Effect of APTES- or MPTS-Conditioned Nanozirconia Fillers on Mechanical Properties of Bis-GMA-Based Resin Composites.

To investigate the effects of 3-aminopropyltriethoxysilane (APTES)- or (3-mercaptopropyl)trimethoxysilane (MPTS)-conditioned nanozirconia fillers on the mechanical properties of Bis-GMA-based resin composites. The conditioned fillers were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and thermodynamic calculations. They were then used to prepare Bis-GMA-based resin composites, whose flexural strength and elastic modulus were evaluated. The Cell Counting Kit-8 (CCK-8) assessed the composites' cytotoxicity. The FTIR spectra of the conditioned fillers showed new absorption bands at 1569 and 1100 cm-1, indicating successful grafting of APTES or MPTS onto nanozirconia. XPS confirmed the Zr-O-Si bonds in the APTES- or MPTS-conditioned fillers at contents of 2.02 and 6.98%, respectively. Thermodynamic calculations reaffirmed the chemical binding between the two silanes and nanozirconia fillers. Composites containing the conditioned nanozirconia fillers had significantly greater flexural strengths (APTES, 121.02 ± 8.31 MPa; MPTS, 132.80 ± 15.80 MPa; control, 94.84 ± 9.28 MPa) and elastic moduli (8.76 ± 0.52, 9.24 ± 0.60, and 7.44 ± 0.83 GPa, respectively) than a control with untreated fillers. The cytotoxicity assay identified no significant cytotoxicity by composites containing the conditioned fillers. Silanes were previously considered to be unable to chemically condition zirconia to bond with resin. Inclusion of APTES- or MPTS-conditioned nanozirconia fillers can improve the mechanical properties of Bis-GMA-based resin composites without obvious cytotoxicity in this study.

Introduction

One of the main reasons for the loss of resin composite restorations, along with secondary caries,[1] is fracture due to mechanical fatigue.[2] Continued research efforts have therefore sought to improve resins’ mechanical properties. Resin-based composites consist of an organic matrix and inorganic filler, in which the filler as the dispersed phase and reinforcement component confer most of the mechanical properties.[3,4] Resin composites have been improved in terms of filler type,[5−7] shape,[8,9] and content,[10] including the addition of reinforcing and toughening components to improve their mechanical properties.[11] The zirconia polycrystal is a kind of mechanically strong, metal oxide ceramic,[12] and nanosized zirconia particles have been used to reinforce and toughen dental resin composites.[13] Note that close bonding of the inorganic fillers and organic matrix can prevent the formation of microcracks.[14] Therefore, to achieve structural integrity and sufficient mechanical properties, it is necessary to condition the inorganic surfaces of nanozirconia fillers to make them lipophilic to bond well with the organic matrix.

Only a few commercial products include nanozirconia fillers to improve the mechanical properties of dental resin composites, almost all of which are developed by the 3M ESPE Company. Representative examples include Filtek Z250 and Filtek Z350, which have individual dispersed zirconia and silica fillers compounded into nanoclusters and then treated with gamma-3-(trimethoxysilyl)propyl methacrylate (γ-MPS).[3] This allows the nanoclusters as a whole to bind closely to the resin matrix through chemical bonding between the γ-MPS-conditioned silica and the resin matrix despite γ-MPS not possessing chemical affinity to zirconia. However, the synthesis of this kind of nanocluster requires some complicated technology such as self-assembly methods,[15] spray-drying techniques,[16] aerosol-assisted technology,[17] and calcination processes,[18] limiting the widespread use of the method. A simple and easy alternative methodology would therefore be attractive.

Coupling agent conditioning can chemically modify the surfaces of inorganic filler particles.[19] For example, γ-MPS has an unsaturated C=C double bond at one end that polymerizes with the organic matrix and a hydroxyl group at the other end that forms a Si–O bond network with the silica filler.[18] However, γ-MPS, which is the most commonly used silane coupling agent for coupling the silica filler and resin matrix in resin-based composites,[20] fails to chemically bond zirconia fillers with the resin matrix due to the scarcity of hydroxyl groups on zirconia’s surface.[21,22] This has discouraged further research on using silanes in the surface conditioning of nanozirconia fillers. Recently, the use of phosphate ester monomers methacryloyloxydecyl dihydrogen phosphate (MDP) and dipentaerythritol pentaacrylate phosphate (PENTA) in improving the bond strength of zirconia ceramics has been established,[23−25] which was attributed to their special bifunctional molecular structures that comprise a phosphate group that can form stable Zr–O–P bonds with zirconia crystals and an unsaturated carbon bond at the other end that can polymerize with the ethylene bond in the resin matrix.[23−26] Furthermore, MDP and PENTA have been found to chemically couple nanozirconia fillers with the resin matrices bisphenol-A diglycidyl ether dimethacrylate (Bis-GMA) and urethane dimethacrylate (UDMA).[27] Nevertheless, the increase of mechanical properties of the resulting resin composites is limited, so any potential clinical application requires further development.

Surfactants are another industrially means to enhance the linkage between inorganic fillers and polymer matrices[28] and so provide another potential way to improve the mechanical properties of acrylic resin composites containing nanozirconia fillers. In contrast with coupling agents, surfactants do not depend on the strength of chemical bonding produced by the coupling interaction. Instead, they use surface modification to prevent the agglomeration of fillers and increase the surface free energy and wettability[29] with the purpose of improving the performance of polymer materials. This is necessary for nanometer-sized inorganic fillers with extremely large surface free energy because nanoparticles tend to spontaneously aggregate.[30] Recently, zirconia nanoparticles have been found to be successfully silanized by 3-aminopropyltriethoxysilane (APTES), and their incorporation enhanced the strength and modulus under bending of glass fiber/epoxy composites to ∼22 and ∼38%, respectively.[31] In addition, it was also found that the inclusion of (3-mercaptopropyl)trimethoxysilane (MPTS)-conditioned zirconia–silica or zirconia–yttria–silica ceramic nanofibers can reinforce the flexural strength and the flexural modulus of the dental composite.[5] However, in the absence of the coupling function, there is no evidence of whether inclusion of nanozirconia fillers conditioned with surfactants can improve the mechanical properties of Bis-GMA-based resin composites.

The current study aimed to evaluate the effects of conditioning nanozirconia fillers with two silane coupling agents, APTES and MPTS, on the mechanical properties of Bis-GMA-based resin composites. The following mechanical parameters were investigated: the flexural strength, the corresponding Weibull analysis, and the elastic modulus. Furthermore, the chemical adsorptions of various coupling agents or surfactants on the surface of nanozirconia fillers were characterized, along with surface wettability. The null hypotheses were as follows: (i) silane coupling agents APTES and MPTS do not chemically adsorb on the surfaces of nanozirconia fillers (ii) and therefore cannot improve the mechanical properties of Bis-GMA-based resin composites.

Results

Fourier Transform Infrared (FTIR) Analysis

Structural formulas of four surface treatment agents (MDP, PENTA, APTES, and MPTS) and their chemical reactions with zirconia or the resin monomer Bis-GMA are shown in Figure .

Figure 1

Structural formulas of four surface treatment agents (MDP, PENTA, APTES, and MPTS) and their chemical reactions with zirconia or the resin monomer Bis-GMA.

The FTIR spectra of nanozirconia fillers before and after conditioning by MDP/PENTA/APTES/MPTS are shown in Figure .

Figure 2

(A) FTIR spectra of (a) unconditioned nanozirconia fillers (control) and nanozirconia fillers conditioned with (b) MDP, (c) PENTA, (d) APTES, and (e) MPTS in the range of 4000 to 400 cm–1. (B) FTIR spectra of nanozirconia fillers conditioned with unconditioned nanozirconia fillers (control) and nanozirconia fillers conditioned with APTES and MPTS in the amplification of the range between 2000 and 800 cm–1. (C) FTIR spectra of nanozirconia fillers conditioned with unconditioned nanozirconia fillers (control) and nanozirconia fillers conditioned with MDP and PENTA in the amplification of the range between 2000 and 800 cm–1.

The P–O stretch was detected in MDP- and PENTA-conditioned nanozirconia fillers at 1165 and 1174 cm–1, respectively. New absorption peaks near 1700 and 3000 cm–1, which were not shown by the unconditioned nanozirconia, indicated the chemical combination of nanozirconia and the phosphate ester monomers MDP and PENTA.

The FTIR spectrum of the APTES-conditioned nanozirconia filler showed an absorption band at 1569 cm–1 and several bands near 3600 cm–1, which may indicate successful grafting of APTES onto the nanozirconia filler. In addition, the wide absorption band near 1100 cm–1 appeared in the FTIR spectrum of the MPTS-conditioned nanozirconia filler, which arose from Si–O bending vibrations and Zr–O–Si stretching vibrations.

X-ray Photoelectron Spectroscopy (XPS) Analysis

Figure shows the XPS O1s spectra of nanozirconia fillers conditioned by MDP, PENTA, APTES, and MPTS. After the spectra were calculated using a peak fitting algorithm, Zr–O–P bonding was detected in MDP-conditioned nanozirconia fillers at higher levels (28.72%) than in PENTA-conditioned nanozirconia fillers (27.26%). Furthermore, Zr–O–Si bonding was detected in APTES- and MPTS-conditioned nanozirconia fillers at a greater content in the latter (6.98%) than in the former (2.02%). The untreated filler showed no XPS peak corresponding to Zr–O–P or Zr–O–Si bonding.

Figure 3

(A) Wide-scan X-ray photoelectron spectra of nanozirconia fillers conditioned with (a) MPTS, (b) APTES, (c) PENTA, (d) MDP, and (e) unconditioned nanozirconia fillers. Peak fitting results of the O1s spectra of (B) the unconditioned nanozirconia filler and fillers conditioned with (C) MDP, (D) PENTA, (E) APTES, and (F) MPTS.

Thermodynamic Calculations

Models for MPTS and APTES in Figure are shown alongside those for their silanol groups and coordination with tetragonal zirconia.

Figure 4

Models of (A) (a) MPTS and (b) its total hydrolysate, (B) (a) APTES and (b) its total hydrolysate, (C) the MPTS–ZrO2 system after optimization, and (D) the APTES–ZrO2 system after optimization.

Hydrolysis of MPTS and APTES reacted in the ethanol phase, whereas silanol groups and ZrO2 clusters reacted at the interface of the ethanol and solid phases. The Gibbs free energies and equilibrium constants (K) of the chemical coordination of MPTS and APTES with tetragonal zirconia are shown in Table .

Table 1

Thermodynamic Calculations of the Chemical Coordination among MPTS, APTES, and Tetragonal Zirconiaa

	ΔG (Ha)	ΔG (kJ/mol)	K
hydrolysis of MPTS	–0.010	–26.948	5.290 × 104
silanol derivative of MPTS coordinated with ZrO2	–0.026	–67.638	7.172 × 1011
hydrolysis of APTES	–0.012	–31.275	3.033 × 105
silanol derivative of APTES coordinated with ZrO2	–0.025	–65.714	3.299 × 1011

ΔG: change in Gibbs free energy, K: equilibrium constant.

According to thermodynamic data, the final Gibbs free energy of the chemical coordination among MPTS and tetragonal zirconia was −94.59 kJ/mol and that of APTES was −96.99 kJ/mol. The final equilibrium constant of the reaction among MPTS and tetragonal zirconia was 3.79 × 1016, and that of APTES was 1.00 × 10.[17] Detailed thermodynamic results are included in the Supporting Information.

Contact Angle Measurement

Figure shows representative images of the contact angle results listed in Table for each group. One-way ANOVA showed that the contact angles of the conditioned ceramic plates were significantly lower than those of the untreated ceramic plates (P < 0.05). There was no significant difference in the surface wettability of MDP–ZrO2 and PENTA–ZrO2 (PMDP-ZrO = 0.185) or APTES–ZrO2 and MPTS–ZrO2 (PAPTES-ZrO = 0.390).

Figure 5

Representative contact angles of (A) unconditioned zirconia ceramic and zirconia ceramic conditioned with (B) MDP, (C) PENTA, (D) APTES, and (E) MPTS.

Table 2

Contact Angles for the Zirconia Ceramics before and after Conditioninga

group	contact angle (deg)
MDP–ZrO2	11.36 ± 1.20c
PENTA–ZrO2	14.01 ± 1.42c
APTES–ZrO2	22.60 ± 3.84b
MPTS–ZrO2	24.50 ± 2.80b
control	33.00 ± 2.78a

The same letter superscript indicates no significant difference between the groups (P > 0.05)..

Scanning Electron Microscope (SEM)

The SEM images in Figure of the five experimental resin composites containing treated or untreated nanozirconia fillers show similar structures without agglomeration.

Figure 6

SEM images in backscattering mode (magnification ×2000) of Bis-GMA-based resin composites with (A) the untreated nanozirconia filler and nanozirconia filler treated with (B) MDP, (C) PENTA, (D) APTES, and (E) MPTS. White pointers point to fillers contained in the prepared resin composites.

Three-Point Bending Strength, Elastic Modulus, and Weibull Analysis

Table lists the means and standard deviations of the flexural strength and elastic modulus of each group.

Table 3

Flexural Strengths (σf), Elastic Moduli, Weibull Moduli (m), and the 95% Confidence Intervals (CIs) of m for the Resin Compositesa

group	σf (MPa)	elastic modulus (GPa)	m	95% CI of m
MDP–ZrO2	117.54 ± 15.58b	7.95 ± 0.63b	8.45	5.49–11.41
PENTA–ZrO2	119.33 ± 11.63b	8.25 ± 0.70b	11.63	7.96–15.30
APTES–ZrO2	121.02 ± 8.31b	8.76 ± 0.52a	14.92	8.22–21.62
MPTS–ZrO2	132.80 ± 15.80a	9.24 ± 0.60a	9.52	6.45–12.60
control	94.84 ± 9.28c	7.44 ± 0.83c	12.07	10.88–13.27

The same letter superscript indicates no significant difference between the groups (P > 0.05)..

One-way ANOVA showed that both properties of the resin composites containing conditioned nanozirconia fillers were significantly higher than those of the composite with the unconditioned filler (P < 0.01). The MPTS–ZrO2 group showed the highest flexural strength among the five experimental resin composites, while there was no difference in the flexural strength of the MDP–ZrO2, PENTA–ZrO2, and APTES–ZrO2 groups (PMDP-ZrO = 0.707, PMDP-ZrO = 0.465, PPENTA-ZrO = 0.722).

LSD tests found no significant difference between the elastic moduli of the MDP–ZrO2 and PENTA–ZrO2 groups (P = 0.256) or between the APTES–ZrO2 and MPTS–ZrO2 groups (P = 0.062). The elastic moduli of the APTES–ZrO2 and MPTS–ZrO2 groups were significantly higher than those of the MDP–ZrO2 and PENTA–ZrO2 groups (P < 0.05).

Table gives the Weibull moduli (m) and 95% confidence intervals (CIs) for the experimental resin composites, and the Weibull distribution plots for flexural strength are presented in Figure . The APTES–ZrO2 group exhibited the highest Weibull modulus.

Figure 7

Weibull distribution plots for Bis-GMA-based resin composites with untreated (A) nanozirconia fillers and fillers conditioned with (B) MDP, (C) PENTA, (D) APTES, and (E) MPTS.

Cytotoxicity Experiments and Morphological Observation

Figure shows cell proliferation curves for the experimental and negative control groups. The OD of the negative control group was higher than those of the experimental groups, indicating better cell proliferation.

Figure 8

Relative cell proliferation of the experimental groups and the negative control group. *p < 0.05 compared with the experimental groups.

Table shows the relative cell growth rate (RGR) of each group. The RGRs of five resin material extracts (from composites containing MDP-treated, PENTA-treated, APTES-treated, MPTS-treated, and untreated nanozirconia fillers) were 80.96, 78.93, 82.29, 84.61, and 88.61% after 120 h, respectively, indicating no obvious cytotoxicity.

Table 4

Relative Growth Rates (RGR) of Resin Composites

	RGR
group	24 h	72 h	120 h
MDP–ZrO2	83.10%	82.18%	80.96%
PENTA–ZrO2	81.65%	79.28%	78.93%
APTES–ZrO2	86.24%	86.61%	82.29%
MPTS–ZrO2	93.35%	89.89%	84.61%
control	94.87%	93.14%	88.61%

Figure shows the morphology of mouse fibroblasts of each group under an inverted microscope. The cells in each group showed normal morphology and similar growth trends, evenly covering the entire bottom of the well plate by the fifth day of culture.

Figure 9

Cell morphology images under an inverted microscope: cell morphology in each group after 1 (A, D, G, J, M, P), 3 (B, E, H, K, N, Q), and 5 (C, F, I, L, O, R) days of cell culture. The resin composites had fillers treated with (A–C) MDP, (D–F) PENTA, (G–I) APTES, (J–L) MPTS, and (M–O) no modifier (control group). (P–R) Results for the negative control (NC) group.

Discussion

Previous studies have demonstrated that zirconia ceramics treated with γ-MPS do not show improved adhesion owing to the scarcity of hydroxyl groups on zirconia’s surface,[32] which prevents γ-MPS from chemically bonding with it. Consequently, few works have considered other kinds of silane applied to zirconia surfaces. Plasma and ultraviolet treatments can increase the amount of hydroxyl groups on the surface of zirconia.[33−35] Therefore, this study exposed zirconia powders to ultraviolet (UV) light for 4 h to increase their surface hydroxyl groups and improve the possibility of chemical combination with silanes. Subsequent characterization by FTIR and XPS aimed to verify the chemical affinity between phosphate ester monomers and nanozirconia filler, as well as the two silanes and UV-treated nanozirconia filler. The FTIR results found the stretching peaks of P–O bonds in the spectra of MDP- or PENTA-conditioned nanozirconia fillers at 1165 and 1174 cm–1, respectively, while the P–O bonding was not identified in the unmodified sample, indicating the effectiveness of surface treatment with MDP or PENTA.[27] Compared with the unmodified nanozirconia fillers, a new absorption peak in the FTIR spectrum of APTES-conditioned nanozirconia fillers appeared at 1569 cm–1, alongside several weak peaks at 3500–4000 cm–1, which can be attributed to the successful grafting of APTES onto nanozirconia. The FTIR spectrum of MPTS-conditioned nanozirconia displays a similar set of absorption peaks in roughly the same area (3500–4000 cm–1) and a broad absorption peak at about 1100 cm–1, which may have arisen owing to the combined action of Si–O bending vibration and Zr–O–Si stretching vibration,[36] affirming the successful grafting of MPTS onto the nanozirconia.

The XPS results were consistent with the FTIR spectra. Peak fitting for the XPS O1s spectra identified Zr–O–P bonding in both MDP- and PENTA-treated nanozirconia, with the surface of the former having a greater coverage (28.72%) than the latter (27.26%), consistent with previous work.[27] The three-dimensional spatial structure of PENTA gives it greater steric resistance during the modification process than the linear structure of MDP (Figure ),[25] resulting in a slightly lower content of Zr–O–P bonds than on MDP-treated nanozirconia. The XPS spectra of the APTES- and MPTS-conditioned fillers showed Zr–O–Si bonds, respectively, at 532.2 and 531.7 eV,[37,38] while XPS identified no peak corresponding to Zr–O–Si in the untreated nanozirconia. The APTES-conditioned nanozirconia had a lower surface covering with Zr–O–Si bonds (2.02%) than did the MPTS-conditioned nanozirconia (6.98%), suggesting that MPTS had a better binding ability with zirconia than APTES at 10% volume concentration.

The FTIR and XPS results are well explained by the thermodynamic calculations for Gibbs free energy and equilibrium constant. Both MPTS and APTES had negative Gibbs free energies for the reaction with zirconia, indicating spontaneous reaction.

Preliminary work has demonstrated that nanozirconia fillers modified by phosphate ester monomers can potentially improve the mechanical properties of Bis-GMA-based and UDMA-based resin composites.[27] This has been attributed to the bifunctional groups (an unsaturated carbon bond at one end and a phosphate group at the other) contained in the molecular structure of the phosphate ester monomers,[24] which allows the nanozirconia filler particles to be well dispersed in the organic matrix medium. Although the FTIR, XPS, and thermodynamic calculation results of this work affirmed that APTES and MPTS successfully grafted onto nanozirconia, they failed to combine with the resin matrix due to a lack of unsaturated C=C bonds in their molecular structures (Figure ). Therefore, whether APTES or MPTS, as surfactants rather than coupling agents, can also improve the dispersion of nanozirconia fillers in the resin matrix and ultimately improve the mechanical properties of the composite resin was considered. The surface wettability results showed contact angles of the four groups of modified zirconia ceramics that were significantly lower than those of the nonmodified zirconia ceramics, indirectly indicating significantly improved lipophilicity of nanozirconia filler particles modified by phosphate ester monomers (MDP and PENTA) or surfactants (APTES and MPTS), which was conducive to their dispersion in the resin matrix medium. However, the contact angles of MDP–ZrO2 and PENTA–ZrO2 were markedly higher than those of APTES–ZrO2 and MPTS–ZrO2, which may be attributed to the chemical reaction between the phosphate ester monomers and the monomer TEGDMA during the measurement process, while the surfactants failed to react with TEGDMA.

The grafting of the four surface treatment agents onto the nanozirconia filler was verified by FTIR, XPS, and thermodynamic calculations. The improved dispersion of the conditioned filler particles in the resin matrix was confirmed by surface wettability tests. However, whether the inclusion of the modified nanozirconia fillers strengthens the mechanical performance of the Bis-GMA-based resin composites required direct study, which was conducted here through the analyses of three-point bending strength, elastic modulus, and Weibull modulus.

The three-point bending strengths and elastic moduli of the composites containing conditioned fillers were significantly improved compared with the one containing the untreated filler. There was no significant difference in flexural strength between the MDP–ZrO2 and PENTA–ZrO2 samples despite the former having a greater extent of Zr–O–P bonding than the latter, consistent with previous results.[27] In addition, without chemical coupling, the nanozirconia filler modified by APTES or MPTS could indeed strengthen the mechanical performances of Bis-GMA-based resin composites. The MPTS–ZrO2 samples’ three-point bending strength was significantly higher than that of the APTES–ZrO2 group, suggesting that MPTS was more suitable for conditioning nanozirconia fillers at the 10% volume concentration employed here. This is consistent with the XPS results that found more extensive Zr–O–Si bonding in nanozirconia conditioned with MPTS rather than APTES. Compared with composites made with unmodified nanozirconia, those with the MPTS-modified filler showed 40% higher three-point bending strength. This was a significantly greater increase than that achieved using the nanozirconia filler modified with phosphate ester monomers (26.3%), indicating that using a coupling agent with bifunctional groups is not the only way to condition nanozirconia fillers to strengthen resin composites. Surfactants represent good alternatives.

Weibull analysis evaluated the reliability of the obtained three-point bending strength results. Generally, a larger Weibull modulus (m) indicates a smaller error range and higher structural integrity (that is a more reliable material).[39,40] The resin composites containing the MPTS-conditioned nanozirconia filler showed the greatest flexural strength and Weibull modulus, further supporting that the surfactant MPTS was the most beneficial to modify nanozirconia to improve the mechanical properties of the Bis-GMA-based resin composite. The SEM image results showed the filler particles in each resin composite as well dispersed without agglomeration.

Biocompatibility is important to the safe clinical application of dental resin composites. As the composites developed here included added modifying agents, it is therefore necessary to evaluate their cytotoxicity. This was done here using L929 mouse fibroblasts, a stable cell line widely recommended for cytotoxicity tests of resin-based materials. Zirconia is considered to be a low-toxic substance with proven favorable biocompatibility, and animal experiments have confirmed that its absorption in the gastrointestinal tract is extremely low.[21] According to the CCK-8 results, the RGR of each experimental group decreased with increasing time, which may be attributed to the unreacted residual monomers (Bis-GMA and TEGDMA) eluted from the dental resin-based materials due to incomplete curing, which can release free radicals or directly damage tissues after entering the mouth, resulting in obvious cytotoxicity.[41,42] Nevertheless, cytotoxicity results after 5 days showed that there was no difference in RGRs between the resin composite containing conditioned nanozirconia fillers and the resin composite with unconditioned fillers, indicating that the five prepared resin composite materials are potentially safe for clinical use.

Conclusions

The results of the present study show, within the study’s limitations, that the null hypothesis can be rejected, and the following conclusions may be drawn:

MDP, PENTA, APTES, and MPTS can chemically bond with nanozirconia fillers.

Such conditioned fillers can potentially improve the mechanical properties of Bis-GMA-based resin composites, without obvious cytotoxicity.

A coupling agent with bifunctional groups is not the only way to condition nanozirconia fillers to strengthen resin composites. Surfactants represent good alternatives. Using APTES- or MPTS-conditioned nanozirconia fillers was better than using MDP- or PENTA-conditioned nanozirconia fillers in improving the mechanical properties of Bis-GMA-based resin composites, and the MPTS-conditioned nanozirconia filler had the best effect.

Materials and Methods

Surface Treatment of Nanozirconia Particles

Nanozirconia particles (Macklin, China, 50 nm) were either treated with a phosphate ester monomer [methacryloyloxydecyl dihydrogen phosphate (MDP) or dipentaerythritol pentaacrylate phosphate (PENTA)],[27,40] or a silane APTES or MPTS. Table shows the specific formulation of the agents and the corresponding surface modification methods.

Table 5

Specific Formulation of the Primers and the Corresponding Surface Modification Method Used in This Studya

primer	manufacturer	formulation of the primer	surface modification method
MDP/PENTA	MDP, DM Healthcare Products, USA; PENTA, Dentsply, USA	10% MDP (PENTA), 88.8% ethanol, 0.3% CQ, and 0.9% EDMAB in weight	Nanozirconia filler was immersed in MDP or PENTA solution by 1 g/mL and then dispersed ultrasonically for 10 min.
			After storage for 12 h at room temperature and protected from light, the mixture was cleaned with ethanol three times and then dried in an oven at 65 °C for 8 h.
APTES/MPTS	APTES, Alfa Aesar, China; MPTS, Sigma-Aldrich, USA	10% APTES (MPTS) and 90% ethanol in volume	The nanozirconia filler was irradiated with ultraviolet for 4 h and then immersed in APTES or MPTS solution by 1 g/5 mL.
			After storage for 8 h at room temperature and protected from light, the mixture was cleaned with ethanol three times and then dried in an oven at 65 °C for 8 h.

Abbreviations: MDP, methacryloyloxydecyl dihydrogen phosphate; PENTA, dipentaerythritol pentaacrylate phosphate; CQ, camphorquinone; EDMAB, ethyl-4-dimethylamino benzoate; APTES, 3-aminopropyltriethoxysilane; MPTS, (3-mercaptopropyl)trimethoxysilane.

Fourier Transform Infrared (FTIR)

The conditioned zirconia powders underwent FTIR spectroscopy (Nicolet 6700, Thermo Scientific, USA) using KBr tablets in transmission mode in the range of 4000 to 400 cm–1 to detect changes in chemical bonds on the zirconia surface. Zirconia powders without conditioning were employed as a control.

X-ray Photoelectron Spectroscopy (XPS)

The zirconia powders were also analyzed using XPS (Escalab 250xi, Thermo Fisher Scientific, UK) with monochromatic Al Kα radiation (photo energy = 1486.6 eV, energy step size = 0.05 eV). The O1s spectra were processed using XPS Peak 4.1 software with the Lorentz–Gauss ratio fixed at 80%.

Modeling of Complexes between Tetragonal Zirconia and Silanes and Thermodynamic Calculations

A tetragonal zirconia crystal model was created using Materials Studio v4.0 (Accelrys, San Diego, CA, USA) according to the Inorganic Crystal Structure Database (Fachinformationszentrum Karlsruhe Information Services, Eggenstein-Leopoldshafen, Germany). The structure of ZrO2 clusters was built according to earlier research.[26] The process of cleaving certain low index faces allowed us to note possible bonding sites for silanes on those faces as silanes react with hydroxyl groups on the ZrO2 surface. Models of MPTS and APTES were constructed according to their molecular formulas and geometrically optimized using the Gaussian 09 software package (Gaussian, Wallingford, CT, USA). The corresponding silanol derivatives are obtained by hydrolysis of MPTS and APTES. Hydrolysis of MPTS and APTES was both simulated in the ethanol phase. The solvent effect was considered using the integral equation formalism polarizable continuum model (IEF-PCM).

The complex of silanol groups and ZrO2 clusters was geometrically optimized, and frequency calculations were performed. High-level calculations found stable optimization geometries and calculated thermodynamic functions of the silane–ZrO2 clusters.

Calculations of Gibbs free energy employed density functional theory with Becke’s 3 parameters (B3) and the Lee–Yang–Parr nonlocal correlation functional (LYP). Atoms of C, H, O, N, Si, and S were studied at the 6-31G** level. For Zr atoms, the valence and core electrons were described using the LanL2DZ basis set and the corresponding relativistic effective core potentials, respectively. The default convergence criteria for optimization were maximum force <0.000450 Hartree/Bohr, root mean square (RMS) force <0.000300 Hartree/Bohr, maximum displacement <0.001800 Hartree/Bohr, and RMS displacement <0.001200 Hartree/Bohr.

All the above calculations related to geometry optimization and thermodynamic functions were performed using Gaussian 09 software (Gaussian, Wallingford, CT, USA). All data were calculated in ambient conditions (1 atm, 298 K).

The zirconia powders were submitted to crystallization firing to obtain ceramic plates (n = 15) of 10 × 10 × 2 mm3. The plates were wet-polished using 1000-, 2000-, 3000-, and 4000-grit silicon carbide abrasive papers consecutively with a rotational polishing device (PG-1, BiaoYu Instrument, Shanghai, China) and ultrasonically cleaned for 30 min. The polished plates were then randomly assigned to five experimental groups (n = 3) to be conditioned by the following primers through immersion for 8 h: 10% MDP (MDP–ZrO2), 10% PENTA (PENTA–ZrO2), 10% APTES (APTES–ZrO2), and 10% MPTS (MPTS–ZrO2). The details are shown in Table . They were then ultrasonically cleaned in ethanol for 5 min and air-dried. Untreated plates were used as a control.

Surface wettability was assessed by the sessile drop method: triethylenegycol dimethacrylate (TEGDMA, Aladdin, China) was placed on the specimen’s surface for 10 s and measured with a contact angle measuring device (Theta Lite, Biolin, Finland).

Resin Composite Preparation

The monomers Bis-GMA and TEGDMA were mixed in a 7:3 ratio. 0.5 wt% CQ and 1 wt% ethyl-4-dimethylamino benzoate (4EDMAB) were then added as the photo-oxidant and photoreductant, respectively.[43] 5 wt% nanozirconia particles treated with MDP, PENTA, APTES, or MPTS were thoroughly mixed with 55 wt% γ-MPS-conditioned silica particles (Sigma-Aldrich, USA) and then added into the resin matrices.[13] The composite containing untreated nanozirconia fillers was employed as a control.

Samples containing the untreated nanozirconia filler and nanozirconia fillers treated with MDP/PENTA/APTES/MPTS were wet-cut into 2 × 2 × 2 mm3 pieces using a low-speed saw (ISOmet1000, Buehler Ltd., Lake Bluff, IL, USA). The cross sections of these samples were coated with gold and examined by SEM (MAIA3 TESCAN, Czech Republic) at an acceleration voltage of 20 kV in backscatter mode to reveal the distributions of inorganic filler particles within them.

Numbered lists can be added as follows: Bar-shaped resin specimens (n = 15) were made using Teflon molds (25 × 2 × 2 mm3), covered with a Mylar film in order to avoid oxygen inhibition. The resin composites were light-cured by four overlapping 20 s exposures of 1200 mW/cm2 (Elipar S10, 3M ESPE, USA) on both sides, according to ISO 4049-2019.[44] The specimens were carefully removed from the mold, polished flat with 1200-grit silicon carbide paper, and then stored in distilled water at 37 °C for 24 h. Subsequently, the specimens were subjected to three-point bending strength testing using a universal testing machine (Instron 3365 ElectroPuls, Instron, USA) at a crosshead speed of 0.5 mm/min until failure. The fracture load and corresponding elastic modulus were recorded. The flexural strength (σf) was calculated as follows: σf = 3Fl/2bh2, where F is the maximum load (N) exerted on the specimen, l is the bracket spacing (the distance between the supports; 20 mm), b is the width (2 mm), and h is the height (2 mm) at the center of the specimen.

For each material specimen (N = 15), the measured flexural strength values were arranged in increasing order and assigned the labels i = 1, 2, 3, ..., N, with i = 1 and i = N denoting the minimum and maximum values of each group. Weibull analysis, based on the σ data, described the failure probability (Pf) as follows: Pf = 1 – exp { – (σf/σθ)}, where σf is the bending strength, σθ is the scale parameter, and m is the Weibull coefficient. The probability of failure may be estimated as follows: Pf = (i – 0.5)/N, where i is the strength ranking in ascending order and N is the number of specimens. The following calculations were linearly transformed from the aforementioned equation using the least-squares method: , where m is the slope and −m ln σθ is the intercept.

Cytotoxicity Experiments and Morphological Observation

Specimens (diameter, 6 mm; thickness, 1.5 mm) of uncured experimental resin composites were prepared using the same method as for three-point bending testing. Ten specimens with a total surface area of 8.478 cm2 for each group were obtained and stored in distilled water at 37 °C for 24 h. Surface area of specimens/volume of medium = 1.25 cm2/mL of specimens of each group were eluted with DMEM (Gibco, USA) according to the manufacturer’s recommendation at 37 °C for 24 h. Then the obtained elutes were passed through a 0.22 μm filter.

L929 mouse fibroblast cells were cultured in DMEM with 10% fetal bovine serum (Gibco, USA) in a humidified incubator at 37 °C under an atmosphere of 5% CO2. Cells were seeded in 96-well plates at a density of 0.5 × 104 cells/well after being digested by trypsin and then cultured for 24 h. After cells were attached to the wells, various elutes obtained from the five experiment groups (MDP–ZrO2, PENTA–ZrO2, APTES–ZrO2, MPTS–ZrO2, control) were added to each well of the 96-well plates when the culture medium supplemented with 10% fetal bovine serum was replaced. After the cells had been cultured for 24, 72, and 120 h, the Cell Counting Kit-8 (CCK-8; Dojindo, Kyushu, Japan) was used to evaluate their viability according to the manufacturer’s instructions. Wells with the CCK-8 solution but without cells were used as a blank control. Subsequently, a microplate reader (SpectraMax M5, Molecular Devices LLC, Sunnyvale, CA, USA) was used to measure the optical density (OD) at a wavelength of 450 nm. Cytotoxicity was evaluated according to the relative growth rate (RGR) of the cells:

The mouse fibroblast L929 cells were cultured in the 24-well plate at a density of 5 × 105 cells/mL as described above. The culture medium supplemented with 10% fetal bovine serum was replaced by various elutes, and the cells were cultured for 24, 72, and 120 h. Morphological alteration of the cells was observed directly using a phase contrast microscope (Nikon, Japan) and photographed.

Statistical Analysis

Statistical comparisons were performed after homogeneity of variance and normality tests. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc least significant difference (LSD) multiple comparisons was used to analyze the three-point bending strength, elastic modulus, contact angle, and OD value. Statistical analysis was performed in SPSS 21.0 statistical software (IBM SPSS Inc., Chicago, IL, USA). Statistical significance was indicated by P < 0.05.