170 JOURNAL OF COSMETIC SCIENCE .s 400 -------------- J � 300� (a)� .tJ � bt)200-� � S 100- ,.C O J-4 � 0 � r:J:J '-IN 0 0 2000 1 4000 1 6000 1 8000 10000 "'d� --- Concentration of albumin( J1 g/15ml) 0.2 0.4 0.6 0.8 Dilution ratios of saliva 1.0 Figure 5. Amount of adsorbed albumin (a) and salivary proteins (b) on HAP powder in 90 minutes as functions of the concentration of albumin and the dilution ratios of saliva. The dilution ratio of unity means undiluted. characteristics similar to those in the albumin solution with regard to the increase in protein adsorption. However, the Mg2 + ions behaved oppositely to the Ca2 + ions, and Na+ ions did not considerably affect protein adsorption. The behavior in the Ca2 + and Mg2 + mixed system was closer to that in the Mg2 + system. These results reveal fundamental differences in the interaction behavior of the tested cations and suggest that the pellicle formation would be enhanced by Ca2 + ions, and that inhibited by Mg2 + ions. The agreement of the results for the three individuals has confirmed the repro­ ducibility of these observations, though the data are not shown here. In the present in vitro experiments, the effects of the reaction times, the protein concentrations, and the metal ions were investigated. None of the in vitro approaches, however, has resulted in a further increase in protein adsorption after reaching saturation, though the pellicle-like film actually accumulates on the enamel surface in the mouth. ADSORPTION OF SALIVARY PROTEINS ON MUCIN-COATED QCM Typical time courses of mass change (�m) on the mucin-coated QCM are shown in Figure 7. The mass increased immediately, responding to the injection of whole saliva and parotid saliva (5 ml) into distilled water (500 ml), and �m reached approximately 200 ng and 130 ng, respectively, by three-time injections for 17 minutes. On the other hand, no change for submandibular saliva occurred to any measurable degree. Then, in order to estimate the surface charge of the adsorbed salivary materials, the PEI and PSS ions were utilized as the standard cationic and anionic polyions, respectively. The

STUDY OF SALIVARY FILMS ON HYDROXYAPATITE 171 � 400 S 350 0 bb 300 'jg' 250 (a) ·a � 200 O 0. 1 0. 2 0. 3 0. 4 Concentration of metal ions(mol/1) 150 r-----------------,,-� (b ) 50..._ ________ ___._ ____ "----� O 0.1 0. 2 0. 3 0.4 Concentration of metal ions(mol/1) Figure 6. The effect of metal ions on the amount of adsorbed albumin (a) and salivary proteins (b). The reaction time is 90 minutes. 0: Ca 6: Mg D: Na ◊: Ca and Mg. electrode covered with the salivary materials was rinsed and transferred into additional freshly distilled water, and then PEI or PSS was injected. The injection of the PEI ions caused a mass increase for the submandibular saliva-coated surface and no mass change for the others. PSS ions, however, behaved oppositely to PEI. Those results suggest that the surface products from parotid and submandibular saliva are cationic and anionic, respectively, and that the surface covered with whole saliva is cationic as a whole, despite a mixture of a number of components. The above-mentioned findings suggest the possibility of alternate adsorption of salivary proteins in the mouth. We then attempted to assemble the pellicle-like structure by alternate adsorption. The results are shown in Figure 8. The mucin-coated gold electrode was immersed alternately in the parotid and the submandibular saliva for 20 minutes, with an intermediate rinse with water. The mass increase demonstrates that the pellicle-like multilayers were constructed from two kinds of salivas. When the electrode was immersed solely in the submandibular, parotid, and whole saliva, however, the mass remained at almost a constant value after the first injection cycle. DISCUSSION The present in situ and in vitro work has confirmed the preferential adsorption of proteins on the HAP surface. These observations have mostly coincided with those of other research-