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Incorporation of an antibacterial and radiopaque monomer in to dental resin system
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) e110–e117
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journal homepage: www.intl.elsevierhealth.com/journals/dema
Incorporation of an antibacterial and radiopaque monomer in to dental resin system Jingwei He a,b,c,∗ , Eva Söderling d , Lippo V.J. Lassila a,b , Pekka K. Vallittu a,b a
Department of Biomaterials Science, Institute of Dentistry and Biocity Turku Biomaterial Research Program, University of Turku, Turku 20520, Finland b Turku Clinical Biomaterials Centre – TCBC, University of Turku, Turku 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
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. The purpose of this study was to prepare antibacterial and radiopaque dental
Received 8 December 2011
resin through adding one polymerizable quaternary ammonium compound with iodine
Received in revised form
as counter anion into Bis-GMA/TEGDMA (50/50, wt/wt) dental resin system, and evaluate
5 April 2012
the degree of monomer conversion, mechanical properties, radiopacity, and antibacterial
Accepted 16 April 2012
effectiveness of the polymer. Methods. 2-Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) (3 wt.% and 5 wt.%) was added into a Bis-GMA/TEGDMA (50/50, wt/wt) resin system with CQ
Keywords:
(0.7 wt.%) and DMAEMA (0.7 wt.%) as photoinitiation system. Degree of monomer conversion
Dental materials
(DC) was determined by FT-IR analysis. The flexural strength (FS) and flexural modulus (FM)
Mechanical properties
of the polymer were measured using a three-point bending set up. Radiographs were taken
Antibacterial effectiveness
to determine the radiopacity of the polymer, and aluminum step-wedge (0.5–4 mm) was used
Radiopacity
as calibration standard. A single-species biofilm model with Streptococcus mutans as the tests organism was used to evaluate the antibacterial property of the polymer. Bis-GMA/TEGDMA without DDMAI was used as control material in all of the tests. Results. ANOVA analysis revealed that there was no significant difference in DC between polymer with and without DDMAI (p > 0.05). Polymer with 3 wt.% DDMAI had higher FS than the control material (p < 0.05), while polymer with 5 wt.% DDMAI had comparable FS with the control material (p > 0.05). Though average FM of control material was lower than that of 3 wt.% DDMAI containing polymer and higher than 5 wt.% DDMAI containing polymer, but the differences were statistically insignificant. By increasing the quantity of DDMAI, the radiopacity of the polymer increased. Of the three formulations, the polymer with 5 wt.% DDMAI inhibited biofilm the formation of the clinically relevant young biofilm. Significance. Incorporation of DDMAI into resin system could endow it with radiopacity and antibacterial effectiveness, and these two properties seem to be improved with increasing the quantities of DDMAI. © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ 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). 0109-5641/$ – see front matter © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dental.2012.04.026
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1.
Introduction
Since methyl methacrylate was firstly used in tooth restoration in 1937, methacrylate monomers having good biocompatibility and adhesive property have been extensively used as dental resin-based materials, e.g., denture base materials [1–3], endodontic sealers [4–6], direct-filling restorative materials [7–10], and dental bonding agents [11,12]. The most commonly used methacrylate monomers in commercial dental resin-based materials are methyl methacrylate (MMA), 2,2-bis[4(2-hydroxy-3-methacryloyloxypropyl)-phenyl]propane (Bis-GMA), 1,6-bis-[2-methacryloyloxyethoxycarbonylamino](UDMA), and tri-ethyleneglycol 2,4,4-trimethylhexane dimethacrylate (TEGDMA) [13]. By free radical polymerization of these monomers, a three dimensional network is formed, and the selection of the monomers influences the properties of dental resin-based materials directly. In order to improve properties of dental resin-based materials, several new methacrylate monomers were introduced into dental resin with the aim of reducing polymerization shrinkage [14–18], decreasing water uptake and solubility [19–21], and improving mechanical properties [22–24]. Besides these properties, dental resin-based materials also need to be radiopaque and antibacterial. However, acrylic polymers formed by commonly methacrylate monomers do not have radiopacity and antibacterial activity intrinsically, so some radiopacifying fillers, e.g., BaO, BaSO4 , TiO2 , SrO, or ZrO2 [25–27], and antibacterial agents, e.g., chlorhexidine, cetylpyridinium chloride, or titania nanoparticles [28–32], are incorporated into dental resin by physical blending. Unfortunately, all the additives mentioned above also bring some unwanted problems. For example, incorporation of radiopaque fillers into dental resin has some adverse effects on the physical and chemical properties of composites materials [33–35] and excessive fillers could cause loss of dimensional stability of the composites materials [36]. The addition of antibacterial agent into dental materials could reduce its mechanical properties even at a small concentration [37], and the releasing agent may exert toxic effects as well as cause short-term antibacterial effectiveness of the material [38]. It was reported that addition of methacrylate compounds containing radioopaque agent or antibacterial agent could also render dental materials relevant function. Davy et al. [39] synthesized iodinate methacrylate and copolymerized it with MMA for the application as X-ray opaque denture base material. Imazato et al. synthesized methacryloyloxydodecyl pyrimidinium bromide (MDPB) and used it as an antibacterial monomer in several kinds of dental materials to give longterm antibacterial effects [40–43]. Though many methacrylate compounds have been taken for the aim of radiopacity or antibacterial activity, there is no report on one methacrylate compound with both of these two functions so far. In this study, a methacrylate monomer containing quaternary ammonium structure, 2-dimethyl-2-dodecyl-1methacryloxyethyl ammonium iodine (DDMAI, structure is shown in Fig. 1), whose minimum inhibition concentration against Streptococcus mutans is 6.25 g/mL [44], was used in
Fig. 1 – Structure of DDMAI.
dental resin as antibacterial agent, because the counter anion of this reactive quaternary ammonium is iodine, so it may also has radiocontrast property. Therefore, radiopacity and antibacterial activity of dental resin with this monomer were studied, and influences of this monomer on monomer conversion and mechanical properties were also examined in this work.
2.
Materials and methods
2.1.
Materials
2-Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) was synthesized in our lab. 2,2-Bis[4(2-hydroxy-3-methacryloyloxypropyl)-phenyl]propane (Bis-GMA) was purchased from Esstech Inc. (Essington, PA, USA), tri-ethyleneglycol dimethacrylate (TEGDMA), camphoroquinone (CQ), and N,N -dimethyl aminoethyl methacrylate (DMAEMA) were purchased from Sigma–Aldrich Co. (St Luois, MO, USA), all of the reagents were used without purification.
2.2.
Methods
The formulation of every dental resin used in this experiment is shown in Table 1. All of them were well blended to obtain a homogenous mixture, and stored at darkness before used.
2.2.1.
Monomer (double bond) conversion
The degree of monomer conversion (DC) was determined by using an FTIR spectrometer (Spectrum One, Perkin-Elmer, Waltham, MA, USA) with an attenuated total reflectance (ATR) accessory. The FTIR 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 60 s with a visible light-curing unit (Curing Light 2500, = 400–520 nm, I ≈ 550 mW cm−2 , 3 M Co.,
Table 1 – Formulation of every experimental resin system. System
Control 3% 5%
Content of every monomer (g) Bis-GMA
TEGDMA
DDMAI
CQ
DMAEMA
49.3 47.8 46.8
49.3 47.8 46.8
0 3 5
0.7 0.7 0.7
0.7 0.7 0.7
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where Gd , Gb , and Ga are the gray value of disc, background and aluminum at the same thickness, respectively.
2.2.4.
Fig. 2 – The absorption peaks at 1636 cm−1 and 1608 cm−1 varied before and after irradiation.
St Paul, MN, USA). The sample was scanned for its FTIR spectrum every 5 s until 15 min after the beginning of irradiation. To determined the percentage of reacted double bonds, the absorbance intensities of the methacrylate C C absorbance peak at 1636 cm−1 , which were decreased after being irradiated (as shown in Fig. 2), 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 every irradiation time was calculated by using the equation
DC(t) =
1 − (Ac c /Aph )t (Ac c /Aph )0
× 100%
where AC C and APh are the absorbance intensity of methacrylate C C at 1636 cm−1 and phenyl ring at 1608 cm−1 , respectively; (AC C /APh )0 and (AC C /APh )t are the normalized absorbance of functional group at the radiation time 0 and t, respectively; DC(t) is the conversion of methacrylate C C as a function of radiation time.
2.2.2.
Three point bending test
Eight specimens were prepared for every sample formulation (size 2 mm × 2 mm × 25 mm). Three point bending test (span 20 mm) was carried out to evaluate the flexural strength and modulus according ISO 10477:92 standard with a material testing machine (model LRX, Lloyd Instrument Ltd., Fareham, England), at a cross-head speed of 1.00 mm/min.
2.2.3.
Radiopacity
Five discs (thickness from 1 mm to 5 mm) were prepared for every resin formulations, and irradiated with X-ray (63 kV, 8 mA, 0.008 s) to get a radiograph. The relative radiopacity was determined by comparison with the opacity exhibited 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 radiopacity (RXO) of disc was calculated by using the equation RXO =
[Gd − Gb ] × 100% [Ga − Gb ]
Biofilm inhibition test
Three discs (2 mm thick and 8 mm in diameter) for every resin formulation were prepared for biofilm inhibition test. Every disc was polished with 4000 grit (FEPA) grinding paper. Biofilm inhibition test of the discs, which were soaked in distilled water for 24 h at room temperature, was also taken to clarify whether the loss of inhibitory effect to mature biofilm was due to none leachable antimicrobial agent left in polymer after being soaked. The inhibition of biofilm formation reflecting plaque accumulation was tested by a modification of the method originally presented by Ebi et al. [45]. The microorganism we used was the type strain S. mutans Ingbritt. It was first grown overnight in Brain Heart Infusion medium (BHI; Difco, MI, 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 cell culture plates. The plates were incubated anaerobically (90% N2 , 5% CO2 , 5% H2 ) at +37 ◦ C for 6 h (young biofilm) and 24 h (mature biofilm). The biofilms were collected with microbrushes (QuickStick, Dentsolv AB, Sweden) from the disc surface exposed to the medium to test tubes containing Tryptic Soy Broth (Difco). The tubes were vortexed and mildly sonicated and then serially diluted for plate culturing of S. mutans. The plates were grown for 3 days anaerobically (80% N2 , 10% CO2 , 10% H2 ) at +37 ◦ C on Mitis salivarius agar (Difco), 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 [46,47].
2.2.5.
Statistical analysis
The results of double bond conversion, three point bending test, and biofilm inhibition test were statistically analyzed with analysis of variance (ANOVA) at the p < 0.05 significance level. Subsequent multiple comparisons were conducted using Tukey’s post hoc analysis.
3.
Results
It could be seen from Fig. 3 that polymerization behavior of resin with and without DDMAI was almost the same, and the final DC of each polymer was quite similar (Table 2). The three point bonding test showed that FS of polymer with 3 wt.% DDMAI was higher (p < 0.05) than that of control material (Fig. 4). When the content of DDMAI increased from 3 wt.% to 5 wt.%, FS of polymer was decreased (p < 0.05), and the value was nearly the same as that of control material (Table 2). FM of 3 wt.% DDMAI containing polymer was higher than that of control material (Fig. 5), where there was no significant difference between them (p > 0.05), and FM of 5 wt.% DDMAI containing polymer was lower than that of control material (Fig. 5), where there was also on significant difference between them (p > 0.05).
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Table 2 – Double bond conversion, flexural strength, flexural modulus, relative X-ray opacity, and S. mutans colonies in young biofilm on the surface of resin with and without DDMAI. Resin system
Properties DC (%)
Control 3% 5%
FS (MPa)
72.1 ± 0.2 72.5 ± 0.4a 72.0 ± 0.4a a
FM (GPa)
93.2 ± 3.6 105.0 ± 5.6b 93.6 ± 4.4a a
2.05 ± 0.16 2.22 ± 0.13a 1.91 ± 0.19b
a,b
RXO (%)
S. mutans colonies (Log CFU/mm2 × 102 )
14.2 22.7 27.8
9.85 ± 0.41a 9.32 ± 0.32a 8.61 ± 0.66b
Lower case letters indicate statistical differences within a column (Tukey’s test, p = 0.05).
Fig. 5 – Flexural modulus of polymers with and without DDMAI. Fig. 3 – Double bond conversion of resins with and without DDMAI.
Fig. 4 – Flexural strength of polymers with and without DDMAI.
The radiograph revealed that radiopacity increased with increasing thickness of polymer and amount of DDMAI in polymer (Figs. 6 and 7). Using the aluminum as radiopacity standard, RXO of polymer with DDMAI was nearly the two times higher than that of control material (Table 2). Fig. 8 shows the number of viable bacteria in the 6-h young biofilm formed on the surface of polymer with and without DDMAI. The number of bacteria on the surface of polymer with 5 wt.% DDMAI was less (p < 0.05) than that of control material. Meanwhile, though the average amount of colonies on the surface of polymer with 3 wt.% DDMAI was less than that of control material and there was no significant difference between them (p > 0.05). Soaking the polymer specimen for 1
Fig. 6 – Radiopacity of polymers (thickness from 1 mm to 5 mm) with and without DDMAI, a aluminum steps (thickness from 0.5 mm to 4 mm) as reference.
day in water did not significantly change the inhibition effect of the polymer containing 5 wt.% DDMAI (Fig. 9).
4.
Discussion
Radiopacity and antibacterial activity are two necessary properties of dental materials, the former render it possible to evaluate quality of treatment by nondestructive techniques [48], and the latter could reduce the possibility of secondary caries occurring [49]. The monomer DDMAI used in this work is a quaternary ammonium compound with iodine as counter anion. Quaternary ammonium compounds are well known
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Fig. 7 – Radiopacity of polymers (thickness from 1 mm to 5 mm) with and without DDMAI.
Fig. 8 – Young biofilm inhibition test against Streptococcus mutans of polymers with and without DDMAI.
effective antibacterial agents, which have already been used in many fields [50,51]. Iodine is an atom with high electronic density, which makes it quite radioopaque, compound with iodine was widely studied as radiocontrast media in modern medicine [52,53]. Therefore, DDMAI could be incorporated into dental resin both as antibacterial agent and radiopacity agent, moreover, the methacrylate group in its structure could immobilize the functional group into polymer backbone and make the acrylic polymer with long-term antibacterial activity and radiopacity [39,45]. However, we found that the miscibility of DDMAI with Bis-GMA/TEGDMA (50 wt/50 wt) system was not well enough, when concentration of DDMAI was more than 5 wt.%, there were some DDMAI could not be miscible with
Fig. 9 – Young biofilm inhibition test against Streptococcus mutans of polymers with and without being soaked in water.
Bis-GMA/TEGDMA (50 wt/50 wt) system. Therefore, we only chose 3 wt.%, and 5 wt.% of DDMAI here. From the results of monomer conversion and three point bending test, it could be seen that adding 3 wt.% and 5 wt.% of DDMAI into Bis-GMA/TEGDMA system had no adverse impact on DC, FS, and FM, even that polymer with 3 wt.% DDMAI had higher FM than control material. It is the beneficial aspect of DDMAI when compared with other antibacterial agent like chlorhexidine, because in some other research, adding even 1 wt.% of antibacterial agent like chlorhexidine into dental resin could reduce mechanical properties [37]. Figs. 6 and 7 show that incorporating DDMAI could increase radiopacity of dental polymer, and the radiopacity increased with increasing thickness of polymer and amount of DDMAI in the polymer. An increase of 100% in RXO was obtained when adding only 5 wt.% DDMAI into Bis-GMA/TEGDMA resin system (Table 2). Though the radiopacity of resin with 3 wt.% and 5 wt.% DDMAI is not necessarily adequate to be used in clinically, it still proves that DDMAI has potential to be used as radiopacity agent in dental and medical materials, if appropriate resin system could be found to be miscible with more DDMAI. Dental plaque is a complex microbial community that develops on tooth surface, embedded in a matrix of polymers [54]. This biofilm also covers dental restorative materials [46]. The present ecological plaque hypothesis emphasizes that non-mutans bacteria may be the key microorganisms responsible for maintaining dynamic stability on the tooth surface
Table 3 – S. mutans colonies in young biofilm on the surface of resin with 5% DDMAI and without DDMAI before and after being soaked in water. Resin system
Properties S. mutans colonies before being soaked (Log CFU/mm2 × 100)
Control 5%
8.66 ± 0.50a, * 7.03 ± 0.23b *
Lower case letters indicate statistical differences within a column (Tukey’s test, p = 0.05). Indicate statistical differences within a row (Tukey’s test, p = 0.05).
∗
S. mutans colonies after being soaked 1 day (Log CFU/mm2 × 102 ) 8.69 ± 0.14a, * 6.84 ± 1.04b *
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[54]. However, S. mutans has a central role in the initiation of dental caries on enamel and root surfaces [55,56]. In our previous study, we found that the minimum inhibition concentration of DDMAI against S. mutans was about 6.25 g/mL [44], so in this study, we added DDMAI into BisGMA/TEGDMA dental resin to see whether it could endow cured resin with antibacterial properties. The biofilm inhibition tests showed that resin with DDMAI had inhibitory effect on young S. mutans biofilm formation, but no inhibitory effect on mature, 24-h biofilm. One explanation to this result could be that the inhibitory effect was the result of unreacted DDMAI leaching out of the polymer, when the test time was short, there were sufficient DDMAI leaching out to show inhibitory effect, just as 5 wt.% DDMAI contained polymer having inhibitory effect on 6 h-young biofilm formation, when the test time become longer, the amount of release DDMAI became too low to show kill bacteria, just as 5 wt.% DDMAI contained polymer having no inhibitory effect on 24 hmature biofilm formation. In order to find out whether the lost of inhibitory effect was caused by this reason, specimens which had been presoaked in water 24 h were also studied in biofilm inhibition test. Fig. 9 and Table 3 revealed that the presoaked polymer specimens with DDMAI still had a significant inhibitory effect on the young biofilm, and there was no statistically significant difference on the inhibitory effect between presoaked and unsoaked specimens. Therefore, the loss of inhibitory effect on mature biofilm formation might not be attributed to the possibility mentioned above. Bactericide-immobilized materials often show the antibacterial effect only against the bacteria which comes into contact with the immobilized antibacterial molecules. At the beginning of biofilm formation, planktonic bacteria reversibly attach to the surface of the material [57], and bactericide immobilized on the surface could come in contact with bacteria and kill them, like resin with DDMAI had an inhibitory effect on young biofilm in this work. When the biofilm maturates, it becomes thicker and the number of bacteria increases and they will be embedded in a matrix of polymers of bacterial and salivary origin [53]. In such biofilm only antimicrobial agents able to penetrate the biofilm will be effective. DDMAI appeared not to be effective when the biofilm is thick. However, it must be kept in mind that in conditions of normal oral hygiene the in vivo biofilm resembles more young biofilm than mature biofilm. Polymer with DDMAI has some radiopacity and antibacterial activity although the effect was not very high. However, the concentration of DDMAI was low because of its low miscibility with Bis-GMA/TEGDMA resin system. Further studies are needed to optimize the concentration of DDMAI to make dental resin with adequate radiopacity and antibacterial activity. Of course, the influences of DDMAI on chemical and physical properties, and biological behavior of dental resin also need to be carefully evaluated.
5.
Conclusion
A quaternary ammonium contained methacrylate monomer DDMAI was used and added into Bis-GMA/TEGDMA resin system as radiopaque and antibacterial agent. There was
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no adverse impact on monomer conversion and flexural strength when incorporating 3 w% and 5 wt.% DDMAI into Bis-GMA/TEGDMA system. Polymer specimens with DDMAI showed better radiopacity than polymer without DDMAI, and the radiopacity increased with increasing the amount of DDMAI in polymer. Only polymer with 5 wt.% DDMAI had inhibition effect on young S. mutans biofilm formation.
Acknowledgments We would like to greatly thank biomedical research technician Oona Hällfors for her help in biofilm inhibition testing. The study belongs to the BioCity Turku Biomaterials Research Program.
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Available online at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Incorporation of an antibacterial and radiopaque monomer in to dental resin system Jingwei He a,b,c,∗ , Eva Söderling d , Lippo V.J. Lassila a,b , Pekka K. Vallittu a,b a
Department of Biomaterials Science, Institute of Dentistry and Biocity Turku Biomaterial Research Program, University of Turku, Turku 20520, Finland b Turku Clinical Biomaterials Centre – TCBC, University of Turku, Turku 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
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. The purpose of this study was to prepare antibacterial and radiopaque dental
Received 8 December 2011
resin through adding one polymerizable quaternary ammonium compound with iodine
Received in revised form
as counter anion into Bis-GMA/TEGDMA (50/50, wt/wt) dental resin system, and evaluate
5 April 2012
the degree of monomer conversion, mechanical properties, radiopacity, and antibacterial
Accepted 16 April 2012
effectiveness of the polymer. Methods. 2-Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) (3 wt.% and 5 wt.%) was added into a Bis-GMA/TEGDMA (50/50, wt/wt) resin system with CQ
Keywords:
(0.7 wt.%) and DMAEMA (0.7 wt.%) as photoinitiation system. Degree of monomer conversion
Dental materials
(DC) was determined by FT-IR analysis. The flexural strength (FS) and flexural modulus (FM)
Mechanical properties
of the polymer were measured using a three-point bending set up. Radiographs were taken
Antibacterial effectiveness
to determine the radiopacity of the polymer, and aluminum step-wedge (0.5–4 mm) was used
Radiopacity
as calibration standard. A single-species biofilm model with Streptococcus mutans as the tests organism was used to evaluate the antibacterial property of the polymer. Bis-GMA/TEGDMA without DDMAI was used as control material in all of the tests. Results. ANOVA analysis revealed that there was no significant difference in DC between polymer with and without DDMAI (p > 0.05). Polymer with 3 wt.% DDMAI had higher FS than the control material (p < 0.05), while polymer with 5 wt.% DDMAI had comparable FS with the control material (p > 0.05). Though average FM of control material was lower than that of 3 wt.% DDMAI containing polymer and higher than 5 wt.% DDMAI containing polymer, but the differences were statistically insignificant. By increasing the quantity of DDMAI, the radiopacity of the polymer increased. Of the three formulations, the polymer with 5 wt.% DDMAI inhibited biofilm the formation of the clinically relevant young biofilm. Significance. Incorporation of DDMAI into resin system could endow it with radiopacity and antibacterial effectiveness, and these two properties seem to be improved with increasing the quantities of DDMAI. © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ 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). 0109-5641/$ – see front matter © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dental.2012.04.026
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1.
Introduction
Since methyl methacrylate was firstly used in tooth restoration in 1937, methacrylate monomers having good biocompatibility and adhesive property have been extensively used as dental resin-based materials, e.g., denture base materials [1–3], endodontic sealers [4–6], direct-filling restorative materials [7–10], and dental bonding agents [11,12]. The most commonly used methacrylate monomers in commercial dental resin-based materials are methyl methacrylate (MMA), 2,2-bis[4(2-hydroxy-3-methacryloyloxypropyl)-phenyl]propane (Bis-GMA), 1,6-bis-[2-methacryloyloxyethoxycarbonylamino](UDMA), and tri-ethyleneglycol 2,4,4-trimethylhexane dimethacrylate (TEGDMA) [13]. By free radical polymerization of these monomers, a three dimensional network is formed, and the selection of the monomers influences the properties of dental resin-based materials directly. In order to improve properties of dental resin-based materials, several new methacrylate monomers were introduced into dental resin with the aim of reducing polymerization shrinkage [14–18], decreasing water uptake and solubility [19–21], and improving mechanical properties [22–24]. Besides these properties, dental resin-based materials also need to be radiopaque and antibacterial. However, acrylic polymers formed by commonly methacrylate monomers do not have radiopacity and antibacterial activity intrinsically, so some radiopacifying fillers, e.g., BaO, BaSO4 , TiO2 , SrO, or ZrO2 [25–27], and antibacterial agents, e.g., chlorhexidine, cetylpyridinium chloride, or titania nanoparticles [28–32], are incorporated into dental resin by physical blending. Unfortunately, all the additives mentioned above also bring some unwanted problems. For example, incorporation of radiopaque fillers into dental resin has some adverse effects on the physical and chemical properties of composites materials [33–35] and excessive fillers could cause loss of dimensional stability of the composites materials [36]. The addition of antibacterial agent into dental materials could reduce its mechanical properties even at a small concentration [37], and the releasing agent may exert toxic effects as well as cause short-term antibacterial effectiveness of the material [38]. It was reported that addition of methacrylate compounds containing radioopaque agent or antibacterial agent could also render dental materials relevant function. Davy et al. [39] synthesized iodinate methacrylate and copolymerized it with MMA for the application as X-ray opaque denture base material. Imazato et al. synthesized methacryloyloxydodecyl pyrimidinium bromide (MDPB) and used it as an antibacterial monomer in several kinds of dental materials to give longterm antibacterial effects [40–43]. Though many methacrylate compounds have been taken for the aim of radiopacity or antibacterial activity, there is no report on one methacrylate compound with both of these two functions so far. In this study, a methacrylate monomer containing quaternary ammonium structure, 2-dimethyl-2-dodecyl-1methacryloxyethyl ammonium iodine (DDMAI, structure is shown in Fig. 1), whose minimum inhibition concentration against Streptococcus mutans is 6.25 g/mL [44], was used in
Fig. 1 – Structure of DDMAI.
dental resin as antibacterial agent, because the counter anion of this reactive quaternary ammonium is iodine, so it may also has radiocontrast property. Therefore, radiopacity and antibacterial activity of dental resin with this monomer were studied, and influences of this monomer on monomer conversion and mechanical properties were also examined in this work.
2.
Materials and methods
2.1.
Materials
2-Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) was synthesized in our lab. 2,2-Bis[4(2-hydroxy-3-methacryloyloxypropyl)-phenyl]propane (Bis-GMA) was purchased from Esstech Inc. (Essington, PA, USA), tri-ethyleneglycol dimethacrylate (TEGDMA), camphoroquinone (CQ), and N,N -dimethyl aminoethyl methacrylate (DMAEMA) were purchased from Sigma–Aldrich Co. (St Luois, MO, USA), all of the reagents were used without purification.
2.2.
Methods
The formulation of every dental resin used in this experiment is shown in Table 1. All of them were well blended to obtain a homogenous mixture, and stored at darkness before used.
2.2.1.
Monomer (double bond) conversion
The degree of monomer conversion (DC) was determined by using an FTIR spectrometer (Spectrum One, Perkin-Elmer, Waltham, MA, USA) with an attenuated total reflectance (ATR) accessory. The FTIR 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 60 s with a visible light-curing unit (Curing Light 2500, = 400–520 nm, I ≈ 550 mW cm−2 , 3 M Co.,
Table 1 – Formulation of every experimental resin system. System
Control 3% 5%
Content of every monomer (g) Bis-GMA
TEGDMA
DDMAI
CQ
DMAEMA
49.3 47.8 46.8
49.3 47.8 46.8
0 3 5
0.7 0.7 0.7
0.7 0.7 0.7
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where Gd , Gb , and Ga are the gray value of disc, background and aluminum at the same thickness, respectively.
2.2.4.
Fig. 2 – The absorption peaks at 1636 cm−1 and 1608 cm−1 varied before and after irradiation.
St Paul, MN, USA). The sample was scanned for its FTIR spectrum every 5 s until 15 min after the beginning of irradiation. To determined the percentage of reacted double bonds, the absorbance intensities of the methacrylate C C absorbance peak at 1636 cm−1 , which were decreased after being irradiated (as shown in Fig. 2), 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 every irradiation time was calculated by using the equation
DC(t) =
1 − (Ac c /Aph )t (Ac c /Aph )0
× 100%
where AC C and APh are the absorbance intensity of methacrylate C C at 1636 cm−1 and phenyl ring at 1608 cm−1 , respectively; (AC C /APh )0 and (AC C /APh )t are the normalized absorbance of functional group at the radiation time 0 and t, respectively; DC(t) is the conversion of methacrylate C C as a function of radiation time.
2.2.2.
Three point bending test
Eight specimens were prepared for every sample formulation (size 2 mm × 2 mm × 25 mm). Three point bending test (span 20 mm) was carried out to evaluate the flexural strength and modulus according ISO 10477:92 standard with a material testing machine (model LRX, Lloyd Instrument Ltd., Fareham, England), at a cross-head speed of 1.00 mm/min.
2.2.3.
Radiopacity
Five discs (thickness from 1 mm to 5 mm) were prepared for every resin formulations, and irradiated with X-ray (63 kV, 8 mA, 0.008 s) to get a radiograph. The relative radiopacity was determined by comparison with the opacity exhibited 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 radiopacity (RXO) of disc was calculated by using the equation RXO =
[Gd − Gb ] × 100% [Ga − Gb ]
Biofilm inhibition test
Three discs (2 mm thick and 8 mm in diameter) for every resin formulation were prepared for biofilm inhibition test. Every disc was polished with 4000 grit (FEPA) grinding paper. Biofilm inhibition test of the discs, which were soaked in distilled water for 24 h at room temperature, was also taken to clarify whether the loss of inhibitory effect to mature biofilm was due to none leachable antimicrobial agent left in polymer after being soaked. The inhibition of biofilm formation reflecting plaque accumulation was tested by a modification of the method originally presented by Ebi et al. [45]. The microorganism we used was the type strain S. mutans Ingbritt. It was first grown overnight in Brain Heart Infusion medium (BHI; Difco, MI, 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 cell culture plates. The plates were incubated anaerobically (90% N2 , 5% CO2 , 5% H2 ) at +37 ◦ C for 6 h (young biofilm) and 24 h (mature biofilm). The biofilms were collected with microbrushes (QuickStick, Dentsolv AB, Sweden) from the disc surface exposed to the medium to test tubes containing Tryptic Soy Broth (Difco). The tubes were vortexed and mildly sonicated and then serially diluted for plate culturing of S. mutans. The plates were grown for 3 days anaerobically (80% N2 , 10% CO2 , 10% H2 ) at +37 ◦ C on Mitis salivarius agar (Difco), 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 [46,47].
2.2.5.
Statistical analysis
The results of double bond conversion, three point bending test, and biofilm inhibition test were statistically analyzed with analysis of variance (ANOVA) at the p < 0.05 significance level. Subsequent multiple comparisons were conducted using Tukey’s post hoc analysis.
3.
Results
It could be seen from Fig. 3 that polymerization behavior of resin with and without DDMAI was almost the same, and the final DC of each polymer was quite similar (Table 2). The three point bonding test showed that FS of polymer with 3 wt.% DDMAI was higher (p < 0.05) than that of control material (Fig. 4). When the content of DDMAI increased from 3 wt.% to 5 wt.%, FS of polymer was decreased (p < 0.05), and the value was nearly the same as that of control material (Table 2). FM of 3 wt.% DDMAI containing polymer was higher than that of control material (Fig. 5), where there was no significant difference between them (p > 0.05), and FM of 5 wt.% DDMAI containing polymer was lower than that of control material (Fig. 5), where there was also on significant difference between them (p > 0.05).
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Table 2 – Double bond conversion, flexural strength, flexural modulus, relative X-ray opacity, and S. mutans colonies in young biofilm on the surface of resin with and without DDMAI. Resin system
Properties DC (%)
Control 3% 5%
FS (MPa)
72.1 ± 0.2 72.5 ± 0.4a 72.0 ± 0.4a a
FM (GPa)
93.2 ± 3.6 105.0 ± 5.6b 93.6 ± 4.4a a
2.05 ± 0.16 2.22 ± 0.13a 1.91 ± 0.19b
a,b
RXO (%)
S. mutans colonies (Log CFU/mm2 × 102 )
14.2 22.7 27.8
9.85 ± 0.41a 9.32 ± 0.32a 8.61 ± 0.66b
Lower case letters indicate statistical differences within a column (Tukey’s test, p = 0.05).
Fig. 5 – Flexural modulus of polymers with and without DDMAI. Fig. 3 – Double bond conversion of resins with and without DDMAI.
Fig. 4 – Flexural strength of polymers with and without DDMAI.
The radiograph revealed that radiopacity increased with increasing thickness of polymer and amount of DDMAI in polymer (Figs. 6 and 7). Using the aluminum as radiopacity standard, RXO of polymer with DDMAI was nearly the two times higher than that of control material (Table 2). Fig. 8 shows the number of viable bacteria in the 6-h young biofilm formed on the surface of polymer with and without DDMAI. The number of bacteria on the surface of polymer with 5 wt.% DDMAI was less (p < 0.05) than that of control material. Meanwhile, though the average amount of colonies on the surface of polymer with 3 wt.% DDMAI was less than that of control material and there was no significant difference between them (p > 0.05). Soaking the polymer specimen for 1
Fig. 6 – Radiopacity of polymers (thickness from 1 mm to 5 mm) with and without DDMAI, a aluminum steps (thickness from 0.5 mm to 4 mm) as reference.
day in water did not significantly change the inhibition effect of the polymer containing 5 wt.% DDMAI (Fig. 9).
4.
Discussion
Radiopacity and antibacterial activity are two necessary properties of dental materials, the former render it possible to evaluate quality of treatment by nondestructive techniques [48], and the latter could reduce the possibility of secondary caries occurring [49]. The monomer DDMAI used in this work is a quaternary ammonium compound with iodine as counter anion. Quaternary ammonium compounds are well known
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Fig. 7 – Radiopacity of polymers (thickness from 1 mm to 5 mm) with and without DDMAI.
Fig. 8 – Young biofilm inhibition test against Streptococcus mutans of polymers with and without DDMAI.
effective antibacterial agents, which have already been used in many fields [50,51]. Iodine is an atom with high electronic density, which makes it quite radioopaque, compound with iodine was widely studied as radiocontrast media in modern medicine [52,53]. Therefore, DDMAI could be incorporated into dental resin both as antibacterial agent and radiopacity agent, moreover, the methacrylate group in its structure could immobilize the functional group into polymer backbone and make the acrylic polymer with long-term antibacterial activity and radiopacity [39,45]. However, we found that the miscibility of DDMAI with Bis-GMA/TEGDMA (50 wt/50 wt) system was not well enough, when concentration of DDMAI was more than 5 wt.%, there were some DDMAI could not be miscible with
Fig. 9 – Young biofilm inhibition test against Streptococcus mutans of polymers with and without being soaked in water.
Bis-GMA/TEGDMA (50 wt/50 wt) system. Therefore, we only chose 3 wt.%, and 5 wt.% of DDMAI here. From the results of monomer conversion and three point bending test, it could be seen that adding 3 wt.% and 5 wt.% of DDMAI into Bis-GMA/TEGDMA system had no adverse impact on DC, FS, and FM, even that polymer with 3 wt.% DDMAI had higher FM than control material. It is the beneficial aspect of DDMAI when compared with other antibacterial agent like chlorhexidine, because in some other research, adding even 1 wt.% of antibacterial agent like chlorhexidine into dental resin could reduce mechanical properties [37]. Figs. 6 and 7 show that incorporating DDMAI could increase radiopacity of dental polymer, and the radiopacity increased with increasing thickness of polymer and amount of DDMAI in the polymer. An increase of 100% in RXO was obtained when adding only 5 wt.% DDMAI into Bis-GMA/TEGDMA resin system (Table 2). Though the radiopacity of resin with 3 wt.% and 5 wt.% DDMAI is not necessarily adequate to be used in clinically, it still proves that DDMAI has potential to be used as radiopacity agent in dental and medical materials, if appropriate resin system could be found to be miscible with more DDMAI. Dental plaque is a complex microbial community that develops on tooth surface, embedded in a matrix of polymers [54]. This biofilm also covers dental restorative materials [46]. The present ecological plaque hypothesis emphasizes that non-mutans bacteria may be the key microorganisms responsible for maintaining dynamic stability on the tooth surface
Table 3 – S. mutans colonies in young biofilm on the surface of resin with 5% DDMAI and without DDMAI before and after being soaked in water. Resin system
Properties S. mutans colonies before being soaked (Log CFU/mm2 × 100)
Control 5%
8.66 ± 0.50a, * 7.03 ± 0.23b *
Lower case letters indicate statistical differences within a column (Tukey’s test, p = 0.05). Indicate statistical differences within a row (Tukey’s test, p = 0.05).
∗
S. mutans colonies after being soaked 1 day (Log CFU/mm2 × 102 ) 8.69 ± 0.14a, * 6.84 ± 1.04b *
d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) e110–e117
[54]. However, S. mutans has a central role in the initiation of dental caries on enamel and root surfaces [55,56]. In our previous study, we found that the minimum inhibition concentration of DDMAI against S. mutans was about 6.25 g/mL [44], so in this study, we added DDMAI into BisGMA/TEGDMA dental resin to see whether it could endow cured resin with antibacterial properties. The biofilm inhibition tests showed that resin with DDMAI had inhibitory effect on young S. mutans biofilm formation, but no inhibitory effect on mature, 24-h biofilm. One explanation to this result could be that the inhibitory effect was the result of unreacted DDMAI leaching out of the polymer, when the test time was short, there were sufficient DDMAI leaching out to show inhibitory effect, just as 5 wt.% DDMAI contained polymer having inhibitory effect on 6 h-young biofilm formation, when the test time become longer, the amount of release DDMAI became too low to show kill bacteria, just as 5 wt.% DDMAI contained polymer having no inhibitory effect on 24 hmature biofilm formation. In order to find out whether the lost of inhibitory effect was caused by this reason, specimens which had been presoaked in water 24 h were also studied in biofilm inhibition test. Fig. 9 and Table 3 revealed that the presoaked polymer specimens with DDMAI still had a significant inhibitory effect on the young biofilm, and there was no statistically significant difference on the inhibitory effect between presoaked and unsoaked specimens. Therefore, the loss of inhibitory effect on mature biofilm formation might not be attributed to the possibility mentioned above. Bactericide-immobilized materials often show the antibacterial effect only against the bacteria which comes into contact with the immobilized antibacterial molecules. At the beginning of biofilm formation, planktonic bacteria reversibly attach to the surface of the material [57], and bactericide immobilized on the surface could come in contact with bacteria and kill them, like resin with DDMAI had an inhibitory effect on young biofilm in this work. When the biofilm maturates, it becomes thicker and the number of bacteria increases and they will be embedded in a matrix of polymers of bacterial and salivary origin [53]. In such biofilm only antimicrobial agents able to penetrate the biofilm will be effective. DDMAI appeared not to be effective when the biofilm is thick. However, it must be kept in mind that in conditions of normal oral hygiene the in vivo biofilm resembles more young biofilm than mature biofilm. Polymer with DDMAI has some radiopacity and antibacterial activity although the effect was not very high. However, the concentration of DDMAI was low because of its low miscibility with Bis-GMA/TEGDMA resin system. Further studies are needed to optimize the concentration of DDMAI to make dental resin with adequate radiopacity and antibacterial activity. Of course, the influences of DDMAI on chemical and physical properties, and biological behavior of dental resin also need to be carefully evaluated.
5.
Conclusion
A quaternary ammonium contained methacrylate monomer DDMAI was used and added into Bis-GMA/TEGDMA resin system as radiopaque and antibacterial agent. There was
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no adverse impact on monomer conversion and flexural strength when incorporating 3 w% and 5 wt.% DDMAI into Bis-GMA/TEGDMA system. Polymer specimens with DDMAI showed better radiopacity than polymer without DDMAI, and the radiopacity increased with increasing the amount of DDMAI in polymer. Only polymer with 5 wt.% DDMAI had inhibition effect on young S. mutans biofilm formation.
Acknowledgments We would like to greatly thank biomedical research technician Oona Hällfors for her help in biofilm inhibition testing. The study belongs to the BioCity Turku Biomaterials Research Program.
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