Research on Silicone Methacrylates Containing Dental Resins With Anti-adhesion Effect Against S. Mutans and Resistance to Mucin Adsorption


 With the purpose of preparing anti-bacterial adhesion dental resin, two silicone methacrylates (SMAs) were synthesized. After being confirmed by the FT-IR and 1H-NMR spectra, SMAs were incorporated into commonly used Bis-GMA/TEGDMA (50/50, wt./wt.) dental resin system with a series of concentration. Physicochemical properties, anti-bacterial adhesion effect, and protein adsorption were tested. The results showed that SMAs had no influence on the double bond conversion of dental resin, and could decrease volumetric shrinkage of dental resin. Because of the increased hydrophobicity and reduced surface free energy, SMAs containing cured resin had resistance to mucin adsorption and anti-adhesion effect against S. mutans. However, flexural properties, water sorption and solubility of dental resin were impaired after introducing SMAs. Therefore, further research should be taken to improve these properties by utilizing appropriate inorganic fillers.


Introduction
Dental resin composites (DRCs), which consist of methacrylate-based resin matrix and silanized inorganic fillers, have been widely as restorative materials in dentistry due to their properties as easy handling and desirable aesthetics [1]. However, compared with other restorative materials such as silver amalgam and glass ionomer cement, DRCs can accumulate more biofilm on the surface because of lacking inherent antibacterial activity [2][3][4], leading to higher occurring possibility of secondar caries, while secondary caries is considered to be one of the main reasons for clinical DRCs restoration failure in long term [5][6][7]. Therefore, how to reduce biofilm formation on the surface of DRCs plays an important role in long-term success rate of restoration.
Besides the characteristics of fillers, properties of DRCs are mainly influenced by the monomeric composition of resin matrix [8]. For the purpose of achieving long-term biofilm inhibition, several methacrylate monomers with antibacterial groups such as quaternary ammonium have been synthesized and incorporated into dental resins as polymerizable antibacterial agents [9][10][11]. These agents immobilize antibacterial groups into polymeric network by covalent bonds, thus could endow dental resins with long-last antibacterial activity [12].
Nevertheless, immobilizable antibacterial groups work only when bacteria contact with them, and acquired pellicle formed on the surface of DRCs would forbid contacting between antibacterial groups and bacteria, leading to the reduction in antibacterial efficiency of polymerizable antibacterial agents [13,14]. The acquired pellicle is a thin protein layer formed by the absorbed biological components such as salivary proteins on DRCs surface [15,16], thus a protein-repellent monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC), was introduced into resin matrix to inhibit the formation of acquired pellicle [13][14][15][16]. Cao et al. [14] revealed that dental resin both with MPC and quaternary ammonium dimethylaminohexadecy methacrylate (DMAHDM) had stronger antibacterial effects than using DMAHDM and MPC alone.
As a reversable adhesion, bacterial adhesion is considered to be the most important step in biofilm formation [17], so modifying DRCs to be anti-adhesion against bacteria should also be an effective way to inhibit biofilm formation. Adhesion capabilities of bacteria have been demonstrated in conjunction with the surface physicochemical properties of biomaterials and bacterial [18][19][20]. It was reported that materials with low surface free energy accumulated less bacteria with high surface free energy, whereas materials with high surface free energy accumulated less bacteria with low surface energy [21]. Bacteria in early plaque such as Streptococcus sanguis, Veillonella parvula, and Streptococcus mutans (S. mutans) are all bacteria with high surface free energy [22,23], thus DRCs with low surface free energy might have inhibition effect on biofilm formation through reducing bacteria adherence.
In this research, two kinds of silicone methacrylates (SMA) were synthesized and incorporated into dental resin system with the aim of reducing surface free energy, and thereby endowing anti-adhesion effect against bacteria. The influence of SMA structure and concentration on physicochemical properties, anti-adhesion effect against S. mutans and mucin adsorption were investigated.

Materials
Two types of branched amino silicone (BAS) (SF1706, methoxyl terminated amino silicone with amino content of 0.46mmol/g; SF1708, methyl terminated amino silicone with amino content of 0.8mmol/g.) were obtained from Momentive Performance Materials Inc., USA.

Synthesis of silicone methacrylates (SMA)
The synthesis route for target compounds was shown in Figure 1. A mixture of 30 g BAS, IEMA (amount was based on the amino content of different BAS), and 50 mL pre-dried acetone was added into a glass flask and stirred at room temperature. The reaction was continued until the infrared absorbance peak of -NCO group (2270 cm-1) vanished in the FT-IR (Vector 33 Model Fourier Transform Infrared Instrument, Bruker Co., Germany) spectra of the sample that taken from the reaction system. The SMA was obtained after removing the acetone by distillation under vacuum. The FT-IR and 1 H-NMR (nuclear magnetic resonance instrument, Avance AV 600MHz, Bruker, Switzerland) spectra of SMA were taken to confirm the structure.

Preparation of experimental dental resin systems
SMA containing dental resin systems were prepared according to the formulations shown in Table 1. A dental resin without SMA was prepared as control.

Measurement of degree of double bond conversion
The degree of double bond conversion (DC) was determined by using a FT-IR spectrometer with an attenuated total reflectance (ATR) accessory. The ATR spectra were collected at a resolution of 4 cm -1 through 4 scans. All the samples were tested in a mold with a thickness of 2.0 mm and a diameter of 6.0 mm. First, the spectrum of the unpolymerized sample was measured. Then, the sample was irradiated with a visible light-curing unit (X-Cure, λ=385-515nm, I∼1000 mWcm −2 , Woodpecker Medical Instrument Co., Ltd., China) for 40s at room temperature. Finally, the spectrum of the polymerized sample was recorded.
In order to calculate the percentage of reacted vinyl, the absorbance intensities of the methacrylate C=C absorbance peak at 1,636 cm -1 , which were decreased after being irradiated, and the absorption peak of phenyl ring at 1608 cm -1 was taken as the internal standard peak. DC was calculated from the percentage of change in the normalized methacrylate C=C absorbance intensity at 1608 cm -1 by using the following equation: (1) where AC=C and Aph are the absorption intensities of methacrylate C=C at 1,636 cm -1 and phenyl ring at 1,608 cm -1 , respectively. (AC=C/Aph)40 and (AC=C/Aph)0 refer to the normalized absorbance of the functional group irradiated for 40s and 0s, respectively. Three replicates (n=3) were performed for the measurement of every resin system.

Measurement of volumetric shrinkage
The volumetric shrinkage was determined according ISO 17304:2013(E). The specimens' densities were measured to determine volumetric shrinkage in accordance with the buoyancy method (Archimedes' principle) by using an analytical balance (FA1104J, Shunyuhengping Scientific Instrument Int., Shanghai, China) with a density test kit. The weight of the specimen was measured in air and water respectively, and the density of the samples was calculated by the following formula: (2) where ρ is the individual density of the sample, M1 is mass of the sample in air, M2 is the mass of the sample in water medium, ρ0 is the density of the water medium at the temperature at the respective time of measurement.
Then, volumetric shrinkage (VS) was calculated according the following formula: Where ρu is the density of unpolymerized specimen, ρc refer to the density of polymerized specimen. Six replicates (n=6) were performed for the measurement of each resin system before and after polymerization, respectively.

Three-point bending test
Bar-shaped specimens were made in half-split stainless steel moulds between transparent Mylar sheets. The resin was cured using a visible light-curing unit as mentioned above for 40 s in five separate overlapping portions from one side of the mould. Sixteen specimens were prepared for every dental resin system and divided into two groups randomly. One group was kept dry in desiccator for 1 week before test, and the other group was immersed in distilled water at 37℃ for one week before test. The three-point bending test (span = 20 mm) was carried out on a universal testing machine (AGS-10KN, Shimadzu Co., Japan) at a cross-head

Measurement of water sorption, solubility, and diffusion coefficient
The resin was filled into a 1.0 mm height cylindrical stainless steel mold with a diameter of 15.0 mm followed by a transparent Mylar sheet. The resin was light cured using a visible light-curing unit as mentioned above for 40 s in twelve separate overlapping portions from one side of the mould. Five specimens (n=5) were prepared for every dental resin system.
The initial mass (M1) of every specimen was measured using an analytical balance with an accuracy of 0.1mg. After that, the specimens were immersed in 30ml of distilled water at 37℃. At fix time intervals, the specimens were taken out, wiped to remove the excess water on the surface, and weighted to get mass (Mt). The equilibrium mass of specimen after 16 days of water immersion was defined as M2. Finally, the specimens were dried in a vacuum oven at 60℃ until getting equilibrium mass (M3). Water sorption (WS) and solubility (SL) were calculated by the following formula.
The water absorption and solubility of resin-based composite have been proved as diffusion-controlled [23]. In a relatively short term (1, 3, 6 and 9h) immersion, when can be calculated using Stefan approximation [24,25]:

Measurement of contact angle and surface free energy (SFE)
The resin was filled into a 1.0 mm height cylindrical stainless steel mold with a diameter of 10.0 mm followed by a transparent Mylar sheet. The resin was light cured using a visible light-curing unit as mentioned above for 40 s in four separate overlapping portions. Every specimen was polished using wet silicon carbide grinding paper (FEPA #4000) in a grinding machine (MPD-1, Guangxiangzhiyang Instrument Ltd., Shanghai, China) to obtain a smooth surface. Subsequently, the polished specimens were ultrasonically cleaned in distilled water for 30 min to remove possible embedded grinding material. After that, specimens were dried in a desiccator before using. Six specimens (n=6) were prepared for every dental resin system. Distilled water and diiodomethane were used as testing liquids for surface free energy calculation. A 2 μL droplet of liquid was placed on the surface of every specimen for 20 s, and equilibrium contact angle was measured at room temperature using a contact angle meter (SDC-100, Dongguan SINDIN Precision Instrument Co., Ltd., Dongguan, China). The surface free energy was calculated using Owens-Wendt approach and the result was directly obtained from the software company with the contact angle meter.

Assessment of mucin adsorption
The specimens for mucin adsorption test were prepared with the same protocol as the specimens for contact angle measurement. Six specimens (n=6) were prepared for every dental resin system. The amount of adsorbed mucin was determined by Bradford method [26].
where y is the concentration of mucin solution, x is the absorbance of mucin solution with Bradford solution.
Finally, the amount of adsorbed mucin per area (AMA) was calculated by the following equation: where yb and ya is the concentration of mucin solution before and after testing, respectively. V is the volume of tested mucin solution. S is the surface area of specimen.

Assessment of bacterial adhesion
Bacterial culture Streptococcus mutans (S. mutans, ATCC 25175) was spread on brain heart infusion (BHI) agar plates. After overnight culture at 37 °C, a single colony was collected into 15 mL centrifuge tubes with 5 mL of BHI medium and incubated under anaerobic conditions at 37 °C for 24 h. The bacterial suspension was diluted to approximately 1 × 10 7 CFU/mL for the bacterial adhesion assay using the McFarland Criterion.

Adhesion assay
The specimens for bacterial adhesion test were prepared with the same protocol as the specimens for contact angle measurement. Two specimens of every dental resin system were prepared for one test, and the test was repeated triplet (totally n=6). After being sterilized with ultraviolet rays on each side, the disc shaped specimen was placed at the bottom of 24-well cell culture plate, which was then filled with 1mL of bacteria suspension. The covered plate was anaerobic incubated at 37℃ for 24h. Subsequently, the sample was rinsed twice with PBS and moved to 5 ml centrifuge tube containing 3 ml phosphate buffered saline (PBS).
After sonication for 15 min and vortexing for 15 s to detach the adhered bacteria, 100 μL of diluted suspension was spread onto BHI agar and incubated at 37 ℃ in 5% CO2 for 24 h. The colonies that grew on BHI agar were counted to assess the ability of resisting bacterial adhesion. The amount of bacteria recovered from surface of control group was setting as 100%.

Statistical analysis
One-way ANOVA analyses of variance were used to examine statistical differences between variables by employing SPSS 25.0 software (IBM SPSS Software，USA)，followed by post hoc Tukey tests ( at a significance level of p = 0.05). Figure 3 were the FT-IR spectra and 1 H-NMR spectra of synthesized SMAs, respectively. All the spectra indicated that both SMAs synthesized as designed.  The results of WS, SL, and DCW were summarized in Table 3, and Figure 6 showed the water sorption behavior of cured resins. Only cured resin with 10 wt.% of SMAs had comparable WS and SL as control (p>0.05), all the other SMAs containing cured resin had higher WS and SL (p<0.05). All of SMAs containing cured resin had higher DCW than control (p<0.05).

Figure 2 and
The results of contact angles and SFE were listed in Table 4. As shown in Table 4

Discussion
With the aim to reduce SFE of DRCs, the strategy of this research was to prepare resin matrix which would have low SFE after polymerization, for resin matrix is the continuous phase in DRCs. For this purpose, methacrylates with silicone chain were synthesized and incorporated into the commonly used resin matrix (Bis-GMA/TEGDMA), because silicone-based materials are low SFE materials that have already been used in several fields for preventing bacteria adhesion, anti-fouling, anti-graffiti, and anti-contamination [27][28][29][30].
The DC is a critical factor that determines the mechanical properties as well as other performance of methacrylate based dental materials [31]. The results of present study indicated that addition of SMAs had no negative effect on DC of dental resin (Table 2), both structure and concentration of SMAs had no influence on DC.
The process of polymerization of methacrylate involves the formation of covalent bonds and reduction in distance between molecules, which would inevitably cause volumetric shrinkage. The volumetric shrinkage would then lead to shrinkage stress, which could induce microleakage, marginal discoloration, postoperative sensitivity, and secondary caries in clinic, eventually causing failure of restoration [32]. Therefore, volumetric shrinkage is a primary defect of methacrylate based dental materials [33][34][35]. The degree of volumetric shrinkage is related to the conversion and concentration of double bond, and studies have reported that for linear methacrylate, when one mole of C=C was converted to C-C, the corresponding volumetric shrinkage was about 22.5 cm 3 [36,37]. Several efforts have been taken to minimize volumetric shrinkage by reducing double bond concentration while optimizing the double bond conversion [32,37,38]. With the same level of DC, SMAs containing dental resins had lower VS than control (Table 2), this should be due to the molecular weight of SMAs. Comparing monomers with the same functionality, volumetric shrinkage would decrease with the increasing of molecular weight, because high molecular weight leads to low double bond concentration [32,[38][39][40]. According to their amino contents, molecular weights of SMA-MEO and SMA-ME could be approximately calculated as 6832 g/mol and 4060 g/mol, respectively, which were much higher than molecular weight of Bis-GMA (512 g/mol) and TEGDMA (286 g/mol). Therefore, it also could be explained that, at the same mass fraction, why SMA-MEO containing resins had lower VS than SMA-ME containing resins.
Mechanical properties of cured resin were revealed to be associated with degree of double bond conversion and chemical structure of monomers [32,41]. With the comparable DC, the reduction of flexural properties after adding SMAs should be due to the branched silicone chains in the structure of SMAs, which could decrease interaction between polymeric chains just like some side alkyl chains [42]. Moreover, adding SMAs into Bis-GMA/TEGDMA would decrease concentration of rigid benzene ring in polymeric network, which could further lead to poor mechanical properties. After water immersion, flexural properties of all cure resins were decreased because of plasticization effect of water molecules [43]. Though mechanical properties of cured resin were reduced significantly after introducing SMAs, this phenomenon would be ameliorated after adding inorganic fillers to prepare DRCs. Because silicone chains in SMAs, especially the methoxyl terminated silicone chains in SMA-MEO, might have effect on improving the miscibility and interface properties between resin matrix and fillers. This should be proved in further study.
Compared with control group, all SMAs containing cured resin had higher hydrophobicity (Table 4), but some of them still possessed comparable or even higher WS (Table 3). This should be attributed to the higher DCW of SMAs containing cured resin, which made the water molecules to penetrate into polymeric networks much more easily. While the higher DCW of SMAs containing cured resin was the result of increased motion of polymer chains [44], that induced by the lower interaction between polymeric chains as mentioned above. The amount and leachability of residual monomers in cured resin determined its SL in water [45], and the amount of residual monomers is correlated with DC [46]. Though having the same DC, cured resin with more than 10 wt.% of SMAs had higher SL, since the decreased interaction between polymeric chains made the residual monomers to be leached out of polymeric network easily.
The development of biofilm always stats from the primary adhesion of bacteria [18,47,48].
Regarding the first colonizers in biofilm, oral streptococci are the most prominent bacteria [47]. Among streptococci, S mutans is a main etiological factor for dental caries [49,50], and have a greatest ability to form biofilm [51], thus S mutans was chosen in this work to study the anti-bacterial adhesion effect of cured resin. What's more, mucin adsorption of cured resin was also investigated in this study, because mucin is one of main components that constitute saliva and acquired pellicle [52,53], and has been reported that can enhance growth, biofilm formation and survival of S mutans [54]. Bovine submaxillary mucin was recommended as the most optimal commercially available mucin source when attempting to replicate saliva based on surface adsorption [53], so bovine submaxillary mucin was used here. The results showed in Figure 7 and Figure 8 indicated that SMAs could not only endow cured resin with anti-bacteria adhesion effect, but also with resistance to mucin adsorption.
It was reported that surfaces with water contact angle lower than 20° and approaching 120° exhibit a reduction in protein adsorption [27,56]. Thus, the resistance to mucin adsorption should be mainly attributed to the increased water contact angle (Table 4) after adding SMAs.
Meanwhile, the reduced surface free energy of SMAs containing cured resin (Table 4) should be the main reason of reduced adhered bacteria. According to the study of Absolom et al. [55], S. mutans with high surface free energy (117 mJ/m 2 ) [22] should adhere worse to surface with low surface free energy.

Conclusion
It could be concluded that the synthesized silicone methacrylates SMAs could reduce volumetric shrinkage of dental resin, endow cured resin with anti-bacterial adhesion effect and resistance to protein adsorption. However, the reduced flexural properties, increased water sorption and solubility after adding SMAs should be improved through incorporating suitable inorganic fillers in further study.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.          Figure 1 Synthesis route of SMAs.   Flexural strength (FS) of cured dental resins: (1) before water immersion; (2) after water immersion. a The same lowercase letter refers to there is no statistical difference between cured resins with and without SMA-MEO (Tukey's test, p=0.05). A The same uppercase letter refers to there is no statistical difference between cured resins with and without SMA-ME (Tukey′s test, p=0.05). # The octothorpe indicates the statistical differences between FSs of the same cured resin before and after water immersion (Tukey's test, p=0.05).

Figure 5
Flexural modulus (FM) of cured dental resins: (1) before water immersion; (2) after water immersion. a The same lowercase letter refers to there is no statistical difference between cured resins with and without SMA-MEO (Tukey's test, p=0.05). A The same uppercase letter refers to there is no statistical difference between cured resins with and without SMA-ME (Tukey′s test, p=0.05). * The asterisk refers to the statistical differences between cured with different SMAs at the same mass ratio in resin system (Tukey′s test, p=0.05). # The octothorpe indicates the statistical differences between FMs of the same cured resin before and after water immersion (Tukey's test, p=0.05).

Figure 6
Water sorption behavior of cured resins.

Figure 7
The results of mucin adsorption on the surface of cured resins. a The same lowercase letter refers to there is no statistical difference between cured resins with and without SMA-MEO (Tukey's test, p=0.05). A The same uppercase letter refers to there is no statistical difference between cured resins with and without SMA-ME (Tukey′s test, p=0.05). * The asterisk refers to the statistical differences between cured with different SMAs at the same mass ratio in resin system (Tukey′s test, p=0.05).

Figure 8
The results of anti-bacterial adhesion test. a The same lowercase letter refers to there is no statistical difference between cured resins with and without SMA-MEO (Tukey's test, p=0.05). A The same uppercase letter refers to there is no statistical difference between cured resins with and without SMA-ME (Tukey′s test, p=0.05). * The asterisk refers to the statistical differences between cured with different