used; this result is also supported by the previously mentioned study.[45] With the use of antibiotics in endodontic treatments, it is aimed to eliminate bacteria and bacterial products that cause periapical lesions and to prevent these products from developing a secondary infection outside the canal system. Systemically taken antibiotics are not effective in the treatment of teeth with necrotic pulp without blood circulation. Local application of antibiotics to the root canal is thought to eliminate the risk of systemic side effects of antibiotics and provide an effective treatment for teeth with necrotic pulp lacking blood supply. TAP plays an important role in root canal disinfection and provides healing in periapical lesions, especially between sessions. Effective healing depends on good penetration of TAP into dentin, cementum, and periradicular tissues. Ensuring this penetration also depends on the selection of the most effective activation procedure. In addition, when used as a canal filling material, it may provide an advantage in the treatment of large periradicular lesions caused by persistent infectious species such as E. faecalis. Therefore, it has been determined that root canal irrigation methods have a significant effect on dentin tubule penetration of medicaments, and there is also a significant difference between medicament types.

   Conclusion Top

According to the findings of our study, the dentinal tubule penetration area was significantly affected by the final irrigation activation procedure, the selection of root canal medicament, and root canal third. Intracanal agitation could enhance the penetration degree of root canal medicaments into dentinal tubules. The ultrasonic technique was superior to other agitation techniques in dentinal tubule penetration of root canal medicaments at one or more sectioned levels from the root apex.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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