STUDY OF SALIVARY FILMS ON HYDROXYAPATITE 173 for the behavior in the whole saliva solutions: Mg2 + ions inhibit the protein adsorption. Divalent cations such as Mg2+ and Ca2 + ions are supposed to react equally with nega­ tively charged ionic species in saliva to form cation-anion complexes. The difference in the effects of these cations on the complex formation was then exam­ ined, as shown in Figure 9. The concentration of the cations added to saliva was 200 mmol/1. Absorbance at 500 nm was monitored while stirring the solution for 240 minutes. The linear increase in absorbance was observed for the Ca2 + -added saliva solution, but no significant changes were observed in the Mg2+-added solution. These phenomena are probably attributed to the difference in the affinities of these cations for the salivary proteins, or to the difference in the solubilities of cation-protein complexes. The point is whether these Ca2 + complexes adsorb directly on the HAP surface to form pellicle. As shown in Figure 6, however, the adsorption of proteins does not increase linearly after reaching the maximum value, despite the further addition of Ca2 + ions. Judging from the results shown in Figures 6 and 9, it is unlikely that the aggregation of the complex formed by the added Ca2 + ions in saliva solution contributes directly to the increase of adsorbed proteins on the HAP surface. The added Ca2+ ions probably increase the available Ca2 + sites on the HAP surface for the adsorption of acidic proteins and also enhance the calcium bridging between these adsorbed proteins. An attempt to assemble the protein films on the QCM electrode has been made by several researchers (37-40). We tried to make use of the QCM to investigate the mechanism of pellicle accumulation. Mandel et al. (41) reported that the parotid gly­ coproteins are mostly cationic and that the submandibular glycoproteins are anionic. The present QCM studies demonstrate that the surface charges of the films deposited from parotid and submandibular saliva are cationic and anionic, respectively, and that the charged proteins adsorb electrostatically on the oppositely charged surface. Thus, QCM, the first-utilized methodology for the study of salivary film, has rationalized the accu­ mulation mechanism. An adsorption and accumulation model based on the present results is illustrated in Figure 10. The Ca2+ and PO/- sites on the HAP surface are supposed to be the major binding sites for the acidic and the basic proteins, respectively. Negatively charged groups like the carboxyl group interact directly with the Ca2 + sites, and the addition of ,-.. 0 0 .,,_.. cd 0. 1 0.08 0.06 0.04 0.02 0 0 H-8-t::::::::::___:W ��H=�H���H 50 100 150 200 Time(minutes) 250 Figure 9. The effect of added divalent metal ions on complex formation with negatively charged salivary species. 0: Ca 6: Mg D: blank.

174 JOURNAL OF COSMETIC SCIENCE (adsorption) (desorption) (accumulation) Saliva Figure 10. Schematic simplified illustration of protein adsorption and desorption via competitive adsorp­ tion and accumulation and via alternate adsorption on the HAP surface in saliva solution. extra Ca2 + ions enhances the indirect binding of negatively charged groups to the HAP by virtue of a calcium bridge. The Mg2 + and Na+ ions also adsorb to HAP, but probably have a much lower affinity for acidic proteins than Ca2 + ions have. This mechanism well explains the results in the acidic protein-like albumin shown previously in Figure 6(a). In whole saliva solutions, however, Mg2 + ions inhibit the adsorption of protein, as shown in Figure 6(6). This result requires another interpretation for the interactions in the whole saliva solutions, where the more complicated interactions are expected to occur. The whole saliva contains not only the acidic but the basic proteins, which adsorb to phosphate sites of HAP. Even in such whole saliva solutions, Ca2 + ions enhance the adsorption of the acidic proteins in the same manner as in the albumin. But Mg2 + ions, which have low affinity to proteins, are rather likely to displace the basic proteins by competitive adsorption. Once the anionic proteins adsorb on the HAP surface, the adsorption of cationic salivary proteins follows, and vice versa, where the surface charges are effectively reversed and the accumulation of salivary components such as proteins and glycoproteins follows. In the mixed whole saliva, however, the alternate adsorption may not proceed easily. Of course, the present results do not necessarily rule out the possi­ bility that protein aggregation in saliva causes the accumulation on the enamel surface. Furthermore, bacteria may also be responsible for the pellicle thickening that is the first step in the formation of the undesirable plaque and colored stain. CONCLUSION In conclusion, this study indicates that the formation of pellicle-like film could be influenced by coexistent metal cations in different manners, and further demonstrates that the development of pellicle-like film could be enhanced by the alternate adsorption of the oppositely charged constituents. In either case, the electrostatic interactions could play an important role. These in vitro results, however, do not necessarily provide the evidence that exactly the same mechanism is involved in the actual oral environments.