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Non-contact profilometry of eroded and abraded enamel irradiated with an Er:YAG laser

Abstract Literature has reported positive results regarding the use of lasers in the control of erosive lesions; however, evaluating whether they are effective in the control of the progression of erosive/abrasive lesions is important. Objectives This study aimed to evaluate the effect of the Er:YAG laser irradiation in controlling the progression of erosion associated with abrasive lesions in enamel. Material and methods Bovine incisors were sectioned, flattened and polished. Forty-eight enamel slabs were subjected to treatment in an intraoral phase. Twelve volunteers used an intraoral appliance containing one slab that was irradiated with an Er:YAG laser (5.2 J/cm2, 85 mJ, 2 Hz) and another non-irradiated slab on each side of the appliance, during one phase of 5 d, under a split-mouth design. Devices were subjected to erosive challenges (1% citric acid, 5 min, 3 times a day) and abrasive challenges one h after (brushing force of 1.5 N for 15 s) randomly and independently on each side of the device. Measurements of enamel loss were performed via 3D optical profilometry (μm). We analyzed data using the Kruskal-Wallis and Mann-Whitney tests and morphological characteristics via scanning electron microscopy. Results Following erosive and abrasive challenges, the group that was irradiated with the Er:YAG laser presented less loss of structure than the non-irradiated group. The group that underwent erosion and irradiation did not exhibit a significant difference from the non-irradiated group. Conclusion Irradiation with the Er:YAG laser did not control the loss of structure of enamel subjected to erosion but did control abrasion after erosion.


Introduction
Dental erosion occurs through the action of intrinsic or extrinsic acids and without the involvement of bacteria 23 . The demineralization caused by erosion is initially characterized by a softening of the surface and is followed by continuous dissolution of enamel crystals, which leads to the loss of hard dental tissue 13 . In this weakened state, dental surfaces are more prone to wear via abrasive action 19 . The most common form of abrasion is brushing, and factors such as the brushing technique and force, the stiffness of the toothbrush bristles and the abrasiveness of the toothpaste used may be involved in this process 16 .
Tooth structure loss due to erosive and abrasive challenges is irreversible, and several strategies have been developed with the aim of preventing damage, including the use of high-intensity lasers 12 . To increase the acid resistance of the enamel, the use of an Er:YAG laser has been proposed 4,7 .
Irradiation with an Er:YAG laser can promote partial denaturation of the enamel matrix, forming a mineral block that makes diffusion of acids within the tissue difficult 28 . Additionally, it might prevent the progression of erosive lesions and therefore minimize the wear caused by abrasion. Another hypothesis regarding the mechanism by which the laser increases the acid resistance of the enamel is that the irradiation temperature between 100 and 650°C can reduce the amount of water and carbonate in the tissue, resulting in increased resistance against acid 8 . Some studies have even suggested that the increasing acid resistance of the enamel is related to morphological changes in the tissue 10,11 . When the enamel and dentin are irradiated with a laser, the surfaces are partially melted and solidified 10,11 , which suggests that the enamel surface would be less permeable.
Because of the susceptibility of enamel to the development of erosive and abrasive lesions, the search for methods capable of controlling such lesions has been intensified. Although the use of lasers to control erosive lesions is widely presented in the literature, there are no studies assessing the use of an Er:YAG laser to control the progression of erosive/abrasive lesions in enamel.
The null hypothesis tested was that the losses of enamel structure following erosive challenges versus erosive challenges associated with abrasive challenges are similar in slabs treated or untreated with an Er:YAG laser.

Experimental design
This in situ, split-mouth, double-blind study with one phase of 5 d was approved by the Ethics Committee of the School of Dentistry of Ribeirão Preto (process number: 2010.1.552.58.7). Forty-eight sound enamel slabs were subjected to the initial erosive challenge, after which they were randomly assigned to 4 groups (n=12). The factors under study were the type of wear at 2 levels (a. erosion and b. erosion associated with abrasion) and Er:YAG laser irradiation at 2 levels (I. irradiated and II. non-irradiated). After treatment, fragments were exposed to erosive wear on one side of the device and erosive wear associated with abrasive wear on the other side of the device during the in situ phase. The response variable was obtained based on enamel loss evaluated with a 3D optical profilometer.
We analyzed morphological characteristics of surface via scanning electron microscopy.

Preparation of enamel slabs
Bovine incisors were freshly extracted and stored in a 0.1% thymol solution at 4°C and were then examined with a stereomicroscope (Leica S6 D Stereozoom, Mycrosystems Leica AG, Heerbrugg, Switzerland) at a magnification of 40×. Teeth with structural anomalies or cracks were discarded 22 . Dental crowns were sectioned with a diamond disk (15HC, Buehler, Lake Bluff, IL, USA) using a sectioning machine (Isomet 1000; Buehler, Lake Bluff, IL, USA), resulting in two enamel slabs per tooth (5×3×2.5 mm). Enamel surfaces of these slabs were flattened in a water-cooled polishing machine for 20 s (Phonix β, Buehler, Lake Bluff, IL, USA) using

Selection of volunteers and the intraoral phase
We selected volunteers (n=12) of both genders with a mean age of 26 years who presented a normal salivary flow, an absence of active caries lesions, and a salivary buffer with a pH between 6.5 and 7.0 and who had the availability to follow the schedule established.
Volunteers with systemic diseases and digestive disorders and those who were pregnant, smokers or on medication that could interfere with salivary secretion were excluded from the study.
Each volunteer had an impression of his/her maxillary arch recorded to produce an intraoral appliance that was constructed in acrylic resin. Four fragments were fixed with two slabs on each side, one of which was irradiated, while the other was nonirradiated, during one phase of 5 d, under a split-mouth design.
Fragments were fixed with wax 1 mm below the edge of the palatal appliance to prevent abrasion by

Statistical analysis
We performed sample size calculation considering a maximum error of 5%, obtaining a sample size of 10.
With an addition to the sample size of 20% considering sample loss, we established n=12 for this study.
We performed analysis of the data obtained through profilometry using SPSS 12.0 (SPSS Inc., Chicago, IL, USA) for Windows with a significance level of 5%. A normality test (Kolmogorov-Smirnov) was performed to check data normality. Because the distribution was not normal, we calculated the mean values, and analyzed the data using the Kruskal-Wallis test with the following factors being used for comparison: laser irradiation (irradiated or non-irradiated) and the type of challenge (erosion or erosion associated with abrasion).
We carried out multiple comparisons using the Mann-Whitney test.

Profilometry analysis
Data analysis revealed statistically significant differences between the groups after the in situ phase.
The results are shown in Table 1.
Following the erosive challenges associated with the abrasive challenges, the group that was irradiated with the Er:YAG laser had significant differences in enamel structure loss compared with the non-irradiated group.
The group that suffered only erosion and was irradiated with the Er:YAG laser did not had a significant difference from the enamel structure loss values obtained in the non-irradiated group. Following the initial erosion and after treatment (non-irradiated or irradiated with the Er:YAG laser), we observed no significant difference in enamel loss between the groups.  Figure 4C).
When the specimens were subsequently subjected to the in situ erosive challenges, those that had previously been irradiated with the Er:YAG laser ( Figure 4E) presented characteristics similar to those of the non-irradiated specimens ( Figure 4F), i.e.,

Irradiated/Eroded and abrasioned in intraoral phase
Sound area -Initial erosion area    eroded enamel 9 , thereby decreasing the abrasive process 19 .
When evaluating the effect of the Er:YAG laser in association with the erosive challenges, we verified that there was no increase in acid resistance due to irradiation. This result is in agreement with the findings of an in vitro study by Reis Dercelli, et al. 18 (2015), who also identified no protective effect of an Er:YAG laser (60 mJ, 2 Hz, 3.92 J/cm 2 ) in the control of enamel wear under erosive challenges with Coca-Cola.
The application of subablative parameters can achieve temperatures between 100 and 650°C, which may lead to a reduction of water and carbonate sufficient to alter the crystallinity of the enamel 8 . Deng and Hsu 7 (2005) observed a reduction of carbonate when enamel specimens were irradiated at energies of 5.1 J/cm 2 , which is similar to the energy levels used in our study (5.2 J/cm 2 ). It could be that the cooling that occurred in this study (3 ml/min) caused a reduction of the surface temperature of the tissue, which led only to changes in morphology that contribute to synergy between irradiation with the Er:YAG laser and fluoride toothpaste. This study used parameters that are below the ablation threshold to avoid mechanical damage to the enamel.
In SEM images, we could observe that eroded, nonirradiated specimens had a demineralization pattern with dissolution of the prisms. Following abrasive procedures, we observed a more homogeneous enamel surface, probably due to the removal of the surface layer of the altered prisms, as described by Rios,et al. 20 (2008), which may have contributed to an increased loss of structure in contrast with the group treated with the Er:YAG laser.

Conclusions
According to the results of this study, although the irradiation with the applied Er:YAG laser did not control the progression of lesions during enamel erosion caused by citric acid, it did control the progression of abrasive lesions in previously eroded enamel.