May 1, 2015
by Prashanthi Vandrangi, PhD; Bettina Basrani, DDS, PhD
Scope of Conventional and Contemporary Endodontic Treatments
The aim of endodontic therapy is to eradicate microbial growth and prevent or treat periradicular disease.1 Successful canal disinfection employs both bio-mechanical and chemo-mechanical means of cleaning. Mechanical preparation, termed bio-mechanical cleaning, prepares the root canal space for delivery of disinfecting irrigants and to remove vital and necrotic tissue and microorganisms.1,2 However, several in vitro micro-computed tomography (µCT) studies have shown anastomoses between canals, fins, accessory canals, and curves that remain untouched by the mechanical instrumentation.2-4 Due to the complex nature of root canal anatomy, mechanical preparation alone cannot sufficiently remove soft tissue debris or kill microorganisms and weakens the structural integrity of the tooth.5,6
Furthermore, areas that are touched by instrumentation files create a smear layer.7 Smear layer is a layer of organic and inorganic material on the canal wall and inside dentinal tubules that can harbor bacteria and pulp tissue remnants.8 It is recommended to remove smear layer in order to allow penetration of irrigants into dentinal tubules, hence eradicating microorganisms and their by-products, and allowing for an adaptive obturation seal.9
Chemo-mechanical cleaning employs irrigation techniques that combine the use of sodium hypochlorite (NaOCl) and ethylene-diamine-tetra-acetic acid (EDTA) solution. It has been shown to dissolve smear layer to enhance the effectiveness of bacterial elimination in the dentinal tubules and to remove hard and soft tissue debris.10 Agitation techniques have shown to increase the effectiveness of irrigation in the apical region and into the corresponding dentinal tubules.11 The most common agitation device used for root canal therapy is ultrasonic activation in conjunction with NaOCl. However, in a histological study that assesses root canal cleanliness, contemporary techniques involving passive ultrasonic irrigation cleaned 80 percent of the canal, whereas conventional techniques cleaned only 29 percent.12
These studies show the necessity of utilizing an effective root canal cleaning method while maintaining the dentin integrity. The GentleWave™ System has been designed to bio-mechanically and chemo-mechanically clean root canals irrespective of canal anatomy using minimal or no instrumentation with water, NaOCl, and EDTA.13,14 In this paper, the mechanism of action and the cleaning and disinfection efficacy of the GentleWave™ System are introduced in a series of pilot studies via histological assessment and via electron microscopy.
Introducing the GentleWave™ System
The GentleWave™ System aims to clean the root canal system through generation and propagation of various physio-chemical mechanisms including a broad spectrum of sound waves, i.e., the multisonic waves. Multisonic waves are initiated at the tip of the GentleWave™ handpiece, which is positioned inside the pulp chamber of the root canal system (Fig. 1). The treatment fluids are supplied continuously via the GentleWave™ console to the handpiece.
The GentleWave™ system is composed of a console and a sterile and single-patient use handpiece. It delivers a stream of treatment solution from the tip of the handpiece into the pulp chamber while excess fluid is simultaneously removed from the chamber by the built-in vented suction through the handpiece into a waste canister inside the console. According to the manufacturer, upon initiation of flow through the treatment tip of the handpiece, the stream of the treatment fluid interacts with the stationary fluid inside the pulp chamber creating a strong shear force, which causes hydrodynamic cavitation in the form of a cavitation cloud. The continuous formation and implosion of thousands of microbubbles inside the cavitation cloud generates an acoustic field with broadband frequency spectrum that travels through the fluid into the entire root canal system. Throughout the treatment, the fluid starts with three percent NaOCl and changes to eight percent EDTA with a water rinse in between. The treatment tip of the handpiece is designed to deflect the stream of treatment fluid in such a way to generate a flow over the orifices of the root canals. This flow induces gentle vortical flow as well as a slight negative pressure within the root canal system. The energy and the vortical flow dissipate as they travel apically into the root canal system. The treatment fluid is degassed to minimize the energy loss and also to ensure energized treatment fluid is delivered throughout the root canal system. Each fluid at equilibrium contains certain amount of dissolved gas. When the fluid is agitated enough, the thermodynamic conditions change and to reach equilibrium in the new conditions, the fluid may release some of the dissolved gas in form of bubbles. The bubbles inside a fluid tend to damp acoustic as well as hydrodynamic energy. As these bubbles are formed inside a root canal and their size becomes comparable to the width of the root canal, the amount of energy loss increases substantially to the point that the volume of root canal system apical to that bubble may not receive any energy/fluid and receive less efficient treatment (as is the case with vapor lock in conventional irrigation and activation).15
The interplay of the Multisonic energy, vortical fluid dynamics, and chemistry of the treatment fluid, result in enhanced dissolution and removal of organic matter, i.e. pulp tissue and biofilm from the root canal system.13 The treatment tip is positioned inside the pulp chamber and is not required to enter canals or orifices, therefore allowing for minimal instrumentation of the root canals leading to saving the integrity and strength of the tooth (Fig. 1B).
Previous Studies Using the GentleWave™ System
Efficiency in Dissolution of Tissue
The tissue dissolution efficacy of the GentleWave™ System was compared with different conventional and contemporary endodontic systems.13 The authors utilized a model of the tooth emulating by cubes of bovine muscle tissue that were immersed in 0.7 mL test tubes as demonstrated in Figure 2. The study was performed at different temperatures and different concentrations of NaOCl.
FIGURE 2. The figure shows the tissue dissolution capability of (A) the GentleWave™ System and (B) active ultrasonic irrigation. Tissue dissolution using the GentleWave™ System was eight times greater than that of all the contemporary and conventional systems tested.
Tissue dissolution using the GentleWave™ System was eight times greater than that of all the irrigation systems including ultrasonic system and End
oVac™ when NaOCl was the irrigant utilized.
Efficiency in Removing Inter-appointment Intra-canal Medicament
The performance of the GentleWave™ System to remove intra-canal medicament, even in the apical third regions of the root canal system was demonstrated.14 Using a complete treatment cycle of seven minutes and 45 seconds, the GentleWave™ System completely removed calcium hydroxide (Ca(OH)2) from the entire root canal system including the apical third regions (Fig. 3).
However, using conventional endodontic treatment, only 61% of the volume of Ca(OH)2 was removed and the apical third of all samples contained remnants of Ca(OH)2 even after instrumentation and 7 minutes and 45 seconds of irrigation. Further, the study demonstrates the GentleWave™ System’s ability to remove Ca(OH)2 using only water within 90 seconds without requiring any additional use of instruments or chemicals.
Pilot Study 1: Removal of biofilm-mimicking gelAim To observe the performance of the GentleWave™ System in simulated molars filled with biofilm-mimicking gel. Pre-treatment Sample Preparation To test the GentleWave™ System’s performance in mandibular molars, the canals of a simulated molar (TrueTooth™ Mandibular Molar Replica 30-001, Dolendo®, SantaBarbara, CA) were filled with a biofilm-mimicking gel that is composed of 1% agarose (ThermoFisher Scientific, Waltham, MA).16 Care was taken to carefully fill the anatomical complex areas of the root canal system. The molars with the biofilm-mimicking gel were allowed to set for 10 minutes until the gel was formed and the GentleWave™ treatment was performed.
GentleWave™ Treatment Cycle
The handpiece was placed on an accessed occlusal surface to deliver the irrigant into the pulp chamber and all the canals were treated simultaneously.13,14
Figure 4 demonstrates the cleaning efficiency of the GentleWave™ System on molars filled with a biofilm mimicking gel. The GentleWave™ System thoroughly cleans the biofilm mimicking gel in a mandibular molar within one minute.
FIGURE 4. The GentleWave™ handpiece is placed on the platform made of impression material. The tip of the GentleWave™ handpiece is visualized in the pulp chamber as the treatment is being performed. The GentleWave™ System thoroughly cleans the blue dyed – agar gel (1%) in a mandibular molar within one minute.
Pilot Study 2: Macroscopic Analysis of Tissue Debris Removal
Aim To compare the GentleWave™ system’s performance with conventional endodontic treatment in ex-vivo human molars using macroscopic visualization with stereo microscope.
Pre-Treatment Sample Preparation
Four extracted human first or second molars (both mandibular and maxillary) with different anatomical characteristics were collected and stored in 1X phosphate buffered solution (PBS) at 4°C until use. Any teeth with decay or fractures below the cemento-enamel junction (CEJ), internal or external root resorption, open apices, or previous root canal therapy were excluded. When present, caries were removed and missing coronal tooth structure was restored using etchant (Etch-Rite, Pulpdent, Watertown, MA), bonding agent (Optibond, Kerr, Orange, CA), and Virtuoso® flowable light-cure composite (Denmat, Lompoc, CA). Following endodontic access, all teeth were firmly secured and sealed within a water-saturated porous medium using an adhesive (McMaster-Carr, Los Angeles, CA) to simulate blood-saturated periapical tissue.14 Reproducible glide paths and working lengths were established using a #10 K-file (MANI K-files, Utsunomiya, Japan) and canals were instrumented with #15/.02 K-files and #15/.04 EndoSequence rotary files (Brasseler,Savannah, GA).
The four teeth were divided into two treatment groups: GentleWave™ System treatment or conventional treatment. For the GentleWave™ treatments, the handpiece was placed on an accessed occlusal surface to deliver the irrigant into the pulp chamber [13-14]. The teeth were minimally shaped up to #15/.04. The GentleWave™ treatment cycle consists of 3% NaOCl for 5 minutes, distilled water for 30 seconds, 8% EDTA for 2 minutes, and distilled water for 15 seconds. For the conventional treatments, WaveOne (#25/.08) (Dentsply, York, PA) instrument was inserted to working length with 3 mL of 5% NaOCl irrigation using a 30G Max-I-Probe needle. Irrigation was performed using 6 mL of 5% NaOCl followed by 3 mL of 17% EDTA using a 30G Max-I-Probe needle at 1 mm from WL.
Post-Treatment Sample Preparation
Each tooth was irrigated and filled overnight with 1 mL of 10 percent buffered formalin to allow for fixation of any remaining pulpal tissue. The roots from the two treatment groups were separated from their crowns using a diamond disc (NTI, Rotary Dental Instruments, Kahla, Germany). The roots were carefully split and stained with 1 percent Eosin solution (Eosin Y – Certified Biological Stain, Fisher Chemical, Waltham, MA) in distilled water using a micro applicator brush for 30 seconds. The stained sections were rinsed with slight agitation and images were acquired using Nikon Eclipse (Nikon Eclipse Ci, Nikon, Melville, New York).
Representative results of distal roots of mandibular molars are shown in Figure 5. The distal root of the molars that underwent conventional treatments demonstrated the presence of tissue throughout the canal wall. However, when treated with the GentleWave™ System, no tissue was observed. Similar images were obtained from the rest of the roots observed.
FIGURE 5. Distal roots of mandibular molars stained with Eosin Y. The teeth were treated using (A) GentleWave™ treatment or (B) Conventional instrumentation with needle irrigation. Tissue debris is visualized when samples were treated with conventional instrumentation with needle irrigation.
Pilot Study 3: Scanning Electron Microscopy Analysis of Root Canal System Having Complex Anatomies
Aim To evaluate the GentleWave™ performance in comparison with conventional endodontic treatment in
ex-vivo human molars with complex anatomies using microscopic visualization with scanning electron microscopy analysis.
Eight mandibular and maxillary molars with complex anatomy were selected for this study and prepared as described above (Pilot study 2). Briefly, teeth were conventionally accessed, patency was established, and working length was determined.
Eight molars were divided into two treatment groups: GentleWave™ System treatments or conventional treatments. For the GentleWave™ System treatments, the canals were minimally instrumented and the GentleWave™ treatment cycle was performed. For the conventional treatments, the canals were instrumented to #25/.08 and the treatment was performed. The treatments were performed as described in Pilot study 2.
Post-Treatment Sample Preparation
The teeth were prepared for SEM evaluation. Fixation with formalin (as discussed in Pilot study 2) was followed by serial graded ethanol dehydration by submerging the samples in 50 percent, 70 percent, 80 percent and 100 percent ethanol solutions. Samples were finally submerged in fresh 100 percent ethanol for 30 minutes. Samples were sputter coated with Au-Pd at 20 mA for two minutes. SEM imaging of the samples at 200× (for debris score; four fields per each region) and 1000× (for smear layer score; eight fields per each region) magnifications were acquired at 15 kV using Hitachi TableTop TM3010 (Krefeld, Germany).
To measure the resultant of debris and smear, the scoring system proposed by Hülsmann et al. (1997) was utilized.7 The criteria for the scoring was as follows –
For debris score — score one: clean canal wall, only very few debris particles; score two: few small conglomerations; score three: many conglomerations, <50 percent of the canal wall covered; score four: >50 percent of the canal wall covered; score five: complete or nearly complete covering of the canal wall by debris.
For smear layer score — score one: no smear layer, orifice of dentinal tubules patent; score two: small amount of smear layer, some open dental tubules; score three: homogeneous smear layer along almost the entire canal wall, very few open dentinal tubules; score four: entire root canal wall covered with homogeneous smear layer, no open dentinal tubules; score five: thick homogenous smear layer covering the entire root canal wall.
SEM micrographs were acquired in the coronal, middle, and apical regions of the root canal system. Two blinded examiners performed evaluations separately. Representative images are shown in Figure 6.
FIGURE 6. The figure shows representative SEM images of mesiobuccal roots in maxillary molars. The images are taken at 30x and zoomed in at 1000X. The scale bar represents 1 mm and 50 µm respectively.
Completely cleaned root canals were found after minimal instrumentation and using the GentleWave™ System. Tissue debris was observed in the root canal system when the teeth underwent conventional endodontic treatments. The characteristic feature of apical regions – anisotropic and lower density of dentinal tubules – were visualized.17 Analysis of the blinded evaluations performed by two calibrated operators is summarized in Figure 7. This study revealed significant differences among the two treatment groups. When the samples were treated with GentleWave™ System, clean canal walls with very few debris particles were observed whereas no smear layer was observed with patent dentinal tubules. The GentleWave™ treatment group presented significantly lower smear layer scores than the conventional group (p<0.001) in the coronal, middle, and apical third of the examined canals.
FIGURE 7. The figure shows the average and standard deviation of the debris and smear score in the coronal, middle, and apical regions of ten molars. When teeth were cleaned using the GentleWave™ System, the debris and smear scores were consistently clean (score=1).
Pilot Study 4: Histological Analysis of Root Canal Systems With Complex Anatomies
Aim To evaluate the GentleWave™ performance in ex-vivo human molars using histological analysis.
Two maxillary molars with lateral fins in the mesiobuccal canals and two mandibular molars with c-shape configurations were selected and treated in a similar way as described above (Pilot study 2). Briefly, the molars were accessed, patency checked, minimally instrumented and treated with the GentleWave System.
The four teeth were treated only with the GentleWave™ System after minimal instrumentation.
Post-Treatment Sample Preparation
Specimens were fixed with 1 ml of 10 percent buffered formalin (as described above in Pilot study 2), were washed in running water for one hour, and decalcified in a solution containing <15 percent wt hydrochloric acid (HCL; Surgipath, Richmond, IL) and <5 percent wt EDTA for two weeks. Following decalcification, samples underwent standard histological processing: dehydrated in ascending grades of ethanol, cleared in xylene, and finally infiltrated. Roots were embedded in paraffin and sectioned cross-sectionally with a rotary microtome (Leica RM2235, Leica, Wetzlar, Germany). Sections were stained using hematoxylin and eosin (H&E). The H&E stained cross-sections were further examined using an optical stereomicroscope (Leica DMLB; Leica, Wetzlar, Germany) and images were acquired using a camera (Leica DFC290; Leica, Wetzlar, Germany).18
Figure 8 demonstrates the cleaning ability of the GentleWave™ System with minimal shaping using histological analysis. In Case A, a clean lateral fin was observed (Fig. 8A). In Case B, the tooth has a c-shaped configuration of the root canal system (Fig. 8B).
FIGURE 8. The figure shows the (A) mesiobuccal canal of a maxillary molar and (B) mandibular molar with a c-shaped configuration. The dotted lines across the radiographic x-rays demonstrate the locations of the respective histological sections. Courtesy of Dr. Wonjun Shon. Scale bar represents 100 µm.
Pilot Study 5: Microscopic Analysis of Biofilm Removal
Aim To evaluate the performance of the GentleWave™ in compari
son with contemporary endodontic treatment on Enterococus Faecalis (E. Faecalis) inoculated ex-vivo human molars using microscopic visualization with SEM.
Four mandibular and maxillary molars were selected and prepared as described (Pilot study 2). The inclusion and exclusion criteria are described in Pilot study 2. Briefly, teeth were conventionally accessed, patency was established, and working length determined. Molars were inoculated with E. Faecalis (ATCC # 19433, Manassas, VA) in sterile brain heart infusion (BHI) (Teknova, Hollister, CA) for five weeks. BHI was replenished biweekly.
The four molars were divided into two treatment groups: GentleWave™ System treatments or contemporary treatments. For the GentleWave™ System treatments, the canals were minimally instrumented and GentleWave™ System treatment was performed as described earlier. For the contemporary treatments, the canals were instrumented to full working length as follows: ProTaper (Dentsply Tulsa Dental Specialties, Tulsa, OK) S1, S2, F1, F2 (350 rpm) followed by EndoSequence #35/.04 (500 rpm). 1 mL of 3 percent NaOCl was used as an irrigant in between each file. The final irrigation was performed with ultrasonic activation using PiezonMasterTM 700 with ESI tip (Electro Medical Systems, Nyon, Switzerland) in Endo mode and was set to maximum power. Each canal was ultrasonically activated using ESI tip for 20 seconds per canal. NaOCl irrigation and ultrasonic activation was repeated until the total elapsed time has reached five minutes. The canals were irrigated with distilled water for 15 seconds and irrigated with 17 percent EDTA for approximately 10 seconds in each canal. EDTA irrigation and ultrasonic activation was repeated until the total elapsed time has reached two minutes. Canals were finally irrigated with distilled water for 15 seconds.19
Post-Treatment Sample Preparation
After treatments, four molars were processed for SEM (as described in Pilot study 3). ResultsRepresentative SEM images are shown in Figure 9.
The results show that even in the apical regions, the root canal system is free of E. Faecalis biofilm when treated with the GentleWave™ System.
Elimination of bacterial load from the root canal system is the main goal of the endodontic treatment.2 Unfortunately, manual and rotary instruments and conventional syringe irrigation are not enough to achieve this goal. Currently, new combination of irrigants and new delivery devices using pressure, vacuum, oscillation, and/or a combination with suction were introduced. Although these contemporary techniques are shown to enhance disinfection, the complete elimination is still a goal. Therefore, the aim of the present study was to report a series of preliminary studies using the new GentleWave™ system to enhance disinfection of the root canal system.
Clinical cases of complex root canal systems show that root canal aberration occurs with a wide variety and variability. Traditionally, even though unique methods of cleaning and shaping complex anatomical regions are employed, persistent infection in the root canal system is common and might cause the endodontic treatment to eventually fail.1-3 It is well known that molars are often the most heavily restored teeth in adult dentition and have been notoriously prone for root canal treatments.20 In this preliminary report, molars with complex anatomies such as anastomoses between the canals and accessory communications, high degree of curvature and c-shaped configurations were selected.
In Pilot study 1, the efficiency of the GentleWave™ System in cleaning complex anatomy of molars by clearing the biofilm-mimicking gel has been demonstrated within 60 seconds. The study has been performed in two simulated molars. Futures studies should be performed to study the mechanism of action of the GentleWave™ System using a high-speed visualization system. Further, it will be interesting to compare the efficiency of the GentleWave™ System with conventional methods in the removal of biofilm-mimicking gel in a larger sample size.
In Pilot study 2, ex-vivo molars were treated with GentleWave™ System or with conventional endodontic treatments. The presence of soft tissue remains was observed in the root canal system when the teeth underwent conventional endodontic treatments via macroscopic histological. Further studies should be planned to replicate these results in a larger sample size.
In Pilot study 3, scanning electron microscopy was used to evaluate the absence of debris and smear layer in molar teeth treated with either conventional or GentleWave™ treatments. The latter showed a higher level of cleanness of the root studied. Future studies are planned to analyze this procedure in a larger sample size and in a different part of the root canal system (other than the apical third). Care was taken to expose the two treatments to the same amount of time of the irrigation in contact with the dentin (five minutes). Although it was shown that the tissue dissolution capacity of the GentleWave™ system surpasses that of contemporary endodontic methods using similar volume of treatment fluids,13 it will be interesting to perform future studies with similar irrigant volume and similar time exposure should be considered to confirm these results.
Pilot study 4 was performed to show the cleaning ability of GentleWave™ using histological analysis. A completely cleaned root canal configuration was observed after minimal instrumentation and using the GentleWave™ System. Future studies are on the way to compare the cleaning ability of GentleWave™ with conventional irrigation systems in c-shape canals and isthmi.
It is well known that currently available endodontic technologies still have difficulty in completely eradicating biofilm.1-3,21,22 The ability of the GentleWave™ System to clean E. Faecalis bacteria from the root canal system of molars was shown in Pilot study 5. The absence of dentinal mud (an accumulation of organic and inorganic debris in the root canal system) has been observed in the electron micrographs of the root canal system when the teeth are treated with the GentleWave™ System in comparison with contemporary endodontic treatments.
A possible reason for the observed cleanliness in the root canal anatomy with open dentinal tubules might be the effective and continuous flow of treatment fluids: NaOCl-Water-EDTA-Water though the GentleWave™ handpiece and the underlying technology. These results demonstrate the efficiency of the GentleWave™ System when compared to conventional endodontic practices. However, the results shown for each pilot study have to be further explored using robust sample sizes.
This report illustrates a viable solution in endodontic treatments using the GentleWave™ System. The system is effective even in areas of anatomical complexities and in the apical-thirds of the root canal system. The results reported in these pilot studies should be interpreted with
caution. Clinical cases are being performed using the GentleWave™ System and the clinical outcomes will demonstrate its benefits. Further studies in removing multi-species biofilm and clinical cases demonstrating faster healing rates are needed to corroborate these results.OH
Dr. Vandrangi has been leading the in-vitro and ex-vivo studies at Sonendo® (Laguna Hills, CA). She holds a MS and PhD in Bioengineering and pursued her post-doctoral work in signaling pathways and the underlying bio-transport phenomena.
Dr. Basrani is an Associate Professor and Co-director M.Sc. Endodontics Program Faculty of Dentistry University of Toronto, Toronto, ON, Canada. She holds a DDS with specialization in Endodontics and a PhD.
Oral Health welcomes this original article.
1. Ricucci D, Siqueira JF. Anatomic and Microbiologic Challenges to Achieving Success with Endodontic Treatment: A Case Report. J Endod. 2008;34: 1249–1254.
2. Cano V, Nair PNR, Henry S, Vera J. Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after “one-visit” endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005;99:231–52.
3. Basrani B, Haapasalo M. Update on endodontic irrigating solutions. Endod Top. 2012;27:74–102.
4. Peters OA, Barbakow F. Effects of irrigation on debris and smear layer on canal walls prepared by two rotary techniques: a scanning electron microscopic study. J Endod. 2000;
5. Zandbiglari T, Davids H, Schäfer E. Influence of instrument taper on the resistance to fracture of endodontically treated roots. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:126-31.
6. Peters OA. Challenges in root canal preparation. J Endod. 2004;30:539–67.
7. Hülsmann M, Rümmelin C, Schäfers F. Root canal cleanliness after preparation with different endodontic handpieces and hand instruments: a comparative SEM investigation. J Endod. 1997;23:301–6.
8. Violich DR and Chandler NP. The smear layer in endodontics – a review. Int Endod J. 2010;43:2–15.
9. Tay FR, Loushine RJ, Lambrechts P, Weller RN, Pashley DH. Geometric Factors Affecting Dentin Bonding in Root Canals: A Theoretical Modeling Approach. J Endod. 2005;31:584–589.
10. Maciel ACdeC and Scelza MFZ. Efficacy of automated versus hand instrumentation during root canal retreatment: an ex vivo study. Int Endod J. 2006;39:779–784.
11. Haapasalo M, Shen Y. Irrigation in Endodontics Endodontics Irrigation Root canal Irrigant. Dent Clin NA. 2010;54:291–312.
12. Van Der Sluis LWM, Shemesh H, Wu MK, and Wesselink PR. An evaluation of the influence of passive ultrasonic irrigation on the seal of root canal fillings. Int Endod J. 2007;40:356–361.
13. Haapasalo M, Wang Z, Shen Y, et al. Tissue dissolution by a novel multisonic ultracleaning system and sodium hypochlorite. J Endod. 2014;40:1178-81.
14. Ma J, Shen Y, Yang Y, et al. In vitro study of calcium hydroxide removal from mandibular molar root canals using a GentleWave™ system. J Endod. 2015;41:553-558
15. Charara K, Friedman S, Sherman A, Kishen A, Malkhassian G, Khakpour M, and Basrani B. Assessment of apical extrusion during root canal procedure with the novel GentleWave system in a simulated apical environment. J Endod. In Press. 2015.
16. Macedo RG, Robinson JP, Verhaagen B, Walmsley AD, Versluis M, Cooper PR, and van der Sluis LM. A novel methodology providing new insights into the ultrasonic removal of a biofilm-mimicking hydrogel from lateral morphological features of the root canal. Int Endod J. 2014; 47: 1040–1051.
17. MjÖr IA and Nordahl I. The density and branching of dentinal tubules in human teeth. Arch Oral Biol.1996;41:401-12.
18. Pinheiro SL, Frasson AD, Bincelli IN, et al. Study of a morphometric model for histological evaluation of the collagen in dentin carious lesions. J Clin Pediatr Dent. 2009;33: 123-126.
19. Yoo YJ, Lee WC, Kim HC, Shon WJ, Baek SH. Multivariate analysis of the cleaning efficacy of different final irrigation techniques in the canal and isthmus of mandibular posterior teeth. Restor Dent Endod. 2013;38:154-9.
20. Carrotte, Endodontics: Part 4 Morphology of the root canal system. Br Dent J. 2004;197:379–383.
21. Paiva SSM, Siqueira JF, Rôças IN, Carmo FL, Leite DCA, Ferreira DC, Rachid CTC, and Rosado AS. Molecular microbiological evaluation of passive ultrasonic activation as a supplementary disinfecting step: a clinical study. J. Endod. 2013;39:190–194.
22. Ordinola-Zapata R, Bramante CM, Aprecio RM, Handysides R, and Jaramillo DE. Biofilm removal by 6% sodium hypochlorite activated by different irrigation techniques. Int Endod J. 2013;47:1365–2591.