Optimizing the formation of the acquired enamel pellicle in vitro for proteomic analysis

Saliva is the major contributor for the protein composition of the acquired enamel pellicle (AEP), a bacteria-free organic layer formed by the selective adsorption of salivary proteins on the surface of the enamel. However, the amount of proteins that can be recovered is even smaller under in vitro condition, due to the absence of continuous salivary flow. Objective: This study developed an in vitro AEP protocol for proteomics analysis using a new formation technique with different collection solutions. Methodology: 432 bovine enamel specimens were prepared (4x4 mm) and divided into four groups (n=108). Unstimulated saliva was provided by nine subjects. The new AEP formation technique was based on saliva resupply by a new one every 30 min within 120 minutes at 37ºC under agitation. AEP was collected using an electrode filter paper soaked in the collection solutions according with the group: 1) 3% citric acid (CA); 2) 0.5% sodium dodecyl sulfate (SDS); 3) CA followed by SDS (CA+SDS); 4) SDS followed by CA (SDS+CA). The pellicles collected were processed for analysis through LC-ESI-MS/MS technique. Results: A total of 55 proteins were identified. The total numbers of proteins identified in each group were 40, 21, 28 and 41 for the groups CA, SDS, CA+SDS and SDS+CA, respectively. Twenty-three typical AEP proteins were identified in all groups, but Mucin was only found in CA and CA+SDS, while three types of PRP were not found in the SDS group. Moreover, a typical enamel protein, Enamelin, was identified in the CA+SDS group only. Conclusion: The new technique of the in vitro AEP formation through saliva replacement was essential for a higher number of the proteins identified. In addition, considering practicality, quantity and quality of identified proteins, citric acid seems to be the best solution to be used for collection of AEP proteins

Proteomic analysis of the acquired enamel pellicle formed on human and bovine tooth: a study using the Bauru in situ pellicle model (BISPM)

The acquired enamel pellicle (AEP) is an organic film, bacteria-free, formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. Objective: This study aimed to compare the proteomic profile of AEP formed in situ on human and bovine enamel using a new intraoral device (Bauru in situ pellicle model – BISPM). Material and Methods: One hundred and eight samples of human and bovine enamel were prepared (4x4 mm). Nine subjects with good oral conditions wore a removable jaw appliance (BISPM) with 6 slabs of each substrate randomly allocated. The AEP was formed during the morning, for 120 minutes, and collected with an electrode filter paper soaked in 3% citric acid. This procedure was conducted in triplicate and the pellicle collected was processed for analysis by LC-ESI-MS/MS. The obtained mass spectrometry MS/MS spectra were searched against human protein database (SWISS–PROT). Results: The use of BISPM allowed the collection of enough proteins amount for proper analysis. A total of 51 proteins were found in the AEP collected from the substrates. Among them, 15 were common to both groups, 14 were exclusive of the bovine enamel, and 22 were exclusive of the human enamel. Proteins typically found in the AEP were identified, such as Histatin-1, Ig alpha-1, Ig alpha 2, Lysozyme C, Statherin and Submaxillary gland androgen-regulated protein 3B. Proteins not previously described in the AEP, such as metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were also identified. Conclusion: These results demonstrate that the proteins typically found in the AEP appeared in both groups, regardless the substrate. The BISPM revealed to be a good device to be used in studies involving proteomic analysis of the AEP

Novel protective salivary peptides and the chitosan nanoparticles delivery system

As a result of its protein composition, saliva performs many essential functions that contribute to oral homeostasis. However, salivary proteins suffer quick degradation in the oral cavity, which decreases the protective potential of that fluid (Chapter 2). Using scanning electron microscopy (SEM), we evaluated the effect of our novel salivary peptides DR9, DR9-DR9, DR9-RR14 and RR14 on the crystallization of calcium oxalate (CaOx). All peptides containing DR9 showed significant inhibitory effect on the crystallization of CaOx, whereas RR14 showed no effect (Chapter 3). Next, we tested the adsorption behavior of our chitosan nanoparticles (CHNP) delivery system to hydroxyapatite (HA) surface and to a protein-coated-HA surface using x-ray photoelectron spectroscopy (XPS) and confocal microscopy. According to our results, CHNP are capable of adsorbing to HA and more evenly to a protein-coated-HA (Chapter 4). Our results bring us closer to developing a reliable delivery system for our protective peptides

A mass spectrometric approach to the proteomic profiling of the Canis lupus familiaris acquired enamel pellicle on hydroxyapatite discs

The acquired enamel pellicle (AEP) is a multi-protein film attached to the surface of teeth, which functions to lubricate the dental surface, form an anti-erosive barrier and exhibits antimicrobial properties. The initiation of AEP formation occurs within seconds of exposure to saliva, a biofluid rich in protein species. While there have been many publications on the formation of human AEP there is little research on the composition of canine AEP during its acquisition. The aim of these studies was to explore the composition of canine AEP formation, utilising hydroxyapatite (HA) discs as a tooth substitute matrix, over time. Qualitative and quantitative proteomics techniques using tandem mass tag labelled peptides and LC-MS/MS were used to follow the formation of canine AEP on hydroxyapatite discs over the course of an hour. Proteins adsorbed to the HA surface included highly abundant proteins in canine saliva, antimicrobial proteins, protease inhibitors and the buffering agent carbonic anhydrase. Greater understanding of the canine AEP deepens fundamental knowledge of the early processes driving bacterial colonisation of the tooth surface and subsequent plaque accumulation

Effect on Pellicle Proteome upon Adsorption of Salivary Peptide on Hydroxyapatite and their effects on Enamel Demineralization

According to WHO, oral health is an important indicator of overall health. Poor oral health cause serious health problems (cardiovascular, stroke, diabetes, liver, and respiratory related), in different age groups, costing billions of dollars. So, proper oral health care management has assumed top priority. Salivary antimicrobial peptides (histatin and statherin), are part of enamel pellicle, required for the oral homeostasis. Current work focused to study the demineralization protection and pellicle proteome modulation capabilities of novel peptide constructs (single and tandem duplicate histatin constructs DR9, DR9-DR9 and histatin-statherin hybrid construct DR9-RR14). For the first time, we provided evidence that, these peptide constructs (low concentration) modulated the pellicle proteome composition as analyzed through nLC-MS. We showed that DR9, and DR9-DR9 provided enamel demineralization (Ca and PO4) protection in presence of parotid saliva pellicle. It is important work from the point-of-view of enamel protection and inhibition of pathological biofilm formation by novel salivary peptides and hence infection in the oral cavity

Evaluation of novel salivary peptides and a new application method for prevention and treatment of oral disease

Some salivary proteins degrade quickly after secretion into the oral cavity, due to proteases present in the oral environment. DR9, the N-terminal domain of statherin is an example of these peptides, which sustains the activity of the original protein. Inspired by naturally occurred DR9 and the evolutionary pathway taken by proteins; we constructed novel peptides by combining the functional domains of different salivary proteins and investigated the functionality of these peptides. Our results revealed that DR9 duplication could increase its functionality and DR9 combination with RR14, the functional domain of histatin, could combine the functions of both peptides. Furthermore, we explored the possibility of using chitosan, an amino-polysaccharide, as a carrier for salivary peptides. Chitosan nanoparticles showed a significant higher killing effect compared to non-particle chitosan against Candida albicans. Moreover, chitosan nanoparticles showed a favorable killing effect in specific pHs, similar to the critical pHs in the oral cavity

Investigating the Therapeutic Potential of Salivary Proteins for Oral Diseases

ABSTRACT Saliva is responsible for the formation of the acquired enamel pellicle (AEP), a protein integument formed as a result of selective adsorption of salivary proteins to the enamel surface. The AEP demonstrates an important role for modulating dental erosion as a result of its physical properties, along with its salivary and exogenous protein composition (Chapter 2). In addition, individual proteins that comprise the AEP have important physiological functions. Histatin 5 (H5) has potent antifungal effect against C. albicans, the yeast responsible for the initiation of oral candidiasis. We designed an in vitro model and found, for the first time, that H5 adhered in the form of pellicle retains its antifungal activity on C. albicans (Chapter 3). As a pellicle precursor protein, H5 demonstrates high affinity for hydroxyapatite, the primary mineral component of enamel. We used atomic force microscopy (AFM) to determine adhesion forces between H5 and the hydroxyapatite surface to be stronger compared to our protein control, albumin. This knowledge can be applied in the design of therapeutic proteins, and the methodology that we developed can be used for measuring adhesion forces between various other proteins and substrates of interest (Chapter 4). Finally, with the development of proteomics instruments, researchers have identified some protein biomarkers, hidden within salivary fluids. These can be used for diagnostic dentistry, in a clinical setting to identify patients’ susceptibility of developing oral diseases. In addition, the delivery proteins with antimicrobial properties via toothpastes or oral rinses can have tremendous therapeutic potential for a multitude of oral diseases (Chapter 5)

Acquired Bracket Pellicle Modulation Via Exposure To Histatin 3

Objectives: To investigate the effect of histatin 3 on the protein pellicle formation on the orthodontic bracket surface. Methods: Our study consisted of 4 sample groups. A sample of human parotid saliva without histatin functioned as a control group. Other groups were immersed with or without histatin 3 and human parotid saliva. Each group was incubated for 2 hours in their respective substrate at 37°C. The acquired pellicle from each group was harvested, then analyzed with SDS-PAGE and LC-ESI-MS/MS. Results: Thirty-nine proteins were identified in the control group, 18 were identified in group 2, and 21 were identified in group 3. Thirteen proteins were common to all groups. Groups immersed in histatin 3 and human parotid saliva showed an increase in the percentage of proteins exhibiting antimicrobial activities and immune response. Conclusions: There were functional differences in the protein pellicle formed on the orthodontic bracket, suggesting that exposure to histatin 3 may alter pellicle formation. However, structural differences were limited due to redundant characteristics of salivary proteins

Caries Management :The Role of Surface Interactions in De- and Remineralization-Processes

Background: Bioadhesion and surface interactions on enamel are of essential relevance for initiation, progression and prevention of caries and erosions. Salivary proteins on and within initial carious and erosive lesions can facilitate or aggravate de- and remineralization. This applies for the pellicle layer, the subsurface pellicle and for proteins within initial carious lesions. Little is known about these proteinaceous structures related to initial caries and erosion. Accordingly, there is a considerable demand for an understanding of the underlying processes occurring at the interface between the tooth surface and the oral cavity in order to develop novel agents that limit and modulate caries and erosion. Objectives and findings: The present paper depicts the current knowledge of the processes occurring at the interface of the tooth surface and the oral fluids. Proteinaceous layers on dental hard tissues can prevent or aggravate demineralization processes, whereas proteins within initial erosive or carious lesions might hinder remineralization considerably and restrict the entry of ions into lesions. Conclusions: Despite the fact that organic–inorganic surface interactions are of essential relevance for de- and remineralization processes at the tooth surface, there is limited knowledge on these clinically relevant phenomena. Accordingly, intensive research is necessary to develop new approaches in preventive dentistry

Electrophoretic analysis of salivary proteins from man and Macaca Fascicularis and immunological identification of salivary proline-rich proteins in the in-vivo, formed pellicle

Illustrations are black and white photographs.Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1979 (Oral Biology).The purpose of this study was the electrophoretic analysis and comparison of salivary proteins from Man and Macaca fascicularis with particular emphasis on the presence of four anionic proteins, known as the major acidic praline-rich proteins (PRP I, PRP II, PRP III, PRP VI). The second part of this study constitutes an immunological approach to identify these salivary praline-rich proteins as constituents of the naturally formed, acquired enamel pellicle. The results obtained from disc gel and slab gel acrylamide electrophoresis indicated that the concentrations of praline-rich proteins (PRPs) present in human glandular secretions were higher than those measured for whole saliva. The latter, however, exhibited a heavily stained component known as serum albumin. Enzymes derived from oral microorganisms have been implicated in the degradation of PRPs in the oral environment leading to the electrophoretic changes observed. Des~ite the great similarities between parotid and submandibular saliva disc gel acrylamide electrophoretograms, at least one qualitative difference could be detected. [TRUNCATED