Impact of the in situ formed salivary pellicle on enamel and dentine erosion induced by different acids

Objective. To investigate and compare the protective impact of the in situ formed salivary pellicle on enamel and dentine erosion caused by different acids at pH 2.6. Methods. Bovine enamel and dentine samples were exposed for 120 min in the oral cavity of 10 healthy volunteers. Subsequently, enamel and dentine pellicle-covered specimens were extraorally immersed in 1 ml hydrochloric, citric or phosphoric acid (pH 2.6, 60 s, each acid n=30 samples). Pellicle-free samples (each acid n=10) served as controls. Calcium release into the acid was determined by atomic absorption spectroscopy. The data were analysed by two-way ANOVA and Tukey's test (α=0.05). Results. Pellicle-covered samples showed significantly less calcium loss compared to pellicle-free samples in all acid groups. The mean (SD) pellicle protection (% reduction of calcium loss) was significantly better for enamel samples [60.9 (5.3)] than for dentine samples [30.5 (5.0)], but revealed no differences among the acids. Conclusion. The efficacy of the in situ pellicle in reducing erosion was 2-fold better for enamel than for dentine. Protection of the pellicle was not influenced by the kind of acid when enamel and dentine erosion was performed at pH 2.6.     

Acquired pellicle as a modulator for dental erosion

Dental erosion is a multifactorial condition that can result in the loss of tooth structure and function, potentially increasing tooth sensitivity. The exposure of enamel to acids from non-bacterial sources is responsible for the progression of erosion. These erosive challenges are counteracted by the anti-erosive properties of the acquired pellicle (AP), an integument formed in vivo as a result of selective adsorption of salivary proteins on the tooth surface, containing also lipids and glycoproteins. This review provides an in-depth discussion regarding how the physical structure of the AP, along with its composition, contributes to AP anti-erosive properties. The physical properties that contribute to AP protective nature include pellicle thickness, maturation time, and site of development. The pellicle contains salivary proteins embedded within its structure that demonstrate anti-erosive properties; however, rather than individual proteins, protein–protein interactions play a fundamental role in the protective nature of the AP. In addition, dietary and synthetic proteins can modify the pellicle, enhancing its protective efficiency against dental erosion. The salivary composition of the AP and its corresponding protein-profile may be employed as a diagnostic tool, since it likely contains salivary biomarkers for oral diseases that initiate at the enamel surface, including dental erosion. Finally, by modifying the composition and structure of the AP, this protein integument has the potential to be used as a target-specific treatment option for oral diseases related to tooth demineralization.     

Pathogenesis and modifying factors of dental erosion

Dental erosion is caused by acidic solutions which come into contact with the teeth. Because the critical pH of dental enamel is approximately 5.5, any solution with a lower pH value may cause erosion, particularly if the attack is of long duration, and repeated over time. Saliva and salivary pellicle counteract the acid attacks but if the challenge is severe, a total destruction of tooth tissue follows. Ultrastructural studies have shown that erosive lesions are seen in prismatic enamel as characteristic demineralization patterns where either the prism cores or interprismatic areas dissolve, leading to a honeycomb structure. In aprismatic enamel the pattern of dissolution is more irregular and areas with various degrees of mineral loss are seen side by side. In dentin the first area to be affected is the peritubular dentin. With progressing lesions, the dentinal tubules become enlarged but finally disruption is seen also in the intertubular areas. If the erosion process is rapid, increased sensitivity of the teeth is the presenting symptom. However, in cases with slower progression, the patient may remain without symptoms even though the whole dentition may become severely damaged. Regarding the role of causative agents, present data does not allow the ranking of different acids with regard to their potential of causing erosion. Neither is there consensus as to how effective fluorides are in preventing the progression of erosive lesions, or how the chemical and structural factors of tooth tissue in general might modify this pathological process.     

Preventive effect of iron gel with or without fluoride on bovine enamel erosion in vitro

Background: The aim of this study was to evaluate the preventive effect in vitro of experimental gel containing iron and/or fluoride on the erosion of bovine enamel. Methods: To standardize the blocks (n = 80), specimens (4 × 4 mm) were previously selected to measure the initial microhardness. The blocks were randomly allocated into four groups of 20 samples each: C (control, placebo gel); F (fluoride gel, 1.23% NaF); Fe (iron gel, 10 mmol/L FeSO₄) and F + Fe (fluoride + iron gel). The gels were applied and removed after 1 minute. The blocks were then submitted to six alternating remineralization and demineralization cycles. The beverage Coca‐Cola^® (10 minutes, 30 mL) was used for demineralization, and artificial saliva (1 hour) for remineralization. The effect of erosion was measured by wear analysis (profilometry). Data were analysed by ANOVA and the Tukey test for individual comparisons (p <0.05). Results: The mean wear (± SD, μm) was C: 0.94 ± 0.22; F: 0.55 ± 0.12; Fe: 0.49 ± 0.11 and F + Fe: 0.55 ± 0.13. When the experimental gels were used, there was statistically significant reduction in enamel wear in comparison with the control (p <0.001). However, the experimental gels did not differ significantly among them. Conclusions: The gels containing iron with or without fluoride are capable of interfering with the dissolution dental enamel in the presence of erosive challenge.     

Protective effect of the in situ formed short-term salivary pellicle

Salivary pellicle, as previously investigated, protects the enamel surface after certain processes of maturation against the influence of acidic agents. The aim of the present study was to investigate the protective effect of the short-term salivary pellicle formed in situ over periods of 3, 60 and 120 min. Six human volunteers used intraoral acrylic splints with bovine enamel samples fixed at the buccal and palatal sites of the maxillary first molars and second premolars. Enamel specimens (n = 252) with and without pellicle were immersed for 60 s in 1.0% citric acid solution under agitation. Knoop surface hardness (KHN) of uneroded polished enamel was measured as a baseline and estimated immediately after erosive treatment reflecting the microhardness loss (DeltaKHN). The amounts of calcium dissolved from the eroded enamel surface were analysed by atomic absorption spectroscopy and scored in mg/l per 10 mm2 of enamel surface area. In addition, the scanning electron microscope was used for the micromorphological examination of the erosive alterations of the enamel surface. The average microhardness loss values after erosion of the enamel samples with buccally/palatally formed pellicle layers were measured as 139.1/144.9 DeltaKHN for 3 min pellicle, 145.9/146.9 DeltaKHN for 60 min pellicle and 141.7/138.6 DeltaKHN for 120 min pellicle. Calcium release values from the specimens with buccal/palatal pellicles were amounted to 15.0/14.9, 16.5/15.9 and 15.3/17.4 mg/l per 10 mm2 for 3, 60 and 120 min-old pellicles, respectively. No significant differences were related to the pellicle formation time and intraoral site (buccal or palatal) in all tested series (ANOVA, P < 0.05). However, significant protection of the enamel surface provided by the pellicle layer was observed on all pellicle-covered surfaces if compared to the non-covered enamel samples (calcium release: 25.6 mg/l per 10 mm2; microhardness loss 187.0 DeltaKHN). These data were in accordance with the morphologic alterations caused by citric acid on the pellicle-covered and pellicle non-covered specimens. It could be concluded that salivary pellicle formed in situ within a period of 3 min offers protection of enamel against citric acid. However, pellicle does not completely inhibit the erosive action of citric acid under the conditions of the present study.     

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牙齿酸蚀症是一种常见的口腔疾病,随着人们生活水平的提高,其发病率在逐年增长.牙釉质在酸性环境下会发生羟基磷灰石溶解反应,磷酸钙盐被溶出,并扩散至牙齿表面,这个过程称之为脱矿.釉质脱矿一旦发生,大部分不可逆转,常会影响到牙齿的健康和美观.防治牙齿酸蚀成为近几年国内外学者研究的热点,而氟化亚锡对牙齿的保护作用也受到学者们的重视.本文对氟化亚锡防治牙齿酸蚀的机制,应用的局限性和安全性,以及验证其防酸蚀效应的实验影响因素进行综述.     

Two-dimensional electrophoresis study of in vitro pellicle formation and dental caries susceptibility

In the present study, a proteomic approach was applied to evaluate the influence of salivary protein composition on in vitro dental pellicle formation and its possible correlation with dental caries. Whole saliva, collected from caries-free and caries-susceptible subjects, was analyzed by two-dimensional electrophoresis, and protein spots were identified by mass spectrometry. Data analysis of salivary protein composition showed a statistically significant correlation between the quantity of acidic proline-rich proteins (PRPs), lipocalin, cystatin SN and cystatin S, and samples from the caries-free group of subjects [decayed, missing or filled teeth (DMFT) = 0]. Samples from subjects with a high DMFT index appear to be correlated with high levels of amylase, immunoglobulin A, and lactoferrin. In vitro pellicle-composition experiments showed the same correlations found for whole saliva. As cystatins are known physiological inhibitors of cathepsins, the higher quantities of lipocalin, and cystatins S and SN found in the samples from the caries-free subjects suggest that inhibition of proteolytic events on other salivary proteins may indirectly provide tooth protection. The correlation between higher levels of the phosphorylated acidic PRPs 1/2 with samples from the caries-free group also suggests a protective role for these proteins.     

Genetic variation may explain why females are less susceptible to dental erosion

Not all individuals at risk for dental erosion (DE) display erosive lesions. The prevalence of DE is higher among male subjects. The occurrence of DE may depend on more than just acidic challenge, with genetics possibly playing a role. The aim of this study was to investigate the association of enamel‐formation genes with DE. One premolar and a saliva sample were collected from 90 individuals. Prepared teeth were immersed in 0.01 M HCl (pH 2.2), and enamel loss (μm) was measured using white light interferometry. DNA was extracted from saliva, and 15 single‐nucleotide polymorphisms were analysed. Allele and genotype frequencies were related to the enamel loss of the specimens. Single‐marker and haplotype analyses were performed using sex as a covariate. Mean enamel loss was higher for male donors than for female donors (P = 0.047). Significant associations were found between enamel loss and amelogenin, X‐linked (AMELX), tuftelin 1 (TUFT1), and tuftelin‐interacting protein 11 (TFIP11). Analyses showed significant associations between variation in enamel‐formation genes and a lower susceptibility to DE in female subjects. The results indicate that susceptibility to DE is influenced by genetic variation, and may, in part, explain why some individuals are more susceptible than others to DE, including differences between female subjects and male subjects.     

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.     

Amino acid composition of acquired enamel pellicle collected in vivo after 2 hours and after 24 hours

Abstract— The adsorption of salivary proteins to dental enamel during pellicle formation has been shown to be a specific process and dependent on the chemical composition of the surfaces. Most studies on the amino acid composition of the acquired enamel pellicle have, however, been performed on the "2-h-pellicle"under controlled experimental conditions. This may have eliminated some natural factors involved in pellicle formation. The aim of the present study was to investigate the effect of extended time of formation and diet on the pellicle formation. Pellicle material was collected from the same subject after 2 and after 24 h when food and beverages were avoided, and after 24 h with the intake of a normal diet. The collected pellicle materials were hydrolyzed and amino acid analyzed. The results showed that pellicle material collected after 24 h and fasting had a chemical composition similar to the "2-h-pellicle", whereas pellicle material collected after 24 h and a normal diet was different, indicating a dietary contribution to pellicle formation or a bacterial degradation of the pellicle.     

人类高分子量唾液粘蛋白抗釉质脱矿的研究

目的 :观察高分子量唾液粘蛋白 (MG1)是否具有抗釉质脱矿作用。方法 :以全唾液、腮腺唾液、颌下 舌下腺唾液和纯化的MG1对实验牙釉质表面进行体外孵育以形成唾液获得性膜 ,观察此膜抗釉质脱矿作用。结果 :全唾液组和腮腺唾液组只有 3 9.7%和 2 1.2 %的最大保护百分度 ,而颌下 舌下腺唾液组和纯化的MG1组能明显抑制脱矿作用 ,其最大保护百分度分别为 96.2 %和 84 .5 %。结论 :唾液获得性膜中的MG1可有效地保护牙釉质抵御有机酸对牙面的短暂脱矿作用。     

Exaggerated abrasion/erosion of human dental enamel surfaces: a case report

An atypical, rapidly proceeding abrasion/erosion of the labial enamel surfaces of the maxillary and mandibular incisors and canines in a 27-yr-old man is reported. Ultrastructural examination of a replica of the teeth showed a practically structureless enamel surface both at the initial examination and after 12 months. However, at the end of the period, minor areas of dentin tubules became visible, indicating that a substantial loss of the tooth substance had taken place. The patient's occupation involved daily environmental contact with proteolytic enzymes. In vitro study of enamel exposed to one of the actual proteolytic enzymes showed dissolution of enamel substance, and it cannot be excluded that enzymatic decomposition of the organic enamel matrix is a contributing cause of the observed exaggerated loss of tooth substance.     

Probiotic bacteria affect the composition of salivary pellicle and streptococcal adhesion in vitro

Introduction:  The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria.
Methods:  Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii , were used as a model.
Results:  Probiotic bacteria that bound to saliva-coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii . Two of the proteins missing from the pellicles made of saliva-treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains.
Conclusion:  This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.     

Interindividual and longitudinal studies of amino acid composition of pellicle collected in vivo

Abstract – The formation of the acquired enamel pellicle is due to the adsorption of salivary proteins to the enamel surface. This adsorption is assumed to be specific and is dependent on the chemical characteristics of the surface. The aim of the present study was to investigate the consistency of the chemical composition of the acquired pellicle collected in vivo. Inter- and intraindividual differences in the chemical composition of pellicle material were examined during 2 yr in three different individuals. The amino acid profiles obtained from pellicle analyses were compared to hydrolyzed whole saliva collected at the same time as the pellicle material. The results showed that the amino acid composition of pellicle was consistent both between and within the individuals. The amino acid profiles obtained from the analyses of the saliva samples were different from the pellicle profiles, illustrating the selective nature of pellicle formation. This supports the contention that the adsorption of salivary proteins to dental enamel is a very specific process.     

Influence of liquid temperature and flow rate on enamel erosion and surface softening

Enamel erosion and softening are based on chemical processes which could be influenced by many factors including temperature and acid flow rate. Knowledge of the influence of these variables could have relevance to research experiments and clinical outcomes. Both parameters were investigated using an ultrasonication and profilometry method to assess erosion depth and surface softening of enamel. The influence of temperature was studied by eroding polished human enamel samples at 4, 20, 35 or 50 degrees C for 2 h. Secondly, different liquid flow conditions were established by varying acid agitation. Additionally, a slow laminar flow and a jet of citric acid, to simulate drinking through a straw, were applied to specimens. Erosion depth increased significantly with acid temperature from 11.0 microm at 4 degrees C to 35.8 microm at 50 degrees C. Surface softening increased much more slowly and plateaued at 2.9 microm to 3.5 microm after 35 degrees C. A strong dependence of erosion on liquid flow was revealed. In unstirred conditions only 8.6 microm erosion occurred, which increased to 22.2 microm with slow stirring and 40.9 microm with fast stirring. Surface softening did not increase correspondingly with its largest extent at slow stirring at 3.4 microm.The implication of these data are: first, the conditions for erosion experiments in vitro or in situ need to be specified for reliable comparisons between studies. Secondly, erosion of teeth by soft drinks are likely to be influenced both by the temperature of the drink and individual drinking habits.     

牙侵蚀与胃食管反流病间的关系

牙侵蚀是指牙体硬组织在无细菌参与的化学作用下的不可逆性丧失,胃食管反流病是常见的胃肠道紊乱性疾病,是牙侵蚀主要的病因之一。本文主要就牙侵蚀的病因、胃食管反流病导致的牙侵蚀机制,牙侵蚀的临床表现,牙侵蚀的预防和治疗等研究进展作一综述。阐明了牙侵蚀是胃食管反流病食管外最重要的表现之一,其诊治需要多科的共同协作,早期诊断胃食管反流病并通过改变生活方式和药物来抑制酸反流,对预防牙侵蚀的发生和干预牙矿质的进一步丢失和损伤极为重要。     

Electron microscopic detection of salivary alpha-amylase in the pellicle formed in situ

Immunological and biochemical analyses have shown that alpha-amylase is an essential component of the acquired pellicle. After adsorption, this enzyme might act as a receptor for bacterial adherence. However, data indicating that amylase is bound to the pellicle surface in vivo and thus available for adhering bacteria are rare. Therefore, the present study focused on alpha-amylase within the pellicle formed in situ, using gold-immunolabeling electron microscopic techniques. Pellicles were formed by intra-oral exposure of enamel specimens for 30 and 120 min in six subjects. The results obtained by transmission electron microscopy indicate that amylase was randomly distributed in the pellicle layer without any preferential localization within the pellicle. Thus, salivary alpha-amylase might be considered as an important structural component that is even involved in the early stages of pellicle formation. The findings of field emission in-lens scanning electron microscopy provided evidence that the enzyme is located on the pellicle surface. It could be concluded that alpha-amylase might act as a receptor for bacterial adherence to the pellicle in vivo.     

唾液获得性膜对不同桥体材料表面自由能的影响

目的：评价唾液获得性膜对不同桥体材料表面自由能的影响。方法：采用接触角测量仪检测4种桥体材料(Co—Cr合金、Au—Pt合金、纯Ti以及Vita95瓷)表面形成唾液获得性膜前后试件的接触角并计算表面能。结果：制备唾液获得性膜后所有受测材料的表面自由能极性分量升高，自由能总量趋于一致。结论：唾液获得性膜会改变修复材料的表面属性，使不同材料表面自由能之间的差异减小。     

Surface etching and subsurface demineralization of dental enamel induced by a strong acid

abstract – Intact enamel surfaces were exposed to 20 ml of 0.1% HCl for various periods. When the acid was unsaturated with respect to both hydroxyapatite and fluorapatite the surface enamel was etched away. With time the liquid close to the enamel became supersaturated with respect to fluorapatite but remained unsaturated with respect to hydroxyapatite. Under this condition subsurface lesions developed. These observations are discussed with reference to the use of diffusion-restricting agents in laboratory caries experiments.     

Iron staining of the acquired enamel pellicle after exposure to tannic acid or chlorhexidine: preliminary report

Abstract – Extrinsic discoloration of teeth following a large consumption of tannin-containing beverages or a prolonged use of chlorhexidine mouthrinses is a well known observation. Tannins as well as chlorhexidine are denaturing agents. Based on preliminary studies revealing the presence of iron in chlorhexidine discolored pellicle material, the ability of iron to stain the integument after pretreatmentwith the two denaturants was studied in a human model. The denaturing effect of an acidic environment was also included. Enamel slabs fixedto acrylie appliances were carried in the oral cavity and alternately exposed to the test solutions in different sequences in vitro. Pretreatment with chlorhexidine or tannic acid led to marked discolorations upon iron application during 5-d tests, whereas the compounds individually had no such effect. A large content of the metal was found in the stained material. Stannous fluoride appeared to reduce the formation of the pigments, and strong oxidation completely bleached the established color. Possible mechanisms underlying the phenomena observed are discussed.