Who is not afraid of the dentist? Or to be more specific. . . who is not afraid of the dental drill? Caries is a common ailment, that is caused by bacteria in dental plaque. Cavities will form when you do not properly remove the plaque on your teeth. The improvement of caries prevention and treatment techniques is a major issue in dentistry. Preparation of cavities prior to filling is presently done by removing infected and non-remineralizable or demineralized tissue by means of mechanical drilling. During mechanical drilling heating takes place and vibrations are induced. This can cause pain sensation in the patient. Moreover, drilling is often destructive: an excess of healthy tissue must be removed to ensure that the cavity is free of bacteria. In addition, the remaining tooth structure is weakened and prone to fractures. This thesis is about the use of non-thermal atmospheric plasmas in dentistry. The goal of the PhD project is to clean dental cavities in a non-destructive and painless way. The idea is to inactivate bacteria in plaque to stop caries. This cleaning can be done by use of a non-thermal atmospheric micro-plasma. A plasma needle, which is a hand operating tool, is used to generate the non-thermal atmospheric micro-plasma. The plasma needle is a portable device consisting of a tungsten needle confined in a Perspex tube. A radio-frequency voltage of 13.56 MHz is applied at the sharpened tip of this needle to ignite the plasma. The plasma is ignited in helium with a little admixture of air at atmospheric pressure. But what is a plasma and why do we want to use a plasma? A plasma, which is generated by an electrical discharge in a gas, contains free electrons and ions, various active species (e.g. atomic or molecular radicals, for example O and OH radicals, and excited molecules) and energetic UV photons. All these species play an important role in sterilization. The sterilizing properties of plasmas are wellknown and extensively described in literature. Non-thermal atmospheric plasmas are for example used in the sterilization of medical instruments, but also in biological warfare (e.g. against Anthrax). Non-thermal atmospheric micro-plasmas operate at room temperature and do not cause pain and bulk destruction of the tissue. Plasmas can treat and sterilize irregular surfaces; therefore they are very suitable for decontaminating dental cavities as a result of caries. The advantage of this novel tissue-saving treatment is that although the plasma treatment itself is a superficial treatment, the active plasma species it produces can penetrate into hollows, such as cavities. In contrast to dental lasers, plasmas can access small irregular cavity and fissure spaces. Moreover, the use of plasmas is relatively inexpensive. In the future plasmas can also be used in other dental interventions than just caries treatment. One can think of the plasma use in root canal treatments, in periodontitis or in superficial cleaning of dental tools. There are many applications possible. First the project started with creating a radio-frequency plasma at the tip of a needle. The first needle was confined in a closed system filled with helium. But this closed system could not be applied in the mouth, so a portable open system was developed. The plasma needle was born. First we characterized the plasma needle in terms of dissipated and emitted power per unit surface (power outflux) (Chapter 3). A calibrated thermal probe was used to determine the power emitted from the plasma towards treated surfaces. Transmission of the emitted power through various media was studied for a broad range of plasma conditions. These data gave insight into various contributions to the power outflux, as well as the penetration depth of the plasma into treated objects. The power outflux is shown to be a very important parameter, which determines the performance of the plasma tool. Then the temperature in the tooth during plasma treatment and the efficiency of the plasma in killing bacteria were investigated (Chapter 4). We established whether the plasma needle can be safely applied to a tooth. Temperature measurements were performed inside the pulpal chamber using extracted human third molars. A thermosensor was inserted into the pulp chamber and the temperature was recorded during plasma needle treatment. The effects of different treatment times on the survival of Escherichia coli were studied. Bacterial viability was substantially reduced after exposure to the plasma. The plasma operating conditions were optimized for bacterial deactivation (Chapter 5). Plasma power, treatment time and needle-to-sample distance were varied. E. coli films plated on agar dishes were used as a model system for this optimization. Plasma treatment of E. coli films results in formation of a bacteria-free void with a size up to 12 mm. 104 – 105 colony forming units are already destroyed after 10 s of treatment. Prolongation of treatment time and usage of high powers do not significantly improve the destruction efficiency: short exposure at low plasma power is sufficient. Furthermore, we studied the effects of temperature increase on the survival of E. coli and compared it with thermal effects of the plasma. The population of E. coli heated in a warm water bath starts to decrease at temperatures above 40 °C. Sample temperature during plasma treatment has been monitored. The temperature can reach up to 60 °C at high plasma powers and short needle-to-sample distances. However, thermal effects cannot account for bacterial destruction at low power conditions. For safe and efficient in vivo application, the sample temperature should be kept low. Thus, plasma power and treatment time should not exceed 150 mW and 60 s, respectively. After this optimization and characterization, we tested the plasma needle for its bactericidal activity against biofilm cultures of a key cariogenic bacterium Streptococcus mutans grown under different sucrose concentrations (Chapter 6). A chlorhexidine digluconate (CHX) rinse was used as a positive antimicrobial reference. Sucrose and frequency of the plasma treatment were shown to have a significant effect on the degree of response to treatment and bactericidal activity. A single plasma treatment of biofilms cultured with no sucrose yielded a bactericidal effect. Single and repeated plasma treatments of biofilms cultured in 0.15 % sucrose only impaired growth. Then we used an easily reproducible model to replicate dental plaque in vitro, the microplate biofilm microcosm (MBM) model (Chapter 7). The effects of the plasma needle and CHX on the growth of MBMs were studied. Plaque biofilms were grown on coverslips in 24-well microplates from enriched human saliva. After treatment, the growth of the biofilms was studied by monitoring the biomass and the pH. Photomicrographs of the structure of the individual biofilms were taken. The differential effect of the treatments among the species was analyzed by the DNA-DNA checkerboard technique. Furthermore, the biofilms were placed in fixative and observed under transmission electron microscope (TEM). The results showed a complex response ofMBMto plasma and CHX treatments. The TEM images showed that the plasma and CHX treatment damaged bacteria in the biofilms. CKB analyses showed that some species were significantly suppressed by the plasma treatment. The CHX treatment visually changed the structure of the biofilms. The pH of the CHX treated samples changed significantly after treatment and went back to the pH value of the blanks. Furthermore, sucrose concentration had a significant influence on the pH, biomass and the species composition of the MBM. Finally, we investigated surface modification of various materials exposed to the plasma needle (Chapter 8). A number of substrates (Perspex and polystyrene) were treated with the plasma needle. The modification of materials was subsequently identified as hydrophilization of the surface, and experimentally validated by water contact angle measurements. Furthermore, we studied the effect of this modification on growth of two bacterial species, E. coli and S. mutans. Bacteria were cultured on treated and non-treated polystyrene 96-wellplates; the growth of E. coli on treated substrates was enhanced, while for S. mutans it was reduced. The results from this PhD project have indicated that the plasma needle is able to inactivate bacteria in a non-destructive and painless way. Therefore, there will be a role for non-thermal atmospheric plasma therapies in dental procedures. However, in this project we did not investigate the effect of plasma on bacteria in vivo. This should be investigated in future studies.
|Qualification||Doctor of Philosophy|
|Award date||17 Oct 2006|
|Place of Publication||Eindhoven|
|Publication status||Published - 2006|