Structural and Morphological Changes in Human Dentin after Ablative and Subablative Er:YAG Laser Irradiation

Abstract

Introduction: This study investigated the influence of Erbium-Doped Yttrium Aluminum Garnet (Er:YAG) laser on microhardness, chemical composition and subsurface morphology of dentin cavity walls.

Methods: Forty sound human premolars were selected and randomly assigned into four groups. Class V cavities were prepared either with an Er:YAG laser (groups 1 and 2; 15 Hz, 250 mJ for enamel, 10 Hz, 200 mJ for dentin) or with a high speed handpiece (groups 3 and 4). The specimens in groups 1 and 3 served as the control, whereas those in groups 2 and 4 were exposed to subablative laser irradiation following cavity preparation (10 Hz, 50 mJ). After bisecting the specimens, one half was subjected to microhardness assessment and the other half was evaluated by SEM-EDS analysis.

Results: Microhardness was significantly greater in the specimens prepared by both ablative and subablative laser irradiation (group 2) than that of the bur-prepared cavities (groups 3 and 4) (P < 0.05). The quantity of calcium ion was significantly greater in cavities prepared by the Er:YAG laser (groups 1 and 2) compared to that of the bur cavities (groups 3 and 4) (P < 0.05). Subablative irradiation improved microhardness and weight percentage of calcium ion in both laser and bur cavities, but the difference was not significant compared to that of the relevant control group (P > 0.05).

Conclusion: Cavity preparation with an Er:YAG laser could be considered as an alternative to the conventional method of drilling, as it enhances the mechanical and compositional properties of lased dentin, especially when combined by subablative irradiation.

Introduction

Despite the protective measures adopted, dental caries is still the most common oral disease in all over the world.^1,2 The growing sugar consumption and the inadequate oral hygiene are considered as the main reasons for the high incidence of dental caries. These factors may also contribute to the formation of secondary caries around restorations. Therefore, searching for a cavity preparation technique that enhances the mineral content of tooth tissue is considered as an issue of interest.

Since the introduction of Ruby laser in 1960, lasers have been applied for different purposes in dentistry. It has been indicated that laser irradiation can enhance the resistance of tooth structure against caries formation and progression through different mechanisms.^3-6 Although various lasers have been employed for the purpose of caries prevention, erbium family lasers including Erbium-Doped Yttrium Aluminum Garnet, (Er:YAG) and Erbium, Chromium-Doped Yttrium Scandium Gallium Garnet (Er,Cr:YSGG) are the only ones that can be employed for removing dentin and enamel structures.^7-9 Cavity preparation by the Er:YAG laser provides advantages such as low noise and vibration, and less pain and stress for the patients, but it is more expensive and time-consuming than the conventional technique with rotary instruments.

The wavelength of the Er:YAG laser (2.94 µm) is greatly absorbed by water and then by hydroxyl groups in hydroxyapatite, making this laser suitable for thermomechanical ablation.^10,11 During irradiation, the water content of enamel and dentin experiences sudden heating and vaporization, which leads to the production of microexplosions and ejection of both organic and inorganic components from dental hard structure.^10,11 Since the energy of Er:YAG laser is mainly absorbed in superficial enamel or dentin tissues, the thermal damage to the pulp or surrounding structures is prevented to a great extent.^12,13

The use of Er:YAG laser for cavity preparation can affect the mineral content of the tooth, which in turn influences its resistance to further caries formation.¹⁴ Previous studies reported contradictory results regarding the effect of Er:YAG laser with ablative or subablative parameters on resistance of lased substrate to acid attack. Some authors¹⁵ reported less development of caries lesion around cavities prepared by the Er:YAG laser than around the bur-prepared cavities, while Hossain et al¹⁶ demonstrated similar knoop hardness in laser and bur cavities. Regarding irradiation with subablative parameters, Apel et al¹⁷ demonstrated some increase in acid resistance following subablative irradiation with erbium family lasers, which failed to achieve significance in the statistical analysis, whereas Liu et al¹⁸ and Castellan et al¹⁹ reported a significant prevention of enamel demineralization. Morphological examination of surfaces treated by the Er:YAG laser revealed roughness and irregularities which provided microretentive patterns^12,20-23, and at the cavity walls, there was almost no smear layer.^10,20,22

Considering the contradictory results of previous authors, the purpose of this in vitro investigation was to determine the influence of ablative and subablative Er:YAG laser irradiation on microhardness, composition and surface morphology of dentin cavity walls and compare the results with that of the conventional method of bur preparation.

Methods

Sound human premolars extracted before orthodontic treatment were collected and stored in normal saline solution until the time of the experiment. The teeth were cleaned with rubber prophylactic cups and examined visually to discard those with hypoplastic areas or other structural defects. Finally, 40 premolars were chosen and stored in 1% thymol solution for 1 week.

The sample was then randomly assigned to four groups of 10 each. Class V cavities 2 mm in width, 2 mm in height and 1.5 mm deep were prepared on the buccal cervical areas of premolar teeth either with an Er:YAG laser (groups 1 and 2) or with a high speed handpiece. The specimens in groups 2 and 4 were also exposed to subablative Er:YAG laser irradiation following cavity preparation in order to assess any benefit of using subablative parameters in increasing caries resistance. The cavity area was delimited by a piece of insulating tape with a central hole, made by a punch. The depth of the cavities was controlled by a periodontal probe.

The laser apparatus used was the Kavo Key Laser 2 (KaVo Dental GmbH, Biberach, Germany). The laser operated in noncontact and focused mode and in association with water stream at 1.5 ml/min flow. The 2051 handpiece was used through a flexible fiber delivery system so that the beam was perpendicular to the irradiated surface. The “very short pulse setting” was chosen throughout the experiment. For enamel ablation, the energy of 250 mJ was used with pulse repetition rate of 15 Hz, whereas 200 mJ and 10 Hz were employed for dentin ablation. The subablative parameters employed were 50 mJ/10 Hz.

After cavity preparation, the roots of the teeth were sectioned 2 mm below the cementoenamel junction and the crowns were mounted in polyester resin. Then, the teeth were bisected longitudinally in a buccolingual direction through the middle of the prepared cavities using a water-cooled diamond saw. One hemisection of each specimen was used for microhardness assessment and the other one was prepared for examination by scanning electron microscopy (SEM).

Microhardness Assessment

The tooth sections destined for microhardness assessment were polished alternatively with no. Six hundred to 1200-grit silicon carbide papers to create a smooth surface for increasing the precision of reading. The specimens were then individually fixed in cylindrical plates with wax. A parallelometer was applied to ensure that the surface was kept parallel to the horizontal plane. A Vickers hardness apparatus (Matsuzawa, model MHT2, Japan) was employed to evaluate microhardness of the specimens using a 500 g load for 20 seconds. Microhardness was assessed at the occlusal, axial, and gingival walls of the cavities at the depth of 30 µm from the surface. The Vickers hardness number (VHN) was recorded and the mean value was calculated for each specimen.

SEM-EDS Examination

One hemisection of each specimen was prepared for SEM-EDS analysis. After a standard preparation technique, the specimens were mounted on aluminum stubs, sputter coated with gold and then examined with a Tescan VEGA II scanning electron microscope (SEM; Tescan, Brno, Czech Republic) operating at an accelerating voltage of 15 kV. Photomicrographs were taken at different magnifications to indicate subsurface morphological changes. The histochemical analysis was done by energy-dispersive x-ray spectroscopy (EDS) and the weight percentage of calcium ion in each specimen was measured. The EDS measurements were taken from the occlusal, axial and gingival walls of the cavities at the depth of 30 µm from the surface, and the mean value was recorded for each specimen.

Statistical Analysis

The average microhardness and calcium ion quantity obtained from different walls of each specimen was calculated and considered for statistical analysis. The Kolmogorov-Smirnov test confirmed the normal distribution of the data. One-way analysis of variance (ANOVA) was run to detect any significant differences in hardness and weight percentage of calcium ion among the study groups, followed by Tukey post hoc test for pairwise comparisons. The statistical analysis was performed by SPSS version 16.0 (SPSS Inc., Chicago) software and the statistical significance was determined at P<0.05.

Results

Microhardness Assessment

The mean and standard deviation (SD) of microhardness values of the study groups is presented in Table 1. ANOVA showed a significant difference in microhardness among the study groups (Table 1). Tukey post hoc test revealed that microhardness was significantly greater in group 2 (laser application for cavity preparation followed by subablative irradiation) than groups 3 and 4 (P<0.05), which were prepared by the bur. No significant difference was found in microhardness either between groups 1 and 2, or between groups 3 and 4 (P>0.05).

Table 1 . The Descriptive Statistics and the Results of Statistical Analysis for Comparison of Microhardness Values Among the Experimental Groups .

	Mean	SD	Pairwise Comparison *
Group 1	708.92	73.43	a, b
Group 2	742.89	108.64	a
Group 3	679.13	80.69	b, c
Group 4	655.32	93.14	b, c
Statistical analysis	P<0.001

*Tukey pairwise comparison test; Different letters indicate statistically significant differences at P<0.05.

SEM-EDS Assessment

Comparison of the weight percentage of calcium ion among the study groups is presented in Table 2. ANOVA revealed a significant difference in the quantity of calcium ion among the groups (Table 2). According to Tukey analysis, the quantity of calcium was significantly greater in groups 1 and 2 compared to groups 3 and 4 (P<0.05). Neither groups 1 and 2 nor groups 3 and 4 displayed statistically significant differences to each other regarding the weight percentage of calcium ion (P>0.05, Table 2).

Table 2 . The Descriptive Statistics and the Results of Statistical Analysis for Comparison of Calcium Ion Quantity Among the Experimental Groups .

	Mean (Weight %)	SD	Pairwise Comparison*
Group 1	29.61	2.12	a
Group 2	30.53	1.36	a
Group 3	22.47	1.02	b
Group 4	22.63	2.15	b
Statistical analysis	P<0.001

*Tukey pairwise comparison test; Different letters indicate statistically significant differences at P<0.05.

Figure 1 illustrates the SEM images taken from cavities prepared by different techniques. Laser irradiation for cavity preparation caused irregularities and alterations in surface morphology. Fusion areas with less opened dentinal tubules were observed after subablative irradiation in both laser and bur cavities.

Figure 1 .

Surface morphology of the cavities prepared by (A) Er:YAG laser, (B) Er:YAG laser followed by subablative irradiation, (C); diamond bur, (D) diamond bur followed by subablative Er:YAG laser irradiation.

Discussion

The application of Er:YAG laser has been suggested as a suitable alternative to conventional cavity preparation instruments for removing dental caries and tooth tissue.²⁴ The outcomes of this study indicate that cavity preparation with the Er:YAG laser instead of rotary instruments can enhance mechanical properties and chemical composition of the lased substrate and possibly increase its resistance against further caries attack.

Laser absorption in tooth tissue provides thermal effects that influence the structural and chemical properties of dental hard tissues.^1,25 The heat produced during laser irradiation leads to vaporization, oxidation of organic components, transformation of acid phosphate to pyrophosphate, and reduction of carbonate content.^3,4,25,26 These changes result in the formation of more stable and less soluble components, which might increase the resistance of irradiated substrate against demineralization.³ The blocking of intra and inter spaces of enamel due to heating of the organic matrix has also been suggested as a possible mechanism for caries prevention.^5,27

Microhardness readings indicate the mineral content of the teeth, and the quantity of calcium ion could be representative for any alteration in the composition of dental hard tissue which affects its permeability and solubility in different media. In the present study, it was found that the quantity of calcium ion was significantly greater in laser- than bur-prepared cavities. Although microhardness of laser cavities was greater than that of bur cavities, but the statistical significance with bur cavities was only obtained when both ablative and subablative parameters (group 2) were employed. The improvement obtained in chemical and mechanical properties of dentin may be related to the vaporization of water and organic components, which increases the percentage of mineral content in the lased substrate.²⁸ In the clinical conditions, it is possible that the modifications occurred in Er:YAG laser ablated dentin act against demineralization and caries formation around restorations.

The findings of this study confirmed the outcomes of Hossain et al¹⁶ who found that the quantities of calcium (Ca weight %) and phosphorus (P weight %) of dentin were significantly greater in laser cavities compared to bur cavities. Perito et al¹⁵ reported less development of secondary caries around the cavities prepared by Er:YAG laser than around bur-prepared cavities. In contrast, Celik et al²⁸ found that cavity preparation with bur or erbium family lasers did not significantly affect the composition and microhardness of dentin structure. Chimello et al¹² reported that microhardness of enamel adjacent to composite resin restorations submitted to high cariogenic challenge was comparable in groups prepared by an Er:AYG laser or a high-speed handpiece. Hossain et al¹⁶ found that the knoop hardness of the cavity floors prepared by Er:YAG laser was comparable to that of the conventional bur cavity. Apel et al²⁹ concluded that cavity preparation by means of an erbium laser would offer no benefits in the sense of resistance to secondary caries formation in the clinical situation. The differences observed between the outcomes of this study and those of previous authors could be attributed to the laser parameters employed such as pulse energy and duration, pulse repetition rate, duration of laser irradiation and application of water cooling.

The application of Er:YAG laser with subablative parameters has been attempted as a way to increase caries resistance by induction of chemical and structural changes on the surface without removing tooth tissue. In this study, the highest microhardness and the greatest quantity of calcium ion were obtained when Er:YAG laser was employed for both cavity preparation (ablative parameters) and surface treatment (subablative parameters). Although subablative Er:YAG laser irradiation increased microhardness and quantity of calcium ion in both laser- and bur-prepared cavities, but the difference was not significant compared to that of the relevant control groups. This is consistent with the outcomes of Rodriguez-Vilchis et al³⁰ who found that acid resistance of enamel did not increase as expected following subablative Er:YAG laser irradiation. Apel et al³¹ indicated no significant difference in calcium solubility between the specimens irradiated with the subablative Er:YAG and Er,Cr:YSGG lasers and that of the control. Another study demonstrated the formation of cracks following subablative erbium laser irradiation, which might act as starting points for acid attack and thus attenuate any positive effect of laser irradiation on caries prevention.³²

Some irregularity and modification in surface texture was observed in cavities prepared with the Er:YAG laser compared to those prepared by the high speed handpiece. In both laser- and bur- prepared cavities, the subablative Er:YAG laser irradiation caused dense and compact regions with less open tubules possibly due to the high percentage of tubular blockage or because of the recrystallization that may occur after surface heating.¹¹ These alterations may be in accordance with the increased mineral content and enhanced composition of the irradiated dentin, as observed in the present investigation. Other studies also indicated topographical alterations including irregularities, craters and cracks in enamel and dentin specimens treated by Er:YAG or Er,Cr:YSGG lasers.^{10,11,20-23,30,33}

The laser settings used in this study were selected in order to produce efficient enamel and dentin ablation, simulating the clinical conditions. Most of the previous studies used lower pulse repletion rates compared to that used in this study, but the number of microexplosions and the rate of ablation are directly dependent on this parameter.^34,35 Therefore, the use of lower pulse repetition rates may make the cavity preparation a time-consuming process. When using Er:YAG laser with subablative parameters, it is very important to use the lowest energy level because higher energies produce microcracks and damage the peripheral and underlying tissues.¹⁸ Therefore, subablative irradiation in this study was carried out with a very low energy level.

Considering the promising outcomes of this study, cavity preparation with Er:YAG laser could be considered as a viable alternative to the conventional application of high speed handpiece and burs, which enhances the mechanical and compositional properties of lased dentin, especially when combined by subablative irradiation. However, alterations in surface morphology and calcium ion quantity can affect the adhesion pattern of restorative materials.^11,36 Therefore, any adverse effect on the bonding interface should be investigated in future studies. Long term studies are also required to elucidate any consequence of the present findings in the clinical situation.

Conclusion

Under the conditions used in this study: (1) Microhardness was significantly greater in cavities prepared by ablative followed by subablative Er:YAg laser irradiation compared to that of the bur-prepared cavities. (2) The application of Er:YAG laser for cavity preparation caused a significant increase in the quantity of calcium ion compared to that of the bur cavities, indicating enhancement in the composition of lased-dentin.

Acknowledgements

The authors would like to thank the vice-chancellor for research of Mashhad University of Medical Sciences for the financial support of this project [grant number 88577]. The results presented in this paper have been taken from a DDS student thesis [thesis number 2515].

Ethical Considerations

This was an in vitro study. The specimens were teeth that had been extracted for orthodontic reasons. Therefore, no ethical code is required.

Conflict of Interest

The authors declare no conflict of interests.

Please cite this article as follows: Moosavi H, Ghorbanzadeh S, Ahrari F. Structural and morphological changes in human dentin after ablative and subablative Er:YAG laser irradiation. J Lasers Med Sci. 2016;7(2):86-91. doi:10.15171/jlms.2016.15.