Synthesis of antibacterial dimethacrylate derived from niacin and its application in preparing antibacterial dental resin system

Journal Pre-proof Synthesis of antibacterial dimethacrylate derived from niacin and its application in preparing antibacterial dental resin system Shuang Li, Xiaolin Yu, Fang Liu, Feilong Deng, Jingwei He PII:

S1751-6161(19)31582-6

DOI:

https://doi.org/10.1016/j.jmbbm.2019.103521

Reference:

JMBBM 103521

To appear in:

Journal of the Mechanical Behavior of Biomedical Materials

Received Date: 17 October 2019 Revised Date:

2 November 2019

Accepted Date: 4 November 2019

Please cite this article as: Li, S., Yu, X., Liu, F., Deng, F., He, J., Synthesis of antibacterial dimethacrylate derived from niacin and its application in preparing antibacterial dental resin system, Journal of the Mechanical Behavior of Biomedical Materials (2019), doi: https://doi.org/10.1016/ j.jmbbm.2019.103521. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Synthesis of antibacterial dimethacrylate derived from niacin and its application in preparing antibacterial dental resin system Shuang Lia,ǂ, Xiaolin Yub, ǂ, Fang Liua, Feilong Dengb,*, Jingwei Hea,* a

College of Materials Science and Engineering, South China University of Technology,

Guangzhou, PR China b

Department of Oral Implantology, Guanghua School of Stomatology, Hospital of

Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, PR China ǂ

The first two authors Shuang Li and Xiaolin Yu contributed equally to this manuscript.

Corresponding Authors: Jingwei He Address: 381 Wushan Rd., College of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China Tel:+86-13538913767 E-mail: [email protected] Feilong Deng Address: 56 Lingyuan Xi Road, Department of Oral Implantology, Guanghua School of Stomatology, Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055, China Tel:

+ 86 20 83862537

Fax:

+86 20 87330446

E-mail: [email protected]

Abstract: In

this

research,

a

bio-based

monomer

1,3-bis(methacryloyloxy)propyl-carbonyl-

hexylpyridinium bromide (QANMA) that derived from niacin was synthesized and incorporated into Bisphenol A glycidyl methacrylate (Bis-GMA)/triethylene glycol dimethacrylate (TEGDMA) (50 wt./50 wt.) with a series of mass fraction as antibacterial agent. The double bond conversion (DC), volumetric shrinkage (VS), mechanical properties, water sorption (WS) and solubility (SL) were investigated among groups with different QANMA concentrations. Antibacterial activity against S. mutans were conducted by bacteria colony counting and bacteria LIVE/DEAD staining. The results showed that QANMA had no influence on DC of dental resin (p>0.05), but would lead to lower volumetric shrinkage (p<0.05). Only dental resin with 10 wt.% and 20 wt.% of QANMA showed obviously antibacterial activity. Mechanical properties, WS and SL of dental resin could be impaired by incorporation QANMA, flexural strength and modulus were decreased with the increasing of QANMA concentration (p<0.05), while WS and SL were increased with the increasing of QANMA concentration (p<0.05). Dental resin with 10 wt.% of QANMA seemed to be the optimal resin system in this research, for it showed significant antibacterial activity and its flexural strength was still met the requirement of ISO standard. This work suggested that bio-based monomer QANMA could be used as antibacterial agent in dental materials, but further optimization experiment and biocompatibility evaluation should be taken in future. Keywords: Niacin derivative; antibacterial activity; dental resin; S. mutans

1. Introduction Dental resin composites, that composed of methacrylate-based resin matrix and silanized inorganic fillers, have been widely used to replace amalgams as restoration for dental cavity because of their easy handling and excellent esthetic properties [1,2]. However, due to lack of antibacterial properties, more bacteria or plaque accumulation on dental resin composites have been reported in vitro or in vivo, when compared with other restorative materials such as amalgams and glass-ionomers [3,4]. Secondary or recurrent caries, that induced by bacteria or plaque accumulation adjacent to the restoration margins, is one of the main reasons for clinical failure of dental resin composites [5,6]. Therefore, endowing dental resin composites with antibacterial activity plays an important role in prolonging service time of composites restorations. In this purpose, several antibacterial monomers, such as quaternary ammonium methacrylates [7-10], methacylates with heterocyclic ring [11,12], and eugenyl methacrylate derived from eugenol [13,14] have been synthesized and incorporated into dental resin composites. As compounds with broad-spectrum antibacterial activity, quaternary ammonium compounds have been widely used in several areas, such as medicine and healthcare products, food applications, and textile products [15-17]. It is well known that antibacterial activity of quaternary ammonium compound is related to its alkyl chain length. The antibacterial activity would increase with increasing of alkyl chain length to a certain level, and then decrease with increasing of alkyl chain length further, that was called “cut off” effect [18]. Moreover, the alkyl chain length of quaternary ammonium compounds would also influence their biocompatibility, such as hemolytic and cytotoxic activities. The shorter the alkyl chain length,

the lower the hemolytic and cytotoxic activities would be [19,20]. Nowadays, bio-based monomers have gained more attention because of their low or no cytotoxicity when applied as dental materials. For example, monomers derived from eugenol have been applied into dental resin composites [21,22] and adhesive [23], which showed antibacterial activity and good cytocompatibility. Isosorbide-based monomers have been used in dental resin composites to reduce polymerization stress [24] and water sorption [25]. Niacin, also known as nicotinic acid, is an organic compound and a form of vitamin B3, which can be obtained from a variety of foods. In the structure of niacin, there is a pyridine ring, which can be quaternized to form a functional group with antibacterial activity, just like the antibacterial group in 12-methacryloyloxydodecylpyridinium bromide (MDPB), the first antibacterial monomer used in dentistry [26]. In this research, a new quaternized pyridine dimethacrylates with short alkyl side chain, that derived from niacin, was synthesized and used to prepare antibacterial dental resin system. The hypothesis was that the new monomer could endow dental resin system with antibacterial activity. 2. Materials and methods 2.1 Materials Glycerol dimethacrylate (GDMA, 90%), nicotinoyl chloride hydrochloride (NCH, 97%), 1-bromohexane (BH, 99%), triethylamine (TEA, 99%), and hydroquinone (HQ, 99%) were purchased from J&K Scientific Ltd., China. Bisphenol A glycidyl methacrylate (Bis-GMA, >85%) and triethylene glycol dimethacrylate (TEGDMA, 95%) were purchased from Essetech Inc., USA. Camphoroquinone (CQ, 99%) and dimethylaminoethyl

methacrylate (DMAEMA, 99%) were purchased from Sigma-Aldrich Co., USA. All of reagents were used without purification. 2.2

Synthesis

of

1,3-bis(methacryloyloxy)propyl-carbonyl-hexylpyridinium

bromide

(QANMA) QANMA was synthesized according to the synthesis route showed in Figure 1. Step 1: 0.15 mol of NCH (26.7 g, Mw=178.0) was dispersed in 100 mL of ultra-dried dichloromethane to form a suspension, and 0.3 mol of TEA (30.36 g, Mw=101.2) was added into the suspension. 0.1 mol of GDMA (22.82 g, Mw=228.2) was dropwisely added into the suspension in the ice bath, after that the reaction mixture was returned to room temperature and stirred for 5 h. Then 100 mL of methanol was added into the reaction mixture, kept stirring until obtaining a clear reaction solution. After removing the solvent by rotary evaporation, the crude product was washed with 4

water three times and rotary evaporated

to remove water to obtain yellow intermediate named 1,3-bis(methacryloyloxy)propylcarbonyl-pyridine (NMA, Mw=333.3). Step2: 100 mL of dioxane was directly added into the same bottle to dissolve NMA. After adding 0.2 mol of BH (33.02, Mw=165.1) and a small amount of HQ, the solution was refluxed at 105

for 4 days. After removing dioxane by rotary evaporation, the crude product

was purified through basic silica gel column chromatograph (first using ethyl acetate to remove impurity, and then using methanol to dissolve product). The methanol solution of product was rotary evaporated under 35

to remove methanol and 27.41 g of QANMA

(Mw=498.4) was obtained as brown liquid with a yield of 55%. Before rotary evaporation, a small amount of HQ was added into the solution, for neat QANMA was very easy to

polymerize during rotary evaporation. The molecular structure of QANMA was confirmed by 1

H-NMR (Nuclear Magnetic Resonance Instrument, Avance AV 400 MHz, Bruker,

Switzerland) spectra using DMSO as solvent. 2.3 Preparation of dental resin systems Dental resin systems were prepared by adding QANMA into Bis-GMA (Mw=512.6) /TEGDMA (Mw=286.3) (50wt./50wt.) with a series of mass fraction (5wt.%, 10wt.%, and 20wt.%), then 0.7wt.% of CQ and 0.7wt.% of DMAEMA were added as initiation system. Dental resin system without QANMA was used as control. All the compounds were weighed and mixed to obtain a homogenous mixture. The prepared dental resin systems were stored in darkness before being used. 2.4 Measurement of double bond conversion (DC) The DC of each resin formulation was monitored by a Fourier Transform Infrared Instrument (FTIR, Vector33, Bruker Co., Germany) with an attenuated total reflectance (ATR) accessory. Prepared resins were analyzed in a mold that was 1.0 mm thick and 5.0 mm in diameter. First, the spectrum of unpolymerized resin, that was placed in the mold, was measured. Then, the resin was induced to polymerize through an upper glass slide for 60 s with a visible light source (Mini LED Curing Lights, λ = 390–510 nm, I ≈ 1250 mW·cm−2, Satelec Inc., France) at room temperature. The cured resin was scanned for its FTIR spectrum after being irradiated. The DC was calculated from the aliphatic C=C peak at 1636 cm-1 and normalized against the phenyl ring peak at 1608 cm-1 according to the formula: (1) where AC=C and APh are the absorbance peak area of methacrylate C=C at 1636 cm-1 and

Phenyl ring at 1608 cm-1, respectively; (AC=C/APh)0 and (AC=C/APh)60 represent the normalized absorbency of the functional group at the radiation time of 0 s and 60 s, respectively; DC represents the percentage of carbon double bonds consumed in the polymerization reaction as a functional of radiation time. For each resin, five trials were performed. 2.5 Measurement of volumetric shrinkage (VS) The volumetric shrinkage was investigated according to the method as shown in literature [27,28]. The specimens’ densities (n=5) were measured to determine volume shrinkage according to Archimedes’ principle with a commercial density determination kit of the analytical balance (FA1104J, Shunyuhengping Scientific Instrument Int., Shanghai, China). The density of unpolymerization sample was measured using a glass dish. First, the mass of glass dish was weighed in air and in water, respectively. The density of the glass dish ρgd was then calculated according to Eq. (2):

(2) where ρw is the density of water at the exactly measured temperature, mgda and mgdw are the mass of the glass dish in air and in water, respectively. Secondly, a certain amount of unpolymerized sample was dispensed into the glass dish, and the total mass of glass dish and sample was weighed in air and in water, respectively. The density of the unpolymerized sample ρ1 was then calculated according to Eq. (3):

(3) where m1a is the mass of the unpolymerized sample and the glass dish measured together in air, mgda is the mass of the glass dish in air, m1w is the mass of the unpolymerized sample and

the glass dish measured together in water, ρgd is the density of the glass dish, ρw is the density of water at the exactly measured temperature. Third, cured samples obtained from DC% measurement were used to investigated density after photopolymerization. The mass of cured sample was weighed in air and in water, and its density ρ2 was calculated according to Eq. (4):

(4) where m2a is the mass of the polymerized sample in air, m2w is the mass of polymerized sample in water, ρw is the density of water at the exactly measured temperature. Finally, VS of sample was calculated according to Eq. (5): (5) 2.6 Measurement of water sorption (WS) and solubility (SL) The bar-shaped specimens (2 mm × 2 mm × 25 mm) were used to measure WS and SL of dental resin systems. Eight specimens of each group were prepared. The initial dry weight (M1) of each specimen was measured with an electronic balance (FA1104J, Shunyuhengping Scientific Instrument Int., Shanghai, China) with an accuracy of 0.1 mg. Then, the specimens were immersed in 30 mL of distilled water and kept at 37°C. At fixed time intervals, the specimens were removed, blotted dry to remove excess water, re-weighed and returned to the water. Equilibrium mass (M2) was obtained at 30 days’ immersion which showed there was no significant change. After that, the specimens were dried at 60°C until their mass kept constant and the result was recorded as M3. The WS and SL were calculated according to the Eq. (6) and (7):

(6)

(7) 2.7 Three-point bending test Sixteen specimens (2 mm × 2 mm × 25 mm) were made for every dental resin system. Eight specimens of each group were kept dry until the start of testing, and the other eight specimens were stored in distilled water at 37

until the start of testing (the storage time in

water was as long as the time for water sorption and solubility test). The three-point bending test (span 20 mm) was carried out to evaluate the flexural strength (FS) and modulus (FM) according ISO 10477:92 standard with a universal testing machine (Model Z010, Zwick GmbH & Co.KG, Germany), at a cross-head speed of 1.00 mm/min. The FS and FM were then calculated according to Eq. (8) and Eq. (9), respectively: (8)

(9) where p is the applied load (N) at the highest point of a load-deflection curve, L is the span length (20 mm), b is the width of test specimens and h is the thickness of test specimens in mm. S is the stiffness (N/m). S=p/d, and d is the deflection corresponding to the load p at a point in the straight-line portion of the trace. 2.8 Measurement of antibacterial activty Bacterial culture Streptococcus mutans (S. mutans, ATCC 25175) was spread on brain heart infusion (BHI)

agar plates. After anaerobic incubation at 37°C for 24 h, a single colony of S. mutans was collected into 10 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 1×107 CFU/mL for bacterial adhesion assay using the McFarland Scale. Bacteria colony counting Each of specimen was placed in one well of a 24-well non-tissue culture plate and 1mL of the diluted bacteria suspension was pipetted into each well. After 24 h anaerobic incubation at 37°C, the specimen was rinsed using PBS and then transferred in a centrifuge tube containing 3 ml PBS separately. After sonication for 15 min followed by vortex for 15 s to detach the adhered bacteria, 100 µL of diluted suspension was spread onto BHI agar and incubated for 48 h at 37°C under an anaerobic condition. The bacteria colony counts of S. mutans were obtained. Bacteria LIVE/DEAD staining The LIVE/DEAD® BacLight™ Bacterial Viability Kit (L13152, Thermo Scientific, USA) was used for live and dead bacteria staining according to the manufacturers' protocol. After bacteria incubation and rinsed by PBS, samples were incubated with 200 µl LIVE/DEAD® BacLight™ solution containing contains SYTO 9 and propidium iodide (PI) whicn prepared according to the product instruction at room temperature in the dark for 15 min. Confocal laser scanning microscopy (CLSM) (Nikon A1 plus, Japan) was used to observe the samples. Mean fluorescence intensity of the captured images was determined by Fiji software [29] to evaluate the live, dead and total bacteria adhesion. 2.9 Statistical analysis

Statistical analysis was performed using SPSS software, version 25.0 (IBM SPSS Software, USA). All the data were expressed as means ± standard deviation (SD). The comparison of six groups was detected by one-way ANOVA followed by a post-hoc Turkey HSD multiple comparisons. The significant level was set at 0.05. 3. Results The 1H-NMR spectrum of QANMA was shown in Figure 2. As shown in Figure 2, all protons in structure of QANMA could find their corresponding peaks in 1H-NMR spectrum, which confirmed that QANMA was successfully synthesized as designed. Because HQ was added in to QANMA as inhibitor, its corresponding peaks were also found in Figure 2. The results of DC and VS were listed in Table 1, all of resin systems had the similar DC (p>0.05), and VS was in the trend of decreasing with the increasing of QANMA concentration in resin system. The WS, SL, FS and FM of cured resin systems were summarized in Table 2. All of QANMA containing resin had higher WS than control group (p<0.05), and resin with 20% of QANMA had the highest WS (p<0.05). Except for resin with 5% of QANMA, which had comparable SL as control group (p>0.05), all the other QANMA containing resin had higher SL than control group (p<0.05), and resin with 20% of QANMA had the highest SL (p<0.05). Before water immersion, FS decreased with the increasing of QANMA concentration in resin system (p<0.05), and only resin with 5% of QANMA had the similar FS as control group (p>0.05). All of QANMA containing resin had comparable FM as control group (p>0.05), except for resin with 20% of QANMA, which had lower FM than control group (p<0.05). After water immersion, FS and FM of all groups decreased significantly (p<0.05), only resin with 20% of QANMA had lower FS than control group (p<0.05), while

only resin with 5% of QANMA had comparable FM as control group (p>0.05), all the other QANMA containing resin had lower FM than control group (p<0.05). The results of S. mutans colony counting assay were shown in Figure 3. S. mutans adherence on the samples with 10% and 20% of QANMA were significantly lower compared with control (p<0.05). Samples with 5% of QANMA did not show significant difference when compared with control (p>0.05). Representative CLSM images of LIVE/DEAD staining of S. mutans were shown in Figure 4, and the results of mean fluorescence intensity of bacteria LIVE/DEAD staining were shown in Figure 5. The results of live bacteria staining were consistent with the results of the bacteria colony counting assay, as the mean fluorescence intensity for live bacteria had similar pattern with colony counts. The mean fluorescence intensity for live bacteria showed a descreasing trend with the increasing of the concentration of QANMA in the resin system. The mean fluorescence intensity for live bacteria on the surface of resin with 5% of QANMA was significantly less than the resin with 10% and 20% of QANMA (p<0.05). In dead bacteria staining, it is noticeable that the mean fluorescence intensity of 20%-QANMA group was significantly higher than that of 5%-QANMA and control group (p<0.05). 4. Discussion According to previous study, quaternary ammonium with hexanyl side chain showed lower hemolysis and toxicity than quaternary ammonium with longer alkyl side chain [19, 20]. With the purpose of endowing dental resin with antibacterial activity without impairing biocompatibility, antibacterial dimethacrylate derived from niacin with hexanyl side chain was synthesized and incorporated into Bis-GMA/TEGDMA resin system.

As a fundamental characteristic of dental resin, DC affects several properties of it. Higher DC always leads to better mechanical properties, lower water sorption, lower solubility, and better biocompatibility [30-33]. According to previous study, incorporation of quaternary ammonium methacrylates into Bis-GMA/TEGDMA had no influence on DC [9,34,35]. In this research, QANMA also had no influence on DC, which was consistent with previous finding. In the structure of QANMA, there is a pyridine ring which could decrease DC because of steric hindrance. However, the alkyl side chain of QANMA could decrease the interaction between polymeric chains and prolong the vitrification time, leading to higher DC [36]. These two effects offset each other and made no variation in DC. As an inherent disadvantage of methacrylate-based dental resin, volumetric shrinkage resulted from the reduction of intermolecular distances after photopolymerization would affect sealing capacity of dental resin composites [37]. Micro-leakage that induced by volumetric shrinkage would lead to high possibility of secondary caries and clinical failure [38,39]. The value of volumetric shrinkage is dependent on double bond conversion and double bond concentration of dental resin, and can be reduced by reducing conversion and concentration of double bond [28,40]. In this research, with the same DC, volumetric shrinkage of dental resin decreased with the increasing of QANMA concentration, this should be attributed to the higher molecular weight of QANMA (Mw=498) when compared with the mean molecular weight (367.4) of Bis-GMA/TEGDMA (50wt./50wt.) resin mixture, which could decrease double bond concentration of dental resin. Therefore, incorporation of QANMA into dental resin could reduce volumetric shrinkage of dental resin. In addition to DC, properties of dental resin are also influenced by several other factors, for

example, the structure of monomer used in dental resin. Mono-methacrylate monomer could increase DC of dental resin, but it might not increase mechanical properties of dental resin, or even induce higher water sorption and solubility, for mono-methacrylate monomer had no contribution on cross-link density of cured resin [11,12,41]. Branched methacrylates might not increase DC of dental composites, but some could increase mechanical properties significantly [42]. Therefore, it is not possible to predict properties of dental resin by DC alone. In this study, though QANMA had no influence on DC of dental resin, flexural strength and modulus of cured resin were reduced when QANMA concentration was over a certain amount. This phenomenon was also observed in some other researches [9,43]. This should be due to the alkyl chain in quaternary ammonium, which could reduce the intermolecular interaction, leading to lower flexural strength and modulus. However, all of QANMA containing dental resin still showed acceptable flexural strength no matter before or after water immersion, except for dental resin with 20 wt.% of QANMA after water immersion, which showed flexural strength lower than 50 MPa that required by ISO 4049:2009 [44]. The flexural strength and modulus of QANMA containing dental resin could be improved by formulation modification and incorporation reinforcing fillers, thus more optimization studies should be done in future. The positive and negative charges in the structure of QANMA can increase hydrophilicity of polymeric network, and higher hydrophilicity always leads to higher water sorption [45]. Therefore, water sorption of cured resin increased with the increasing of QANMA in this study. Though having the comparable DC, solubility of cured resin increased with the increasing of QANMA concentration, this might be attributed to the increased water sorption

and decreased intermolecular interaction as mentioned above, making the unreacted monomer to be eluted more easily [46]. The results of antibacterial tests showed that the resin with 10 wt.% and 20 wt.% of QANMA exhibited a significant reduction in S. mutans colony counts after 24 h incubation. Furthermore, the resin with 20 wt.% of QANMA showed significantly more dead bacteria than 5%QANMA group. This suggested that QANMA containing resin was a promising approach to combat secondary or recurrent caries. As a novel quaternized pyridine with short alkyl chain, QANMA is belonged to quaternary ammonium compounds (QACs). Its antimicrobial effect is associated with strong affinity and damaging interactions between the negatively charged head groups of acidic phospholipids in bacteria membranes and the positively charged quaternary nitrogen of the QACs [47,48]. Furthermore, the antimicrobial potential of QACs was affected by the polarity and steric properties [49]. Though QANMA had lower antibacterial activity than some other QACs, for in some research [9,34,35], dental resin with 5 wt.% of polymerizable QACs have already showed significant antibacterial activity, but its shorter alkyl chain length and niacin-based structure might lead to better biocompatibility. There are some researches have showed that niacin-based biomaterials had good biocompatibility. For example, biocompatibility of magnetite nanorods used as drug precursor could be improved by coating with niacin [50]. Coordination polymer based on niacin showed no cytotoxic effects on normal cell lines of human body when used as anticancer agents [51]. Even though, biocompatibility of QANMA containing dental materials should be proved in further study. 5. Conclusion

It could be concluded that the synthesized niacin-based monomer 1,3-bis(methacryloyloxy)propyl-carbonyl-hexylpyridinium bromide (QANMA) could endow Bis-GMA/TEGDMA dental resin with antibacterial activity when its concentration was over 10 wt.% in the resin system. Though QANMA could lead to lower volumetric shrinkage, it would also bring out drawbacks like higher water sorption and solubility as well as lower flexural strength and modulus. However, flexural strength of QANMA containing dental resin was still in the range of ISO standard requirement.

Acknowledgement This work was funded by the National Natural Science Foundation (No.81970974, No.81801012), China Postdoctoral Science Foundation (2019M653234) and the Young Teacher Training Program of Sun Yat-sen University (19ykpy84).

References [1]. Ferracane JL. Resin composite-state of the art. Dental Materials 2011;27:29-38. [2]. Meereis CTW, Münchow EA, Rosa WLO, Silva AF, Piva E. Polymerization shrinkage stress of resin-based dental materials: A systematic review and meta-analyses of composition strategies. Journal of the Mechanical Behavior of Biomedical Materials 2018;82:268-281. [3]. Beyth N, Domb AJ, Weiss EI. An in vitro quantitative antibacterial analysis of amalgam and composite resins. Journal of Dentistry 2007;35:201-206. [4]. Svanberg M, Mjör IA, Ørstavik D. Mutans Streptococci in plaque from margins of amalgam, composite, and glass-ionomer restorations. Journal of Dental Research

1990;69:861-864. [5]. Deligeorgi V, Mjor IA, Wilson NH. An overview of reasons for the placement and replacement of restorations. Primary Dental Care 2001;8:5-11. [6]. Ferracane JL. Resin-based composite performance: are there somethings we can’t predict. Dental Materials 2013;29:51-58. [7]. Imazato S, McCabe JF. Influence of incorporation of antibacterial monomer on curing behavior of a dental composite. Journal of Dental Research 1994;73:1641-1645. [8]. Liang X, Huang Q, Liu F, He J, Lin Z. Synthesis of novel antibacterial monomers (UDMQA) and their potential application in dental resin. Journal of Applied Polymer Science 2013;6:3373-3381. [9]. Liang X, Söderling E, Liu F, He J, Lassila JVJ, Vallittu PK. Optimizing the concentration of quaternary ammonium dimethacrylate monomer in bis-GMA/TEGDMA dental resin system for antibacterial activity and mechanical properties. Journal of Materials Science: Materials in Medicine 2014;25:1387-1393. [10]. He J, Söderling E, Lassila LVJ, Vallittu PK. Preparation of antibacterial and radio-opaque dental resin with new polymerizable quaternary ammonium monomer. Dental Materials 2015;31:575-582. [11]. Luo W, Huang Q, Liu F, Lin Z, He J. Synthesis of antibacterial methacrylate monomer derived from thiazole and its application in dental resin. Journal of the Mechanical Behavior of Biomedical Materials 2015;49:61-68. [12]. Zhu W, Lao C, Luo S, Liu F, Huang Q, He J, Lin Z. Mechanical and antibacterial properties of benzothiazole-based dental resin materials. Journal of Biomaterials Science,

Polymer Edition 2018;29:635-645. [13]. Almaroof A, Rojo L, Mannocci F, Deb S. A resin composite material containing a eugenol derivative for intracanal post cementation and core build-up restoration. Dental Materials 2015;32:149-160. [14]. Almaroof A, Niazi SN, Mannocci F, Deb S. Evaluation of dental adhesive systems incorporating an antibacterial monomer eugenyl methacrylate (EgMA) for endodontic restorations. Dental Materials 2017;33:e239-e254. [15]. Moran J, Addy M, Jackson R, Newcombe RG. Comparative effects of quaternary ammonium mouthrinses on 4-day plaque regrowth. Journal of Clinical Periodontology 2000;27:37-40. [16]. Kenawy ER, Worley SD, Broughton R. The chemistry and application of antimicrobial polymers: a state-of-art review. Biomacromolecules 2007;8:1359-1384. [17]. Simoncic B, Tomsic B. Structure of novel antimicrobial agents for textiles-a review. Textile Research Journal 2010;80:1721-1737. [18]. He J, Söderling E, Österblad M, Vallittu PK, Lassila LVJ. Synthesis of methacrylate monomers with antibacterial effects against S. Mutans. Molecules 2011;16:9755-9763. [19]. King A, Chakrabarty S, Zhang W, Zeng X, Ohman DE, Wood LF, et al. High antimicrobial effectiveness with low hemolytic and cytotoxic activity for PEG/Quaternary copolyoxetanes. Biomacromolecules 2014;15:456-467. [20]. Zhu W, Liu F, He J. Effect of polymerizable quaternary ammonium monomer MEIM-x’s alkyl chain length and content on bone cement’s antibacterial activity and physicochemical properties. Journal of the Mechanical Behavior of Biomedical Materials.

2018;87:279-287. [21]. Rojo L, Vazquez B, Parra J, Bravo AL, Deb S, Roman JS. From nature products to polymeric derivatives of “eugenol”: A new approach for preparation of dental composites and orthopedic bone cements. Biomacromolecules 2006;7:2751-2761. [22]. Almaroof A, Rojo L, Mannocci F, Deb S. A resin composite material containing an eugenol derivative for intracanal post cementation and core built-up restoration. Dental Materials 2016;32:149-160. [23]. Almaroof A, Niazi SA, Rojo L, Mannocci F, Deb S. Evaluation of dental adhesive systems incorporating an antibacterial monomer eugenyl methacrylate (EgMA) for endodontic restorations. Dental Materials 2017;33:e239-e254. [24]. Herrera-González AM, Pérez-Mondragón AA, Cuevas-Suárez CE. Evaluation of bio-based monomers from isosorbide used in the formulation of dental composite resins. Journal of the Mechanical Behavior of Biomedical Materials 2019;100:103371. [25]. Kim JS, Park HW, Lee JH, Lee SH, Cho JK, Shin S. Synthesis of a novel isosorbide-based dental material with improved water sorption. European Polymer Journal 2019;112:629-635. [26]. Imazato S, Russell RRB, McCabe JF. Antibacterial activity of MDPB polymer incorporated in dental resin. Journal of Dentistry 1995;23:177-181. [27]. He J, Caroushi S, Vallittu PK, Lassila LVJ. Effect of low-shrinkage monomers on the physicochemical

properties

of

experimental

composite

resin.

Acta

Biomaterialia

Odontologica Scandinavica 2018;4:30-37. [28]. Luo S, Liu F, He J. Preparation of low shrinkage stress dental composite with

synthesized dimethacrylate oligomers. Journal of the Mechanical Behavior of Biomedical Materials 2019;94:222-228. [29]. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 2012;9:676-682. [30]. Par M, Gamulin O, Spanovic N, Bjelovucic R, Tarle Z. The effect of excitation laser power in Raman spectroscopic measurements of the degree of conversion of resin composites. Dental Materials 2019;35:1227-1237. [31]. Alshali RZ, Silikas N, Satterthwaite JD. Degree of conversion of bulk-fill compared to conventional resin-composites at two time intervals. Dental Materials 2013;29:e213-e217. [32]. Pearson GJ, Longman CM. Water sorption and solubility of resin-based materials following inadequate polymerization by a visible-light curing system. Journal of Oral Rehabilitation 1989;16:57-61. [33]. Durner J, Obermaier J, Draenert M, Ilie N. Correlation of the degree of conversion with the amount of elutable substances in nano-hybrid dental composites. Dental Materials 2012;28:1146-1153. [34]. He J, Söderling E, Lassila LVJ, Vallittu PK. Incorporation of an antibacterial and radiopaque monomer in to dental resin system. Dental Materials 2012;28:e110-e117. [35]. He J, Söderling E, Vallittu PK, Lassila LVJ. Investigation of double bond conversion, mechanical properties, and antibacterial activity of dental resins with different alkyl chain length quaternary ammonium methacrylate monomers (QAM). Journal of Biomaterials Science, Polymer Edition 2013;5:565-573.

[36]. He J, Söderling E, Lassila LVJ, Vallittu PK. Synthesis of antibacterial and radio-opaque dimethacrylate monomers and their potential application in dental resin. Dental Materials 2014;30:968-976. [37]. Yin M, Guo S, Liu F, He J. Synthesis of fluorinated dimethacrylate monomer and its application in preparing Bis-GMA free dental resin. Journal of the Mechanical Behavior of Biomedical Materials 2015;51:337-344. [38]. Podgórski M. Synthesis and characterization of novel dimethacrylates of different chain lengths as possible dental resins. Dental Materials 2010;26:e188-e194. [39]. Yin M, Liu F, He J. Preparation and characterization of Bis-GMA free dental resin system

with

synthesized

dimethacrylate

monomer

TDDMMA

derived

from

tricyclo[5.2.1.0(2,6)]-decanedimethanol. Journal of the Mechanical Behavior of Biomedical Materials 2016;57:157-163. [40]. Fu W, Wang L, He J. Evaluation of mechanical properties and shrinkage stress of thiol-ene-methacrylate dental composites with synthesized fluorinated allyl ether. Journal of the Mechanical Behavior of Biomedical Materials 2019;95:53-59. [41]. He J, Liu F, Luo Y, Jia D. Properties of 2,2-Bis[p-(2’-hydroxy-3’-methacryloxypropoxy)phenyl]propane/Isobornyl (Meth)acrylate copolymers. Journal of Applied Polymer Science 2012;126:1527-1531. [42]. Yu X, He J, Li S, Liu F, Yang J, Deng F. Preparation of experimental resin composites with an anti-adhesion effect against S. mutans using branched silicon methacrylate. Journal of the Mechanical Behavior of Biomedical Materials 2020;101:103414. [43]. Xu X, Wang Y, Liao S, Wen ZT, Fan Y. Synthesis and characterization of antibacterial

dental monomers and composites. Journal of Biomedical Materials Research Part B 2012;100B:1151-1162. [44]. ISO 4049: 2009 Dentistry — Polymer-based restorative materials, International Organization for Standardization, Geneva. [45]. Miettinen VM, Narva KK, Vallittu PK. Water sorption, solubility and effect of post-curing of glass fibre reinforced polymers. Biomaterials 1999;20:1187-1194. [46]. Huang Q, Lin Z, Liang X, Liu F, He J. Preparation and characterization of antibacterial dental resin with UDMQA-12. Advances in Polymer Technology 2014;33:21395. [47] Buffet-Bataillon S, Tattevin P, Bonnaure-Mallet M, Jolivet-Gougeon A, Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds − a critical review. International Journal of Antimicrobial Agents 2012;39:381-389. [48] Asri LATW, Crismaru M, Roest S, Chen Y, Ivashenko O, Rudolf P, et al. A Shape-adaptive, antibacterialcoating of immobilized quaternary-ammonium compounds tethered on hyperbranched polyurea and its mechanism of action. Advanced Functional Materials 2014;24:346-355. [49] Tischer M, Pradel G, Ohlsen K, Holzgrabe U. Quaternary ammonium salts and their antimicrobial potential: targets or nonspecific interactions? Chemmedchem 2012;7:22-31. [50]. Attallah OA, Khalil WKB, Abdel Mottaleb MMS, Girgis E. Nicotinic acid coated magnetite nanorods evaluation by lipid profile analysis and genetic tests. Journal of Nanoscience and Nanotechnology 2018;18:872-878. [51]. Etaiw SEH, Fayed TA, El-bendary MM, Marie H. Three-dimensional coordination polymers based on trimethyltin cation with nicotinic and isonicotinic acids as anticancer

agents. Applied Organometallic Chemistry 2018;32:e4066.

Figure Captions Figure 1. Synthesis route of 1,3-bis(methacryloyloxy)- propyl-carbonyl-hexylpyridinium bromide (QANMA). Figure 2. 1H-NMR spectra of QANMA. Figure 3. S. mutans colony counts on the surfaces of specimens after 24 h incubation. The same lower-case letter indicated that there was no statistical difference. Figure 4. CLSM images of LIVE/DEAD staining of S. mutans on the surface of specimens, the live bacteria were stained green, and the dead bacteria were stained red. (A) control group, (B) 5%QANMA, (C) 10%QANMA, (D) 20%QANMA. Figure 5. The results of mean fluorescence intensity of bacteria LIVE/DEAD staining. The same lower-case letter indicated that there was no statistical difference.

Table 1. Double bond conversion (DC) and volumetric shrinkage (VS) of experimental dental resin systems.

a

Resin system

DC (%)

VS (%)

Control

64.4 ± 2.6a

10.3 ± 0.6a

5%QANMA

65.5 ± 3.3a

9.2 ± 0.4b

10%QANMA

66.4 ± 3.1a

8.9 ± 0.5b

20%QANMA

65.0 ± 1.8a

7.1 ± 1.2c

The lower case letters indicated statistical differences within a column (Tukey’s test, p=0.05)

Table 2 Water sorption (WS), solubility (SL), flexural strength (FS) and modulus (FM) of experimental dental resin systems FS (MPa) Resin system

a

A

WS (%)

SL (%)

FM (GPa)

Before water

After water

Before water

After water

immersion

immersion

immersion

immersion

Control

4.5 ± 0.3a

2.0 ± 0.3a

121 ± 11a,A

86 ± 9a,B

2.61 ± 0.23a,A

2.11 ± 0.19a,B

5% QANMA

6.9 ± 0.6b

2.5 ± 0.4a,b

119 ± 4a,A

84 ± 7a,B

2.64 ± 0.09a,A

1.90 ± 0.14a,b,B

10% QANMA

7.6 ± 0.7b

3.2 ± 0.7b

108 ± 4b,A

77 ± 9a,B

2.43 ± 0.12a,A

1.73 ± 0.20b,B

20% QANMA

9.6 ± 0.4c

4.1 ± 0.7c

92 ± 6c,A

47 ± 9b,B

2.14 ± 0.14b,A

1.18 ± 0.10c,B

The lower case letters indicated statistical differences within a column (Tukey’s test, p=0.05) The upper case letter indicated statistical differences between FSs or FMs of the same resin system before and after water immersion (Tukey’s

test, p=0.05)

Conflicts of interest statement

Dear Editor,

The authors have declared that there is no competing interests exist.

Sincerely

Jingwei He