The LC-MS analysis was performed using a LC-Orbitrap-FTMS system consisted of an Accela 600 HPLC system combined with an Exactive (Orbitrap) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) including on-line DAD (200–600 nm) and UV at 280 nm analysis. The chromatographic separation of the compounds from red propolis was performed using an ACE^® C18 columns (100 × 4.6 mm; 3 µm) from (Hichrom, Reading United Kingdom) and a mobile phase (0.1% of formic acid in H2O: 0.1% of formic acid in Acetonitrile; A:B) in flow rate of 300 μl/min. The gradient elution was programmed as follows: 0–6 min linear gradient 30–45% of B, 6–14 min linear gradient 45–75% of B, 14–20 min linear gradient 75–100% of B, 20–51 min at 100% of B for elution of the guttiferones and cleaning of the column, 51–54 min decreasing in B to 45%, 54–55 linear gradient 30% of B, 55–60 min isocratic condition with 30% of B to re-equilibration of the column for next run. The injection volume was 10 µl. The MS detection range was from 100 to 1200 m/z set at 30,000 resolutions and the scanning was performed under ESI negative polarity mode with capillary temperature was 250°C.

An ultra-performance liquid chromatography coupled with a photodiode array (UPLC-PDA) analysis was performed using an UPLC-PDA system from Shimadzu (Tokyo, Japan). The UPLC-PDA equipment consisted of the modules: degasser (model DGU-20A3R), two high-pressure pumps (model LC-20ADXR), auto-injector (model SIL-20AXR), oven chromatographic column (Prominence^® CTO-20A), photodiode array detector (model SPD-M20A), a controller (model CBM-20A), and Shimadzu Labsolution software. The chromatographic separation of flavonoids from red propolis was performed using a Kinetex^® C18- column (150 mm × 4.6 mm; 5 µm) and a mobile phase that consisted of solvent A (Milli-Q water) and solvent B (acetonitrile) pumped at flow rate of 0.3 ml/min. The initial elution gradient consisted of 70% water (A) and 30% acetonitrile (B) (v/v). The column was eluted by varying the percentage of (B) as follows: 0–2 min 30% B, 2–5 min 36% B, 5–8 min 46% B, 8–11 min 52% B, 11–14 min 52% B, 14–17 min 57% B, 17–20 min 62% B, 20–24 min 62% B, 24–28 min 68% B, 28–32 min 72% B, 32–36 min 90% B, 36–42 min 97% B, 42–50 min 100% B, 50–55 min 100% B, 55–57 min acetonitrile was reduced to 30% and this condition was maintained up to 60 min. Volumetric working solutions of analytical standards of the daidzein, liquiritigenin, pinobanksin, isoliquiritigenin, formononetin, pinocembrin, and biochanin A from Sigma-Aldrich (St. Louis, MO, United States) was prepared at 200 μg/ml (ethanolic solutions). After that, the working solutions were diluted to obtain a calibration curve at the concentrations: 7.50, 5.00, 2.50, 1.00, 0.50 and 0.15 μg/ml (n = 3). This calibration curve was used for the quantification of the listed flavonoids in the EABRP dried extract. The EABRP lyophilized extract (EABRP dried extract) were solubilized in ethanol (HPLC grade) to obtain stock solution at 10 mg/ml. Then, 250 µl of EABRP stock solution was pipetted to 5 ml volumetric flasks (n = 3) to obtain work solutions at 500 μg/ml. The injection volume of each sample was 2.0 µl. The UPLC-PDA method was previously validated to the quantification of markers from Brazilian red propolis (do Nascimento et al., 2016). The analysis was carried out at 280 nm.

The EBRP and EABRP were tested against Streptococcus mutans CCT 3440 from Tropical Cultures Collection (CCT). The strain was provided by André Tosello Foundation (Campinas, São Paulo, Brazil). The stock solutions of EBRP and EABRP were prepared at 30 mg/ml in acetone and diluted, first to 10 mg/ml in 1.0% dimethyl sulfoxide (DMSO) and after to 2500 μg/ml (1.0% DMSO). This dilution process was performed to minimize the acetone’s antibacterial action in the assay. The final solutions of EBRP and EABRP (2500 μg/ml in 1.0% DMSO) was made in the same day of the broth microdilution assay. The S. mutans CCT 3440 strain was activated in BHA (brain heart infusion agar) for 18–20 h at 37 ± 1°C in a microaerobic environment. The broth microdilution assay was performed to determination the minimal inhibitory concentration (MIC) of BRP extracts. The broth microdilution assay was carried out in microplates (96 wells) and the procedure according to the Clinical and Laboratory Standards Institute (CLSI, 2012) and Rufatto et al. (Celerino de Moraes Porto et al., 2018). The microplates were prepared with 80 µl of brain heart infusion broth (BHI) and 80 µl of EBRP and EABRP at 2500 μg/ml in each well groups from line A of the microplates, respectively. Serial two-fold dilutions was carried out to obtaining ethanol Brazilian red propolis and ethyl acetate Brazilian red propolis extracts at range from 1000 μg/ml to 7.81 μg/ml, respectively (n = 6). The inoculum was prepared by direct colonies suspension in sterile saline after strain activation period. A bacterial suspension was standardized equivalent to 0.5 McFarland standard suspension by turbidity comparison, resulting in a bacterial suspension containing approximately 1 × 10⁸ colony-forming units (CFU)/ml. The standardized inoculum suspension containing about 1 × 10⁶ CFU/ml was performed by dilution (1:20) of the 1 × 10⁸ CFU/ml bacterial suspension in sterile saline. Exactly 20 µl of the standardized inoculum suspension were added in each well, resulting a final bacterial concentration of 1 × 10⁵ CFU/ml, according CLSI (CLSI, 2012). The negative controls were A (inoculum + medium) and B (inoculum + medium + 1.0% DMSO). The positive control was inoculum + medium + 0.12% chlorhexidine gluconate. The sterility control test was performed for BHI medium. The microplates were incubated in ambient air at 35 ± 2°C for 20 h. After microplates’ incubation, 5.0 µl of each well was pipetted and plated in Petri dishes containing sterile BHA for minimal bactericidal concentration (MBC) determination after new incubation period of 24 h at 35 ± 2°C. Exactly 20 µl of resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one) solution at 0.15 mg/ml and pH 7.4 (50 mM) were added in each well of the microplates that were incubated for more 2 h at 35 ± 2°C for observation of the bacterial growth. MIC values were defined as the lowest concentration where the well’s color remained purple. The MBC values were defined as the lowest concentration where it did not colony growth visually.

The radical scavenging capacity of the extracts were mensurated by DPPH ^• method following the procedure described by Oliveira et al. (2020), with some modifications. Stock solutions of EBRP and EABRP were performed at 1.0 mg/ml in absolute ethanol. Aliquots of EBRP were transferred to volumetric flask of 5.0 ml, and then 2.0 ml of DPPH solution (39.432 μg/ml; in ethanol) was added obtaining final concentrations of 0.5, 1.0, 1.5, 2.0, 5.0, 10.0, 15.0, 20.0 and 25.0 μg/ml, respectively (n = 3). The EABRP samples were obtained in the same way, but to final concentrations of 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 μg/ml (n = 3). The scavenging reaction was performed in dark at 25°C for 30 min. After reaction time, the absorbance readings were performed with a spectrophotometer (Model UV-1240, Shimadzu, Kyoto, Japan) at 518 nm and the solvent ethanol was used as blank. The percentage of radical-scavenging capacity (%RSC—DPPH^•) of samples was calculated as follows: %RSC  =   ( ( A D P P H   –   A s a m p l e )   /   A D P P H )  *  100 a 2 + b 2 where A _DPPH is the absorbance of the diluted DPPH solution (2.0 ml of DPPH at 39.432 μg/ml to 5 ml of ethanol); A _sample is the absorbance of the tested sample at specific concentration. The IC₅₀ was determined for EBRP and EABRP through of linear equations from its calibration curves y = ax + b, plotted by extract concentration vs. %RSC; where: x is the equivalent concentration to IC₅₀ of %RSC; y is the numeric value 50; a is the angular coefficient (slope) of specific line; b is the linear coefficient of specific line. The positive control was trolox (6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydrochromene-2-carboxylic acid) (Sigma-Aldrich) at final concentration range from 0.25 to 4.0 μg/ml in ethanol.

The total flavonoid content (TFC) was determined by AlCl₃ method (Woisky and Salatino, 1998), with some modifications. Therefore, were performed methanolic stock solutions of 5% AlCl₃ (w/v), EABRP at 10 mg/ml and quercetin (Sigma-Aldrich) at 1.0 mg/ml, respectively. The assay was performed by add of aliquots of 10 mg/ml EABRP and 100 µl of 5% AlCl₃ in a 5 ml volumetric flask (methanol). The final concentration range of EABRP was from 50 to 200 μg/ml (n = 3). The solutions absorbances were measured in a UV-Vis spectrophotometer set at the 425 nm wavelength after 30 min of incubation in dark room at 25°C. Total flavonoids content was calculated using the quercetin calibration plot (Y = 0.0711x + 0.0027, R ² = 0.999) and expressed as mg quercetin equivalent (QE) g⁻¹ of dried EABRP extract.

Five disc-shaped samples to each experimental group were prepared with the aid of a stainless metallic matrix (h = 1.0 mm, Ø = 5.0 mm) and then were photoactivated for 20 s, as describe previously. The antibacterial activity of experimental composites was determined in relation to the Streptococcus mutans CCT 3440 strain. The bacterial strain was activated in sterile BHI medium at 37°C for 24 h, and then sowed in BHA medium and incubated at 37°C for 18 h. The inoculum concentration was adjusted in sterile physiologic solution from colonies isolated in BHA. The inoculum was adjusted to 10⁶ UFC/ml for the determination of antibacterial activity by direct contact test.

The antibacterial activity of experimental composite RP25 was also determined by direct contact test (Tavassoli Hojati et al., 2013; Zhang et al., 2017). Therefore, 10 μl of suspended bacteria (10⁶ UFC/ml) were placed on each disc-shaped sample (ø = 5 mm; thickness = 1.0 mm) individually positioned in sterile microtubes (n = 5). The disc-shaped sample in contact with inoculum were maintained laminar flow, for 1 h, until the entire liquid suspension evaporated to obtain a thin layer of bacteria. After this period, 200 μl of sterile physiologic solution was added to the each microtubes and the bacterial suspension was homogenized in vortex. To estimate colony formation, bacterial suspension from each specimen was diluted in sterile physiologic solution at 1:10 (10⁻¹) proportion, then 25 μl of this dilution was inoculated in BHA plates for 24 h at 37 ± 1°C in a microaerobic environment. After incubation, the total colonies were counted (n = 5) and the determination of UFC/ml were performed to each group, using the equation: UFC / ml  =  Nº of colonies x  40  x  10 Where: Nº of colonies is the total colonies present the plate after direct contact test; 40 is the correction factor to 1 ml (25 μl × 40 = 1 ml); 10 is the modulus of dilution used (10⁻¹) to UFC/ml count. The group RC (commercial composite) was used as negative control. The group RS (commercial composite with the solvent HEMA) was used to evaluate the solvent effect in enriched composite resins formulations. A microtube with inoculum without composites was used as cell control. Antibacterial activity was expressed by antibacterial ratio, where: r  ( % ) =   [ ( b  −  c )   /  b ]  x  100 Where r (%) is the antibacterial ratio; b is the average UFC/ml recovered for bacterial control; c is the average of recovered UFC/ml for the experimental group.

The in vitro cytotoxicity assay was performed for determined the cytotoxicity of BRP enriched composite resin against 3T3 fibroblasts. The composite samples were prepared as describe previously, and 24 h later the samples were immersed in pure Dulbecco’s Modified Eagle Medium (DMEM) medium, incubated for 24 h at 37°C, 5% CO₂ and 95% relative humidity to obtain the extracts (composite resins extracts in DMEM). The extracts were prepared at the ratio of 1.0, 2.0, 3.0, 5.0 and 6.0 cm² of composite/mL of medium to the experimental groups analyzed RC, RS and RP25 (n = 5), according to ISO 10993-12 (International Organization for Standarization, 2006). The samples were taken from the culture medium after the specified time and the obtained extracts were filtered in 0.22 μm membranes (Millipore, Molsheim, France) to ensure sterile conditions.

Fibroblasts of the 3T3 strain (embryonic fibroblasts of mice) were sown in DMEM medium supplemented with 10% bovine fetal serum (SBF), L-glutamine (2.0 μM) and penicillin/streptomycin (0.02 μg/ml) and were kept at 37°C, 5% CO₂ and 95% relative humidity. The cells were transferred to 96-well plates (200 μl/well—7.0 × 10³ cells/ml of complete culture medium) and incubated for 24 h in the same conditions as described previously. After this period, the supernatant was removed and each well with cells received 180 μl of fresh complete DMEM and 20.0 μl of the extract, and the cells were incubated for 24 h under the same conditions of temperature, CO₂ and humidity. The negative control group (cell control) received only fresh complete DMEM. Cell viability was determined by the MTT assay [3-bromide (3,5-dimetiltiazole-2-yl)-2,5-diphenyltrazzole] 24 h after exposure of cells to composite resins extracts in DMEM. The cells were washed with phosphate buffered saline (PBS) and 23 μl of an MTT solution (5.0 mg/ml in PBS) were added to each well as well as 23 μl of an MTT solution (5.0 mg/ml in PBS), then the cells were incubated again for 4 h under the same conditions. The supernatant was discarded and 150 μl dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. After 10 min the optical density of the resulting solution was read at 540 nm in a spectrophotometer (Model UV-1240, Shimadzu, Kyoto, Japan). The mean values of optical density obtained from cells exposed to DMEM were used as a negative control reference (100% cell survival). The cytotoxicity of the tested samples was expressed as the percentage of cell viability in relation to the negative control (100%), (n = 5). The in vitro cytotoxicity was determined by decrease of the cell viability as the equation: Cell viability %  =   ( 100  x  O D 540  sample )   /   O D 540   c e l l   c o n t r o l   where OD_540sample is the optical density of cells after sample treatment and OD_{540 cell control} is the optical density of cells control group.

Rectangular samples were made for each experimental group (n = 10) in a stainless-steel matrix (2 mm × 2 mm x 25 mm) according to the specifications of ISO 4049 (International Organization for Standarization, 2000) and then were photoactivated for 20 s, as describe previously. The photoactivation process was carryout according to the ISO 4049 specifications: one moment of photoactivation for each of the three points on top and bottom samples surfaces. After cure, the samples were kept in distilled water at 37°C for 24 h before test. The test was performed in a Microtensile/Semi-universal OM100 test machine (Odeme Dental Research, SC, BRA) with two rods (2 mm in diameter), mounted parallel with 20 mm between its centres, and a third rod (2 mm in diameter) centred between, and parallel to, the other two. The loading ratio and speed applied on samples was of 50 N/min and 0.75 ± 0.25 mm/min, respectively, according to ISO 4049 until the sample fracture. Flexural strength (FS) was expressed in MPa by equation: FS  =   3 F l   /   2 b h 2 where F is the maximum load, in newtons, exerted on the sample; l is the distance, in millimeters, between the supports; b is the width, in millimeters, of the specimen measured immediately prior to testing and h is the height, in millimeters, of the specimen measured immediately prior to testing.

Five disc-shaped samples were made for each group, with the aid of a stainless metallic matrix (h = 1.0 mm, Ø = 5.0 mm) and then were photoactivated for 20 s, as describe previously. The samples were stored in distilled water at 37°C for 24 h before test. Five indentations were performed on the irradiated surface of each sample at the center and more four regions with a load of 50 g for 5 s using an HMV-2000 microhardness (Shimadzu, Tokyo, Japan). The average of five indentations was taken as KHN to each of four samples on each experimental group and the average of four KHN in each experimental group was taken as the KHN to the group. The Knoop microhardness (KHN) was determined by equation: K H N   =   P   /   C L 2 where P is the load applied on the sample (Kgf); L is the length of the longest diagonal of the indentation (mm²) and C is the indentation constant (0.07028).

The post-cure depth was determined according to ISO 4049 procedure specifications (International Organization for Standarization, 2000). Five cylindric-shaped samples per group were produced with the aid of a cylindrical stainless metallic matrix (h = 6.0 mm, Ø = 2.0 mm) and then were photoactivated for 20 s, as describe previously. Then, the samples were immediately removed from the matrix and the uncured material at the bottom surface was scraped with a plastic spatula. The height of the cylindric-shaped samples was measured with a micrometer (with an accuracy of 0.01 mm) after scraping. The post-cure depth to each experimental groups was determined as the mean of each group samples.

The degree of conversion of the experimental groups was analyzed by Fourier transform infrared spectroscopy (FTIR; IRAffinity-1 Model, Shimadzu, Kyoto, Japan) in attenuated total reflection (ATR) mode. The DC% was measuring by change of height of the band at 1638 cm⁻¹ (corresponding with the aliphatic carbon double bond absorbance). The aromatic carbon double bond absorbance at 1608 cm⁻¹ was used as standard control. Each sample spectrum was acquired with 64 scans, resolution of 4.0 cm⁻¹ and using the absorbance mode. Disc-shaped samples were made with the aid of a stainless metallic matrix (h = 1.0 mm, Ø = 5.0 mm) and then were photoactivated for 20 s, as describe previously. Were analyzed non-polymerized samples (n = 3), immediately polymerized samples (n = 3) and samples with 24 h after polymerization (n = 3) to each experimental group and theirs respective FTIR spectra were obtained. The degree of conversion was calculated as follows: ( DC% ) =   [ 1 − ( ( R c u r e d )   /   ( R u n c u r e d ) ) ] ] x  100 where: R = (band at 1638 cm⁻¹)/(band at 1608 cm⁻¹).

The water sorption (Wsp) and water solubility (Wsl) in water of the experimental resins were determined according to ISO 4049 specifications (International Organization for Standarization, 2000). Five samples (h = 1.0 mm, Ø = 15.0 mm) per experimental group were prepared in a stainless metallic matrix and were photoactivated at nine points on the upper surface, each point for 20 s. After photoactivation, the samples were arranged in a light-proof container for 24 h and transferred to a desiccator containing silica gel dehydrated (Fischer Scientific^®, Leicester, United Kingdom), this set was kept at 37°C in an oven. After 22 h, all groups were transferred to a second desiccator, where they were kept for 2 h at 37°C. At the end of the cycle, each sample was weighed on an analytical balance with precision of 0.01 mg (Ohaus^® DV314C Discovery, Pine Brook, United States) and replaced in the initial desiccator. The cycles were repeated until a constant mass was reached M1 (μg), so that the weight variability was not greater than 0.1 mg in 24 h period. The samples were measured with digital micrometer (accurate to 0.01 mm, DIGIMESS-110-250, Digimess^® Precision Instruments Ltda, SP, BR) in order to determine the area (mm²) and volume (mm³). Then, the samples were immersed in water at 37 ± 1°C (Fanem^® Water Bath 1100, SP, BR), for 7 days. After this period, excess water was removed, and each sample again weighed. The wet mass was recorded as M2 (μg). The design cycles were repeated, as previously described, until the stable weight was reached, corresponding to M3 (μg). The mean values of Wsp and Wsl of each sample were calculated in micrograms per cubic millimeter using equations, respectively: Wsp  =   ( M 2   –  M 3 ) / V Wsl  =   ( M 1 − M 3 ) / V

Disc-shaped samples were made with the aid of a stainless metallic matrix (h = 1.0 mm, Ø = 5.0 mm) and then were photoactivated for 20 s, as describe previously. The average surface roughness (Ra) was determined by an atomic force microscopy (AFM) (Model Multiview 4000TM, Nanonics, Jerusalem, Israel) combined with optical microscope (Model BXFM Olympus, Tokyo, Japan). The AFM system was acoustically isolated, and the instrument was stabilized on an active table of movements. The topography of the samples was determined (256 × 256 pixels) in tapping mode with a scan ratio of 0.3–1.0 Hz in an area of 20 × 20 μm. The probe has a diameter <10 nm, 300 µm cantilever, 30° bend angle, 32 kHz resonance frequency and a Cr coating. Twelve regions per experimental group were analyzed and the average roughness (Ra) was determined using the Software WSxM (Nanotec, Madrid, Spain). The roughness average was calculated as the absolute mean of the heights of the irregularities along the profile as the equation: R a = 1 N ∑ i = 1 N | z i − z ¯ | Where: N is the number of sample points, z_i is the height of each sample point and Z ¯   is the average height of the sample points.

Five disc-shaped samples were prepared in a stainless metallic matrix (h = 1.0 mm, Ø = 5.0 mm) and powdered to obtain uniform powder. Thermogravimetry analysis (TGA) was performed in a TGA-51H model equipment (Shimadzu, Kyoto, Japan), from 4.5 mg ± 6.6% of each sample, packed in alumina crucible. The heating ratio was 10°C min⁻¹ in the range of 25–900°C in nitrogen atmosphere with flow of 50 cm³ min⁻¹. The equipment was calibrated under the same conditions with calcium oxalate monohydrate pattern. Differential scanning calorimetry analysis (DSC) was performed in DSC model 60 plus (Shimadzu, Kyoto, Japan) from 2.5 mg ± 4.0% of each sample packed in hermetically sealed alumina crucible. The heating ratio was 5.0°C min⁻¹ in the range of 30–600°C in nitrogen atmosphere and flow of 50 cm³ min⁻¹. The equipment was calibrated under the same conditions with indium and zinc.

The cytotoxicity of BRP enriched composite resins was determined by MTT assay against fibroblasts 3T3. In this assay were observed the effect of addition of BRP in the commercial composite resins and the effect of composite resins dose on the cytotoxicity. The usual dose of composite resin for this assay was defined as ratio of 3.0 cm² of composite resin/mL (surface area/volume), conforming ISO 10993-12 (International Organization for Standarization, 2006). The result shows that the groups RP25, RC and RS no presented cytotoxicity to 3T3 fibroblasts, once that after 24 h of exposition to composite resins extracts the cell viability were bigger than 70% Figure 4. Nevertheless, was observed statistically significant difference on the cell viability between experimental groups when analyzed the addition of BRP to the composite resin (p < 0.0001). The dose effect was not statistically significant (p = 0.0611). Comparing the results for groups RP25, RC and RS at ratio of 3.0 cm² is possible observed that the cells show bigger viability after exposition to RP25 than after to RC and to RS exposition (p < 0.05), respectively, as shows Table 5.

BRP enriched composite resins exhibit flexural strength above of 100 MPa, according to the ISO 4049 specifications (minimal FS of 80 MPa). Formulations RP10, RP15, RP25 and RS are statistically similar (p > 0.05) and RC shows the biggest FS 125.57 MPa ± 11.49 (p < 0.05) Figure 5A. KHN for RS, RP10, RP15 and RP25 were 19.4 ± 1.5, 18.6 ± 1.3, 18.2 ± 1.3 and 18.8 ± 1.1, respectively Figure 5B. The KHN for commercial composite resin (RC) was 24.8 ± 1.0. The addiction of HEMA, a methacrylate monomer solvent used as diluent in composite resin development was responsible by decrease of the mechanical properties of BRP enriched composite resins compared with RC (p < 0.05) once that promotes the decrease of viscosity in comparation. Decrease of viscosity in composite resins is caused by decreased of filler proportion in the composite.

Composites enriched with red propolis showed a cure depth compatible with the commercial composites used as a basis in this study, the bulk composites (with increment of ≥ 4.0 ± 0.1 mm), except RP25. The post-cure depth of RP25 and RP15 was 2.74 ± 0.06 mm and 3.80 ± 0.30 mm, respectively, both smaller than RC (5.86 ± 0.03 mm), RS (5.69 ± 0.05 mm) and RP10 (4.48 ± 0.34 mm) composites Figure 6A. The post-cure depth among the enriched composites was inversely proportional to the red propolis concentration: RP10 > RP15 > RP25. This phenomenon can be explained because the increase of red propolis concentration at composites led to decrease of its translucency, decreasing the capacity of irradiation of the curing light through sample. The Shapiro-Wilk normality test exhibit a non-normal distribution of post-cure depth results; therefore, the Kruskal-Wallis nonparametric test was performed. There was not statistically significant difference between three formulations of enriched composite resins (p > 0.05), but there was statistically significant difference between: RP25 and RC (p = 0.0002); RC and RP15 (p = 0.0171). The statistical analysis demonstrated that there was not significant difference between RC and RP10 (p = 0.2546). In this way, RP10 can be considered a bulk enriched composite resin.

This study analyzed the degree of conversion of BRP enriched composite resins by FTIR spectroscopy in twice moments: immediately after light curing photoactivation and 24 h after photoactivation. Was observed that the addition of EARP-HEMA to the commercial composite promoted a significant increase in the DC% of enriched composites when compared to the RC Figure 6B. When analyzing each moment separately, is possible verify that at first moment RS, RP10, RP15 and RP25 are statistically similar each other (p > 0.05) showing DC% among 76.95 ± 2.24% to 79.81 ± 0.39% and have bigger DC% than RC with 60.17 ± 3.09% (p < 0.0001). At the second moment this result profile remains: RS, RP10, RP15 and RP25 are statistically similar each other (p > 0.05) showing DC% from 80.70 ± 4.07% to 83.96 ± 2.87% and have bigger DC% than RC with 69.58 ± 4.51% (p < 0.029).

The BRP enriched composite resins, RC and RS controls exhibited values conforming ISO 4049 specification for Wsp (≤40.0 μg/mm³) Figure 6C. RS, RP10, RP15 and RP25 were statistically like each other (p > 0.05), but smaller than of RC (p < 0.028). The Wsl for BRP enriched composite resins was conforming ISO 4049 (≤7.5 μg/mm³) Figure 6D. The Shapiro-Wilk test demonstrated that Wsl data have non-normal distribution, therefore was conducted the Kruskal-Wallis test (p < 0.05) for this assay. Was observed that RP25 showed a median of 3.9 μg/mm³, exhibiting a bigger Wsl than RC with 0.68 μg/mm³ (p = 0.0109). The groups RC, RS, RP10 and RP15 no showed statistically significant difference (p > 0.05) each other.

Topographic images obtained in AFM, representative of the average surface roughness (Ra) of the experimental groups are presented in Figure 7A. The values of Ra for BRP enriched composite resins are presented in Figure 7B and show that all groups presented average surface roughness below 21 nm. RS, RP10, RP15 and RP25 exhibited small Ra than RC (p < 0.0001), being RC presented the lowest Ra (6.59 ± 1.94 nm), followed for RP15 (14.48 ± 1.63 nm). There was no significant difference (p > 0.05) between the Ra values of groups RS (19.24 ± 1.87 nm), RP10 (20.58 ± 3.86 nm) and RP25 (20.76 ± 2.31 nm).

Was performed the thermal analysis for BRP enriched composite resins for determination of its thermal events of degradation that explain the degree of formation of covalent bonds in the composite polymeric chains. The more resistant to thermal degradation below 600°C, the greater the number of covalent bonds existing in the three-dimensional structure of the composite. The TGA, DTG and DSC thermograms are shown in Figure 8. The thermogravimetric profiles of the studied groups were similar, where all groups presented two events of mass loss, one between 334–379°C and the other between 448–455°C. All composite resins formulations analyzed presented thermal resistance between 300–350°C, since no group had mass loss of up to 5% below these temperatures Figure 8A. The values of thermogravimetric events can be observed in Table 6, where it is observed that the addition of EARP-HEMA led to increased thermal resistance of composite resins.

All samples presented similar DSC profile between each other, with two events of thermal decomposition of the polymer matrix in 368°C and 407°C, and the beginning of thermal degradation of the composite stars at ∼ 560°C Figure 8B. It was observed that the addition of EARP-HEMA led to a increase in the heat variation involved in the thermal decomposition of the polymer matrix of enriched composite resins. The values of DSC events can be observed in Table 7.