Root Canal Wall Dentin Structure in Uninstrumented but Cleaned Human Premolars: A Scanning Electron Microscopic Study

Root Canal Wall Dentin Structure in Uninstrumented but Cleaned Human Premolars: A Scanning Electron Microscopic Study

Basic Research—Technology Root Canal Wall Dentin Structure in Uninstrumented but Cleaned Human Premolars: A Scanning Electron Microscopic Study Zheju...

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Basic Research—Technology

Root Canal Wall Dentin Structure in Uninstrumented but Cleaned Human Premolars: A Scanning Electron Microscopic Study Zhejun Wang, DDS, PhD, Ya Shen, DDS, PhD, and Markus Haapasalo, DDS, PhD Abstract Introduction: Conventional endodontic treatment includes instrumentation of the canals in most cases to size #25/.06 or larger, which changes the original canal wall anatomy. In recent years, energy-driven equipment, such as photon-induced photoacoustic streaming (Fotona LLC, Dallas, TX) and a multisonic GentleWave system (Sonendo Inc, Laguna Hills, CA), have been introduced to facilitate cleaning of minimally instrumented canals or even uninstrumented canals. The purpose of this study was to examine root canal wall anatomy in premolar teeth cleaned by a noninstrumentation method after #10 K-file patency examination. Methods: Twenty-four freshly extracted human premolars were accessed, and patency was established by a #10 K-file. Seventeen teeth were treated by the GentleWave system using 3% sodium hypochlorite, and 7 untreated teeth served as negative controls. The dentin surface in the coronal, middle, and apical thirds of the root canal was examined by scanning electron microscopy after tooth splitting. The canal wall structures were assessed using a predefined scale of 4 parameters: calcospherites, surface irregularities, dentinal tubule openings, and tissue debris. Results: A clean surface of mineralized dentin was exposed with no organic tissue remnants or debris left in the root canal system, including the isthmus areas between the 2 canals. The uninstrumented root canals showed an irregular dentin structure in many areas, including previously unreported fingerlike projections. The isthmus areas had no or only a few dentinal tubule openings. The dentin structures were well preserved in the test group, whereas in the untreated control teeth tissue debris covered most of the dentin surface. Conclusions: Root canal wall dentin in premolars cleaned with a noninstrumentation method showed a wide structural variety, especially in the middle and apical region. No organic tissue remnants or dentin debris were detected. (J Endod 2018;-:1–7)

Key Words Calcospherites, debris, dentin, GentleWave, microstructure, noninstrumentation, tissue remnants

T

he root canals of huSignificance man teeth are inherHuman premolars can be completely cleaned free ently complex systems of organic matter with a specific noninstrumentawith many irregular struction method. This can greatly contribute to saving tures (1, 2). The anatomic the root structure. The absence of dentin debris complexity of the root because of noninstrumentation cleaning may facilcanal system enables itate a better antimicrobial effect of the treatment. bacteria to hide and multiply (3), and despite instrumentation of high quality and the use of different irrigating solutions, debris often remains in specific areas of the canal (4). When adequate cleaning cannot be achieved, the sealing of portals of communication with periapical tissues is more difficult, and the persisting intracanal bacteria in the root canal system may eventually cause failure of the endodontic treatment (5). Conventional endodontic treatment includes instrumentation of the canals in most cases to a minimum size of #25/.06 or #30/.04 and often larger than these. This has been necessary for 2 main reasons: to optimize cleaning by irrigation, especially of the apical root canal, and to support the making of a high-quality root filling. As a result of canal preparation, much of the root canal wall area is flattened by the instruments, and original wall structures remain only in areas that are beyond the reach of the files. In recent years, energy-driven equipment, such as photon-induced photoacoustic streaming (Fotona LLC, Dallas, TX) and the multisonic GentleWave (GW) system (Sonendo Inc, Laguna Hills, CA), have been introduced to facilitate cleaning of minimally instrumented canals or even uninstrumented canals (6). A noninstrumentation cleaning method has the advantage of saving the tooth structure, but by leaving canal wall structures untouched, it might also create a greater challenge for the removal of tissue remnants and biofilm by irrigation in the complex microanatomic landscape. The details of dentin morphology have been investigated by several previous studies (7–12). Some of these studies examined the microscopic structure of tubular dentin using coronal dentin (7, 8, 10); others focused on root canal dentin morphology and the root dentin structure after instrumentation (9, 11, 12), when much of the original root canal structure has already been changed. There are no reports of the shapes and structures of the root canal dentin wall after the use of the newly introduced, energy-driven noninstrumentation cleaning methods.

From the Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, The University of British Columbia, Vancouver, British Columbia, Canada. Address requests for reprints to Prof Markus Haapasalo, Division of Endodontics, Oral Biological and Medical Sciences, UBC, Faculty of Dentistry 2199 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.01.014

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Basic Research—Technology Recent histologic and scanning electron microscopic (SEM) studies in extracted human molars have indicated that the GW system, using the GW molar instrument, creates a clean root canal space and no erosion of dentin could be detected (13, 14). There are no studies so far using the GW instrument for premolar and anterior teeth or the anatomy of the root canal dentin surface after the cleaning procedure. The goal of the present study was to evaluate the surface morphology of the root canal wall dentin after GW cleaning without instrumentation in human premolars using scanning electron microscopy. The shape and the amount of calcospherites, surface irregularities, dentinal tubule openings, and tissue debris were examined.

SEM Analysis After cleaning, external grooves were made on the buccal-lingual surface of the roots with a 0.17-mm-thick diamond disc (Brasseler Inc, Savannah, GA), and the roots were split into 2 halves by a single-edge razor blade and a hammer. Thus, the sample size was 14 and 34 for the negative control and test groups, respectively. The dentin halves were subjected to increasing concentrations of ethanol (50%, 70%, 80%, and 100%) for serial dehydration. The dehydrated specimens were sputter coated with iridium using a Leica EM MED020 Coating System (Leica Microsystems Inc, Concord, Canada) for SEM analysis. The surface of the root canal wall after treatment was imaged by scanning electron microscopy (Helios Nanolab 650; FEI, Eindhoven, Netherlands).

Materials and Methods

Image Evaluation An SEM image at 50 magnification was taken at each of the coronal, middle, and apical thirds of the root to obtain an overall view. The presence and absence of calcospherites, surface irregularities, dentinal tubule openings, and tissue debris were rated and scored using a modified predefined scale system (16). The scoring system used in the present study is described in Table 1. SEM images at the magnifications of 200 and 1000 were used for scoring. Specific areas of dentin were observed for qualitative analysis at greater magnification ranging from 4000 to 20,000 whenever there was a need to determine the type of tissue (ie, organic or inorganic). The scoring of surface irregularities and tissue debris was performed using SEM images at 200 magnification, and the scoring of calcospherites and dentinal tubule openings were performed using 1000 magnification. A total of 288 images (48 samples  3 canal thirds  2 magnifications) were analyzed by 2 examiners who were blinded to the group distribution to examine and score the images. The percentage of the count of each score level among the total sample size in each group (34 in the noninstrumentation group and 14 in the negative control group) was calculated to show the distribution of each score. Extra SEM images (in addition to the 288 images) with magnification higher than 4000 were taken when necessary to identify specific dentin microstructures and to separate mineralized irregular structures from organic tissue remnants and inorganic debris.

Tooth Collection Twenty-four single-rooted permanent premolar teeth extracted for orthodontic reasons were collected and visually and radiographically examined. All samples were intact teeth with no previous root canal treatment, extensive coronal restoration, root caries, root resorption, or open apices. The protocol of this study was approved by the ethics committee of the university (certificate H12-02430). All samples were accessed according to standard endodontic procedures. A #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) was inserted into the canals to confirm patency, but no filing was performed. Radiographs and examination under a stereomicroscope showed that root development was completed, and none of the teeth had an open apex. Eight of the 24 teeth had 2 joining canals in 1 root. To maintain the original root canal microstructure, no instrumentation was performed in this study. Samples were randomly divided into 2 different groups with 7 and 17 samples in each group, respectively. Seven untreated premolars were used as a negative control with 2 samples having 2 joining canals. The remaining 17 teeth were cleaned by the GW premolar instrument with 6 samples having 2 joining canals.

Cleaning Protocol The GW system consists of a console and a treatment instrument whose mechanism has been previously described (6, 15). According to the manufacturer, a degassed stream of fluid generates a broad spectrum of sound waves that travel through the fluid into the root canal system (15). After the access cavity was created, the tip of the treatment instrument was placed in the space of the access opening and the pulp chamber of the teeth and sealed with a block-out resin material (Kool-Dam; Pulpdent, Watertown, MA). The treatment consisted of 3% sodium hypochlorite for 5 minutes followed by distilled water for 15 seconds. The sodium hypochlorite solution was dispensed by the treatment instrument at a flow rate of 45 mL/min. Irrigation using EDTA was not done because the canals were not instrumented.

Statistical Analysis Statistical analysis was performed using SPSS for Windows, Version 16.0 (SPSS Inc, Chicago, IL). The weighted coefficient kappa was used to measure interobserver reproducibility. The differences in the scores between groups were analyzed by the Kruskal-Wallis test and the MannWhitney test at a significance level of P < .05.

Results Score distribution attributed to each parameter on the three thirds of the canals is summarized in Figure 1. The kappa value for the

TABLE 1. Scale of Values Assigned to Different Parameters Evaluated Scale

2

1

2

3

4

Calcospherites

Absent

Surface irregularities Dentinal tubule openings Tissue debris

Absent

Covering less than 50% of the root canal surface Covering less than 25% of the root canal surface 25%–50% open

Covering 50%–75% of the root canal surface Covering 25%–75% of the root canal surface 50%–75% open

Covering over 75% of the root canal surface Covering over 75% of the root canal surface 75%–100% open

Covering less than 25% of the canal surface

Covering 25%–75% of the canal surface

Covering over 75% of the canal surface

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0%–25% open Absent

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Figure 1. A 4-level scoring system showing the percentage distribution of each score level in the coronal, middle, and apical thirds of the teeth in the noninstrumentation and untreated control groups for (A) calcospherites, (B) irregular surface structures, (C) dentinal tubule openings, and (D) tissue debris.

interobserver reproducibility on the scoring was higher than 0.93 for all 4 parameters (calcospherites, surface irregularity, dentinal tubule openings, and tissue debris), indicating high agreement between the 2 examiners. The teeth treated by the GW system (noninstrumentation) were clean with no presence of organic tissue remnants or biofilm in the root canal. In contrast, a large amount of pulpal tissue remnants and predentin was observed in all three thirds of the root canal from the untreated control group (Fig. 2).

Calcospherites A significantly higher score in calcospherite coverage was observed in the noninstrumentation cleaning group than the control group (P < .001) (Fig. 1A). No signs of instrumentation or erosion were observed (Fig. 3). Prominent calcospherites, characterized by the presence of grooves and depressions, were present as domelike structures on the surface of the root canal wall. Some calcospherites were coalesced as flattened shapes (Fig. 3A and B). Highermagnification images showed a well-defined tubular structure in each single calcospherite (Fig. 3C) and the peritubular mineral network surrounding the tubules (Fig. 3D). More calcospherites were observed in the coronal and middle thirds than in the apical third (Fig. 1A). In the untreated control teeth, calcospherites could only occasionally be observed because of the soft tissue remnants covering the dentin surface (Fig. 1A). No calcospherites were observed in the isthmus areas in teeth with 2 canals (Fig. 3E and G and Fig. 4A and E). Surface Irregularity There was no statistically significant difference in dentin irregularities between the noninstrumentation cleaning group (test group) and the untreated negative controls (P > .05) (Fig. 1B). Amorphous calcified structures were detected in isthmus areas in the test group. Interlaced mineralized fibers were detected in the middle of the root JOE — Volume -, Number -, - 2018

(Fig. 4A–C). Nanosized mineralized crystals were present on the surface of each fiber bundle (Fig. 4D). No signs of organic matter (ie, remnants of pulp tissue or predentin) were observed in the test group. The middle and apical portions of the root had a higher incidence of surface irregularities than the coronal portion (Fig. 1B). The closeup view of the irregularity showed rod-shaped structures (Fig. 4F) embedded in a pool of mineralized crystals (Fig. 4G and H). Pulp stones embedded in the dentin walls were observed in the middle root of 2 samples (Fig. 4E, I, and J). Resorption lacunae were observed on the stone surface (Fig. 4L), and fingerlike dentin projections were present next to the pulp stone (Fig. 4K). The #10 K-file had occasionally produced a narrow, flattened dentin surface in a small area of the canal during the patency check (Fig. 4E).

Dentinal Tubule Openings The test group revealed significantly more areas with over 50% opening tubules (score 3–4) than the negative control group (P < .001) (Fig. 1C). Interestingly, it was noticed that dentin in the isthmus areas had a different, irregular surface structure without dentin canal openings (Fig. 3E and G). A narrow anastomosis was present between 2 main canals in 1 sample in the test group (Fig. 3E). A highmagnification SEM image revealed that the anastomosis was completely clean with no soft tissue remnants (Fig. 3F). The dentin surface in this transition zone showed a clean, irregular structure without dentinal tubule openings (Fig. 3G and H). Tissue Debris All 34 samples in the noninstrumentation test group were clear of any organic or inorganic debris and pulpal remnants (Figs. 1D and 2). A considerable amount of pulp debris was present in the untreated control group. Tissue debris was extensively present in the coronal portion of the root in the control group (Fig. 2). Root Canal Wall Dentin Structure

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Figure 2. A comparison of the root canal wall between the noninstrumentation and control groups. In the control group, small conglomerations were observed in random orientation in the coronal third of the root; large-sized pulp tissue debris blocked the middle third of the root. The root canal wall was covered by mediumsized debris particles and organic tissue in the apical third. In the noninstrumentation group, all parts of the root canal wall were clean without any sign of tissue debris.

Discussion The great structural variability makes the root canal challenging for cleaning. In conventional endodontics, instrumentation creates enough space for the irrigants to be delivered into the anatomic complexity of the root canal system (eg, lateral canals, isthmus area, apical ramification, and transverse anastomoses) (17). These structures may have a significant implication for biomechanical properties of the teeth (12). Most previous studies have examined the root canal surface after instrumentation (11, 12, 16), contributing to an idea that the root canal wall should be smooth and clean with a small portion of untouched areas. Foschi et al (18) showed a smear- and debris-free dentin surface in the coronal and middle thirds after Mtwo (VDW, Munich, Germany) and ProTaper (Dentsply Tulsa Dental Specialities, Tulsa, OK) preparation. Calcospherites were observed in limited uninstrumented areas. Costa et al (11) used a size #15 K-file to remove the pulp tissue and abrasive papers to flatten the root canal dentin surfaces before observing the root dentin morphology using scanning electron microscopy. It has obviously been difficult to accomplish the preservation of the original dentin structure and adequate cleaning of the root canal system at the same time. The GW system has been designed to deliver a broad spectrum of sound waves within the irrigant to effectively clean the root canal system 4

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with minimum or no instrumentation. Molina et al (13) reported that the GW system thoroughly cleans the root canal system even in the apical thirds. The mechanism of the cleaning by the GW system is attributed to the interplay between propagating multisonic energy and fluid dynamics (19). The system disperses fluids from the tip of the handpiece into the pulp chamber. Generally, the cavitation effect consists of countless vacuum microbubbles that explode and release their energy when meeting a target surface (20). The cleaning of surfaces is a result of locally released energy, shear forces, and hydrodynamic cavitation in the fluid (21). Whether this happens in GW cleaning has not been directly shown, but indirect evidence (eg, soft tissue dissolution/disappearance using water only as the liquid with the GW system) has led to the suggestion of the presence of cavitation (6). With the advantage of noninstrumentation cleaning, the present study is the first one to gain insights into the original microstructural nature of root canal dentin in premolars. Untouched root canal walls with calcospherites were observed. The grooves in the calcospherites are dentinal tubules for the odontoblast processes (22). It was interesting to notice that the surface irregularities were mostly located on the mesial and distal root canal walls (Fig. 4D and H). Because of the irregularity of the structures, a high SEM magnification up to 20,000 was used in order to identify whether the surface layers of

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Figure 3. The structure of calcospherites (low magnification: A and C, high magnification: B and D) and the effect of cleaning in the anastomosis (low magnification: E, high magnification: F) and isthmus (low magnification: G, high magnification: H) areas in the GW noninstrumentation group. (A) Calcospherites were dome-shaped structures on the root canal wall. (B) Grooves and depressions were observed in and between different calcospherites. (C) A well-defined tubular structure on a single calcospherite and (D) the peritubular mineral network surrounding the dentinal tubules. (E) An anastomosis was present between the 2 main canals. A completely clean anastomosis area with no tissue remnants. (F) A few flat calcospherite structures can be seen at 1 end of the anastomosis. (G) A clean transition zone from 1 canal on the left to the isthmus in the middle and second canal on the right. A transition zone from calcospherites to a more structureless isthmus zone. (H) A flat, irregular profile without dentinal tubule openings.

these structures were of organic or inorganic nature. The high magnification clearly showed small crystals covering the surfaces completely, confirming the inorganic nature of the surfaces. Interestingly, dentinal JOE — Volume -, Number -, - 2018

tubule openings were not observed on the mesial and distal wall dentin of the main root canals (Fig. 4C, G, and K). Vertical root fracture, which can occur in any tooth, occurs in the buccolingual direction, not

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Figure 4. SEM micrographs of irregular surface structures observed in teeth in the noninstrumentation group after GW cleaning. Images in the same row are from the same sample. Magnification increases from the left to right. (A) The isthmus area in which 2 canals bifurcate in the middle of the root. (B) Networklike interlaced bundles with no dentinal tubule openings. (C) A higher magnification of the interlaced bundles. (D) Nanosized mineralized crystals were covering the bundles, confirming the inorganic nature of their surface. (E) Pulp stones embedded in wall dentin in the middle root. (F) Rod-shaped structures embedded in the canal wall. (G and H) A close-up view reveals that the structures in F are covered with mineral crystals. (I) Several pulp stones in another tooth sample. (J) Higher magnification of a pulp stone shows fingerlike projections above the stone. (K) Higher magnification of the projections next to the pulp stone. No dentinal tubule openings can be seen. (L) Resorption lacunae on the surface of the pulp stone.

mesiodistally, even though in premolars the dentin is usually thicker buccally and lingually than on mesial and distal surfaces (23). Whether the irregular dentin and the lack of dentinal tubule openings on mesial and distal surfaces protects against vertical root fracture in this direction is not known. Further identification and characterization of such crystalline structures deeper into the dentin may require the use of a transmission electron microscope. Other irregular, mineralized structures such as embedded pulp stones were found in the middle and apical thirds of the root canals more commonly than in the coronal third. Irregular dentin is known to be related to age, irritation from previous dental treatment, or trauma, which may stimulate tertiary dentin formation (24, 25). However, in the present study, many teeth were intact, indicating that irregular dentin surface structures are a normal part of human premolar root canal surface anatomy. Traditional instrumentation may pack debris into fins, anastomoses, and untouched canal irregularities by a rotating or reciprocating motion (26). Biofilm and infected tubules may remain unaffected by irrigation if covered with debris (27). Although the clinical evidence is lacking, it is reasonable to assume that the remaining debris may in some cases have an adverse effect on the outcome of the root canal treatment (28). Thus, irrigation and activation of the irrigation solutions are crucial for further improvement of canal cleanliness and disinfection of the root canal system (27). In the present study, although bacterial biofilm would not be expected in samples of the intact, noninfected teeth, the noninstrumentation method used appeared to be efficient enough to remove all predentin and other organic matter

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from the root canals while not creating dentin debris regularly observed after conventional instrumentation. No comparison was made in the present study to other cleaning methods. This is because in conventional chemomechanical cleaning of the root canals much dentin is removed in the main canal. Therefore, it would no longer have been possible to examine intact wall dentin structures. Instead, untreated premolars without any mechanical or chemical cleaning served as negative controls. The control and experimental groups were not the same size. However, both groups were large enough to allow adequate use of proper statistical methods to be used. Moreover, because the root canal wall of the samples was in a semicircle shape, it was not always possible for the electron beam of the scanning electron microscope to reach perpendicular to the side walls to obtain the images of some small canal areas next to the fractured root dentin surfaces. In conclusion, surface irregularities were abundant in different parts of the root canals of premolars cleaned with a noninstrumentation method. More irregular structures were found in the middle and apical thirds than the coronal third of the root canal. No calcospherites were detected in the isthmus areas or narrow anastomoses between the 2 main canals. In addition, no dentinal tubule openings were observed in these areas. Pulp stones and long, vertical irregular calcification, both attached to root canal wall dentin, were present in some teeth. Because even the structurally most irregular areas were completely clean of organic matter (tissue remnants) and dentin debris, the results indicate that it is possible to completely clean root canals without instrumentation.

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Basic Research—Technology Acknowledgments Supported by Sonendo Inc, and start-up funds were provided by the Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada. One of the authors (M.H.) has a commercial interest in 1 of the products used in the present study.

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