J. Cosmet. Sci.) 55, 163-176 (March/April 2004) Salivary films on hydroxyapatite studied by an in vitro system for investigating the effect of metal ions and by a quartz-crystal microbalance system for monitoring layer-by-layer film formation Y. TANIZAWA, N.JOHNA, Y. YAMAMOTO, and N. NISHIKAWA, Analytical Research Center, Lion Corporation, Hirai, Edogawa-ku, Tokyo 132-0035, Japan Accepted for publication January 26, 2004. Synopsis The salivary film or the acquired pellicle is a protein film formed initially on the enamel surface of teeth. Such a film plays an important role in enamel protection, but is also an initial substructure for the formation of plaque and the cosmetically undesirable colored stain. The composition and the structure of the film are still essentially unknown because of the difficulty of its isolation for characterization. The purpose of this study was to investigate the effect of some metal cations on the salivary film or the pellicle formation, and also to clarify the mechanism of development. First, using infrared spectroscopy (IR) and X-ray photoelec­ tron spectroscopy (XPS), the in situ-farmed film in the mouth was confirmed to contain selectively adsorbed well-known proteins. Then, in vitro studies have demonstrated that Ca2 + ions enhance film formation at the initial stage in virtue of Ca bridging and, interestingly, that Mg2 + ions oppositely inhibit the formation. Furthermore, the quartz-crystal microbalance (QCM), utilized successfully for the first time to study the salivary film, has shown the possibility of an alternate accumulation mechanism by which the surface charges on the film are effectively reversed by the opposite charged proteins. INTRODUCTION Extrinsic dental stains are cosmetically undesirable colored deposits (1,2) that lie on the surface of the teeth. The prevention and the removal of the stains by abrasive toothpastes have been investigated (3-6). The dental stains are often associated with culculus, plaque (7), and adsorbed salivary film materials termed the acquired enamel pellicle (8), though the mechanism of formation is still speculative. In some cases, substances like tea and coffee incorporated into the pellicle produce the staining as a result of their inherent colors. The pellicle materials are also stained as the result of aging and chemical interactions (3,9). Therefore, in the present work attention has been directed to the salivary film or pellicle. The secretion from human salivary glands contains a number of proteins or glycoproteins having a strong affinity for enamel and hydroxyapatite (HAP). These proteins selectively 163

164 JOURNAL OF COSMETIC SCIENCE adsorb on the HAP surface, followed by the pellicle formation (10). The ultrastructural localization of acidic proline-rich proteins (PRPs), histatin and statherin, has been demonstrated to exist in the acquired pellicles (11). A group of PRPs is one of the major organic compounds in saliva (12). These proteins selectively adsorb on tooth surfaces (10) and delay acid diffusion (13), and, at the same time, prevent epitactic crystallization on the enamel surface (14). A review (15) has gone into detail about saliva and the dental pellicle. A number of researchers have previously studied the pellicle by a variety of analytical techniques such as infrared spectroscopy (IR) (16), X-ray photoelectron spectroscopy (XPS) (17), laser microscopy (18), auger electron spectroscopy (19), wavelength dispersive spectroscopy (20), and ellipsometry (21). Among those techniques, XPS is useful in characterizing the outermost surface of materials. The previous XPS study, however, determined only the amount of limited elements like Ca, P, 0, C, and N on the saliva-coated enamel (17). Amino acid analysis has also been performed (9,12,22), whereby the composition of a two-hour pellicle was characterized by a high content of serine, glycine, and glutamic acid instead of proline, which is a major amino acid in the whole saliva. Nevertheless, amino acid analysis has given little information about specific proteins adsorbed on the dental enamel or about the adsorption mechanism. In the present in situ study, we have confirmed that the salivary proteins selectively adsorb on HAP. However, we primarily had been interested in the mechanism whereby the salivary proteins adsorb on HAP and grow into the accumulated pellicle layers. Therefore, we have quantitatively investigated the in vitro-formed salivary film on HAP, and clarified the effects of some metal ions added to saliva on the formation of the film. Furthermore, the use of a quartz-crystal microbalance (QCM) mass-sensitive detection tool in the nanogram level has provided evidence that the electrostatic interactions between cationic and anionic proteins are involved in the alternate adsorption and the accumulation of the salivary film. We have used the QCM successfully for the first time in the field of oral science, and found it a powerful technique. MATERIALS AND METHODS IN SITU FORMATION OF SALIVARY FILM ON HAP DISKS An in situ device, comprised of a silicone rubber appliance and HAP disks simulating tooth enamel, was used for the generation of salivary film in the mouth. The HAP disks were sintered-pure hydroxyapatite (Ca/P molar ratio: 1.67 relative density: over 99% 7 mm in diameter and 4 mm in thickness), obtained from Asahi Optical Co., Ltd, Tokyo, Japan. The disks were polished using a rotary polisher to give a mirror finish. The degrees of roughness (Ra) measured by an atomic force microscope (AFM) were of the order of 30--40 nm. The devices were placed in the mouth of each volunteer to collect the salivary film material. During the collection no food or beverage was taken. After given times, the devices were removed from the mouths, and then the HAP disks were rinsed with distilled water, dried, and followed by examination using IR and XPS. CHARACTERIZATION OF IN SITU-FORMED SALIVARY FILM ON HAP DISKS Attenuated total reflection infrared spectroscopy (ATR-IR) was employed to characterize the products. All infrared spectra were obtained on a JEOL JIR2000 spectrometer ,,