Abstract
Objectives
The aim of the present work was to perform the first in vitro evaluation of a new interfacial bond-promoting material-and-method concept for on-demand long term bacteria inhibition in dental restoration procedures.
Methods
The bioactivity, mechanical bonding strength and photocatalytic bactericidal properties, induced by low dose ultraviolet-A (UV-A) irradiation of dental adhesives containing crystalline titania nanoparticles (NPs), were analyzed.
Results
Dental adhesives with a NP content of 20 wt% were shown to be bioactive in terms of spontaneous hydroxylapatite formation upon storage in simulated body fluid and the bioactivity was found to be promoted by chemical etching of the adhesives. The mechanical bonding strength between the adhesives and a HA tooth model was shown to be unaffected by the NPs up to a NP content of 30 wt%. Elimination of Staphylococcus epidermidis in contact with the adhesives was found to depend both on UV photocatalytic irradiation intensity and time. Efficient elimination of the bacteria could be achieved using a UV-A dose of 4.5 J/cm 2 which is about 6 times below the safe maximum UV dose according to industry guidelines, and 20 times below the average UV-A dose received during an ordinary sun bed session.
Significance
The combined features of bioactivity and on-demand bactericidal effect should open up the potential to create dental adhesives that reduce the incidence of secondary caries and promote closure of gaps forming at the interface towards the tooth via remineralization of adjacent tooth substance, as well as prevention of bacterial infections via on-demand UV-A irradiation.
1
Introduction
Dental caries is a prevalent oral disease. The development of caries is a complex interaction between the tooth and the acid producing bacteria colonized in dental plaque. It is also influenced by host factors, like saliva composition, dietary carbohydrates and oral hygiene . Today, resin-based composites are used as both adhesives and restorative materials and constitute the first choice for restorations procedures. The advantages of resin composite systems to the previously used amalgam include reduced removal of dentin in cavity restoration preparation, strengthening of the remaining tooth and better aesthetics through the control of the material color . In addition to composite restorations, adhesive dental materials are widely used in other dental applications such as orthodontics and fixation of amalgam, crowns, fixed partial dentures and veneers to the surface of teeth.
Unfortunately, there are significant problems with present dental restorations; it has been estimated that as much as half of all restorations are replacements, with the major reason for replacements being secondary (recurrent) caries . Similar problems occur in orthodontic applications; it has been indicated that the adhesive used to fix the bracket on the enamel actually enhances the adhesion and accumulation of bacteria . Despite improvements in dental adhesives, the bonded interface is still the weakest area of composite restorations . Part of this problem may be due to polymerization shrinkage, which is considered to result in the formation of a gap between the filling material and the tooth when the contraction stress exceeds the adhesive bonding strength in the dentin–adhesive–resin structure . However, there is dispute as to whether polymer shrinkage, marginal deterioration or marginal gap width are the main causes of secondary caries . What is undisputed is that the bacteria are responsible for caries development, and therefore it has been suggested that the development of dental adhesive materials that suppress bacterial activity at the tooth–composite interface may be the most effective route for reducing secondary caries .
While the clinical performance of dental composite materials has greatly improved in terms of restoration durability, bonding strength, and aesthetics, it has only been recently that increased focus has been put on achieving antibacterial properties in these materials. For example, the incorporation of soluble antimicrobial agents, such as chlorhexidine, in the resin matrix have been investigated . Clear inhibition of bacteria has been shown, but the release kinetics is difficult to control and long term effect is not to be expected. An advantage with having a releasing agent is that the antibacterial effect can extend beyond the immediate region of the dental composite, but the drawback is that often the mechanical properties of the composite are degraded . Another strategy is to develop contact antibacterial dental materials in which the antibacterial agent is immobilized in the resin matrix and is not released. In this case the antibacterial effect is limited to bacteria that come in direct contact with the material. For example, silver has been incorporated in orthodontic adhesives and filler composites and showed good bacterial inhibition. Methacryloyloxy dodecyl pyridinium bromide (MDPB) has been successfully used for arresting the residual bacteria after the cavity preparation and for bacteria that penetrate into the gap between the restoration material and cavity wall . Similarly, cetylpyridinium chloride (CPC), a well known antiplaque agent, has been immobilized in an adhesive matrix and showed antibacterial effect .
Photocatalysis of titanium dioxide, TiO 2 , has well known pathogenicidal effect; inhibition of viruses, Gram-positive and Gram-negative bacteria, and even cancer cells have been demonstrated by photocatalysis of TiO 2 . Due to the high redox reaction ability of the photocatalysis products, TiO 2 can also be used as a bactericide for applications such as drinking water and indoor environments . The mechanism behind its action is the destruction of the cell membrane or cell wall and causing of leakage or structural damage of the cell . The use of TiO 2 as an antibacterial agent in dental adhesive materials has, to the best of our knowledge, not been investigated previously.
For the anatase crystalline form of TiO 2 , irradiation with ultraviolet light (UV) having a wavelength less than 385 nm can excite electrons above the material’s band gap of 3.2 eV and thus generate an electron–hole pair. Reacting with a suitable electron acceptor, like oxygen, the excited electron will produce superoxide ions (O 2 − ). The positive hole in the valence band can react with H 2 O or OH − to produce hydroxyl radicals ( OH). Other reactive oxygen species, like hydroxyl peroxide H 2 O 2 and singlet oxygen can also be generated. The reactive oxygen species and radicals can diffuse and decompose nearby organic molecules into CO 2 and H 2 O .
Another potential area in which the performance of dental adhesives may be enhanced is bioactivity. In the present context bioactivity is defined as interfacial bonding of a material to bone tissue by means of formation of a biologically active hydroxylapatite (HA) layer on the material surface . Bioactive materials have been proven to promote bone formation and to form a stable bond to bone and prediction of a material’s bioactivity can be made both in vivo and in vitro . In the present study the spontaneous formation of HA on the material surface upon storage in a simulated body fluid (SBF) is taken as evidence of bioactivity . A bioactive dental material which forms HA on the surface would have several benefits including remineralization of adjacent tooth substance, potential closure of gaps between material and tooth and potentially better bond strength over time (less degradation of bond). In general, today’s dental restorative materials are not considered to be bioactive . Currently, only a few attempts to develop bioactive dental materials have been published based on either calcium aluminate (ceramics) or via addition of calcium phosphates to resin based materials .
The aim of the present work was to perform the first in vitro evaluation of a new interfacial bond-promoting material-and-method concept for on-demand long term bacteria inhibition in dental restoration procedures. For this purpose we investigate the performance of a novel dental adhesive containing TiO 2 nanoparticles as photocatalytic bactericidals, establish the bioactive nature of such a material, and study the effect of the nanoparticle content on the mechanical strength of the adhesive. We show for the first time that it is possible to integrate bactericidal effects with bioactivity in one material-and-method system for dental restorations.
2
Materials and methods
2.1
Photocatalytic activity of TiO 2 nanoparticles and nanoparticle/adhesive mixture
Commercially available TiO 2 nanoparticles were used in this study (P25, lot number 4166031598, Degussa AG, Frankfurt/Main). Degussa P25 nanoparticles were used as they have a high photocatalytic activity and are recognized as the standard by which other titania powders are measured . According to the manufacturer and several previous studies , the P25 powder consists of particles with average sizes between ∼25 and 85 nm, and contains anatase and rutile phases in a ratio of about 3:1. Electron microscopy studies performed by us (not shown) support previous findings regarding the size of the particles with the major part of the particles confined in aggregates. For the photocatalytic activity studies, 50 ± 5 mg of the powder were dispersed in 45 ml of rhodamine B solution (5 × 10 −6 M, pH ∼ 6, Sigma). Dyes such as rhodamine B are often used to assess the efficiency of photocatalysts since free radicals generated from the photocatalytic process degrade the dye molecules, which can be easily monitored using UV–vis absorption to measure the loss of color of the solution. The dispersion was irradiated with UV light (Philips, TL/10 UV-A, 360–380 nm, 15 W) while stirring with a magnetic bar. The UV light intensity was 1.0 mW/cm 2 , measured using an UV light meter (UV-340, Lutron) at the surface of the dispersion. Aliquots were collected at various time intervals to monitor the time dependence of rhodamine concentration using a UV–vis spectrophotometer (UV-1650pc, SHIMADZU). Samples were centrifuged to remove the nanoparticles and the absorbance at 554 nm was measured.
The nanoparticle-containing adhesives (hereafter referred to as the NP adhesives) were made by mixing the TiO 2 nanoparticles with the commercially available dental adhesive Adper TM Scotchbond TM 1XT (3M ESPE, St. Paul, Germany) to four different batches containing 5, 10, 20 and 30 wt% of P25 particles. The NP adhesives were then spread in a thin layer on plastic disks having an area of 1.2 cm 2 and subsequently light-cured according to the adhesive manufacturer’s instructions. A control sample consisting of the pure adhesive (0% P25) was prepared in an identical manner to the NP adhesives. Prior to testing the photocatalytic reaction rates, the samples were pre-soaked overnight in a rhodamine B solution to reduce effects of absorption of the dye into the samples. Subsequently, samples were placed into 5 ml of rhodamine B solution (5 × 10 −6 M, pH ∼ 6) and irradiated with the UV light (Philips, TL/10 UV-A, 360–380 nm, 15 W) while gently agitating. The intensity of the UV light at the sample surface was 1.1 mW/cm 2 . Aliquots were collected at various time intervals to monitor the time dependence of rhodamine concentration by measuring the absorbance at 554 nm.
2.2
Bactericidal tests with UV irradiation
Gram-positive Staphylococcus epidermidis (CCUG 18000A) was employed as model bacteria in the bactericidal tests. S. epidermidis is part of the human skin flora, has a rather low requirement of nutrition and forms biofilms having a high degree of antibiotic resistance , which makes S. epidermidis an interesting target for non-antibiotic bactericidal treatment development. In addition, this bacterium is a common cause of medical device-associated infections . S. epidermidis was inoculated into 40 ml of LB broth culture medium (Difco, Becton, Dickinson and Company, Sweden) and placed in a sterilized 200 ml flask and overnight cultured at 37 °C while gently agitating. Bacteria were collected by centrifugation (5000 rpm, 8 min, EBA 30 Hettich, Tuttlingen, Germany) and re-suspended in 300 μl of sterile distilled water. Bacterial suspensions (30 μl each) were spread on the sample plates to be tested. The sample plates were prepared as described above in the photocatalytic activity testing using 0 wt% NP (pure adhesive control), 10 wt% NP and 20 wt% NP, except that the samples covered an area of 4 cm 2 on glass plates. The NP adhesives were imaged by Scanning Electron Microscopy (SEM) with a LEO 1550 SEM (Zeiss, Oberkochen, Germany) using the in-lens detector and 10 kV acceleration voltage.
Two different UV light intensities were used to study the bactericidal properties of the NP adhesives listed in Table 1 ; a low intensity UV light source (Philips, TL/10 UV-A, 360–380 nm, 15 W) producing a UV intensity at the sample surface of 1.2 mW/cm 2 , and a stronger UV light source (Philips, HPA S400, 400 W) used together with a filter that blocks short wavelength UV light below 320 nm, thus producing a UV-A intensity at the sample surface of 7.5 mW/cm 2 . The given UV intensities were measured at the sample surfaces with a UV light meter (UV-340, Lutron). The low intensity UV source was employed for 30 or 120 min, while the high intensity UV source irradiated the samples for 7 or 10 min. The controls for the low and high intensity UV tests were only exposed to ambient indoor fluorescent lighting conditions for 120 and 10 min, respectively. For the 120 min irradiated samples, a drop of water was spread on the bacteria-containing area every 30 min to compensate for loss of water due to evaporation. After all tests (7, 10, 30 and 120 min), the bacteria were collected by rinsing with 20 μl of water to re-suspend the bacteria from the surface. This procedure was carried out twice on each sample surface. The collected bacteria were then transferred to a piece of blood agar culture (Columbia bloodagar base (Acumedia, USA) with 5% defibrinated horse blood, National Veterinary Institute, SVA, Sweden) and cultured overnight at 37 °C prior to a check for colony formation units (cfu).
| Surface | Sample name | UV irradiation time [min] |
|---|---|---|
| Glass plate | I low -0 | 0 |
| I low -30 | 30 | |
| I low -120 | 120 | |
| I high -0 | 0 | |
| I high -7 | 7 | |
| I high -10 | 10 | |
| 10% P25 TiO 2 NP in adhesive | II low -0 | 0 |
| II low -30 | 30 | |
| II low -120 | 120 | |
| 20% P25 TiO 2 NP in adhesive | III low -0 | 0 |
| III low -30 | 30 | |
| III low -120 | 120 | |
| III high -0 | 0 | |
| III high -7 | 7 | |
| III high -10 | 10 | |
The samples and UV irradiation times used in the bactericidal test were as listed in Table 1 . Tests were performed in triplicates for each nanoparticle concentration under study and each UV intensity used.
2.3
Mechanical testing of tensile bond strength of NP adhesive
In order to study the effect of addition of TiO 2 nanoparticles on the bond strength of the NP adhesive towards hydroxylapatite, tensile strength measurements were performed on samples consisting of a hydroxylapatite tablet bonded with the NP adhesive to a dental filling composite (Filtek Z250, 3M ESPE, St. Paul, Germany). This arrangement mimics the bonding of a composite restoration filler to the tooth.
The analyzed NP adhesives were made in an identical manner to those used for the photocatalytic activity measurements to three different batches containing 10 wt%, 20 wt%, and 30 wt% of P25 particles. Pure adhesives (0% nanoparticle content) were used as controls.
The adhesive was spread over a circular area, 4 mm in diameter, onto the top surface of the HA tablet, cf. Fig. 1 a . The polymerizations of the adhesive and the dental filling material were performed using light curing according to the instructions of the manufacturers. The bonded structures were stored in water for at least 30 min before the tensile tests. The tensile strength measurements were carried out using a universal mechanical tester (Instron 5544, Instron, USA) with two custom made holders, Fig. 1 b, using a crosshead speed of 1 mm/min.
The mechanical tests were performed with n = 12 and a one-way ANOVA analysis of the data was performed to evaluate if the nanoparticle content in the adhesive significantly influences the bonding strength between the HA tablet and the NP adhesive.
2.4
Bioactivity tests
To assess the bioactivity of the NP adhesive, three different types of NP adhesives containing 20 wt% TiO 2 nanoparticles were tested: (i) as-synthesized samples with no further surface treatment; (ii) samples that were lightly abraded with 1200 grit silicon carbide sandpaper (3M, USA) after light curing, and iii) samples that were chemically etched with acetone (99.5%, Sigma–Aldrich Company Ltd., Germany) after light curing. Polishing and etching were used as methods to expose nanoparticles to the surface by removing the resin coating. Pure adhesives (0% nanoparticle content) were used as negative controls. Three samples were tested under each of the three conditions listed above.
Subsequent to the surface treatments (if any), the samples were cleaned by rinsing with deionized water. After cleaning, the samples were immediately immersed in 50 ml centrifuge tubes containing 45 ml of pre-heated (60 °C) simulated body fluid (SBF, Dulbecco’s Phosphate Buffered Saline, containing calcium chloride and magnesium chloride, Sigma–Aldrich Company Ltd.) and incubated at 37 °C for 7 days. The samples were then removed from the SBF and thoroughly rinsed with deionized water. After drying with nitrogen, a small piece of each sample was removed for inspection with a SEM. The samples were sputter coated with an approximately 10 nm gold/palladium layer for improved imaging (Au/Pd sputter coater, Polaron SC7640, Thermo VG Scientific, England) and SEM images were recorded with a LEO 1550 SEM (Zeiss, Oberkochen, Germany) using the in-lens detector and 10 kV acceleration voltage.
2
Materials and methods
2.1
Photocatalytic activity of TiO 2 nanoparticles and nanoparticle/adhesive mixture
Commercially available TiO 2 nanoparticles were used in this study (P25, lot number 4166031598, Degussa AG, Frankfurt/Main). Degussa P25 nanoparticles were used as they have a high photocatalytic activity and are recognized as the standard by which other titania powders are measured . According to the manufacturer and several previous studies , the P25 powder consists of particles with average sizes between ∼25 and 85 nm, and contains anatase and rutile phases in a ratio of about 3:1. Electron microscopy studies performed by us (not shown) support previous findings regarding the size of the particles with the major part of the particles confined in aggregates. For the photocatalytic activity studies, 50 ± 5 mg of the powder were dispersed in 45 ml of rhodamine B solution (5 × 10 −6 M, pH ∼ 6, Sigma). Dyes such as rhodamine B are often used to assess the efficiency of photocatalysts since free radicals generated from the photocatalytic process degrade the dye molecules, which can be easily monitored using UV–vis absorption to measure the loss of color of the solution. The dispersion was irradiated with UV light (Philips, TL/10 UV-A, 360–380 nm, 15 W) while stirring with a magnetic bar. The UV light intensity was 1.0 mW/cm 2 , measured using an UV light meter (UV-340, Lutron) at the surface of the dispersion. Aliquots were collected at various time intervals to monitor the time dependence of rhodamine concentration using a UV–vis spectrophotometer (UV-1650pc, SHIMADZU). Samples were centrifuged to remove the nanoparticles and the absorbance at 554 nm was measured.
The nanoparticle-containing adhesives (hereafter referred to as the NP adhesives) were made by mixing the TiO 2 nanoparticles with the commercially available dental adhesive Adper TM Scotchbond TM 1XT (3M ESPE, St. Paul, Germany) to four different batches containing 5, 10, 20 and 30 wt% of P25 particles. The NP adhesives were then spread in a thin layer on plastic disks having an area of 1.2 cm 2 and subsequently light-cured according to the adhesive manufacturer’s instructions. A control sample consisting of the pure adhesive (0% P25) was prepared in an identical manner to the NP adhesives. Prior to testing the photocatalytic reaction rates, the samples were pre-soaked overnight in a rhodamine B solution to reduce effects of absorption of the dye into the samples. Subsequently, samples were placed into 5 ml of rhodamine B solution (5 × 10 −6 M, pH ∼ 6) and irradiated with the UV light (Philips, TL/10 UV-A, 360–380 nm, 15 W) while gently agitating. The intensity of the UV light at the sample surface was 1.1 mW/cm 2 . Aliquots were collected at various time intervals to monitor the time dependence of rhodamine concentration by measuring the absorbance at 554 nm.
2.2
Bactericidal tests with UV irradiation
Gram-positive Staphylococcus epidermidis (CCUG 18000A) was employed as model bacteria in the bactericidal tests. S. epidermidis is part of the human skin flora, has a rather low requirement of nutrition and forms biofilms having a high degree of antibiotic resistance , which makes S. epidermidis an interesting target for non-antibiotic bactericidal treatment development. In addition, this bacterium is a common cause of medical device-associated infections . S. epidermidis was inoculated into 40 ml of LB broth culture medium (Difco, Becton, Dickinson and Company, Sweden) and placed in a sterilized 200 ml flask and overnight cultured at 37 °C while gently agitating. Bacteria were collected by centrifugation (5000 rpm, 8 min, EBA 30 Hettich, Tuttlingen, Germany) and re-suspended in 300 μl of sterile distilled water. Bacterial suspensions (30 μl each) were spread on the sample plates to be tested. The sample plates were prepared as described above in the photocatalytic activity testing using 0 wt% NP (pure adhesive control), 10 wt% NP and 20 wt% NP, except that the samples covered an area of 4 cm 2 on glass plates. The NP adhesives were imaged by Scanning Electron Microscopy (SEM) with a LEO 1550 SEM (Zeiss, Oberkochen, Germany) using the in-lens detector and 10 kV acceleration voltage.
Two different UV light intensities were used to study the bactericidal properties of the NP adhesives listed in Table 1 ; a low intensity UV light source (Philips, TL/10 UV-A, 360–380 nm, 15 W) producing a UV intensity at the sample surface of 1.2 mW/cm 2 , and a stronger UV light source (Philips, HPA S400, 400 W) used together with a filter that blocks short wavelength UV light below 320 nm, thus producing a UV-A intensity at the sample surface of 7.5 mW/cm 2 . The given UV intensities were measured at the sample surfaces with a UV light meter (UV-340, Lutron). The low intensity UV source was employed for 30 or 120 min, while the high intensity UV source irradiated the samples for 7 or 10 min. The controls for the low and high intensity UV tests were only exposed to ambient indoor fluorescent lighting conditions for 120 and 10 min, respectively. For the 120 min irradiated samples, a drop of water was spread on the bacteria-containing area every 30 min to compensate for loss of water due to evaporation. After all tests (7, 10, 30 and 120 min), the bacteria were collected by rinsing with 20 μl of water to re-suspend the bacteria from the surface. This procedure was carried out twice on each sample surface. The collected bacteria were then transferred to a piece of blood agar culture (Columbia bloodagar base (Acumedia, USA) with 5% defibrinated horse blood, National Veterinary Institute, SVA, Sweden) and cultured overnight at 37 °C prior to a check for colony formation units (cfu).
