|Year : 2015 | Volume
| Issue : 2 | Page : 114-119
A scanning electron microscopy evaluation of the cleanliness of un-instrumented areas of canal walls after root canal preparation
Abdullah J Dohaithem1, Nick Tovar2, Paulo G Coelho2, Saad Alnazhan3, Sultan Almansouri1, Arwa Bafail4
1 Dental Department, Endodontic Division, King Fahd Armed Forces Hospital, Jeddah, Saudi Arabia
2 Department of Biomaterials and Biomimetics, New York University College of Dentistry, New York, United States
3 Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
4 College of Dentistry, Taibah University, Medina, Saudi Arabia
|Date of Web Publication||20-Apr-2015|
Abdullah J Dohaithem
Endodontics Division, Dental Center, King Fahd Armed Forces Hospital, P. O. Box 9862, Jeddah - 21159
Source of Support: None, Conflict of Interest: None
Introduction: Cleanliness of the canal space is the ultimate goal of its preparation. Nevertheless, some portion of the canal walls are left un-instrumented during preparation. Therefore, the aim of this ex vivo study was to evaluate the cleanliness of un-instrumented canal walls after root canal preparation for the presence or absence of debris and smear layer. Materials and Methods: A total of 24 single-rooted extracted teeth were prepared with the crown-down technique using Protaper universal rotary file system. Micro-computed tomography (micro-CT) was used to scan the specimens before and after instrumentation. The un-instrumented area was measured and localized. The roots were split longitudinally and then subjected to scanning electron microscopy (SEM). The presence of debris and smear layer in the instrumented and un-instrumented areas of the canal were evaluated by analysing the SEM images with a five-score evaluation system based on the reference photographs. Results: High levels of root canal cleanliness (≤ score 2) were found for the instrumented areas were detected (P = 0.003). Conclusions: Under the condition of this study, un-instrumented areas of the canal were less clean in comparison to instrumented portion.
Keywords: Debris, micro-CT, root canal preparation, scanning electron microscopy, smear layer, un-instrumented canal wall
|How to cite this article:|
Dohaithem AJ, Tovar N, Coelho PG, Alnazhan S, Almansouri S, Bafail A. A scanning electron microscopy evaluation of the cleanliness of un-instrumented areas of canal walls after root canal preparation. Saudi Endod J 2015;5:114-9
|How to cite this URL:|
Dohaithem AJ, Tovar N, Coelho PG, Alnazhan S, Almansouri S, Bafail A. A scanning electron microscopy evaluation of the cleanliness of un-instrumented areas of canal walls after root canal preparation. Saudi Endod J [serial online] 2015 [cited 2019 Feb 21];5:114-9. Available from: http://www.saudiendodj.com/text.asp?2015/5/2/114/155449
| Introduction|| |
The ultimate goal of endodontic treatment is to either prevent the development of apical periodontitis or to create conditions that allow the periradicular tissue to heal.  To achieve this goal, the root canal system should be shaped, disinfected, and obturated in three dimensions, followed by the placement of a restoration that seals the coronal access. Root canal instrumentation produces a smear layer that contains both organic and inorganic material and may contain bacteria and its by-products; the formation of this smear layer is almost inevitable during root canal instrumentation.  Whether or not the smear layer should be removed remains controversial. Some authors suggest that maintaining the smear layer blocks the dentine tubules and limits penetration by bacteria and toxins by altering the permeability of the dentine, , while others believe that the smear layer, being a loosely adherent structure, should be completely removed from the surface of the root canal wall because it can harbor bacteria and provide an avenue for leakage leading to the potential failure of treatment. ,,
The introduction of rotary file systems has resulted in significant progress in the mechanical preparation of root canal space. However, several studies has shown that rotary file systems have a limited area of action leaving unprepared areas. ,,,,
During the last decade, high-resolution micro-computed tomography (micro-CT) has emerged as an effective methodology for experimental endodontology; it allows for non-destructive, quantitative analyses of different variables such as volume, surface area, cross-sectional shape, taper, and the proportion of instrumented surface of the canal wall. ,,, Histologic methods also have been used for root canal instrumentation evaluation but it is considered archaic when compared to current micro-CT.  Nevertheless, they give significant data that cannot generally be acquired; thus, they should be considered as an essential complimentary method to be utilized.
Additionally, scanning electron microscopy (SEM) has proved to be a valuable method for the assessment of debris and smear layer. Several investigators evaluated the efficacy of various irrigation protocols and file systems in the elimination of debris and smear layer using this method. ,, However, the un-instrumented areas on the canal walls were assessed based on the surface regularity, abrupt change on the continuity of root canal walls, and partial or total pre-dentine removal.
Thus, the present study was designed to locate un-instrumented areas of the canal walls using micro-CT, subsequently evaluating its cleanliness in comparison with instrumented portion using SEM.
| Materials and methods|| |
Selection and preparation of specimens
Twenty-four freshly extracted single-rooted human teeth were collected. Teeth were externally cleaned with pumice and stored in 0.01% sodium hypochlorite (NaOCl) at 4°C before use. Each root was radiographed in the buccolingual and mesiodistal directions to evaluate the shape of the root canal and to detect any possible obstructions. The inclusion criteria were single-rooted teeth with a straight root canal and an intact pulp chamber. The pulp chambers were accessed conventionally using water-cooled diamond burs in a high-speed hand piece. A size 10 K-file (DENTSPLY Maillefer, Ballaigues, Switzerland) was inserted through the canal and 1 mm beyond the apical foramen to establish apical patency. The working length was set at 1 mm short of the apical foramen, which was sealed from the outside using an impression compound (Kerr, Orange, CA).
Root canal instrumentation
Cleaning and shaping was carried out using the Protaper rotary file system (DENTSPLY, Maillefer, Ballaigues, Switzerland) driven by the X-Smart electric motor (DENTSPLY, Maillefer, Ballaigues, Switzerland) at 300 rpm in a crown-down technique according to manufacturer instructions. Prior to instrumentation, the canal orifices were pre-flared with the SX and S-1 files and the root canal was instrumented with an F2 finishing size file. The pulp chamber was filled with 5.25% NaOCl throughout instrumentation, and 2 mL of 5.25% NaOCl was used to irrigate the canal between each instrument with a syringe and 23-gauge needle. After instrumentation, the canal was irrigated with 10 mL of 5% NaOCl, followed by 5 mL of 17% ethylenediaminetetraacetic acid (EDTA). Each set of instruments was used to prepare three canals before being discarded.
Quantitative micro-CT with three-dimensional surface analysis was performed before and after instrumentation. Root samples were scanned (micro-CT 40, Scanco Medical, Basserdorf, Germany) with a slice resolution of 36 μm. Micro-CT slices were imaged at an X-ray energy level of 70 kVp and a current of 114 μA. Integration time was 200 ms with a total scanning time of approximately 60 min (175 mAs). All data were exported in Digital Imaging and Communications in Medicine (DICOM) format and imported into the Amira software program (Visage Imaging GmbH, Berlin, Germany) for evaluation. Amira software extracted surface area measurements from the gray value distribution of the segmented image for the root canal surface area before instrumentation and after instrumentation [Figure 1]. This allowed for the quantification of the changes in surface area following instrumentation.
|Figure 1: Three-dimensional reconstructed models of the root canal. (a) Red color indicates preoperative surfaces, (b) green color indicates postoperative surfaces, and (c) superimposing (a) and (b) illustrates the amount and location of un-instrumented areas (red spots)|
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Scanning electron microscopy
Crowns were sectioned at the cement enamel junction with a diamond bur. The roots were grooved buccally and lingually without penetrating the canal space with a fissure carbide bur. The roots were then split longitudinally by placing a cement spatula in the grooves and applying pressure. The surface was imaged by SEM (Hitachi 3500, Pleasantown, CA) at various magnifications under an accelerating voltage of 15 kV in secondary electron (SE) mode to observe the surface topography. Samples were sputter coated with gold (EMItech K350, Fall River, MA) for 3 minutes prior to imaging. Representative sections of instrumented and un-instrumented portions of the canal from the apical third were evaluated at 400x and 1000x magnification.
Selection of representative sections
The SEM beam was centered on a predetermined spot based on the micro-CT scan images at 10x magnification. Images of the selected area were then taken at 400x and 1000x magnification [Figure 2].
|Figure 2: Scanning electron micrographs of the apical aspect of root canals. (a, b) Debris and the smear layer are absent in the cleaned portion. Dentin tubules are visible, and the score = 1 for both images. (c) Debris is visible in the un-instrumented portion, and the score = 2. (d) Smear layer is absent, dentin tubules are visible in the un-instrumented area of some samples, and the score = 1|
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SEM image analysis and scoring
The cleanliness of instrumented and un-instrumented portions of the canals was evaluated visually using the 5-step scale method introduced by Hulsmann et al.  Two different and calibrated examiners independently scored each of the images, which were coded and randomly arranged so that the examiners were blinded to the area from which a given sample originated. When both examiners independently agreed on a score, it was recorded. When there was disagreement, both examiners discussed the sample and its scoring, and an agreed upon score was reached. The presence of debris was evaluated from images at 400x magnification using a scale of 5 scores as follows: Score 1 - clean root canal wall and only a few small debris particles, score 2 - a few small agglomerations of debris, score 3 - many agglomerations of debris covering less than 50% of the root canal wall, score 4 - more than 50% of the root canal walls were covered with debris, and score 5 - complete or nearly complete coverage of the root canal wall with debris. The presence of a smear layer was evaluated from images at 1000x magnification using a scale of 5 scores. The scoring system for the smear layer is described in [Table 1]. The results were then dichotomized into "clean canal wall" that included scores 1 and 2 and "smear layer and debris present" that included scores 3, 4, and 5.
|Table 1: Grading score of the smear layer in the scanning electron microscopy evaluation |
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The non-parametric Chi-square test was performed using statistical package for social sciences software 17.0 (SPSS, Chicago, Il) to detect any statistical differences in the presence of debris and smear layer between instrumented and un-instrumented groups. The level of significance was set at P ≤ 0.05.
| Results|| |
The rate of initial independent agreement of both examiners was 85% and 80% for the debris and smear layer scores, respectively. In no case was the difference between the examiners more than one level of scoring. The mean un-instrumented area of the canal surface was 39%; in all specimens, some areas of un-prepared canal walls were detected.
Debris evaluation of the instrumented root canal surfaces resulted in "clean" debris scores of 1 or 2 in 18 out of 24 samples (71%) compared to 7 out of 24 samples (29%) in the un-instrumented areas, (X 2 (1) =8.4, P = 0.003 [Figure 3]).
|Figure 3: Debris and smear layer scores of instrumented and un-instrumented walls of the canal|
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Evaluation of the smear layer on the instrumented root canal surfaces resulted in scores of 1 or 2 in 19 out of 24 samples (79%) compared to 7 out of 24 (29%) in the un-instrumented area, (X 2 (1) =10.2, P = 0.001 [Figure 3]). There was a significant difference between the instrumented and un-instrumented surfaces in terms of the presence or absence of debris and a smear layer.
| Discussion|| |
The canal anatomy plays a pivotal role when assessing the amount of un-instrumented area on the canal surface after preparation. Canals graded as "wide" had significantly larger un-instrumented areas amounting to 43-49% of their total area compared to their "constricted" counterparts. ,,,, In the present study, the mean un-instrumented surface area of the canal wall was 39%. The evaluation of debris and the presence of smear layer require high magnification levels that are achievable only through the use of SEM. Thus, the standard technique for evaluating post-operative root canal cleanliness is by imaging the root canal walls with an SEM.  Numerous studies have investigated the cleanliness of un-instrumented areas of the root canal under SEM, using longitudinal sections of extracted teeth. , However, un-instrumented areas on the canal walls were assessed based on the surface regularity, abrupt change on the continuity of root canal walls and partial or total pre-dentine removal. Moreover, no precondition was given how to select the area of the root canal wall, which was submitted for scoring. It has to be taken into consideration that such selection might be biased, as cleaner portions might be preferred for scoring. In the present study un-instrumented areas was assessed based on micro-CT images. The advantage of this technique is that it allows the creation of three-dimensional replication of the root canal system, additionally enables the evaluation of the extent of un-instrumented canal area accurately.  In addition, the use of micro-CT images gives a better view of the prepared and unprepared canals. 
Two parameters were used for cleanliness assessment: Debris and the smear layer. Reduction of bacteria and the removal/presence of tissue were more clinically relevant but difficult to assess.  Debris is defined as dentin chips, tissue remnants and particles that are loosely attached to the root canal wall. The smear layer is defined by the American Association of Endodontists Glossary of Endodontic Terms  as "a surface film of debris retained on dentin and other surfaces after instrumentation with either rotary instruments or endodontic files; consists of dentine particles, remnants of vital or necrotic pulp tissue, bacterial components and retained irrigant." McComb and Smith  observed a smear layer covering the openings of dentin tubules, so they concluded that this feature was encountered only in the areas of the canal wall that had been contacted by the instruments. In the present study, the smear layer was observed on some areas of the un-instrumented canal wall as well [Figure 4]. This may be explained by the rotation and resulting centrifugal force of the instrument in the canal, which may lead to circumferential scattering of the smear layer particles on the canal surface. Previous studies ,, used the same scoring system as the present study showed that debris scores of 3 to 5 were recorded in the apical thirds of the root canal in 40% to 73% of the samples. Furthermore, it showed that smear layer scores of 3 to 5 were reported for the apical third of the root canal in 48% to 95% of the samples. Though, it was not revealed if the designated area examined was instrumented or un-instrumented. The results of this study indicate that even when teeth were thoroughly instrumented and irrigated, significant amounts of debris and a smear layer remained in the prepared and unprepared root canals, which is in agreement with other studies.  The apical canal region is difficult to clean, which was also shown in the present study. This observation could be attributed to the smaller apical canal diameter obstructing the penetration of root canal irrigants and chelating agents, resulting in limited contact with the root canal wall.  Despite the recent improvements in endodontic instruments and instrumentation techniques, data show that cleaning and shaping of the canal space is not effective in removing all organic and inorganic remnants from the root canal system. ,,, A potential limitation of the present study is the selection of teeth; it is almost impossible to obtain identical shapes of root canals in natural teeth. However, it is essential to use natural teeth in studies such as this. Another important fact that needs to be addressed is that irrigating solution and technique plays an important role in debris and smear layer removal.  The penetration of the irrigants solution to the apical third of the canals depends on the final size of the instrument used.  Mandorah  reported no difference of the cleanliness of the rotary prepared canals if the irrigation needle tip was placed at full or half of the working length. Comparison between the results obtained by different authors and the present study is difficult because of different research protocols. However, the results of the present study support, to some extent, those obtained by several authors. ,,, Finally, it is worth emphasizing that caution should be exercised in the interpretation of the findings and their extrapolation clinically of in-vitro and ex-vivo studies. In conclusion under the conditions of the present study, there was a significant difference in cleanliness between the instrumented and un-instrumented surfaces of the canal wall after root canal preparation, which confirm the need for additional improvements in mechanical and chemical disinfection to compensate for areas that are inaccessible to rotary instrumentation.
|Figure 4: (a) Pulpal debris and (b) a smear layer were visible in some samples of the un-instrumented portion of the apical area (score = 5 for both images)|
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| Conclusions|| |
Under the condition of this study, un-instrumented areas of the canal were less clean in comparison to instrumented portion.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]