Morten Rykke

Rate of pellicle formation in vivo

The acquired enamel pellicle is thought to be the result of a selective adsorption of salivary proteins and to be involved in the protection of the enamel surfaces. The chemical composition of the 2-h acquired enamel pellicle is fairly well established. However, the rate of formation and the amino acid composition of the initially formed enamel pellicle have been little investigated. The aim of this study was therefore to examine the rate of pellicle formation and the amino acid composition of the initially formed enamel pellicle. Samples of human enamel surfaces were carried in the mouth for various periods of time (2.5 min to 10 h). Rate of pellicle formation was indicated as a function of oral exposure time and the time necessary to remove the proteinaceous film from the surfaces by argon ion sputtering. The chemical composition of the initially acquired pellicle was examined by amino acid analyses of pellicle material collected in vivo from enamel surfaces 15 min and 1 h after pumicing, respectively. The pellicle reached an initial thickness in about 2-3 min, at which level it stayed for about 30 min. The thickness of the acquired pellicle then increased to about three times the initial thickness and stayed at that level for the rest of the experimental period (10 h). Amino acid analyses of pellicle material collected after 15 min and after 1 h were different in that the amino acid profiles of the 15-min pellicle only contained traces of proline and arginine. It may be argued that the pellicle formation proceeds in two stages owing to the adsorption of protein aggregates and that the chemical compositions of the pellicles of the two stages differ.

Micelle-like structures in human saliva

Abstract The acquired enamel pellicle can easily be visualized by scanning electron microscopy (SEM) at low magnifications, and some researchers have described the surface morphology of the acquired pellicle as being predominantly globular based on scanning and transmission electron microscopic examinations. This is consistent with the adsorption of salivary protein aggregates in the form of globular or micelle-like structures comprising amphiphilic salivary proteins. The aim of the present study was to demonstrate the presence of and to characterize possible globular structures of human saliva. Parotid saliva was collected from three healthy individuals and examined by transmission electron microscopy, photon correlation spectroscopy (PCS) and zeta potential determinations. TEM examinations (negative staining with a 2% ammonium molybdate solution) demonstrated globular structures in parotid saliva in the size range 150–200 nm. The globular structures appeared to be more clustered (comprising 6–10 globules) with increasing time after sampling. The PCS analyses likewise demonstrated particles in parotid saliva with an initial size in the range 100–150 nm. Unimodal analysis showed that the mean hydrodynamic diameter of the salivary particles increased to about 450 nm 50 min after sampling. Size distribution processor (SDP) analyses, furthermore, indicated the presence of three different size fractions of the salivary particles; one in the size range of about 10 nm, one in the size range of 40–110 nm and one of 240–500 nm. Initial addition of 0.5 ml of 50 mM pyrophosphate solution (PP) to the saliva samples seemed to inhibit the increase in size of the globular structures, which were then less than 50 nm. Addition of PP to the saliva samples 120 min after sampling, disintegrated the globular structures, as the unimodal mean of the particle size decreased from about 450 nm to 100–150 nm. The PCS intensity (counts s −1 ) also decreased. Zeta potential determinations indicated an overall net negative surface charge of the salivary globular structures of about −13 to −17 mV. The study clearly demonstrated the presence of negatively charged globular structures of human parotid saliva in the size range of about 100–500 nm, consistent with micelle-like structures consisting of amphiphilic salivary proteins. Calcium seems furthermore to be of importance in maintaining the structure of these salivary particles.

Zeta Potentials of Human Enamel and Hydroxyapatite as Measured by the Coulter® DELSA 440

The zeta potential of human enamel is of physiological importance for interactions between enamel surfaces and the surrounding aqueous medium of saliva. The zeta potentials of both enamel and hydroxyapatite (HA) have been examined previously by various techniques. In this study, we examined the zeta potential of human enamel and HA using the Coulter® DELSA 440, which, by a laser, makes independent Doppler shift measurements of moving particles in an electric field at 4 different angles, providing advantages over previous techniques. The enamel and HA particles were suspended directly in different phosphate buffers, or first incubated for 2 hrs in parotid (PS) or whole saliva (HWS) and then suspended in the same buffers. The enamel and HA particles exhibited an overall net surface potential of -15 to -30 mV, depending on the buffer content. Incubation in PS and HWS gave less negative potentials of -8 to -14 mV. In our previous studies, the salivary micelle-like structures (SMSs), seen in TEM of parotid saliva, were observed to have a zeta potential of -9 mV (Rykke et al., 1996). The zeta potential determinations in this study support the concept of an adsorption of mostly SMSs to the enamel surfaces, with a change of the zeta potential of the enamel and HA toward that of the SMSs.

Evidence for presence of micelle-like protein globules in human saliva

Abstract It is known that recently formed acquired enamel pellicles show a globular structure. We have suggested that these globules may be salivary micelles, related to the casein micelles found in milk. The aim of the present study was to examine whether human whole saliva contains micelle-like globules. Micelle-like spherical globules with a diameter of 100–300 nm were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and these had an amino acid profile similar to the pellicle formed after 2 h. The globules solubilized lipid-soluble dyes and could be collected from whole saliva after acidification to pH 3 with lactic acid by centrifugation at 3000 × g for 10 min. Milk micelles could be obtained by a similar procedure. Micelles of this kind have a large surface area and a high density of negative charges on the surface. The presence of micelles in saliva and on the enamel could affect the calcium equilibrium in saliva and thus be of physiological significance.

Quantitative and qualitative analyses of human salivary micelle-like globules

In the present study we examined the protein proportion and amino acid profile of the salivary micelle-like globules (SMGs) of human whole saliva and parotid saliva (HWS, HPS). Saliva and SMG samples from each subject (clarified HWS and HPS from 6 subjects, and unclarified HWS from 3 subjects) were analysed for amino acids using standard acid hydrolysis procedures. HPS, clarified HWS and the respective supernatant samples (remaining after removal of the SMGs) were also measured for protein using the micro-Kjeldahl method. SMGs from clarified and unclarified HWS made up 4.7% and 19.7%, respectively, of the total salivary protein based on amino acid analyses. With the micro-Kjeldahl method SMGs from clarified HWS made up 7.3% of the total saliva protein. SMGs isolated from HPS were found in only small amounts. The amino acid profile for the SMGs was strikingly similar to that known for the 2-h pellicle, and differed significantly from HWS or HPS. The results support previous morphological studies indicating that the SMGs represent a major component of the newly formed pellicle.

Zeta potentials of human salivary micelle-like particles

Abstract Zeta potential determinations of recently demonstrated salivary micelle-like structures may provide information about their adsorption potentials to enamel surfaces. The aim of this study was to determine the zeta potentials of the salivary micelle-like structures at different pH levels and to determine their isoelectric points (IEPs). The zeta potentials were determined by measuring the electrophoretic mobilities of the salivary particles in an electric field using an electrophoretic light scattering method. Parotid saliva was collected from four individuals and analyzed separately. The zeta potentials were measured at different pH by acidification with lactic acid. Zeta potential determinations of casein micelles in milk were included as a control. The zeta potential measurements showed an overall net negative surface charge of the micelle-like structures of about −9 mV at physiological pH. On acidification of the saliva the zeta potential gradually increased from pH 5 and at pH 3 the particles became positive. The IEPs ranged from pH 2.9 to pH 3.1. The zeta potential determinations in the present study are consistent with the assumption that the salivary particles exhibit a micelle-like structure which may interact electrostatically with enamel surfaces.

The potential of liposomes as dental drug delivery systems.

The potential of liposomes as a drug delivery system for use in the oral cavity has been investigated. Specifically targeting for the teeth, the in vitro adsorption of charged liposomal formulations to hydroxyapatite (HA), a common model substance for the dental enamel, has been conducted. The experiments were performed in human parotid saliva to simulate oral-like conditions. It was observed, however, that precipitation occurred in tubes containing DPPC/DPTAP or DPPC/DPPG-liposomes in parotid saliva with no HA present, indicating that constituents of parotid saliva reacted with the liposomes. The aggregation reactions of liposome-parotid saliva mixtures were examined by turbidimetry and by atomic force microscopy. Negatively charged DPPC/DPPS and DPPC/PI-liposomes were additionally included in these experiments. The initial turbidity of positive DPPC/DPTAP-liposomes in parotid saliva was very high, but decreased markedly after 30 min. AFM images showed large aggregates of micelle-like globules known to be present in saliva. The turbidity of the various negatively charged liposome and parotid saliva mixtures stayed relatively constant throughout the measuring time; however, their initial turbidities were different; mixtures with DPPC/DPPG-liposomes were the most turbid and DPPC/DPPA-liposomes the least. Pyrophosphate (PP) was added to the various liposome-parotid saliva mixtures to examine the effect of Ca(2+) on the interactions. The effect of PP treatment of the negatively charged liposome-parotid saliva mixtures was most pronounced with DPPC/DPPG-liposome mixtures where it caused a sudden drop in turbidity. For positive DPPC/DPTAP liposome and parotid saliva mixtures, the effect of PP was minimal. These experiments showed that saliva constituents may interact with liposomes. An appropriate liposomal drug delivery system intended for use in the oral cavity seems to be dependent on the liposomal formulation. Based on the present results, negatively charged DPPC/DPPA-liposomes seem to be most suitable for use in the oral cavity as they were found to be the least reactive with the components of parotid saliva.

A clinical and scanning electron microscopic study of a new restorative material for use in posterior teeth

Eighty-two restorations with a new posterior restorative material showed good wear resistance on the occlusal surfaces after 2 years of service. The loss of substance averaged 30-50 micron, which is below the clinically detectable level. Inferior contact points were frequently registered, reflecting the problems with inadequate matrix systems. Clinically relevant problems were all related to the quality of the contact area.

Protein adsorption to hydroxyapatite and to calcium fluoride in vitro and amino acid analyses of pellicle formed on normal enamel and on calcium-fluoride-covered enamel in vivo

Fluoride treatment of enamel has been reported to result in the formation of a layer of a CaF2-like material on the enamel surface. Protein adsorption to enamel is a specific process dependent on the nature of the surface, and little is known about protein adsorption to CaF2. Albumin and lysozyme were adsorbed to hydroxyapatite (HA) and CaF2 powder in vitro, and protein adsorption patterns constructed. In vivo pellicle was collected from three volunteers from fluoride-treated enamel and from normal enamel, and the amino acid compositions analyzed separately. The results showed that CaF2 took up small amounts of proteins as compared with HA. When the CaF2 was pretreated with a phosphate buffer, pH 6.8, the protein adsorption increased markedly. The amino acid analyses showed no major differences in the amino acid compositions between pellicle collected from CaF2-covered enamel and pellicle collected from normal enamel. This lack of difference is presumably due to the adsorption of phosphate ions to the CaF2 crystals and hence changed surface properties.

Polymer coated liposomes for dental drug delivery--interactions with parotid saliva and dental enamel.

The interactions between pectin coated liposomes and parotid saliva and dental enamel were studied to investigate their potential to mimic the protective biofilm formed naturally on tooth surfaces. Different pectin coated liposomes with respect to pectin type (LM-, HM- and AM-pectin) and concentration (0.05% and 0.2%) were prepared. Interactions between the pectin coated liposomes and parotid saliva were studied by turbidimetry and imaging by atomic force microscopy. The liposomes were adsorbed to hydroxyapatite (HA) and human dental enamel using phosphate buffer and parotid saliva as adsorption media. A continuous flow was imposed on the enamel surfaces for various time intervals to examine their retention on the dental enamel. The results were compared to uncoated, charged liposomes. No aggregation tendencies for the pectin coated liposomes and parotid saliva were revealed. This makes them promising as drug delivery systems to be used in the oral cavity. In phosphate buffer the adsorption to HA of pectin coated liposomes was significantly lower than the negative liposomes. The difference diminished in parotid saliva. Positive liposomes adsorbed better to the dental enamel than the pectin coated liposomes. However, when subjected to flow for 1h, no significant differences in the retention levels on the enamel were found between the formulations. For all formulations, more than 40% of the liposomes still remained on the enamel surfaces. At time point 20 min the retention of HM-pectin coated and positive liposomes were significantly higher. It was concluded that pectin coated liposomes can adsorb to HA as well as to the dental enamel. Their ability to retain on the enamel surfaces promotes the concept of using them as protective structures for the teeth.