- Email: [email protected]
Development of an antimicrobial resin—A pilot study
d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Development of an antimicrobial resin—A pilot study Catherine Fan, Lianrui Chu, H. Ralph Rawls, Barry K. Norling, Hector L. Cardenas, Kyumin Whang ∗ Division of Research, Department of Comprehensive Dentistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX, United States
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. To demonstrate that silver nanoparticles (AgNPs) could be synthesized in situ in
Received 24 June 2010
acrylic dental resins.
Accepted 1 November 2010
Methods. Light-cure (LC; bisphenol A glycidyl methacrylate, tetraethyleneglycol dimethacrylate, bisphenol A ethoxylate dimethacrylate blend) and chemical-cure systems (CC; orthodontic denture resin) were used to synthesize AgNPs using different concentrations of
Keywords:
Ag benzoate (AgBz).
Silver nanoparticles
Results. Rockwell hardness for LC resins showed that resins could be cured with up to 0.15%
Antimicrobial
AgBz, while the hardness of CC resins were unaffected in the concentrations tested. UV–Vis
Dental materials
spectroscopy and transmission electron microscopy confirmed the presence of AgNPs in
Controlled release
both LC and CC resins. Generally, CC resins had better distribution of and much smaller
Orthopedic
AgNPs as compared to LC resins overall. In some samples, especially in LC resins, nanoclus-
Craniofacial
ters were visible. An in vitro release study over four-weeks showed that CC resins released the most Ag+ ions, with release detected in all samples. However, LC resins only released Ag+ ions when AgBz concentration was greater than 0.1% (w/w). AgNP-loaded CC resins made with 0.2 and 0.5% (w/w) AgBz were tested for antibacterial activity in vitro against Streptococcus mutans, and results showed 52.4% and a 97.5% bacterial inhibition, respectively. Further work is now warranted to test mechanical properties and to optimize the initiator system to produce commercially useful dental and medical resins. Significance. Success in this work could lead to a series of antimicrobial medical and dental biomaterials that can prevent secondary caries and infection of implants. © 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
There is a need for effective broad-spectrum antimicrobial resin materials in dentistry and medicine. In dentistry, there is a 50–60% rate of dental caries after restorative treatment [1,2] or in areas around orthodontic bracket bonding agents where effective tooth brushing is difficult [3]. In addition, 50%
∗
of patients who wear complete or partial dentures experience problems with stomatitis [4]. In medicine, there is a 1–3% rate of infection after orthopedic surgery [5,6] and a 5% rate of infection when poly(methyl methacrylate) (PMMA) was used in cranioplasty [7] despite attempts to create a sterile environment. Unfortunately, current standards of treatment such as the use of antimicrobial mouthwashes, proper tooth-brushing technique, and the use of prophylactic systemic antibiotics
Corresponding author at: Division of Research, Department of Comprehensive Dentistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., MSC 7914, San Antonio, TX 78229-3900, United States. Tel.: +1 210 567 3674; fax: +1 210 567 3669. E-mail address: [email protected] (K. Whang). 0109-5641/$ – see front matter © 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2010.11.008
d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
have limited success or side-effects due to problems with patient compliance and the development of antibiotic resistant strains of bacteria. Thus, a broad-spectrum antimicrobial resin is needed. A widely investigated, effective, biocompatible, broadspectrum antimicrobial agent is silver (Ag). Ag ions have been reported to inactivate important enzymes and affect the replication mechanism of the DNA in bacteria [8]. Ag in nanoparticulate form (AgNP), which would release Ag ions more effectively and therefore have better bactericidal activity due to its high surface area-to-volume ratio [9], have been shown to attach to the outer membrane and affect permeability as well as induce structural changes in the cell—ultimately leading to cell death. In addition, Ag does not cause resistant bacterial strains to develop. Thus, Ag has already been used to provide household commodities with antibacterial effects, and many commercial products for kitchenware, washing machines, clothes, toiletries, stationery, etc. are available [10,11]. For dental applications, various forms of Ag or Ag-ion containing fillers have been used such as Ag ion-implanted SiO2 [12], Ag-containing silica glass [13], Ag-zeolite [14], Agapatite [15], and Ag-supported zirconium phosphate [16]. All of these approaches have demonstrated antibacterial activity in vitro. However, Ag-zeolite and Ag-apatite decreased mechanical properties at loadings necessary for antibacterial effects [15], and approaches that do not release Ag ions, such as Agsupported zirconium phosphate, only kill bacteria that come in contact with the surface and any protein adsorption on the surface would reduce the antibacterial effect. Furthermore, there are increasing concerns about potential side effects of directly using nanoparticles in vivo. Another major problem for the use of AgNPs is the difficulty in dispersing and homogeneous incorporation into the resin. AgNPs have been synthesized in many different ways such as chemical reduction of Ag+ ions in aqueous solutions with or without stabilizing agents, thermal decomposition in organic solvents, chemical and photoreduction in reverse micelles, and radiation and chemical reduction. However these methods invariably fail to provide Ag dispersions and that affects proper incorporation, mechanical properties, and release kinetics [17]. Thus, for effective dental and medical application, a more effective method of incorporating AgNP into acrylic resins and delivering Ag+ ions is needed. Recently, Kumar et al. used the natural oxidative drying process of oils to synthesize metal nanoparticles in the oil media without any reducing or stabilizing agents. The AgNPs were synthesized in situ as the paint dried and cured, thus eliminating the problem of non-uniform NP dispersion as well as the need for harsh chemicals. These AgNP paints showed excellent antimicrobial properties against S. aureus and E. coli [9]. We have modified this approach to generate AgNP in situ in polymethyl methacrylate and bisphenol A glycidyl methacrylate (Bis-GMA)-based resins for dental and medical applications. The goal of this work is to demonstrate the concept and to determine if this modified method can produce AgNP in situ in these resins, release Ag+ ions, and provide effective antimicrobial activity.
2.
323
Materials and methods
AgNPs were formed in situ in dental resins using Ag benzoate (AgBz), and the effect of AgBz concentration and curing method (light-curing vs. chemical-curing) on the degree of cure, nanoparticle size and formation, in vitro release of Ag ions and in vitro antibacterial activity were determined. Transmission electron microscopy (TEM) was used to observe the AgNPs, UV/Vis spectroscopy to further determine the presence of AgNPs, clusters of AgNPs and release of Ag+ ions, and Rockwell15T hardness to measure degree of cure. In addition, the antibacterial activity of these novel resins was assessed in vitro with Streptococcus mutans.
2.1.
Synthesis of AgNP-loaded resins
Light-cured (LC) resins were made by blending varying concentrations (0, 0.002, 0.02, 0.1, 0.15 and 0.2%, w/w, of total monomer) of AgBz (Sigma–Aldrich) in dimethylaminoethyl methacrylate (DMAEMA; 2%, w/w, of total monomer; Sigma–Aldrich), camphorquinone (CQ: 1%, w/w, of monomer blend; Sigma–Aldrich), and GTE (a blend of 37.5% bisphenol A glycidyl methacrylate (Bis-GMA), 25.0% tetraethyleneglycol dimethacrylate (TEGMA) and 37.5% bisphenol A ethoxylate dimethacrylate (Bis-EMA; EssTech)). The blend was then poured into a mold (3/8 diameter × 1/16 thick) between two glass slides and light-cured on each side for 40 s using a Demetron Optilux 401 curing light with the light guide held against the glass slide. Chemically cured (CC) specimens were made by dissolving different concentrations (0, 0.002, 0.02, 0.2 and 0.5%) of AgBz into 2% DMAEMA and then into liquid orthodontic monomer (Dentsply), and subsequently mixing with PMMA powder, as described in the manufacturer’s guidelines. This PMMAbased resin blend was immediately poured into molds pressed between two glass slides and allowed to chemically cure for 60 min or until tested.
2.2.
Rockwell hardness
The Rockwell15T hardness of cured specimens was measured with a 15T 1/16 ballpoint indenter with a 15 kg force. Three measurements were made on different areas of each of the specimens to verify that they were cured evenly.
2.3.
Transmission electron microscopy (TEM)
Cured specimens were cut into 100 nm thin slices using a microtome, placed on copper grids, and observed using TEM (Jeol JEM-1230 transmission electron microscope).
2.4.
Ultraviolet–visible (UV/Vis) spectroscopy
Specimens were also cured in plastic cuvettes and UV/Vis spectra from 200 to 800 nm were taken (SmartSpec 3000 spectrophotometer, Bio-Rad) using the control (0% AgBz) as a blank. For CC specimens, an uncured control specimen with 0% AgBz was used as the blank for 0 min specimens, and a 60 min cured control specimen with 0% AgBz was used as the blank for
324
d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
60 min specimens to ensure that any absorbance due to the resin was properly accounted for.
2.5.
In vitro Ag release study
Specimens were placed into glass vials with 5 mL of sterile deionized water (n = 5). At certain intervals (1 day, 4 days, 1 week, 2 weeks, 4 weeks) 1 mL of the water was extracted and its UV/Vis absorption was measured from 200 to 800 nm. The control specimens contained 0% AgBz. The UV/Vis scan of the control was taken using sterile deionized water as the blank while the readings for all the other specimens were taken using the control as the blank. At each time period the water was replaced to maintain sink conditions.
2.6. Inhibitory effects of AgNP-loaded resin discs on the growth of S. mutans Based on the in vitro release data and a pilot growth inhibition assay showing minimal effect from the 0.2% AgBz LC specimens (data not shown), the growth inhibition assay was only done with CC resins made with 0 (negative control), 0.2 and 0.5% AgBz. S. mutans (TACC 25175) was grown on TSBY (Trypticase Soy Broth with 0.5% yeast extract) agar plates in an anaerobic chamber with a mixed gas (N2 = 85%, H2 = 5, and CO2 = 10%) and specimens were placed on the bacteriacontaining agar and anaerobically inoculated at 37 ◦ C for 5 days to determine their efficacy in inhibiting bacterial growth by identifying zones of inhibition. To estimate colony formation, 20 l of different concentrations of the bacteria (about 10−1 , 10−2 , 10−3 , 10−4 , 10−5 and 10−6 /ml) were homogenously spread onto each area of the surface on the gelled TSBY agar plates using sterile spreaders. The colony formation was determined by counting colonies from suitable dilutions for each specimen.
2.7.
Statistics
Rockwell hardness, in vitro release and % bacterial inhibition data were compared between groups using ANOVA with
Fig. 1 – Photograph of LC and CC resins with different AgBz concentrations.
Newman–Keuls’s post hoc test. For release data, comparisons were made at each time point.
3.
Results
3.1.
Synthesis of AgNP-loaded resins
As the AgBz concentration increased, it became more difficult to cure the specimens. The highest AgBz concentration that could be incorporated and light-cured was 0.15% (w/w). However, for chemically cured resins, specimens with as much as 0.6% (w/w) AgBz were curable. Fig. 1 shows that as the AgBz concentration increased, the amber color of the resins became darker due to the plasmon effect of the AgNPs [18]. Also the CC resins were lighter in color than the light-cured resins at the same AgBz concentration.
3.2.
Rockwell hardness
Fig. 2 shows that Rockwell hardness of LC resins decreased significantly (p < 0.05) when AgBz reached concentrations of 0.1% (w/w) and above. Above 0.2%, the resins did not cure well. However, the hardness of CC resins did not change significantly
Fig. 2 – Rockwell15T hardness of LC and CC resins made with different concentrations of AgBz.
d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
325
Fig. 3 – TEM of LC (a–f) and CC (g–j) resins with different AgBz concentrations. Bar is 100 nm. (a) and (f) were taken at 80,000× and the rest were taken at 300,000×. (a) LC with 0% negative control, (b) LC with 0.002% AgBz, (c) LC with 0.02% AgBz, (d) LC with 0.1% AgBz, (e) LC with 0.15% AgBz, (f) LC with 0.2% AgBz, (g) CC with 0.002% AgBz, (h) CC with 0.02% AgBz, (i) CC with 0.2% AgBz and (j) CC with 0.5% AgBz. Specimens containing greater than 0.2% AgBz could not be fully cured.
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d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
UV/Vis Spectra of Ag-Loaded Resins 1.6 LC 0.002% AgBz 1.4
LC 0.02% AgBz LC 0.1% AgBz
1.2
LC 0.15% AgBz CC 0.002% AgBz
Absorbance
1
CC 0.02% AgBz CC 0.2% AgBz
0.8
CC 0.5% AgBz
0.6 0.4 0.2
800
780
760
740
720
700
680
660
640
620
600
580
560
540
520
500
480
460
440
420
400
380
360
340
320
300
280
260
240
220
200
0 -0.2 -0.4
Wavelength (nm)
Fig. 4 – UV/Vis spectra of LC and CC resins cured with different concentrations of AgBz. Note the characteristic peak of AgNPs at 400 nm and the broader peak beginning at 500 nm that may be due to NP clusters.
from the 0% control, but this may have been mainly due to the large standard deviations caused by pores that are formed in the mixing process of the different components of the chemical cure resin. Still, the CC resins were significantly harder than the LC resins above 0.2% AgBz.
3.3.
Transmission electron microscopy
TEM images (Fig. 3b–f) show that there are sparse individual particles and large nanoparticle clusters in the LC resins. The higher the AgBz concentration, the greater the number of nanoparticles and the larger the nanoparticle clusters. Some clusters were as large as 50–70 nm. Nanoparticles were generally between 10 and 20 nm, but with the 0.2% specimens, there were also many smaller particles.
With the CC resins however there were distinctly more nanoparticles and they were distributed more widely (Fig. 3g–j). There were not as many nanoparticle clusters. The individual particles from all specimens usually ranged from 2 to 18 nm in size but it was apparent that the CC specimens had smaller nanoparticles. This might explain the lighter amber color of the AgNP-loaded CC resins. Finally, as the AgBz concentration increased, so did the number of visible nanoparticles.
3.4.
Ultraviolet–visible spectroscopy
It is known that spherical AgNPs have a UV absorption band with a peak centered around 400 nm [19]. All LC specimens showed a peak centered around 400 nm (Fig. 4). However, there
Release of Ag ions from Ag-loaded resins Cumulative UV/Vis Absorbance @ 223 nm
4
LC 0.01% AgBz LC 0.02% AgBz LC 0.1% AgBz CC 0.002% AgBz CC 0.02% AgBz CC 0.2% AgBz
3.5 3 2.5 2 1.5 1 0.5 0 0
7
14
21
28
-0.5
Time (days)
Fig. 5 – UV absorbance at 223 nm vs. time showing cumulative Ag ion release from LC and CC resins made with different concentrations of AgBz.
d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
327
was also a broad peak at 500 nm and tailing out to 800 nm that consistently broadened as AgBz concentration was increased. This may be due to the formation of Ag nanoclusters with a range of sizes [20]. The UV–Vis spectra for the CC specimens show a band peak at about 450 nm (Fig. 4). As the AgBz concentration increased, a broad band with a peak beginning at around 500 nm became visible. This band also became visible after 24 h for most of the specimens as more NPs were formed. However, unlike the two distinct bands in the LC specimens, the bands in the CC specimens were very diffuse and overlapped, producing a very broad peak at about 450 nm.
3.5.
In vitro Ag release study
Fig. 5 shows the UV/Vis absorbance measuring the released Ag ions in water versus time. Bands characteristic of Ag+ ions peak at about 223 nm [21]. For LC specimens, Ag+ ion release was only measured with resins made with 0.1% AgBz. However for CC specimens, Ag ion release was detected for every concentration, and as the AgBz concentration increased, the peak became more intense. However, over time Ag ion release gradually decreased.
3.6. Inhibitory effects of AgNP-loaded resin discs on the growth of S. mutans Fig. 6a shows the presence of the resin disks on agar and Fig. 6b shows the agar with the discs removed after 5 days of incubation at 37 ◦ C. A clear zone of inhibition is visible in A1 and B1 where resins with 0.5% AgBz were used. A2 and B2 show a vague zone for resins made with 0.2% AgBz, and A3 and B3 show no zones of inhibition when AgBz was not used. This correlates with Fig. 6c, which shows the percent inhibition of the growth of S. mutans. Resins made with 0.5% AgBz showed 97.5% inhibition and those made with 0.2% AgBz inhibited 52.4% of S. mutans growth as compared to 0% for negative controls. These results demonstrate that 0.5% silver containing resin inhibited almost all of the bacterial growth. Even resins containing only 0.2% silver were able to inhibit more than 50% of the bacterial growth.
4.
Discussion
An effective broad-spectrum antimicrobial resin for dental and medical use that does not promote antibiotic resistant strains of bacteria is needed. We have developed a method of synthesizing silver nanoparticles (AgNP) in situ in dental and medical resins. Hardness tests showed that in light cured (LC) specimens, as the concentration of silver benzoate (AgBz) increases the degree of cure decreases. This is probably due to Ag+ ions being reduced and generating atom clusters and nanoparticles during the curing, and thus being in competition with the free radical polymerization process. However, it is interesting that this did not seem to affect the degree of cure for chemically cured (CC) specimens to the same extent, and while the maximum concentration of AgBz with which LC resins could be cured was 0.2%, CC resins could be cured with 0.6%. It is also
Fig. 6 – Inhibitory effect of resin disk containing AgNP on the growth of Streptococcus mutans. Panel a shows the resin specimens and panel b shows the agar with the specimens removed and panel c shows the actual growth inhibition. (a1) Resin made with 0.5% AgBz. (a2) Resin made with 0.2% AgBz. (a3) Resin made with 0% AgBz (negative control). (b1) shows a transparent ring suggesting growth of S. mutans was inhibited. (b2) Shows a semi-transparent ring demonstrated partial inhibition. In contrast, (b3) shows normal bacterial growth. Resin containing 0.5% AgBz inhibited 97.5% and those made with 0.2% AgBz inhibited 52.4% of the growth of S. mutans as compared to the negative controls, respectively.
interesting that the color of LC resins were darker than CC resins for the same AgBz concentration, which may be due to the larger particle sizes that were seen with TEM. It appears that the slower CC process allows a greater number of AgNP nucleation sites to form, thereby generating more particles, a better dispersion of the particles, and smaller particle sizes. The UV–Vis spectra of the resins further supported the TEM data that indicated that the spherical particles are AgNPs, since the UV band at 400 nm is characteristic of spherical AgNPs. The narrower bands of the LC resins also support TEM observations that the particles are larger than those formed by CC resins. The broader bands that begin after 500 nm but are not as visible in the CC resins, also support the TEM data
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d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
in that there seems to be more clustering of the NPs in the LC specimens. The release study showed that continued release of Ag+ ions was only observed with 0.1% AgBz LC specimens, while CC specimens showed release with as low as 0.002% AgBz. The 0.1% LC specimens may have released Ag+ ions only because of a visible crack since 0.15% LC specimens did not release any Ag+ ions (data not shown). The release of Ag+ ions from CC specimens may be due to the smaller particle size and more homogeneous distribution of the NPs and/or the higher porosity that exists in CC resins as compared to LC resins. Only the 0.2% and 0.5% AgBz CC resins (with the most Ag+ ion release) were tested in the in vitro bacterial growth inhibition assay. A zone of inhibition of S. mutans is clearly visible for the 0.5% and barely perceptible for the 0.2% specimens. This correlated to 97.5% and 52.4% inhibition of S. mutans growth. In short, this pilot study demonstrated a novel process that generates AgNPs in situ using the resin’s own curing process to form antimicrobial resins for dental and medical use. Further studies are needed to investigate the effect of initiator and amine concentrations on maximum AgBz loading concentration, mechanical properties, in vitro release properties, and in vitro antibacterial effects. In addition, the effect of light curing vs. chemical curing and PMMA vs. BisGMA-based resin on Ag ion release needs to be further investigated. Success in this work could lead to a series of antimicrobial medical and dental biomaterials that can prevent secondary caries and implant-centered infection.
references
[1] Mjor IA, Toffenetti F. Secondary caries: a literature review with case reports. Quintessence Int 2000;31:165–79. [2] Wilson NH, Burke FJ, Mjor IA. Reasons for placement and replacement of restorations of direct restorative materials by a selected group of practitioners in the United Kingdom. Quintessence Int 1997;28:245–8. [3] Mjor IA. Glass-ionomer cement restorations and secondary caries: a preliminary report. Quintessence Int 1996;27:171–4. [4] Budtz-Jorgensen E. Oral mucosal lesions associated with the wearing of removable dentures. J Oral Pathol 1981;10:65–80. [5] Antti-Poika I, Josefsson G, Konttinen Y, Lidgren L, Santavirta S, Sanzen L. Hip arthroplasty infection. Current concepts. Acta Orthop Scand 1990;61:163–9.
[6] Harris WH, Sledge CB. Total hip and total knee replacement (2). N Engl J Med 1990;323:801–7. [7] Cho YR, Gosain AK. Biomaterials in craniofacial reconstruction. Clin Plast Surg 2004;31:377–85, v. [8] Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT, et al. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:2346–53. [9] Kumar A, Vemula PK, Ajayan PM, John G. Silver-nanoparticle-embedded antimicrobial paints based on vegetable oil. Nat Mater 2008;7:236–41. [10] Pyrek KM. Battling biofilm: surface science, antimicrobials help combat medical device-related infections. Infection Control Today; 2002. [11] Simpson K. Using silver to fight microbial attack. Plast Additiv Compound 2003;5:32–5. [12] Yamamoto K, Ohashi S, Aono M, Kokubo T, Yamada I, Yamauchi J. Antibacterial activity of silver ions implanted in SiO2 filler on oral streptococci. Dent Mater 1996;12: 227–9. [13] Kawashita M, Tsuneyama S, Miyaji F, Kokubo T, Kozuka H, Yamamoto K. Antibacterial silver-containing silica glass prepared by sol–gel method. Biomaterials 2000;21: 393–8. [14] Hotta M, Nakajima H, Yamamoto K, Aono M. Antibacterial temporary filling materials: the effect of adding various ratios of Ag–Zn–zeolite. J Oral Rehabil 1998;25:485–9. [15] Syafiuddin T, Hisamitsu H, Toko T, Igarashi T, Goto N, Fujishima A, et al. In vitro inhibition of caries around a resin composite restoration containing antibacterial filler. Biomaterials 1997;18:1051–7. [16] Yoshida K, Tanagawa M, Atsuta M. Characterization and inhibitory effect of antibacterial dental resin composites incorporating silver-supported materials. J Biomed Mater Res 1999;47:516–22. [17] Sondi I, Goia DV, Matijevic E. Preparation of highly concentrated stable dispersions of uniform silver nanoparticles. J Colloid Interface Sci 2003;260:75–81. [18] Bohren CF, Huffman DR. Absorption and scattering of light by small particles. New York: Wiley; 1983. [19] Petit C, Lixon P, Pileni M-P. In situ synthesis of silver nanoclusters in AOT reverse michelles. J Phys Chem 1993;97:12974–83. [20] Zhang Z, Patel RC, Kothari R, Johnson CP, Friberg SE. Stable silver clusters and nanoparticles prepared in polyacrylate and inverse micellar solutions. J Phys Chem B 2000;104:1176–82. [21] Sato K, Yoshinari T, Kintaichi Y, Haneda M, Hamada H. Remarkable promoting effect of rhodium on the catalytic performance of Ag/Al2 O3 for the selective reduction of NO with decane. Appl Catal B: Environ 2003;44:67–78.
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Development of an antimicrobial resin—A pilot study Catherine Fan, Lianrui Chu, H. Ralph Rawls, Barry K. Norling, Hector L. Cardenas, Kyumin Whang ∗ Division of Research, Department of Comprehensive Dentistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX, United States
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. To demonstrate that silver nanoparticles (AgNPs) could be synthesized in situ in
Received 24 June 2010
acrylic dental resins.
Accepted 1 November 2010
Methods. Light-cure (LC; bisphenol A glycidyl methacrylate, tetraethyleneglycol dimethacrylate, bisphenol A ethoxylate dimethacrylate blend) and chemical-cure systems (CC; orthodontic denture resin) were used to synthesize AgNPs using different concentrations of
Keywords:
Ag benzoate (AgBz).
Silver nanoparticles
Results. Rockwell hardness for LC resins showed that resins could be cured with up to 0.15%
Antimicrobial
AgBz, while the hardness of CC resins were unaffected in the concentrations tested. UV–Vis
Dental materials
spectroscopy and transmission electron microscopy confirmed the presence of AgNPs in
Controlled release
both LC and CC resins. Generally, CC resins had better distribution of and much smaller
Orthopedic
AgNPs as compared to LC resins overall. In some samples, especially in LC resins, nanoclus-
Craniofacial
ters were visible. An in vitro release study over four-weeks showed that CC resins released the most Ag+ ions, with release detected in all samples. However, LC resins only released Ag+ ions when AgBz concentration was greater than 0.1% (w/w). AgNP-loaded CC resins made with 0.2 and 0.5% (w/w) AgBz were tested for antibacterial activity in vitro against Streptococcus mutans, and results showed 52.4% and a 97.5% bacterial inhibition, respectively. Further work is now warranted to test mechanical properties and to optimize the initiator system to produce commercially useful dental and medical resins. Significance. Success in this work could lead to a series of antimicrobial medical and dental biomaterials that can prevent secondary caries and infection of implants. © 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
There is a need for effective broad-spectrum antimicrobial resin materials in dentistry and medicine. In dentistry, there is a 50–60% rate of dental caries after restorative treatment [1,2] or in areas around orthodontic bracket bonding agents where effective tooth brushing is difficult [3]. In addition, 50%
∗
of patients who wear complete or partial dentures experience problems with stomatitis [4]. In medicine, there is a 1–3% rate of infection after orthopedic surgery [5,6] and a 5% rate of infection when poly(methyl methacrylate) (PMMA) was used in cranioplasty [7] despite attempts to create a sterile environment. Unfortunately, current standards of treatment such as the use of antimicrobial mouthwashes, proper tooth-brushing technique, and the use of prophylactic systemic antibiotics
Corresponding author at: Division of Research, Department of Comprehensive Dentistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., MSC 7914, San Antonio, TX 78229-3900, United States. Tel.: +1 210 567 3674; fax: +1 210 567 3669. E-mail address: [email protected] (K. Whang). 0109-5641/$ – see front matter © 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2010.11.008
d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 322–328
have limited success or side-effects due to problems with patient compliance and the development of antibiotic resistant strains of bacteria. Thus, a broad-spectrum antimicrobial resin is needed. A widely investigated, effective, biocompatible, broadspectrum antimicrobial agent is silver (Ag). Ag ions have been reported to inactivate important enzymes and affect the replication mechanism of the DNA in bacteria [8]. Ag in nanoparticulate form (AgNP), which would release Ag ions more effectively and therefore have better bactericidal activity due to its high surface area-to-volume ratio [9], have been shown to attach to the outer membrane and affect permeability as well as induce structural changes in the cell—ultimately leading to cell death. In addition, Ag does not cause resistant bacterial strains to develop. Thus, Ag has already been used to provide household commodities with antibacterial effects, and many commercial products for kitchenware, washing machines, clothes, toiletries, stationery, etc. are available [10,11]. For dental applications, various forms of Ag or Ag-ion containing fillers have been used such as Ag ion-implanted SiO2 [12], Ag-containing silica glass [13], Ag-zeolite [14], Agapatite [15], and Ag-supported zirconium phosphate [16]. All of these approaches have demonstrated antibacterial activity in vitro. However, Ag-zeolite and Ag-apatite decreased mechanical properties at loadings necessary for antibacterial effects [15], and approaches that do not release Ag ions, such as Agsupported zirconium phosphate, only kill bacteria that come in contact with the surface and any protein adsorption on the surface would reduce the antibacterial effect. Furthermore, there are increasing concerns about potential side effects of directly using nanoparticles in vivo. Another major problem for the use of AgNPs is the difficulty in dispersing and homogeneous incorporation into the resin. AgNPs have been synthesized in many different ways such as chemical reduction of Ag+ ions in aqueous solutions with or without stabilizing agents, thermal decomposition in organic solvents, chemical and photoreduction in reverse micelles, and radiation and chemical reduction. However these methods invariably fail to provide Ag dispersions and that affects proper incorporation, mechanical properties, and release kinetics [17]. Thus, for effective dental and medical application, a more effective method of incorporating AgNP into acrylic resins and delivering Ag+ ions is needed. Recently, Kumar et al. used the natural oxidative drying process of oils to synthesize metal nanoparticles in the oil media without any reducing or stabilizing agents. The AgNPs were synthesized in situ as the paint dried and cured, thus eliminating the problem of non-uniform NP dispersion as well as the need for harsh chemicals. These AgNP paints showed excellent antimicrobial properties against S. aureus and E. coli [9]. We have modified this approach to generate AgNP in situ in polymethyl methacrylate and bisphenol A glycidyl methacrylate (Bis-GMA)-based resins for dental and medical applications. The goal of this work is to demonstrate the concept and to determine if this modified method can produce AgNP in situ in these resins, release Ag+ ions, and provide effective antimicrobial activity.
2.
323
Materials and methods
AgNPs were formed in situ in dental resins using Ag benzoate (AgBz), and the effect of AgBz concentration and curing method (light-curing vs. chemical-curing) on the degree of cure, nanoparticle size and formation, in vitro release of Ag ions and in vitro antibacterial activity were determined. Transmission electron microscopy (TEM) was used to observe the AgNPs, UV/Vis spectroscopy to further determine the presence of AgNPs, clusters of AgNPs and release of Ag+ ions, and Rockwell15T hardness to measure degree of cure. In addition, the antibacterial activity of these novel resins was assessed in vitro with Streptococcus mutans.
2.1.
Synthesis of AgNP-loaded resins
Light-cured (LC) resins were made by blending varying concentrations (0, 0.002, 0.02, 0.1, 0.15 and 0.2%, w/w, of total monomer) of AgBz (Sigma–Aldrich) in dimethylaminoethyl methacrylate (DMAEMA; 2%, w/w, of total monomer; Sigma–Aldrich), camphorquinone (CQ: 1%, w/w, of monomer blend; Sigma–Aldrich), and GTE (a blend of 37.5% bisphenol A glycidyl methacrylate (Bis-GMA), 25.0% tetraethyleneglycol dimethacrylate (TEGMA) and 37.5% bisphenol A ethoxylate dimethacrylate (Bis-EMA; EssTech)). The blend was then poured into a mold (3/8 diameter × 1/16 thick) between two glass slides and light-cured on each side for 40 s using a Demetron Optilux 401 curing light with the light guide held against the glass slide. Chemically cured (CC) specimens were made by dissolving different concentrations (0, 0.002, 0.02, 0.2 and 0.5%) of AgBz into 2% DMAEMA and then into liquid orthodontic monomer (Dentsply), and subsequently mixing with PMMA powder, as described in the manufacturer’s guidelines. This PMMAbased resin blend was immediately poured into molds pressed between two glass slides and allowed to chemically cure for 60 min or until tested.
2.2.
Rockwell hardness
The Rockwell15T hardness of cured specimens was measured with a 15T 1/16 ballpoint indenter with a 15 kg force. Three measurements were made on different areas of each of the specimens to verify that they were cured evenly.
2.3.
Transmission electron microscopy (TEM)
Cured specimens were cut into 100 nm thin slices using a microtome, placed on copper grids, and observed using TEM (Jeol JEM-1230 transmission electron microscope).
2.4.
Ultraviolet–visible (UV/Vis) spectroscopy
Specimens were also cured in plastic cuvettes and UV/Vis spectra from 200 to 800 nm were taken (SmartSpec 3000 spectrophotometer, Bio-Rad) using the control (0% AgBz) as a blank. For CC specimens, an uncured control specimen with 0% AgBz was used as the blank for 0 min specimens, and a 60 min cured control specimen with 0% AgBz was used as the blank for
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60 min specimens to ensure that any absorbance due to the resin was properly accounted for.
2.5.
In vitro Ag release study
Specimens were placed into glass vials with 5 mL of sterile deionized water (n = 5). At certain intervals (1 day, 4 days, 1 week, 2 weeks, 4 weeks) 1 mL of the water was extracted and its UV/Vis absorption was measured from 200 to 800 nm. The control specimens contained 0% AgBz. The UV/Vis scan of the control was taken using sterile deionized water as the blank while the readings for all the other specimens were taken using the control as the blank. At each time period the water was replaced to maintain sink conditions.
2.6. Inhibitory effects of AgNP-loaded resin discs on the growth of S. mutans Based on the in vitro release data and a pilot growth inhibition assay showing minimal effect from the 0.2% AgBz LC specimens (data not shown), the growth inhibition assay was only done with CC resins made with 0 (negative control), 0.2 and 0.5% AgBz. S. mutans (TACC 25175) was grown on TSBY (Trypticase Soy Broth with 0.5% yeast extract) agar plates in an anaerobic chamber with a mixed gas (N2 = 85%, H2 = 5, and CO2 = 10%) and specimens were placed on the bacteriacontaining agar and anaerobically inoculated at 37 ◦ C for 5 days to determine their efficacy in inhibiting bacterial growth by identifying zones of inhibition. To estimate colony formation, 20 l of different concentrations of the bacteria (about 10−1 , 10−2 , 10−3 , 10−4 , 10−5 and 10−6 /ml) were homogenously spread onto each area of the surface on the gelled TSBY agar plates using sterile spreaders. The colony formation was determined by counting colonies from suitable dilutions for each specimen.
2.7.
Statistics
Rockwell hardness, in vitro release and % bacterial inhibition data were compared between groups using ANOVA with
Fig. 1 – Photograph of LC and CC resins with different AgBz concentrations.
Newman–Keuls’s post hoc test. For release data, comparisons were made at each time point.
3.
Results
3.1.
Synthesis of AgNP-loaded resins
As the AgBz concentration increased, it became more difficult to cure the specimens. The highest AgBz concentration that could be incorporated and light-cured was 0.15% (w/w). However, for chemically cured resins, specimens with as much as 0.6% (w/w) AgBz were curable. Fig. 1 shows that as the AgBz concentration increased, the amber color of the resins became darker due to the plasmon effect of the AgNPs [18]. Also the CC resins were lighter in color than the light-cured resins at the same AgBz concentration.
3.2.
Rockwell hardness
Fig. 2 shows that Rockwell hardness of LC resins decreased significantly (p < 0.05) when AgBz reached concentrations of 0.1% (w/w) and above. Above 0.2%, the resins did not cure well. However, the hardness of CC resins did not change significantly
Fig. 2 – Rockwell15T hardness of LC and CC resins made with different concentrations of AgBz.
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Fig. 3 – TEM of LC (a–f) and CC (g–j) resins with different AgBz concentrations. Bar is 100 nm. (a) and (f) were taken at 80,000× and the rest were taken at 300,000×. (a) LC with 0% negative control, (b) LC with 0.002% AgBz, (c) LC with 0.02% AgBz, (d) LC with 0.1% AgBz, (e) LC with 0.15% AgBz, (f) LC with 0.2% AgBz, (g) CC with 0.002% AgBz, (h) CC with 0.02% AgBz, (i) CC with 0.2% AgBz and (j) CC with 0.5% AgBz. Specimens containing greater than 0.2% AgBz could not be fully cured.
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UV/Vis Spectra of Ag-Loaded Resins 1.6 LC 0.002% AgBz 1.4
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Fig. 4 – UV/Vis spectra of LC and CC resins cured with different concentrations of AgBz. Note the characteristic peak of AgNPs at 400 nm and the broader peak beginning at 500 nm that may be due to NP clusters.
from the 0% control, but this may have been mainly due to the large standard deviations caused by pores that are formed in the mixing process of the different components of the chemical cure resin. Still, the CC resins were significantly harder than the LC resins above 0.2% AgBz.
3.3.
Transmission electron microscopy
TEM images (Fig. 3b–f) show that there are sparse individual particles and large nanoparticle clusters in the LC resins. The higher the AgBz concentration, the greater the number of nanoparticles and the larger the nanoparticle clusters. Some clusters were as large as 50–70 nm. Nanoparticles were generally between 10 and 20 nm, but with the 0.2% specimens, there were also many smaller particles.
With the CC resins however there were distinctly more nanoparticles and they were distributed more widely (Fig. 3g–j). There were not as many nanoparticle clusters. The individual particles from all specimens usually ranged from 2 to 18 nm in size but it was apparent that the CC specimens had smaller nanoparticles. This might explain the lighter amber color of the AgNP-loaded CC resins. Finally, as the AgBz concentration increased, so did the number of visible nanoparticles.
3.4.
Ultraviolet–visible spectroscopy
It is known that spherical AgNPs have a UV absorption band with a peak centered around 400 nm [19]. All LC specimens showed a peak centered around 400 nm (Fig. 4). However, there
Release of Ag ions from Ag-loaded resins Cumulative UV/Vis Absorbance @ 223 nm
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Fig. 5 – UV absorbance at 223 nm vs. time showing cumulative Ag ion release from LC and CC resins made with different concentrations of AgBz.
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was also a broad peak at 500 nm and tailing out to 800 nm that consistently broadened as AgBz concentration was increased. This may be due to the formation of Ag nanoclusters with a range of sizes [20]. The UV–Vis spectra for the CC specimens show a band peak at about 450 nm (Fig. 4). As the AgBz concentration increased, a broad band with a peak beginning at around 500 nm became visible. This band also became visible after 24 h for most of the specimens as more NPs were formed. However, unlike the two distinct bands in the LC specimens, the bands in the CC specimens were very diffuse and overlapped, producing a very broad peak at about 450 nm.
3.5.
In vitro Ag release study
Fig. 5 shows the UV/Vis absorbance measuring the released Ag ions in water versus time. Bands characteristic of Ag+ ions peak at about 223 nm [21]. For LC specimens, Ag+ ion release was only measured with resins made with 0.1% AgBz. However for CC specimens, Ag ion release was detected for every concentration, and as the AgBz concentration increased, the peak became more intense. However, over time Ag ion release gradually decreased.
3.6. Inhibitory effects of AgNP-loaded resin discs on the growth of S. mutans Fig. 6a shows the presence of the resin disks on agar and Fig. 6b shows the agar with the discs removed after 5 days of incubation at 37 ◦ C. A clear zone of inhibition is visible in A1 and B1 where resins with 0.5% AgBz were used. A2 and B2 show a vague zone for resins made with 0.2% AgBz, and A3 and B3 show no zones of inhibition when AgBz was not used. This correlates with Fig. 6c, which shows the percent inhibition of the growth of S. mutans. Resins made with 0.5% AgBz showed 97.5% inhibition and those made with 0.2% AgBz inhibited 52.4% of S. mutans growth as compared to 0% for negative controls. These results demonstrate that 0.5% silver containing resin inhibited almost all of the bacterial growth. Even resins containing only 0.2% silver were able to inhibit more than 50% of the bacterial growth.
4.
Discussion
An effective broad-spectrum antimicrobial resin for dental and medical use that does not promote antibiotic resistant strains of bacteria is needed. We have developed a method of synthesizing silver nanoparticles (AgNP) in situ in dental and medical resins. Hardness tests showed that in light cured (LC) specimens, as the concentration of silver benzoate (AgBz) increases the degree of cure decreases. This is probably due to Ag+ ions being reduced and generating atom clusters and nanoparticles during the curing, and thus being in competition with the free radical polymerization process. However, it is interesting that this did not seem to affect the degree of cure for chemically cured (CC) specimens to the same extent, and while the maximum concentration of AgBz with which LC resins could be cured was 0.2%, CC resins could be cured with 0.6%. It is also
Fig. 6 – Inhibitory effect of resin disk containing AgNP on the growth of Streptococcus mutans. Panel a shows the resin specimens and panel b shows the agar with the specimens removed and panel c shows the actual growth inhibition. (a1) Resin made with 0.5% AgBz. (a2) Resin made with 0.2% AgBz. (a3) Resin made with 0% AgBz (negative control). (b1) shows a transparent ring suggesting growth of S. mutans was inhibited. (b2) Shows a semi-transparent ring demonstrated partial inhibition. In contrast, (b3) shows normal bacterial growth. Resin containing 0.5% AgBz inhibited 97.5% and those made with 0.2% AgBz inhibited 52.4% of the growth of S. mutans as compared to the negative controls, respectively.
interesting that the color of LC resins were darker than CC resins for the same AgBz concentration, which may be due to the larger particle sizes that were seen with TEM. It appears that the slower CC process allows a greater number of AgNP nucleation sites to form, thereby generating more particles, a better dispersion of the particles, and smaller particle sizes. The UV–Vis spectra of the resins further supported the TEM data that indicated that the spherical particles are AgNPs, since the UV band at 400 nm is characteristic of spherical AgNPs. The narrower bands of the LC resins also support TEM observations that the particles are larger than those formed by CC resins. The broader bands that begin after 500 nm but are not as visible in the CC resins, also support the TEM data
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in that there seems to be more clustering of the NPs in the LC specimens. The release study showed that continued release of Ag+ ions was only observed with 0.1% AgBz LC specimens, while CC specimens showed release with as low as 0.002% AgBz. The 0.1% LC specimens may have released Ag+ ions only because of a visible crack since 0.15% LC specimens did not release any Ag+ ions (data not shown). The release of Ag+ ions from CC specimens may be due to the smaller particle size and more homogeneous distribution of the NPs and/or the higher porosity that exists in CC resins as compared to LC resins. Only the 0.2% and 0.5% AgBz CC resins (with the most Ag+ ion release) were tested in the in vitro bacterial growth inhibition assay. A zone of inhibition of S. mutans is clearly visible for the 0.5% and barely perceptible for the 0.2% specimens. This correlated to 97.5% and 52.4% inhibition of S. mutans growth. In short, this pilot study demonstrated a novel process that generates AgNPs in situ using the resin’s own curing process to form antimicrobial resins for dental and medical use. Further studies are needed to investigate the effect of initiator and amine concentrations on maximum AgBz loading concentration, mechanical properties, in vitro release properties, and in vitro antibacterial effects. In addition, the effect of light curing vs. chemical curing and PMMA vs. BisGMA-based resin on Ag ion release needs to be further investigated. Success in this work could lead to a series of antimicrobial medical and dental biomaterials that can prevent secondary caries and implant-centered infection.
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