Bioadhesion on Textured Interfaces in the Human Oral Cavity-An In Situ Study.

Extensive biofilm formation on materials used in restorative dentistry is a common reason for their failure and the development of oral diseases like peri-implantitis or secondary caries. Therefore, novel materials and strategies that result in reduced biofouling capacities are urgently sought. Previous research suggests that surface structures in the range of bacterial cell sizes seem to be a promising approach to modulate bacterial adhesion and biofilm formation. Here we investigated bioadhesion within the oral cavity on a low surface energy material (perfluorpolyether) with different texture types (line-, hole-, pillar-like), feature sizes in a range from 0.7-4.5 µm and graded distances (0.7-130.5 µm). As a model system, the materials were fixed on splints and exposed to the oral cavity. We analyzed the enzymatic activity of amylase and lysozyme, pellicle formation, and bacterial colonization after 8 h intraoral exposure. In opposite to in vitro experiments, these in situ experiments revealed no clear signs of altered bacterial surface colonization regarding structure dimensions and texture types compared to unstructured substrates or natural enamel. In part, there seemed to be a decreasing trend of adherent cells with increasing periodicities and structure sizes, but this pattern was weak and irregular. Pellicle formation took place on all substrates in an unaltered manner. However, pellicle formation was most pronounced within recessed areas thereby partially masking the three-dimensional character of the surfaces. As the natural pellicle layer is obviously the most dominant prerequisite for bacterial adhesion, colonization in the oral environment cannot be easily controlled by structural means.

Mechanical characterization of in vitro-formed short-term salivary pellicle.

Human saliva consists of approximately 98% water and a variety of electrolytes and proteins. Those proteins can be selectively adsorbed onto the enamel surface. The cuticular material formed on the enamel surface is termed acquired salivary pellicle (ASP), which is critical for the health of oral mucosa and teeth. The ASP is composed of a inner layer and a outer layer. The lubricating properties of ASP are closely associated with the inner layer. The aim of this research is to characterize the structural and mechanical properties of the inner layer of ASP. In this paper, enamel specimens were immersed for 1 min in human saliva. The ASP formed in vitro within 1 min was studied using a nanoindenter. The results show that the thickness of the inner layer of ASP is approximately 18 nm. Moreover, the inner layer is a heterogeneous pellicle with a gradient in density. From the surface of the inner layer to the enamel surface, the density and mechanical properties gradually increase. The research results may be helpful to extend the understanding of mechanical properties of salivary pellicle and to the oral hygiene industry for diagnose oral diseases.

Intraoral electric potential via oral bacterial power generation -A novel mechanism of biofilm formation.

In the early stages of biofilm accumulation, the electric charge of the dental enamel and pellicle surfaces is known to be involved. We therefore investigated the relationship between oral hygiene and intraoral electric potential (IoP) in 45 male participants using a double-blind study. IoP, but not body surface electric potential, was loosely correlated with oral hygiene condition (Oral Hygiene Index; OHI). IoP was also loosely correlated with smartphone use; however, there was no significant correlation between smartphone use and OHI. IoP elevation might be caused by OHI elevation resulting from biofilm formation as an internal factor, with smartphone use as an external factor. This in vitro study revealed the generating capacity of Streptococcus mutans accompanied by biofilm accumulation using a microbial fuel cell. These results suggest that IoP elevation is caused by biofilm accumulation induced by power generation of oral bacteria, resulting in elevation of OHI.

The mucosal pellicle - An underestimated factor in oral physiology.

Bioadhesion and bio-adsorption of proteins, glycoproteins and other biomolecules are ubiquitous phenomena in the oral cavity. While the protective role of the adsorbed salivary biomolecules on teeth (the acquired enamel pellicle) is well established, it has yet to be defined whether comparable processes occur on the desquamating oral soft tissues. The general term for these layers is pellicle, but due to the different characteristics of the coated surfaces the enamel pellicle and mucosal pellicle are their own entities. There is considerable information on the enamel pellicle, whereas only limited data are available on the mucosal pellicle. This can be attributed to the difficult standardized preparation of this biological structure. Based on the present knowledge the abundant and characteristic components of the mucosal pellicle include secreted soluble mucins (MUC5B, MUC7), membrane-associated epithelial mucins (MUC1), and to a lesser degree CA VI, sIgA, and cystatin. However, it seems to be of completely different ultrastructure as compared with the enamel pellicle. Since it is comprised of larger glycoproteins retaining water, it might be considered as a hydrogel, and it appears to have a lower tenacity than the enamel pellicle. Maturation and turnover are influenced by the delivery of salivary proteins, by the flow of saliva and the underlying desquamating oral epithelium. Its probable functions include lubrication and moisture retention. In general, the mucosal pellicle can be regarded as an underestimated key player in oral physiology.

Salivary acquired pellicle-inspired DpSpSEEKC peptide for the restoration of demineralized tooth enamel.

Salivary acquired pellicle (SAP) is a layer of proteins and glycoproteins of salivary origin that tightly coat the tooth surface. Statherin is an important part of the SAP. The initial six-peptide sequence DpSpSEEK (where pS denotes phosphorylated serine) of the N-terminus of statherin can be immobilized on a hydroxyapatite (HAP) surface and the negatively charged domains of the DpSpSEEK side chain can catch free Ca2+ in saliva due to the charge adsorption effect. In order to prepare more functional materials based on DpSpSEEK, we designed a cysteine-labeled peptide sequence DpSpSEEKC, which could conjugate other macromolecules by forming a sulfur-based linkage. In this work, we measured the adsorption of DpSpSEEKC to HAP by various methods. We also coated DpSpSEEKC on a demineralized tooth enamel surface to evaluate its biomineralization capacity. The DpSpSEEKC-coated samples were characterized after immersion in artificial saliva for 2 weeks. The results showed that DpSpSEEKC has a strong adsorption capacity to HAP and could induce remineralization on the demineralized tooth enamel surface due to its carboxyl and phosphate groups. Compared with the control samples, the mechanical properties of the DpSpSEEKC-coated samples were obviously improved. In conclusion, DpSpSEEKC can provide a potential method for restoring demineralized tooth enamel.

The Susceptibility to Dental Erosion Differs among Individuals.

Studies of wine tasters and patients with self-induced vomiting have revealed that 30-50% of individuals at high risk do not develop erosive lesions. The aim was to investigate this apparent individual susceptibility to enamel erosion. Two enamel specimens were made from each of 3 premolars from 8 persons (donors). Six acrylic mouth appliances were worn by 6 volunteers (carriers). One specimen from each donor was mounted on each appliance. The carriers wore the appliances for 9 days. The appliances were immersed in 0.01 M HCl for 3 min twice per day to imitate a vomiting/reflux situation. The enamel specimens were analysed by a white-light interferometer to measure enamel loss (in micrometres). The enamel loss varied significantly both between the donor teeth (p = 0.009) and the carriers (p = 0.004). The lesion in the specimen with the largest amount of enamel loss was 4 times as deep as in the specimen with the lowest. In 1 carrier, all specimens displayed enamel loss above the mean, including the specimen from the donor with the most resistant enamel. The variation in susceptibility to erosion among individuals appears to be influenced both by the sustainability of the enamel and by factors in the oral environment. This could explain the variation in prevalence and severity of dental erosions among patients exposed to similar acidic challenges. The results suggest that for certain individuals, only minimal acidic challenges may be sufficient to cause damage to the teeth, while others may never develop dental erosions despite extensive exposure to acid.

Salivary pellicles equalise surfaces' charges and modulate the virulence of Candida albicans biofilm.

INTRODUCTION: Numerous environmental factors influence the pathogenesis of Candida biofilms and an understanding of these is necessary for appropriate clinical management. AIMS: To investigate the role of material type, pellicle and stage of biofilm development on the viability, bioactivity, virulence and structure of C. albicans biofilms. METHODS: The surface roughness (SR) and surface free energy (SFE) of acrylic and titanium discs was measured. Pellicles of saliva, or saliva supplemented with plasma, were formed on acrylic and titanium discs. Candida albicans biofilms were then generated for 1.5 h, 24h, 48 h and 72 h. The cell viability in biofilms was analysed by culture, whilst DNA concentration and the expression of Candida virulence genes (ALS1, ALS3 and HWP1) were evaluated using qPCR. Biofilm metabolic activity was determined using XTT reduction assay, and biofilm structure analysed by Scanning Electron Microscopy (SEM). RESULTS: Whilst the SR of acrylic and titanium did not significantly differ, the saliva with plasma pellicle increased significantly the total SFE of both surface. The number of viable microorganisms and DNA concentration increased with biofilm development, not differing within materials and pellicles. Biofilms developed on saliva with plasma pellicle surfaces had significantly higher activity after 24h and this was accompanied with higher expression of virulence genes at all periods. CONCLUSION: Induction of C. albicans virulence occurs with the presence of plasma proteins in pellicles, throughout biofilm growth. To mitigate such effects, reduction of increased plasmatic exudate, related to chronic inflammatory response, could aid the management of candidal biofilm-related infections.

In vitro salivary pellicles from adults and children have different protective effects against erosion.

OBJECTIVES: We aimed at analyzing the protective effects of salivary pellicles, formed with saliva from adults or children, on enamel from permanent or deciduous teeth. MATERIALS AND METHODS: Ninety human enamel specimens (45 permanent premolars and 45 deciduous canines) were ground, and the outer 200 µm of enamel was removed. We divided the teeth into three further subgroups: no salivary pellicle (control), adult salivary pellicle (AP), and child salivary pellicle (CP). We collected stimulated saliva from adults and children and placed 160 µl of either saliva on enamel specimens from AP and CP, respectively. Control specimens received no saliva. Specimens were stored at 37 °C for 2 h and then submitted to an erosive challenge (10 mL; 1 % citric acid; pH 3.6; 25 °C, 1 min). Pellicle formation and erosion was repeated for a total of 4 cycles. After every cycle, relative surface reflection intensity (rSRI) and surface microhardness (rSMH) were calculated. RESULTS: On permanent enamel, AP presented significantly better protective effects, with less rSMH loss (p < 0.001) and less rSRI loss (p < 0.001). On deciduous enamel, CP presented significantly better protective effects than AP and control (p < 0.05), for both measured parameters. CONCLUSION: We conclude that pellicles from adults and children promote different erosion protective effects, where adult pellicle provides better protection for permanent enamel, and child pellicle promotes better protection on deciduous enamel. CLINICAL RELEVANCE: The present results provide a better understanding toward the protective effect of salivary pellicle against dental erosion and brings light to one more factor involved in the erosion of deciduous teeth.

Sequence of stannous and sodium fluoride solutions to prevent enamel erosion.

OBJECTIVES: Investigate the timing of stannous (SnF2) and sodium fluoride (NaF) application with and without salivary pellicle to prevent enamel erosion. METHODS: Human buccal molar enamel samples (n=120, REC ref 12/LO/1836) were randomly assigned to three groups testing SnF2 and NaF basic fluoride formulation and commercial mouthrinses with and without the presence of human saliva. Samples were randomly allocated to 2 subgroups: immersion in either fluoride for 1 min either before or after citric acid immersion (0.3%, pH 3.2, 10 min), and the cycle repeated 5 times. For human saliva group, samples were immersed in 80 ml of natural saliva for 24 h prior to the experiment. Analysis was done using non-contacting profilometry and microhardness change. Data were not normal and were log transformed. A linear model tested statistical differences between the groups. RESULTS: SnF2 application before erosion statistically reduced step height compared to application after erosion for all groups (solutions: 6.5 µm (±1.2), 7.5 µm (±0.8); p=0.01, mouthrinses: 3.2 µm (±0.6), 4.2 µm (±0.7); p<0.0001, mouthrinses with saliva: 2.5 µm (±0.4), 3.1 µm (±0.6); p=0.002, before and after respectively). In contrast, application of NaF before erosion increased step height compared to application after, but this was only statistically significant for the saliva group (before: 5.6 µm (±0.3) and after: 4.9 µm (±0.3); p=0.023). Presence of saliva increased microhardness change (p<0.0001). Within this group, greatest microhardness change was observed when SnF2 was applied before erosion and when NaF was applied after erosion (SnF2: 156.6KHN (±32.8), 123KHN (±20.1); p=0.02. NaF: 119.5KHN (±33.5), 218KHN (±24.9), before, and after respectively). CONCLUSION: SnF2 reduced step height formation overall when compared to NaF, but particularly when applied before citric acid immersion. In contrast, NaF reduced step height when applied after citric acid immersion, but only in the presence of saliva. CLINICAL SIGNIFICANCE: Stannous fluoride can be recommended over sodium fluoride to patients at risk of dental erosion and is optimally applied before erosion occurs. If sodium fluoride is to be used in the presence of saliva it is optimally applied after erosion has occurred.

Erosion Prevention Potential of an Over-the-Counter Stabilized SnF2 Dentifrice Compared to 5000 ppm F Prescription-Strength Products.

OBJECTIVE: To determine the relative ability of various F-containing products to protect enamel against the initiation and progression of tooth surface loss due to erosive acid challenges. METHODS: Cores of enamel were prepared from extracted human teeth, soaked in pooled human saliva (pellicle formation), and then treated in a 1:3 slurry (product:saliva) of either OTC level (1100 ppm F) or prescription level (5000 ppm F) products, followed by a standardized erosion cycling procedure (five days of cycling) that included 10-minute challenges with an erosive dietary acid (1% citric acid at pH 2.3) applied 60 minutes after each dentifrice treatment (repeated four times per day). Enamel surface loss was measured using transverse microradiography. Two studies were conducted. Study 1 included: A) 1100 ppm F as NaF; B) 1100 ppm F as stabilized SnF; C) 5000 ppm F as NaF; and D) 5000 ppm F as NaF + acidulated phosphate. Study 2 included: 1) 1100 ppm F as stabilized SnF; 2) 5000 ppm F as NaF + tricalcium phosphate; and 3) 1100 ppm F as NaF. RESULTS: Study 1: Treatment B (1100 ppm F as SnF), where specimens lost only 8.0 µm of the enamel surface, was significantly more effective than Treatments A, C, and D at protecting enamel against the initiation and progression of erosive acid damage (p < 0.05). Specimens treated with product A exhibited 22.8 (1.25) µm (mean ± sem) of enamel loss; 20.0 (0.71) µm of enamel loss with treatment C and 24.0 (1.4) µm of enamel loss with Treatment D. Study 2 also demonstrated significantly greater erosion protection with the stabilized SnF2 dentifrice (p < 0.05), with only 5.8 (1.93) µm of tooth surface loss, while groups 2 and 3 lost 19.8 (0.75) µm and 18.0 (2.16) µm, respectively. CONCLUSION: Results from both studies demonstrated the OTC dentifrice formulated with stabilized SnF2 provides significantly greater protection against erosive acid attack compared to some of the most popular prescription level (5000 ppm F) fluoride treatments available.

Plasma proteins in the acquired denture pellicle enhance substrate surface free energy and Candida albicans phospholipase and proteinase activities.

AIM: The objective of the present study was to determine if blood plasma proteins could change the proteome of the acquired denture pellicle by label-free quantitative proteomics. As pellicle proteome modulates the interaction between substrates and Candida cells, we investigated its effect on the surface free energy (SFE) of the coated resin and on Candida albicans phospholipase and aspartyl proteinase activities. METHODS: Poly(methylmethacrylate) discs were exposed to saliva (control) or saliva enriched with blood plasma (experimental group). The pellicle proteome was analyzed by mass spectrometry coupled with liquid chromatography. SFE was determined by acid-base technique. After biofilm formation, phospholipase and proteinase activities were determined accordingly to classic plate methods. Data were analyzed by two-way anova and Tukey test (P < 0.05). RESULTS: α-Amylase, cystatins, mucins, and host-immune system proteins were the main proteins identified in the control group. Fibrinogen and albumin were observed only in the experimental group. Coated discs of the experimental group presented an increased SFE (P < 0.05). For both enzymes tested, the experimental group showed higher proteolytic activity (P < 0.001). CONCLUSION: Blood plasma changes the proteome of the acquired denture pellicle, increasing surface free energy and the activity of Candida albicans phospholipase and aspartyl proteinase.

The interactions between attrition, abrasion and erosion in tooth wear.

Tooth wear is the result of three processes: abrasion (wear produced by interaction between teeth and other materials), attrition (wear through tooth-tooth contact) and erosion (dissolution of hard tissue by acidic substances). A further process (abfraction) might potentiate wear by abrasion and/or erosion. Knowledge of these tooth wear processes and their interactions is reviewed. Both clinical and experimental observations show that individual wear mechanisms rarely act alone but interact with each other. The most important interaction is the potentiation of abrasion by erosive damage to the dental hard tissues. This interaction seems to be the major factor in occlusal and cervical wear. The available evidence is insufficient to establish whether abfraction is an important contributor to tooth wear in vivo. Saliva can modulate erosive/abrasive tooth wear, especially through formation of pellicle, but cannot prevent it.

The potential of saliva in protecting against dental erosion.

Saliva is the most relevant biological factor for the prevention of dental erosion. It starts acting even before the acid attack, with an increase of the salivary flow rate as a response to the acidic stimuli. This creates a more favorable scenario, improving the buffering system of saliva and effectively diluting and clearing acids that come in contact with dental surfaces during the erosive challenge. Saliva plays a role in the formation of the acquired dental pellicle, a perm-selective membrane that prevents the contact of the acid with the tooth surfaces. Due to its mineral content, saliva can prevent demineralization as well as enhance remineralization. These protective properties may become more evident in hyposalivatory patients. Finally, saliva may also represent the biological expression of an individual's risk for developing erosive lesions; therefore, some of the saliva components as well as of the acquired dental pellicle can serve as potential biomarkers for dental erosion.

The pellicle and erosion.

All tooth surfaces exposed to the oral environment are naturally coated by the acquired salivary pellicle. The pellicle is composed of adsorbed macromolecular components from saliva, gingival crevicular fluid, blood, bacteria, mucosa and diet. The pellicle (formed in situ/in vivo) functions as a semipermeable network of adsorbed salivary macromolecules and provides partial protection against acidic challenges; however, it cannot completely prevent demineralization of the tooth surface. The physiological pellicle reduces calcium and phosphate release from the enamel, and much less from the dentinal surface. With high probability, calcium- and phosphate-binding peptides and proteins adsorbed in the basal pellicle layer are of main relevance for the erosion-reducing effects of the natural salivary pellicle. Improvement of the pellicle's protective properties by dietary components (e.g. polyphenolic agents) might be a promising erosion-preventive approach that, however, needs validation by in situ experiments.

Efficacy of citric acid denture cleanser on the Candida albicans biofilm formed on poly(methyl methacrylate): effects on residual biofilm and recolonization process.

BACKGROUND: It is well known that the use of denture cleansers can reduce Candida albicans biofilm accumulation; however, the efficacy of citric acid denture cleansers is uncertain. In addition, the long-term efficacy of this denture cleanser is not well established, and their effect on residual biofilms is unknown. This in vitro study evaluated the efficacy of citric acid denture cleanser treatment on C. albicans biofilm recolonization on poly(methyl methacrylate) (PMMA) surface. METHODS: C. albicans biofilms were developed for 72 h on PMMA resin specimens (n = 168), which were randomly assigned to 1 of 3 cleansing treatments (CTs) overnight (8 h). CTs included purified water as a control (CTC) and two experimental groups that used either a 1:5 dilution of citric acid denture cleanser (CT5) or a 1:8 dilution of citric acid denture cleanser (CT8). Residual biofilms adhering to the specimens were collected and quantified at two time points: immediately after CTs (ICT) and after cleaning and residual biofilm recolonization (RT). Residual biofilms were analyzed by quantifying the viable cells (CFU/mL), and biofilm architecture was evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Denture cleanser treatments and evaluation periods were considered study factors. Data were analyzed using two-way ANOVA and Tukey's Honestly Significant Difference (HSD) test (α = 0.05). RESULTS: Immediately after treatments, citric acid denture cleansing solutions (CT5 and CT8) reduced the number of viable cells as compared with the control (p < 0.01). However, after 48 h, both CT groups (CT5 and CT8) showed biofilm recolonization (p < 0.01). Residual biofilm recolonization was also detected by CLSM and SEM analysis, which revealed a higher biomass and average biofilm thickness for the CT8 group (p < 0.01). CONCLUSION: Citric acid denture cleansers can reduce C. albicans biofilm accumulation and cell viability. However, this CT did not prevent biofilm recolonization.

Salivary pellicles.

The salivary pellicle is a thin acellular organic film that forms on any type of surface upon exposure to saliva. The role of the pellicle is manifold, and it plays an important role in the maintenance of oral health. Its functions include not only substratum protection and lubrication, but also remineralization and hydration. It also functions as a diffusion barrier and possesses buffering ability. Not only the function, but also the formation, composition and stability of the pellicle are known to be highly influenced by the physicochemical properties of both substrata and ambient media. In this chapter, we discuss these aspects of salivary pellicles, an area where research has boomed in the past years partly because of the application of experimental techniques often reserved for more traditional surface science studies.

Lubrication.

Saliva is capable of decreasing friction force by at least 2 orders of magnitude when in between hydrophobic surfaces. This ability to lubricate is key to oral health, food processing and taste perception. In this paper different mechanisms of saliva lubrication are reviewed, and their interconnection is demonstrated using a simple physical framework. The current understanding of the roles of the molecular structure and physicochemical properties of major salivary proteins and protein complexes on lubrication is summarised and critically evaluated.

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.

Erosion protection comparison of stabilised SnF2 , mixed fluoride active and SMFP/arginine-containing dentifrices.

PURPOSE: To investigate the relative erosion protection potential of marketed dentifrices formulated with either stabilised stannous fluoride (SnF2 ), sodium fluoride (NaF) and/or sodium monofluorophosphate (SMFP) using an established laboratory erosion cycling model. METHODS: Sound enamel cores from extracted, human enamel were cleaned, ground and polished, soaked in pooled saliva (pellicle formation) and treated with a 1:3 slurry of dentifrice and saliva. Specimens were subjected to daily challenges with 1% citric acid, a potentially damaging acid found in common food and drinks. Marketed dentifrices compared were: (1) a stabilised stannous fluoride product formulated with 1,100 ppm F as SnF2 ; (2) a cavity protection product containing 1,100 ppm F as NaF; (3) a cavity protection product comprising a mixed active fluoride system with 1,000 ppm F as SMFP + 450 ppm F as NaF; and (4) a sensitivity product containing 1,450 ppm F as SMFP + 8% arginine bicarbonate. RESULTS: Specimens from Group 1 demonstrated an average loss of 5.5 (±1.2) µm of tooth surface enamel; Groups 2, 3 and 4 lost an average of 18.3 (±0.9) µm, 16.0 (±2.0) µm and 17.1 (±1.1) µm, respectively, of tooth surface enamel. Group 1 provided a statistically significant difference in protection compared with the other products. CONCLUSIONS: These results suggest that the marketed dentifrice formulated with stabilised SnF2 may provide enhanced protection of exposed tooth surfaces against dietary acid attack compared with the other products tested.

Comparison of the possible protective effect of the salivary pellicle of individuals with and without erosion.

The acquired pellicle adheres to tooth surfaces and has been suggested to provide differing degrees of protection against acidic erosion. This study investigated whether pellicle formed on enamel blocks in patients suffering dietary dental erosion modified the effect of an in vitro simulated dietary challenge, in comparison with pellicle formed on enamel blocks in healthy subjects and to no-pellicle enamel samples. Sixty subjects recruited from dental erosion clinics were compared to healthy age-matched controls. Subjects wore a custom-made maxillary splint holding human enamel blocks for 1 h during which the acquired enamel pellicle was formed. Enamel blocks were removed from the splints and a simulated dietary erosive challenge of 10 min was performed. In addition the challenge was performed on 30 enamel samples without pellicle. Profilometry showed no statistical difference between samples from the erosion subjects with a mean step height of 1.74 µm (SD 0.88) and median roughness (Sa) of 0.39 µm (interquartile range, IQR 0.3-0.56) and the controls with 1.34 µm (SD 0.66) and 0.33 µm (IQR 0.27-0.38), respectively. The control samples without pellicle had Sa of 0.44 µm (IQR 0.36-0.69) and these differences were statistically significant compared to those from the healthy subjects (p = 0.002). Mean (SD) microhardness reduction with a 100-gram load for the erosion group was 113.5 (10) KHN, for healthy subjects was 93 (15.4) KHN and for the enamel samples without pellicle 139.6 (21.8) KHN and all groups were statistically different. The microhardness and roughness data suggested the pellicle influenced erosion under these study conditions.