Root surface caries is an economically important disease in Western industrialised nations and appears likely to become more so in future due to demographic changes and improvements in oral hygiene. There are a variety of model systems which have been used to study root surface caries including; in situ studies, experimental laboratory animals, the use of pure acids in vitro and the use of bacterial cultures in vitro. However the published data fail to present a clear and definitive picture of the role that different bacterial species play or of the biochemical stages in the demineralisation of root surfaces by bacteria. Therefore it was decided to develop a novel in vitro model to study this problem. The first system investigated was the Millicell-HA tissue culture insert (Millipore, U.K.), which consisted of a polystyrene cylinder sealed at one end with a 0.45mum pore-size membrane filter. These units are relatively cheap, easy to handle, provide a stable surface to support bacteria and also keep the bacteria separate from their broth supply to facilitate collection of samples for analysis. In chapter 3 a series of experiments is described in which three bacterial species - Streptococcus mutans, Lactobacillus casei and Actinomyces viscosus - were selected for study on the basis of previous scientific reports. Relatively reproducible films of the three test species were cultured in Millicell- HA inserts, with no significant differences between the retrievable viable counts of each species whether as pure or mixed cultures. Furthermore scanning electron micrographs confirmed that both S. mutans and A, viscosus could be cultured as healthy biofilms in the inserts, although technical difficulties precluded this conclusion for L. casei. Moreover there appeared to be a maximum attainable population density of approximately 108 c.f u./cm2, which was independent of the initial inoculum density. However, when the incubation period was extended it was found that S. mutam passed from the Millicell-HA inserts into the nutrient broth phase by day 4 of incubation, although the population densities of both L. casei and A. viscosus remained stable for 14 days. It was therefore decided to discontinue work on the Millicell-HA model system. The second system investigated was the Ultrafree-CL ultrafiltration unit (Millipore, U.K.), which is structurally similar to the Millicell-HA insert but available with smaller diameter pores. Chapter 4 characterised the properties of the model, which included the following; (i) reproducible films of the three test species could be grown as pure or mixed cultures with no significant differences between the final viable counts and (ii) S. mutans was retained by the filter membrane with a pore size of 0.22mum for around 7 - 8 days and for at least 21 days by a 0.1 mum pore size. Since transverse sections of S. mutans films in Ultrafree-CL units with 0.22mum pores showed bacteria invading the membrane filter structure it is likely that this is the route of spread. Populations of L. casei remained stable within the Ultrafree-CL units for up to 21 days, whilst A. viscosus viable counts tended to decline between days 6 and 21 days. Furthermore there appeared to be a maximum population density of approximately 107 c.f.u/cm2, which was independent of the viable counts in the initial inoculum. However, when the films were cultured with sucrose solution or distilled water instead of Todd Hewitt broth (T.H.B.) there were substantial decreases in viable counts, although these could be reversed by returning the films to T.H.B. This suggested that transfer from sucrose back to T.H.B. could allow one film to be used for a series of studies with few adverse effects on the overall viable counts. In chapter 5, preliminary experiments are described which sought to determine whether the test bacterial species could cause demineralisation of root surfaces or hydroxyapatite powder within the Ultrafree-CL model system. The data indicated that they did indeed cause calcium release from mineral, with the rank order of S mutans ~ L casei > A. viscosus, whilst lactate was usually the predominant anion. These results concur in general with the available literature. However, there was substantial variation in both the acid anion production and calcium release data between repeat experiments which indicated that modifications to the model system may be required before it can be successfully employed for demineralisation experiments. In chapter 6 a range of modifications to the Ultrafree-CL system are described which would help to reduce the variation to acceptable levels and allow the model to be employed to investigate root surface caries further.
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