Chemical analysis and scanning electron microscopy of acquired pellicle formed in vivo on stannous fluoride treated enamel

Abstract – Stannous fluoride (SnF₂) has been shown to be an effective caries preventive agent. After topical treatment of enamel surfaces, two reaction products have been demonstrated to precipitate on the surfaces, a larger type of globules, probably a calcium fluoride like product, and a smaller type of globules, probably a tin phosphate. The aim of the present study was to examine the amino acid composition and the formation of the acquired pellicle on SnF₂-treated enamel in vivo. The chemical composition was examined by amino acid analysis of pellicle material collected in vivo from SnF₂-treated enamel surfaces. Pellicle formation was examined by scanning electron microscopy on SnF₂-treated enamel fragments carried in the mouth for 2 h. The results showed that pellicle material was formed in abundant amounts and covered the globular surfaces following the SnF₂ treatment. The chemical analyses showed amino acid profiles with high content of acidic and neutral amino acids. The profiles were different from known amino acid profiles obtained from analyses of pellicle material collected from untreated enamel surfaces.     

Chemical analysis and scanning electron microscopy of acquired pellicle formed in vivo on stannous fluoride treated enamel.

Stannous fluoride (SnF2) has been shown to be an effective caries preventive agent. After topical treatment of enamel surfaces, two reaction products have been demonstrated to precipitate on the surfaces, a larger type of globules, probably a calcium fluoride like product, and a smaller type of globules, probably a tin phosphate. The aim of the present study was to examine the amino acid composition and the formation of the acquired pellicle on SnF2-treated enamel in vivo. The chemical composition was examined by amino acid analysis of pellicle material collected in vivo from SnF2-treated enamel surfaces. Pellicle formation was examined by scanning electron microscopy on SnF2-treated enamel fragments carried in the mouth for 2 h. The results showed that pellicle material was formed in abundant amounts and covered the globular surfaces following the SnF2 treatment. The chemical analyses showed amino acid profiles with high content of acidic and neutral amino acids. The profiles were different from known amino acid profiles obtained from analyses of pellicle material collected from untreated enamel surfaces.     

Proteomic profiles of the acquired enamel pellicle formed in vitro,in situ,or in vivo

This study compared the protein profile of the acquired enamel pellicle (AEP) formed under three conditions: in vitro, in situ, and in vivo. Nine volunteers participated in all procedures. In the in vitro condition, the volunteers donated saliva, in which specimens were incubated to form the AEP. In the in situ condition, the volunteers used an oral device containing specimens where the AEP was formed. In the in vivo condition, the AEP was collected from the volunteers own teeth. All AEPs were formed for 120 min, collected and processed by mass spectrometry. Overall, a total of 321 proteins were identified, among which 37 proteins are commonly considered typical in the AEP. For each of the in vitro, in situ, and in vivo conditions, respectively, 66, 174, and 170 proteins were identified. For the in vitro condition, 17 pellicle-typical proteins were not identified. Furthermore, several proteins with important functions within the AEP presented differences in expression in the three conditions. The qualitative profile of the proteins, especially the typical ones, is different in the in vitro condition. In addition, there are important quantitative differences that may interfere when attempting to extrapolate in vitro results to an in situ and in vivo condition.     

Further studies on the composition of the acquired enamel pellicle

Abstract – In the present study pellicle material was collected from human teeth 2, 4 and 6 hr after cleaning. The material obtained was examined by gel filtration, ion exchange chromatography on CM-Sephadex and DEAE-Sephadex with subsequent amino acid analyses of the major anionic component. No major changes were observed to occur either in the overall composition or in the main anionic component of the pellicle during the first 6 hr.     

Further studies on the composition of the acquired enamel pellicle

In the present study pellicle material was collected from human teeth 2, 4 and 6 hr after cleaning. The material obtained was examined by gel filtration, ion exchange chromatography on CM-Sephadex and DEAE-Sephadex with subsequent amino acid analyses of the major anionic component. No major changes were observed to occur either in the overall composition or in the main anionic component of the pellicle during the first 6 hr.     

Desorption of acquired enamel pellicle in vivo by pyrophosphate

Soluble pyrophosphate (PP) has been introduced in dentifrices to inhibit the formation of dental calculus. The mechanism of inhibition is probably an adsorption of the pyrophosphate ions to the Ca-sites on the enamel surfaces and a blocking of the active sites for crystal growth. It has been shown in a recently published study that PP reduced the protein adsorption to hydroxyapatite (HA) in vitro and also inhibited the pellicle formation in vivo. The aim of the present study was to examine the desorption potential of pyrophosphate on the acquired enamel pellicle in vivo. Enamel fragments were carried in the mouth to collect pellicle material and some of the enamel surfaces were then treated with PP. Pellicle formation was examined by SEM of the enamel surfaces. The results showed that pyrophosphate desorbed the acquired enamel pellicle effectively. The clinical consequences of this effect is unknown, but it could possibly explain some aspects of hypersensitivity of teeth observed in some individuals using dentifrices containing PP.     

Desorption of acquired enamel pellicle in vivo by pyrophosphate

Abstract – Soluble pyrophosphate (PP) has been introduced in dentifrices to inhibit the formation of dental calculus. The mechanism of inhibition is probably an adsorption of the pyrophosphate ions to the Ca-sites on the enamel surfaces and a blocking of the active sites for crystal growth. It has been shown in a recently published study that PP reduced the protein adsorption to hydroxyapatite (HA) in vitro and also inhibited the pellicle formation in vivo. The aim of the present study was to examine the desorption potential of pyrophosphate on the acquired enamel pellicle in vivo. Enamel fragments were carried in the mouth to collect pellicle material and some of the enamel surfaces were then treated with PP. Pellicle formation was examined by SEM of the enamel surfaces. The results showed that pyrophosphate desorbed the acquired enamel pellicle effectively. The clinical consequences of this effect is unknown, but it could possibly explain some aspects of hypersensitivity of teeth observed in some individuals using dentifrices containing PP.     

Influence of in vivo formed salivary pellicle on enamel erosion.

This study assessed the protective effect of the salivary pellicle formed in vivo during 24 h or 7 days against demineralization of bovine enamel caused by citric acid. In addition, the influence of acid treatment on the behavior of the pellicle was investigated. Enamel specimens with and without in vivo pellicles were immersed in citric acid (0.1, 1.0%) over 30, 60, and 300 s, and processed for scanning (SEM) and transmission electron microscopy (TEM), as well as for measurement of surface microhardness (SMH). Specimens coated with the in vivo formed pellicles revealed less extensive erosive demineralization of the enamel surface compared to uncovered enamel specimens. SEM analysis and SMH results did not indicate distinct differences between erosive surface alterations on enamel slabs covered with 24-hour pellicles and on those covered with 7-day pellicles. TEM analysis showed that the pellicle layer was dissolved in part from the enamel surface due to acid exposure. However, pellicle residues could be detected by TEM in all specimens, even after 5-min exposure to 1.0% citric acid. It is concluded that the in vivo salivary pellicle can resist the acidic action to some extent and provides protection to the underlying enamel surface against erosive destruction caused by short-term action of citric acid.     

Transaminases in the acquired pellicle

Objective

Transaminases (AST, aspartate amino transferase; ALT, alanine amino transferase) are relevant enzymes in physiology and pathology of the human organism. The aim of the present in situ study was to demonstrate the presence of these enzymes in the enamel pellicle.

Methods

Bovine enamel slabs were fixed on buccal sites of individual upper jaw splints and worn for 3, 30 and 120 min by 5 subjects to allow pellicle formation. The in situ pellicles were tested for AST and ALT. Enzyme activities were measured photometrically via determination of the products pyruvate and oxalacetate using lactate-dehydrogenase and malate-dehydrogenase, respectively.

Results

Enzymatic AST- as well as ALT-activities are present in the acquired pellicle within 3 min. The enzyme activities exposed at the pellicles’ surfaces increased slightly with the pellicle formation time (ANOVA, AST: n.s., ALT: p = 0.021). However, the two enzymes show considerable intraindividual and interindividual variability. The mean AST-activity of the pellicle amounted to 1.07 ± 0.81 mU/cm² (ALT 1.18 ± 0.52 mU/cm²). The ALT-activity of the centrifuged saliva was 26.62 ± 11.09 mU/ml (AST 35.98 ± 29.35 mU/ml).

Conclusions

AST as well as ALT are present in the in situ pellicle layer and may contribute to the intrinsic maturation of pellicle proteins.     

Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva

Abstract – Two hour in vivo formed enamel pellicle samples and paraffin wax-stimulated saliva samples were collected from 10 volunteers for analyses of glucosyltransferase activity (GTF). GTF activity was recorded by monitoring incorporation of radioactivity from ¹⁴C-glucose labeled sucrose into glucan. Pellicle and saliva samples from all 10 subjects demonstrated GTF activity. The GTF activity in the pellicle samples was highest in subjects, with high GTF activity-producing adhesive glucan in saliva.     

Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva

Two hour in vivo formed enamel pellicle samples and paraffin wax-stimulated saliva samples were collected from 10 volunteers for analyses of glycosyltransferase activity (GTF). GTF activity was recorded by monitoring incorporation of radioactivity from 14C-glucose labeled sucrose into glucan. Pellicle and saliva samples from all 10 subjects demonstrated GTF activity. The GTF activity in the pellicle samples was highest in subjects with high GTF activity producing adhesive glucan in saliva.     

Protective effect of the in situ pellicle on dentin erosion - an ex vivo pilot study

AIM: The acquired pellicle is well known as an anti-erosive proteinaceous layer on enamel, but its protective properties on dentin have not been investigated in detail until now. The aim of the present ex vivo study was to evaluate the erosive effects on pellicle coated dentin. METHODS: Bovine dentin slabs were exposed to the oral cavity of one subject for 120 min for in situ pellicle formation. Subsequently, the slabs were incubated with HCl (pH 2.3) in vitro for 5 min and erosive calcium-release was measured photometrically. In addition, the acid treated specimens were evaluated by transmission electron microscopy (TEM). Pellicle free samples served as controls. RESULTS: Calcium erosion from the pellicle coated dentin slabs amounted to 23.5+/-2.9 microg Ca/min (pellicle free samples: 32.2+/-4.2 microg Ca/min). The difference was statistically significant (p < or = 0.05). In pellicle coated as well as in uncoated dentin samples, TEM-evaluation showed a demineralised dentinal surface layer which thickness ranged between 3 and 6 microm. The pellicle itself was partially dissolved but not removed by hydrochloric acid treatment. CONCLUSION: The protective properties of the acquired pellicle against an erosive challenge of the dentinal surface are limited. The dentinal pellicle functions like an ion permeable network rather than a barrier.     

Enzymes in the acquired enamel pellicle

The acquired pellicle is a biofilm, free of bacteria, covering oral hard and soft tissues. It is composed of mucins, glycoproteins and proteins, among which are several enzymes. This review summarizes the present state of research on enzymes and their functions in the dental pellicle. Theoretically, all enzymes present in the oral cavity could be incorporated into the pellicle, but apparently enzymes are adsorbed selectively onto dental surfaces. There is clear evidence that enzymes are structural elements of the pellicle. Thereby they exhibit antibacterial properties but also facilitate bacterial colonization of dental hard tissues. Moreover, the immobilized enzymes are involved in modification and in homeostasis of the salivary pellicle. It has been demonstrated that amylase, lysozyme, carbonic anhydrases, glucosyltransferases and fructosyltransferase are immobilized in an active conformation in the pellicle layer formed in vivo. Other enzymes, such as peroxidase or transglutaminase, have been investigated in experimental pellicles. Despite the depicted impact of enzymes on the formation and function of pellicle, broader knowledge on their properties in the in vivo-formed pellicle is required. This might be beneficial in the development of new preventive and diagnostic strategies.     

An in vivo model for the identification of serum proteins in the acquired subgingival pellicle

The present study describes an in vivo model for the collection of the subgingival pellicle adsorbed to tooth surface, and the identification of some serum proteins within this layer. Clean dentin slabs were prepared from freshly extracted teeth, and then placed subgingivally for 2 h. The dentin slabs with their adsorbed pellicle layer were processed for transmission electron microscopy. Thin sections were made from the specimens, and treated with antisera to human immunoglobulins and albumin. The reactions were visualized by means of protein A-gold complex, which allowed semiquantification of the serum proteins. The indicator proteins were all identified within the pellicle material, but their amounts and distribution varied. Albumin demonstrated higher amounts in the pellicle layer than other proteins, followed by IgA, IgG, and IgM in descending order. The model described seems useful for studying the acquired subgingival pellicle under varying degrees of disease and health.     

Small molecular weight proteins/peptides present in the in vivo formed human acquired enamel pellicle

Objective

The aim of this study was to investigate the type and the nature of peptides present in the in vivo formed human acquired enamel pellicle.

Design

Pellicle material was collected from 10 volunteers and subjected to sample preparations consisting of centrifugal filtration using a 10 kDa molecular weight cut-off membrane and high-resolution gel filtration chromatography. The fractions containing peptides <10 kDa obtained by both methods were analyzed by LC-ESI-MS/MS.

Results

78 natural pellicle peptides with molecular weights ranging from 766.9 Da to 3981.4 Da were identified originating from 29 different proteins.

Conclusions

The number of peptides present in acquired enamel pellicle appears to be large and this is likely to enhance the functional spectrum of this protein film. The presence of small peptides in pellicle may be functionally important since structure/function studies of many salivary proteins have shown that specific domains within these native proteins retain or even exhibit enhanced biological activities. The data present the basis for determining the precise function of these pellicle peptides and for gaining insights into the role pellicle plays in the oral cavity.     

Scanning electron-microscopic study of pellicle and plaque formation on tetracycline-impregnated dentin

Previous experiments have shown that tetracyclines may react with hydroxyapatite, e.g. in enamel and dentin, without losing their antimicrobial capacity. The present paper examines the pattern of pellicle and plaque formation on doxycycline-treated dentin by the use of scanning electron microscopy (SEM). From newly extracted human teeth were prepared standardized dentin slabs, half of which were soaked in aqueous solutions of doxycycline HCl, 10 mg/ml (pH 2.5) for 10 min. Seven volunteers carried doxycycline-impregnated specimens ligated to the buccal surface of a maxillary molar for 2 h, 8 h, 24 h and 120 h, respectively. Untreated control specimens were ligated to the contralateral teeth. After removal from the oral cavity, the dentin slabs were briefly rinsed in water, allowed to air dry and processed for SEM. SEM assessment of the specimens showed that doxycycline-impregnation resulted in a superficial etching of the dentin, a reduced rate of pellicle formation as well as an impairment of pellicle adhesion, and a retarded bacterial plaque formation on the dentin surfaces.