Effect of plasma on composition of human enamel and cementum pellicle

Abstract – Slabs of human enamel and cementum were incubated with plasma alone or with various mixtures of plasma and saliva. Proteins and glycoproteins that adsorbed to the surface of the slabs in 0 to 60 min were labeled by lactoperoxidase-catalyzed ¹²⁵I-iodination and by mild periodate oxidation followed by NaB³H₄ reduction. The labeled components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography or fluorography. From plasma alone, a 58 and a 66 kDa protein (probably albumin) were adsorbed to the enamel surface in relatively equal amounts, but no ¹²⁵I-labeled components were detected on the cementum surface in the absence of saliva. Adding 10% saliva to the incubation mixture promoted the adsorption of the 58 and 66 kDa components to cementum. In addition, another set of proteins, including components of 44, 47, 29, and 25 kDa, was adsorbed to both cementum and enamel in the presence of saliva. These six proteins were the major ¹²⁵I-labeled species in all of the pellicles formed from mixtures of plasma and saliva. The electrophoretic mobility of the major 120 and 140 kDa ³H-labeled sialoglycoproteins adsorbed to both cementum and enamel was similar to that of the low-molecular-weight mucin of submandibular/sublingual saliva.     

Effect of plasma on composition of human enamel and cementum pellicle

Slabs of human enamel and cementum were incubated with plasma alone or with various mixtures of plasma and saliva. Proteins and glycoproteins that adsorbed to the surface of the slabs in 0 to 60 min were labeled by lactoperoxidase-catalyzed 125I-iodination and by mild periodate oxidation followed by NaB3H4 reduction. The labeled components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography or fluorography. From plasma alone, a 58 and a 66 kDa protein (probably albumin) were adsorbed to the enamel surface in relatively equal amounts, but no 125I-labeled components were detected on the cementum surface in the absence of saliva. Adding 10% saliva to the incubation mixture promoted the adsorption of the 58 and 66 kDa components to cementum. In addition, another set of proteins, including components of 44, 47, 29, and 25 kDa, was adsorbed to both cementum and enamel in the presence of saliva. These six proteins were the major 125I-labeled species in all of the pellicles formed from mixtures of plasma and saliva. The electrophoretic mobility of the major 120 and 140 kDa 3H-labeled sialoglycoproteins adsorbed to both cementum and enamel was similar to that of the low-molecular-weight mucin of submandibular/sublingual saliva.     

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.     

Immobilisation and activity of human alpha-amylase in the acquired enamel pellicle

Amylase is an important salivary component and structural element of the acquired enamel pellicle. Aim of the study was to establish a method for precise and direct determination of pellicle bound amylase activity in order to analyse kinetics and activity of the immobilised enzyme. Six bovine enamel slabs (5mm diameter) were fixed on individual maxillary trays and worn by five subjects for different times (3, 30 and 120 min) on buccal and palatal sites on different days. Slabs were removed from the trays and rinsed with aqua dest. Afterwards, pellicle bound amylase activity was determined directly with a photometric method using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltotriosid (GalG2CNP) as substrate yielding the coloured product chloronitrophenolate (CNP). All investigated pellicles exhibited immobilised amylase activity. Mean activity was 1.39 +/- 187 mU/cm(2) (n=87, range 0.14-11.5 mU/cm(2)). Product formation of CNP by immobilised amylase was linear over time. Pellicle bound amylase showed a Michaelis type kinetic (Km = 3.3 x 10(-3) M). Immobilised activity on buccal surfaces ranged between 0.25 and 11.1 mU/cm(2) (palatal slabs: 0.14-3.06 mU/cm(2)). Thirty minutes pellicles formed on buccal sites exhibited significantly higher immobilised amylase activity (2.85 +/- 3.65 mU/cm(2)) than palatal ones (0.63 +/- 0.32 mU/cm(2)). Amylase activity showed great intraindividual variability when comparing same positions on different days. CONCLUSION: Pellicle bound amylase activity can be determined directly with GalG2CNP and shows a Michaelis Menten kinetic. Enzyme activity of the amylase immobilised in the in situ pellicle reveals great intra- and interindividual differences.     

Distribution of fluoride across human dental enamel, dentine and cementum

This was determined across the entire width of sections from 20 mandibular premolars, containing enamel, coronal dentine, root dentine and cementum. An abrasive technique was used to sample all three dental tissues in a single experiment. In the profiles of fluoride distribution, fluoride concentration was thus precisely related to the position of the tissue sample. There was a marked increase in the fluoride content of coronal and root dentine, at least until the age of about 50 years. There had been uptake of fluoride by the root dentine and cementum throughout the life of the tooth. There was no evidence of any change in the fluoride content of enamel with age.     

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.     

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.     

At-home bleaching system: effects on enamel and cementum

An analysis by scanning electron microscopy was undertaken to investigate the effects of bleaching on enamel-cementum surface morphology. Teeth were investigated in five groups: control; vital bleached for 5, 15, and 30 days; and 37 percent phosphoric acid gel etched for 20 seconds. Only the gel-etched group exhibited etching patterns. Shade change in vital bleached teeth was noted in the group vital bleached for 30 days.     

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.     

Effect of bleaching agents on inorganic components of human dentin and cementum.

The effect of bleaching agents on the inorganic composition of human dentin and cementum was examined. Intact teeth were crushed, pulverized, and separated to dentin and cementum powders. The pulverized tissues were exposed to treatments with 30% H2O2, 3% H2O2, 2% sodium perborate in 30% H2O2, 2% sodium perborate in 3% H2O2, and 2% sodium perborate in bidistilled water for periods of 15 min and 1, 24, and 72 h. The degree of dissolution and the percentage of inorganic material for both dentin and cementum were measured. Thirty percent H2O2 and 2% sodium perborate in 30% H2O2 treatments significantly increased the solubility of dentin and cementum. The degree of dissolutions and the percentage of inorganic material remaining in the undissolved dentin and cementum increased with time progression. The greatest increase occurred with 30% H2O2 and 2% sodium perborate in 30% H2O2 after 24- and 72-h treatments. It is concluded that 30% hydrogen peroxide treatment may cause alteration in the chemical structure of the dentin and cementum making them more susceptible to degradation.     

Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle

Salivary carbonic anhydrase (CA VI) appears to protect teeth from caries via mechanisms other than direct regulation of salivary pH and buffering capacity. To elucidate whether CA VI acts in the local microenvironment of the tooth surface, we studied the location and activity of the enzyme in the human enamel pellicle. The study was performed using a specific rabbit antiserum to human CA VI in conjunction with immunostaining and immunoblot techniques. CA activity was demonstrated using a histochemical staining method. CA VI immunostaining of extracted teeth having in vivo formed pellicle showed that the enzyme is present in the enamel pellicle. Immunostaining for salivary alpha-amylase, which is known to be present in the pellicle, showed a similar staining pattern. The presence of CA VI in the enamel pellicle was confirmed by immunoblotting of in vivo formed pellicle proteins. In vitro studies showed that CA VI binds to polished enamel surfaces from both saliva and solutions of purified enzyme. The intensity of the CA VI immunostaining on the enamel surface was dependent on the concentration of the applied enzyme. The histochemical staining of in vitro formed enamel pellicle confirmed that the bound enzyme retains its enzymatic activity. The presence of active CA VI in the human enamel pellicle suggests that it may accelerate the removal of acid by functioning locally in the pellicle layer on dental surfaces.     

Impact of acquired enamel pellicle modification on initial dental erosion

The acquired enamel pellicle that forms on the tooth surface serves as a natural protective barrier against dental erosion. Numerous proteins composing the pellicle serve different functions within this thin layer. Our study examined the effect of incorporated mucin and casein on the erosion-inhibiting potential of the acquired enamel pellicle. Cyclic acidic conditions were applied to mimic the erosive environment present at the human enamel interface during the consumption of soft drinks. One hundred enamel specimens were prepared for microhardness tests and distributed randomly into 5 groups (n = 20) that received the following treatment: deionized water, humidity chamber, mucin, casein, or a combination of mucin and casein. Each group was exposed to 3 cycles of a 2-hour incubation in human saliva, followed by a 2-hour treatment in the testing solution and a 1-min exposure to citric acid. The microhardness analysis demonstrated that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. The addition of individual proteins did not statistically impact the function of the pellicle. These data suggest that protein-protein interactions may play an important role in the effectiveness of the pellicle to prevent erosion.     

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.     

The protective nature of pellicle towards toothpaste abrasion on enamel and dentine

OBJECTIVES: To determine the protective nature of pellicle towards toothpaste abrasion. METHODS: The enamel region of human enamel-dentine blocks was indented with a Knoop diamond and the profile across the enamel-dentine junction was measured. Blocks were either exposed to deionised water or placed onto intra-oral appliances and worn in the mouth to produce in situ pellicles. This was followed by a 10-day period of tooth brushing experiments. Each day, specimens were brushed with a slurry of either Toothpaste A (RDA=90) or Toothpaste B (RDA=204) for 25 cycles (10s) on a brushing machine. This was repeated three times per day for a total of 750 brushing cycles. Between brushing cycles specimens were returned to water or in situ. The geometry of the Knoop indents and the enamel-dentine profile were re-measured and the enamel and dentine wear calculated. Specimens were also prepared for TEM analyses. RESULTS: The mean enamel wear (microm) for Toothpastes A and B (water) was 0.23 and 0.06, and for Toothpastes A and B (in situ) was 0.03 and 0.08, respectively. The mean dentine wear (microm) for Toothpastes A and B (water) was 5.08 and 6.03, and for Toothpastes A and B (in situ) was 1.94 and 1.70, respectively. For Toothpaste A, the presence of in situ pellicle significantly (p<0.05) reduced enamel and dentine wear compared to water and for Toothpaste B, dentine wear was significantly reduced compared to water. After tooth brushing, residues of the in situ pellicle layer could be detected on enamel and dentine surfaces by TEM analysis. CONCLUSIONS: The study has demonstrated for the first time that the presence of pellicle can significantly reduce toothpaste abrasion.     

Dentinogenesis imperfecta type III with enamel and cementum defects

Ground sections and scanning electron microscopic studies of primary teeth from a 2-year-old child whose teeth showed absence of enamel and large pulp chambers are examined. The findings suggest a diagnosis of odontodysplasia in a case classified as dentinogenesis imperfecta type III.