Smear layer removal efficacy of combination of herbal extracts in two different ratios either alone or supplemented with sonic agitation: An in vitro scanning electron microscope study.

OBJECTIVES: The study aimed to evaluate the effectiveness of the combination of two natural extracts in varying ratios for removal of smear layer either alone or supplemented with sonic agitation.
MATERIALS AND METHODS: Fifty extracted single-rooted teeth were collected, disinfected and decoronated below the cementoenamel junction to obtain standardized root length of 10 mm. Root canals were instrumented using rotary files at working length 1 mm short of the apex. Specimens were divided into six groups according to the irrigation protocol as follows: Group A - Distilled water, Group B - 17% ethylenediaminetetraacetic acid, Group C - Herbal extracts in 1:1 ratio, Group D - Herbal extracts in 1:1 ratio supplemented with sonic agitation, Group E - Herbal extracts in 2:1 ratio, Group F - Herbal extracts in 2:1 ratio supplemented with sonic agitation. Specimens were longitudinally sectioned and evaluated under scanning electron microscope for smear layer removal efficacy. Obtained scores were statistically analyzed using one-way analysis of variance and post-hoc test.
RESULTS: Among all, Group B showed the best results followed by Group F. Remaining other groups showed inferior outcome (P < 0.05).
CONCLUSION: The combination of two extracts in 2:1 ratio was slightly better than 1:1 ratio and the smear layer removal efficacy was further improved when accompanied with sonic agitation.

INTRODUCTION

Root canal therapy is aimed to completely eradicate microbes and three dimensionally seal the endodontic system. Chemo-mechanical debridement of the root canal system is the most accepted method to achieve complete disinfection.[1]

Mechanical preparation inadvertently forms an amorphous layer termed as “smear layer” over prepared dentinal walls that may hold the microorganisms as well. Thus, elimination of smear layer from the root canal walls is highly desirable to obtain long-term endodontic success.[2]

Chelating agents such as 17% ethylenediaminetetraacetic acid (EDTA),[3]10% citric acid,[4] tannin, and maleic acid[5] are used for smear layer removal. Literature also supports the use of 17% EDTA solution followed by sodium hypochlorite (NaOCl) as final flush.[6] Citric acid is a weak organic acid, belonging to the chelating agent's category has been claimed to be as efficacious as EDTA in the removal of smear layer.[78]

Efficacy of irrigating solutions can be effectively increased by incorporation of surfactant as it reduces the surface tension, thus, increasing the wettability of the prepared surface. High wettability also enables the irrigating solution for the deeper penetration into the dentinal tubules. Furthermore, studies have shown that irrigating solutions with detergents eradicate bacteria in direct contact faster than agents without detergent. It may be related to weakening of cohesive forces in extracellular polymeric substances and bacterial membranes.[3]

Synthetic chemical endodontic irrigating solutions with their huge list of adverse effects have led the researchers to explore various natural substitutes for the same.[9] Extracts of Azadirachta indica, Ocimum sanctum, Syzygium aromaticum, Cinnamomum zeylanicum and Citrus aurantifolia have been explored as a possible endodontic irrigating solution.[101112] C. aurantifolia contains 88% water, 6–8% citric acid, 2% potassium citrate, and other substances. Owing to the citric acid as a basic constituent in C. aurantifolia, researchers have evaluated it for its possible role in the endodontic smear layer removal; though, the results were disagreeable.[1213] Furthermore, the role of natural fruit juices including C. aurantifolia on the exposed root dentin surfaces in the elicitation of dentinal hypersensitivity has been confirmed and is linked with the removal of existing smear layer on the exposed dentin.[14]

Sapindus mukorossi (Sapindaceae), commonly known as “ritha,” “aritha” or “soapnut” is found throughout India. The major constituents of its fruit's pericarp are saponins (10–11.5%), sugars (10%), and mucilage. Higher content of saponins results in greater surfactant action. Hence, the extract of soapnut is utilized to clean the skin of oily secretion and is even used as a cleanser for washing hair and as a hair tonic. The powdered seeds have also been employed in the treatment of dental caries, arthritis, common cold, constipation, and nausea.[15]

Agitation techniques improve the cleaning efficacy of irrigating solutions by enhancing the irrigation dynamics. This results in the maximum elimination of dentin debris, remnant pulp tissues and biofilms from the root canal wall. Studies have concluded that there is a minimal extrusion of the irrigating solution beyond apex when using sonic agitation as compared to other agitation techniques.[16] Furthermore, the efficacy of smear layer removal was better as compared to manual agitation.[17]

In the light of above context, we hypothesized that the combination of C. aurantifolia and Sapindus mukorrossi extracts accompanied with sonic agitation during final wash should result in better smear layer removal. Hence, the aim of this study is to test the unique combination of two herbal extracts in two different ratios for the elimination of endodontic smear layer either alone or supplemented with sonic agitation.

MATERIALS AND METHODS

Fifty intact single rooted human permanent teeth having a single canal and fully developed apices, indicated for extraction due to orthodontic/periodontal reasons were selected for the study. The teeth were cleaned using ultrasonic scaler followed by sterilization under autoclave as suggested in previous studies.[1819] Thereafter, teeth were stored in distilled water until use. The teeth were decoronated below the cementoenamel junction using the diamond disc to obtain standardized root length of 10 mm. Working length was measured 1 mm short of the apical foramen and glide path was established using size 15 K file (Mani Inc., Japan). All the samples were randomly divided into six groups out of which Group A (negative control) and Group B (positive control) consisted of 5 samples each and remaining 4 experimental groups had 10 samples, respectively [Table 1].

Table 1

Benefits and risks in a clinical trial

Root canals were prepared till apical size 35, 0.06 taper using K3XF™ nickel titanium files (SybronEndo Corp., Orange, USA) in Crown down manner. During instrumentation, each root canal was irrigated using 2 ml of test solution corresponding to its group. The apical foramen of each root was coated with cyanoacrylate glue before embedding the roots into a polyvinyl-siloxane impression material filled transparent tube to obtain the closed system. The extracts of C. aurantifolia and S. mukorrossi were procured directly from the manufacturer (Navchetana Kendra, New Delhi, India) and the aqueous solution of the combination was prepared in different ratios in the laboratory. The 1:1 ratio solution of the combination was prepared in a dilution of 0.1 g/ml of C. aurantifolia and 0.1 g/ml of S. mukorrossi extract dissolved in distilled water. Similarly the 2:1 ratio of the combination was prepared in a dilution of 0.12 g/ml of C. aurantifolia and 0.06 g/ml of S. Mukorossi extract dissolved in distilled water. The prepared solutions were filtered using Whatman filter paper and the final irrigating solution obtained.

For all the groups, the final rinse irrigation was done according to group protocol [Table 1]. Each specimen was irrigated using 3 ml of corresponding irrigating solution using 30-G side vent needles (Max-I-Probe™, Dentsply, New Delhi, India) with the flow rate of approximately 5 ml/min. Irrigation was supplemented with sonic agitation using EndoActivator® (Dentsply Tulsa Dental Specialities, Tulsa, USA) in Group D and Group F. For all the groups, root canal of specimens was finally rinsed with sterile distilled water and dried using sterile absorbent paper points.

Two longitudinal grooves were prepared on the buccal and lingual surfaces of each root using a diamond disc, avoiding penetration into the canal. The roots were split into two halves with a chisel and coded according to groups. The coded specimens were then mounted on metallic stubs with carbon strip backing and subsequently examined independently by two observers under scanning electron microscope (SEM). After general evaluation of the canal wall, three SEM photomicrographs were taken at magnification of ×1000 for evaluation of smear layer at the center of the coronal, middle, and apical thirds of each specimen [Figure 1]. Cleanliness was evaluated using 5-point scoring system by Hülsmann et al.[7] [Table 2] and the results were tabulated.

Figure 1

Figure shows representative scanning electron microscope images (×1000) of different groups (A-F). The alphabet and numeric value in the figure label depict the group, and area of the root canal imaged, respectively as follows: 1 – Coronal third, 2 – Middle third, 3 – Apical third

Table 2

Benefits and risks in a clinical trial

The collected data were subjected to one-way analysis of variance to compare the means of the groups. The post-hoc test (Tukey) was performed to find the interrelationship between different groups of significant difference (P < 0.05; confidence interval 95%).

RESULTS

Group B showed the best outcome and showed statistically significant difference from other groups except Group F at all three levels that were comparable to Group B (P < 0.05). There was statistically insignificant difference in the smear layer removal efficacy among the Group C and Group E. However, Group F differed significantly from Group D at middle and apical third sections of the root (P < 0.001) [Figures 2–4].

Figure 2

Smear layer score in coronal third (P < 0.05)

Figure 4

Smear layer score in apical third (P < 0.05)

Smear layer score in middle third (P < 0.05)

DISCUSSION

C. aurantifolia has been previously tested for smear layer removal capabilities and found unsatisfactory.[12] The negative outcome may be related to the poor wettability of the solution. The present study employed C. aurantifolia and S. mukorrossi in two different ratios to determine the effect of surfactant in the removal of smear layer. The results portrayed 2:1 concentration to be more effective. Reduced effect of 1:1 solution may be related to the reduced effective concentration of the citric acid in the prepared irrigant. Other different proportions of the tested extract combinations with further reduced concentration of S. mukorossi may be evaluated to determine the optimal proportion.

Emulsification activity of the bio-surfactants like S. mukorrossi is one of the important properties that dissolve the water-insoluble substances/hydrocarbons.[20] This property may be primarily responsible for the better smear layer removal effect when used in combination with lime extract.

The present experiment utilized an aqueous solution of the extract combination; however, the effect of ethanolic extract solutions may be further evaluated to confirm the smear layer removal capabilities of the tested solution.

The root canal system behaves as a closed end channel, being apically surrounded by bone socket. Therefore, our study also utilized the closed apical model to mimic the clinical situation. Sonic agitation was opted over ultrasonic method in this study as EndoActivator consist of polymer based tips that do not damage the prepared dentinal walls also, the higher frequency generated by ultrasound can result in the greater extrusion of debris. In this trial, the sonic agitation performed better in Group F (2:1 concentration); however, 1:1 concentration accompanied with sonic agitation, that is, Group D failed to do so. Hence, the results partially coincide with the previous research,[19] but the primary factor responsible for the better outcome remains the concentration of the combination extract solution.

The optimal contact time of the irrigant is essential to obtain adequate canal cleaning effect. Although, the closed apical model provided good contact time for the irrigating solution, the results of the study were not as good as for 17% EDTA group except the Group F, which showed comparable results. This may be related to the presence of reduced available concentration of citric acid in the tested solutions. Five minutes contact time for all irrigants was decided in this trial considering the clinical scenario.

SEM images of all the groups showed the presence of debris over the prepared dentin surfaces that may probably be linked to the residues of other organic components of the solution and the nonuse of any debris dissolving solution like NaOCl.

Complete apical penetration and constant replenishment of irrigating solution are essential to achieve thorough cleaning and may be achieved using apical negative pressure irrigation systems. Further researches may be designed to explore the effect of such devices in improving the efficacy of the tested solution. Also the effect of other adjuncts such as canal brush[21] and self-adjusting file system[22] may be evaluated.

Although, the therapeutic use of both the extracts owing to its various medicinal values is well-established, the chemical analysis of the combination of the extracts needs to be established to confirm the biocompatibility issues.

CONCLUSION

Within the limitations of the present in vitro research, it can be concluded that the experimental solution showed mixed results. The 2:1 ratio was slightly superior to 1:1 and sonic activation improved the effectiveness of the solution. Further researches with the different combination ratios, contact time and extract type may be designed to validate the research outcome.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.