RESUMO
OBJECTIVES: This study characterized the proteome profile of the acquired pellicle formed in vivo on enamel. Changes in this proteome profile after exposure to lactic or citric acid were also evaluated. METHODS: Volunteers (n=8) were subjected to dental prophylaxis. After 2 h to allow the formation of the acquired pellicle, the teeth were isolated with cotton rolls and 1 mL of citric acid (1%, pH 2.5) or lactic acid (0.1 M pH 4.8) or deionized water was gently applied with a pipette on the anterior teeth (both maxillary and mandibular) for 10 s. In sequence, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days following a crossover protocol. Proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to Proteome Discoverer software. Searches were done using SWISS-PROT and TrEMBL databases for human proteins. RESULTS: In total, seventy-two proteins were present in all groups and were submitted to quantitative analysis (SIEVE). Some of these proteins were increased more than two-fold after exposure to the acids. Among them, cystatin-B was increased 20- and 13-fold after exposure to citric and lactic acids, respectively. Additionally, some proteins were identified in only one of the groups (18, 5, and 11 proteins for deionized water, citric and lactic acids, respectively). CONCLUSIONS: Our results open new insights regarding potentially acid-resistant proteins that could be added to dental products to prevent acidic dissolution of the teeth.
Assuntos
Esmalte Dentário/química , Película Dentária/química , Película Dentária/efeitos dos fármacos , Proteínas/química , Proteínas/isolamento & purificação , Proteoma/análise , Adolescente , Adulto , Ácido Cítrico/química , Ácido Cítrico/farmacologia , Estudos Cross-Over , Cistatina B/farmacologia , Cárie Dentária/prevenção & controle , Esmalte Dentário/efeitos dos fármacos , Solubilidade do Esmalte Dentário/efeitos dos fármacos , Película Dentária/metabolismo , Feminino , Humanos , Ácido Láctico/química , Ácido Láctico/farmacologia , Proteoma/química , Proteoma/metabolismo , Proteômica/métodos , Desmineralização do Dente/prevenção & controle , Erosão Dentária/prevenção & controle , Água/química , Adulto JovemRESUMO
OBJECTIVES: For the first time, this study characterized the proteome of the acquired pellicle formed on human dentine. The changes in this proteome after exposure to lactic or citric acid were also evaluated. METHODS: Volunteers (n=9) wore a mandibular device containing 6 specimens of human root dentine. After the device remained in the volunteers' oral cavities for 10min or 2h to allow the formation of the acquired pellicle in situ, the specimens were immersed in citric acid (1%, pH 2.5) or lactic acid (0.1M, pH 4.8) or deionized water for 20s. In sequence, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days following a crossover protocol. After harvest, proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to Proteome Discoverer software. Searches were done using SWISS-PROT and TrEMBL databases for human proteins. RESULTS: In total, 223 distinct proteins were identified in the dentine acquired pellicle in each of the different conditions. Exposure to citric acid dramatically reduced the number of identified proteins. This did not occur for lactic acid. Acid-resistant proteins, such as mucins, were identified after pellicle was exposed to lactic or citric acid. CONCLUSIONS: These proteins could be related to protective effect of tooth homeostasis. Moreover, in the future, they could be candidates to the development of a supplemental therapy for the prevention and treatment of dental caries and dental erosion. CLINICAL SIGNIFICANCE: This study indicates some acid-resistant proteins that could be used in dental products to prevent dental caries and erosion.
Assuntos
Ácido Cítrico/farmacologia , Película Dentária/efeitos dos fármacos , Película Dentária/metabolismo , Dentina/efeitos dos fármacos , Ácido Láctico/farmacologia , Proteoma/metabolismo , Adolescente , Adulto , Esmalte Dentário/efeitos dos fármacos , Esmalte Dentário/metabolismo , Película Dentária/química , Dentina/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Mandíbula/anatomia & histologia , Dente Serotino , Mucinas/metabolismo , Proteoma/química , Adulto JovemRESUMO
A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52...
The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free...