1.

Introduction

Etching is one of the most fundamental steps in the restoration of teeth by adhesion of composite resin. Acid etching on dentin with phosphoric acid is effective for removing the smear layer and also for enlarging the dentin tubules or decalcifying the superficial intertubular dentin.¹ The smear layer is the accumulated layer of mechanically polished debris. Etching is utilized as a pretreatment before adhesion of composite resin for removal of the smear layer. The technique exposes fresh tooth surfaces and facilitates direct contact of the adhesive and hydrogen bonding. Etching also enhances mechanical bonding through an interlocking effect in the roughened interface between the resin and the tooth.²

With the development of new adhesive materials, alternatives for dental structure conditioning have appeared as well. One of these innovations for substrate surface treatment is the use of erbium-doped yttrium-aluminum-garnet (Er:YAG) laser irradiation.³ Laser irradiation on dental hard tissue causes morphological and chemical changes. The extent of these changes is affected by the absorption characteristics of the tissues, so that the changes vary according to the type of laser and dental tissue.⁴

Dentists have yet to reach consensus regarding the correct parameters for Er:YAG laser use. The benefits of dentin laser etching prior to adhesive procedures are still controversial and the results of several studies are conflicting.^{5, 6, 7, 8, 9} Some studies report that the adhesion strength of laser-irradiated dentin is lower than that of nonirradiated dentin^{10, 11, 12} due to the weaker mechanical properties of dentin after laser irradiation.¹² Some authors have also discussed the possible denaturation of collagen fibrils.^{3, 12} We modified the laser-irradiation parameters used in a previous investigation³ to determine whether it was possible to etch dentin without causing significant damage on inorganic and organic dentin components.

For the in vitro study, the chemical characteristics of teeth following specimen preparation must be carefully considered. An important factor that could affect the chemical content of teeth is the disinfection/sterilization method used to prepare the extracted teeth specimens.^{13, 14} Several studies have evaluated the effects of sterilization methods on tooth tissues. As steam autoclaving is available in dental clinics; it is the easiest sterilization method.¹⁵ Despite its damage to collagen,¹³ autoclaving sterilization is still used in some studies.¹⁶ In other studies the methods used for tooth decontamination and storage treatment are not mentioned.⁶

The influence of tooth sterilization methods has been evaluated by analytical tools such as Fourier transform infrared spectroscopy (FTIR),¹⁷ bond strength study,¹⁸ and Raman spectroscopy.¹⁴ However, there are no previous reports of X-ray fluorescence microanalysis used to analyze sterilization effects on dentin components.

X-ray fluorescence is a nondestructive analytical technique based on the atomic emission of a given material’s components when irradiated. The most common irradiation method utilizes an electron beam coupled with a scanning electron microscope (SEM).^{19, 20} The interaction of the electron beam with the sample surface causes X-rays to be emitted by the atoms and ions in the top few micrometers of the sample surface. An electron is ejected from an inner shell of the atom; when an outer shell electron takes the place of the missing electron, energy is emitted in the form of X-ray radiation.²¹

Compositional changes due to laser irradiation were evaluated by atomic analytical studies in previous reports. Rohanizadeh showed through X-ray fluorescence analysis of dentin that Nd:YAG laser irradiation reduced the calcium/phosphorus $(\mathrm{Ca}∕\mathrm{P})$ ratio in comparison to nonirradiated specimens.⁴ Lin ¹⁹ used SEM-energy-dispersive X-ray spectroscopy (EDX) to evaluate Nd:YAG laser irradiation of dentin at a range from $150\mathrm{mJ}$ /pulse, $10\mathrm{pps}$ , $4\mathrm{s}$ , to $150\mathrm{mJ}$ /pulse, $30\mathrm{pps}$ , $4\mathrm{s}$ . The authors verified that the $\mathrm{Ca}∕\mathrm{P}$ ratio of the irradiated area increased proportionally with the elevation in irradiation energy.

This analytical tool has also been used to study the chemical composition of dentin after Er:YAG laser irradiation.^{20, 22} Hossain ²⁰ showed through atomic analysis by SEM-EDX that the quantities of Ca and P (weight percent) were increased in cavity floors prepared by the erbium, chromim doped yttrium, scandium, gallium, garnet (Er,Cr:YSGG) laser. However, in these studies, the laser parameters employed to prepare the cavities used higher energies than those used to etch the dentin. Analysis was conducted using a combination of SEM and X-ray fluorescence. Therefore, to the best of our knowledge, X-ray fluorescence microanalysis and chemical mapping of the dentin surface after etching have not yet been completed.

The aim of this in vitro study was to investigate, using energy-dispersive X-ray fluorescence spectrometry (EDXRF), the effects of Er:YAG laser irradiation on dentin components. The effects of decontamination and storage processes on dentin components were also evaluated.

2.

Materials and Methods

3.

Results and Discussion

All experimental samples showed lower $\mathrm{Ca}∕\mathrm{P}$ weight ratios than those of stoichiometric hydroxyapatite, which is calculated as 2.15. This observation indicates that the dental hydroxyapatite (as biological hard tissue) is nonstoichiometric, as reported in the literature.²³ Statistical comparisons of Ca and P content as well as $\mathrm{Ca}∕\mathrm{P}$ weight ratio were performed for normal and treated specimens in all experimental groups (Table 2, horizontal comparisons). Statistical analysis showed that the amounts of Ca (Ca weight %) and P (P weight %) decreased significantly in the autoclaved specimens and in those treated with phosphoric acid (A̱CG), as compared with the autoclaved untreated specimens (Table 3, rows 1 and 2). Therefore, for this group the weight ratio $(\mathrm{Ca}∕\mathrm{P})$ increased significantly (Table 3, row 4). However, no significant differences were found between the quantities of Ca and P, in gram atom percentage, before and after treatments in the specimens stored in thymol and treated with phosphoric acid (ḆCG) $(\mathrm{P}>0.05)$ .

Table 2

Mean and standard deviations (n=15) of calcium and phosphorus percentages and Ca∕P weight ratios obtained by X-ray fluorescence, before (normal) and after (treated) dentin surface treatment.  *

Table 3

Results of Mann–Whitney unpaired-sample test.

Lower quantities of phosphorus were found in the acid-etched specimens that had been autoclaved than in those treated with thymol (Table 3, row 3). The $\mathrm{Ca}∕\mathrm{P}$ weight ratio increased significantly for the acid-etched specimens that had previously been autoclaved as compared to those treated with thymol (Table 3, row 5).

Differences among etching treatments were found only in the autoclaved specimens, as shown by the Tukey–Kramer test. Calcium levels were lower in the acid-etched specimens than in those subjected to laser irradiation of $120\mathrm{mJ}$ $(\mathrm{P}<0.01)$ (Table 2, column 3). Phosphorus levels were lower in the acid-etched specimens than in those subjected to laser irradiation of $80\mathrm{mJ}$ $(\mathrm{P}<0.05)$ , $120\mathrm{mJ}$ $(\mathrm{P}<0.001)$ , and $180\mathrm{mJ}$ $(\mathrm{P}<0.01)$ (Table 2, column 5). The $\mathrm{Ca}∕\mathrm{P}$ weight ratio was higher in the acid-etched specimens than in those that were laser-irradiated $(\mathrm{P}<0.01)$ (Table 2, column 7).

The results of the elemental analysis in the present study showed that the amounts of calcium (Ca weight %) and phosphorus (P weight %) were reduced in the autoclaved specimens treated with 37% phosphoric acid $\left(15\mathrm{s}\right)$ . The calcium-to-phosphorus weight ratio also increased significantly. Acid etching dissolved peritubular and intertubular dentin, significantly removing chemical compounds containing Ca and P. Heat and pressure effects ( $121°\mathrm{C}$ and pressure of $1.1\mathrm{kgf}∕{\mathrm{cm}}^2$ ) likely explain the intense effect observed in autoclaved specimens as opposed to those treated with thymol. For this group (A̱CG), the results are in agreement with previous studies obtained by dispersive Raman spectroscopy.¹⁴ White ¹⁷ used FTIR spectroscopy to investigate the sterilization effects of steam autoclaving on whole tooth roots. They found that this treatment induced a loss of mineral and collagen components (as shown by reduction in amine and phosphate peaks) from the sample surface. Notably, dentin tissue releases its organic material at temperatures ranging from $100\text{to}400°\mathrm{C}$ and begins to lose carbonate at $100°\mathrm{C}$ .²⁴

When acid-etching treatment with phosphoric acid was compared to Er:YAG laser irradiation in specimens that had previously been autoclaved, it was observed that the acid removed more calcium than laser treatment with $120\mathrm{mJ}$ $(\mathrm{P}<0.01)$ . Acid removed more phosphorus than laser irradiation with $80\mathrm{mJ}$ $(\mathrm{P}<0.05)$ , $120\mathrm{mJ}$ $(\mathrm{P}<0.001)$ , and $180\mathrm{mJ}$ $(\mathrm{P}<0.01)$ . The $\mathrm{Ca}∕\mathrm{P}$ weight ratio also increased significantly in the acid-treated specimens, as compared to those that were laser-treated. This trend was not observed for the thymol-treated specimens, further demonstrating the impact of high-temperature autoclave treatment. Our study also showed that the amounts of Ca and P (weight %) increased in dentin irradiated with an Er:YAG laser. This result probably stems from the evaporation of organic components.^{4, 20}

With regard to Ca and P content, the mapping of treated dentin showed an elemental uniformity in the control group (CG) specimens, when comparing groups A and B [Figs. 1a and 2a (Ca) and 3a and 4a (P)]. Isolated regions with blue spots in the GI and GII areas were observed for both decontamination treatments (groups A and B). This is shown in Figs. 1b, 2b, 1c, 2c (Ca) and 3b, 4b, 3c, 4c, (P) and indicates lower calcium and phosphorus concentration than that found in the CG area. A significant demineralization (dark blue areas), as shown in Figs. 1d (Ca) and 3d (P), was found in the GIII̱A area when compared to the GIII̱B area, as shown in Figs. 2d (Ca) and 4d (P).

Fig. 1

Calcium mapping results obtained by X-ray fluorescence of specimens in group A with each subgroup of treatment: (a) CG, (b) GI, (c) GII, and (d) GIII.

Fig. 2

Calcium mapping results obtained by X-ray fluorescence of specimens in group B with each subgroup of treatment: (a) CG, (b) GI, (c) GII, and (d) GIII.

Fig. 3

Phosphorus mapping results obtained by X-ray fluorescence of specimens in group A with each subgroup of treatment: (a) CG, (b) GI, (c) GII, and (d) GIII.

Fig. 4

Phosphorus mapping results obtained by X-ray fluorescence of specimens in group B with each subgroup of treatment: (a) CG, (b) GI, (c) GII, and (d) GIII.

Elemental mapping by EDXRF showed that the autoclaving treatment caused greater demineralization after acid etching and laser irradiation than treatment with thymol solution. Acid etching produced demineralization that was chemically homogeneous compared to that induced by laser treatment, as shown by the yellow areas in the mapping. Surface distributions of calcium and phosphorus were more homogeneous in the acid-treated specimens of the group decontaminated with thymol than in the autoclaved samples. Laser treatment produced irregular patterns of Ca and P distribution throughout the dentin surface; $180\mathrm{mJ}$ of laser energy produced higher demineralization in autoclaved specimens compared to lower laser energies and compared to the specimens treated with thymol. Previous reports of Er:YAG laser etching on dentin, subjected to SEM analysis, showed a scaly, flaky surface of highly irregular shape with partially opened dentin tubules. In contrast, acid-etched dentin retained a smoother surface with opened dentin tubules.^{3, 5, 8, 9} Our results are in agreement with those previous reports; we found irregular patterns of elemental chemical distribution throughout the dentin surface after Er:YAG laser irradiation.

The surface produced by laser etching is also acid-resistant. According to Secilmis,²⁵ laser irradiation of dental hard tissues modifies the $\mathrm{Ca}∕\mathrm{P}$ ratio and leads to the formation of a more stable and less acid-soluble structure. The alterations in chemical composition may affect the permeability and solubility characteristics of dentin, as well as the adhesion of dental materials to dentin.

In summary, EDXRF analysis proved an adequate research tool to study the changes that occurred in dentin following decontamination or storage and laser or chemical etching. Sample preparation is fairly simple, since it does not require a specific tooth size or dehydration procedure. The measurements can be performed under normal atmospheric conditions, without the need for high vacuum. Finally, because this technique is nondestructive, samples can be used in multiple analyses.

4.

Conclusions

The results of EDXRF measurements showed that the amounts of Ca (Ca weight percent) and P (P weight percent) decreased significantly in the specimens that were autoclaved and treated with phosphoric acid (A̱CG). The results suggest that thymol storage treatment is advised for in vitro studies. Er:YAG laser etching at lower laser energies did not produce significant changes in dentin components. The EDXRF mapping data support the hypothesis that the acid etching on dentin produced a more chemically homogeneous surface, yielding a more favorable surface for the diffusion of adhesive monomers. These findings elucidate the chemical structure of dentin after laser etching, facilitating the development of effective guidelines for laser use.