Abstract The use of cocaine and its main derivative, crack, can cause some systemic effects that may lead to the development of some oral disorders. Objective To assess the oral health of people with a crack cocaine use disorder and identify salivary protein candidates for biomarkers of oral disorders. Methodology A total of 40 volunteers hospitalized for rehabilitation for crack cocaine addiction were enrolled; nine were randomly selected for proteomic analysis. Intraoral examination, report of DMFT, gingival and plaque index, xerostomia, and non-stimulated saliva collection were performed. A list of proteins identified was generated from the UniProt database and manually revised. Results The mean age (n=40) was 32 (±8.88; 18–51) years; the mean DMFT index was 16±7.70; the mean plaque and gingival index were 2.07±0.65 and 2.12±0.64, respectively; and 20 (50%) volunteers reported xerostomia. We identified 305 salivary proteins (n=9), of which 23 were classified as candidate for biomarkers associated with 14 oral disorders. The highest number of candidates for biomarkers was associated with carcinoma of head and neck (n=7) and nasopharyngeal carcinoma (n=7), followed by periodontitis (n=6). Conclusions People with a crack cocaine use disorder had an increased risk of dental caries and gingival inflammation; less than half had oral mucosal alterations, and half experienced xerostomia. As possible biomarkers for 14 oral disorders, 23 salivary proteins were identified. Oral cancer and periodontal disease were the most often associated disorders with biomarkers.

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

ABSTRACT The “RichBlend” protocol was designed for facial filling and collagen biostimulation, by means of a mixture of calcium hydroxyapatite (CaHA), hyaluronic acid (AH) and autologous platelet concentrates. This work reports the case of a 53-year-old patient with cutaneous photoaging, loss of facial volume, multiple rhythms in the frontal and periorbital regions, also marked skin flaccidity, especially the eyelid. The treatment was done with botulinum toxin (65 U) and the “RichBlend” protocol. Venipuncture was performed and the blood was centrifuged to obtain i-PRF (injectable platelet-rich fibrin) and plasma gel. After venipuncture and blood centrifugation, i-PRF and plasma gel were obtained. CaHA (Radiesse®) was diluted: a) in saline solution + i-PRF (hyperdilution) for biostimulationof the lower third of the face; and b) in AH (Juvederm Ultraplus XC®) + plasma gel, for hydrolifting on the forehead and dark circles, malar and temples. Plasma gel was applied to the nasogenian grooves and then the entire face was properly massaged. The “RichBlend” protocol rejuvenated the patient, as it promoted filling, volumizing, collagen formation (biostimulation), reduction of flaccidity, in addition to skin whitening. Since HA and CaHA are high-cost products, their mixture with autologous platelet concentrates, in liquid or gel form, allows the use of a greater amount of filled and biostimulator material on the face, at a more affordable cost.

RESUMO O protocolo “RichBlend” foi idealizado para preenchimento facial e bioestimulação de colágeno, por meio da mistura de hidroxiapatita de cálcio (CaHA), ácido hialurônico (AH) e concentrados plaquetários autólogos. Este trabalho relata o caso de um paciente de 53 anos, com fotoenvelhecimento cutâneo, perda de volume facial, múltiplas rítides nas regiões frontal e periorbital, apresentando também acentuada flacidez cutânea, especialmente palpebral. Foi feito o tratamento com toxina botulínica (65 U) e protocolo “RichBlend”. Foi realizada a venopunção e o sangue foi centrifugado para obtenção da i-PRF (fibrina rica em plaquetas injetável) e do plasma gel. Após venopunção e centrifugação sanguínea, obtiveram-se a i-PRF e o plasma gel. A CaHA (Radiesse®) foi diluída: a) em soro + i-PRF (hiperdiluição) para bioestimulação do terço inferior da face; e b) em AH (Juvederm Ultraplus XC®) + plasma gel, para hidrolifting na fronte e preenchimentos de olheira, malar e têmporas. Plasma gel foi aplicado nos sulcos nasogenianos e, em seguida, toda a face foi devidamente massageada. O protocolo “RichBlend” rejuvenesceu o paciente, pois promoveu preenchimento, volumização, formação de colágeno (bioestimulação), redução da flacidez, além do clareamento cutâneo. Uma vez que o AH e a CaHA são produtos de alto custo, sua mistura com os concentrados plaquetários autólogos, na forma líquida ou gel, permite a utilização de uma maior quantidade de material preenchedor e bioestimulador na face, com custo mais acessível.

Abstract The initial characteristics of white spot lesion (WSLs), such as the degree of integrated mineral loss (ΔZ), depth and pattern of mineral distribution, have an impact on further demineralization and remineralization. However, these lesion parameters have not been evaluated in WSLs produced from microcosm biofilms. Objective: This study characterized artificial white spot lesions produced on human enamel under microcosm biofilm for different experimental periods. Methodology: In total, 100 human enamel specimens (4x4mm) were assigned to 5 distinct groups (n=20/group) differing according to the period of biofilm formation (2, 4, 6, 8 or 10 days). Microcosm biofilm was produced on the specimens from a mixture of human and McBain saliva at the first 8h. Enamel samples were then exposed to McBain saliva containing 0.2% sucrose. WSLs formed were characterized by quantitative light-induced fluorescence (QLF) and transverse microradiography (TMR). Data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p<0.05). Results: A clear time-response pattern was observed for both analyses, but TMR was able to better discriminate among the lesions. Regarding QLF analysis, median (95%CI; %) changes in fluorescence ∆Z were -7.74(-7.74:-6.45)a, -8.52(-8.75:-8.00)ab, -9.17(-10.00:-8.71)bc, -9.58(-10.53:-8.99)bc and -10.01(-11.44:-9.72)c for 2, 4, 6, 8, and 10 days, respectively. For TMR, median (95%CI; vol%.µm) ∆Z were 1410(1299-1479)a, 2420(2327-2604)ab, 2775(2573-2899)bc, 3305(3192-3406)cd and 4330(3972-4465)d, whereas mean (SD; µm) lesion depth were 53.7(12.3)a, 71.4(12.0)a, 103.8(24.8)b, 130.5(27.2)bc, 167.2(39.3)c for 2, 4, 6, 8 and 10 days, respectively. Conclusion: The progression of WSLs formed on human enamel under microcosm biofilm can be characterized over 2-10 days, both by QLF and TMR analyses, although the latter provides better discrimination among the lesions.

Abstract A new sugarcane-derived cystatin (CaneCPI-5) showed anti-erosive properties when included in solutions and strong binding force to enamel, but the performance of this protein when added to gel formulations and its effect on surface free energy (SFE) requires further studies. Objective 1) to evaluate the protective effect of gels containing different concentrations of CaneCPI-5 against initial enamel erosion (Experiment 1); and 2) to analyze the SFE (γS) after treating the enamel surface with CaneCPI-5 solution (Experiment 2). Methodology In Experiment 1, 75 bovine enamel specimens were divided into five groups according to the gel treatments: placebo (negative control); 0.27%mucin+0.5%casein (positive control); 0.1 mg/mL CaneCPI-5; 1.0 mg/mL CaneCPI-5; or 2.0 mg/mL CaneCPI-5. Specimens were treated with the gels for 1 min, the AP was formed (human saliva) for 2 h and the specimens were incubated in 0.65% citric acid (pH=3.4) for 1 min. The percentage of surface hardness change (%SHC) was estimated. In Experiment 2, measurements were performed by an automatic goniometer using three probing liquids: diiodomethane, water and ethylene glycol. Specimens (n=10/group) remained untreated (control) or were treated with solution containing 0.1 mg/mL CaneCPI-5, air-dried for 45 min, and 0.5 µL of each liquid was dispensed on the surface to measure contact angles. Results Gels containing 0.1 and 1.0 mg/mL CaneCPI-5 significantly reduced %SHC compared to the other treatments (p<0.05). Treated enamel showed significantly lower γS than control, without changes in the apolar component (γSLW), but the polar component (γSAB=Lewis acid-base) became more negative (p<0.01). Moreover, CaneCPI-5 treatment showed higher γS - (electron-donor) values compared to control (p<0.01). Conclusions Gels containing 0.1 mg/mL or 1.0 mg/mL CaneCPI-5 protected enamel against initial dental erosion. CaneCPI-5 increased the number of electron donor sites on the enamel surface, which may affect AP formation and could be a potential mechanism of action to protect from erosion.

Abstract Vaccinium macrocarpon (cranberry) is a fruit that has an inhibitory effect on matrix metalloproteinases (MMPs) present in dentin and saliva. The inhibition of MMPs has been shown to prevent dentin erosion. The aim of this study was to analyze the effect of cranberry juice on the reduction of dentin erosion in vitro. Specimens of bovine dentin (4×4×2 mm) were randomized and divided into 4 groups (n = 17/group): distilled water (C-control, pH 7.2); green tea extract solution containing 400 µm epigallo-catechin-gallate (EGCg, positive control, pH 4.5); 10% cranberry extract (CrE, pH 3.9), and cranberry juice (CrJ, Cranberry JuxxTM, pH 2.8). Specimens were submitted to erosive pH cycles for 5 days. Each day, four demineralizations were carried out with 0.1% citric acid (90 s). After the acid challenges, specimens were rinsed and kept in treatment solutions for 1 min; afterwards, they were rinsed and stored in artificial saliva for 1 h at 37°C (or overnight at the end of each day). After the experimental period of 5 days, dentin loss was evaluated by contact profilometry. Data were analyzed by ANOVA and Tukey’s test (p < 0.05). Dentin loss (µm ± SD) was significantly lower for all treatments (EGCg = 9.93 ± 2.90; CrE = 12.10 ± 5.44; CrJ = 11.04 ± 5.70) compared to control (21.23 ± 11.96), but it did not significantly differ from each other. These results indicate that the commercial cranberry juice, despite its low pH, is able to reduce dentin erosion, which might be due to the ability of cranberry components to inhibit MMPs.

Abstract: Purpose: to evaluate the effect of dentifrice pH and fluoride concentration ([F]) on fluoride uptake on the biofilm and nails of children from a non-fluoridated area. Methods: two hundred and twenty-eight two- to four-year-old children were randomly allocated into 3 groups according to the type of dentifrice: G1: 1100 μg F/g, pH 4.5 (n = 76); G2: 750 μg F/g, pH 4.5 (n = 74); and G3: 1100 μg F/g, pH 7.0 (n = 78). Nails were collected at 4, 8, and 12 months after starting dentifrice use and biofilm was collected 5 and 60 minutes after toothbrushing. The concentrations of F in nails and biofilm were analyzed by HMDS facilitated diffusion. Data were analyzed by Kruskal-Wallis/Mann-Whitney's test and the comparison between biofilm collection times was done using Wilcoxon test (p £ 0.05). Results: a significant reduction of [F] in biofilm was observed 60 minutes after toothbrushing, regardless of the dentifrice used. However, 5 minutes after toothbrushing, G1 had a significantly higher [F] compared to G2 and G3, and 60 minutes after toothbrushing, [F] was significantly higher for G1 and G2 compared to G3. G1 and G3 had significantly higher [F] in the nails compared to G2. Conclusion: a lower dentifrice concentration is a relevant factor for the reduction of excessive fluoride intake. The use of a low-F acidified dentifrice combines the reduction of fluoride uptake with caries prevention by leading to greater incorporation of F into the biofilm over time.

Abstract Autologous platelet concentrates (APCs) are promising therapeutic agents in facial rejuvenation since they are a great source of cytokines, growth factors and other biologically active substances. Obtained from the patient’s blood, they have the advantages of reducing immunological reactions, making the procedure safer, well tolerated, with minimal adverse effects and lower cost. Currently, they are used for facial rejuvenation both in combination with microneedling and in mesotherapy techniques, as well as to treat facial acne scars, melasma and wounds after laser ablative treatments. This review summarizes current knowledge on the use of APCs, ranging from basic concepts related to their composition and mechanisms of action to up-to-date information on their clinical efficacy. Methodology MEDLINE (PubMed) was searched from inception through 2021 for English language publications on APCs for facial rejuvenation. Results A total of 100 files were found. Based on the available literature, APCs for skin rejuvenation are safe and well tolerated. The most studied product is the first-generation material, platelet-rich plasma (PRP). Conclusions The results are in general favorable, but the quality of the studies is low. The second and third generation products, platelet-rich fibrin (PRF) and injectable platelet-rich fibrin (i-PRF), respectively, are easier to be obtained and, at least in vitro , seem to induce greater collagen production than PRP, especially under lower relative centrifugation forces, but to date only a few clinical trials evaluating these products exist. More high-quality trials with appropriate follow-up are necessary to provide adequate evidence that may help to improve the treatment regimens with APCs. Many aspects should be considered when designing clinical trials to evaluate APCs, such as the patients’ characteristics that best predict a favorable response, the optimal number of sessions and the interval between them, the characteristics of the studies and the development of better instruments to evaluate skin aging.

Abstract We evaluated the efficacy of xylitol varnishes on the remineralization of newly erupted permanent and deciduous teeth in vitro and in situ. Human enamel specimens were randomly allocated to 8 groups (n = 15/group). Artificial caries lesions were produced and enamel alterations were quantified by surface/subsurface hardness and transverse microradiography. The blocks were then treated with the following varnishes: DuraphatTM; 20 wt% xylitol (146 μm) varnish; 20 wt% milled xylitol (80 μm) varnish, and placebo varnish, and removed after 6 h of immersion in artificial saliva. In vitro the blocks were subjected to pH-cycles for 8 days. In situ fifteen subjects wore palatal appliances containing four pre-demineralized and treated enamel specimens, for 5 days. Data were statistically analyzed by ANOVA/Tukey and Kruskal-Wallis/Tukey’s test (p < 0.05). The %SHR in both studies was significantly increased by xylitol and Duraphat™ varnishes when compared to placebo. Considering subsurface remineralization, only the xylitol varnishes were able to significantly reduce the enamel lesion. Xylitol varnishes can be promising alternatives to promote enamel remineralization of newly erupted permanent and deciduous teeth.

Abstract The loss of continuity of the nerve structure interrupts the transmission of nerve impulses and leads to the disorganization of functional activities. Many methods, as the use of neurogenic factors, aid in the process of neural regeneration by accelerating or improving peripheral nerves neoformation. The adipose tissue is abundant in the human body, and it has presented promising results in the regeneration of peripheral nerves. We carried out a randomized controlled study in 9 months, using 45 male Wistar rats, 80 days old, and the sciatic nerve was chosen for analysis. The control animals were divided into three groups – Initial group (IG), Final group (FG), and denervated group (DG) – with seven animals each. The experimental groups, with twelve animals each, were polyethylene tube filled with fat (EGF) and polyethylene tube without filling (EGwf). All groups, except IG, were submitted to 10 sessions of hyperbaric oxygen treatment of 1h 45 min in alternating days. Functional evaluation by walking-track was assessed using the Catwalk XT® software and tissues were harvested and stained with 1% toluidine blue for histological analysis. Quantitative data were first analyzed with the Kolmogorov Smirnov normality test. Comparison between the four groups was analyzed by ANOVA followed by Tukey Test. We concluded that hyperbaric oxygen therapy had positive results on morphometric and functional parameters. However, no significant differences were found regarding the use of autologous fat graft.

Abstract Identifying the risk factors for dental caries is vital in epidemiology and clinical practices for developing effective preventive strategies, both, at the individual and collective levels. Different causality/determination models have been proposed to understand the development process of dental caries. In the present review, we designed a model inspired by the world-known social determinants models proposed in the 90s and more recently in the 10s, wherein the contextual factors are placed more externally and encompass the individual factors. The contextual factors included those related to the cultural and societal values, as well as the social and health government policies. The individual factors were classified into the following categories: socioeconomic (social class, occupation, income, and education level), demographic characteristics (age, sex, and ethnicity), behavioral factors (non-use of fluoride dentifrice, sugar consumption, poor oral hygiene, and lack of preventive dental care), and biological factors (recent caries experience/active caries lesions, biofilm retentive factors, developmental defects of the enamel, disabilities, saliva amount and quality, cariogenic biofilm). Each of these variables was addressed, while focusing on the current evidence from studies conducted in Latin American and Caribbean countries (LACC). Based on the proposed model, educational aspects were addressed, and individual caries risk assessment and management decisions were proposed; further, implications for public health policies and clinical practice were described. The identification of modifiable risk factors for dental caries should be the basis for multi-strategy actions that consider the diversity of Latin American communities.

Resumo Este estudo teve como objetivo comparar quantitativamente a diferença da expressão de proteínas na progressão da patogênese pulpar, bem como descrever as funções biológicas das proteínas identificadas no tecido pulpar. As amostras foram obtidas de seis pacientes atendidos na Faculdade de Odontologia de Araçatuba e divididas em três grupos: polpa normal - dentes extraídos por indicação ortodôntica; polpa inflamada e polpa necrótica - pacientes diagnosticados com pulpite irreversível e periodontite apical crônica, respectivamente. Após o preparo proteômico prévio, as amostras de polpa dentária foram processadas para análise proteômica quantitativa livre de marcadores em um sistema nanoACQUITY UPLC-Xevo QTof MS. A diferença de expressão entre os grupos foi calculada usando o software Protein Lynx Global Service através do algoritmo de Monte Carlo. Um total de 465 proteínas humanas foram identificadas em todos os grupos. As proteínas mais expressas no grupo polpa inflamada em relação ao grupo polpa normal foram hemoglobinas, peroxirredoxinas e imunoglobulinas, enquanto as menos expressas foram as tubulinas. Os níveis de expressão de albuminas, imunoglobulinas e alfa-2-macroglobulina foram maiores no grupo polpa necrótica do que no grupo de polpa inflamada. Quanto à análise qualitativa, as funções proteicas mais prevalentes no grupo polpa normal foram vias metabólicas e energéticas; no grupo polpa inflamada: comunicação celular e transdução de sinal; e regulação e reparo de DNA / RNA, enquanto no grupo polpa necrótica as proteínas foram associadas à resposta imune. Assim, a análise proteômica mostrou diferenças quantitativas e qualitativas na expressão de proteínas em diferentes tipos de condições pulpares.

Abstract This study aimed to quantitatively compare the difference in protein expression in the progression of pulp pathogenesis, as well as to describe the biological functions of proteins identified in pulp tissue. Samples were obtained from six patients treated at the Araçatuba School of Dentistry and were divided into three groups: normal pulp - from teeth extracted for orthodontic indication; inflamed pulp and necrotic pulp - from patients diagnosed with irreversible pulpitis and chronic apical periodontitis, respectively. After previous proteomic preparation, dental pulp samples were processed for label-free quantitative proteomic analysis in a nanoACQUITY UPLC-Xevo QTof MS system. The difference in expression between the groups was calculated using the Protein Lynx Global Service software using the Monte Carlo algorithm. A total of 465 human proteins were identified in all groups. The most expressed proteins in the inflamed pulp group in relation to the normal pulp group were hemoglobin, peroxiredoxins and immunoglobulins, whereas the less expressed were the tubulins. Expression levels of albumins, immunoglobulins and alpha-2-macroglobulin were higher in the necrotic pulp group than in the inflamed pulp group. As for the qualitative analysis, the most prevalent protein functions in the normal pulp group were metabolic and energetic pathways; in the inflamed pulp group: cellular communication and signal transduction; and regulation and repair of DNA/RNA, while in the necrotic pulp group proteins were associated with the immune response. Thus, proteomic analysis showed quantitative and qualitative differences in protein expression in different types of pulp conditions.

Abstract Objective This in vitro study evaluated the effect of commercial whitening dentifrices on erosive tooth wear (ETW) of bovine enamel samples, in comparison with commercial regular dentifrices. Methodology Sixty bovine crowns were embedded in acrylic resin, polished and then had their baseline profile determined. They were randomly assigned to 5 groups (n=12/group), according to the type of commercial dentifrice to be tested: GI – Crest Anti-cavity Regular; GII – Crest 3D White; GIII – Colgate Total 12 Clean Mint; GIV – Colgate Optic White; GV – Placebo (negative control, fluoride-free dentifrice). The samples were submitted to daily erosive and abrasive challenges for 3 days. The erosive challenges were performed 3 times a day by immersing the specimens in 0.1% citric acid solution (pH 2.5) for 90 s. Each day after the first and last erosive challenges, the specimens were subjected to the abrasive challenge for 15 s, using a toothbrushing machine (Biopdi, São Carlos, SP, Brazil), soft toothbrushes and slurry (1:3 g/ml) of the tested toothpastes (1.5 N). The specimens were kept in artificial saliva between the challenges. The final profile was obtained and the ETW (µm) was calculated. Data were analyzed by Kruskal-Wallis and Dunn’s tests (p<0.05). Results All dentifrices tested significantly reduced the enamel wear in comparison with the Placebo, except GIII. The median (95% CI) ETW was 1.35 (1.25-1.46)bc for GI, 1.17 (1.01-1.34)cd for GII, 1.36 (1.28-1.45)ab for GIII, 1.08 (1.04-1.14)d for GIV and 2.28 (2.18-2.39)a for GV. Conclusion When dentifrices from the same manufacturer were compared, the whitening dentifrices led to similar or less wear than the regular ones.

Abstract 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.