Effect of pyrophosphate on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Abstract – This study showed that pyrophosphate (PP) desorbed an acidic protein (albumin) from hydroxyapatite (HA) more effectively than phosphate and that HA pretreated with PP took up markedly less protein than untreated HA. The protein which adsorbed to PP-treated HA was more loosely bound than that adsorbed to untreated HA. In vivo experiments showed that pellicle formed more slowly on PP-treated etched enamel than on untreated controls. The possible clinical implications of these findings are discussed.     

Effect of pyrophosphate on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

This study showed that pyrophosphate (PP) desorbed an acidic protein (albumin) from hydroxyapatite (HA) more effectively than phosphate and that HA pretreated with PP took up markedly less protein than untreated HA. The protein which adsorbed to PP-treated HA was more loosely bound than that adsorbed to untreated HA. In vivo experiments showed that pellicle formed more slowly on PP-treated etched enamel than on untreated controls. The possible clinical implications of these findings are discussed.     

Effect of silicone oil on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Abstract – Silicone oil has been introduced in a dentifrice for smokers because of its effect as a polishing agent. Silicone oils are hydrophobic in character and have low surface tensions and good wetting properties. Due to the low surface tension, silicone oils may spread readily on solid surfaces and cover them with a thin, water-repellant film. Introduced via dentifrices silicone oil may thus well be able to adsorb to enamel surfaces and to interfere with surface characteristics such as protein adsorption. The aim of the present study was to examine the effect of silicone oil on protein adsorption to hydroxyapatite (HA) in vitro and on pellicle formation in vivo. The effect on protein adsorption to HA in vitro was studied by adsorption of albumin to either untreated or silicone oil treated HA powders. Ion exchange chromatography was also used with either untreated or silicone oil treated HA as bed materials. The effect on pellicle formation in vivo was studied using enamel fragments carried in the mouth to acquire pellicle material. The chemical composition of the acquired pellicle was studied by collection and chemical analysis of pellicle material formed on enamel surfaces in vivo. The study showed that silicone oil treated HA took up less protein and that the adsorbed protein was bound to hydroxyapatite by a different mechanism as compared to untreated controls. The results indicated that hydrophobic interactions could be involved in binding of proteins to silicone oil treated hydroxyapatite. Silicone oil treated enamal fragments carried in the mouth showed a slower rate of pellicle formation as compared to untreated fragments. The amino acid composition of the acquired pellicle collected in vivo from silicone oil treated enamel surfaces was also different from pellicle material collected from untreated enamel.     

Effect of silicone oil on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Silicone oil has been introduced in a dentifrice for smokers because of its effect as a polishing agent. Silicone oils are hydrophobic in character and have low surface tensions and good wetting properties. Due to the low surface tension, silicone oils may spread readily on solid surfaces and cover them with a thin, water-repellent film. Introduced via dentifrices silicone oil may thus well be able to adsorb to enamel surfaces and to interfere with surface characteristics such as protein adsorption. The aim of the present study was to examine the effect of silicone oil on protein adsorption to hydroxyapatite (HA) in vitro and on pellicle formation in vivo. The effect on protein adsorption to HA in vitro was studied by adsorption of albumin to either untreated or silicone oil treated HA powders. Ion exchange chromatography was also used with either untreated or silicone oil treated HA as bed materials. The effect on pellicle formation in vivo was studied using enamel fragments carried in the mouth to acquire pellicle material. The chemical composition of the acquired pellicle was studied by collection and chemical analysis of pellicle material formed on enamel surfaces in vivo. The study showed that silicone oil treated HA took up less protein and that the adsorbed protein was bound to hydroxyapatite by a different mechanism as compared to untreated controls. The results indicated that hydrophobic interactions could be involved in binding of proteins to silicone oil treated hydroxyapatite. Silicone oil treated enamel fragments carried in the mouth showed a slower rate of pellicle formation as compared to untreated fragments. The amino acid composition of the acquired pellicle collected in vivo from silicone oil treated enamel surfaces was also different from pellicle material collected from untreated enamel.     

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.     

Effect of sodium lauryl sulfate on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Sodium lauryl sulfate (SLS) is widely used as a synthetic detergent in dentifrices. It has been shown to have high affinity for hydroxyapatite (HA), and the binding mechanism has been proposed to be electrostatic, involving the negative sulfate terminals of the SLS and the calcium sites on the HA. The binding of SLS to HA may thus well interfere with the protein adsorption to HA. The aim of the present study was to examine the effect of SLS on protein adsorption in vitro and on pellicle formation in vivo. The effect on protein adsorption was studied using ion exchange chromatography. The effect on pellicle formation was studied using enamel fragments carried in the mouth. The study showed that SLS-treated HA adsorbed less protein than untreated HA. Protein adsorbed to SLS-treated HA was more firmly bound to HA as compared to untreated HA. SLS-treated enamel fragments carried in the mouth showed a slower rate of pellicle formation than non-treated enamel.     

Effect of sodium lauryl sulfate on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Abstract – Sodium lauryl sulfate (SLS) is widely used as a synthetic detergent in dentifrices. It has been shown to have high affinity for hydroxyapatite (HA), and the binding mechanism has been proposed to be electrostatic, involving the negative sulfate terminals of the SLS and the calcium sites on the HA. The binding of SLS to HA may thus well interfere with the protein adsorption to HA. The aim of the present study was to examine the effect of SLS on protein adsorption in vitro and on pellicle formation in vivo. The effect on protein adsorption was studied using ion exchange chromatography. The effect on pellicle formation was studied using enamel fragments carried in the mouth. The study showed that SLS-treated HA adsorbed less protein than untreated HA. Protein adsorbed to SLS-treated HA was more firmly bound to HA as compared to untreated HA. SLS-treated enamel fragments carried in the mouth showed a slower rate of pellicle formation than non-treated enamel.     

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.     

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.     

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.     

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.     

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.     

Protective influence of experimentally formed salivary pellicle on enamel erosion. An in vitro study

The purpose of this study was to evaluate dental erosion in 0.1 and 1.0% citric acid in vitro by several different methods and to assess the protective potential of experimentally formed salivary pellicle (24 h in vitro). Bovine enamel slabs were embedded in epoxy resin and polished. Erosion was performed in citric acid for 1, 5 or 10 min and recorded as microhardness loss, as changes of surface roughness (R(a), R(t) and R(zDIN)) and as calcium release. Additionally, erosive alterations were observed with scanning electron microscopy. Significant microhardness loss on non-pellicle-covered specimens was measured after 1-min exposure to 0.1% citric acid. Microhardness loss was time- and concentration-dependent. Salivary pellicle significantly inhibited both microhardness loss, except after 10-min immersion in 1.0% citric acid, and significantly reduced the increase of surface roughness. There were, however, no significant differences in calcium release between pellicle-covered and non-covered enamel. The results support the general conclusion that salivary pellicle effectively protects enamel surface against short-term erosion in organic acids.     

Characterization of in vivo salivary-derived enamel pellicle

Salivary proteins and glycoproteins that participate in the formation of 2-h in vivo enamel pellicle were determined utilizing polyacrylamide gel electrophoresis [sodium dodecyl sulphate (SDS)-PAGE and anionic PAGE]/Western transfer analyses, and specific radiolabelling/SDS-PAGE fluorography. The sensitivity of these methods permitted the identification of individual members of different salivary protein families. The major components of this pellicle were salivary alpha-amylase, cysteine-containing phosphoprotein (CCP or cystatins), salivary mucin and sIgA. Glycosylated amylase was present in larger quantity than the non-glycosylated species. Only CCP1 (cystatin SA-I) of the cysteine-containing phosphoprotein family was identified. The higher molecular-weight salivary mucin (MG1), but not the lower molecular-weight species (MG2), was detected. These results extend earlier observations regarding the selective nature of salivary protein adsorption to enamel surface by demonstrating that only specific members of salivary protein families are involved in 2-h in vivo enamel pellicle formation. The findings also suggest that individual family members may have different functions in the mouth.     

Structural studies on calcium fluoride formation and uptake of fluoride in surface enamel in vitro

abstract — The structural changes in human enamel during exposure to an acetate buffer, pH 4, containing 150 parts/10⁶ fluoride have been studied using scanning electron microscopy and polarized light microscopy. After exposure for 2 h the enamel surface was covered by a fine-granular layer of calcium fluoride. The underlying enamel was highly eroded with an increased pore volume in the outer layer. Following an equilibration period of 3 months the uptake of fluoride in the apatite lattice had resulted in a highly mineralized, 100-μm-thick surface layer rich in fluoride covering a subsurface porous zone with an unchanged fluoride content. The deeply located, caries-like porous zone may have provided calcium and phosphate for saturation of the liquid     

In vitro remineralization of in vivo and in vitro formed enamel lesions

Thin sections of natural white spot enamel lesions (WS) and of artificial in vitro lesions (VL) were remineralized simultaneously in vitro. The sections, clamped in a PMMA holder, were microradiographed at baseline and after remineralization in a calcium- and phosphate-containing solution (pH = 7.0; 1 ppm F) after 2 and 4 weeks. All data were analyzed with respect to baseline. The results show that the lesion depth values did not change significantly during 2 and 4 weeks of remineralization. The mineral accumulation (change in DeltaZ), however, was substantial and significant in WS and VL. In WS the change in mineral accumulation was roughly proportional to the amount of mineral at baseline. The WS accumulated more than two times the amount of mineral than VL in the same periods of remineralization. After 4 weeks of remineralization the maximum mineral value Vmax in the surface layer of the WS was nearly up to the sound enamel level thick approximate87 vol%. This study shows that the technique and calculation procedure described make this single section method attractive for longitudinal demineralization-remineralization studies in vitro or in situ. Both WS and VL samples obviously remineralized in vitro similarly with respect to the baseline. Furthermore, this in vitro work indicates that remineralization inhibitors present in saliva, and previously penetrated into the enamel tissue, do not influence remineralization later on.     

Fluoride profiles in dental plaque in vivo formed on fluoride pre-treated human enamel

Using a novel device capable of generating plaque in vivo on a natural enamel substrate, it has been possible to determine fluoride profiles from the saliva-plaque interface towards the enamel surface. Fluoride profiles in dental plaques tended to fall from the saliva-plaque interface towards the enamel. The device also offered the possibility of examining fluoride distributions after pre-treatment of the enamel with fluoride in vitro. Fluoride profiles were determined in plaque generated in vivo on enamel surfaces, which had been previously treated with a 900-ppm fluoride solution. The results showed the previously reported fall from the plaque surface, but in addition, a further rise towards the enamel surface was seen. The data imply that enamel loaded with fluoride can release some of this fluoride back into the plaque and may act as a fluoride reservoir.     

Structural studies on calcium fluoride formation and uptake of fluoride in surface enamel in vitro.

The structural changes in human enamel during exposure to an acetate buffer, pH 4, containing 150 parts/10(6) fluoride have been studied using scanning electron microscopy and polarized light microscopy. After exposure for 2 h the enamel surface was covered by a fine-granular layer of calcium fluoride. The underlying enamel was highly eroded with an increased pore volume in the outer layer. Following an equilibration period of 3 months the uptake of fluoride in the apatite lattice had resulted in a highly mineralized, 100-micrometer-thick surface layer rich in fluoride covering a subsurface porous zone with an unchanged fluoride content. The deeply located, caries-like porous zone may have provided calcium and phosphate for saturation of the liquid.     

Kinetics of acquisition and loss of calcium fluoride by enamel in vivo

Two in vivo experiments were performed, concerning (1) the kinetics of the acquisition of calcium fluoride on enamel during daily rinses with a solution of 0.023% F as sodium fluoride, and (2) the loss of calcium fluoride from enamel slabs which had been topically treated with a neutral solution containing 0.9% F as sodium fluoride. Enamel slabs were carried in the mouth by 6 volunteers for 8 days in both experiments. Sound and etched enamel were included. (1) During mouthrinses moderate amounts of fluoride were acquired by sound enamel, and more as calcium fluoride than as fluoridated apatite, whereas on etched enamel, more fluoride was deposited as fluoridated apatite. On etched enamel there was also a tendency that the deposition of calcium fluoride levelled out whereas the incorporation of firmly bound fluoride continued. This may indicate that calcium fluoride was transformed into fluoridated apatite, probably through remineralization during pH cycling in plaque covering the etched enamel. (2) After single topical application, it was found that etched enamel initially took up more calcium fluoride than sound enamel, but also lost more during the 1st day of in vivo exposure. The loss of calcium fluoride was arrested after 1-2 days, on sound enamel at 70% and on etched enamel at 40% of the original level. It is suggested that the increased amounts of firmly incorporated fluoride in enamel originated from calcium fluoride on enamel, and that calcium fluoride is an important and clinically significant source of fluoride ions on enamel.     

Effect of two organic phosphonates on protein adsorption in vitro and on pellicle formation in vivo

Abstract – Organic phosphonates have been introduced in dentifrices to reduce the formation of dental calculus. They may conceivably act as calcium sequestrants or crystal growth inhibitors, interfering directly with the calcium ions on the hydroxyapatite (HA) and enamel surfaces. The aim of the present study was to examine the effect of two organic phosphonates on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo. The effect on protein adsorption in vitro was studied by adsorption of albumin to either untreated or phosphonate treated HA powder. Ion exchange chromatography was also performed with columns with untreated and phosphonate treated HA as bed materials and with linear gradients of either phosphate or phosphonates. The effect on pellicle formation in vivo was studied by scanning electron microscopy on untreated and phosphonate treated enamel fragments which had been carried in the mouth to acquire pellicle materials. The present study showed that phosphonate-treated HA took up less protein. The adsorbed protein was, furthermore, less, firmly bound to phosphonate treated hydroxyapatite. Phosphonate-treated enamel fragments carried in the mouth also exhibited a slower rate of pellicle formation as compared to the untreated enamel fragments.