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Preparation of antibacterial and radio-opaque dental resin with new polymerizable quaternary ammonium monomer Jingwei He a,b,c,∗ , Eva Söderling d , Lippo V.J. Lassila a,b,d , Pekka K. Vallittu a,b,d,e a
Department of Biomaterials Science, Institute of Dentistry and Biocity Turku Biomaterial Research Program, University of Turku, Lemminkäisenkatu 2, Turku 20520, Finland b Turku Clinical Biomaterials Centre-TCBC, University of Turku, Itäinen Pitkäkatu 4 B, Turku FI-20520, Finland c College of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China d Institute of Dentistry, University of Turku, Turku 20520, Finland e City of Turku Welfare Division, Oral Health Care, Turku 20101, Finland
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. A new polymerizable quaternary ammonium monomer (IPhene) with iodine anion
Received 24 August 2014
was synthesized and incorporated into Bis-GMA/TEGDMA (50/50, wt/wt) to prepare antibac-
Received in revised form
terial and radio-opaque dental resin.
3 December 2014
Methods. IPhene was synthesized through a 2-steps reaction route, and its structure was con-
Accepted 10 February 2015
firmed by FT-IR and 1 H-NMR spectra. IPhene was incorporated into Bis-GMA/TEGDMA (50/50,
Available online xxx
wt/wt) with a series of mass fraction (from 10 wt.% to 40 wt.%). Degree of monomer conversion (DC) was determined by FT-IR analysis. Polymerization shrinkage was determined
Keywords:
according to the variation of density before and after polymerization. The flexural strength,
Dental resin
modulus of elasticity, and fracture energy were measured using a three-point bending set
Synthesis
up. Radiograph was taken to evaluate the radio-opacity of the polymer. A single-species
Antibacterial activity
biofilm model with Streptococcus mutans (S. mutans) as the tests organism was used to evalu-
Radio-opacity
ate the antibacterial activity of the polymer. Bis-GMA/TEGDMA resin system without IPhene
Double bond conversion
was used as a control group.
Mechanical property
Results. FT-IR and 1 H-NMR spectra of IPhene revealed that IPhene was the same as the
Polymerization shrinkage
designed structure. ANOVA analysis showed that when mass fraction of IPhene was more than 10 wt.%, the obtained resin formulation had lower DC, polymerization shrinkage, FS, and FM than control resin (p < 0.05). Polymers with 20 wt.% and 30 wt.% IPhene had higher fracture energies than control polymer (p < 0.05). IPhene containing samples had higher radio-opacity than control group (p < 0.05), and radio-opacity of IPhene containing sample increased with the increasing of IPhene mass fraction (p < 0.05). Only polymers with 30 wt.% and 40 wt.% of IPhene showed antibacterial activity (p < 0.05).
∗ Corresponding author at: Corresponding author at: Turku Clinical Biomaterials Centre-TCBC, University of Turku, Itäinen Pitkäkatu 4 B, FI-20520 Turku, Finland. Tel.: +358 465968930. E-mail address: hejin@utu.fi (J. He).
http://dx.doi.org/10.1016/j.dental.2015.02.007 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: He J, et al. Preparation of antibacterial and radio-opaque dental resin with new polymerizable quaternary ammonium monomer. Dent Mater (2015), http://dx.doi.org/10.1016/j.dental.2015.02.007
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Significance. IPhene could endow dental resin with both antibacterial and radio-opaque activity when IPhene reached 30 wt.% or more. Though sample with 30 wt.% of IPhene had lower FS and FM than control group, its lower volumetric shrinkage, higher fracture energy, higher radio-opacity, and antibacterial activity still made it having potential to be used in dentistry. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Dental composites, which consist of resin matrix and inorganic filler, are popular restorative materials because of their natural tooth color and direct-filling capabilities [1]. Dental composites have been reported to be used in more than 95% of all anterior tooth direct restorations and in about 50% of all posterior tooth direct restorations [2]. However, dental composite restorative materials have been reported to accumulate more bacteria or plaque than other restorative materials in vitro [3–6] or in vivo [7,8] because of their lack of antibacterial activity, and bacteria or plaque accumulation adjacent to the restoration margins may lead to secondary caries in vivo and shorten the life of composite restoration. Quaternary ammonium compounds, which are active against a broad spectrum of micro-organisms such as Grampositive and Gram-negative bacteria, fungi, and certain types of viruses [9], are well known antibacterial agents and have already been used in fields like water treatment, medicine, food applications, and textile products [9–11]. Quaternary ammonium compounds with polymerizable groups can immobilize the antibacterial quaternary ammonium group into the polymer backbone and give long-term antibacterial activity to the polymer [12]. In dentistry, Imazato firstly applied a polymerizable quaternary ammonium named methacryloyloxydodecyl-pridinium bromide (MDPB) as an antibacterial agent to prepare long-term antibacterial dental restorative materials [13–16]. Besides the antibacterial activity, quaternary ammonium compounds with iodine anion (I− ) also have radio-opacity [17], which is an important property for dental materials, because of the high electronic density of iodine [18,19]. Though radioopacity of dental materials can be implemented by adding radio-opaque inorganic fillers, there still exist some dental materials like E-glass fiber reinforced composites and flowable resin composites that have insufficient radio-opacity [20]. Therefore, radio-opaque dental resin can be used to solve this problem. In our previous study, a polymerizable quaternary ammonium named 2-dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) was synthesized and used to prepare antibacterial and radio-opaque dental resin system [17]. Unfortunately, DDMAI had miscible problem with hydrophobic methacrylate monomer tri-ethyleneglycol dimethacrylate (TEGDMA), and only 5 wt.% of DDMAI could be added into 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropyl)phenyl]propane (Bis-GMA)/TEGDMA resin system. Thus, the antibacterial activity and radio-opacity of DDAMI containing polymer were not obvious.
In this work, a new polymerizable quaternary ammonium monomer (IPhene) with iodine anion was synthesized and incorporated into Bis-GMA/TEGDMA dental resin with different mass ratio. The hypotheses are (I): IPhene could be mixed well with Bis-GMA/TEGDMA at high mass ratio; (II): IPhene could endow Bis-GMA/TEGDMA with both antibacterial and radio-opaque activity. Double bond conversion, polymerization shrinkage, flexural strength and modulus, antibacterial activity, radio-opacity of IPhene containing resin formulations were investigated and compared with resin formulation without IPhene.
2.
Materials and methods
2.1.
Materials
N-methyl diethanol amine (MDEA), 1-iodododecane, dibutyltin dilaurate (DBTDL), TEGDMA, camphoroquinone (CQ), N,N -dimethylaminoethylmethacrylate (DMAEMA), and 3-isopropenyl-␣,␣-dimethylbenzyl isocyanate (IDI) were purchased from Sigma–Aldrich Co. (St Luois, MO, USA). Bis-GMA was applied from Esstech Inc. (Essington, PA, USA). All of the compounds were used without further purification.
2.2.
Methods
2.2.1. Synthesis of polymerizable quaternary ammonium monomer (IPhene) As shown in Fig. 1, IPhene was synthesized through a 2-steps route. FT-IR (Spectrum One, Perkin-Elmer, Waltham, MA, USA) and 1 H-NMR (AV 400 MHz, Bruker Co., Germany) spectra of intermediate products and IPhene were obtained to confirm their structures. The FT-IR spectra were recorded with 32 scans at a resolution of 4 cm−1 . The chemical shifts of 1 H-NMR spectra were reported in ppm on ı scale with tetramethylsilane as the internal reference and CDCl3 as the solvent.
2.2.1.1. Synthesis of intermediate product N,N-bis(2hydroxyethyl)-N-methyldodecyl ammonium iodide (HQAI-12). A mixture of MDEA (0.05 mol), 1-iodododecane (0.051 mol), and 30 mL acetone were stirred at reflux. After 24 h reaction, the acetone was removed by distillation under vacuum. The obtained raw product was washed with ethyl ether and filtered for several times. Then the white intermediate product HQAI-12 was dried under vacuum at 35 ◦ C for 48 h. The results of spectroscopic studies for HQAI-12 are as follows: IR (neat): (cm−1 ) 3294, 2956, 2927, 2855, 1381, 1090, 720. 1 H-NMR (CDCl3 , 400 MHz): ı 4.22, 4.09, 3.77–3.89, 3.57–3.62, 3.38, 1.80, 1.31–1.42, 0.94–0.95.
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accessory. The FT-IR spectra were recorded with one scan at a resolution of 4 cm−1 . All the samples were analyzed in a mold that was 2 mm thick and 6 mm in diameter. First, the spectrum of the unpolymerized sample was measured. Then, the sample was irradiated for 180s with a visible light-curing unit (Curing Light 2500, = 400–520 nm, I ≈ 550 mW cm−2 , 3 M Co., St Paul, MN, USA). The sample was scanned for its FTIR spectrum every 5 s until 15 min after the beginning of irradiation. Each group was measured for 5 times. To determined the percentage of reacted double bonds, the absorbance intensities of the C C absorbance peak at 1636 cm−1 , which were decreased after being irradiated, and an internal phenyl ring standard peak at 1608 cm−1 , were calculated using a baseline method. The ratios of absorbance intensities were calculated and compared before and after polymerization. The DC at each irradiation time was calculated by using the equation
DC(t) = Fig. 1 – Synthesis route of IPhene.
2.2.1.2. Synthesis of final product IPhene. Acetone used here ˚ molecular sieves for 2 weeks. A mixture was dried over 4 A of HQAI-12 (0.04 mol), IDI (0.08 mol), 20 mL acetone, and 2 droplets of DBTDL were stirred at 45 ◦ C. The reaction was continued until the infrared absorbance peak of the –NCO group (2270 cm−1 ) disappeared in the FT-IR spectra of the samples taken from the reaction medium. After removing the acetone by distillation under vacuum, the product was washed with diethyl ether to remove DBTDL. Then the yellow viscose liquid was dried under vacuum at 35 ◦ C for 48 h to obtain IPhene. The results of spectroscopic studies for IPhene are as follows: IR (neat): (cm−1 ) 3284, 3083, 2956, 2925, 2855, 1710, 1629, 1600, 1578, 1521, 1485, 1221, 1173, 1091, 724. 1 H-NMR (CDCl3 , 400 MHz): ı 7.31–7.49, 6.15, 5.36, 5.10, 4.47, 3.38–3.82, 2.16, 1.66, 1.54, 1.28–1.38, 0.91.
2.2.2.
Preparation of resin formulation
The formulation of each dental resin used in this work is shown in Table 1. All of them were well blended to obtain a homogenous mixture, and stored at darkness before used.
2.2.3.
Double bond conversion
The degree of double bond conversion (DC) was determined by using an FT-IR spectrometer (Spectrum One, Perkin-Elmer, Waltham, MA, USA) with an attenuated total reflectance (ATR)
Table 1 – Formulation of every experimental resin system. System
Control 10%IPhene 20%IPhene 30%IPhene 40%IPhene
TEGDMA
IPhene
CQ
DMAEMA
49.3 44.3 39.3 34.3 29.3
49.3 44.3 39.3 34.3 29.3
0 10 20 30 40
0.7 0.7 0.7 0.7 0.7
0.7 0.7 0.7 0.7 0.7
(AC=C /Aph )0
× 100%
(1)
where AC C and Aph are the absorbance intensity of C C at 1636 cm−1 and phenyl ring at 1608 cm−1 , respectively; (AC C /Aph )0 and (AC C /Aph )t ate the normalized absorbance of functional group at the radiation time 0 and t, respectively; DC(t) is the conversion of C C as a function of radiation time.
2.2.4.
Polymerization shrinkage
Volumetric polymerization shrinkage of dental resin was measured according to the method reported previously [21]. The specimen’s densities (n = 5 per group) were measured to determine polymerization shrinkage using Archimedes’ principle with a commercial Density Determination Kit of the analytical balance Mettler Toledo X (Mettler Instrument Co., Highstone, NJ, USA). The kit provides a special holder for the specimens, a special container to keep the water, a computer, and appropriate software. The specimens were weighed in air and in water, and the density was directly calculated in gram per cubic centimeter by the software of the Mettler Toledo XS balance according to the equation: d=
Ma × (dw − da ) + da Ma − Mw
(2)
where d density of sample, Ma weight of sample in air, Mw weight of sample in water, dw density of water at the exactly measured temperature in ◦ C according to the density table of distilled water, da air density (0.0012 g/cm3 ). An internal balance correction factor (0.99985) of the Mettler Toledo XS balance software took the air buoyancy of the adjustment weight into account. The volumetric polymerization shrinkage S was calculated from the densities according to the equation:
Content of every monomer (g) Bis-GMA
1 − (AC=C /Aph )t
S=
dafter − dbefore × 100% dafter
(3)
where dafter is the density of sample after curing, dbefore is the density of sample before curing. All of polymerized samples were irradiated samples obtained from double bond conversion measurement.
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2.2.5.
Three point bending test
Eight specimens were prepared for each sample formulation (size 2 × 2 × 25 mm3 ). For the 40%IPhene group, the specimens were irradiated for 90 s at every part until all the parts were irradiated. For all the other groups, the irradiation time for every part was 60 s. The irradiation time was decided according to the curve of DC versus time obtained from the double bond conversion measurement. Three-point bending test (span 20 mm) was carried out to evaluate the flexural strength (FS) and (FM) according ISO 4049 standard with a material testing machine (model LRX, Lloyd Instrument Ltd., Fareham, England), at a cross-head speed of 1.00 mm/min. The energy of fracture U(ε) was also calculated as the area under stress–strain curve [22] according to the following equation:
εb
U(ε) =
(4)
(ε)dε
Fig. 2 – FT-IR spectrum of IPhene.
0
where and ε are stress and strain, respectively, and εb is the strain at break.
2.2.6.
Radio opacity
Three discs with 3 mm thickness were prepared for each resin formulations, and irradiated with X-ray (63 kV, 8 mA, 0.008 s) to get a radiograph. The relative radio opacity was determined by a standard aluminum step-wedge (0.5–4 mm) exposed on the same radiograph. A free image editing software ImageJ was used to measure the gray value of the sample and aluminum in the resulting image. The relative radio opacity (RXO) of disc was calculated by using the equation RXO =
G − G d b Ga − Gb
× 100%
(5)
where Gd and Ga are the gray value of sample disc and aluminum at the same thickness, respectively, and Gb is the gray value of background.
2.2.7.
Biofilm inhibition test
Four disc shaped samples (2 mm thick and 8 mm in diameter) for each resin formulation were prepared for biofilm inhibition test. Each disc was polished with 4000 grit (FEPA) grinding paper. The inhibition of biofilm formation reflecting plaque accumulation was tested by a modification of the method originally presented by Ebi et al. [23]. The microorganism we used was the reference strain Streptoccus mutans (S. mutans) Ingbritt. It was first grown overnight in Brain Heart Infusion medium (BHI; Becton Dickinson and Company, Sparks, MD, USA). In the morning the cells were washed with phosphate-buffered saline (5000 × g, 10 min) and then they were suspended in BHI containing 1% sucrose (A550 = 0.05). This suspension (500 L) was pipetted onto the experimental discs placed in the wells of 24-well cell culture plates. The plates were incubated anaerobically (90% N2, 5% CO2 , 5% H2 ) at 37 ◦ C for 24 h. The biofilms were collected with microbrushes (QuickStick® , Dentsolv AB, Saltsjö-Boo, Sweden) from the disc surface exposed to the medium to test tubes containing Tryptic Soy Broth (Becton Dickinson and Company). The tubes were vortexed and mildly sonicated and then serially diluted for
plate culturing of S. mutans. The plates were grown for three days anaerobically (80% N2 , 10% CO2 , 10% H2 ) at +37 ◦ C on Mitis salivarius agar (Becton Dickinson and Company), the colonies were counted under a stereomicroscope and results expressed as colony-forming units (CFU)/disc surface. The biofilm collection method has been tested in our earlier studies and it is highly reproducible [7,24].
2.2.8.
Statistical analysis
All of results were statistical analyzed with analysis of variance (ANOVA) at the p < 0.05 significance level by software SPSS 13.0 (SPSS Inc., Chicago, CA, USA, 2004). Subsequent multiple comparisons were conducted using Tukey’s post hoc analysis.
3.
Results
IPhene was synthesized via a two-steps route (Fig. 1), and its structure was confirmed by the FTIR (Fig. 2) and 1 H-NMR (Fig. 3) spectra. The curves of DC versus time for all resins were shown in Fig. 4, it could be noticed that there was a delay in the conversion kinetics for the IPhene containing samples, and the delay was prolonged with the increasing of IPhene concentration. Except for the resin with 10 wt.% IPhene, which had the same DC as control resin (Table 2, p > 0.05), all the other IPhene containing resins had lower DC than control resin (Table 2, p < 0.05). All of IPhene containing samples had lower volumetric shrinkage than control resin except for the sample contained 10 wt.% IPhene (Table 2, p < 0.05), which had nearly the same volumetric shrinkage as control resin (Table 2, p > 0.05). The results of three point bending test (Table 2) showed that only FS of polymer with 10 wt.% IPhene was comparable with that of control polymer (p > 0.05), and all the other FS and FM of IPhene containing polymers were lower than that of control polymer (p < 0.05). Polymers with 30 wt.% and 40 wt.% IPhene had the lowest FS and FM in all of the experimental polymers. Fig. 5 depicts the stress–strain curves of the polymers derived from three point bending test with the values of
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Fig. 3 – 1 H-NMR spectrum of IPhene.
energy of fracture provided in Table 2. Compared with the control polymer, polymers with 10 wt.% and 40 wt.% IPhene had nearly the same fracture energies (p > 0.05), while polymers with 20 wt.% and 30 wt.% IPhene had higher fracture energies (p < 0.05). In all of polymers, polymer with 30 wt.% IPhene had the highest fracture energy. Fig. 6 was the radiograph of experimental polymers and aluminum steps. The radiograph and RXO (Table 2) of samples revealed that IPhene containing polymers had higher radio-opacity than control polymer (p < 0.05), and radioopacity increased with the increasing of IPhene concentration (p < 0.05). The amounts of S. mutans counts formed on the surfaces of polymers were shown in Table 2. From the results, it could be seen that the amounts of bacteria recovered from the surfaces of control polymer, polymer with 10 wt.% IPhene, and polymer with 20 wt.% IPhene were nearly the same (p > 0.05), which were more than the amounts of bacteria recovered from the surface of polymers with 30 wt.% and 40 wt.% IPhene (p < 0.05).
Fig. 4 – Curves of DC versus time of resins with and without IPhene.
Table 2 – Double bond conversion, polymerization shrinkage, flexural strength and moduls, energy of fracture, relative X-ray opacity (RXO), S. mutans colonies in 24 h biofilm of polymers with and without IPhene. Resin systems
Control 10%IPhene 20%IPhene 30%IPhene 40%IPhene
DC (%, n = 5)
72.9 73.2 71.5 70.3 69.7
± ± ± ± ±
0.5a 0.3a 0.2b 0.4c 0.3c
Shrinkage (%, n = 5)
8.8 8.4 4.6 3.9 3.4
± ± ± ± ±
0.3a 0.6a 0.3b 0.9b,c 0.5c
Flexural strength (MPa, n = 8) 109.1 103.4 94.6 83.5 79.8
± ± ± ± ±
5.3a 4.1a 5.2b 2.8c 5.3c
Flexural modulus (GPa, n = 8) 2.67 2.38 2.28 1.65 1.69
± ± ± ± ±
0.27a 0.16b 0.17b 0.11c 0.14c
Energy of fracture (MPa, n = 8) 5.37 6.10 8.07 9.72 3.46
± ± ± ± ±
1.79a 1.87a,b 1.38b 1.15c 1.01a
RXO (%, n = 3)
10.2 25.8 43.9 60.3 84.9
± ± ± ± ±
2.4a 1.4b 1.9c 0.6d 1.4e
S. mutans colonies ((LogCFU/mm2 ) × 102 , n = 4) 13.99 14.00 14.33 3.63 5.40
± ± ± ± ±
0.34a 0.14a 0.52a 5.14b 1.97b
The same lower case letter indicate there is no statistical difference within a column (Tukey’s test, p = 0.05)
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Fig. 5 – The stress–strain curve of each polymer.
4.
Discussion
According to previous study, it could be concluded that quaternary ammonium monomer would endow dental resin with antibacterial activity when its alky side chain was dodecyl or longer [12,25,26]. However, prolong the alkyl side chain would decrease the concentration of iodine in the monomer [27], which could decrease the radio-opacity of monomer. Therefore, polymerizable quaternary ammonium with dodecyl was only synthesized in present study. From the results of FTIR (Fig. 2) and 1 H-NMR (Fig. 3) it could be confirmed that IPhene was synthesized successfully as designed. Different from the monomer DDMAI studied before [17], IPhene had no miscibility problem with Bis-GMA/TEGDMA resin system, so high mass ratio of IPhene in Bis-GMA/TEGDMA could be obtained here.
It should be noticed that the reactable double bond in IPhene was from ␣-Methylstyrene, and the reactivity of double bond in ␣-Methylstyrene was reported to be lower than that of double bond in methacrylate in free radical polymerization [28]. Therefore, the delay in conversion kinetics for the IPhene containing samples and the lower DCs of them might be attributed to the lower reactivity double bond in IPhene. However, lower polymerization rates of IPhene containing polymers might help them to reduce the contraction stress during polymerization, for higher polymerization rate always leads higher polymerization shrinkage stress [29]. As the major drawback of methacrylate-based dental materials, polymerization shrinkage could bring on marginal gaps between the tooth and the material, which would lead to recurrent caries because of the pathogenic bacteria invasion of gaps [25]. Therefore, to some extent, possibility of secondary caries could be reduced if polymerization shrinkage of dental materials decreased. The polymerization shrinkage of methacryate monomers with the same functionality was directly influenced by molecular weight and DC, higher molecular weight and lower DC would lead to lower polymerization shrinkage [30,31]. The lower polymerization shrinkage of IPhene containing resins in this study should be attributed to their lower DC, in addition, the higher molecular weight of IPhene (817) when compared with Bis-GMA (512) and TEGDMA (286). Just like some other quaternary ammonium monomers, IPhene would decrease the FS and FM when its concentration was beyond a certain limit [32], even though there are two benzene groups in the structure of IPhene which could have enhancement effect on polymeric network. This might be attributed to the alkyl side chain of IPhene, which could decrease the interaction between polymer chains and lead lower FS and FM. However, it was interesting to notice that the energy required to break the specimens was higher for the specimens with 20 wt.% and 30 wt.% IPhene, and these specimens showed plastic deformation prior to fracture (Fig. 5).
Fig. 6 – Radiograph of experimental polymers and aluminum steps. Please cite this article in press as: He J, et al. Preparation of antibacterial and radio-opaque dental resin with new polymerizable quaternary ammonium monomer. Dent Mater (2015), http://dx.doi.org/10.1016/j.dental.2015.02.007
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The higher fracture energy and plastic deformation of polymers with 20 wt.% and 30 wt.% IPhene might lead to higher fracture toughness [22], which is an important criterion in dental materials longevity [33]. Though FS and FM of polymers with 20 wt.%, 30 wt.%, and 40 wt.% were lower than those of control polymer, their values still met the ISO (4049:1988) requirements for dental resin-based restorative materials in respect to FS and FM, where the FS should be above 50 MPa and FS > [(FM × 0.0025) + 40]. With the increasing of IPhene concentration, radio-opacity of IPhene containing polymer increased and the polymer showed biofilm inhibition effect when the IPhene concentration reached 30 wt.% or more. These results revealed that the hypothesis of this study that IPhene could endow Bis-GMA/TEGDMA with both antibacterial and radio-opaque activity could be accepted. Besides the alkyl side chain length of quaternary ammonium, quaternary amine charge density of quaternary ammonium containing polymer is another important factor for its antibacterial activity [34]. It was reported that the positively-charged ammonium group could interact with negatively-charged bacterial membrane to disrupt membrane functions, alter the balance of essential ions (i.e., K+ , Na+ , Ca2+ , and Mg2+ ), interrupt protein activity and damage bacterial DNA [34,35]. Increasing quaternary ammonium concentration could increase the charge density of a polymer and improve the antibacterial activity [27,34,36]. That is why only polymers with 30 wt.% and 40 wt.% IPhene showed antibacterial activity in this research. Moreover, the lower DCs of polymers with 30 wt.% and 40 wt.% IPhene might be another reason for their higher antibacterial activity. The lower DC might lead to more leachable unreacted antibacterial IPhene left in the polymers, which could leach out of the polymers to kill bacteria. However, DCs of polymers with 30 wt.% and 40 wt.% IPhene were only 2–3% lower than control polymer, whether there were sufficient amount of IPhene leaching out of the polymers to kill bacteria should be confirmed in further study. Above all, incorporation of synthesized IPhene into Bis-GMA/TEGDMA dental resin could bring out lower polymerization shrinkage, antibacterial activity, and radio-opacity, which were benefit for dental application. However, some disadvantages like lower DC, FS, and FM of IPhene containing dental resin should be solved, and much more studies concerned about biocompatibility, exact antibacterial mechanism of IPhene containing polymer, and oral condition resistance of IPhene containing polymer should be taken in future.
5.
Conclusion
Within the limitation of this study, the following can be concluded: (1) A novel polymerizable quaternary ammonium IPhene was synthesized and confirmed by FT-IR and 1 H-NMR spectra. (2) Incorporation of IPhene into Bis-GMA/TEGDMA dental resin could reduce its DC, polymerization shrinkage, FS and FM. Only 30 wt.% and 40 wt.% of IPhene could endow dental resin both with antibacterial and radio-opaque activity. Though FS and FM of polymers with IPhene
7
were lower than control polymer, they still met the ISO requirements for dental resin-based restorative materials in respect to FS and FM.
Acknowledgments We would like to greatly thank biomedical research technician Oona Hällfors for her help in biofilm inhibition testing. Thanks for the supporting of Fundamental Research Funds for Central Universities (2014ZM0006), China. The study belongs to the BioCity Turku Biomaterials Research Program (www.biomaterials.utu.fi).
references
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Preparation of antibacterial and radio-opaque dental resin with new polymerizable quaternary ammonium monomer Jingwei He a,b,c,∗ , Eva Söderling d , Lippo V.J. Lassila a,b,d , Pekka K. Vallittu a,b,d,e a
Department of Biomaterials Science, Institute of Dentistry and Biocity Turku Biomaterial Research Program, University of Turku, Lemminkäisenkatu 2, Turku 20520, Finland b Turku Clinical Biomaterials Centre-TCBC, University of Turku, Itäinen Pitkäkatu 4 B, Turku FI-20520, Finland c College of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China d Institute of Dentistry, University of Turku, Turku 20520, Finland e City of Turku Welfare Division, Oral Health Care, Turku 20101, Finland
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. A new polymerizable quaternary ammonium monomer (IPhene) with iodine anion
Received 24 August 2014
was synthesized and incorporated into Bis-GMA/TEGDMA (50/50, wt/wt) to prepare antibac-
Received in revised form
terial and radio-opaque dental resin.
3 December 2014
Methods. IPhene was synthesized through a 2-steps reaction route, and its structure was con-
Accepted 10 February 2015
firmed by FT-IR and 1 H-NMR spectra. IPhene was incorporated into Bis-GMA/TEGDMA (50/50,
Available online xxx
wt/wt) with a series of mass fraction (from 10 wt.% to 40 wt.%). Degree of monomer conversion (DC) was determined by FT-IR analysis. Polymerization shrinkage was determined
Keywords:
according to the variation of density before and after polymerization. The flexural strength,
Dental resin
modulus of elasticity, and fracture energy were measured using a three-point bending set
Synthesis
up. Radiograph was taken to evaluate the radio-opacity of the polymer. A single-species
Antibacterial activity
biofilm model with Streptococcus mutans (S. mutans) as the tests organism was used to evalu-
Radio-opacity
ate the antibacterial activity of the polymer. Bis-GMA/TEGDMA resin system without IPhene
Double bond conversion
was used as a control group.
Mechanical property
Results. FT-IR and 1 H-NMR spectra of IPhene revealed that IPhene was the same as the
Polymerization shrinkage
designed structure. ANOVA analysis showed that when mass fraction of IPhene was more than 10 wt.%, the obtained resin formulation had lower DC, polymerization shrinkage, FS, and FM than control resin (p < 0.05). Polymers with 20 wt.% and 30 wt.% IPhene had higher fracture energies than control polymer (p < 0.05). IPhene containing samples had higher radio-opacity than control group (p < 0.05), and radio-opacity of IPhene containing sample increased with the increasing of IPhene mass fraction (p < 0.05). Only polymers with 30 wt.% and 40 wt.% of IPhene showed antibacterial activity (p < 0.05).
∗ Corresponding author at: Corresponding author at: Turku Clinical Biomaterials Centre-TCBC, University of Turku, Itäinen Pitkäkatu 4 B, FI-20520 Turku, Finland. Tel.: +358 465968930. E-mail address: hejin@utu.fi (J. He).
http://dx.doi.org/10.1016/j.dental.2015.02.007 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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Significance. IPhene could endow dental resin with both antibacterial and radio-opaque activity when IPhene reached 30 wt.% or more. Though sample with 30 wt.% of IPhene had lower FS and FM than control group, its lower volumetric shrinkage, higher fracture energy, higher radio-opacity, and antibacterial activity still made it having potential to be used in dentistry. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Dental composites, which consist of resin matrix and inorganic filler, are popular restorative materials because of their natural tooth color and direct-filling capabilities [1]. Dental composites have been reported to be used in more than 95% of all anterior tooth direct restorations and in about 50% of all posterior tooth direct restorations [2]. However, dental composite restorative materials have been reported to accumulate more bacteria or plaque than other restorative materials in vitro [3–6] or in vivo [7,8] because of their lack of antibacterial activity, and bacteria or plaque accumulation adjacent to the restoration margins may lead to secondary caries in vivo and shorten the life of composite restoration. Quaternary ammonium compounds, which are active against a broad spectrum of micro-organisms such as Grampositive and Gram-negative bacteria, fungi, and certain types of viruses [9], are well known antibacterial agents and have already been used in fields like water treatment, medicine, food applications, and textile products [9–11]. Quaternary ammonium compounds with polymerizable groups can immobilize the antibacterial quaternary ammonium group into the polymer backbone and give long-term antibacterial activity to the polymer [12]. In dentistry, Imazato firstly applied a polymerizable quaternary ammonium named methacryloyloxydodecyl-pridinium bromide (MDPB) as an antibacterial agent to prepare long-term antibacterial dental restorative materials [13–16]. Besides the antibacterial activity, quaternary ammonium compounds with iodine anion (I− ) also have radio-opacity [17], which is an important property for dental materials, because of the high electronic density of iodine [18,19]. Though radioopacity of dental materials can be implemented by adding radio-opaque inorganic fillers, there still exist some dental materials like E-glass fiber reinforced composites and flowable resin composites that have insufficient radio-opacity [20]. Therefore, radio-opaque dental resin can be used to solve this problem. In our previous study, a polymerizable quaternary ammonium named 2-dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) was synthesized and used to prepare antibacterial and radio-opaque dental resin system [17]. Unfortunately, DDMAI had miscible problem with hydrophobic methacrylate monomer tri-ethyleneglycol dimethacrylate (TEGDMA), and only 5 wt.% of DDMAI could be added into 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropyl)phenyl]propane (Bis-GMA)/TEGDMA resin system. Thus, the antibacterial activity and radio-opacity of DDAMI containing polymer were not obvious.
In this work, a new polymerizable quaternary ammonium monomer (IPhene) with iodine anion was synthesized and incorporated into Bis-GMA/TEGDMA dental resin with different mass ratio. The hypotheses are (I): IPhene could be mixed well with Bis-GMA/TEGDMA at high mass ratio; (II): IPhene could endow Bis-GMA/TEGDMA with both antibacterial and radio-opaque activity. Double bond conversion, polymerization shrinkage, flexural strength and modulus, antibacterial activity, radio-opacity of IPhene containing resin formulations were investigated and compared with resin formulation without IPhene.
2.
Materials and methods
2.1.
Materials
N-methyl diethanol amine (MDEA), 1-iodododecane, dibutyltin dilaurate (DBTDL), TEGDMA, camphoroquinone (CQ), N,N -dimethylaminoethylmethacrylate (DMAEMA), and 3-isopropenyl-␣,␣-dimethylbenzyl isocyanate (IDI) were purchased from Sigma–Aldrich Co. (St Luois, MO, USA). Bis-GMA was applied from Esstech Inc. (Essington, PA, USA). All of the compounds were used without further purification.
2.2.
Methods
2.2.1. Synthesis of polymerizable quaternary ammonium monomer (IPhene) As shown in Fig. 1, IPhene was synthesized through a 2-steps route. FT-IR (Spectrum One, Perkin-Elmer, Waltham, MA, USA) and 1 H-NMR (AV 400 MHz, Bruker Co., Germany) spectra of intermediate products and IPhene were obtained to confirm their structures. The FT-IR spectra were recorded with 32 scans at a resolution of 4 cm−1 . The chemical shifts of 1 H-NMR spectra were reported in ppm on ı scale with tetramethylsilane as the internal reference and CDCl3 as the solvent.
2.2.1.1. Synthesis of intermediate product N,N-bis(2hydroxyethyl)-N-methyldodecyl ammonium iodide (HQAI-12). A mixture of MDEA (0.05 mol), 1-iodododecane (0.051 mol), and 30 mL acetone were stirred at reflux. After 24 h reaction, the acetone was removed by distillation under vacuum. The obtained raw product was washed with ethyl ether and filtered for several times. Then the white intermediate product HQAI-12 was dried under vacuum at 35 ◦ C for 48 h. The results of spectroscopic studies for HQAI-12 are as follows: IR (neat): (cm−1 ) 3294, 2956, 2927, 2855, 1381, 1090, 720. 1 H-NMR (CDCl3 , 400 MHz): ı 4.22, 4.09, 3.77–3.89, 3.57–3.62, 3.38, 1.80, 1.31–1.42, 0.94–0.95.
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accessory. The FT-IR spectra were recorded with one scan at a resolution of 4 cm−1 . All the samples were analyzed in a mold that was 2 mm thick and 6 mm in diameter. First, the spectrum of the unpolymerized sample was measured. Then, the sample was irradiated for 180s with a visible light-curing unit (Curing Light 2500, = 400–520 nm, I ≈ 550 mW cm−2 , 3 M Co., St Paul, MN, USA). The sample was scanned for its FTIR spectrum every 5 s until 15 min after the beginning of irradiation. Each group was measured for 5 times. To determined the percentage of reacted double bonds, the absorbance intensities of the C C absorbance peak at 1636 cm−1 , which were decreased after being irradiated, and an internal phenyl ring standard peak at 1608 cm−1 , were calculated using a baseline method. The ratios of absorbance intensities were calculated and compared before and after polymerization. The DC at each irradiation time was calculated by using the equation
DC(t) = Fig. 1 – Synthesis route of IPhene.
2.2.1.2. Synthesis of final product IPhene. Acetone used here ˚ molecular sieves for 2 weeks. A mixture was dried over 4 A of HQAI-12 (0.04 mol), IDI (0.08 mol), 20 mL acetone, and 2 droplets of DBTDL were stirred at 45 ◦ C. The reaction was continued until the infrared absorbance peak of the –NCO group (2270 cm−1 ) disappeared in the FT-IR spectra of the samples taken from the reaction medium. After removing the acetone by distillation under vacuum, the product was washed with diethyl ether to remove DBTDL. Then the yellow viscose liquid was dried under vacuum at 35 ◦ C for 48 h to obtain IPhene. The results of spectroscopic studies for IPhene are as follows: IR (neat): (cm−1 ) 3284, 3083, 2956, 2925, 2855, 1710, 1629, 1600, 1578, 1521, 1485, 1221, 1173, 1091, 724. 1 H-NMR (CDCl3 , 400 MHz): ı 7.31–7.49, 6.15, 5.36, 5.10, 4.47, 3.38–3.82, 2.16, 1.66, 1.54, 1.28–1.38, 0.91.
2.2.2.
Preparation of resin formulation
The formulation of each dental resin used in this work is shown in Table 1. All of them were well blended to obtain a homogenous mixture, and stored at darkness before used.
2.2.3.
Double bond conversion
The degree of double bond conversion (DC) was determined by using an FT-IR spectrometer (Spectrum One, Perkin-Elmer, Waltham, MA, USA) with an attenuated total reflectance (ATR)
Table 1 – Formulation of every experimental resin system. System
Control 10%IPhene 20%IPhene 30%IPhene 40%IPhene
TEGDMA
IPhene
CQ
DMAEMA
49.3 44.3 39.3 34.3 29.3
49.3 44.3 39.3 34.3 29.3
0 10 20 30 40
0.7 0.7 0.7 0.7 0.7
0.7 0.7 0.7 0.7 0.7
(AC=C /Aph )0
× 100%
(1)
where AC C and Aph are the absorbance intensity of C C at 1636 cm−1 and phenyl ring at 1608 cm−1 , respectively; (AC C /Aph )0 and (AC C /Aph )t ate the normalized absorbance of functional group at the radiation time 0 and t, respectively; DC(t) is the conversion of C C as a function of radiation time.
2.2.4.
Polymerization shrinkage
Volumetric polymerization shrinkage of dental resin was measured according to the method reported previously [21]. The specimen’s densities (n = 5 per group) were measured to determine polymerization shrinkage using Archimedes’ principle with a commercial Density Determination Kit of the analytical balance Mettler Toledo X (Mettler Instrument Co., Highstone, NJ, USA). The kit provides a special holder for the specimens, a special container to keep the water, a computer, and appropriate software. The specimens were weighed in air and in water, and the density was directly calculated in gram per cubic centimeter by the software of the Mettler Toledo XS balance according to the equation: d=
Ma × (dw − da ) + da Ma − Mw
(2)
where d density of sample, Ma weight of sample in air, Mw weight of sample in water, dw density of water at the exactly measured temperature in ◦ C according to the density table of distilled water, da air density (0.0012 g/cm3 ). An internal balance correction factor (0.99985) of the Mettler Toledo XS balance software took the air buoyancy of the adjustment weight into account. The volumetric polymerization shrinkage S was calculated from the densities according to the equation:
Content of every monomer (g) Bis-GMA
1 − (AC=C /Aph )t
S=
dafter − dbefore × 100% dafter
(3)
where dafter is the density of sample after curing, dbefore is the density of sample before curing. All of polymerized samples were irradiated samples obtained from double bond conversion measurement.
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2.2.5.
Three point bending test
Eight specimens were prepared for each sample formulation (size 2 × 2 × 25 mm3 ). For the 40%IPhene group, the specimens were irradiated for 90 s at every part until all the parts were irradiated. For all the other groups, the irradiation time for every part was 60 s. The irradiation time was decided according to the curve of DC versus time obtained from the double bond conversion measurement. Three-point bending test (span 20 mm) was carried out to evaluate the flexural strength (FS) and (FM) according ISO 4049 standard with a material testing machine (model LRX, Lloyd Instrument Ltd., Fareham, England), at a cross-head speed of 1.00 mm/min. The energy of fracture U(ε) was also calculated as the area under stress–strain curve [22] according to the following equation:
εb
U(ε) =
(4)
(ε)dε
Fig. 2 – FT-IR spectrum of IPhene.
0
where and ε are stress and strain, respectively, and εb is the strain at break.
2.2.6.
Radio opacity
Three discs with 3 mm thickness were prepared for each resin formulations, and irradiated with X-ray (63 kV, 8 mA, 0.008 s) to get a radiograph. The relative radio opacity was determined by a standard aluminum step-wedge (0.5–4 mm) exposed on the same radiograph. A free image editing software ImageJ was used to measure the gray value of the sample and aluminum in the resulting image. The relative radio opacity (RXO) of disc was calculated by using the equation RXO =
G − G d b Ga − Gb
× 100%
(5)
where Gd and Ga are the gray value of sample disc and aluminum at the same thickness, respectively, and Gb is the gray value of background.
2.2.7.
Biofilm inhibition test
Four disc shaped samples (2 mm thick and 8 mm in diameter) for each resin formulation were prepared for biofilm inhibition test. Each disc was polished with 4000 grit (FEPA) grinding paper. The inhibition of biofilm formation reflecting plaque accumulation was tested by a modification of the method originally presented by Ebi et al. [23]. The microorganism we used was the reference strain Streptoccus mutans (S. mutans) Ingbritt. It was first grown overnight in Brain Heart Infusion medium (BHI; Becton Dickinson and Company, Sparks, MD, USA). In the morning the cells were washed with phosphate-buffered saline (5000 × g, 10 min) and then they were suspended in BHI containing 1% sucrose (A550 = 0.05). This suspension (500 L) was pipetted onto the experimental discs placed in the wells of 24-well cell culture plates. The plates were incubated anaerobically (90% N2, 5% CO2 , 5% H2 ) at 37 ◦ C for 24 h. The biofilms were collected with microbrushes (QuickStick® , Dentsolv AB, Saltsjö-Boo, Sweden) from the disc surface exposed to the medium to test tubes containing Tryptic Soy Broth (Becton Dickinson and Company). The tubes were vortexed and mildly sonicated and then serially diluted for
plate culturing of S. mutans. The plates were grown for three days anaerobically (80% N2 , 10% CO2 , 10% H2 ) at +37 ◦ C on Mitis salivarius agar (Becton Dickinson and Company), the colonies were counted under a stereomicroscope and results expressed as colony-forming units (CFU)/disc surface. The biofilm collection method has been tested in our earlier studies and it is highly reproducible [7,24].
2.2.8.
Statistical analysis
All of results were statistical analyzed with analysis of variance (ANOVA) at the p < 0.05 significance level by software SPSS 13.0 (SPSS Inc., Chicago, CA, USA, 2004). Subsequent multiple comparisons were conducted using Tukey’s post hoc analysis.
3.
Results
IPhene was synthesized via a two-steps route (Fig. 1), and its structure was confirmed by the FTIR (Fig. 2) and 1 H-NMR (Fig. 3) spectra. The curves of DC versus time for all resins were shown in Fig. 4, it could be noticed that there was a delay in the conversion kinetics for the IPhene containing samples, and the delay was prolonged with the increasing of IPhene concentration. Except for the resin with 10 wt.% IPhene, which had the same DC as control resin (Table 2, p > 0.05), all the other IPhene containing resins had lower DC than control resin (Table 2, p < 0.05). All of IPhene containing samples had lower volumetric shrinkage than control resin except for the sample contained 10 wt.% IPhene (Table 2, p < 0.05), which had nearly the same volumetric shrinkage as control resin (Table 2, p > 0.05). The results of three point bending test (Table 2) showed that only FS of polymer with 10 wt.% IPhene was comparable with that of control polymer (p > 0.05), and all the other FS and FM of IPhene containing polymers were lower than that of control polymer (p < 0.05). Polymers with 30 wt.% and 40 wt.% IPhene had the lowest FS and FM in all of the experimental polymers. Fig. 5 depicts the stress–strain curves of the polymers derived from three point bending test with the values of
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Fig. 3 – 1 H-NMR spectrum of IPhene.
energy of fracture provided in Table 2. Compared with the control polymer, polymers with 10 wt.% and 40 wt.% IPhene had nearly the same fracture energies (p > 0.05), while polymers with 20 wt.% and 30 wt.% IPhene had higher fracture energies (p < 0.05). In all of polymers, polymer with 30 wt.% IPhene had the highest fracture energy. Fig. 6 was the radiograph of experimental polymers and aluminum steps. The radiograph and RXO (Table 2) of samples revealed that IPhene containing polymers had higher radio-opacity than control polymer (p < 0.05), and radioopacity increased with the increasing of IPhene concentration (p < 0.05). The amounts of S. mutans counts formed on the surfaces of polymers were shown in Table 2. From the results, it could be seen that the amounts of bacteria recovered from the surfaces of control polymer, polymer with 10 wt.% IPhene, and polymer with 20 wt.% IPhene were nearly the same (p > 0.05), which were more than the amounts of bacteria recovered from the surface of polymers with 30 wt.% and 40 wt.% IPhene (p < 0.05).
Fig. 4 – Curves of DC versus time of resins with and without IPhene.
Table 2 – Double bond conversion, polymerization shrinkage, flexural strength and moduls, energy of fracture, relative X-ray opacity (RXO), S. mutans colonies in 24 h biofilm of polymers with and without IPhene. Resin systems
Control 10%IPhene 20%IPhene 30%IPhene 40%IPhene
DC (%, n = 5)
72.9 73.2 71.5 70.3 69.7
± ± ± ± ±
0.5a 0.3a 0.2b 0.4c 0.3c
Shrinkage (%, n = 5)
8.8 8.4 4.6 3.9 3.4
± ± ± ± ±
0.3a 0.6a 0.3b 0.9b,c 0.5c
Flexural strength (MPa, n = 8) 109.1 103.4 94.6 83.5 79.8
± ± ± ± ±
5.3a 4.1a 5.2b 2.8c 5.3c
Flexural modulus (GPa, n = 8) 2.67 2.38 2.28 1.65 1.69
± ± ± ± ±
0.27a 0.16b 0.17b 0.11c 0.14c
Energy of fracture (MPa, n = 8) 5.37 6.10 8.07 9.72 3.46
± ± ± ± ±
1.79a 1.87a,b 1.38b 1.15c 1.01a
RXO (%, n = 3)
10.2 25.8 43.9 60.3 84.9
± ± ± ± ±
2.4a 1.4b 1.9c 0.6d 1.4e
S. mutans colonies ((LogCFU/mm2 ) × 102 , n = 4) 13.99 14.00 14.33 3.63 5.40
± ± ± ± ±
0.34a 0.14a 0.52a 5.14b 1.97b
The same lower case letter indicate there is no statistical difference within a column (Tukey’s test, p = 0.05)
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Fig. 5 – The stress–strain curve of each polymer.
4.
Discussion
According to previous study, it could be concluded that quaternary ammonium monomer would endow dental resin with antibacterial activity when its alky side chain was dodecyl or longer [12,25,26]. However, prolong the alkyl side chain would decrease the concentration of iodine in the monomer [27], which could decrease the radio-opacity of monomer. Therefore, polymerizable quaternary ammonium with dodecyl was only synthesized in present study. From the results of FTIR (Fig. 2) and 1 H-NMR (Fig. 3) it could be confirmed that IPhene was synthesized successfully as designed. Different from the monomer DDMAI studied before [17], IPhene had no miscibility problem with Bis-GMA/TEGDMA resin system, so high mass ratio of IPhene in Bis-GMA/TEGDMA could be obtained here.
It should be noticed that the reactable double bond in IPhene was from ␣-Methylstyrene, and the reactivity of double bond in ␣-Methylstyrene was reported to be lower than that of double bond in methacrylate in free radical polymerization [28]. Therefore, the delay in conversion kinetics for the IPhene containing samples and the lower DCs of them might be attributed to the lower reactivity double bond in IPhene. However, lower polymerization rates of IPhene containing polymers might help them to reduce the contraction stress during polymerization, for higher polymerization rate always leads higher polymerization shrinkage stress [29]. As the major drawback of methacrylate-based dental materials, polymerization shrinkage could bring on marginal gaps between the tooth and the material, which would lead to recurrent caries because of the pathogenic bacteria invasion of gaps [25]. Therefore, to some extent, possibility of secondary caries could be reduced if polymerization shrinkage of dental materials decreased. The polymerization shrinkage of methacryate monomers with the same functionality was directly influenced by molecular weight and DC, higher molecular weight and lower DC would lead to lower polymerization shrinkage [30,31]. The lower polymerization shrinkage of IPhene containing resins in this study should be attributed to their lower DC, in addition, the higher molecular weight of IPhene (817) when compared with Bis-GMA (512) and TEGDMA (286). Just like some other quaternary ammonium monomers, IPhene would decrease the FS and FM when its concentration was beyond a certain limit [32], even though there are two benzene groups in the structure of IPhene which could have enhancement effect on polymeric network. This might be attributed to the alkyl side chain of IPhene, which could decrease the interaction between polymer chains and lead lower FS and FM. However, it was interesting to notice that the energy required to break the specimens was higher for the specimens with 20 wt.% and 30 wt.% IPhene, and these specimens showed plastic deformation prior to fracture (Fig. 5).
Fig. 6 – Radiograph of experimental polymers and aluminum steps. Please cite this article in press as: He J, et al. Preparation of antibacterial and radio-opaque dental resin with new polymerizable quaternary ammonium monomer. Dent Mater (2015), http://dx.doi.org/10.1016/j.dental.2015.02.007
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The higher fracture energy and plastic deformation of polymers with 20 wt.% and 30 wt.% IPhene might lead to higher fracture toughness [22], which is an important criterion in dental materials longevity [33]. Though FS and FM of polymers with 20 wt.%, 30 wt.%, and 40 wt.% were lower than those of control polymer, their values still met the ISO (4049:1988) requirements for dental resin-based restorative materials in respect to FS and FM, where the FS should be above 50 MPa and FS > [(FM × 0.0025) + 40]. With the increasing of IPhene concentration, radio-opacity of IPhene containing polymer increased and the polymer showed biofilm inhibition effect when the IPhene concentration reached 30 wt.% or more. These results revealed that the hypothesis of this study that IPhene could endow Bis-GMA/TEGDMA with both antibacterial and radio-opaque activity could be accepted. Besides the alkyl side chain length of quaternary ammonium, quaternary amine charge density of quaternary ammonium containing polymer is another important factor for its antibacterial activity [34]. It was reported that the positively-charged ammonium group could interact with negatively-charged bacterial membrane to disrupt membrane functions, alter the balance of essential ions (i.e., K+ , Na+ , Ca2+ , and Mg2+ ), interrupt protein activity and damage bacterial DNA [34,35]. Increasing quaternary ammonium concentration could increase the charge density of a polymer and improve the antibacterial activity [27,34,36]. That is why only polymers with 30 wt.% and 40 wt.% IPhene showed antibacterial activity in this research. Moreover, the lower DCs of polymers with 30 wt.% and 40 wt.% IPhene might be another reason for their higher antibacterial activity. The lower DC might lead to more leachable unreacted antibacterial IPhene left in the polymers, which could leach out of the polymers to kill bacteria. However, DCs of polymers with 30 wt.% and 40 wt.% IPhene were only 2–3% lower than control polymer, whether there were sufficient amount of IPhene leaching out of the polymers to kill bacteria should be confirmed in further study. Above all, incorporation of synthesized IPhene into Bis-GMA/TEGDMA dental resin could bring out lower polymerization shrinkage, antibacterial activity, and radio-opacity, which were benefit for dental application. However, some disadvantages like lower DC, FS, and FM of IPhene containing dental resin should be solved, and much more studies concerned about biocompatibility, exact antibacterial mechanism of IPhene containing polymer, and oral condition resistance of IPhene containing polymer should be taken in future.
5.
Conclusion
Within the limitation of this study, the following can be concluded: (1) A novel polymerizable quaternary ammonium IPhene was synthesized and confirmed by FT-IR and 1 H-NMR spectra. (2) Incorporation of IPhene into Bis-GMA/TEGDMA dental resin could reduce its DC, polymerization shrinkage, FS and FM. Only 30 wt.% and 40 wt.% of IPhene could endow dental resin both with antibacterial and radio-opaque activity. Though FS and FM of polymers with IPhene
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were lower than control polymer, they still met the ISO requirements for dental resin-based restorative materials in respect to FS and FM.
Acknowledgments We would like to greatly thank biomedical research technician Oona Hällfors for her help in biofilm inhibition testing. Thanks for the supporting of Fundamental Research Funds for Central Universities (2014ZM0006), China. The study belongs to the BioCity Turku Biomaterials Research Program (www.biomaterials.utu.fi).
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