The carbohydrate composition of experimental salivary pellicles

Abstract. The carbohydrate compositions of pellicles formed in vivo and others formed in vitro from submandibular, parotid, and mixed (submandibular-parotid) saliva were determined using gas-liquid chromatography. Samples of the total pellicles as well as the relatively acid-soluble supernates and acid-insoluble sediments were collected, analyzed, and compared. The differences in carbohydrate composition between the supernates and sediments, particularly in the in vivo and in vitro mixed salivary pellicles, suggest that the total pellicle is composed of more than one component. In each instance obvious differences in composition between the pellicle and the saliva from which it was formed indicated that pellicle formation is a highly selective process.
Glucose: galactose ratios of approximately 1:1 were found in each of the total pellicles. These data suggest that at least one pellicle component is an unusual glycoprotein in that it contains high levels of glucose.
The total submandibular salivary pellicle and both its fractions were remarkably similar in carbohydrate composition to the counterparts formed from parotid saliva alone. The data strongly suggest that there are present in both submandibular and parotid saliva similar glycoproteins that are selectively deposited onto etched enamel as the initial pellicle.     

External radiolabelling of components of pellicle on human enamel and cementum

Enamel and cementum pellicles form by different adsorption of salivary and serum components to the tooth surface. The authors compared the constituents of surface pellicle formed on human enamel and cementum under three conditions: (1) natural pellicle, present on extracted teeth, which was formed by prolonged exposure to human salivary and serum components in vivo; (2) short-term in-vivo pellicle, formed by exposing enamel and cementum slabs to the oral environment for 0-60 min; (3) in-vivo pellicle, formed by incubating enamel and cementum slabs in a 1:1 mixture of parotid and submandibular/sublingual saliva for 0-60 min. Pellicle composition was characterized by external radiolabelling techniques specific for exposed carbohydrate (sialic acid and galactose) and amino-acid (tyrosine) residues. There were differences between cementum and enamel in the electrophoretic profiles of natural-pellicle components; notably, a major 180 kda 3H-labelled sialoglycoprotein, unique to the cementum pellicle, had the same electrophoretic mobility as the low-molecular-weight mucin from human submandibular/sublingual saliva. After alkaline-borohydride treatment, 3H-labelled natural-pellicle oligosaccharides chromatographed in the di- to tetrasaccharide region of a Bio-Gel P-2 column. The most prominently labelled components of short-term enamel and cementum pellicles in vivo and in vitro had the same electrophoretic mobility as the low-molecular-weight salivary mucin. The pellicle components formed in vitro, unlike those formed for the same period of time in vivo, were rapidly desorbed from the cementum, but not from the enamel surface. We conclude that: (1) external labelling techniques are useful for obtaining a profile of pellicle components; (2) submandibular/salivary mucins are major constituents of salivary pellicles on tooth surfaces; (3) glycoproteins that carry low-molecular-weight, sialic-acid-containing saccharides are important determinants of pellicle surface properties [corrected].     

Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro

Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.     

Adsorbed salivary acidic proline-rich proteins contribute to the adhesion of Streptococcus mutans JBP to apatitic surfaces

Experimental pellicles formed on hydroxyapatite (HA) beads from parotid or submandibular saliva promoted the adhesion of Streptococcus mutans JBP cells to a greater extent than did pellicles prepared from buffer, human plasma, or serum. The nature of the salivary components responsible was studied by the preparation of pellicles from fractions of parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Two groups of fractions promoted attachment of the organism. Components migrating in the high-molecular-weight mucin fraction were most effective, but a later-eluting fraction also possessed adhesion-promoting activity. Subfractionation of the latter material indicated that the adhesion-promoting activity was associated with the acidic proline-rich proteins (PRPs). Pellicles prepared from 10-20-micrograms/mL solutions of pure PRP-1 were effective in promoting attachment of S. mutans JBP cells. PRP-3 was less effective, while human salivary statherin, fibrinogen, fibronectin, type 1 collagen, and the amino-terminal tryptic peptide derived from PRP-1 were ineffective. The quantities of 150-residue and 106-residue PRPs and of statherin, which became incorporated into experimental pellicles prepared from saliva, were estimated with use of radiolabeled protein tracers. The data obtained suggest that these proteins compete for similar binding sites on HA, and that their ratios in saliva would therefore influence the quantity of the larger PRPs that become incorporated into the pellicle. Such competition may contribute to the variability observed in the adhesion-promoting activities of different saliva samples.     

Adsorption of human salivary proteins to hydroxyapatite: a comparison between whole saliva and glandular salivary secretions.

The protein compositions of in vitro pellicles formed from whole saliva and parotid and submandibular secretions were determined by use of synthetic hydroxyapatite as a model for dental enamel. The adsorbed and unadsorbed protein fractions were analyzed by amino acid analysis and both anionic and cationic discontinuous polyacrylamide gel electrophoresis. For further characterization of the in vitro pellicle, the adsorbed fractions were subjected to gel filtration on Sephadex G-100 and reversed-phase chromatography on C18 columns. Amylase, acidic and glycosylated proline-rich proteins, statherins, and histatins were identified in the parotid-derived pellicle. Detailed analysis of the statherin-containing fractions resulted in the observation of several statherin-like proteins. The use of cationic gel electrophoresis allowed for the identification of histatin 3 and histatin 5, which have not been previously detected in pellicle formed in vitro. The protein composition of submandibular-derived pellicle was similar to that of parotid-derived pellicle except for the presence of cystatins and the absence of glycosylated proline-rich proteins. In contrast, in vitro pellicle derived from whole saliva exhibited a vastly different composition, consisting primarily of amylase, acidic proline-rich proteins, cystatins, and proteolytically-derived peptides. The results indicate that acidic phosphoproteins as well as neutral and basic histatins from pure secretions selectively adsorb to hydroxyapatite, whereas in whole saliva some of these proteins are proteolytically degraded, dramatically changing its adsorption pattern.     

Concerning the composition and source of the acquired enamel pellicle of human teeth

Natural pellicle was collected from sound, clean enamel of extracted, erupted, permanent human teeth. Short-term experimental pellicles were formed in the mouth and from ductal saliva which had been Millipore^®-filtered to remove any contaminating bacteria. Since much of the organic matter from the enamel surface was soluble in the 2 per cent hydrochloric acid used for collection of the pellicles, all the samples analyzed in this study were of “total” pellicle, including both the acid-soluble and the acid-insoluble pellicle. Unfiltered and filtered saliva samples were analyzed for comparison with the pellicles.

Total natural pellicle had a fairly constant amino-acid composition which was quite different from the analyses of insoluble pellicle reported in the literature and different from the experimental pellicles and saliva analyzed. The total natural and experimental pellicles did not contain appreciable amounts of collagen, enamel protein, eukeratin nor haemoglobin but did exhibit some characteristics of a “soft” keratin.

Formation of the experimental pellicles from saliva was a selective process. The in vivo and in vitro experimental pellicles were similar in composition, indicating that all the constituents necessary for the formation of a short-term pellicle were present in saliva and that bacteria or their products were not essential.     

Experimental salivary pellicles formed on the surface of self-curing resin

The aim of this study was to identify the salivary components present in the pellicles formed on self-curing resin and to investigate the qualitative variations in adsorbed salivary pellicle compositions according to different exposure time to saliva. Experimental pellicles were formed by the incubation of polymerized resin particles with fresh human parotid or submandibular-sublingual saliva for either 20 min or 2 h. Pellicles were extracted using formic acid and lyophilized, they were then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting to identify the adsorbed salivary components. The amino acid profiles of the 2 h-pellicles were analysed and compared with those of fresh glandular salivas. There was a difference in the 2 h-pellicle components on the self-curing resin compared with those of other dental materials as well as tooth enamel. The amino acid profiles of the 2 h-pellicles were also different from those of fresh glandular salivas. In the case of submandibular-sublingual saliva, the components of the 2 h-pellicle showed a different pattern compared with those of the 20 min-pellicle. However, there was no significant difference between the components of the 2 h- and 20 min-pellicles in the case of parotid saliva. A distinct difference was found in the surface binding affinities of immunoglobulin (IgA) from different glandular salivas. The findings of this study provide information concerning the initial bacterial adhesion on the surfaces of self-curing resin.     

Human minor and major gland saliva proteins and ability to mediate Actinomyces naeslundii adherence

Bacteria-binding components and the ability to mediate bacterial adhesion to the tooth surface have been thoroughly studied in major salivary gland secretions. Our knowledge on the bacteria binding activity in minor gland saliva is, however, limited. In this study, proteins were examined in parallel in minor (palatal, buccal and labial) and major (parotid and submandibular/sublingual) salivary gland secretions in one subject using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The adherence of early colonizing Actinomyces naeslundii to pellicles formed from the secretions on hydroxyapatite beads was also examined. Amylase, IgA, proline-rich proteins and the high-molecular-weight glycoproteins, agglutinins, were detected in all saliva tested. Carbohydrate-reactive antibodies recognized the low-molecular weight mucin, MUC 7 in submandibular/sublingual saliva only. A. naeslundii strain 12104 adhered to all pellicles and especially to the buccal gland saliva pellicles. Strain LY7 adhered in highest numbers to the submandibular/sublingual saliva pellicles. It also bound in considerable numbers to parotid and palatal saliva pellicles but not to the ones formed from buccal and labial gland saliva. Our findings indicate that several bacteria-binding components are secreted in both minor and major gland saliva. The adherence-promoting ability of the various gland secretions differs, however.     

Protective properties of salivary pellicles from two different intraoral sites on enamel erosion

The purpose of this study was to investigate the protective effect and ultrastructure of salivary pellicles formed in vivo near the orifices of the ducts of parotid and submandibular/sublingual salivary glands. Pellicles were formed by exposing bovine enamel slabs to the oral environment at the buccal aspect of the upper first molars and at the lingual aspect of the lower incisors in 3 subjects over periods of 24 h. Enamel specimens with and without 24-hour pellicles were immersed in citric acid (0.1 and 1%) for periods ranging from 30 s to 5 min, and processed for measurement of surface microhardness (SMH) and transmission electron microscopy (TEM). In comparison to uncovered enamel specimen significantly less decrease in SMH due to acid exposure was observed in pellicle-coated enamel specimens. Pellicles formed at the buccal aspect of the upper molars were less effective in protecting the enamel against acid-induced softening as compared to pellicles formed at the lingual aspect of the lower incisors only after 5 min exposure in 1% citric acid. TEM analysis showed that pellicle layers were dissolved continuously due to acid exposure. However, even after 5 min exposure to 1% citric acid, a residual pellicle layer could be detected on the enamel surface. In conclusion, site-dependent differences of buccally and lingually in vivo formed 24-hour pellicles have minor importance concerning the pellicle-induced protection of the enamel surface against erosive changes.     

Experimental salivary pellicles on the surface of orthodontic materials.

The purpose of this study was to define the composition of salivary pellicles that form on the surfaces of orthodontic materials and to further investigate whether qualitative differences exist between the composition of adsorbed salivary pellicles that form on 3 different orthodontic materials: stainless steel bracket metal, elastomeric ligature ring, and bracket bonding resin. Experimental pellicles were formed by incubating these materials in fresh human parotid or submandibular-sublingual saliva for 2 hours. Pellicles were extracted with sodium dodecyl sulfate buffer and lyophilized. They were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the adsorbed salivary components. Remarkable differences in the profiles of pellicle components were found, dependent on the type of orthodontic materials. The pellicle components on the bracket metal were almost the same as those found on the elastomeric ligature ring. Salivary protein adsorption patterns to bonding resin showed different features. Distinct differences were also found between the surface-binding affinities of the same salivary proteins from different glandular salivas. These results may be explained on the basis that binding sites for specific proteins on the surfaces of the materials are covered by molecules of submandibular-sublingual saliva, probably mucins. The results of this study provide valuable information concerning initial bacterial adhesion to the surfaces of orthodontic materials, as well as information that could be used in the development of orthodontic materials with enhanced surface properties.     

Quantitative immunochemistry of salivary proteins adsorbed in vitro to enamel and cementum from caries-resistant and caries-susceptible human adults

Salivary proteins adsorbed to powdered enamel and cementum from parotid and submandibular saliva (1:1) of caries-resistant (CR) and caries-susceptible (CS) subjects were examined qualitatively by polyacrylamide disc electrophoresis and quantitatively by immunochemical procedures. The electrophoretic patterns showed no consistent difference between enamel and cementum or between CR and CS samples. Quantitatively, there were no significant differences between CR and CS samples, but significant differences between enamel and cementum. On the basis of ng/mm2, enamel adsorbed five times as much acidic proline-rich protein and cysteine-containing protein as cementum and nearly twice as much lysozyme. There were no significant differences in adsorption of albumin and lactoferrin. The lack of difference in the concentration of pellicle proteins in CR and CS subjects may be related to similar findings with parotid and submandibular saliva. The influence of the major differences in surface pellicle proteins between enamel and cementum on the properties of their respective pellicles is uncertain.     

Adhesion of oral streptococci to experimental bracket pellicles from glandular saliva.

The aim of this study was to evaluate the functions of bracket pellicles as the binding receptors for Streptococcus mutans and Streptococcus gordonii. Four different types of orthodontic brackets were used: stainless steel, monocrystalline sapphire, polycrystalline alumina, and plastic. The bracket pellicles were formed by incubating orthodontic brackets with fresh submandibular-sublingual saliva or parotid saliva for 2 hours. The pellicles were extracted, and their components were confirmed by gel electrophoresis, immunodetection, and amino acid composition analysis. The roles of the bracket pellicles in the adhesion of oral streptococci were evaluated by incubating tritium-labeled streptococci with pellicle-transfer blots. The results showed that the salivary components adhered selectively according to type of bracket and glandular saliva. The selective adsorption was also proven by the amino acid composition profiles. Among the several salivary proteins, MG2, alpha-amylase, and the acidic proline-rich proteins provided the binding sites for S gordonii. However, none of these proteins in the bracket pellicles contributed to the adhesion of S mutans. These findings suggest that numerous salivary proteins can adhere selectively to the orthodontic brackets, and some of them contribute to the binding of S gordonii.     

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.     

Levels of pre-kallikrein in resting and stimulated human parotid and submandibular saliva

Salivary tissue kallikrein is stored in an active form in human salivary glands. Pre-kallikrein has been demonstrated in mixed saliva, but it is not clear if the various salivary glands contribute equally. This study set out to determine if pre-kallikrein is present in human parotid and submandibular salivas at rest, whether levels change during stimulation, and to compare the pattern of pre-kallikrein and kallikrein secretion with that of total protein. Resting and citric acid-stimulated parotid and submandibular, and gum-stimulated parotid saliva samples were collected from 6 healthy subjects. Salivary flows were determined gravimetrically. Total protein concentration and kallikrein enzymic activity were assayed using standard techniques. Pre-kallikrein was assayed following trypsinisation of duplicate samples. Pre-kallikrein was present in parotid and submandibular ductal saliva. Proportions of pre-kallikrein and active kallikrein were similar in salivas secreted at rest and during stimulation, and both outputs mirrored protein output in both major glands. Gum-stimulated parotid saliva showed lower activity than resting, and no differences were seen between resting and stimulated submandibular samples.     

Saliva mediated adherence, aggregation and prevalence in dental plaque of Streptococcus mutans, streptococcus sanguis and actinomyces spp. in young and elderly humans

Salivary components in the pellicle mediate bacterial adherence to the tooth. Such components may also aggregate bacteria in saliva and prevent them becoming established in dental plaque. In the present study, the adherence and aggregation of Streptococcus mutans strain Ingbritt, S. sanguis strain 10556 and Actinomyces viscosus strain 19246 mediated by parotid and whole saliva from groups of young and elderly people were examined. Significant differences were found between test strains, salivary secretions and age groups. S. sanguis 10556 and A. viscosus 19246 generally adhered more strongly than S. mutans Ingbritt, which adhered better to pellicles from parotid saliva than from whole saliva. Strain 19246 bound in higher numbers to parotid saliva pellicles from elderly compared to young individuals. Strain 10556 adhered better to whole saliva than parotid saliva pellicles, and the difference was significant among the young individuals, indicating reduced adherence ability in elderly whole saliva. The streptococci were aggregated by parotid and whole saliva, and S. sanguis aggregation was less with whole saliva from the elderly than from the young participants. Besides a correlation between whole saliva aggregation of S. mutans and proportions of bacteria in plaque, no correlations were found for the individual binding properties of saliva and prevalence of bacteria in vivo. However, the level of saliva-mediated adherence in vitro was in the following order: S. mutans < Actinomyces S. sanguis, which corresponded to their isolation frequency in plaque. These findings emphasize the importance of initial adherence to salivary receptors in bacterial colonization on teeth. Further studies are needed to reveal if individual patterns in the in vitro binding characteristics of saliva lead to variation of colonization in vivo.     

The contribution of oral minor mucous gland secretions to the volume of whole saliva in man

To determine the contribution of minor mucous gland secretions to total saliva by a direct method, flow rates of both unstimulated and sour lemon drop (SLD)-stimulated saliva were initially determined in 15 subjects. The right and left lingual nerves were then anaesthetized to halt submandibular and sublingual secretion, and both parotid ducts were cannulated. The only remaining saliva in the mouth was that secreted by minor salivary glands. Unstimulated and SLD-stimulated minor mucous gland secretions were then collected and the median percentage contributions to whole saliva were calculated to be 8 and 7 per cent, respectively. Comparable results were obtained on 3 subjects using an indirect method similar to that of Schneyer (1956). With the left parotid duct cannulated, subjects maintained a constant, SLD-stimulated, left parotid flow rate of 1 ml/min and the remaining mixed saliva was collected to determine its flow rate. The right parotid and the submandibular and sublingual glands were then also cannulated and the flow rate from these glands determined whilst that from the left parotid was maintained at 1 ml/min. The contribution from minor mucous glands was the difference between the flow rate of mixed saliva and the combined flow rate from the right parotid, submandibular and sublingual glands.     

Hydroxyapatite-reactive salivary protein revealed by iso-electrofocusing electrophoresis.

Salivary protein involvement in the formation of acquired enamel pellicle, so far, has been discussed in terms of hydroxyapatite (HA)-reactive salivary proteins only from the parotid gland. This study was undertaken to seek this type of protein in the human whole (mixed) saliva and to investigate its normal and pathological variations. Several kinds of hydroxyapatite, either biogenous or synthesized by solid phase reaction, were used as a powder (250 mesh). HA was incubated with concentrated whole saliva at 25 degrees for 30 min. After centrifugation and filtration salivary proteins were analysed on a Multiphor isoelectrofocusing gel electrophoresis. The control salivary proteins were separated into three major groups; acidic (A1-A8), neutral neutral (N1-N4), and basic (B1-B3) isoelectric point (pI). In the HA incubated sample, one of the major neutral bands (NI) preferentially disappeared at about pI 7.5. This NI band was missing or scarce in the parotid saliva and had an amino acid composition rich in glycine, lysine, serine, glutamic acid, aspartic acid, and histidine. This protein was considered to be one of the major HA-reactive proteins in human whole saliva.     

Preparation and application of monoclonal antibodies against sialoglycoprotein purified from human submandibular-sublingual saliva

Sialoglycoprotein (SGP) having relatively high molecular weight was purified from human submandibular sublingual saliva by anion exchange chromatography on Q-Sepharose Fast Flow followed by gel filtration on Superose 6 prep grade. The molecular weight of SGP was estimated to be about 440,000 and the isoelectric points obtained by IEF-PAGE, ranged from 5.2 to 5.8. The purified SGP contained high compositions of glutamic acid, proline, glycine and aspartic acid which were calculated as about 57% of the total amino acids, and about 15% of sugars such as N-acetylneuraminic acid, galactose, fucose, mannose, N-acetylgalactosamine and N-acetylglucosamine. A panel of 23 murine monoclonal antibodies (MAbs; 8 IgG1, 15 IgM) directed against SGP was prepared. Although three protein components were detected by SDS PAGE, SGP specific MAb reacted intensely with a 60 kilodalton of protein component. Bacteroides gingivalis 381 showed high binding ability to SGP, but this interaction was inhibited by SGP specific MAb. To detect SGP in the pellicle formed on fragments of human dental enamel, SGP specific MAb were used. During the first hour of formation, rapid increase of SGP detected was observed, and it remained relatively unchanged between 1 and 24 h after beginning of pellicle formation. This work has suggested that SGP purified from submandibular-sublingual saliva is a main component of salivary pellicle on tooth surface and plays an important role in the interaction with oral bacteria including Bacteroides gingivalis.     

Effects of salivary or serum pellicles on the Candida albicans growth and biofilm formation on soft lining materials in vitro

summary The effects of salivary or serum pellicle on Candida albicans growth, biofilm formation and cavitation on the soft lining materials were examined. Both saliva and serum pellicles reduced the antifungal effects of soft liners. The fungal biofilm formation on these materials varied depending upon both the materials tested and protein-coats, and the pellicles which significantly enhanced the biofilm formation. Similarly, the pellicles enhanced the firm colonization and hyphal invasion of the yeasts on the specimens, although the cavitation appeared to be regulated by the plasticizer used. These results suggest that the interactions between proteinaceous pellicle, tissue conditioners and fungi are complex. They also suggest that denture pellicles facilitate fungal plaque formation onto soft lining materials through several mechanisms such as reduction of the antifungal effects of soft liners, facilitation of biofilm formation, firm colonization and hyphal invasion. In addition, the composition of the materials is also involved in the susceptibility to the fungi.     

Adsorption of salivary and serum proteins, and bacterial adherence on titanium and zirconia ceramic surfaces

Objectives: The aim of this study was to determine the pattern of salivary and serum proteins present in pellicles formed on titanium (Ti) and zirconia ceramic (ZrO₂) surfaces, and the ability of bacterial cells to adhere to the experimental pellicles. In addition, the protein profiles and bacterial binding properties of pellicles on Ti and ZrO₂ were compared to those formed on hydroxyapatite (HA) surface.
Methods: The pellicles were formed in vitro by incubating the materials with whole saliva, serum or saliva+serum. Protein composition in each of the pellicles was investigated by SDS-PAGE and immunodetection. The adherence of radiolabeled Streptococcus mutans and Actinomyces naeslundii to uncoated surfaces and experimental pellicles was determined by means of scintillation counting. Statistical analyses were done using ANOVA and Tukey's test at significance level at P <0.05. In general, the electrophoretic analysis of the pellicles formed on HA, Ti and ZrO₂ revealed few qualitative differences of the composition of proteins of the pellicles formed on HA, Ti and ZrO₂ surfaces. Pellicle components identified included amylase, IgA, IgG, albumin, fibronectin and fibrinogen. The number of S. mutans cells adhered to uncoated Ti and ZrO₂ was significantly higher than those adhered to HA ( P <0.05). In contrast, lower number of A. naeslundii cells adhered to uncoated Ti and ZrO₂ than to HA ( P <0.05). However, the presence of saliva and saliva+serum pellicles greatly reduced the number of S. mutans cells bound to each of the surfaces. The data showed that Ti and ZrO₂ display similar pellicle protein composition and bacterial binding properties; however, significant differences were observed when both materials were compared to HA.