Scanning electron microscopic evaluation of smear layer removal at the apical third of root canals using diode laser, endoActivator, and ultrasonics with chitosan: An in vitro study.

AIM: The aim of this study was to compare the effect of diode laser, endoActivator, and passive ultrasonics for smear layer removal at the apical third from root canals with 0.2% chitosan.
MATERIALS AND METHODS: A total of 40 mandibular premolars were decoronated to establish a working length of 12 mm and shaped with ProTaper rotary files up to size F3. In Group A, canals were irrigated with 1 ml of 0.2% chitosan. In Group B, canals were initially irrigated with 0.8 ml of 0.2% chitosan and the remaining 0.2 ml was activated with diode laser. In Group C, canals were irrigated with 1 ml of 0.2% chitosan which was activated with endoActivator. In Group D, canals were irrigated with 0.2% chitosan and activated with passive ultrasonics. All samples were finally flushed with 3 ml of distilled water. The percentage of smear layer removal was analyzed with a scanning electron microscope examination at ×1000 and ×3000. Data were analyzed using Statistical Package for the Social Sciences version 16.0 (SPSS Inc., Chicago, IL, USA) at a significance level of P < 0.05.
RESULTS: The mean value for Group B when compared to Group C for the removal of smear layer was higher, but there was no statistically significant difference between the two groups (P < 0.068 and P < 0.295). Both Group B and Group C showed a statistically significant difference (P < 0.001) when compared to Group A and Group D for the removal of smear layer.
CONCLUSION: Diode laser and endoActivator with 0.2% chitosan proved better in the removal of the smear layer when compared to passive ultrasonic irrigation.

INTRODUCTION

Primary endodontic infections are caused by necrotic pulp tissue colonized by microorganisms.[1] The ultimate goal of endodontic treatment is to control microbial factor in complex root canal anatomy, especially in the apical third. The smear layer is potentially infected, and its removal allows more efficient penetration of intracanal medications into dentinal tubules and a better interface between the filling material and root canal walls.[2] Success of endodontic treatment depends on complete disinfection and debridement of the root canal. Instrumentation alone cannot achieve total elimination of bacteria and debris in all canals due to which effective irrigation is mandatory.[3]

Activation of irrigants proved to enhance the efficacy of root canal irrigants, not only within the root canal but also in anatomical complexities of the root canal system and dentinal tubules.[4] As per various studies, sonics, ultrasonics, and lasers are widely researched as irrigant activation methods.[56]

Chitosan is recently introduced material with excellent properties such as biocompatibility, biodegradability, and antimicrobial activity.[7] The chelating behavior of chitosan favored smear layer removal as it acted on an inorganic portion of smear layer.[8] Even in a low concentration such as 0.2% chitosan solution effectively removed the smear layer from the root canal.[7] The properties of chitosan as a root canal irrigating solution activated by different irrigant activation systems is yet to be investigated.

Passive ultrasonic irrigation (PUI) utilizes ultrasonic wave energy that is transmitted from a tip or file to the irrigant.[5] EndoActivator uses sonic activation of irrigants, and its activator tip produces clouds of debris within a pulp chamber by vigorous intracanal fluid agitation. Cavitation and acoustic streaming significantly improve the disruption of the smear layer and biofilm.[9] One single positive and negative node along the polymer-based tips is observed in sonic and subsonic activation, while multiple positive and negative nodes along the length of a metal instrument are observed in ultrasonics. Greater extrusion of debris is due to the high frequency generated by ultrasound. The efficiency of a sonic protocol is achieved by moving tip up and down in short vertical strokes along with vibration that synergistically produces a hydrodynamic phenomenon.[10]

Diode laser has shown promising results in smear layer removal and root canal disinfection in endodontics to achieve reduced permeability and microleakage.[11] The diode laser is recommended for endodontic treatment because its wavelength is within the infrared range where thin and flexible fibers can be used. Previous reports have demonstrated the bactericidal effects of 810-nm wavelength and 980-nm wavelength diode lasers.[12]

The need of this in vitro study is to evaluate the efficacy of diode laser, endoActivator, and passive ultrasonics with 0.2% chitosan in removing the smear layer at the apical third. The null hypothesis was that there is no significant difference in the efficacy of various irrigation activation systems using 0.2% chitosan in removing the smear layer at the apical third.

MATERIALS AND METHODS

Forty adult human noncarious, single-rooted mandibular premolars extracted for orthodontic purposes or for being periodontally compromised, with straight single canal and mature apices were selected. Teeth with curved roots (more than 5° curvature), cracks, or fracture lines were excluded. Sample size was calculated assuming the highest mean difference of 1.5 between any of the four groups using formula, n = 2Z12S2/d2

Where

n = minimum sample size

Z = value associated with alpha and beta

S = standard deviation of mean test intervention and mean control intervention

d = absolute precision.

Sample preparation

The teeth were stored in 10% formalin solution until use. The root surfaces were cleaned and then decoronated using a diamond disc under water irrigation to obtain a standardized root length of 12 mm. Root canal instrumentation was initiated with ISO hand K-files up to #20, followed by ProTaper rotary files up to size F3. About 2 ml of 3% NaOCl was used as an irrigant after every successive instrument. The irrigants were delivered with a disposable syringe of 27G needle placed 1 mm short of the working length. After cleaning and shaping, samples were randomly divided according to the activation system into four different groups of ten specimens each using simple lottery method of randomization.

Group A (Control group) – Canals irrigated with 1 ml of 0.2% chitosan (Chitorigant, Everest Biotech, Bengaluru) using syringe irrigation

Group B (Diode Laser) – Canals were initially irrigated with 0.8 ml of 0.2% chitosan and the remaining 0.2 ml was activated with diode laser (Biolase Epic X, Irvine, California, USA) for 20 s cycle

Group C (EndoActivator) – Canals were irrigated with 1 ml of 0.2% chitosan with endoActivator (Dentsply Sirona, New York, USA) for a cycle of 30 s

Group D (PUI) – Canals were irrigated with 0.2% chitosan with passive ultrasonic activation (Sybron Endo-North Ryde, Australia) with 25 no. ultrasonic tip for a cycle of 30 s.

The root canals were finally irrigated with 3 ml of distilled water to terminate the action of irrigating solutions, dried, and prepared for scanning electron microscope (SEM) analysis.

Scanning electron microscopic analysis

The roots were grooved along the buccal and lingual planes using a diamond disc at low speed. The roots were then split longitudinally with chisel and mallet. One-half of each specimen sample was selected and prepared for SEM (Leica Stereoscan 440i SEM) analysis. The selected samples were progressively dehydrated using graded concentrations of aqueous ethanol (90%) for 24 h at each concentration. After dehydration, samples were placed in a vacuum chamber and sputter coated with a 30-nm gold layer.

The dentinal wall of root canals was examined at the apical thirds under a magnification of ×1000 and ×3000. Photomicrographs of the root canals were taken at the apical level for scoring in a calibrated single-blind manner, and SEM images were examined by three specialists with respect to the amount of smear layer remaining on the dentine walls, according to the rating system developed by Gutmann et al.[13]

Score 1 – little or no smear layer; covering <25% of the specimen; most tubules were visible and patent or almost complete laser melting. Score 2 – little to moderate or patchy mounts of smear layer; covering 25%–50% of the specimen; many tubules visible and patent or laser melting. Score 3 – moderate amounts of scattered of aggregated smear layer; covering 50%–75% of the specimen; minimal to no tubule visibility or patency or scattered laser melting.

Score 4 – heavy smear layer; covering >75% of the specimen; no tubule orifices were visible or patent or no visible laser melting.

RESULTS

The present in vitro SEM study compared the efficacy of three irrigation activation systems, diode laser, endoActivator, and ultrasonics using 0.2% chitosan, in the removal of smear layer at the apical third of root canal. Observations were noted based on the photomicrographs from SEM and the results were tabulated. According to Gutmann rating system, mean score of 3.8 was seen in Group A [Figures 1 and 2a] whereas Group B showed a mean score of 1.2 [Figures 1 and 2b], Group C showed a mean score of 1.7 [Figures 1 and 2c], and Group D showed a mean score of 2.8 [Figures 1 and 2d].

Figure 1

Scanning electron microscope images of remaining smear layer scores at apical level: (a) control group, (b) diode laser, (c) endoActivator, and (d) passive ultrasonic irrigation visualized at ×1000

Figure 2

Scanning electron microscope images of remaining smear layer scores at apical level: (a) control group, (b) diode laser, (c) endoActivator, and (d) passive ultrasonic irrigation visualized at ×3000

At the apical third level, ×1000 and ×3000 magnifications: Group B (mean value 1.2) followed by Group C (mean value 1.7) had the least smear layer scores with no statistically significant difference between them (P = 0.068). This was followed by Group D (mean value 2.8) and Group A (mean value 3.8) with a significant difference between Group A and all other groups, respectively (P < 0.001) [Table 1 and Graph 1].

Table 1

Comparison of remaining smear layer scores at apical level among Group A, Group B, Group C, and Group D visualized at ×1000 and ×3000 under standard error of mean using ANOVA F-test

Groups	×1000				×3000
	Mean	SD	ANOVA F-test	P, significance	Mean	SD	ANOVA F-test	P, significance
Control (Group A)	3.8	0.42	70.391	<0.001	3.6	0.51	41.867	<0.001
Diode laser (Group B)	1.2	0.42			1.3	0.48
EndoActivator (Group C)	1.7	0.48			1.7	0.48
Ultrasonics (Group D)	2.8	0.42			2.6	0.51

P<0.05: Statistically significant, P<0.001: Highly significant. SD: Standard deviation

Graph 1

Representation of smear layer removal scores at × 1000

Intergroup comparison for smear layer removal was done by Tukey's post hoc test, and highly statistically significant differences were found between efficacies of smear layer removal. Mean interexaminer kappa reliability was found to be moderate; k = 0.657 at ×1000, while k = 0.730 at ×3000. Diode laser was proven to be better than endoActivator but statistically not significant [Table 2 and Graph 2].

Table 2

Intergroup comparison of remaining smear layer scores at the apical level among Group A (control group), Group B (diode laser), Group C (endoActivator), and Group D (ultrasonics) visualized at ×1000 and ×3000 under standard error of mean using Tukey’s post hoc test

Comparison groups	×1000		×3000
	Mean difference	P, significance	Mean difference	P, significance
Control versus diode laser	2.6	<0.001	2.3	<0.001
Control versus endoActivator	2.1	<0.001	1.9	<0.001
Control versus ultrasonics	1.0	<0.001	1.0	<0.001
Diode laser versus endoActivator	0.5	0.068	0.4	0.295
Diode laser versus ultrasonics	1.6	<0.001	1.3	<0.001
EndoActivator versus ultrasonics	1.1	<0.001	0.9	0.002

P>0.05: Not significant, P<0.05: Statistically significant, P<0.001: Highly significant

Graph 2

Intergroup comparison of control, diode laser, endoActivator, and passive ultrasonic irrigation at ×3000

DISCUSSION

The goal of root canal instrumentation is to achieve optimal canal sanitization based on the combined use of mechanical instrumentation and two or several irrigating solutions and obtain a continuous taper. To improve the speed, efficiency, and predictability of the treatment; recently, mechanized or automated systems are preferred. Ni-Ti instrument's super elasticity allows less transportation and a decreased incidence of canal aberrations with more centered canal preparations.[14] In a landmark article, specifically devoted to root canal irrigation, Louis Grossman stated: “mechanical instrumentation should be followed by irrigation of canal in order to wash out fragments of pulp tissue and dentinal shavings.”[10]

Whenever dentin is cut, the mineralized tissues are shattered and not shredded or cleaved to produce considerable quantities of debris. This is made up of collagen, spread over the inner surface to form smear layer. The electron microprobe with SEM attachment was used for the identification of smear layer, and it was made of particles ranging from <0.5 to 15 μm. Penetration depth of the smear plugs into dentinal tubules may reach up to 40 μm.[15] The presence of smear layer was evaluated at ×1000 and opening of dentinal tubules was evaluated at ×3000.[16]

Syringes and needles resulted in ineffective irrigation, particularly in anastomosis between canals, fins, and the most apical part with its high percentage of ramifications leading to recurrent infection.[10] The results obtained in this study showed that conventional irrigation using saline with needles or syringes performed the least in removing debris and smear layer as saline is neither an organic tissue solvent nor does it have a smear layer removing efficacy, it can only flush out loose debris from the root canal system.

The use of sonics, lasers, PUI devices, and techniques has been reported to improve disinfection of the root canal.[17] In the current study, findings regarding smear layer removal showed that treatment with diode laser, followed by endoActivator and ultrasonics produced a debris-free surface, whereas syringe irrigation did not remove the smear layer. Diode laser brought about superior dentinal tubule disinfection compared to conventional and ultrasonically activated irrigation .[18]

Diode laser is a solid-state semiconductor laser that is designed to emit light over numerous wavelengths in continuous or pulsed modes. In endodontics, wavelengths between the 800 and 980 nm range are used, which are poorly absorbed by water[19] and provide access to formerly unreachable parts of the tubular network, as it has better depth of penetration (500 μm) when compared to irrigating solutions (100 μm). Root canal irradiation by diode laser results in opening of dentinal tubule orifices by vaporization of smear layer.[20] The optic fibers used in diode laser have fine diameters (200–320 μm) which enable effective delivery of laser light at the apical third of the root canal.[21] It balances between absorption and penetration to bring energy below the dentinal surface without tending to damage it.[22]

Sonic and ultrasonic systems improved smear layer removal only in a straight coronal portion of a curved root canal.[23] In the present study, there was no statistically significant difference between diode laser and endoActivator [Graphs 1 and 2], but diode laser resulted in complete laser melting and thus enhanced removal of smear layer than endoActivator.

EndoActivator uses sonic energy and has effectively removed smear layer from the apical third of root canals after instrumentation. On placing irrigant into canal and chamber, passively fitting tips are activated at 10,000 cycles/min for 30–60 s.[23] Cleaner canals were obtained as higher frequency resulted in higher flow velocity which aided in the killing of bacteria and debridement of necrotic tissue and debris more efficiently than PUI and control group.[10]

PUI uses an ultrasound-activated instrument and induces acoustic microstreaming into the root canal filled with irrigant around the tip.[6] Geurisoli DM mentioned in his study that researchers found ultrasonics to increase the bactericidal action.[24] Ultrasonic irrigation is capable of removing smear layer more effectively than syringe irrigation.[25]

Partial deacetylation of chitin produces chitosan which comprises glucosamine and n-acetylglucosamine. It is antimicrobial, biocompatible, biodegradable, and bioadhesive with no reported toxicity. It acts by suppressing the growth of bacteria, impairing exchanges with a medium, chelating transition metal ions, and inhibiting enzymes as these eliminate dentin calcium ions.[1] Its efficacy depends on dentin hardness, pH, concentration of the material, root canal length, penetration depth of material, and application time. 0.2% chitosan produced cleaner dentinal walls and removed smear layer efficiently with minimal chelation and minimal erosion of intraradicular dentin when compared to routinely used chelating agents such as 15%–17% ethylenediaminetetraacetic acid and 10% citric acid.[15]

CONCLUSION

Within the limitations of the current study, all tested groups were able to remove the smear layer from prepared root canals to different degrees except the control group. Smear layer was removed more efficiently by the activation of irrigant and with machine-assisted irrigation devices. Diode laser has increased the success rate of endodontic therapy due to its ability to remove smear layer and root canal microbes. This study clearly shows the advantages of laser treatments over currently used conventional irrigation methods and techniques while using 0.2% chitosan as an irrigating solution.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.