Highlights
- •
Ag doping of TiO 2 results in a band gap shift towards the visible spectrum.
- •
Small quantities of Ag–TiO 2 have a bactericidal effect on S. mutans under visible light conditions.
- •
Although reduced, the bactericidal effect was still observed when Ag–TiO 2 was embedded in polymer.
Abstract
Objective
The objectives of this in vitro study were to produce a filled resin containing Ag–TiO 2 filler particles and to test its antibacterial properties.
Methods
Ag–TiO 2 particles were manufactured using the ball milling method and incorporated into an epoxy resin using a high speed centrifugal mixer. Using UV/vis spectrophotometry investigations were performed to assess how the photocatalytic properties of the Ag–TiO 2 particles are affected when encased in resin. Adopting the bacteria colony counting technique, the antibacterial properties of Ag–TiO 2 particles and Ag–TiO 2 containing resins were assessed using Streptococcus mutans under varying lighting conditions.
Results
Ag doping of TiO 2 results in a band gap shift towards the visible spectrum enabling Ag–TiO 2 to exhibit photocatalytic properties when exposed to visible light. Small quantities of Ag–TiO 2 were able to produce a bactericidal effect when in contact with S. mutans under visible light conditions. When incorporated into the bulk of an epoxy resin, the photocatalytic properties of the Ag–TiO 2 particles were significantly reduced. However, a potent bactericidal effect was still achieved against S. mutans .
Significance
Ag–TiO 2 filled resin shows promising antimicrobial properties, which could potentially be used clinically.
1
Introduction
A truly antibacterial resin could find a number of clinical dental applications in both restorative dentistry and orthodontics. In restorative dentistry it could be used as a filling or denture base material, whilst in orthodontics it could be used as a bracket or bracket bonding material. Demineralisation of enamel is still one of the main complications of orthodontic treatment, particularly with fixed appliances . The first sign of demineralisation may be the development of white spot lesions (WSL) on the enamel surface around the bracket margins, which if left unchecked can progress to cavitation. This whole process occurs more rapidly in orthodontic patients when compared to non-orthodontic patients . Preventing enamel demineralisation and the formation of WSL is an important consideration for clinicians, as the lesions are unaesthetic and potentially irreversible. One cross sectional study showed that up to 50% of individuals undergoing fixed appliance therapy had non-developmental WSL compared with just 25% of controls . Although the progression from WSL to cavitation is low (cavitation occurs in only 2% of WSL), the high incidence of WSLs is a significant factor where orthodontic treatment is being performed to improve aesthetics .
A Cochrane review assessing the effectiveness of methods used in the prevention of WSL formation concluded there was only evidence to support the use of daily 0.05% sodium fluoride mouthrinses in order to reduce their prevalence and severity . Unfortunately it is often the case that patients at the highest risk of developing WSL are those least likely to comply with oral hygiene and mouth rinsing regimes. Therefore less patient dependent modes of delivery are required. Although the same Cochrane review concluded that the use of glass-ionomer cement for bracket bonding reduces the prevalence and severity of WSL, both glass-ionomer and resin modified glass-ionomer cements (RMGIC) have not gained widespread acceptance as orthodontic bonding agents, due to the reportedly lower shear bond strength and a lack of familiarity when compared to resin bonding systems and the associated acid etch technique .
A number of new technologies, principally fillers and coatings, have recently become available with potential antimicrobial properties. Although such coatings have the potential to be effective, they are also likely to be abraded and lost over time, or may corrode within the oral environment. For example if orthodontic brackets were to be coated with an antibacterial material it is unlikely it would be maintained on the bracket surface for the duration of a course of treatment .
Titanium dioxide (TiO 2 ), particularly in its nanoparticle form, has generated a great deal of interest over recent years, as it has numerous potential applications. A number of studies have demonstrated it to be an effective light activated photocatalyst, with strong bactericidal activity. However, its main disadvantage is its wide band-gap, meaning it is only really bactericidal when it absorbs UV light. With UV light being only a very small fraction of the solar spectrum (<5%), it means its activity is severely reduced under dark or visible light conditions, as would be found within the oral cavity . In addition, traditional TiO 2 photocatalysis is effective only upon irradiation with UV-light at levels that could cause damage to human cells. As a result, researchers have conducted extensive studies on doping, sensitisation and covering the surface of the TiO 2 with dyes, in order to extend light absorption to the visible range. Doping TiO 2 with transition metal ions and/or anions is commonly used to this effect. This method creates intra-band gap states close to the conduction or valence band edges that induce visible-light absorption at the sub-band gap energies . Ag+ modification of TiO 2 induces a decrease in the band gap energy, allowing visible light to activate the material’s photocatalytic activity. This opens the possibility that such technology could be adopted within dental biomaterials and help prevent bacterial colonisation of intraoral appliances. Examples might include polymeric orthodontic brackets, bonding resins, dentures and intracoronal polymeric restorations. If the active particles were retained within the bulk of a polymer as well as at the surface, then any wear would simply expose more particles and thereby continue to confer an antimicrobial effect.
In this study the aims were to examine the effects of incorporating doped titanium dioxide nanoparticles into a polymer. The specific objectives were to investigate:
- 1.
Free radical release at different silver doping concentrations of TiO 2 nanopowder.
- 2.
The effect of varying the light conditions has on free radical release.
- 3.
Free radical release when Ag–TiO 2 particles are incorporated into a bulk polymer.
- 4.
The effect of silver doped TiO 2 on bacterial growth, both as a powder and when incorporated within a bulk resin.
2
Materials and methods
2.1
Production of Ag–TiO 2 powders
Silver doped photocatalytic TiO 2 powders were prepared by ball milling from commercial TiO 2 (P25) powders. Four different suspensions of Ag–TiO 2 , were produced ( Table 1 ). After drying, the powders were calcined at 400 °C for 90 min in a high temperature oven (Heratherm Oven, Thermo Scientific, UK) in order to decompose the silver salt and permit diffusion of the silver ions (Ag + ). Following calcination, the course powder produced was further ground using a centrifugal laboratory mixing and grinding machine (DAC150 Speedmixer, Hauschild Engineering, High Wycombe, UK).
| Sample | AgNO 3 | Na 2 CO 3 |
|---|---|---|
| 1 = 2% Ag | 2.3 ml | 2.5 ml |
| 2 = 4% Ag | 4.6 ml | 5.0 ml |
| 3 = 6% Ag | 6.9 ml | 7.5 ml |
| 4 = 8% Ag | 9.2 ml | 10 ml |
To assess how the silver loading affected the band gap energy of the TiO 2, the powder samples were placed in a UV/vis Spectrophotometer (Lambda 35 UV/vis Spectrophotometer, Perkin Elmer, Massachussets, USA) and the readings plotted to show absorption at wavelengths ranging from 200 to 500 nm. Optical spectra were analysed using the Tauc model as reported by Impellizeri et al. . Using this data the band gap shift for the TiO 2 and each of the four Ag–TiO 2 powders (2, 4, 6, and 8%) was determined.
2.2
Testing Ag–TiO 2 powder for free radical release under different lighting conditions
The Ag–TiO 2 powders were next tested for free radical release under visible light conditions. 0.05 g of samples 1–4 ( Table 1 ) of Ag–TiO 2 powder and TiO 2 powder were added to beakers containing 10 ml of Methylene Blue dye used as a photobleaching indicator of photcatalysis . These aqueous solutions were left stirring for 4 h under visible light conditions of 2000 lux with the light intensity measured using a digital light lux meter (Model DT-1300, CEM, Shenzen, China). Three samples of 50 μl of each solution were collected every 30 min for 4 h and dispensed into a microplate and stored under dark conditions. The microplate was then positioned in the spectrophotometer to measure the absorbance of the solutions in each of the wells. The same procedure was carried out under dark and UV light (355 nm) conditions.
2.3
Incorporation of silver doped titanium dioxide into an unfilled resin and testing for free radical release
The 6% Ag doped TiO 2 powder was shown to have the most effective photocatalytic properties under visible light conditions and was subsequently incorporated into the polymeric samples. These samples were made using 3.5 g of uncured epoxy resin, 1 g of curing agent and 3.6 g of 6% Ag doped TiO 2 powder to produce a 44% by weight Ag–TiO 2 resin sample. The uncured epoxy and doped powder were placed into the centrifugal laboratory mixing and grinding machine and spun at 3000 rpm for 5 min to ensure adequate dispersion of the powder within the resin. The curing agent was then added and the specimen mixed at 2500 rpm for a further 30 s. The final mixed resin was then poured into a plastic mould measuring 3.5 cm in diameter and 0.5 cm thickness. At this degree of filler loading it was possible to incorporate the powder in the resin, mix it and pour it into the moulds.
To test for free radical release the 6% Ag loaded Ag–TiO 2 resin disk was then placed in a 100 ml glass beaker containing 10 ml of Methylene Blue dye and stored under visible light conditions of 2000 lux. Three samples of 50 μl of each solution were collected every 30 min for 4 h using an Eppendorf micropipette and placed into a microplate well, which was stored under dark conditions. At the end of collection, the microplate was positioned in the spectrophotometer, which had previously been calibrated to a wavelength of 655 nm (visible light setting), in order to measure the absorbance of each of the Methylene Blue solutions. An average of the 3 readings was calculated for each solution and plotted on a line graph showing the reduction of Methylene Blue dye with time in the presence of Ag–TiO 2 resin.
To determine the effect of an increased surface area of available Ag–TiO 2 particles in the resin on free radical release, a further 6% Ag doped Ag–TiO 2 resin disk was sectioned into 8 smaller segments, thereby increasing the surface area from 25 cm 2 to 39 cm 2 .
2.4
Antibacterial testing
In order to test the likely effectiveness of the Ag + doped TiO 2 powders against cariogenic bacteria, Streptococcus mutans strain NG8 was chosen. 0.05, 0.1 and 0.2 g of 6% Ag–TiO 2 powder was placed into three separate wells, with a fourth well left empty to act as a negative control. To each of the four wells, 0.9 ml of sterile PBS (phosphate-buffered saline: 10 mM KH 2 PO 4 –K 2 HPO 4 at pH 7.2 containing 0.15 M NaCl) was added along with 0.1 ml of S. mutans cell suspension (OD600 1.0). The plate was then positioned on a rocking platform (Stuart See-saw rocker, Bibby Scientific Ltd., Staffs, UK) under an LED light for 4 h and at a distance that provided an intensity of 1500 lux. A LED light was used to ensure no heat was given off. A separate well containing 0.05 g of 6% Ag–TiO 2 powder in 0.9 ml of PBS and 0.1 ml S. mutans culture was also placed under dark conditions for the same time period in order to act as a control.
At 4 h, the plate was removed from the light source and 0.1 ml samples were removed from the wells. Each was serially 10-fold diluted in PBS and portions (20 μl) were spotted onto BHY agar (37 g/l Brain Heart Infusion, 5 g/l yeast extract, 15 g/l agar) plates. These were incubated for 16 h at 37 °C in a candle jar and colonies were counted in order to estimate the viable numbers of S. mutans cells expressed as colony forming units (CFU). The experiments were carried out in duplicate.
The same experimental procedure was carried out using an Ag–TiO 2 resin sample, fabricated as describes in Section 2.3 . A 6% Ag–TiO 2 , filled resin disk, 3.5 cm in diameter, was cut into 5 mm 2 sections. One 5 mm sample was then placed in a well containing 0.9 ml of sterile PBS, together with 0.1 ml of S. mutans cell suspension, positioned on a rocking platform and exposed to LED light of 1500 lux intensity for 4 h. A second 5 mm section was placed in a well containing bacterial suspension in PBS on a rocking platform and covered with a foil lid, preventing light access and acting as a control.
2
Materials and methods
2.1
Production of Ag–TiO 2 powders
Silver doped photocatalytic TiO 2 powders were prepared by ball milling from commercial TiO 2 (P25) powders. Four different suspensions of Ag–TiO 2 , were produced ( Table 1 ). After drying, the powders were calcined at 400 °C for 90 min in a high temperature oven (Heratherm Oven, Thermo Scientific, UK) in order to decompose the silver salt and permit diffusion of the silver ions (Ag + ). Following calcination, the course powder produced was further ground using a centrifugal laboratory mixing and grinding machine (DAC150 Speedmixer, Hauschild Engineering, High Wycombe, UK).
| Sample | AgNO 3 | Na 2 CO 3 |
|---|---|---|
| 1 = 2% Ag | 2.3 ml | 2.5 ml |
| 2 = 4% Ag | 4.6 ml | 5.0 ml |
| 3 = 6% Ag | 6.9 ml | 7.5 ml |
| 4 = 8% Ag | 9.2 ml | 10 ml |
To assess how the silver loading affected the band gap energy of the TiO 2, the powder samples were placed in a UV/vis Spectrophotometer (Lambda 35 UV/vis Spectrophotometer, Perkin Elmer, Massachussets, USA) and the readings plotted to show absorption at wavelengths ranging from 200 to 500 nm. Optical spectra were analysed using the Tauc model as reported by Impellizeri et al. . Using this data the band gap shift for the TiO 2 and each of the four Ag–TiO 2 powders (2, 4, 6, and 8%) was determined.
2.2
Testing Ag–TiO 2 powder for free radical release under different lighting conditions
The Ag–TiO 2 powders were next tested for free radical release under visible light conditions. 0.05 g of samples 1–4 ( Table 1 ) of Ag–TiO 2 powder and TiO 2 powder were added to beakers containing 10 ml of Methylene Blue dye used as a photobleaching indicator of photcatalysis . These aqueous solutions were left stirring for 4 h under visible light conditions of 2000 lux with the light intensity measured using a digital light lux meter (Model DT-1300, CEM, Shenzen, China). Three samples of 50 μl of each solution were collected every 30 min for 4 h and dispensed into a microplate and stored under dark conditions. The microplate was then positioned in the spectrophotometer to measure the absorbance of the solutions in each of the wells. The same procedure was carried out under dark and UV light (355 nm) conditions.
2.3
Incorporation of silver doped titanium dioxide into an unfilled resin and testing for free radical release
The 6% Ag doped TiO 2 powder was shown to have the most effective photocatalytic properties under visible light conditions and was subsequently incorporated into the polymeric samples. These samples were made using 3.5 g of uncured epoxy resin, 1 g of curing agent and 3.6 g of 6% Ag doped TiO 2 powder to produce a 44% by weight Ag–TiO 2 resin sample. The uncured epoxy and doped powder were placed into the centrifugal laboratory mixing and grinding machine and spun at 3000 rpm for 5 min to ensure adequate dispersion of the powder within the resin. The curing agent was then added and the specimen mixed at 2500 rpm for a further 30 s. The final mixed resin was then poured into a plastic mould measuring 3.5 cm in diameter and 0.5 cm thickness. At this degree of filler loading it was possible to incorporate the powder in the resin, mix it and pour it into the moulds.
To test for free radical release the 6% Ag loaded Ag–TiO 2 resin disk was then placed in a 100 ml glass beaker containing 10 ml of Methylene Blue dye and stored under visible light conditions of 2000 lux. Three samples of 50 μl of each solution were collected every 30 min for 4 h using an Eppendorf micropipette and placed into a microplate well, which was stored under dark conditions. At the end of collection, the microplate was positioned in the spectrophotometer, which had previously been calibrated to a wavelength of 655 nm (visible light setting), in order to measure the absorbance of each of the Methylene Blue solutions. An average of the 3 readings was calculated for each solution and plotted on a line graph showing the reduction of Methylene Blue dye with time in the presence of Ag–TiO 2 resin.
To determine the effect of an increased surface area of available Ag–TiO 2 particles in the resin on free radical release, a further 6% Ag doped Ag–TiO 2 resin disk was sectioned into 8 smaller segments, thereby increasing the surface area from 25 cm 2 to 39 cm 2 .
2.4
Antibacterial testing
In order to test the likely effectiveness of the Ag + doped TiO 2 powders against cariogenic bacteria, Streptococcus mutans strain NG8 was chosen. 0.05, 0.1 and 0.2 g of 6% Ag–TiO 2 powder was placed into three separate wells, with a fourth well left empty to act as a negative control. To each of the four wells, 0.9 ml of sterile PBS (phosphate-buffered saline: 10 mM KH 2 PO 4 –K 2 HPO 4 at pH 7.2 containing 0.15 M NaCl) was added along with 0.1 ml of S. mutans cell suspension (OD600 1.0). The plate was then positioned on a rocking platform (Stuart See-saw rocker, Bibby Scientific Ltd., Staffs, UK) under an LED light for 4 h and at a distance that provided an intensity of 1500 lux. A LED light was used to ensure no heat was given off. A separate well containing 0.05 g of 6% Ag–TiO 2 powder in 0.9 ml of PBS and 0.1 ml S. mutans culture was also placed under dark conditions for the same time period in order to act as a control.
At 4 h, the plate was removed from the light source and 0.1 ml samples were removed from the wells. Each was serially 10-fold diluted in PBS and portions (20 μl) were spotted onto BHY agar (37 g/l Brain Heart Infusion, 5 g/l yeast extract, 15 g/l agar) plates. These were incubated for 16 h at 37 °C in a candle jar and colonies were counted in order to estimate the viable numbers of S. mutans cells expressed as colony forming units (CFU). The experiments were carried out in duplicate.
The same experimental procedure was carried out using an Ag–TiO 2 resin sample, fabricated as describes in Section 2.3 . A 6% Ag–TiO 2 , filled resin disk, 3.5 cm in diameter, was cut into 5 mm 2 sections. One 5 mm sample was then placed in a well containing 0.9 ml of sterile PBS, together with 0.1 ml of S. mutans cell suspension, positioned on a rocking platform and exposed to LED light of 1500 lux intensity for 4 h. A second 5 mm section was placed in a well containing bacterial suspension in PBS on a rocking platform and covered with a foil lid, preventing light access and acting as a control.
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