Effect of laser-activated irrigation on smear layer removal and sealer penetration: An in vitro study.

INTRODUCTION: The present study was carried out with the objective of evaluation of efficacy of two root canal irrigants (LARGAL ULTRA and Biopure mixture of tetracycline, acid, and detergent [MTAD]) in smear layer removal and sealer penetration along the canal walls and to apprise any change in their efficacy when they were activated with two different types of lasers (neodymium-doped yttrium aluminum garnet [Nd:YAG] and erbium-doped yttrium aluminum garnet [Er:YAG]). The analysis was done with the help of confocal laser scanning microscope.
MATERIALS AND METHODS: The curved root canals (>20°) from 140 extracted human molars (negative control, n = 20) were prepared to size #25.06 with NaOCl irrigation. Teeth were divided into two experimental groups with sample size 60 each (Group I, II). Groups further divided into subgroups IA, IB, and IC and IIA, IIB, and IIC with sample size twenty each. Group IA, Largal Ultra irrigation without LAI; Group IB, Largal Ultra irrigation with Nd:YAG; Group IC, Largal ultra with Er:YAG; Group IIA, MTAD irrigation without LAI; Group IIB, MTAD irrigation with Nd:YAG; Group IIC, MTAD irrigation with Er:YAG. Teeth were obturated with gutta-percha and fluorescent dye labeled sealer. Transverse sections at 3 mm from root apex were examined with confocal laser scanning microscopy, and the percentage of sealer penetration into dentinal tubules was measured. STATISTICAL ANALYSIS: Analysis of variance calculated (e.g., one-way between-subjects ANOVA) and Tukey's test was performed for each mean comparison.
RESULTS: Difference between control and Group IIc (65.48 ± 0.57) was highest, followed by control and Group Ic (54.92 ± 0.57) and minimum between Group Ib and Group IIa (0.23 ± 0.57). Order of percent diffusion in the present study was: Group IIc > Group Ic > Group IIb > Group Ib ≈ Group IIa > Group Ia > Control (P <.001).
CONCLUSIONS: It was inferred that, among the two irrigating solutions tested, Biopure MTAD is more proficient than Largal Ultra. Laser activation of final irrigating solution has been shown to allow better sealer penetration, with Er:YAG laser being more potent than Nd:YAG laser. Copyright:

INTRODUCTION

While performing chemomechanical preparation of root canal, a layer containing predentin, mineralized dentin, pulp residues, and bacteria is formed, which is called as smear layer.[1] Smear layer removal has been recommended because it is assumed to weaken the effect of disinfecting agents and degrade the quality of the root canal filling by impeding the penetration of endodontic sealers into the dentinal tubules.[2] Various modalities have been used to remove smear layer including chemical agents, ultrasonics, and laser irradiation.[34]

Two novel root canal irrigants recently introduced into the market are Largal Ultra and Biopure Mixture of Tetracycline, Acid and Detergent (MTAD). Biopure MTAD is a mixture of 3% doxycycline hyclate (Tetracycline isomer), 4.25% citric acid, and 0.5% Tween-80 (detergent). It has been introduced as a final irrigant, for the simultaneous elimination of inorganic smear layer and disinfection of root canal system. Largal Ultra (Septodont, Paris, France) contains 15% EDTA solution as a disodium salt, O.75% of Cetyl-tri-methyl ammonium bromide (Cetrimide). This cetrimide was added to reduce the surface tension of the irrigant facilitating the better penetration of chelator into the root canal dentin. Syringe irrigation is a standard procedure followed but unfortunately, it is not effective in the apical part of the root canal.[5] Laser-activated irrigation (LAI) has been introduced as a powerful method for root canal irrigation. The laser irradiation produces transient cavitation in the liquid by strong absorption of the laser energy.[6] The null hypothesis tested in the present study was that the smear layer removal and percentage of sealer penetration into dentinal tubules along the canal walls is not affected by the use of different final irrigating solutions and by neodymium-doped yttrium aluminum garnet (Nd:YAG)) and erbium-doped yttrium aluminum garnet (Er:YAG) laser activation at microscopic levels.

MATERIALS AND METHODS

Sample collection

One hundred and forty freshly extracted human molars that had mesiobuccal roots (maxillary or mandibular molars) or distobuccal roots (maxillary molars) with >20° of curvature with fully formed apices were used in the current study. The teeth were thoroughly washed under running tap water to remove blood and saliva. They were then rendered free of calculus and other soft tissue debris using hand scalers. The samples were stored in aqueous solution containing 0.001% thymol at room temperature for no longer than 6 months. This was done to avoid samples from being dehydrated and becoming brittle.

Specimen preparation

The access cavities were made and pulp chamber opened using Endo-Access bur under water spray. Canals were located and straight line access to root canal was gained and confirmed using #10 K-files (Dentsply Maillefer, Zurich, Switzerland). Working length was determined using #10 K-file inserted into the canal until it was visible at the apical foramen. The working length was established 1 mm short of this length. Reference cusp height was adjusted so that the working length of each tooth sample was 19 mm. Root canals were prepared using Mtwo rotary file system (VDW, Munich, Germany) up to size #25.06. The canals were irrigated with 1 mL of 5% NaOCl between successive files during instrumentation. Then, the canals were flushed with 10 ml of distilled water. After canal preparation, apexes were sealed with Glass Ionomer Cement to prevent the leakage of final irrigating solution past the apex. All irrigation in the present study was performed using 30G irrigation needles (Vista-Probe; Vista Dental, Racine, WI), and they were used with an up-and-down motion to 1–2 mm short of the working length. Twenty teeth that were not irrigated during preparation were used as negative controls.

Distribution of samples

Control Group (n = 20) not irrigated during preparation.

Experimental teeth (n = 120) were divided into

Group I (n = 60) – Largal ultra as final irrigant

Subgroup IA – No laser activation-

Subgroup IB – Nd: YAG laser activation

Subgroup IC – Er: YAG laser activation

Group II (n = 60)-Biopure MTAD as final irrigant

Subgroup IIA – No laser activation

Subgroup IIB – Nd: YAG laser activation

ubgroup IIC – Er: YAG laser activation.

Laser application

Neodymium-doped yttrium aluminum garnet

A Nd:YAG laser with the wavelength of 1064 nm at the standardized settings of 1 Watt and 25 Hz was used for 10 s. R24 Handpiece of Nd:YAG laser was selected which was having the flexible fiberoptic endodontic tip of 320 μm diameter. The fiberoptic tip was set into the prepared canals, 3 mm short of the apex through a stopper. Then, the laser was activated with the help of a foot switch at MicroShortPulse (MSP) for 10 s. During irradiation, the laser fiber was used with constant motion in apicocoronal direction and was kept 3 mm away from the apex.

Erbium-doped yttrium aluminum garnet

Er:YAG laser with the wavelength of 2940 nm at the standardized settings of 1 Watt, 25 mJ, and 40 HZ was used for 10 s. R14 contact handpiece of Er:YAG laser was selected to which radial firing X-PULSE endodontic fiber tip was attached. The fiber tip was 14 mm long having the diameter of 600 μm. The tip was kept stable in the coronal third of the canal, as it was not necessary to move the tip. Then, the laser was activated with the help of a foot switch at MSP for 10 s. Laser was activated only after the insertion of fiberoptic into the canal.

Root canal obturation

After the final irrigation, each canal was flushed with 5 mL of distilled water and then dried with paper points. All canals were obturated with gutta-percha and AH Plus sealer (Dentsply DeTrey, Konstanz, Germany) using lateral condensation technique. For fluorescence under confocal laser scanning microscopy (CLSM), sealer was mixed with 0.1% fluorescent rhodamine B isothiocyanate. Master cone gutta-percha of size No. 25 was tested in canal for tug-back at apical preparation. A thin mix of resin sealer was made according to the manufacturer's instructions. This paste (sealer) was then thoroughly applied into the canal to the level of 1 mm short of working length with #25 lentulo spirals. The consistency of mix was kept constant in all the samples. The master cone was coated with sealer and positioned into the canal. Thereafter, accessory cones were laterally compacted using Ni–Ti finger spreaders, until they could not be introduced >5 mm into the canal. Then, the cones were sealed up to the orifice with the help of spirit lamp and finger pluggers. Finally, the access cavities were sealed with temporary filling material (Cavit; ESPE, Seefeld, Germany). Subsequently, the teeth were kept in incubator at 37°C and 100% humidity for 24 h to allow the sealer to set.

CLSM investigation

Then, the samples were prepared and evaluated with CLSM. Each tooth was embedded in an acrylic block, and 500 μm thick transverse sections of each mesiobuccal or distobuccal roots were obtained with a slow-speed, water cooled diamond saw at 3 mm from the apex. All the sections were polished with silicone carbide abrasive stone. Then, the samples were mounted on glass slides and examined using CLSM with excitation by a He/Ne G laser (543 nm). The samples were observed using a ×2.5 numeric aperture, 0.075) oil lens with additional zooms of ×2 (total magnification, ×50). The images were acquired and analyzed using Zeiss LSM Image Examiner Software (Carl Zeiss) [Figure 1,2]. In each image, the circumference of the root canal was measured with the measuring tool software. Next, areas along the canal circumference into which the sealer penetrated the dentinal tubules with any distance were outlined and measured. The percentage of any canal wall where sealer had penetrated was calculated by dividing outlined length by the canal circumference.

Figure 1

(a) Control (b) Largal Ultra without laser activation (c) MTAD without laser activation

Figure 2

(a) Largal Ultra with Nd:YAG laser activation (b) Largal Ultra with Er:YAG laser activation (c) MTAD with Nd:YAG laser activation (d) MTAD with Er:YAG laser activation

Statistical tools employed

Analysis of variance was calculated (e.g., one-way between-subjects ANOVA). Tukey's test was performed for each mean comparison. Checked to see, if Tukey's score is statistically significant with Tukey's probability/critical value table taking into account appropriate dfwithin and number of treatments.

RESULTS

Percent diffusion ranged between 0 and 72.70. In control group, it was 4.33 ± 1.56, in Group IA 20.71 ± 1.95, in Group IB 41.24 ± 1.83, in Group IC 59.25 ± 1.77, in Group IIA 41.01 ± 1.80, in Group IIB 49.96 ± 2.05, and in Group IIC, it was found to be 69.81 ± 1.70 [Table 1 and Bar Diagram 1]. Analysis of variance and above box plot shows statistically significant difference in percent diffusion among the groups (P < 0.001). Percent diffusion values of controls were found to be of lower order and that of Group IIC of higher order. The box plot shows Percent diffusion values of controls were found to be of lower order and that of Group IIC of higher order. The box plot shows overlap of interquartile values of percent diffusion of Group IB and Group IIB [Table 2 and Bar Diagram 2]. Difference between control and Group IIC (65.48 ± 0.57) was highest, followed by control and Group IC (54.92 ± 0.57) and minimum between Group IB and Group IIA (0.23 ± 0.57) [Table 3 and Bar Diagram 3].

Table 1

Comparison of percent diffusion in different groups

Group	Number of samples	Mean±SD	Minimum	Maximum
Control	20	4.33±1.56	0.00	5.99
Group Ia	20	20.71±1.95	18.03	23.76
Group Ib	20	41.24±1.83	38.31	43.78
Group Ic	20	59.25±1.77	57.05	62.90
Group IIa	20	41.01±1.80	38.24	43.74
Group IIb	20	49.96±2.05	47.03	53.68
Group IIc	20	69.81±1.70	67.21	72.70

SD: Standard deviation

Bar Diagram 1

Above box plot shows comparison of percent diffusion in different groups

Table 2

Analysis of variance for percent diffusion

Comparison	Sum of squares	df	Mean square	F	P
Between groups	59995.06	6	9999.176	3035.771	<0.001
Within groups	438.07	133	3.294
Total	60433.13	139

Bar Diagram 2

Above box plot shows Analysis of variance for percent diffusion of different groups

Table 3

Between group comparison of percentage diffusion (Tukey HSD test)

Comparison	Mean difference	SE	P
Control versus Group Ia	16.38	0.57	<0.001
Control versus Group Ib	36.91	0.57	<0.001
Control versus Group Ic	54.92	0.57	<0.001
Control versus Group IIa	36.68	0.57	<0.001
Control versus Group IIb	45.63	0.57	<0.001
Control versus Group IIc	65.48	0.57	<0.001
Group Ia versus Group Ib	20.53	0.57	<0.001
Group Ia versus Group Ic	38.54	0.57	<0.001
Group Ia versus Group IIa	20.30	0.57	<0.001
Group Ia versus Group IIb	29.25	0.57	<0.001
Group Ia versus Group IIc	49.10	0.57	<0.001
Group Ib versus Group Ic	18.01	0.57	<0.001
Group Ib versus Group IIa	0.23	0.57	1.000
Group Ib versus Group IIb	8.72	0.57	<0.001
Group Ib versus Group IIc	28.57	0.57	<0.001
Group Ic versus Group IIa	18.24	0.57	<0.001
Group Ic versus Group IIb	9.29	0.57	<0.001
Group Ic versus Group IIc	10.56	0.57	<0.001
Group IIa versus Group IIb	8.95	0.57	<0.001
Group IIa versus Group IIc	28.80	0.57	<0.001
Group IIb versus Group IIc	19.85	0.57	<0.001

SE: Standard error

Bar Diagram 3

Box plot showing in between group comparison of percentage diffusion

Order of percent diffusion in the present study was:

Group IIc > Group Ic > Group IIb > Group Ib ≈ Group IIa > Group Ia > Control

DISCUSSION

In this study, two novel root canal irrigants with and without laser activation were juxtaposed to evaluate smear layer removal and penetration of sealers in the apical thirds of root canal walls. The two irrigating solutions used were Largal Ultra and Biopure MTAD and they were activated using two types of lasers, i.e., Nd:YAG and Er:YAG. Simon J in 1994[7] recognized the apical area as the critical zone for instrumentation. Studies demonstrate remaining debris even after removal of smear layer with both conventional and activated irrigation techniques.[8] Due to these reasons, this area was focused in the present study. Authors believe that the smear layer, being a loosely adherent structure, should be removed completely as it can foster bacteria providing an avenue for leakage which compromise the impervious seal of root canal filling.[9] Biopure MTAD is available in two components, to be mixed just before use according to manufacturer's instructions.[10] According to various studies, the tetracycline part of MTAD removes the smear layer and other debris, 4.25% citric acid acts as a chelator and detergent Tween-80 reduces the surface tension of the irrigant, thereby facilitating its penetration.[4] Largal Ultra (Septodont, Paris, France) contains 15% EDTA solution as a disodium salt, O.75% of cetyl-tri-methyl ammonium bromide (Cetrimide) and sodium hydroxide to adjust the pH value to 7.4. The additives in Largal Ultra further are presumed to decrease the viscosity and the surface tension. It removed the smear layer, opened the dentinal tubules, and left only minimal debris.[11] In recent years, lasers have begun to be used to increase the effectiveness of irrigating solutions. Although the technological advances in laser systems have broadened the options, at the present time, Er:YAG, Nd:YAG, and Er,Cr:YSGG are the most widely used laser types in endodontics.[1] The concept of LAI is based on cavitation. Because of the high absorption of water by the mid-infrared wavelength of lasers, the cavitation process generates vapor-containing bubbles, which explode and implode in a liquid environment.[12] Er:YAG laser has been identified to create photoacoustic shock waves within the irrigating solutions introduced in the canal. The containment of the shockwaves by root canal walls thoroughly streams these solutions, augmenting their effectiveness. The canals are left clean and free of smear layer. The reason for the greater efficacy of Er:YAG laser than Nd:YAG can be attributed to the fact that Er:YAG lasers are well established for hard-tissue preparation, these wavelengths are highly absorbed by water, which facilitates better tissue removal through thermomechanical ablation.[13] On the other hand, Nd:YAG laser is shown to get absorbed by mineral structures such as phosphates and carbonate hydroxyapatite, and disrupts crystal structures by thermomechanical action. This results in the melting and resolidification processes on dentin surface, which reduces dentinal permeability by promoting almost complete occlusion of dentinal tubules before root canal filling. The lesser sealer penetration along the canal walls in the specimens which were irradiated with Nd:YAG subgroup than Er:YAG subgroup might be speculated due this occlusion of dentinal tubules. Although the temperatures on the root surfaces were not monitored in our study, some studies demonstrated that the healthy surrounding periodontal tissues are not damaged thermally if the laser equipment is used with a correct adjustment and temperature increase of the root canals remains <5°C.[1415]

Temperature elevation is a function of output intensity and irradiation time. The setting parameters for the present study were based on those safely used for laser application in endodontic treatment for permanent teeth. In addition, to prevent excessive temperature rising and damage to periodontal tissues during irradiation, the delivery fiber tip of Nd:YAG laser was constantly moved in apicocoronal direction.[16] Moreover, in Er:YAG laser, the fiber tip was kept up to the coronal third of the canals only as it was not necessary to insert the laser tip to the apex because the cavitation bubbles produced assisted in cleaning the apical region.[17]

Apart from the presence of smear layer, other factors which could affect the penetration of sealers into dentinal tubules include the presence of sclerotic dentin, less tubule density, or occluded tubules. Compaction forces of the spreader while obturation interrupts the formation of a continuous layer of sealer between gutta-percha and the root canal wall leaving certain areas without sealer after the filling procedure. These factors can be taken as limitation of the present study.[18]

Over the years, investigators have used different techniques to evaluate penetrability of sealers into dentinal tubules such as light microscopy[19] and scanning electron microscopy.[20] Stereomicroscopy and transmission electron microscopy have also been used in some studies. However in the present study, confocal microscopy was opted as it offers several advantages over abovementioned techniques. These include the ability to control depth of field, elimination, or reduction of background information away from the focal plane (which leads to image degradation), and the capability to collect serial optical sections even from thick specimens. CLSM does not require any special specimen processing, and observations can be made under environmental conditions. This means that CLSM observations have lower potential to produce technique artifacts.[21]

The results obtained from the present study have clinical relevance and provide clinicians with information about the use of various root canal irrigating solutions with and without laser activation for better smear layer removal and hence promoting better sealer penetration into the tubules giving an impervious seal.

CONCLUSIONS

Under the present experimental conditions and limitations, Biopure MTAD seemed to present with better sealer penetration due to better smear layer removal efficacy than Largal Ultra. Laser activation of final irrigating solution proved to be beneficial in promoting sealer penetration into dentinal tubules along the root canal walls. Moreover, activation of irrigating solutions with Er:YAG laser has shown to give better results than Nd:YAG laser activation. However, none of them allowed complete smear layer removal and sealer penetration in the apical thirds of the curved root canals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.