Ultrastructural analysis of uninstrumented root canal areas following various irrigation regimens

Abstract Introduction During endodontic treatment smaller or larger areas of root canal wall remain non-instrumented. This can affect prognosis of endodontic treatment as some bacteria may be left behind. The purpose of this study was to evaluate the morphology of non-instrumented areas of the root canal wall using scanning-electron-microscopy (SEM) after completed instrumentation and various irrigation regiments. Materials and Methods Eighteen single-rooted extracted teeth were divided into the six groups. One tooth in each group represented a control sample. In all samples only one half of the canal was instrumented using ISO 40 hand files. Control samples were subjected to an irrigation protocols without instrumentation. Irrigants used were physiological saline, 3% sodium hypochlorite and 15% of ethylene-diamine-tetra-acetate. Irrigation protocol included using each of these irrigants alone, or a combination of NaOCl and EDTA, as well as their combination with final irrigation using NaOCl or chlorhexidine. Then after, roots were sectioned longitudinally and prepared for SEM. Results Saline irrigation left pulpal debris on uninstrumented areas of the canal wall. Irrigation with 3% NaOCl left behind canal wall with different forms of calcospherites. However, after EDTA irrigation dentin appeared as an undulating surface with open tubules without a smear layer. The combination of NaOCl and EDTA showed remnants of calcospherites and open slightly widened dentinal tubules. Final irrigation with NaOCl on the uninstrumented areas showed enlarged dentinal tubules along with dentinal erosion, while after final irrigation with CHX clean dentin and open dentinal tubules without smear layer were noted. Conclusion From the morphological point of view, the most favorable effect of irrigation on both uninstrumented and uninstrumented canal walls was achieved after irrigation with NaOCl and EDTA or NaOCl, EDTA and chlorhexidine as the final irrigant.


INTRODUCTION
One of the basic preconditions for successful endodontic treatment is adequate instrumentation of the root canal. However, satisfactory instrumentation and irrigation is difficult to achieve due to the very specific and complex root canal morphology, as well as limited effect of instruments [1]. Morphological variations of the root canal system and inability of endodontic instruments to reach all parts of root canal wall make cleaning of complete root canal practically impossible [1,2]. Micro-computerized tomography has confirmed that some areas of root canal walls remain untouched after instrumentation [3][4][5][6]. These areas may contain bacteria and compromise endodontic treatment [7]. In addition, the presence of smear layer and debris as a result of instrumentation is significant clinical problem [2,8]. This layer often contains bacteria and blocks dentinal tubules, which significantly decrease the effect of used irrigant affecting the quality of obturation and the outcome of endodontic treatment [8,9].
Due to limited effectiveness of endodontic instruments in root canal cleaning, it is necessary to use appropriate chemical agents during and after instrumentation. Their role is to eliminate and reduce any remaining microorganisms as well as remove smear layer [8][9][10]. Even though there is no general consensus about removing smear layer immediately before obturation, most endodontists agree that if it is not removed, it could disintegrate and lead to microleakage due to the low quality of the bond strength between the sealer and root canal walls [9,10].
The aim of this study was to use a SEM analysis to evaluate the morphology of uninstrumented areas of the root canal walls following mechanical instrumentation and application of various irrigation regimens.

MATERIALS AND METHODS
The material used in this research included 18 freshly extracted intact human maxillary single-rooted teeth without any visible damage (root caries, cracks, internal or external resorption, etc.). According to the irrigation regimens, all teeth were divided into six groups, with one tooth in each group representing a control specimen. The teeth samples were kept for eight hours in 0.5% NaOCl solution to facilitate removal of organic debris. After rinsing teeth under running water, they were immersed in saline solution and refrigerated until the beginning of the experiment.
Prior to canal instrumentation, using a diamond disc, longitudinal grooves were created on the facial and lingual surfaces of the root, without penetrating it, in order to facilitate the fracture. The crowns were amputated and discarded, while the remaining debris was removed using running water. Following pulp extirpation, one tooth from each group (two control samples) underwent different irrigation regimens only without previous instrumentation. All root canals were checked for patency and working length was determined by shortening the distance to the anatomical foramen by 1 mm. The apex was sealed with a pink wax piece.
The root canals of experimental teeth were instrumented using the step back technique to the instrument size 40 (NiTi files I-FLEX, IMD, USA). Only one half of each root canal, either the facial or the lingual half, was particularly marked and instrumented [11]. During the instrumenta-tion, care was taken that endodontic instruments did not come in contact with the oposing side of the canal wall that represented the "uninstrumented" half. The control sample from each group was used for comparison of uninstrumented areas of the canal with the uninstrumented main root canal following identical irrigation regimens. The amount of the irrigant used for each irrigation regimen was identical and carefully controlled, and the total time of chemomechanical preparation was 10 min.

RESULTS
After irrigation with saline pulpal debris covering dentin of uninstrumented areas of the canal was noticed. Irrigation with 3% NaOCl left behind dentin with different forms of calcospherites. When EDTA was used alone for irrigation dentin was present as an undulating surface and open tubules without a smear layer were visible. Combination of NaOCl and EDTA for irrigation left remnants of calcospherites and open and slightly widened dentinal tubules. If NaOCl was used as the final irrigant (after NaOCl and EDTA) enlarged dentinal tubules were noted along with dentinal erosion, while if the final irrigant was CHX, clean dentin and open dentinal tubules without smear layer were noted. SEM findings on uninstrumented and instrumented areas are shown in Table 2 and Figures 1-7. Wall morphology of uninstrumented areas in instrumented root canals did not show any differences compared to the morphology of uninstrumented canals (control samples) following all irrigation regimens.

DISCUSSION
The aim of this study was to analyze the morphology of uninstrumented areas of the root canal walls after canal instrumentation using SEM. Several studies used microcomputerized tomography to determine the presence of uninstrumented surfaces in the main root canal by calculating the area that remains intact after instrumentation (canal volume before and after instrumentation, distance between canal surface before and after instrumentation in μm, the size of a specific area, the width of the canal, taper, etc.) [3][4][5][6]. On the other hand, SEM analysis allows visualization of root canal walls, their cleanliness, dentinal tubules covered with smear layer, as well as complete dentin morphology at ultrastructural level [13][14][15].
In the current study the control samples included uninstrumented canals after performed irrigation regimens. That way it was possible to compare the morphology of uninstrumented canals with uninstrumented surfaces of instrumented canals. According to Peters et al. after biomechanical instrumentation, both hand or rotary, approximately 35% of the canal wall remains untouched by the instruments [16]. In addition, other studies have also confirmed the presence of uninstrumented surfaces, especially in the apical third of the root canal, where any irregularities on canal walls (grooves and depressions) prevent contact between the wall and instrument [17,18]. Endodonic instruments are mostly designed to fit into the conical root configuration, which leaves untreated regions in oval and flat canals [16]. Beside complex canal morphology [19], limitation of instrumentation techniques Ravna površina dentinskog zida sa uklonjenim razmaznim slojem i dentinskim debrisom, otvoreni dentinski kanalići, intertubularni dentin očuvan [20], instrument taper [21] or file alloy properties [22] add to impossibility to instrument all canal walls.
In the current study we assumed that uninstrumented surfaces in the main root canal actually exist, which is why biomechanical instrumentation was performed with the intention of leaving half of the root canal uninstrumented. On the other hand, instrumented areas of the root canal showed surfaces with expected morphology and more-less      . NaOCl + EDTA + NaOCl. a) uninstrumented area -apronounced reduction in calcospherites with a funnel-like widening on dentinal tubules, no organic debris; b) instrumented area -root canal wall with removed debris and smear layer, but with intratubular dentin which has worn away. Dentin erosion in some areas connects two or more orifices of the dentin tubules. Slika 5. NaOCl + EDTA + NaOCl. a) neinstrumentisana površina -izrazita redukcija kalcisferita sa levkasto proširenim dentinskim kanalićima, odsutan organski debris; b) instrumentisana površina -zid kanala korena sa uklonjenim debrisom i razmaznim slojem, ali i sa istanjenim intratubularnim dentinom. Dentinska erozija na nekim mestima spaja dva ili više otvora dentinskih kanalića. clean wall surfaces following certain irrigation regimens as reported in other studies [13][14][15]. In our study we analyzed only the coronal and middle third of the canal, while the apical third was excluded due to its complexity and possible presence of a smear layer even after irrigation that could influence the interpretation of obtained results.
Uninstrumented areas of the canal were difficult to notice prior to irrigation with NaOCl that removed organic debris and exposed conical and wedge-shaped calcospherites. Structures that were found on uninstrumented areas included pulpal tissue remnants, odontoblastic extensions, but no smear layer was found. In the current study 3% NaOCl solution was used and completely removed organic debris. In studies where canals were irrigated with 0.5% NaOCl solution, dentin of uninstrumented areas was not completely cleaned of organic debris [23].
According to the findings of many studies, NaOCl irrigation is exceptionally important because it dissolves organic tissue very efficiently. Even though it has an inadequate surface tension and cannot reach narrow canals, NaOCl can effectively clean uninstrumented areas of the main canal that consist of predentin, necrotic pulpal tissue and a bacterial biofilm [7,24].
Following irrigation regimens IV, V, and VI (NaO Cl+EDTA; NaO Cl+EDTA+NaO Cl; NaO Cl +EDTA+CHX) uninstrumented surfaces showed more or less reduced calcospherites that was also confirmed in other studies. However, some studies have not found calcospherites after the same irrigation regimens [11,18].
In the current study, following irrigation regimen V (NaOCl+EDTA+NaOCl), erosion of intertubular and peritubular dentin occurred on both uninstrumented and instrumented surfaces. Most likely NaOCl was not able to prevent demineralizing effect of EDTA on peritubular and intertubular dentin due to its slow degradation [25]. In addition, there was an interaction between EDTA and NaOCl that manifested in sudden decrease in the amount of free chlorine causing loss of NaOCl activity and inability to dissolve soft tissue within the canal [26]. In our study no organic debris was noted after this irrigation regimen, but many authors do not recommend the use of NaOCl as the final irrigant (after EDTA) due to possible dentinal erosion [25,27].
The literature reports interaction between irrigants that can be manifested as mutual inactivation, coloring of dentin or creation of harmful precipitation [28][29][30]. Therefore, flushing canals with sterile water between each irrigant is recommended, as well as drying the canal prior to introduction of a new irrigant [26,29]. In the current study, these recommendations were followed in order to prevent any unwanted interactions between the irrigants and obtain desired result [11].
Following irrigation regimen VI (NaOCl+EDTA+CHX) no dentinal erosion was noted. According to the literature, when EDTA and CHX come into contact EDTA anion is neutralized with CHX cation and there is no further reduction in dentin [30]. In addition, antimicrobial effect of CHX against Enterococcus faecalis and Candida albicans as well as its substantivity (prolonged effect) support its use as the final irrigant in endodontic treatment [24].

CONCLUSION
Taking into consideration limitations of all in vitro studies, the following can be concluded: The morphology of uninstrumented areas of main root canal is similar to the morphology of those parts of the canal endodontic instruments cannot reach (narrowings, lateral canals, anastomosis, invagination of the root canal, etc.).
The presence of uninstrumented areas in the root canal during endodontic instrumentation is inevitable due to the complex morphology of the canals and indicates the importance of irrigants use during instrumentation.
Even though this was not the primary aim of this study, the most favorable effect of irrigation (including instrumented and uninstrumented areas of the canal) was noted following the irrigation regimen: NaOCl+EDTA, or even better using NaOCl+EDTA+CHX as the final irrigant.