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 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.     

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

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.     

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.     

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.     

Protein adsorption to hydroxyapatite and to calcium fluoride in vitro and amino acid analyses of pellicle formed on normal enamel and on calcium-fluoride-covered enamel in vivo

Fluoride treatment of enamel has been reported to result in the formation of a layer of a CaF2-like material on the enamel surface. Protein adsorption to enamel is a specific process dependent on the nature of the surface, and little is known about protein adsorption to CaF2. Albumin and lysozyme were adsorbed to hydroxyapatite (HA) and CaF2 powder in vitro, and protein adsorption patterns constructed. In vivo pellicle was collected from three volunteers from fluoride-treated enamel and from normal enamel, and the amino acid compositions analyzed separately. The results showed that CaF2 took up small amounts of proteins as compared with HA. When the CaF2 was pretreated with a phosphate buffer, pH 6.8, the protein adsorption increased markedly. The amino acid analyses showed no major differences in the amino acid compositions between pellicle collected from CaF2-covered enamel and pellicle collected from normal enamel. This lack of difference is presumably due to the adsorption of phosphate ions to the CaF2 crystals and hence changed surface properties.     

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.     

十二烷基硫酸钠抗牙菌斑作用的实验研究

十二烷基硫酸钠（ｓｏｄｉｕｍｄｏｅｃｙｌｓｕｌｆａｔｅ，ＳＤＳ）常作为去垢剂广泛地应用于牙膏之中。已证实ＳＤＳ与羟基磷灰石（ＨＡ）有高度亲合力，通过ＳＤＳ与ＨＡ的结合，干扰蛋白质对ＨＡ的吸附，从而影响牙面获得性膜的形成。为进一步了解ＳＤＳ的抑菌和抗菌斑作用，本研究利用口腔恒化器和扫描电镜及同位素标记液闪检测技术，对ＳＤＳ的抗菌斑作用进行了观察。结果显示，经ＳＤＳ处理后的牙釉质表面没有成熟的牙菌斑形成，仅有少数细菌散在附着，其放射性明显少于对照组。提示ＳＤＳ具有抗菌斑作用，是牙膏中的天然抗菌斑成分之一。     

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 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.     

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.     

Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite

The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.     

Compositional analysis of human acquired enamel pellicle by mass spectrometry

Relatively little is known about the formation of the acquired enamel pellicle other than that it involves the selective adsorption of specific proteins from oral fluids. Previous studies on the identification of pellicle components have relied largely on immunological or enzymatic detection and have been hampered by the fact that only minute quantities of pellicle can be removed from tooth surfaces. The present work describes an improved method of harvesting pellicle that combines mechanical and chemical removal; this approach was used to investigate systematically the desorption of in vitro pellicle components with different solutions. Eleven major in vitro pellicle proteins were identified by using a combination of electrophoretic separation and matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. A similar analysis of in vivo-formed pellicle revealed the presence of intact statherin, lysozyme, albumin and amylase. Further analysis of in vivo pellicle by liquid chromatography-electrospray ionization mass spectrometry suggested the presence of numerous low molecular-weight fragments of precursor proteins. The protein composition of in vitro whole-salivary pellicle adsorbed to hydroxyapatite and that of in vivo enamel pellicle differed for proline, the result of a reduction in the content of acidic proline-rich proteins in the in vivo samples. Unique features of the oral environment such as enzymatic activities or mineral surface properties may account for these differences between in vivo and in vitro pellicle formation.     

In vitro studies on the effect of sodium tripolyphosphate on the interactions of stain and salivary protein with hydroxyapatite

OBJECTIVES: To study properties of sodium tripolyphosphate (STP) relevant to inhibition or removal of dental stain in vitro. METHODS: The effects of STP and other phosphates on adsorption of a dietary chromogen (black tea polyphenol) and salivary protein to hydroxyapatite (HA) powder were studied by analysing loss of protein or tea stain from solutions mixed with HA or HA pre-treated with the test agents. The effects on desorption of protein and stain from HA were studied by analysis of water or solutions of test agents mixed with HA or HA pre-treated with saliva or tea solution. RESULTS: At concentrations and pH representative of those likely to occur in the mouth, STP inhibited adsorption of salivary protein and black tea polyphenol to, and desorbed these substances from, HA surfaces. Adsorption and desorption of protein and stain were not influenced by pH of the STP solutions but adsorption varied with concentration. STP showed equivalent effectiveness with respect to salivary protein adsorption and desorption as a longer-chain condensed phosphate. The inhibitory activity of HA-bound STP on adsorption of salivary protein and stain resisted extensive washing. CONCLUSIONS: STP is likely to be an effective agent for inhibiting and removing dental stain, whether bound directly to mineralised surfaces or indirectly via salivary pellicle.     

In situ studies of pellicle formation on hydroxyapatite discs

The formation of acquired enamel pellicle on hydroxyapatite (HA) discs of known surface area carried in the mouth was studied; discs were carried in the mouth for 30 s, 1, 5, 10 and 20 min. Similar amounts of protein were found on the discs at each time-point, as determined by ninhydrin analyses. The amounts of amylase and lysozyme detected remained stable after 5 min of exposure of the discs to the mouth. Assay of the discs for fructosyl- and glucosyltransferase activities revealed that fructosyltransferase activity increased up to 1 min of exposure to the mouth and decreased when kept in the mouth for longer periods; glucosyltransferase activity, in contrast, increased the longer the discs were kept in the mouth. This in situ model provides insight into the activities of various enzymes during the first 20 min of pellicle formation. The effects of rinsing with sucrose and sugar alcohols on pellicle formation on the discs were also explored. The discs were placed in the mouth for 30 s, 1, 5, 10 and 20 min, preceded by rinsing with either distilled deionized water, sucrose, sorbitol, xylitol or phosphate-buffered saline. Western blot analyses of disc eluates with antiserum/antibody preparations to various salivary components revealed distinct patterns of deposition of bacterial and salivary components depending on the composition of the rinse. These studies confirm that salivary molecules and bacteria are deposited on apatitic surfaces in a selective manner and reveal that pellicle formation may be influenced by composition of diet. It is apparent that this in situ model could be used in screening potential antiplaque agents.     

Intrinsic enzymatic crosslinking and maturation of the in situ pellicle

AIM: The acquired enamel pellicle is a proteinaceous layer formed on all solid substrata exposed to the oral cavity. It has been supposed that the pellicle undergoes maturation after protein adsorption. The aim of the present study was to investigate enzyme activities with an impact on intrinsic maturation processes in in situ formed pellicles. METHODS: Bovine enamel specimens were exposed to the oral cavity in six subjects to allow in situ pellicle formation over 3, 30 and 120 min. The slabs were fixed on the buccal and palatal surfaces of individual splints fixed with silicone impression material. After rinsing with deionised water, the pellicle samples were tested fluorimetrically for transglutaminase, protease and elastase activity. Phosphatase activities were tested photometrically. Separate samples were used for each of the enzymes tested. RESULTS: Transglutaminase was detected in in situ pellicle (16.7+/-21.2 mU/cm(2)) as was alkaline phosphatase activity (0.87+/-0.99 mU/cm(2)). For both enzymes, there was no correlation of enzyme activities with time or localisation of pellicle formation. Acidic phosphatase- and protease-activities were not detectable. Only traces of elastase activity were found in 57% of the samples. CONCLUSION: Transglutaminase and phosphatase activity are detectable within in situ pellicle. Enzymatic crosslinking and dephosphorylation appear more important for intrinsic maturation of the acquired enamel pellicle than proteolysis.     

Plaque formation in vivo and bacterial attachment in vitro on permanently hydrophobic and hydrophilic surfaces.

Highly hydrated polyethylene oxide (PEO) films represent one type of surface modification which may interfere with biofilm formation. Protein adsorption and saliva-mediated bacterial adherence were investigated in vitro on normal and hydrophobized glass surfaces and on glass surfaces with immobilized PEO films. More protein and bacteria bound to untreated compared to hydrophobized and PEO-treated glass. Pellicle and plaque formation was also studied in vivo on ceramic crown surfaces either untreated, hydrophobized or with immobilized PEO films. Pellicle and plaque formation was similar on the untreated ceramic and PEO surfaces. Less plaque seemed to collect on these surfaces compared to adjacent normal tooth surfaces. Almost no plaque accumulated on the hydrophobic crown surface and it was virtually devoid of stainable pellicle. Even after 7 days in the mouth without oral hygiene this surface was very hydrophobic and the disclosing solution could not spread.     

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.