Long-Term Dentin Bonding Performance of Universal Adhesives: The Effect of HEMA Content and Bioactive Resin Composite

Citation: Wu, D.; Yao, Y.; Cifuentes-Jimenez, C.C.; Sano, H.; Álvarez-Lloret, P.; Yamauti, M.; Tomokiyo, A. Long-Term Dentin Bonding Performance of Universal Adhesives: The Effect of HEMA Content and Bioactive Resin Composite. J. Funct. Biomater. 2024,15, 379. https://doi.org/10.3390/ jfb15120379 Academic Editors: Tatjana Maravic, Tijana Lainovi´c and Uroš Josi´c Received: 7 November 2024 Revised: 10 December 2024 Accepted: 11 December 2024 Published: 16 December 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Long-Term Dentin Bonding Performance of Universal Adhesives: The Effect of HEMA Content and Bioactive Resin Composite Di Wu 1, Ye Yao 1, Carolina Cecilia Cifuentes-Jimenez 2, Hidehiko Sano 1, Pedro Álvarez-Lloret 3, Monica Yamauti 1,4,*and Atsushi Tomokiyo 1 1Department of Restorative Dentistry, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan; [email protected] (D.W.); [email protected] (Y.Y.); [email protected] (H.S.); [email protected] (A.T.) 2Department of Stomatology, Faculty of Dentistry, University of Granada, Campus de Cartuja, s/n, 18011 Granada, Spain; [email protected] 3Department of Geology, Faculty of Geology, University of Oviedo, Campus de Llamaquique, s/n, 33005 Oviedo, Spain; pedroalvar[email protected] 4Department of Biomedical and Applied Sciences, School of Dentistry, Indiana University Indianapolis, Indianapolis, IN 46202, USA *Correspondence: [email protected] Abstract: This study investigated the effects of resin composites (RCs) containing surface pre-reacted glass ionomer (S-PRG) filler on the dentin microtensile bond strength ( µ TBS) of HEMA-free and HEMA-containing universal adhesives (UAs). Water sorption (WS) and solubility (SL), degree of conversion (DC), and ion release were measured. The UAs BeautiBond Xtreme (BBX; 0% HEMA), Modified Adhesive-1 (E-BBX1; 5% HEMA), Modified Adhesive-2 (E-BBX2; 10% HEMA), and two 2-step self-etch adhesives (2-SEAs): FL-BOND II (FBII; with S-PRG filler) and silica-containing adhesive (E-FBII) were used. Teeth were restored with Beautifil Flow Plus F00 with S-PRG filler (BFP) and flowable resin composite with silica filler (E-BFP). µ TBS was evaluated after 24 h and 6 months of water storage. WS and SL measurement followed ISO 4049:2019; spectroscopy measured DC; ICP-MS evaluated ion release. BBX and FBII presented the highest DC. The adhesives did not comply with the WS ISO requirements, but the bonding resin of 2-SEAs complied with the SL threshold. BFP released more ions than E-BFP. BFP positively affected the µ TBS of UAs, regardless of HEMA concentration after 24 h, comparable to the 2-SEAs. The 6 months µ TBS decrease depended on the adhesive and RC combination. HEMA did not affect the µTBS of UAs, while bioactive resins had a positive impact. Keywords: universal adhesive; S-PRG fillers; HEMA; degree of conversion; water sorption and solubility; ions release; bond strength 1. Introduction Universal adhesives (UAs) are widely recognized as adhesive systems that can be applied in etch-rinse and self-etch modes to various dental tissues and materials utilized in direct or indirect restorative techniques [ 1 ]. Due to their multifunctional features, their composition is more complex than the preceding all-in-one self-etch adhesive systems. When developing single-bottle UAs, an ideal mixture of acrylic resin monomers with differing hydrophilicity and hydrophobicity, solvent water, silane, and functional monomers needed to be blended and function well in combination, ideally polymerized to produce a durable bonded interface [ 2 ]. However, achieving an ideal composition is highly technique-sensitive since all adhesive compounds blend, even though they are not always miscible [3]. A functional monomer, 2-hydroxyethyl methacrylate (HEMA), is frequently added to UAs. HEMA is highly soluble in other solvents and water; its hydrophilic and co-solvent nature improves stability and helps to keep hydrophobic and hydrophilic monomers in a homogenous solution by reducing phase separation [ 4 ]. Phase separation occurs when J. Funct. Biomater. 2024,15, 379. https://doi.org/10.3390/jfb15120379 https://www.mdpi.com/journal/jfb J. Funct. Biomater. 2024,15, 379 2 of 18 the solvent evaporates at moist bonding interfaces, especially when using adhesives like UAs that contain high water concentrations [ 1 ]. Moreover, HEMA’s polar characteristics and water-solubility properties increase wettability and promote resin monomer diffusion into the dentin collagen fibril network [ 5 ]. However, uncured HEMA also lowers water vapor pressure in the adhesive and may make it more challenging to evaporate during the air-drying step, impairing polymerization [ 6 ]. Due to its high hydrophilicity, HEMA readily absorbs water in its uncured and polymerized state [ 7 ]. High water sorption can cause a decrease in mechanical properties and contribute to the degradation of dental polymer matrices [ 5 ]. A previous investigation has also suggested that HEMA hampers the interactions between the phosphate groups of 10-MDP and hydroxyapatite (HAp), which could undermine the bond strength of UAs containing substantial amounts of HEMA [ 8 ]. To increase the durability of the composite restoration, manufacturers and researchers have attempted to reduce the hydrophilicity of UAs by adjusting the concentrations of HEMA to increase the longevity of the composite restoration [ 9 ]. UAs could still be considered a one-step adhesive [2]. The so-called controversial bioactivity has been another property addressed in dental restorative materials for a stable dentin-resin bonding [ 1 ]. The latest definition of bioactivity from FDI divides the mechanisms into three levels: purely biological, mixed biological/chemical, or strictly chemical (e.g., through ion release from bioactive glass fillers [ 10 ]). Recently, significant interest has been drawn to using glass particles that demonstrate various effects by releasing multiple ions [ 11 ]; among them, the silanated pre-reacted glass ionomer (S-PRG) filler has been widely used in commercial products. According to the manufacturer, the S-PRG filler is a multilayered, ultrafine glass particle with a SiO 2 coating on the outer layer, a pre-reacted glass-ionomer phase in the middle, and a glass core that could be released into dental hard tissues and enhance their mineralization [12]. The cured adhesive layers in single-step adhesives may act as semi-permeable membranes that allow water diffusion from the bonded hydrated dentin to the intermixed zone between the adhesive and the composite [ 13 ]. A recent study reported that multi-ions released by S-PRG-filled resin composite might permeate tooth substrates through adhesives, hindering tooth demineralization around composite restorations [ 14 ]. To combine the advantages of UAs and the properties of S-PRG fillers, new resin-based products have been developed and require investigation. Therefore, this study aimed to investigate the effects of a resin composite (RC) containing S-PRG filler on the dentin bond strength of HEMA-free and HEMA-containing UAs over a period of 6 months. Two different two-step self-etch adhesives (2-SEAs) were used as reference groups. Additionally, the water sorption and solubility of the materials, as well as their degree of conversion, were investigated. The hypotheses were that (1) there would be no significant differences in the degree of conversion of materials; (2) there would be no difference in the water sorption and solubility of adhesive systems and RCs; (3) there would be no difference in the ion release of adhesives and RCs; (4) the storage period, adhesive types, RCs, and their combinations would not affect the dentin bond strength to dentin. 2. Materials and Methods 2.1. Study Design and Ethical Considerations This study represents quantitative, qualitative, and prospective in vitro research. The experimental factors were adhesive type (5 levels), restorative resin (2 levels), and storage period (2 levels—only for bond strength). The response variables were degree of conversion, water sorption and solubility, and bond strength. The research protocol and the use of human extracted teeth was approved by the Institution’s Human Research Ethics Committee in the Graduate School of Dental Medicine, Hokkaido University (Protocol #2018/9). The teeth were preserved in an aqueous solution of 0.5% chloramine-T at 4 ◦ C and used within 6 months of extraction. J. Funct. Biomater. 2024,15, 379 3 of 18 2.2. Degree of Conversion of Adhesives and Resin Composites The materials used in the study are listed in Table 1, along with their manufacturers, abbreviations, and instructions. A modular confocal Raman spectrograph was used to investigate the degree of conversion (DC) of the adhesives under non-polymerized and polymerized conditions. Raman spectra were obtained using a JASCO NRS-5100 (Jasco Inc., Easton, MD, USA) spectrometer with a charge-coupled detector (1024 × 256 pixels) cooled by a Peltier-effect module. Two drops of each adhesive were poured into a circular Teflon mold (10.0 mm diameter × 4.0 mm depth) and placed under magnification on an XYZ stage. A near-infrared diode laser (785 nm) kept at 500 mW was employed to induce the Raman scattering, focusing the laser beam with a 20 × lens (optical microscope). Spectra were acquired in a range between 1000 and 1800 cm −1 using an exposure time of 5 s and 10 accumulations with an average spectral resolution of 1.6 cm −1 . The adhesive samples were analyzed while non-polymerized to obtain the uncured monomer spectra. Subsequently, each adhesive sample was light-activated using an LED curing unit with light irradiance of 1200 mW/cm 2 (G-Light Prima-II Plus; GC Corp., Tokyo, Japan) following the manufacturer’s instructions, and the polymerized measurements were taken. Instrument calibration was determined before data acquisition by comparison with the silicon standard spectrum to set the reference position at 520 cm−1. Table 1. Commercial names, batch numbers, abbreviations, compositions, and manufacturer’s instruction of the materials used in the study commercial. Adhesives Abbreviations Compositions Manufacturer’s Instructions BeautiBond Xtreme (Shofu Inc., Kyoto, Japan, Lot. 122012) (Universal Adhesive) BBX Acetone, distilled water, Bis-GMA, carboxylic acid monomer, phosphoric acid monomer, TEGDMA, acid resistant silane coupling agent, others. Apply bonding adhesive to the dentin surface. Apply gentle air for 3 s and strongly air until dry. Light cure for 5 s. Modified Adhesive 1 (Shofu Inc., Kyoto, Japan, Lot. 230714) (Universal Adhesive) E-BBX1 HEMA (5 wt%), acetone, distilled water, Bis-GMA, carboxylic acid monomer, phosphoric acid monomer, TEGDMA, acid resistant silane coupling agent, others. Apply bonding adhesive to the dentin surface. Apply gentle air for 3 s and strongly air until dry. Light cure for 5 s. Modified Adhesive 2 (Shofu Inc., Kyoto, Japan, Lot. 230714) (Universal Adhesive) E-BBX2 HEMA (10 wt%), acetone, distilled water, Bis-GMA, carboxylic acid monomer, phosphoric acid monomer, TEGDMA, acid resistant silane coupling agent, others. Apply bonding adhesive to the dentin surface. Apply gentle air for 3 s and strongly air until dry. Light cure for 5 s. FL-BOND II (Shofu Inc., Kyoto, Japan, Lot. 092130) (Two-step, self-etch adhesive) FBII Primer: water, ethanol, carboxylic acid monomer, phosphoric acid monomer and initiator Adhesive: SPRG based on fluoroboroaluminosilicate glass, UDMA, TEGDMA, HEMA, polymerization initiator. Apply primer to the dentin surface and leave for 10 s. Apply gentle air until dry. Apply bonding adhesive to the dentin surface. Light cure for 10 s. Silica-containing adhesive (Shofu Inc., Kyoto, Japan, Lot. HFLB01) (Two-step, self-etch adhesive) E-FBII UDMA, HEMA, TEGDMA, polymerization initiator, silica filler, others Primer: water, ethanol, carboxylic acid monomer, phosphoric acid monomer and initiator. Adhesive: silica filler, UDMA, TEGDMA, HEMA, polymerization initiator, others. Apply primer to the dentin surface and leave for 10 s. Apply gentle air until dry. Apply bonding adhesive to the dentin surface. Light cure for 10 s. Resin Composites Abbreviations Compositions Manufacturer’s Instructions BEAUTIFIL Flow Plus F00 (Shofu Dental, Kyoto, Japan, Lot. 122012) (Flowable restorative resin composite) BFP Bis-GMA, TEGDMA, S-PRG filler based on fluoroboroaluminosilicate glass, polymerization initiator, pigments, others. Apply the material in layers with a needle tip to build up a 4 mm thickness resin block. Each layer is no more than 2 mm thick, and light cure for 10 s. Silica-containing flowable resin composite (Shofu Dental, Kyoto, Japan, Lot. HBFP02) (Flowable restorative resin composite) E-BFP Bis-GMA, TEGDMA, silica filler, polymerization initiator, pigments, others. Apply the material in layers with a needle tip to build up a 4 mm thickness resin block. Each layer is no more than 2 mm thick, and light cure for 10 s. Abbreviations: HEMA, 2-Hydroxyethyl Methacrylate; Bis-GMA, bisphenol A diglycidyl methacrylate; TEGDMA, triethylene glycol dimethacrylate; S-PRG, surface pre-reacted glass-ionomer; UDMA, urethane dimethacrylate. Resin composite samples were prepared using a circular bipartite Teflon mold (10.0 mm diameter × 1.0 mm thick) placed on top of a glass slide and analyzed using J. Funct. Biomater. 2024,15, 379 4 of 18 a Fourier transform infrared (FTIR) JASCO 6200 (Jasco Inc., Easton, MD, USA) spectrometer equipped with a diamond-tip attenuated total reflection (ATR) accessory (ATR Pro ONE; Jasco Inc.). The samples were placed on the ATR glass holder, covering the surface before and after polymerization. All spectra were acquired in absorbance mode between 600 and 4000cm −1 spectral range, with a resolution of 2cm −1 and 124 scan accumulations. Three samples of each uncured adhesive/composite material were analyzed. The DC values were calculated by determining the area ratio of the absorbance aliphatic C=C at 1638 cm −1 and the internal reference peak of aromatic C=C at 1608 cm −1 [ 15 ]. A region of the spectra between 1590 and 1660 cm −1 was selected and baseline corrected; after spectrometric analyses, the DC was calculated as follows: DC (%)area = [1−Cured C =C 1638 cm−1/Cured C =C 1608 cm−1 Uncured C =C 1638 cm−1/Uncured C =C 1608 cm−1]×100 The area peak values were resolved using curve-fitting software Peakfit v4.12 (Systat Software, Chicago, IL, USA). The second derivative method was utilized for the measurements of each peak within the spectral region. The degree of smoothing was set at 20% (Savitzky–Golay algorithm), and a mixed Gaussian-Lorentzian function was used to adjust the peak profiles (i.e., curve shape and width). Curve fitting was accepted when r 2 reached values up to 0.995. 2.3. Water Sorption and Solubility A metal mold (15.0 ± 0.1 mm diameter, 1.0 ± 0.1 mm thick) was used to prepare resin disks (n= 6), and a silicone rubber mold (6.0 ± 0.1 mm diameter, 1.0 ± 0.1 mm thick) was used to produce adhesive disks (n= 6). The measurement and calculation of water sorption (WS) and solubility (SL) followed the guidelines specified in ISO 4049:2019 [ 16 ]. After polymerization, the samples were promptly transferred to a desiccator and moved to a pre-conditioning oven set at 37 ◦ C. All samples were dried in a silica gel desiccator for 22 h at 37 ± 2 ◦ C and 2 h at 23 ± 2 ◦ C. The samples underwent weightings at 24 h intervals until a stable mass (m1) was achieved, indicated by a fluctuation of less than 100 µ g over 24 h for 3 days. The dimensions of the samples were assessed utilizing a digital caliper, with values rounded to the nearest 0.01 mm, and these measurements were used to calculate the volume (V: mm 3 ) of each sample. Subsequently, the samples were individually introduced into hermetically sealed glass vials, each containing 10 mL (for adhesive disk) and 20 mL (for resin disk) of deionized water with a pH of 7.2, and maintained at a temperature of 37 ◦ C. Following a storage period of 7 days, the vials were removed from the oven and allowed to equilibrate at ambient temperature for 20 min. The samples were washed using running water and gently wiped using soft absorbent paper. Subsequently, the samples were weighed using an analytical balance, identified as m2. After a storage period of 7 days at ambient conditions, the samples were subjected to a drying process within a desiccator that contained newly replenished silica gel. As previously explained, the samples were weighed daily until they attained a stable mass (m3). The mass measured after the initial desiccation process (m1) was used to compute the mass variation at regular intervals during 7 days of water storage. The kinetics of water absorption during the entire duration of water storage were determined by plotting changes in mass against the storage period. WS and solubility SL values were determined during 7 days of water storage. The calculations were performed using the formulas WS = (m2−m3)/Vand SL = (m1−m3)/V[16]. 2.4. Measurement of Ion Release The 7-day immersing solutions of adhesives and resin disks from the SL test were used to measure ion release (n= 6). The immersion solutions (0.5 mL) were examined using an inductively coupled plasma-mass spectrometer (ICP-MS; 8900 Triple Quadrupole, Agilent Corp, Santa Clara, CA, USA) to quantify the levels of B, Na, Al, Si, and Sr ions. This analysis utilized a pre-established calibration curve that relates the spectrometry values to the standard concentration of multiple elements (XSTC-622; Seishin Trading, Kobe, Japan). J. Funct. Biomater. 2024,15, 379 5 of 18 The release of F ions was examined using an ion chromatography device (Dionex™ ICS 1600; Thermo Fisher Scientific Inc., Waltham, MA, USA) attached to a high-speed anion chromatography column (TSKgel guard column SuperIC-AHS; Tosoh Corp., Tokyo, Japan). To achieve pH stabilization, 0.7 mM Na 2 CO 3 buffer (37991-13; Kanto Chemical, Tokyo, Japan) was introduced into the solution. The calibration curve was constructed by diluting the anion mixed standard solution (01849-96, Kanto Chemical, Tokyo, Japan) with ultrapure water [17]. 2.5. Microtensile Bond Strength (µTBS) and Failure Mode Eighty sound human molars were randomly selected for the µ TBS test, checking for the absence of cavities, fissures, or fractures using a stereoscope (Moticam 1080; Shimadzu Corp., Kyoto, Japan). Flat, occlusal dentin surfaces were exposed using a gypsum model trimmer (Model Trimmer MT 10; J. Morita MFG. Corp., Tokyo, Japan) under water coolant. The surfaces were then checked with a stereoscope to ensure no enamel was remaining on the surfaces. The smear layer was standardized for 60 s using 600-grit silicon carbide paper (Fuji Star Type DDC, Sankyo Rikagaku Co., Saitama, Japan) under high-flowing water polishing. Then, they were randomly divided into five groups according to the dental adhesives: Beautibond Xtreme (BBX), Modified Adhesive 1 (E-BBX1), Modified Adhesive 2 (E-BBX2), FL-Bond II (FBII), silica-containing adhesive (E-FBII), which were applied following the manufacturer’s instructions (Table 1). Each bonded group (n= 16) was further divided into two subgroups (n= 8) according to the flowable resin composite used (Beautifil Flow Plus F00 [BFP] and silica-containing flowable resin composite [EBFP]). Each flowable resin composite was applied incrementally (1 mm increment) on dentin surfaces to build up the resin blocks (4 mm thick) and light-cured with a blue LED light-curing unit (G-Light Prima-II Plus; GC Corporation, Tokyo, Japan) with light irradiance of 1200 mW/cm 2 . According to the manufacturer’s instructions, when UAs were utilized, the curing time was 5 s, and the curing time of the bonding resin of the two-step self-etch adhesives was 10 s. The flowable resin composite was light-cured for 10 s per layer. The µ TBS test followed the Academy of Dental Materials’ recommendations for the non-trimmed µ TBS testing [ 18 ]. After 24 h (24 h) of storage in distilled water at 37 ◦ C, all teeth underwent longitudinal sectioning in two perpendicular directions across the bonded interface. A low-speed diamond saw (Isomet ® 1000; Buehler Ltd., Lake Bluff, IL, USA) with water cooling was used for this procedure, resulting in beam sticks with a cross-sectional area of about 1 mm 2 . Following the sectioning procedure, half of the beams were immediately subjected to testing, while the other half were stored for 6 months (6 m) in distilled water at 37 ◦ C. According to the specified storage period (24 h or 6 m), each beam was fixed to a jig utilizing a cyanoacrylate glue (Model Repair II Blue; Dentsply-Sankin, Tokyo, Japan) installed in a tabletop testing machine (EZ-S test; Shimadzu Corp., Tokyo, Japan) to be tested under tension at a 1.0 mm/min crosshead speed. The bond strength value of each beam was calculated and expressed in megapascals (MPa). Then, the mean of the bond strength obtained from all beams in each tooth was calculated, and “tooth” was used as the statistical measure. The fractured beams were observed with a stereoscope equipped with a digital camera system (Moticam 1080; Shimadzu Corp., Tokyo, Japan) at a magnification of 100 × . Failure modes were categorized as follows: Adhesive failure (A): failure that took place in the adhesive area; cohesive failure in resin composite (CC): failure that occurred within the resin composite; cohesive failure in dentin (CD): failure that occurred within dentin; and mixed failure (M): failure from the adhesive into resin composite and/or dentin area [ 9 ]. Additionally, representative beams, after fracture, from each group (n= 24) were placed on aluminum stubs, and sputter-coated for 120 s with Pt-Pd using an ion sputtering machine (E-1030; Hitachi Ltd., Tokyo, Japan). The fractured surfaces were observed using a field emission scanning electron microscope (SEM; S-4800, Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 10 kV. Figure 1illustrates the adhesives’ and resin composites’ sample preparation for each testing method. J. Funct. Biomater. 2024,15, 379 6 of 18 J. Funct. Biomater. 2024, 15, x FOR PEER REVIEW 6 of 21 µTBS test followed the Academy of Dental Materials’ recommendations for the nontrimmed µTBS testing [18]. After 24 h (24 h) of storage in distilled water at 37 °C, all teeth underwent longitudinal sectioning in two perpendicular directions across the bonded interface. A low-speed diamond saw (Isomet® 1000; Buehler Ltd., Lake Bluff, IL, USA) with water cooling was used for this procedure, resulting in beam sticks with a cross-sectional area of about 1 mm2. Following the sectioning procedure, half of the beams were immediately subjected to testing, while the other half were stored for 6 months (6 m) in distilled water at 37 °C. According to the specified storage period (24 h or 6 m), each beam was fixed to a jig utilizing a cyanoacrylate glue (Model Repair II Blue; Dentsply-Sankin, Tokyo, Japan) installed in a tabletop testing machine (EZ-S test; Shimadzu Corp., Tokyo, Japan) to be tested under tension at a 1.0 mm/min crosshead speed. The bond strength value of each beam was calculated and expressed in megapascals (MPa). Then, the mean of the bond strength obtained from all beams in each tooth was calculated, and “tooth” was used as the statistical measure. The fractured beams were observed with a stereoscope equipped with a digital camera system (Moticam 1080; Shimadzu Corp., Tokyo, Japan) at a magnification of 100×. Failure modes were categorized as follows: Adhesive failure (A): failure that took place in the adhesive area; cohesive failure in resin composite (CC): failure that occurred within the resin composite; cohesive failure in dentin (CD): failure that occurred within dentin; and mixed failure (M): failure from the adhesive into resin composite and/or dentin area [9]. Additionally, representative beams, after fracture, from each group (n = 24) were placed on aluminum stubs, and spuer-coated for 120 s with Pt-Pd using an ion spuering machine (E-1030; Hitachi Ltd., Tokyo, Japan). The fractured surfaces were observed using a field emission scanning electron microscope (SEM; S-4800, Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 10 kV. Figure 1 illustrates the adhesives’ and resin composites’ sample preparation for each testing method. Figure 1. Schematic illustration of sample preparation of adhesives and resin composites for the measurement of degree of conversion, water sorption and solubility, ions release, microtensile bond strength to dentin, and failure mode. Figure 1. Schematic illustration of sample preparation of adhesives and resin composites for the measurement of degree of conversion, water sorption and solubility, ions release, microtensile bond strength to dentin, and failure mode. 2.6. Statistical Analysis All data were tested for normality using the Shapiro–Wilk and homogeneity of variance using Levene’s tests. The level of significance was set at α = 0.05. The DC (%) data were normally distributed (p ≥ 0.05), and the variance was homogenous (p ≥ 0.05); therefore, a one-way ANOVA and independent sample t-test were performed for the adhesives and resin composites. Post hoc multiple comparisons were performed using the Bonferroni test. As the WS, SL, and ion release date of the adhesives and resin composites were not normally distributed, the Kruskal–Wallis and Mann–Whitney U tests were conducted, followed by post hoc multiple comparisons with a Bonferroni adjustment. Since the µ TBS data were normally distributed (p= 0.056) and the variance was homogenous (p= 0.493), the subsequent analyses were carried out using a three-way ANOVA (factors: adhesives, composites, and storage period) and Sidak post hoc tests. The statistical analysis was conducted using the statistical software package for medical science (SPSS Ver.27 for Windows, SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Degree of Conversion Table 2and Figure 2describe the DC of adhesive systems and resin composites. A significant difference in the DC of adhesives was observed (p= 0.011). E-BBX2 showed the lowest DC (82.63 ± 1.65%), which was inferior to the highest DC presented by BBX (97.22 ± 1.09%). No statistically significant differences were found between the DCs of resin composites (p= 0.68). J. Funct. Biomater. 2024,15, 379 7 of 18 Table 2. Degree of conversion (DC: %) expressed as Mean (SD) of adhesives systems and resin composites (n= 3). Adhesive Systems (Abbreviation) DC BBX 97.22 (1.09) A E-BBX1 93.34 (2.96) A,B E-BBX2 82.63 (1.65) B FBII 92.87 (5.40) A,B E-FBII 87.62 (6.41) A,B Resin Composites (Abbreviation) DC BFP 79.78 (1.27) a E-BFP 74.72 (3.29) a Different uppercase letters indicate statistically significant differences between adhesives (p< 0.05), and the same lowercase letters refer to statistically significant similarity in resin composites (p> 0.05). BBX; BeautiBond Xtreme; E-BBX1: Modified Adhesive 1; E-BBX2: Modified Adhesive 2; FBII: FL-BOND II; E-FBII: Silica-containing adhesive; BFP: BEAUTIFIL Flow Plus (F00); E-BFP: silica containing flowable resin. J. Funct. Biomater. 2024, 15, x FOR PEER REVIEW 8 of 21 Figure 2. Curve-fiing analysis for average spectra from adhesives and resin composite. Area values for the two reference peaks were calculated: 1608 cm−1 (internal standard aromatic carbon double bond, C=C) and 1638 cm−1 (methacrylate C=C). (A) BBX; BeautiBond Xtreme; (B) E-BBX1: Modified Adhesive 1; (C) E-BBX2: Modified Adhesive 2; (D) FBII: FL-BOND II; (E) E-FBII: Silica-containing adhesive; (F) BFP: BEAUTIFIL Flow Plus (F00); (G) E-BFP: silica containing flowable resin. 3.2. Water Sorption and Solubility The WS and SL values (µg/mm3) measured for the adhesives and resin composites tested are shown in Table 3. The WS values of all adhesives did not comply with the ISO 4049:2019 threshold (≤40 µg/mm3). No significant differences in WS were detected between BBX, E-BBX1, E-BBX2, and FBII (p > 0.05). E-FBII produced the lowest WS (64.37 ± 1.86 µg/mm3), and this value was significantly lower than those of BBX (165.70 ± 65.03 Figure 2. Curve-fitting analysis for average spectra from adhesives and resin composite. Area values for the two reference peaks were calculated: 1608 cm −1 (internal standard aromatic carbon double bond, C=C) and 1638 cm −1 (methacrylate C=C). (A) BBX; BeautiBond Xtreme; (B) E-BBX1: Modified Adhesive 1; (C) E-BBX2: Modified Adhesive 2; (D) FBII: FL-BOND II; (E) E-FBII: Silica-containing adhesive; (F) BFP: BEAUTIFIL Flow Plus (F00); (G) E-BFP: silica containing flowable resin. J. Funct. Biomater. 2024,15, 379 8 of 18 3.2. Water Sorption and Solubility The WS and SL values ( µ g/mm 3 ) measured for the adhesives and resin composites tested are shown in Table 3. The WS values of all adhesives did not comply with the ISO 4049:2019 threshold ( ≤ 40 µ g/mm 3 ). No significant differences in WS were detected between BBX, E-BBX1, E-BBX2, and FBII (p> 0.05). E-FBII produced the lowest WS (64.37 ± 1.86 µ g/mm 3 ), and this value was significantly lower than those of BBX (165.70 ± 65.03 µ g/mm 3 ), E-BBX1 (127.58 ± 13.21 µ g/mm 3 ), and E-BBX2 (150.62 ± 41.13 µ g/mm 3 ). The SL values of BBX, E-BBX1, and E-BBX2 also did not comply with that ISO requirement ( ≤ 7.5 µ g/mm 3 ). The SL of FBII (4.73 ± 1.76 µ g/mm 3 ) and E-FBII (4.06 ± 1.41 µ g/mm 3 ) were significantly lower than those of BBX (71.54 ± 32.86 µ g/mm 3 ) and E-BBX1 (51.96 ± 13.20 µ g/mm 3 ) (p< 0.05), but they did not show a significant difference compared to E-BBX2 (71.17 ± 9.46 µ g/mm 3 ). No significant difference in SL was detected among BBX, E-BBX1, and E-BBX2 (p> 0.05). The values of WS and SL in both resins complied with the ISO 4049:2019. The WS and SL of BFP (31.29 ± 1.19 µ g/mm 3 and 2.99 ± 0.45 µ g/mm 3 ) are significantly higher than those of E-BFP (21.3 ±0.72 and 1.4 ±0.28 µg/mm3) (p< 0.05). Table 3. Water sorption (WS) and solubility (SL) expressed as Mean (SD) in ( µ g/mm 3 ) of adhesives systems and resin composites (n= 6). Adhesive Systems WS SL BBX 165.70 (65.03) A71.54 (32.86) A E-BBX2 150.62 (41.13) A71.17 (9.46) A E-BBX1 127.58 (13.21) A51.96 (13.20) AB FBII 81.11 (3.27) AB 4.73 (1.76) B E-FBII 64.37 (1.86) B4.06 (1.41) B Resin Composites WS SL BFP 31.29 (1.19) a2.99 (0.45) a E-BFP 21.30 (0.72) b1.40 (0.28) b Different uppercase letters in each column indicate significant differences between the adhesive, and different lowercase letters indicate significant difference between resin composites (p < 0.05). BBX; BeautiBond Xtreme; E-BBX1: Modified Adhesive 1; E-BBX2: Modified Adhesive 2; FBII: FL-BOND II; E-FBII: Silica-containing adhesive; BFP: BEAUTIFIL Flow Plus (F00); E-BFP: silica containing flowable resin. 3.3. Measurement of Ion Release The results of ion release from each material are shown in Table 4. F ion was only detected in the materials with S-PRG fillers (FBII [0.165 µ g/mL] and BFP [0.575 µ g/mL]). FBII had a higher B ion release than the other adhesives (p< 0.05), and a higher Si ion release than BBX and E-BBX1 (p< 0.05). E-BBX1 showed significantly higher Na ion release than BBX (p< 0.05). The resin composite BFP released more B, Si, Sr, and F ions than E-BFP (p< 0.05). The concentrations of Al ions were all below the detection limit (<0.01 µg/mL). Table 4. Ion release results are expressed as median (minimum/maximum) in µ g/mL of adhesives systems and resin composites (n= 6). Adhesive Systems B Na Si Sr F BBX 1.60000 B (1.45000/2.64000) 2.06000 B (1.67000/3.51000) 0.08700 C (0.07000/0.20200) 0.00325 (0.00060/0.00350) <detection limit B E-BBX1 2.35000 B (2.06000/2.55000) 3.27000 A (2.89000/3.68000) 0.11950 BC (0.08300/0.17600) 0.00390 (0.00240/0.00450) <detection limit B E-BBX2 2.22500 B (1.81000/2.83000) 3.07500 A (2.64000/4.01000) 0.13750 ABC (0.10300/0.47300) 0.00285 (0.00260/0.00320) <detection limit B FBII 3.41500 A (1.96000/4.04000) 2.67500 AB (2.38000/3.20000) 0.55500 A (0.47600/0.84500) 0.01005 (<detection limit/0.12000) 0.16500 A (<detection limit/0.27000) J. Funct. Biomater. 2024,15, 379 9 of 18 Table 4. Cont. Adhesive Systems B Na Si Sr F E-FBII 1.72000 B (1.37000/2.97000) 2.53000 AB (2.29000/3.25000) 0.40950 AB (0.35200/0.70400) 0.00005 (<detection limit/0.10800) <detection limit B Resin Composites B Na Si Sr F BFP 3.53500 a (3.1300/3.8300) 2.66000 (2.32000/3.31000) 0.46300 a (0.30800/0.76300) 0.75900 a (0.32600/1.53000) 0.57500 a (0.30000/1.00000) E-BFP 1.57500 b (0.60500/1.94000) 2.24500 (0.87900/2.74000) 0.25300 b (0.12500/0.43600) 0.00205 b (0.00100/0.00280) <detection limit b The concentrations of Al ions were all below the detection limit (<0.01 µ g/mL). Different uppercase/lowercase letters in each column indicate significant differences among the adhesives/resin composites (p< 0.05). BBX; BeautiBond Xtreme; E-BBX1: Modified Adhesive 1; E-BBX2: Modified Adhesive 2; FBII: FL-BOND II; E-FBII: Silica-containing adhesive; BFP: BEAUTIFIL Flow Plus (F00); E-BFP: silica containing flowable resin. 3.4. µTBS and Fracture Mode The µ TBS results are shown in Table 5. A three-way ANOVA revealed significant main effects of adhesive (F = 2.803, p= 0.028), storage period (F = 10.113, p= 0.002), and composite (F = 18.865, p< 0.001) on the bond strength. However, the interaction of these three factors was not statistically significant (F = 1.949; p= 0.106). Furthermore, statistically significant interactions were found between storage period and composites (F = 12.764, p< 0.001). Simple main effects analyses showed that the 24 h µ TBS of BFP used with all adhesives was significantly higher than that of E-BFP (p< 0.001), but they did not statistically differ after 6 m of storage (p> 0.05). The highest bond strength results were produced when BFP was used to restore the teeth regardless of the adhesive utilized at 24 h testing (p< 0.05). The µ TBS of BFP utilized with E-BBX1, E-BBX2, and FBII was significantly reduced after 6 m of water storage (p< 0.001). Pairwise comparisons showed that at 24 h, E-BBX1 and E-BBX2 restored with BFP [50.61 ± 10.10 MPa and 49.73 ± 7.42 MPa, respectively] showed higher bond strengths than those restored with E-BFP (p< 0.001 and p= 0.006, respectively). Table 5. Microtensile bond strength ( µ TBS) is expressed as Mean (SD) in MPa (n= 8). For each adhesive system, two different restorative resin composites were tested after 24 h and 6 months of storage in distilled water. Adhesive System Resin Composite 24 h 6 Months BBX BFP 42.37 (4.60) Aa1 41.72 (3.24) Aa1 E-BFP 35.38 (4.12) Aa1 35.87 (11.19) Aa1 E-BBX1 BFP 50.61 (10.10) Aa1 39.65 (6.66) Ba1 E-BFP 36.15 (5.04) Ab1 41.39 (9.86) Aa1 E-BBX2 BFP 49.73 (7.42) Aa1 40.17 (7.32) Ba1 E-BFP 39.91 (4.01) Ab1 39.59 (4.94) Aa1 FBII BFP 50.60 (8.41) Aa1 35.35 (5.02) Ba1 E-BFP 44.24 (5.42) Aa1 41.99 (5.59) Aa1 E-FBII BFP 42.55 (8.04) Aa1 41.06 (8.71) Aa1 E-BFP 35.73 (6.42) Aa1 34.77 (8.87) Aa1 Different uppercase letters indicate significant differences between 24 h and 6 m) for the same adhesive-resin combination (p< 0.05); different lowercase letters indicate significant differences between resin composites within each column (24 h or 6 m) for the same adhesive (p< 0.05); different numbers indicate significant differences between adhesives within each column for the same resin composite (p< 0.05). BBX; BeautiBond Xtreme; E-BBX1: Modified Adhesive 1; E-BBX2: Modified Adhesive 2; FBII: FL-BOND II; E-FBII: Silica-containing adhesive; BFP: BEAUTIFIL Flow Plus (F00); E-BFP: silica containing flowable resin. J. Funct. Biomater. 2024,15, 379 16 of 18 Funding: This study was partially supported by Japanese Science and Technology Agency: JST SPRING (Grant Number JPMJSP2119); Japan Society for the Promotion of Science: JSPS Kakenhi Grant Number JP24K12924, and the European Regional Development Fund (ERDF)—Next Generation/EU program. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Hokkaido University Hospital Ethics Committee (protocol code 2018/09 and date of approval 1 February 2019). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 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