Antibacter ia l D e n t a l A d hes iv e t h r ough Photocataly s is o f T it aniu m D i oxide Yanl in g C a i Degr e e p r oje c t i n a p pli e d b i o technolo g y , M a st e r o f S c ien c e ( 2 y e ar s ) , 2 0 09 Examensarbe t e i t i l l ämp a d b i o tekn i k 3 0 h p t i l l m a sterexame n , 2 0 09 Biolo g y E d ucati o n C e nt r e a n d D e partme n t o f e n gineeri n g s c i enc e s , U p ps a l a U n iversity Superviso r : K e n W elch Table of Contents Summary ……………………………………………………………… 2 Introduction …………………………………………………………… 3 Results ………………………………………………………………… 10 Discussion …………………………………………………………… 18 Conclusions and Future Experiments………………………………… 22 Materials and Methods ……………………………………………… 23 Acknowledgements …………………………………………………… 28 References …………………………………………………………… 29 1 Summary The system of dental adhesive and restorative composite is considered to be the first choice in the treatment of dental decay. However, because of the shrinkage of the resin composite material and possible residual bacteria in the dental cavity, secondary caries occurs in about 40% of the cases. Much research has been done with the aim of achieving antibacterial properties with both the restorative resin composite and dental adhesives, using strategies such as agent releasing materials and particle mixtures. However, because of the problems of short release times, poor effectiveness in killing bacteria and reduction of bonding strength after incorporation of agents, commercial realizations of this research have been rare. In this project, a novel dental adhesive was made by mixing TiO nanoparticles with a 2 commercially available dental adhesive. A photocatalytic reaction occurs when TiO is 2 irradiated under UV light. A series of radicals produced decompose organic molecules and can thus kill nearby bacteria. Several characterization tests were done to determine if this adhesive can kill the bacteria sufficiently and, at the same time, maintain its functionality as a bonding agent. Antibacterial tests showed that an adhesive composed of 20 wt% nanoparticles had a good bacterial killing effect. The mechanical properties of the nanoparticle/adhesive mixture were not significantly changed compared to the pure commercial adhesive. Additionally, the material was found to be not bioactive after one week of PBS treatment. 2 Introduction Dental caries treatment and the challenge    Dental caries, also known as tooth decay, 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 salivary dysfunction, dietary carbohydrates, and so on (Selwitz et al, 2007). Without proper care, caries will progress until cavitations occur (Featherstone, 2000). In clinical practice, the dental cavity is treated by restorative dentistry. Dental amalgam has been used as restorative material for more than a century. Since the 1990s, system of adhesive and resin-based composite became the first choice for restorations instead of dental amalgam. The advantages of the adhesive and resin composite system include the reduced removal of dentin in cavity preparation, flexibility of the material to fit the cavity and better aesthetics through the control of the material color (Roeters, 2004). Many improvements in the material, technique and instruments have been made (Sarrett, 2005). There are two main types of adhesives used today – total-etch and self-etch. With total-etch, the standard procedure for repair of caries is as follows: remove the infected dentin by means of drilling, etch the surface of the cavity wall and rinse, spread two thin layers of adhesive with a specific applicator, light cure the adhesive (polymerization of the resin components initiated by the radicals produced by the photoinitiator), press the restorative material into the cavity and light cure (or let set if self-curing), polish the surface of the restorative bulk and the interface of the dentin-adhesive-resin (3M ESPE, 2004; 3M ESPE, 1998). Self-etch adhesives (see Fig. 1) are also popular in dental clinics because the acidic adhesive can perform the function of etchant. An advantage is that there is no problem of water retention in the etched dentine surface, which often occurs in the rinsing step when the dentist uses a separate etching step (Moszner, 2005). 3 Figure 1. Self-etching dental adhesive monomer with acid group. (Picture resource: Moszner, 2005) However, there are significant problems with present dental restorations such as secondary (recurrent) caries and the resulting need for restoration replacement (Mjör, 1997; Peris, 2007). A main case is 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 (see Fig. 2) (Davidson, 1997). Figure 2. Left: Leakage caused by the polymerization shrinkage and secondary caries. Right: Scanning electron microscopy (SEM) picture of the open dentinal tubules. (Picture source: http://www.uthscsa.edu/mission/spring00/dentresearch.htm; Kubinek, 2007.) Clinical data shows that the average lifetime of a resin restoration is about 8 years and adhesion failure occurs in more than 39% of restorations, most of which are accompanied with secondary caries (Sarrett, 2005). Similar problems occur in 4 orthodontic applications. Evidence shows that the adhesive used to fix the bracket on the enamel actually help the adhesion and accumulation of bacteria (Imazato, 2003). Although it cannot be conclusively shown that the microleakage along the cavity wall is the primary cause of secondary caries (Cenci, 2008), secondary caries are believed to be reduced by minimizing the marginal gap and ensuring better sealing of the exposed dentinal tubules due to the removal of the lesion (Sarrett, 2005; Bergenholtz, 2000; Love, 2002). Dental adhesives  Dental adhesives have a wide range of applications, mainly in orthodontics and restoration. In a typical orthodontic device, a dental adhesive can be used to fix the brackets clamping the teeth. In restorative treatment, the use of dental adhesive and filling composite is the first choice for repair of dental caries. Dental adhesive can also be used for root surface desensitization and bonding of amalgam, porcelain and porcelain veneers on the surface of teeth (Nicholson, 1998; 3M ESPE, 2004). The basic components of the dental adhesive include the resin matrix and photoinitiator. The photoinitiator produces initiator radicals to start polymerization of the resin matrix, which will form a solid network to supply required mechanical and bonding properties (Nicholson, 1998). A traditional photoinitiator used in dental material is camphoroquinone (CQ, see Fig. 3), a visible light sensitive diketone which can absorb light at a wavelength of about 470 nm and produce radicals for polymerization initiation (Cook, 1992). The adhesive exhibits good attachment to both enamel and dentine, of which the mineral phase (hydroxyapatite, Ca (PO ) (OH) ) is 10 4 6 2 approximately 97% and 76%, respectively. Clinically, acidic etchant is used before adhesive application to create higher microporosity within the cavity surface to increase the available surface area for bonding. This etching step allows the adhesive to penetrate into the surface and form inter-locking “resin tags” after curing (Nicholson, 1998). 5 Figure 3. Left: Structure of the camphoroquinone (CQ), also known as 1, 7, 7-trimethylbicyclo (2.2.1) heptan-2, 3-dione. Right: the photoinitiating process of CQ (Picture resource: sci-toys.com/scichem/jqp066/25208.html; Moszner, 2005) The adhesive used in this project was AdperTM ScotchbondTM 1XT Adhesive, manufactured by 3M ESPE. The resin matrix components include HEMA (hydroxyethylmethacrylate), BisGMA (bisphenylglycidyl dimethacrylate), and dimethacrylates (see Fig. 4). A patented photoinitiator system produces initiator radicals that start the polymerization when irradiated under high intensity light at about 470nm (3M ESPE, 2004). HEMA Figure 4. Structure of commonly used monomers for dental adhesives (Picture resource: Moszner, 2005) Antibacterial property of dental adhesive  Because of the high probability of secondary caries, much attention has been focused on improvement of the filling composite and dental adhesive (Imazato, 2003), including the antibacterial effect of the material and inhibition of demineralization (Ahn, 2009). 6 Self-etching dental adhesives contain an acidic functional group in the resin (See Fig. 1), leading to a pH of around 2 to 3 that provides an antibacterial effect due to the low pH (Feuerstein, 2007). However, it is not sufficiently effective to kill the bacteria. A common way to achieve an antibacterial effect is to use an agent-releasing material (Imazato, 2003). Many kinds of agents have been incorporated into the adhesive resin, such as sodium fluoride, dodecylamine, bipyridine and so on (Imazato, 2003). The following paragraphs describe some agents that have been recently investigated. Silver nanoparticles (with diameter less than 5nm) have been added to an orthodontic adhesive. As an antibacterial agent, silver ions are selectively toxic to prokaryotic microorganisms without harming eukaryotic cells. Due to the reduced surface free energy (SFE), bacteria adhesion is decreased and bacterial suspensions containing a silver filled adhesive showed slower bacterial growth (Ahn, 2009). Silver colloids have also been added to restoration composites and showed antibacterial effects (Kassaee, 2008). Chlohexidine (Fig. 5) has proved to inhibit the host-derived degradation of collagen, which is the main organic component of the dentin and enamel. By adding 2 wt% CDA (chlorhexidine diacetate) to the resin adhesive, the adhesive can slowly release chlohexidine and has shown good effect in adhesive-dentin interface preservation (Hiraishi, 2008). MDPB (methacryloyloxy dodecyl pyridinium bromide, see Fig. 5) has been successfully used in adhesives for arresting the residual bacteria after the cavity preparation and for adhesives that penetrate into the gap between the restoration material and cavity wall. The bonding strength of the adhesive was not significantly influenced by the addition of MDPB (Imazato, 2003). The commercially available adhesive Clearfil Protect Bond contains 5% MDPB (Imazato, 2006). CPC (cetylpyridinium, see Fig. 5) is a well known antiplaque agent, which can destroy the cell wall of the bacterial by interrupting the negatively charged cell wall due to the positive charge of CPC. CPC has been added to an adhesive and showed good antibacterial effect. However, this strategy suffers from short release time, about 180 days, with the highest release in the first week (Al-Musallam, 2006). To solve this problem, CPC has been immobilized in a resin and tested. The results showed that bacteria on the surface of the CPC-resin were completely inhibited. However, there is no effect on bacteria not attached to the surface (Namba, 2009). 7 Although several agents have shown antibacterial properties, none claim to have long-lasting effect or completely killing of bacteria. Furthermore, some of the agents even decrease the bonding strength of the adhesive. dodecylamine Chlohexidine MDPB bipyridine CPC Figure 5. Several antibacterial agents that have been investigated in previous research. Photocatalysis of Titanium dioxide  The portion of the electromagnetic spectrum containing ultraviolet light (UV) is subdivided into different regions. In the near to middle UV region we have UVA (400nm- 320nm), UVB (320nm-280nm) and UVC (280nm-100nm). Although extensive exposure to UVA can lead to damage of collagen fibers, it is generally harmless under limited dosages, and does not cause sunburns like UVB does. UVB and shorter wavelengths cause direct damage to DNA and are therefore much more harmful to living organisms. The short wave UVC is generally blocked by ozone in the upper atmosphere, and is widely used for sterilization purposes in medical facilities and disinfection of drinking water as the high energy of the light results in damage to organisms. For the anatase crystalline form of titanium dioxide, irradiation with UVA light having a wavelength less than 385nm can excite electrons above the material’s band gap of 3.2eV and thus generate an electron-hole pair. Reacting with a suitable electron acceptor, like oxygen, the excited electron will produce superoxide ions (O -). The 2 positive hole in the valence band can react with H O or OH- to produce hydroxyl 2 radicals (·OH). Other reactive oxygen species, like hydroxyl peroxide H O and singlet 2 2 8 oxygen can also be generated. The reactive oxygen species and radicals can diffuse and decompose nearby organic molecules into CO and H O. This photocatalytic property 2 2 has been used in solar cells, anti-fouling surfaces, self-cleaning coatings and so on (Maness, 1999). Due to the high redox reaction ability of the photocatalysis products, TiO can be used 2 as a bactericide for the drinking water and indoor environments (Li, 2008). The mechanism is the destruction of the cell membrane or cell wall and causing leakage or structural damage of the cell (Maness, 1999). Research has demonstrated the killing of viruses, Gram-positive and Gram-negative bacteria, and even cancer cells by the photocatalysis of TiO . TiO can even be metal doped to change the band gap energy 2 2 and render TiO photocatalytic under visible light (Li, 2008). 2 Aims of this project  The aim of this project was to investigate a novel dental adhesive made by mixing TiO 2 nanoparticles with a commercially available dental adhesive, AdperTM ScotchbondTM 1XT Adhesive manufactured by 3M ESPE. Initially, three different candidate nanoparticles (NP) were assessed for photocatalytic activity. Included in this goal was the selection of an appropriate assessment technique for photocatalytic activity. Then the TiO nanoparticles showing the best photocatalytic 2 properties were mixed into the commercial dental adhesive. The antibacterial effect was tested using Staphylococcus epidermidis. Characterization of the nanoparticle/ adhesive mixture (referred to as NP/adhesive in this report) was performed, including bioactivity and mechanical strength (tensile bond strength). 9