**2. Results**

Figure 1 shows the results of CeO2 particles' characterization. In Figure 1A, the X-ray diffraction analysis of the powder shows the crystallinity pattern of a monoclinic phase of CeO2 (powder diffraction file, ICDD-PDF 37-1468). In Figure 1B, the chemical composition analyzed by Fourier Transform Infrared Spectroscopy (FTIR) displays the peaks related to Ce–O bonds at 400 cm<sup>−</sup>1. Figure 1C,D presents the results of size distribution analysis. The histogram shows a non-normal distribution of size (Figure 1C). The 10th percentile showed a particle diameter of 1.15 μm. The median (50th percentile) was 14.98 μm, the 90th percentile was 31.32 μm, and the mean particle size was around 16 μm (Figure 1D).

Figure 2 presents the qualitative assessment via micro-Raman of the materials' surfaces containing 0.36 (A) and 0.76 (B) vol.% of CeO2. The image consists of a 2-D array of measured spectra, which means that the distribution of the chemical composition can be investigated. After mapping the polymer's surfaces, the integration of the corresponding CeO2 peak (464 cm<sup>−</sup>1) was used for analysis and graphs generation. In the graphs, from blue to yellow, more CeO2 is identified. It was observed that the higher the load of CeO2 in the resin, the larger the area under the curve (peak) in micro-Raman spectra for this oxide, generating more yellow regions in the graph. In these analyses, more areas in yellow were presented in the adhesive with 0.76 vol.% of CeO2 in comparison to the group with 0.36 vol.%.

**Figure 1.** CeO2 particles' characterization: (**A**) X-ray diffraction analysis shows the pattern of the monoclinic phase of crystallinity for CeO2; (**B**) FTIR analysis displays the peaks related to Ce-O bonds at 400 cm<sup>−</sup>1; (**C)** and (**D**) show the non-normal distribution size of CeO2 and the values of size distribution.

**Figure 2.** Micro-Raman analysis of the dental adhesive surfaces containing 0.36 (**A**) and 0.76 (**B**) vol.%ofCeO2.BothimagesdisplaytheintensityoftheCeO2peakat464cm<sup>−</sup>1. The higher the load of CeO2

 in the dental adhesive, the more areas in yellow are observed.

Figure 3 indicates the results of the radiopacity of each experimental adhesive resin containing a different percentage of volume fraction of CeO2. In Figure 3A, an illustrative radiograph displays the location of the dental adhesive layer underneath the composite restorative material. The arrows indicate the radiolucent areas corresponding to the adhesive layer. The difference of radiopacity between the composite resin and the adhesive layer can be clearly observed. In Figure 2B, the mean and standard deviation of radiopacity is expressed in mm of aluminum. The control group, which is an unfilled adhesive, showed the lower mean value of radiopacity, without statistical difference for the groups with 0.36 vol.% and 0.72 vol.% of CeO2. From the addition of 1.44 vol.% of CeO2, there was increased radiopacity of the adhesive in comparison to the control group (*p* < 0.05). The group with 5.76 vol.% of CeO2 showed the highest value of radiopacity among all groups (*p* < 0.05). The incorporation of 4.32 vol.% and 5.76 vol.% presented values of more than 1 mm of aluminum.

**Figure 3.** The dental radiograph displays the layer of dental adhesive applied under the composite resin (**A**) The arrows guide the visualization of the radiolucent adhesive layer. (**B**) The radiopacity of the experimental dental adhesives according the to the increasing concentration of cerium oxide. Different letters indicate statistical differences among groups (*p* < 0.05).

Figure 4 shows the results of the degree of conversion analysis of the experimental dental adhesives. The uncured adhesive samples were directly dispensed on the attenuated total reflection (ATR) device of FTIR to analyze the conversion of carbon-carbon double bonds in the aliphatic chain. The values ranged from 61.52 (±0.33) % for the control group to 47.90 (±1.64) % for 5.76 vol.% of CeO2. From the incorporation of 2.88 vol.% of CeO2, the degree of conversion reduced in comparison to the control group (*p* < 0.05). The lowest value of the degree of conversion was found for the highest load of CeO2 incorporated in the adhesive (*p* < 0.05).

**Figure 4.** Degree of conversion of the experimental dental adhesives containing CeO2. Different letters indicate statistical differences among groups (*p* < 0.05).

## **3. Discussion**

In this in vitro study, CeO2 particles were explored as potential radiopacifier for dental adhesives since high atomic weight and density can provide a suitable level of radiopacity. CeO2 was first chemically characterized by XRD, Raman, and FTIR to be incorporated for the first time in a dental adhesive resin. In the current investigation, CeO2 successfully increased the radiopacity of the adhesive, maintaining a suitable degree of monomer conversion.

CeO2 particles used in this study presented a monoclinic crystalline phase with particular chemical groups and the average particle size of around 16 μm. This oxide was incorporated in an experimental adhesive resin formulated as previously reported, with conventional dental methacrylate monomers [19,20]. The presence of a radiopacifying agen<sup>t</sup> is essential to enable the identification of restorative materials and dissimilarity from pathological processes in the adjacent areas [21]. Recurrent caries and marginal gaps are very often the reasons for the replacement of composite restorations [22]. The misdiagnosis and treatment decision for unnecessary replacement of restorations lead to additional loss of sound tooth tissue, with an increased cost and discomfort for the patient [23]. Therefore, the restorative materials should have optimal radiopacity for accurate radiographic di fferentiation for existing restorations and recurrent dental caries, supporting clinical follow-ups. Substantial changes in radiopacity are compulsory by the International Organization for Standardization (ISO) for dental restorative materials [24]. The compliance with the ISO 4049 requires a minimum radiopacity of restorative materials higher than that of dentin and greater or equivalent thickness of aluminum (with ≥98% purity) [24]. In the present study, the increased load of CeO2, by the percentage of volume, was added to an experimental dental adhesive, providing a material with proper radiopacity. Our results have shown that CeO2 at 4.32 vol.% had radiopacity higher than 1 mm of Al.

Besides the increase of radiopacity, the addition of inorganic fillers in monomeric blends may improve polymers properties, such as the elastic modulus, tensile strength, fracture toughness, Knoop hardness, and stability against solvents [25]. However, the higher filler addition can also decrease the degree of monomer's conversion [19]. The degree of conversion is a valuable chemical property for polymers, and it may be associated with the stability of the restoration over time [20]. Therefore, a reliable polymerization is desired for adhesive resins to reduce hydrolytic degradation in the clinical setting [26,27]. Here, we observed that the incorporation of CeO2 at a load that reaches the high radiopacity level reduced the degree of conversion of the dental polymer. Previous studies have reported similar outcomes in the investigations of radiopacifying agents containing di fferent kinds of heavy metals [28]. Marins et al. [29] evaluated the addition of niobium pentoxide (Nb2O5) nanoparticles to the dental adhesive as radiopacifiers and observed that the degree of conversion decreased with the addition of particles at percentage mass fraction equal or greater than 10%. In other studies, Nb2O5 and Ta2O5 showed a decrease in the degree of conversion up to 5 wt.% [19,20]. Our results are also in agreemen<sup>t</sup> with those of Amirouche-Korichi et al. [28], where progressive decreases of the degree of conversion were linearly related to the filler contents.

The outcome observed for the degree of conversion may be attributed to the high refractive index of CeO2 (approx. ղ = 2.2 to 2.8) [30], which may have decreased the accessibility of light energy inside the polymer. The limited mobility of monomer chains by the incorporation of the opaque fillers has also been considered as a contributing factor for the decrease in the monomer's conversion [31]. Another consideration for observed decay in the degree of conversion with regard to the dental adhesive with loadings higher than 2.88 vol.% relies on particle size. The fillers used in our study are micro fillers with an average size of around 16 μm. Previous reports support the e ffect of particle size in microns and highlight that the volume occupied by the particles may compromise the polymerization rates of the material [32–34]. Further evaluation of the polymerization behavior of nanosized CeO2 could be interesting to address this subject. Moreover, it would be noteworthy to evaluate the e ffect of micro-sized CeO2 in the adhesive with thinner samples or in situ in dentin.

Moreover, we sugges<sup>t</sup> further biological studies on dental adhesives containing CeO2 in micro and nanoscale, since this oxide has emerged as a noteworthy agen<sup>t</sup> for bioactive (such as sca ffolds and bioglasses) and antimicrobial materials [35–37]. In this context, a dental adhesive with such properties could be a promising strategy to assist in decreasing the incidence of recurrent caries and improving the remineralization process after selective removal of carious dentin. In this study, despite the decreased degree of conversion observed by increasing CeO2 addition, all groups showed values around 50%, which is in accordance with commercial dental adhesives [38]. Therefore, CeO2 may be a promising alternative filler for biopolymers.

#### **4. Materials and Methods**

#### *4.1. X-ray Di*ff*raction Analysis of CeO2*

CeO2 particles purchased were analyzed via X-ray di ffraction to detect the crystalline phases of the powder. di ffractometer (PW 1730/1 model, Philips, Santa Clara, CA, USA) was operated at 40 kV and 40 mA with CuKa radiation. The scanning rate used was 0.058/min, with 2 s of time-steps at 0.02◦ each, from 5◦ to 60◦ [18].

#### *4.2. FTIR Analysis of CeO2*

Fourier Transform Infrared Spectroscopy (FTIR) was used to chemically characterize the powder of CeO2. The analysis was performed using the spectrophotometer Vertex 70 (Bruker Optics, Ettlingen, Germany) with an attenuated total reflectance device (ATR). CeO2 powder was placed on the ATR, and the analysis performed using 20 scans, 4 cm<sup>−</sup><sup>1</sup> of resolution in 5000 to 400 cm<sup>−</sup>1, and Opus 6.5 software (Bruker Optics, Ettlingen, Germany).

#### *4.3. Particle Size Distribution of CeO2*

CeO2 particles were dispersed in water with sonication for 60 s. Then, particle size was analyzed via laser di ffraction particle size analyzer (CILAS 1180, Cilas, Orleans, France) according to a previous study [18].

#### *4.4. Preparation of Dental Adhesives*

The adhesive resins were formulated by mixing 50 wt.% bisphenol A glycol dimethacrylate (BisGMA), 25 wt.% triethylene glycol dimethacrylate (TEGDMA), and 25 wt.% 2-hydroxyethyl methacrylate (HEMA). As a photoinitiator system, camphorquinone and ethyl 4-dimethylaminobenzoate were added at 1 mol%, each one in the base resin. As polymerization inhibition, 0.01 wt.% of butylated hydroxytoluene was incorporated. CeO2 was added at 0.36, 0.72, 1.44, 2.88, 4.32, and 5.76 vol.%. The resins adhesives were hand-mixed for 5 min, sonicated during 180 s, and hand-mixed again for 5 min. One group without CeO2 was used as control.

#### *4.5. Qualitative Analysis of CeO2 into Dental Adhesives*

To identify the presence of CeO2 in dental adhesives, two groups containing this filler were evaluated via micro-Raman Spectroscopy (Senterra, Bruker Optics, Ettlingen, Germany). One sample from the group with 0.36 vol.% and another from the group with 5.76 vol.% were prepared using a polyvinylsiloxane mold. The samples were photoactivated for 20 s on each side using a light-emitting diode dental curing unit (Radii Cal, SDI, Melbourne, Australia) with 1200 mW/cm2. An area of 110 μm × 110 μm of the surfaces was analyzed via Opus 7.5 software (Opus 7.5, Bruker Optics, Ettlingen, Germany). The analyses were performed with a wavelength of 785 nm, with 5 s and two co-additions. The peak correspondent to CeO2 (464 cm<sup>−</sup>1) was used for integration.

#### *4.6. Radiopacity Evaluation*

The adhesive resins were evaluated for their radiopacity, according to the International Organization of Standardization (ISO) 4049/2009 guidelines [24]. Five samples per group (n = 5) with disc-shaped were prepared with 10.0 mm (±0.5 mm) in diameter and 1.0 mm (±0.1 mm) in thickness with photoactivation for 20 s on each side. The images were made using a phosphor plate for the digital system (VistaScan; Dürr Dental GmbH & Co. KG, Bietigheim-Bissingen, Germany) at 70 kV, 8 mA, and 0.4 s of exposure time. A focus-film distance of 400 mm was used in the assays. One specimen per group was positioned on the film for each X-ray exposition. An aluminum step-wedge (99.12 wt.% of aluminum, thickness from 0.5 mm to 5.0 mm, in increments of 0.5 mm) was exposed with the samples for each image specimens in all images. The images were saved in TIFF, analyzed using Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA), and the mean and standard deviation of the grey levels (pixel density) were measured.

#### *4.7. Degree of Conversion*

To analyze the degree of conversion of the adhesives, FTIR-ATR was used according to a previous study [39]. Three drops per group (n = 3, 3 μL each one) were directly dispensed on ATR diamond crystal. An adjustable holder stand was used to fix the light-curing unit and to standardize the distance between its tip and the top of the samples. Two spectra were acquired per group from 4000 to 400 cm<sup>−</sup><sup>1</sup> with a resolution of 4 cm<sup>−</sup>1. The first spectrum was obtained before the photoactivation. Then, the samples were photoactivated for 20 s and analyzed again. To calculate the monomer conversion, the peak related to the carbon-carbon (C=C) double bond in the aromatic ring of BisGMA (1610 cm<sup>−</sup>1) was used as an internal standard. The peak related to C=C in the aliphatic chains (1640 cm<sup>−</sup>1) was used along with the C=C at 1610 cm<sup>−</sup><sup>1</sup> according to the following equation:

$$\text{Degree of conversion} \ (\%) = 100 \times \left( \frac{\text{I\\_1\'{640} - \text{current}/\text{I\\_1}\,1610 - \text{current}}{\text{I\\_1}\,1640 - \text{uncured}/\text{I\\_1}\,610 - \text{uncure}} \right)$$

## *4.8. Statistical Analysis*

CeO2 characterization was descriptively analyzed, as well as the identification of CeO2 in the polymerized adhesives. Shapiro-Wilk test was used to evaluate the data normality. The data of radiopacity was evaluated via ANOVA on ranks and Dunn's test. The data of the degree of conversion was analyzed via one-way ANOVA and Tukey's post-hoc test. Both analyses were performed at 0.05 level of significance (*p* < 0.05).
