In vitro evaluation of periapical safety of various laser-activated irrigation protocols using a quantitative assessment method

Abstract

Context:

Successful root canal treatment (RCT) depends on effective irrigant activation to enhance disinfection and minimize the risk of apical periodontitis. Laser-activated irrigation (LAI) has shown promise; however, its periapical safety remains to be fully evaluated.

Aims:

The aim of this study was to assess and compare the periapical safety of erbium:yttrium-aluminum-garnet (Er: YAG) and diode laser irrigation protocols with passive ultrasonic irrigation (PUI) during RCT.

Settings and Design:

This was a quantitative in vitro experimental study.

Methods:

Sixty-six extracted human single-rooted teeth were standardized and fixed into a 96-well plate containing 300 μL of a pH indicator solution. Teeth were randomly allocated to three groups: PUI, diode laser, and Er: YAG laser. Irrigation with 5.25% sodium hypochlorite (NaOCl) was activated using the designated technique. Apical extrusion of NaOCl was quantified using ELISA-based spectrophotometry.

Statistical Analysis Used:

Kruskal–Wallis and post hoc pairwise comparison tests were performed (P < 0.05).

Results:

All groups showed apical extrusion. The Er: YAG group had significantly less extrusion than the diode and PUI groups (P < 0.05).

Conclusions:

Within the study’s limitations, Er: YAG and diode LAI reduced apical NaOCl extrusion compared to PUI, indicating improved periapical safety. Further clinical studies are needed.

INTRODUCTION

Chemo-mechanical preparation of root canal system is essential in achieving canal disinfection and successful root canal treatment.[1] Although advancements in mechanical instrumentation have improved canal shaping and biofilm disruption, they often have complex anatomy areas, such as isthmuses, lateral canals, and apical deltas, untouched, allowing residual microorganisms to persist.[2,3] Consequently, the adjunctive use of chemical irrigation, such as sodium hypochlorite (NaOCl), remains indispensable for effective debridement.[2,4]

Notably, the antibacterial effect of irrigating solutions alone against the spectrum of bacteria is limited, and their ability to adequately penetrate dentinal tubules and anatomical complexities is also restricted.[3] However, the effectiveness of these irrigants can be significantly enhanced when activated using energy-based agitation techniques, such as passive ultrasonic irrigation (PUI),[5,6] laser-activated irrigation (LAI),[7] or a combination of both.[8]

These machine-assisted irrigant activation techniques increase the temperature and kinetic energy of the irrigant, thereby enhancing its chemical and biological effects, as well as improving contact with canal walls.[7,8,9,10] Both PUI and LAI operate through mechanisms such as acoustic streaming and cavitation, which facilitate the disruption of biofilms and removal of debris from inaccessible canal areas.[10,11]

LAI effectively promotes irrigant penetration into anatomically challenging areas such as lateral canals and deep dentinal tubules-regions often unreachable by conventional irrigation techniques.[12,13,14] Among the various laser systems, the erbium:yttrium-aluminum-garnet (Er: YAG) laser has demonstrated superior efficacy in removing debris and smear layer due to its efficient photoacoustic streaming action.[3] The diode laser, while less effective in smear layer removal, exhibits greater penetration depth into dentinal tubules, enhancing its antimicrobial potential and contributing to the reduction of postoperative pain.[15]

A critical concern with laser activation is the risk of apical irrigant extrusion beyond the foramen, which can lead to adverse effects such as periapical tissue damage, postoperative pain, or neurotoxicity.[2,3,4] While numerous studies have evaluated the antimicrobial efficacy of various LAI protocols, relatively few have investigated their periapical safety profiles. Furthermore, there is limited consensus regarding the most effective and safest laser parameters for irrigant activation in endodontics. Therefore, this study aims to evaluate and compare the periapical safety of Er: YAG and diode LAI protocols in terms of apical extrusion, using a quantitative in vitro model, and benchmark their performance against the widely used PUI technique.

METHODS

Following ethical approval from the Research Ethics Committee at University of Sharjah, a total of 66 extracted human single-rooted teeth were obtained from the university dental hospital and examined radiographically. Only intact teeth with straight roots and a single canal were included. Teeth with accessory canals, cracks, internal or external resorption, open apices, calcification, or prior endodontic treatment were excluded.

Teeth were immersed in 5.25% NaOCl for 48 h to remove organic debris, then scaled with a periodontal scaler to eliminate residual calculus. They were then rinsed with distilled water and stored in 10% formalin until use. This storage protocol was adapted from the methodology described by Helvacıoğlu Kıvanç et al.[4] To standardize sample length, all crowns were decoronated to achieve a uniform root length of 12 mm.

Root canals were accessed and prepared using the ProTaper Gold rotary system (Dentsply, Maillefer, Ballaigues, Switzerland) to a final size of F3. Irrigation with 5.25% NaOCl was performed between each file using syringe irrigation. External root surfaces were double-coated with a moisture-isolating nail varnish (Rimmel, London, UK). A #15 K-file (Antaeos, Vereinigte Dentalwerke, GmbH and Co., Munich, Germany) was used to ensure apical patency with 1 mm of apical extrusion.

Prior to the experiment, samples were randomized into three groups of 22 teeth each based on the irrigation activation technique: PUI, Er: YAG laser, or diode laser. Each tooth was vertically positioned through custom-made holes in the lid of a 96-well plate and fixed in position using flowable composite resin. An illustration of the sample setup is provided in Figure 1.

Figure 1.

Schematic representation of sample preparation workflow. Extracted tooth samples were first decoronated and standardized in length, followed by root canal instrumentation. The external surfaces were coated with a moisture-isolating material to prevent leakage. Subsequently, roots were mounted vertically in a 96-well plate containing a pH indicator solution to facilitate the detection of apical irrigant extrusion

Preparation and calibration of the dye indicator

The pH indicator was prepared by blending 150 g of red cabbage with 150 mL of tap water (1:1 ratio) for 1 min. The mixture was filtered to obtain a clear solution and stored overnight at room temperature.

A preliminary calibration experiment was conducted to correlate NaOCl concentration with optical density. Known volumes of 5.25% NaOCl were added to the indicator solution in control wells, and changes in optical density were recorded after 24 h of incubation using an ELISA microplate reader, allowing the construction of a standard calibration curve.

Experimental irrigation protocols

Each well of the 96-well plate was filled with 300 μL of pH indicator solution, fully immersing root apices. Root canal irrigation was performed using 3 mL of 5.25% NaOCl per sample and activated according to the assigned protocol. Procedures were repeated in duplicate to enhance reproducibility, refreshing the irrigant after each activation cycle.

An Er: YAG laser system (Fotona, Slovenia), with a wavelength of 2940 nm, was used following the photon-induced photoacoustic streaming (PIPS) protocol. A 600/9 PIPS tip was placed in the coronal access cavity following Olivi et al.[16] In the diode laser group, an 810 nm diode laser (Elexxion, Germany) equipped with a 200 μm fiber-optic tip was employed. The laser was operated in continuous wave mode at a power of 1 W, with the fiber tip introduced 4 mm short of the apex, then gradually withdrawn in a gentle spiraling motion.[17] For the PUI group, activation was performed using the Ultra X cordless ultrasonic device (Eighteeth, China), with the ultrasonic tip positioned 2 mm short of the working length, following the protocol adapted from Yang et al.[18] A summary of the laser and ultrasonic activation settings is provided in Table 1.

Table 1.

Protocols for the activation systems used in the study

Protocol	Number of cycles (s)	Tip placement	Power	Frequency	Wavelength	Water and air setting
Er: YAG	2 (10)	Coronal access cavity	0.3 W	15 Hz	2940 nm	Off
Diode	3 (10)	4 mm short of apex	1 W	0	810 nm	Off
PUI	3 (30)	2 mm short of apex	N/A	45 KHz	N/A	N/A

PUI: Passive ultrasonic irrigation, Er: YAG: Erbium: yttrium-aluminum-garnet, N/A: Not available

Data analysis

Color changes resulting from apically extruded NaOCl were detected and quantified using ELISA-based spectrophotometry, which measured changes in the optical density of the pH indicator solution. The data were statistically analyzed using IBM SPSS Statistics (version 29.0.0.0; IBM Corp., Armonk, NY, USA). The one-sample Kolmogorov–Smirnov test was conducted to assess the normality of data distribution. As the data was not normally distributed, nonparametric tests were employed. The Kruskal–Wallis test was applied to determine significant differences among the three experimental groups, followed by post hoc pairwise comparisons. A significance level of P < 0.05 was used for all statistical analyses. The findings are presented in Table 2.

Table 2.

Kruskal–Wallis test and pairwise comparison of the groups

Kruskal–Wallis test				Pairwise comparison test
Group (s)	Median (IQR)	χ2 (df)	P*	Pairwise comparison	P*
Diode	0.3355 (0.12)	38.84 (3)	<0.001	Diode versus Er: YAG	<0.001
Er: YAG	0.4553 (0.18)			Diode versus PUI	1.00
PUI	0.3058 (0.16)			PUI versus Er: YAG	<0.001

*Significant at level 0.05. The nonnormality assumption is fulfilled. PUI: Passive ultrasonic irrigation, Er: YAG: Erbium: yttrium-aluminum-garnet, IQR: interquartile range

RESULTS

The calibration experiment using the dye indicator demonstrated an inverse relationship between NaOCl concentration and optical density [Figure 2]. As the volume of extruded NaOCl increased, a corresponding decrease in optical density was observed, confirming the indicator’s reliability for quantitative assessment.

Figure 2.

(a) Calibration curve of the pH indicator showing the inverse linear relationship between NaOCl concentration and optical density, (b and c) Optical density measurements indicating apical extrusion of NaOCl across the three activation techniques: diode laser, Er: YAG laser, and ultrasonic in Experiment 1 (b) and Experiment 2 (c)

Statistical analysis using the Kruskal–Wallis test revealed a significant difference among the three groups [P < 0.05, Table 2]. Post hoc pairwise comparisons showed that the Er: YAG group exhibited significantly higher optical density values than both the diode laser and PUI groups (P < 0.05), indicating the least amount of apical extrusion. The diode laser group showed intermediate optical density values, while the PUI group exhibited the lowest, suggesting the greatest degree of extrusion. However, the difference between the diode laser and PUI groups was not statistically significant [P > 0.05, Table 2].

To confirm reproducibility, the experiment was repeated, and similar results were obtained, supporting the consistency and reliability of the findings [Figure 2].

DISCUSSION

Periapical extrusion of NaOCl during root canal irrigation is a well-documented clinical complication, potentially leading to severe consequences including acute pain, swelling, ecchymosis, tissue necrosis, and delayed periapical healing.[4,19] Therefore, the selection of irrigation protocols must balance disinfection efficacy with periapical safety. This study quantitatively compared periapical extrusion of NaOCl using three activation techniques: PUI, Er: YAG, and diode laser, within an in vitro model.

All three techniques produced measurable apical extrusion, as indicated by changes in the optical density of the pH indicator. This observation is consistent with previous studies associating extrusion risk with positive pressure delivery and irrigant activation when apical resistance is insufficient.[19,20]

The findings of the present study are in agreement with those of Helvacıoğlu Kıvanç et al.,[4] who found no significant difference in extrusion between diode and PUI, even with higher diode power settings. Similarly, findings of a newly published study by Abbas et al.[17] quantified apically extruded bacteria from infected root canals and reported the highest extrusion levels in the PUI group, compared to the Er: YAG and diode laser groups. These findings are in line with the present results, although the prior study used saline as the irrigant instead of 5.25% NaOCl.

In contrast, İnce Yusufoglu et al.[21] reported no significant difference between the Er: YAG and PUI techniques. However, a key methodological variation was the Er: YAG application duration, over four times longer than in the present study, potentially accounting for their higher extrusion values.

Additionally, Karasu et al.[10] reported contradictory findings, in which Er: YAG activation resulted in greater extrusion compared to the diode laser and PUI groups. This discrepancy may be attributed to their use of higher laser power setting (1 W vs. 0.3 W in our study) and a closer tip placement (3 mm vs. 12 mm from the apex), underscoring the sensitivity of LAI outcomes to parameter adjustments.

Although PUI exhibited the highest level of irrigant extrusion in the present in vitro model, Peeters et al.[22] no observable apical extrusion in clinical cases using this technique. This discrepancy could be explained by the natural back pressure from periapical tissues in vivo, which limits irrigant extrusion beyond the apex.

The extent of irrigant extrusion is influenced by multiple interrelated factors, complicating direct comparisons across studies. These include apical preparation size, canal taper, irrigant delivery method and depth, activation technique, tip placement, irrigant concentration, and anatomical or pathological conditions such as open apices or periapical bone loss.[23,24] Even minor variations in laser settings, such as pulse duration, energy output, or frequency, can significantly alter hydrodynamic effects and, consequently, the likelihood of extrusion.[7,10]

Limitations of the study

A primary limitation of this study is the absence of simulated periapical tissues, which fails to replicate the resistance offered by periapical structures in a clinical setting. This is a common shortcoming of in vitro models and may result in an overestimation of extrusion risk.[25] Additionally, while optical density measurements effectively compare relative differences in extrusion, they do not precisely quantify the actual volume or concentration of extruded NaOCl.

Further methodological constraints include potential variability in the optical response of the red cabbage pH indicator at higher pH levels, as well as susceptibility to environmental factors such as light exposure, oxygen, or microbial contamination which may affect its stability and accuracy. The sensitivity of ELISA spectrophotometer in detecting minute quantities of low-concentration NaOCl, along with possible human errors during sample handling, activation, and micropipetting may also have contributed to variability in the results.

Clinical implications and future directions

The results of this study suggest that LAI using the Er: YAG laser with the PIPS protocol is associated with the least periapical extrusion, supporting its potential for safer endodontic irrigation. Although diode lasers demonstrated intermediate performance, further optimization of parameters may enhance their safety profile. While PUI remains widely used due to its accessibility and established efficacy, clinicians should be aware of its potential to extrude irrigants, especially in anatomically compromised cases.

Future studies should aim to replicate these findings in clinical or animal models incorporating periapical tissues and investigate additional outcomes such as tissue response, antimicrobial efficacy, and postoperative symptoms. Comparative assessments of different LAI power settings, tip designs, and activation durations would also contribute to developing standardized and safe irrigation protocols.

CONCLUSIONS

This in vitro study demonstrated that all three tested activation techniques: PUI, diode laser, and Er: YAG laser resulted in apical extrusion of NaOCl, with significant differences observed between them. Er: YAG laser produced the least extrusion, followed by diode laser, while PUI exhibited the highest levels. These findings suggest that Er: YAG LAI may offer superior periapical safety compared to conventional activation methods. However, due to inherent limitations of in vitro models, particularly the absence of simulated periapical tissue, further clinical research is warranted to validate these results under real-world conditions.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.