Specimen Preparation

A total of 20 teeth with natural NCCLs located in the cervical region of the buccal surface were used in the experiment. They were randomly divided into two groups (N=10); G1, to be restored with an adhesive system containing 10-MDP, and G2, to be restored with an adhesive system containing methacrylamide. After extraction, the teeth were cleaned with sterile gauze and saline solution. Any remaining periodontal tissues were removed with the aid of periodontal curettes. After cleaning, the teeth were stored in saline at 4°C.

Artificial defects (ADs) in the shape of Class V cavities were created in the lingual surface of the same tooth with a CVDentus (C1, 1.0 × 4.0 mm) cylindrical diamond tip coupled with an ultrasound device (CVDent 1000, CVDVale, São Carlos, Brazil) under continuous water cooling. ADs were prepared in sound dentin with dimensions and shape approximately the same as those of the corresponding NCCL, serving as a control.¹⁵

Then, dental specimens containing the NCCLs and ADs were obtained from each tooth. The teeth were sectioned with a diamond disk at low speed under water cooling in the following sequence: first, just above the lesion to remove the crown, and then, just below the lesion to remove the remaining two-thirds of the root. Finally, a section was made along the long axis of the tooth to separate specimens containing the NCCLs from the ADs. Once the dental specimens were obtained, they were ready to be employed in the sequence of analyses described below (Figure 1).

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Adhesive System Application

The composition of the adhesive systems used in this study and the recommended mode of application are described in Table 1. Light-curing was done with the Translux Power Blue unit (Heraeus Kulzer, Hanau, Germany) at 1000 mW/cm². The light-curing time for each adhesive system was according to the manufacturer's instructions.

Table 1 Composition and Mode of Application of Adhesive Systems Used in the Experiment

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Dentin Mineral Composition

Mineral composition analysis of the dentin in ADs (control) and NCCLs was performed with micro-Raman (MR) spectroscopy. The analysis was conducted with a micro-Raman spectrometer (Bruker Optik GmbH, Ettlingen, Germany), equipped with a Senterra confocal microscope, whose operation is based on infrared light scattering, ie, the source of light irradiation (laser with invisible wavelength) excites the studied matter. In this interaction, the Raman effect is obtained, which allows studying vibrations at the molecular level.

Specimens' spectra were measured at three different points on the dentin surface. All measurements were collected at a resolution of 4 cm⁻¹ in the spectral region between 3500 and 450 cm⁻¹. Each spectrum was obtained from an average of 60 readings to decrease the signal-to-noise ratio, with a laser wavelength of 785 nm, power of 100 mW, and objective gain of 100×. In addition to the high number of readings, the signal pattern was improved by decreasing detector temperature to −84°C. In all readings, the surface area selected for measurement, the support mirror used, and manual focusing followed by autofocusing were performed following the same standardized procedures.

All spectra were placed on the same baseline and normalized with the aid of Opus spectroscopy software (Bruker Optics, Ettlingen, Germany). Additionally, origin software (OriginPro 8 Corp, Northampton, MA, USA) was used to obtain numerical quantifications of MR spectra by integrating each curve of the band at 961 cm⁻¹ (phosphate) to calculate the respective area and mean of the three distinct points measured in the sound dentin of the AD specimens and in the sclerotic dentin of the natural NCCLs.

Adhesive/Dentin Chemical Interactions

Specimens' spectra were measured with Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) both before and after being submitted to the adhesive treatment. The technique provides the optical absorption bands of the sample, which are considered the fingerprint of specific molecules. Information on the chemical modifications within the specimen is expressed by means of changes and/or emergence of new peaks.

The experiments were performed with a Nicolet Spectrometer (MTEC Photoacoustics, Ames, IA, USA) equipped with a MTEC 200 photoacoustic cell model. This equipment allows monitoring the absorption of the substance of interest at specific specimen depths, providing the distribution profile of the substances along the thickness studied. All spectra were collected at a resolution of 8 cm⁻¹, with scanning speed of 0.5 cm/s. The spectral region of the measurements lies in the energy range between 4000 and 400 cm⁻¹. After the specimen was inserted, the photoacoustic cell was filled with helium to minimize interference on the optical absorption spectra of oxygen and water molecules present in the air and on the surface of the specimen.

To determine the depth of the analysis in the present experimental condition, FTIR-PAS test measurement depth was defined by the thermal diffusion length. The thermal diffusivity of the adhesive was measured by the thermal lens technique¹⁶ as described by Oliveira and others.¹⁵ The inspection depth of the technique for the readouts taken in this study was about 6 μm for G1 and 4 μm for G2. Since the adhesive film depth can range from 2-3 μm,^17,18 this technique enabled reading not only the hybrid region, but also the dentin under the adhesive.

To evaluate the spectrum of the adhesive system, a disc of pure adhesive was prepared, applying 1 mL of the material on a histological glass slide. After photoactivation for 20 seconds, the disc was inserted into the measuring equipment. This is an important step to differentiate the composition of the adhesive from that of the dental structure and to verify the differences between photoacoustic absorption peaks of the adhesive and dentin.

Collected data were transferred to the origin software. Graphs were generated for each tooth individually, from which the average spectrum of ADs and natural NCCLs dentin specimens were calculated. Bands identified as chemical interactions between the dentin and the adhesive system were selected, and the respective intensities in the ADs and natural NCCLs before and after the application of the adhesive were compared.

Degree of Demineralization

After FTIR-PAS analysis, all cavities were filled with composite resin (Filtek Z250, 3M ESPE, St Paul, MN, USA) and subsequently sectioned for MR scanning analysis. Dental specimens were cut longitudinally with a sectioning machine (Isomet 1000 Precision Saw, Buehler, Lake Bluff, IL, USA) using a diamond disc (Diamond Wafering Blade, Series 15LC, Arbon size ½, 12.7 mm, Buehler) under water cooling.

MR spectra were obtained by scanning the inner region of the composite resin toward the deeper layers of the dentin in NCCLs and ADs. All spectra were obtained under the same conditions as previously described for mineral composition analysis. Raman spectra were acquired at 1-μm intervals in 20-μm long lines at a distance of 10 μm between lines. Scans were individually analyzed, and spectra reading presenting any flaws, possibly due to the presence of bubbles, were excluded from the analysis. All spectra were placed on the same baseline and normalized with the aid of the Opus spectroscopy software, and analyses were conducted with the origin software. Degree of demineralization (DD), as a function of location, was determined at the bands at 961 cm⁻¹ (PO₄ of the dentin) in relation to the band at 1458 cm⁻¹ (CH₂), according to the equation:

!--$$\text{DD}=\left(1-\frac{961c{m}^{-1}intensityinterface/1458c{m}^{-1}intensityinterface}{961c{m}^{-1}intensitydentin/1458c{m}^{-1}intensitydentin}\right)\times 100\%$$-->

Interface Morphology

Analysis of the adhesive system/dentin substrate interface morphology was performed with scanning electron microscopy (SEM). Sample treatment was performed according to the protocol suggested by Monticelli and others.¹⁹ After MR analysis, the samples were embedded in acrylic resin and polished with a wet sandpaper sequence (220, 400, 600, 1200, 1800, 2000 grit). Then the specimens were submitted to demineralization with 37% phosphoric acid for 10 seconds, deproteinization with 2% sodium hypochlorite for 1 minute, and dehydration with 100% alcohol for 2 minutes in an ultrasonic vat (Bio-Free-Gnatus, Ribeirão Preto, Brazil) and air jets. Specimens were sputter-coated with gold and evaluated with SEM (Shimadzu, Model SS-550 Superscan, Kyoto, Japan) with a magnification of 1000×.

Collagen Fiber Exposure

To examine collagen fiber exposure at the adhesive system/dentin interface, four additional teeth were prepared according to the protocol described above, up to the application of the adhesive system, and histologically analyzed under light optical microscopy.

Immediately after application of the adhesive system, samples were fixed in Karnovsky's solution (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.3) for 48 hours and washed in running water for 4 hours. The samples were placed in a decalcifying solution (20% sodium citrate and 50% formic acid) for 25 days. After that, the specimens were washed in running water for 4 hours, dehydrated in an increasingly concentrated alcohol sequence, and embedded in paraffin. Histological sections, 6 μm thick, were serially made using a tungsten carbide blade coupled with a microtome (Leica RM2265, Leica Microsystems, Wetzlar, Germany).

Goldner-modified Masson Trichrome^19-21 was used to stain the specimens. Before staining, the samples were prepared according to the protocol proposed by Wang and Spencer.²⁰ The sections were first stained with Weigert's iron hematoxylin solution for 5 minutes, immersed in Masson solution for 10 minutes, and rinsed twice in 0.2% acetic acid solution. Afterward, they were kept in mordant solution for 5 minutes and washed again with 1% acetic acid. Finally, they were stained with a light green solution for 5 minutes and rinsed twice with 0.2% acetic acid. For sample dehydration, the sections were immersed in 96% and 100% alcohol twice for 1 minute. Specimens were immersed twice in xylol for 3 and 5 minutes and mounted within histological slides. Sections were examined and photographed at a magnification of 100× under a light optical microscope (Olympus BX41, Tokyo, Japan).

Statistical Analysis

Statistical analysis was performed using the R i386 3.0.2 software (R statistical software, R Foundation for Statistical Computing, Vienna, Austria). The areas of the band at 961 cm⁻¹ (PO₄) for the phosphate present in the dentin, the intensities related to the chemical interactions found with FTIR-PAS, and DD were submitted to the Shapiro-Wilk and Student t-test (p<0.05).

Adhesive/Dentin Chemical Interactions

Figure 2A shows the spectrum of the pure adhesive system containing the 10-MDP monomer (G1) with its organic and inorganic functional groups: methacrylate monomer (carbonyl C=O [1720 cm⁻¹], CH₂CH₃ [1457 cm⁻¹]), BIS-GMA (C=C [1638 cm⁻¹], C-O-C [1140 cm⁻¹], [CH₃]₂-C [1300 cm⁻¹], C₆H₄ [840 cm⁻¹]), and load (SiO₂ [1105 cm⁻¹]). Figure 2B shows the dentin spectra of the natural NCCLs and ADs. The bands associated with mineral and organic composition were observed at the following wavenumbers: phosphate (1179 cm⁻¹), amide I (1650 cm⁻¹), amide II (1550 cm⁻¹), amide III (1240 cm⁻¹).

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Figure 3A and B illustrate the NCCL spectra before and after application of the adhesive system containing the 10-MDP monomer (G1). Circles and bars indicate changes in spectra, suggesting chemical interactions between the dentin and the adhesive system. Table 2 shows the bands and functional groups identified as possible chemical interactions between the dentin and the adhesive system containing 10-MDP monomer and the means and standard deviations of the intensities obtained from the ADs and natural NCCLs characterizing chemical interactions.

Table 2 Means and Standard Deviations (SDs) of the Intensities of the Bands That Characterize Chemical Interactions in ADs and Natural NCCLs With the Adhesive Containing the 10-MDP Monomer (G1)

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Although no statistically significant differences were observed between the two groups, mean values for natural NCCLs in all band intensities were numerically higher than those of ADs.

Figure 4A shows the photoacoustic absorption spectra of the pure adhesive containing the methacrylamide monomer (G2), with the following functional groups: CH₂ (1456 cm⁻¹), CH₃ (1373 cm⁻¹), PO₂ (1265 cm⁻¹), CH (1139 cm⁻¹), C-O-C (1040 cm⁻¹), and Al-OH (920 cm⁻¹). Figure 4B shows the dentin in the natural NCCLs before and after application of the adhesive system, suggesting a change in the inorganic components such as phosphate and calcium. However, no indication of chemical interactions can be observed.

Dentin Mineral Composition

Means and standard deviations of the measured spectra obtained from the integrated areas at the band at 961 cm⁻¹ (PO₄) on the surface of the ADs and natural NCCLs are shown in Table 3. The Student t-test showed that the mineral content in dentin in natural NCCLs was significantly higher than that found in the ADs (p<0.05).

Table 3 Means and Standard Deviations of the Integrated Areas of the Band at 961cm⁻¹ (PO₄) of ADs and Natural NCCLs in arbitrary units (a.u.)

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Degree of Demineralization (DD)

MR scanning analysis of the natural NCCLs and ADs with the adhesive systems used are represented by Figures 5 and 6. It can be observed that, in G1, the behavior in natural NCCLs (Figure 5A) and ADs (Figure 5B) was similar. However, in G2, the dentin in the ADs (Figure 6A) underwent deeper demineralization than did the natural NCCLs (Figure 6B).

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Means of the DD (%) of the dentin using the systems G1 and G2 are presented in Table 4. The DD in G1 had similar behavior in the first 2 μm of the hybrid layer, whereas from 3 μm, a statistically significant difference (p<0.05) between the ADs and natural NCCLs was observed. In G2, DD presented no statistically significant differences between ADs and natural NCCLs.

Table 4 Degree of Demineralization (%) in ADs and NCCLs in Groups G1 and G2

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Interface Morphology

Scanning electron microscopy analysis showed differences in the adhesive system/dentin substrate interface between G1 and G2 (Figure 7). In G1, images demonstrated similar projections of the adhesive within the demineralized dentin in ADs (Figure 7A) and natural NCCLs (Figure 7B), although the sclerotic aspect of the dentin with greater mineralization can be identified in the natural NCCLs. In G2, when applied to ADs (Figure 7C), the methacrylamide-containing adhesive provided greater inter- and peritubular demineralization, with well-defined projections within the dentin. On the other hand, the interface in the natural NCCLs (Figure 7D) was indefinite and more obliterated because of the higher degree of mineralization, compromising the formation of a hybrid layer due to irregular primer diffusion and reduced adhesive infiltration, leading to failure of the restoration (indicated by the arrow in Figure 7D).

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Collagen Fiber Exposure

Photomicrographs of the samples stained with Goldner's Modified Masson's Trichrome are shown in Figure 8. The dentin appears stained in green, the exposed unprotected collagen is evidenced in red/pink, while pure adhesive is unstained.^22,23

In G1, histomorphological analysis demonstrated a thin layer (pink) of exposed collagen in the dentin of the ADs (Figure 8A). In the natural NCCLs (Figure 8B), collagen was hardly evident. In G2, the photomicrographs illustrate deeper demineralization of the dentin and more pronounced collagen exposure (dark red) in the ADs (Figure 8C) as well as natural NCCLs (Figure 8D).