Scanning Electron Microscopic Evaluation of Efficacy of 17% Ethylenediaminetetraacetic Acid and Chitosan for Smear Layer Removal with Ultrasonics: An In vitro Study.

INTRODUCTION: The main aim of root canal treatment is cleaning, shaping and then obturating three dimensionally to prevent reinfection. This includes chemicomechanical cleansing by instrumentation and the use of irrigating solutions. Therefore, the purpose of this study was to compare the smear layer removal from root canal dentine subjected to two root canal irrigants, 17% EDTA and 0.2% Chitosan, a new irrigant using Scanning Electron Microscope.
METHODOLOGY: 40 single rooted premolars were decoronated followed by instrumentation with I Race files and intermediate irrigation with 3% sodium hypochlorite and activation with ultrasonics. Then the samples were longitudinally sectioned and place in the respective test solutions and their controls for 5 minutes. Scanning Electron Microscopic evaluation was further carried out.
RESULTS: The results of the present study indicates that the Chitosan which was proved effective in removing smear layer.
CONCLUSION: A moderate concentration of 0.2% chitosan removes the smear layer with greater efficiency.

Introduction

The main aim of root canal treatment is cleaning, shaping, and then obturating three dimensionally to prevent reinfection.[1] Once the dental pulp undergoes pathological changes, the root canal system becomes susceptible to infection caused by several bacterial species, with their toxins and by-products. In the root canal system, microorganisms invade the anatomic irregularities and also invade dentinal tubules and can reinfect the root canals if they are left viable after inadequate root canal treatment.[1] The primary goal in biomechanical preparation is debridement. This includes chemico-mechanical cleansing by instrumentation and the use of irrigating solutions. Irrigation is presently one of the best methods for the removal of tissue remnants and dentin debris during instrumentation. The process of irrigation flushes all the loose, necrotic, contaminated materials before they are inadvertently pushed deeper into the canal and tissues at the apex.[2] Thus, the use of irrigating solution during root canal preparation is essential for successful endodontic treatment.[3] The interaction between physical–chemical properties with mechanical factors intensifies the cleaning of the root canal in teeth with vital and necrotic pulps.[4] Disinfection and shaping of the root canal is interactive. When an irrigating solution is selected for clinical use, compatibility in terms of the physical–chemical properties, antibacterial efficacy, dissolution of the tissue, cleaning effect, chelating action, and tissue tolerance must be considered.[4] Mader et al. in 2001 have reported that only mechanical instrumentation alone leaves around 40%–50% of the root canal walls untouched; therefore, irrigating solutions should be used for superior disinfection during canal preparation.[5] Debridement of root canal walls produces a micropellicle of debris called a smear layer that was first described by that was first described by McComb and Smith (1975).[6] They reported that the smear layer consisted of dentin as in the coronal smear layer and the remnants of odontoblastic processes, pulp tissue, and bacteria.[7] Chemically, it is composed of two phases, organic mainly composed of dentinal collagen residues and glycosaminoglycans from the organic extracellular matrix. For the second, seemingly predominant inorganic phase, it serves as a matrix.[6] Violich et al. (1970) first reported smear layer using scanning electron microscope (SEM).[8] The smear layer was described as “organic matter trapped within translocated inorganic dentin which usually adheres to the root canal walls and also might partially or completely occlude dentinal tubules.”[8] The composition is dependent on the endodontic procedures used, also on the type and sharpness of the cutting instruments, and whether the dentin is dry or wet when cut (Barnes 1974, Gilboe et al. 1984, Cameron 1988).[8] Smear layer is divided into two parts: first, superficial layer and second, the material packed into the dentinal tubules. It has a surface thickness of 1–2 μm and debris was found to be in the tubules to a depth of 40 μm, due to action of burs and instruments.[9]

The question of retaining or removing the smear layer remains controversial. According to some authors, maintaining the smear layer may obstruct the dentinal tubules and limit the penetration of bacteria or bacterial toxin by altering dentinal permeability.[10] Others have a belief that the smear layer should be completely removed from the surface of the root canal wall being a loosely attached structure as it can harbor bacteria and provide an avenue for leakage.[11] It may also affect disinfection of dentinal tubules by preventing irrigants and other intracanal medicaments from penetrating the dentinal tubules. However, a meta-analysis of leakage studies and recent systemic review concluded that the removal of smear layer improves the fluid tight seal of the root canal system.[12] Current methods of smear layer removal include the chemical, ultrasonic, and laser techniques.

Although instrumentation can remove most of the contents in the main root canal area, irrigation plays a very important role in all areas of the root canal system, in particular those parts that are inaccessible for instrumentation.[13] Historically, countless aqueous solutions have been described as root canal irrigants including inert substances such as highly toxic and allergic biocides such as formaldehyde or sodium chloride (saline).[14] Currently, irrigants ideally should have broad antimicrobial spectrum and dissolve necrotic pulp tissue remnants, inactivate endotoxin, prevent the formation of the smear layer during instrumentation, or dissolve after it has formed. Furthermore, as they come in contact with the vital tissues, they should be systemically noncaustic, nontoxic to the periodontal tissues and have little or no potential to cause an anaphylactic reaction.[14] Investigations on a number of chemicals have been done as irrigants to remove the smear layer such as sodium hypochlorite, chelating agents, and organic acids.[1516] Sodium hypochlorite is the most commonly used intracanal irrigant because of its antimicrobial activity and tissue-dissolving ability. It inactivates endotoxins and also disintegrates endodontic biofilms.[17] It is used at a concentration ranging from 0.5% to 6%.[18] It causes periapical inflammation at high concentrations, and at low concentrations, it is ineffective against some specific microorganisms. NaOCl does not impart antimicrobial substantivity and its effect is for short duration.[19]

The use of chelating agents and acids removes the smear layer from the root canal because the components of them are small particles having a large surface-mass ratio, making them highly soluble in acids.[720] Specifically, in case of root dentin, the agent reacts with the ions of calcium in the hydroxyapatite crystals. This process can cause changes in the microstructure of dentin and in the calcium: potassium ratio.[21] The efficiency of these agents depends on the root canal length, the penetration depth, application time, dentin hardness, pH, and concentration of the material.[22] Various chemical agents such as ethylenediaminetetraacetic acid (EDTA), citric acid, MTAD, EGTA, and CDTA have been used for the removal of smear layer. EDTA is a nonspecific divalent cation chelator that has six potential sites for binding metal ions, it forms stable complexes with Ca ions, and demineralizes the root canal surface.[23] It has been reported that EDTA decalcifies dentin to a depth of 20–30 μm in 5 min (Von Fher and Naygard Ostby 1957); however, Fraser (1974) calculated that 0.02 ml of EDTA decalcified only about 0.35 mm2 of dentin.[24] EDTA with different compositions is used in endodontic therapy to facilitate identification of canals, to disinfect and clean the dentinal wall, and to reduce possible microleakage.[23] Moreover, EDTA causes increased dentin permeability, which augments the action of medicaments.[24] Severe peri- and intratubular erosions were seen in root canal dentin after 10 min irrigation with a liquid EDTA chelator.[21]

Chitosan is a natural, glucosamine, and n-acetylglucosamine's cationic aminopolysaccharide copolymer obtained by the alkaline and the partial deacetylation of chitin which is obtained from shells of crustaceans and shrimps. Chitosan possesses high chelating capacity[22] and has properties of biocompatibility, bioadhesion, biodegradability, and antimicrobial activity.[23] In 2012, De-Deus et al.[22] evaluated the effect of chitosan on microhardness of root dentin and revealed no significant differences among 0.2% chitosan, 10% citric acid, and 15% EDTA solutions in the reduction of root dentin microhardness. On the other hand, studies on chitosan’ chelating ability and the smear layer removal are scarce in literatures.

Therefore, this study was designed to compare the efficacy for smear layer removal from root canal dentin subjected to two root canal irrigants, 17% EDTA, and a new irrigant 0.2% chitosan, using SEM.

Methodology

Forty, freshly extracted, single-rooted, human permanent teeth with closed apex were obtained from the department of orthodontics and stored in physiological saline under refrigeration. The teeth were sectioned transversely at the cementoenamel junction using a diamond disc (Diaflex disc; HORICO DENTAL), and the crowns were discarded. The working length of each root canal was established 1 mm short of the apical foramen with #15 K-file after gauging with #10 K-file. The specimens were cleaned and shaped using crown-down technique. The canals were prepared to apical file size of 35/0.04 using iRace rotary files following the standard root canal treatment protocol, irrigating with 1 ml each of 3% NaOCl and saline alternatively between each file with the use of ultrasonics. After cleaning and shaping, each tooth was cleaved longitudinally into two halves using a low-speed diamond disc. Specimens were randomly divided into two groups of twenty halves each for final treatment. They were further divided into two subgroups for the current study. One-half of these specimens in both the groups was subjected to final rinse with either of the two experimental solutions, namely 17% EDTA or chitosan, 5 ml for 5 min with ultrasonic. The contrary halves were taken as control. Root canals were rinsed with 5 ml of distilled water for removal of possible dentin chips and subsequently dried with absorbent paper points.

The surfaces of the sectioned specimen were coated with nail varnish, leaving only the root canal dentin to get exposed to the chelating agent. Each half of a sample was immersed in a beaker with 5 ml aliquots of each of the test solutions for 5 min and its contrary half was immersed in 5 ml of saline for 5 min and maintained under constant agitation using a stirrer. Then, the samples were taken out of the beaker.

The SEM examination for each specimen, one representative area at apical third, was photographed with SEM at ×3000 and ×1000 magnifications for evaluation of presence or absence of smear layer. The degree of evaluation was scored in a blind manner based on a three-grade scale by an examiner who was not informed about the true nature and purpose of this study.

The root canal cleanliness was qualitatively assessed at the apical region of each root half of each specimen using a graded scale from 0 to 2 to assess the quality of smear layer removal according to Kanter et al. (0 – completely opened tubules with complete smear layer removal, 1 – partially opened tubules, and 2 – no open tubules).

Results

The present in vitro SEM study compared the efficacy of two chelating agents, 17% EDTA and 0.2% chitosan, in removal of smear layer from root canal dentin. Observations were noted based on the photomicrographs from the SEM and results were tabulated.

In Group I, 0.2% chitosan, in concern with the smear layer removal at the apical third, irregularly enlarged open dentinal tubules were visible with most of the specimens free of smear layer [Figure 1]. Group II (17% EDTA), with regard to smear layer removal at the apical third, 17% EDTA did not completely remove the superficial smear layer and some of the dentinal tubular orifices were clogged.

Figure 1

Group I – 0.2% Chitosan, irregularly enlarged open dentinal tubules were visible with most of the specimens free of smear layer at ×3000 and ×1000

Intragroup comparison for smear layer removal was done by applying Mann–Whitney U–test, and high statistically significant difference was found between efficacies of smear layer removal by chitosan compared to that of the control group saline (Group I) using ultrasonic irrigation technique under SEM [Table 1]. Similarly, high statistically significant difference was found in the Group II, 17% EDTA compared to saline (Group II) on smear layer removal using ultrasonic irrigation technique under SEM [Table 2]. On intergroup comparison for smear layer removal, statistically significant difference was found between efficacy of 0.2% chitosan (Group I) compared to 17% EDTA (Group II) on smear layer removal using ultrasonic irrigation technique under SEM [Table 3].

Table 1

Statistical analysis indicated that chitosan was more effective than saline (Group I) on smear layer removal using ultrasonic irrigation technique under scanning electron microscope (P <0.001**)

Table 2

Statistical analysis indicated that 17% ethylenediaminetetraacetic acid was more effective than saline (Group II) on smear layer removal using ultrasonic irrigation technique under scanning electron microscope (P <0.001**)

Table 3

Statistically significant difference was found between effectiveness of 0.2% chitosan and 17% ethylenediaminetetraacetic acid on smear layer removal by using ultrasonic irrigation technique under scanning electron microscope (P <0.001**) which indicates that chitosan is superior

Discussion

Microscopic examination of the root canals shows that they are irregular and complex, with lateral canals, many cul-de-sacs and fins. Numerous dentinal tubules open onto the root canal surface additionally. In the root canal system, the microorganisms present invade the anatomic irregularities, dentinal tubules, and if they remain viable can reinfect the root canals after inadequate root canal treatment.[1] Although most of the canal contents in the main root canal area are removed by the instruments, irrigation plays an indispensible role in all areas of the root canal system, particularly in the areas inaccessible for instrumentation.[13]

Whenever dentin is cut using rotary or hand instruments, the mineralized tissues are shattered and not shredded or cleaved to produce considerable quantities of debris. Much of this is made up of very small particles of mineralized matrix of collagen, which is spread over the inner surface to form what is called the smear layer. The electron microprobe with SEM attachment was used for identification of smear layer, which was first reported by Erick et al. and showed that smear layer was made of particles ranging from <0.5 to 15 μm.[8] It has been shown that the smear layer consists of two confluent components; the superficial smeared layer on the surface of canal walls, the thickness of which is estimated to be about 1–2 μm and the smeared material (smear plugs) which is packed into the dentinal tubules. Mader et al. have reported that the penetration depth of the smear plugs into the dentinal tubules varies and may reach up to 40 μm.[9]

To date, no single irrigant has been demonstrated to possess the ability of dissolving organic pulpal material and predentin as well as demineralizing the inorganic calcified portion of the canal wall. Hence, combination of various irrigants has been recommended to accomplish these goals.[16] Chelating agents improve the chemomechanical debridement in root canal treatment by removing the smear layer as well as demineralizing and softening dentin.[22] Chelator preparations could be either liquid type or paste type. Many studies have shown that, though paste-type chelating agents have a lubricating effect, they cannot remove the smear layer effectively when compared to liquid chelator.[8] Due to these reasons, liquid preparations have been considered in this in vitro study. The efficacy of a chelating agent depends on the root canal length, the penetration depth of the material, the application time, dentin hardness, pH, and the concentration of the material.[22]

In the current study, the findings regarding smear layer removal showed that treatment with NaOCl followed by 17% EDTA and 0.2% chitosan produced a debris-free surface, whereas that with NaOCl and saline did not remove smear layer. These results were expected because NaOCl removes only the organic components of the smear layer, while a decalcifying agent removes the greater amount of the smear layer (inorganic), providing clean surfaces. Yamada et al. (1983) demonstrated that 10 ml of EDTA and 10 ml of NaOCl combination is the most efficient volume for smear layer removal. It was said that NaOCl (2.5%–5%) and EDTA (10%–17%) are particularly effective in the removal of organic and inorganic debris without weakening the tooth (Zehnder et al., 2007).[24] The effectiveness of MTAD is enhanced when low concentrations of NaOCl are used to completely remove the smear layer with no significant changes in the structure of dentinal tubules (Torabinejad).

As per the observations made in samples of this study, chitosan worked better at the apical third [Figure 1] than 17% EDTA [Figure 2 and Graph 1]. The tubules in this study were seen open but not widely enlarged which implies that there was no significant change in the structure of dentinal tubules as similarly shown by Saghiri et al. as per similar studies.[21] A neutral EDTA solution has the ability to reduce the noncollagenous proteins (NCPs) component and mineral of dentin as described in recent studies. Because the content of NCPs is less in the apical third, the degree of chelation of EDTA is low in this part.[22]

Figure 2

Group II (17% ethylenediaminetetraacetic acid) did not completely remove the superficial smear layer and some of the dentinal tubular orifices were clogged at ×3000 and ×1000

Graph 1

Efficacy of 0.2% chitosan, 17% ethylenediaminetetraacetic acid, normal saline (Group I), and normal saline (Group II) on smear layer removal using ultrasonic irrigation technique under scanning electron microscope showed that chitosan had significant results compared to ethylenediaminetetraacetic acid and its respective control group

The effects of EDTA and citric acid solutions on the microhardness and roughness of human root canal dentin were recently studied. Significant differences in the microhardness of dentin were found between test groups, and the citric acid group had the lowest microhardness but increased surface roughness. A similar study by Antonio M. Cruz-Filhoon on microhardness showed that EDTA and citric acid had the highest overall effect without a significant difference in decrease in dentin microhardness.[23]

The results of the present study indicate that the chitosan which was proved effective in eradicating Enterococcus faecalis, that is, primarily as an antibacterial, also carries the property of chelation and thus can be said a calcium-depleting endodontic irrigant. Furthermore, chitosan with minimal chelation produced cleaner dentinal walls with minimal erosion of intraradicular dentin and so removed smear layer efficiently. There is uncertainty at this point as to the use of strong or weak decalcifying agents in conjunction with root canal preparation. Strong agents completely remove the smear layer but bear the disadvantage of attacking the dentin and affect its mechanical integrity. Consequently, a moderate rate of decalcifying effect might represent a good choice in case the preservation of dentin is desired.

The goal of the present work was restricted to the direct quantitative comparison of the chelating ability of chitosan with EDTA. The application of these results to the clinical conditions is not straightforward. The limitations of the current method are that the chelator solution was applied to a horizontal dentin surface, which is different from clinical situation, in which the contact is affected by the vertical position of the tooth and the intrinsic anatomic variability of the root canal system. Moreover, a rinsing procedure was not performed which is not in line with the clinical situation.

Future studies can be carried out in providing a better understanding of the mechanism of chelator-induced dentin degradation and its effect on the sealing ability of root fillings and adaptation as well as its possible influence on root strength. Chitosan can be further tested for its effects on dentinal properties such as wettability, microhardness, and roughness.

Conclusion

It can be concluded that a moderate concentration of 0.2% chitosan removes the smear layer with greater efficiency than 17% EDTA at apical third of the root canals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.