In vitro and in vivo studies of salivary films at solid/liquid interfaces

Sammanfattning: A conditioning film, the pellicle, of which many salivary proteins are important constituents, covers the surfaces present in the mouth. The pellicle forms in a selective adsorption process, and it has protective and lubricating functions as well as an influence on the adherence of oral microbes that ultimately leads to the development of dental plaque. Understanding the interactions responsible for the selective pellicle formation would make it possible to strive at creating a pellicle that serves its protective and lubricating functions and also promotes a healthy biofilm for the benefit of the individual. The aim of this research was to characterize the adsorption of salivary proteins to different types of substrates, to evaluate the influence of different protein-surface interactions on the adsorption process, and also to assess substrate dependent differences in film composition. Furthermore, as it is well known that complexes form between different salivary proteins and mucins (large glycoproteins) in bulk saliva, the aim was also to study interactions between mucins and other pellicle constituents at the solid/liquid interface. Additionally, the effects of a surfactant, sodium dodecyl sulphate (SDS), on the protein films were investigated, to evaluate the stability of the films and also the possibilities to completely remove the in vivo formed pellicle. The adsorption experiments were performed in vitro using null ellipsometry, by which parameters such as adsorbed amount per unit area and average layer thickness can be obtained. Hydrophilic and hydrophobized silica were used as model substrates. The adsorption behaviour of the cationic, antimicrobial proteins lactoferrin, lactoperoxidase, lysozyme, and histatin 5 indicated that the adsorption on hydrophilic substrates was mainly driven by electrostatics, while on hydrophobized substrates hydrophobic interactions also influenced the adsorption process. Furthermore, it was shown that 12 sequential alternating adsorption of the anionic salivary mucin MUC5B and lactoperoxidase resulted in the build-up of multilayered structures on the surface. The rate of build-up was influenced by the surface characteristics. Sequential adsorption studies showed that neither MUC5B nor human whole saliva (HWS) was able to exchange substantial amounts of the pre-adsorbed anionic pellicle proteins acidic proline-rich protein 1 (PRP-1) or statherin, respectively. The resistance of the adsorbed mixed protein films to SDS elution depended on surface properties as well as on the number of layers adsorbed and adsorbed components. Pre-adsorbed PRP-1 was to some extent protected from SDS elution by the sequential adsorption of MUC5B to the PRP-1 film. Pellicles formed on natural tooth enamel were collected in vivo and investigated using two-dimensional gel electrophoresis (2-DE). Mechanicallyassisted SDS elution was used to collect the in vivo formed pellicle. The effectiveness of the collection procedure was validated in vitro by means of mechanical removal in combination with HCl treatment. The results indicated that rubbing the tooth surfaces with fibre pellets soaked in 0.5 % (w/v) SDS was sufficient to completely remove the pellicle from human enamel. In addition, 2-DE analysis of pellicles formed in vitro on human enamel and the dental materials titanium and poly (methyl methacrylate) (PMMA) showed differences in composition when compared to each other, revealing that the pellicle is influenced by the substrate properties.