Abstract
Objectives
To demonstrate that silver nanoparticles (AgNPs) could be synthesized in situ in acrylic dental resins.
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 Ag benzoate (AgBz).
Results
Rockwell hardness for LC resins showed that resins could be cured with up to 0.15% AgBz, while the hardness of CC resins were unaffected in the concentrations tested. UV–Vis spectroscopy and transmission electron microscopy confirmed the presence of AgNPs in both LC and CC resins. Generally, CC resins had better distribution of and much smaller AgNPs as compared to LC resins overall. In some samples, especially in LC resins, nanoclusters 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.
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 or in areas around orthodontic bracket bonding agents where effective tooth brushing is difficult . In addition, 50% of patients who wear complete or partial dentures experience problems with stomatitis . In medicine, there is a 1–3% rate of infection after orthopedic surgery and a 5% rate of infection when poly(methyl methacrylate) (PMMA) was used in cranioplasty 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 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, broad-spectrum antimicrobial agent is silver (Ag). Ag ions have been reported to inactivate important enzymes and affect the replication mechanism of the DNA in bacteria . 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 , 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 .
For dental applications, various forms of Ag or Ag-ion containing fillers have been used such as Ag ion-implanted SiO 2 , Ag-containing silica glass , Ag-zeolite , Ag-apatite , and Ag-supported zirconium phosphate . 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 , and approaches that do not release Ag ions, such as Ag-supported 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 . 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 .
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
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 Rockwell 15T 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 g lycidyl 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 PMMA-based 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 Rockwell 15T 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 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 (N 2 = 85%, H 2 = 5, and CO 2 = 10%) and specimens were placed on the bacteria-containing 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 μl 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 Newman–Keuls’s post hoc test. For release data, comparisons were made at each time point.
2
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 Rockwell 15T 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 g lycidyl 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 PMMA-based 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 Rockwell 15T 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 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 (N 2 = 85%, H 2 = 5, and CO 2 = 10%) and specimens were placed on the bacteria-containing 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 μl 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 Newman–Keuls’s post hoc test. For release data, comparisons were made at each time point.
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