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Saliva and Dental Pellicle-A Review

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Advances in Dental Research

Saliva and Dental Pellicle-A Review U. Lendenmann, J. Grogan and F.G. Oppenheim

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Saliva and Dental Pellicle -- A Review

U. Lendenmann' *, J. Grogan1, F.G. Oppenheim1 2

'Department of Periodontology and Oral Biology, Boston University

Goldman School of Dental Medicine, 700 Albany Street, Boston, MA

021 18. Corresponding author, urs.lendenmann@. Present

address, Ivoclar-Vivadent AG, Research and Development,

Bendererstrasse 2, FL-9494 Liechtenstein. department of Biochemistry,

Boston University School of Medicine.

AdvDent Res 14:22-28, December, 2000

Phe acquired enamel pellicle is an organic film covering the surfaces of teeth. When this film was first discovered, it was thought to be of embryologic origin. Only in the middle of this century did it become clear that it was acquired after tooth eruption. Initially, the small amounts of material that could be obtained have virtually limited the investigation of pellicle proteins to amino acid analysis. Nevertheless, this technique revealed that the pellicle is mainly proteinaceous and is formed by selective adsorption of salivary proteins on tooth enamel. Later, immunologic techniques allowed for the identification of many salivary and fewer non-salivary proteins as constituents of pellicle. However, to this date, isolation and direct biochemical characterization of in vivo pellicle protein have not been possible, because only a few micrograms can be obtained from a single donor. Therefore, the composition and structure of the acquired enamel pellicle are still essentially unknown. Information on the functions of pellicle has been obtained mainly from in vitro experiments carried out with saliva-coated hydroxyapatite and enamel discs. It was found that pellicle protects enamel by reducing demineralization upon acid challenge. Improved pellicle harvesting procedures and analysis by state-of-the-art proteomics with mass spectroscopy approaches promise to make major inroads into the characterization of enamel pellicle.

Introduction

The acquired enamel pellicle provides a focal point for a variety of basic science questions. The research areas of protein adsorption, mineralization/demineralization, bacterial adherence, and antimicrobial activity all have a direct relevance to the full understanding of pellicle function. In addition, the known deterioration of oral hard tissues in the absence of saliva implies a major role of pellicle in tooth protection (Guchelaar et al., 1997). The objective of this review is to present knowledge that has been obtained since the acquired enamel pellicle was discovered in 1839. The steps leading to general acceptance that pellicle is acquired after tooth eruption are presented in a brief historical overview. This summary is followed by a review of the literature pertaining to the histologic and biochemical analysis of pellicle with emphasis on the methods which have emerged recently-- for example, the use of mass spectrometric tools in the investigation of in two-formed pellicle. The last section focuses on our current understanding of acquired enamel pellicle function and provides examples of investigational approaches.

membrane was thought to be of embryologic origin. However, organic films covering adult teeth were recognized not to be Nasmyth's membrane, because they could also be obtained from the surfaces of amalgam and synthetic fillings (Chase, 1926). In 1930, Korff (cited in Zuber, 1948) pointed out that the so-called Nasmyth's membrane or cuticula dentis of the mature tooth was not a tissue derived from histologic elements but consisted of inorganic and organic foreign debris which was deposited on exposed enamel by mediation of saliva. In 1949, Frank clearly distinguished two types of enamel membranes. Prior to and immediately after eruption, teeth are covered with a transient cellular epithelium which is succeeded by an amorphous structure. Both membranes can be "floated" from the tooth surface by acid treatment. The amorphous membrane can also be found on abraded teeth and enamel, providing evidence that it cannot be of embryologic origin (Frank, 1949). In a review article, Dawes separated the many previously described organic structures covering teeth into those of embryologic origin and those which are acquired only after eruption. Since then, it has been accepted that embryologic integuments are lost after eruption of the teeth, and are replaced by an acellular, essentially bacteria-free, membrane commonly referred to as the acquired enamel pellicle (Dawes et al, 1963).

Collection Methods

Well-defined collection techniques are essential for obtaining pellicle samples free of contamination with saliva, plaque, or other elements present in the oral cavity. Therefore, methods used for the collection of pellicle are described here. Initially, as indicated by Nasmyth's discovery, pellicle was obtained from extracted teeth that were made plaque-free' by being brushed under tap water. Subsequently, teeth were either etched by hydrochloric acid or demineralized with EDTA. Upon such treatment, a membrane "cuticula dentis" floated off which could be readily collected (Schiile, 1951;Leach et al, 1967;Armstrong, 1968; Mayhall, 1970). The material obtained by this procedure is henceforth referred to as "old pellicle", to distinguish it from other preparations. Old pellicle may be affected by the extraction of the tooth, which can cause loss of material and contamination with blood. Additionally, plaque can contribute bacterial material to old pellicle. Therefore, methods were developed to collect freshly acquired enamel pellicle. These techniques usually start with a professional toothcleaning including pumicing. Subjects are asked to refrain from eating and drinking. After 2 hrs, teeth are isolated, rinsed with water, and dried with air. Investigators collect pellicle by curetting the coronal two-thirds of the buccal surfaces with a sealer. A whitish material, sometimes coming off in flakes, can be collected on glass wool in a Pasteur pipette connected to a suction device (Sonju and Rolla, 1973). Alternatively, the material collected on the sealer can be wiped off onto a PVDF membrane (Sonju Clasen et al, 1997). Recently, methods have been used in which teeth are directly scrubbed with filter papers or sponges. In one case, Whatman Number 1 paper previously washed free of alcian-blue-positive substances was

History of Pellicle Research

Key Words

In 1839, Alexander Nasmyth first described the organic film covering tooth surfaces. He wrote: "...I observed detached portions of membrane floating on the surface of the solution in which human teeth had been submitted to the action of acid" and described this membrane as "the persistent dental capsule" (Nasmyth, 1839). For a long time, Nasmyth's

Acquired enamel pellicle.

Presented at the 16th International Conference on Oral Biology (ICOB), "Saliva in Health and Disease", held in Chantilly, Virginia, USA, April 9-12, 2000, sponsored by the International Association for Dental Research and supported by Unilever Dental Research

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22

used (Embery et al, 1986). Dental

mini-sponge applicators soaked in

2% SDS have also been used to

collect in vivo pellicle (Carlen et al,

1998). Our laboratory has developed

a technique using hydrophilic PVDF

membranes held with cotton pliers

(Yao et al, 2001) which also proved

to be efficient and was well-

accepted by the patient compared

with mechanical scaling. Individual

collections from 20 buccal tooth

surfaces typically yield from 10 to 15

ug of protein, which is comparable

with the yields of the scaling

technique (Al-Hashimi and Levine,

1989). Material collected with this method in vivo 2 hrs after a toothcleaning will be referred to as "fresh pellicle7'.

Asx

Ser Glx Pro Gly Ala Cys Val Met He Leu Tyr Phe His Lys Arg

Amino acid residue

Fig. 1 -- Amino acid analysis of acquired enamel pellicles. The abundance of individual amino acid residues is

given in mols ? 1000 mols~1 of total amino acids. White bars represent old pellicles obtained by acid treatment of

Formation of Pellicle

extracted teeth. Black bars represent freshly formed two-hour pellicles. From left to right, the bars give data reported by Leach et al. (1967), Armstrong and Hayward (1968), Mayhall (1970), Belcourt et al. (1974), Sonju and

Ultrastructural studies indicate that Rolla (1973), Al-Hashimi and Levine (1989), Rykkeand Sonju (1991), and Zehnder et al. (2000).

a detectable pellicle layer is

adsorbed onto enamel slabs as early

as one minute after exposure to the

oral environment (Hannig, 1999). Several publications suggest

Chemical Composition

that pellicle formation reaches equilibrium between adsorption and de-sorption of protein within 2 hrs. No further increase in

of the Acquired Enamel Pellicle

amino acid quantities was found after 90 min in analyses of Histochemical staining suggested that pellicle was of a

samples collected after 30, 60, 90, and 120 min (Sonju and proteinaceous nature. Such results were confirmed by the

Rolla, 1973). Equilibrium was observed after 90 min by x-ray observation that pellicle was lost upon incubation of teeth with

photoelectron spectroscopic studies (Kuboki et al, 1987). An proteolytic enzymes (Dobbs, 1932; Meckel, 1965). Indeed, the

argon-sputtering experiment showed that pellicle formation chemical composition of old pellicle samples was reported to

appeared to be complete after 45 min, and no increase was be 46% amino acids, 2.7% hexosamines, and 14% total

observed within 10 hrs (Skjorland et al, 1995). Bacteria were carbohydrates (Armstrong, 1967).

found on enamel splints worn in the oral cavity after 4 hrs (Lie,

Only preliminary information is available about lipids in

1975). Hence, a two-hour pellicle is virtually free from plaque pellicle. The major phospholipids in pellicle are

accumulation.

phosphatidylcholine, phosphatidylethanolamine, and

sphingomyelin. Glycolipids and neutral lipids were found as

Ultrastructure of Pellicle

well (Slomiany et al, 1986). To date, only the presence of phosphatidylcholine has been confirmed by other researchers

Electron microscopy has been used extensively to investigate the (Kautsky and Featherstone, 1993).

structure of the acquired enamel pellicle. The majority of the

studies report the thickness of pellicle to range between 30 and 100 Carbohydrate composition nm on self-cleansing surfaces (Tinanoff et al, 1976; Hannig, 1989). Samples of "old pellicle'7 floated from extracted teeth

Scanning and transmission electron microscopy revealed that stained positive with PAS (Schiile, 1964). Therefore, pellicle

pellicle is not a homogeneous film covering enamel or was assumed to consist of mucins, and investigators tried

hydroxyapatite discs but exhibits distinct structures. An extensive to analyze carbohydrates. In old pellicle, glucose, mannose,

study was carried out by Lie (Lie, 1977). He attached galactose, and glucosamine could be identified by paper

hydroxyapatite splints to the dentition and examined samples after various time periods of pellicle formation. After 2 hrs, granular structures with diameters between 25 and 125 nm were observed. Sometimes, these globules were connected to the hydroxyapatite surface via stalk-like structures. After 24 hrs, the globules appeared to be covered by a fibrillar pellicle reaching a thickness of 550-900 nm. Globular structures in the fibrillar pellicle were smaller, with diameters between 5 and 70 nm. After 12 hrs, the pellicle surface became relatively smooth, and globular and fibrillar structures were not observed anymore. However, a granular structure was observed, and electron-dense lamination lines could be found in the middle of pellicle (Lie, 1977). Other researchers also observed such globular and granular structures (Hannig, 1989; Schiipbach et al, 1996). Transmission electron microscopic images additionally show that pellicle is not limited to the tooth surface but penetrates enamel in a filamentous manner (Meckel, 1965; Tinanoff et al, 1976; Hannig, 1989). This so-called subsurface pellicle is particularly pronounced on approximal surfaces (Leach and Saxton, 1966). Plaque can be covered by a

chromatography. In samples of supragingival calculus, ribose was found as well, which was suggested to be of bacterial origin (Schiile, 1964). Mayhall reported a carbohydrate composition of old pellicle of 20% glucose, 27% galactose, 9% manose, 18% fucose, 18% glucosamine, and 14% galactosamine (Mayhall and Butler, 1976). Fresh pellicle contains high proportions of glucose as well. Sonju reported 67% glucose, 18% glucosamine, 9% galactose, and 6% mannose (Sonju, 1975). The high content of glucose in pellicle was initially not well-understood, because this sugar is rare in glycoproteins. Since in old and fresh pellicle, readily soluble material is washed away during collection, most of the sugars found are likely to derive from macromolecules or glycosylated proteins. The experimental work of Hay helped to clarify the origin of carbohydrate in pellicle (Hay, 1969). He compared the sugars present in proteins of whole saliva supernatant which specifically bind to hydroxyapatite with those present in pellicle. Protein bound to hydroxyaptatite contained large quantities of galactose, some glucose and

pellicle looking similar to that of enamel (Tinanoff et al, 1976).

mannose, and again a large quantity of fucose, indicating

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Adv Dent Res 14:22-28, December, 2000

Saliva and Dental Pellicle

23

300

acquired enamel pellicle (Leach et ah,

1967). This could be a consequence

250

of plaque being covered with a

pellicle similar to that of enamel, as

200

suggested by electron microscopic

150

data (Tinanoff et ah, 1976). An amino

acid composition similar to that of

? 100

S

50

enamel pellicle was even found on the tissue-bearing sides of maxillary dentures (Edgerton and Levine,

30

1992). These results suggest that

He Phe Val Leu Met Ala Gly Cys Tyr Pro Thr Ser His Glx Asx Lys Arg most oral surfaces bathed with saliva

acquire a protein coating with a

composition similar to that of

enamel pellicle. Hence, amino acid

analysis alone is not adequate to

identify differences in the

composition of organic films formed

on oral surfaces.

Nevertheless, a comparison of

pellicle with salivary secretions

Hydrophobic

Neutral

Hydrophilic

yields interesting results. Fig. 2A

shows amino acid compositions of

Fig. 2 -- (A) Amino acid compositions of two-hour pellicle, human whole saliva, human submandibular/sublingual saliva, and human parotid saliva. The data were adapted from an earlier study (AlHashimi and Levine, 1989). Amino acids are arranged from the left to the right in decreasing order of hydrophobicity (Eisenberg, 1984). (B) Hydrophobic, neutral, and hydrophilic amino acids in pellicle, whole saliva, submandibular/sublingual saliva, and parotid saliva.

fresh pellicle, whole saliva, submandibular/sublingual saliva, and parotid saliva. All three types of saliva contain dramatically more proline but less leucine, alanine,

the presence of salivary glycoproteins. In contrast, the predominant sugar in enamel pellicle material was glucose, while little galactose and no detectable fucose were observed. These results suggested that the majority of glucose present in pellicle does not originate from salivary protein.

and tyrosine than pellicle. Furthermore, pellicle contains significantly more hydrophobic and significantly less neutral amino acid than whole saliva or submandibular/sublingual saliva (Fig. 2B). These differences suggest that the composition of pellicle protein is significantly different from that of saliva.

Amino acid composition

The chemical characterization of pellicle protein has relied heavily on amino acid analysis. Therefore, many authors who investigated the acquired enamel pellicle have reported compositions (Fig. 1). No significant differences were observed between old and fresh pellicle. In a few studies, unusual amino acids and other components were reported in pellicle. For example, muramic acid, an amino sugar derivative specific to bacterial cell walls not found in animal proteins, was identified (Armstrong and Hayward, 1968). This finding is of interest because it suggests a useful marker for contamination of pellicle samples with bacterial plaque.

Many investigators attempted to use amino acid analysis to identify variability in pellicle composition. For example, amino acid compositions of pellicle collected from the buccal sides of upper molars, upper incisors, and lower anterior teeth were virtually identical (Sonju and Rolla, 1973; Sonju, 1975). Statistically significant were the findings of higher serine and glycine but lower tyrosine content for pellicle samples derived from deciduous teeth compared with permanent teeth (Sonju Clasen et al.f 1997), but the overall amino acid compositions were very similar. A longitudinal study involving three subjects showed that amino acid compositions of two-hour acquired enamel pellicle were consistent among three different subjects over a period of 24 months (Rykke et ah, 1990). Furthermore, pellicles formed for 2 hrs and 24 hrs when food and beverage intake was avoided were similar. In contrast, 24hour pellicles exhibited significant variability when subjects consumed a normal diet. This suggests that dietary components can contribute to in vivo pellicle (Rykke and Sonju, 1991). Presently, it is impossible to judge whether significant differences in the amino acid composition could be expected in the above cases, because, surprisingly, the amino acid composition of plaque collected 24 hrs after a toothcleaning and at least 2 hrs after a meal was found to be similar to that of

Proteins in Pellicle

Adsorption of salivary proteins by hydroxyapatite

The minute quantities of in vivo pellicle that could be obtained made attempts to isolate and characterize pellicle proteins difficult. Therefore, many researchers used hydroxyapatite as an in vitro substitute for enamel to study adsorption of salivary proteins. Although hydroxyapatite has long been used for chromatographic protein purifications, the nature of the interactions leading to specific binding is still poorly understood. Investigations carried out with synthetic proteins showed that the strongest binding to hydroxyapatite is mediated by phosphoserine, followed by glutamic and aspartic acid. However, these properties do not easily allow for predictions for binding of natural proteins, because even the cationic protein lysozyme was found to bind to hydroxyapatite (Bernardi and Kawasaki, 1968). When whole-saliva supernatant was incubated with enamel powder, gel electrophoresis of proteins adsorbed to the powder and those remaining in the supernatant shows significant differences in banding patterns. This indicated that, among salivary proteins, some exhibited high and some low affinity toward enamel. Qualitatively similar results were obtained when the same experiment was carried out with hydroxyapatite (Hay, 1967). Known salivary proteins exhibiting high affinity to hydroxyapatite are therefore called "pellicle precursor proteins". This term also takes into account the complex modifications of intact salivary proteins which may occur before or after their incorporation into the pellicle structure.

Identification of proteins in pellicle

Whether a pellicle precursor protein is actually a pellicle protein can be confirmed only if it can be identified in pellicle. Table 1 presents an overview of the proteins which could be identified in pellicle. Major salivary proteins in pellicle are secretory IgA, acidic proline-rich protein (PRP), cystatin SA-I,

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24

Lendenmann et al.

Adv Dent Res 14:22-28, December, 2000

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