Orientador: Adriana Franco Paes Leme / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T17:14:30Z (GMT). No. of bitstreams: 1 Masson_Nadia_M.pdf: 2480697 bytes, checksum: 8edab99b43557ac621635c6c1d19b060 (MD5) Previous issue date: 2012 / Resumo: A película adquirida é um filme protéico resultante da adsorção seletiva de proteínas presentes na saliva total à superfície dental. Devido ao seu contato com a superfície de esmalte, a película adquirida no esmalte desempenha um papel importante nos estágio iniciais da formação da placa dental por meio da modulação da aderência bacteriana. Tem sido reportado que o tratamento do esmalte com produtos de alta concentração de fluoreto leva a formação de uma camada de mineral do tipo fluoreto de cálcio ("CaF2") em sua superfície. Sabe-se que a adsorção protéica ao esmalte é um processo específico e dependente da natureza da superfície e pouco se conhece da influência da camada de "CaF2" na composição da película salivar. A proposta deste estudo foi avaliar a composição da película salivar formada in vitro quando da aplicação de flúor fosfato acidulado no esmalte. Para tal, blocos de esmalte bovino foram aleatoriamente divididos em 3 grupos de tratamento. Cada bloco foi exposto ao tratamento com água destilada (controle negativo), solução de ácido fosfórico (controle ativo - H3PO4 0,1 M pH 3,5) ou solução de flúor fosfato acidulado (NaF 0,5 M em H3PO4 0,1 M pH 3,5) por 4 minutos. Os blocos foram então lavados, secos e imersos em saliva humana por 2 horas para a formação da película salivar. A película adquirida de cada grupo foi extraída, submetida ao protocolo de digestão com tripsina e os peptídeos gerados foram analisados por cromatografia líquida de fase reversa acoplada a uma interface com ionização por nanoelectrospray em um espectrômetro de massas LTQ Velos Orbitrap. Após processamento e análise dos dados gerados pelo espectrômetro de massas utilizando o programa Proteome Discoverer e ScaffoldQ+ foram identificadas 56 proteínas. Cada grupo de tratamento apresentou uma quantidade similar de proteínas totais identificadas e 17,8% das proteínas totais estavam presentes em todos os grupos de tratamento. Doze proteínas foram exclusivas do grupo tratado com água destilada, 11 exclusivas do grupo tratado com ácido fosfórico e 12 do grupo tratado com flúor fosfato acidulado. A quantificação relativa por contagem de espectros demonstrou que quando da aplicação de flúor fosfato acidulado no esmalte a abundância de algumas proteínas diminui, dentre elas a proteína histatina-1. Entretanto, a abundância da proteína de S100-A9, confirmada por western blot, aumentou quando a superfície de esmalte foi tratada com flúor fosfato acidulado. Os dados sugerem que a modificação causada no esmalte pela aplicação de flúor fosfato acidulado influencia a composição da película adquirida no esmalte podendo ter um impacto na posterior colonização bacteriana inicial / Abstract: The acquired pellicle is a protein film resulting from the selective adsorption of proteins present in whole saliva onto tooth surfaces. Because of its contact with enamel surfaces, the enamel pellicle plays an important role in the initial stages of plaque formation by modulating bacterial attachment. High concentrated fluoride product treatment of enamel has been reported to result in the formation of a layer of a CaF2-like material ("CaF2") on the enamel surface. Protein adsorption to enamel is a specific process dependent on the nature of the surface, and little is known about the influence of this "CaF2" layer on pellicle composition. The purpose of this investigation was to gain further insights into the in vitro pellicle components when APF application is performed. Bovine enamel blocks were randomly divided in 3 groups. Each block was exposed to distilled water (negative control), or phosphoric acid (active control - 0.1 M H3PO4 pH 3.5) or acidulated phosphate fluoride (APF) (0.5 M NaF in 0.1 M H3PO4 pH 3.5) solution for 4 minutes. Blocks were then washed, dried and immersed in human saliva for 2 hours for enamel pellicle formation. AEP from each group was collected, subjected to trypsin digestion protocol and the peptides generated were analyzed by reverse-phase liquid chromatography coupled with nanoelectrospray ionization in a LTQ Velos Orbitrap mass spectrometer. After analyses of the data by Proteome Discoverer e ScaffoldQ+ programs 56 proteins were identified. Each treatment group presented a similar amount of total identified protein and 17.8% of total proteins were present in all four groups. Twelve proteins were exclusively in the group treated with water, 11 proteins were exclusively in the group treated with phosphoric acid and another 12 proteins were only present in the discs treated with APF solution. Relative proteomic quantification showed that the abundance of some proteins decreases with APF application, such as histatin-1. However, the concentration of S100 - A9, confirmed by immunoblotting analyses, increases when enamel surface was treated with APF solution. These data suggest that the modification caused on enamel surface by APF application influences the composition of AEP and may have an impact on initial bacterial attachment / Mestrado / Cariologia / Mestra em Odontologia

A erosão dentária apresenta uma etiologia multifatorial, com isto, existem várias possibilidades preventivas e terapêuticas. A saliva é um dos mais importantes fatores biológicos envolvidos, tendo um papel protetor contra desafios erosivos, por contribuir na formação da película adquirida. Já a incorporação de proteínas á película adquirida, através da sua adição a produtos odontológicos, como soluções para bochecho ou géis, por exemplo, pode afetar sua habilidade em proteger contra a erosão. Experimentos preliminares revelaram que uma cistatina clonada recentemente a partir da cana-de-açúcar, denominada CaneCPI-5, tem uma grande força de interação com o esmalte, sendo capaz de protegê-lo contra a erosão inicial. O objetivo do presente estudo foi avaliar o efeito de soluções ou géis contendo CaneCPI-5, em diferentes concentrações, na proteção contra a erosão inicial do esmalte n vitro. Foram confeccionados 150 blocos de esmalte bovino (4 X 4 mm). Para cada um dos veículos a ser testado (solução ou gel) foram constituídos 5 grupos, sendo um grupo controle, constituído por água deionizada ou gel placebo e 4 grupos experimentais. Para as soluções, os grupos experimentais foram constituídos de: Mucina 0,27% + Caseína 0,5%, CaneCPI-5 0,025 g/L, CaneCPI-5 0,1 g/L e CaneCPI-5 1,0 g/L. Para os géis, os grupos experimentais foram: Mucina 0,27% + Caseína 0,5%, CaneCPI-5 0,1 g/L, CaneCPI-5 1,0 g/L e CaneCPI-5 2,0 g/L O tratamento dos espécimes com as soluções foi feito por 2 h a 30°C, sob agitação, enquanto com os géis foi feito por 1 min. Saliva estimulada foi coletada de 3 voluntários para formação da película adquirida (durante 2 h) sobre os espécimes. Os espécimes foram então incubados em solução de ácido cítrico 0,65% (pH = 3,4) por 1 min a 30ºC sob agitação constante. Cada espécime foi assim tratado uma vez ao dia durante 3 dias. As análises de microdureza de superfície (SHM) foram feitas e as alterações na SMH (SHM baseline SMH pós-erosão; %SHC) foram calculadas nos dias 1 e 3. Os dados foram analisados pelos teste de Kruskal-Wallis, seguido pelo teste de Dunn (p<0,05). As soluções contendo CaneCPI-5 a 0,1 e 1,0 g/l reduziram significativamente %SHC em comparação aos demais tratamentos, para ambos os períodos experimentais, sem diferença entre si. Para os géis resultados semelhantes aos das soluções foram obtidos apenas no primeiro dia de tratamento. Após 3 dias, entretanto, o efeito protetor foi perdido. Em conclusão, o tratamento com solução contendo CaneCPI-5 a 0,1 g/L parece ser uma boa alternativa para prevenir a erosão inicial do esmalte, o que deve ser confirmado em estudos utilizando condições experimentais mais similares às situações clínicas. / Dental erosion has a multifactorial etiology, thus, there are several preventive and therapeutic possibilities. Saliva is one of the most important biological factors involved, having a protective role against erosive challenges and contributing to the formation of the acquired enamel pellicle. The incorporation of proteins in the acquired enamel pellicle, by their addition to dental products such as mouthwashes or gels, for example, can affect its ability to protect against erosion. Preliminary experiments from our group have shown that a sugar cane-derived protein that was recently cloned, known as CaneCPI-5 has a strong interaction force with the enamel and is capable of protecting it against initial erosion. The aim of this study was to evaluate the effect of solutions or gels containing CaneCPI-5, in different concentrations, on the protection against initial enamel erosion in vitro. Bovine enamel blocks (n=150) (4X4 mm) were prepared. For each of the vehicles tested (solution or gel) 5 groups (one control, constituted by deionized water or placebo gel and four experimental) were constituted. For the solutions, the experimental groups were constituted of: 0.27% mucin + 0.5% casein, 0.025 g/L CaneCPI-5, 0.1 g/L CaneCPI-5 or 1.0 g/L CaneCPI-5. For the gels, they were: 0.27% mucin + 0.5% casein, 0.1 g/L CaneCPI-5, 1.0 g/L CaneCPI-5 or 2.0 g/L CaneCPI-5. The specimens were treated with the solutions for 2 h at 30°C under stirring or with the gels for 1 min. Stimulated saliva was collected from three volunteers and the acquired enamel pellicle was formed for 2 h. Then the specimens were incubated in a solution of 0.65% citric acid (pH = 3.4) for 1 min at 30°C under stirring. Each specimen was treated once a day for 3 days. Surface hardness analysis (SHM) was made and changes in SMH (SHM baseline - posterosion SMH; %SHC) was calculated on days 1 and 3. Data were analyzed by Kruskall-Wallis and Dunn´s tests (p<0.05). The solutions containing 0.1 and 1.0 g/L CaneCPI-5 significantly reduced %SHC when compared with the other treatments, for both experimental periods, without significant difference between them. As for the gels, similar results were obtained but only at the first day of treatment. After 3 days, the protective effect was lost. In conclusion, the treatment with solution containing 0.1g/L CaneCPI-5 seems to be a good alternative to prevent initial enamel erosion, what needs to be confirmed using experimental conditions that more closely resemble the clinical situation.

A erosão dentária apresenta uma etiologia multifatorial, com isto, existem várias possibilidades preventivas e terapêuticas. A saliva é um dos mais importantes fatores biológicos envolvidos, tendo um papel protetor contra desafios erosivos, por contribuir na formação da película adquirida. Já a incorporação de proteínas á película adquirida, através da sua adição a produtos odontológicos, como soluções para bochecho ou géis, por exemplo, pode afetar sua habilidade em proteger contra a erosão. Experimentos preliminares revelaram que uma cistatina clonada recentemente a partir da cana-de-açúcar, denominada CaneCPI-5, tem uma grande força de interação com o esmalte, sendo capaz de protegê-lo contra a erosão inicial. O objetivo do presente estudo foi avaliar o efeito de soluções ou géis contendo CaneCPI-5, em diferentes concentrações, na proteção contra a erosão inicial do esmalte n vitro. Foram confeccionados 150 blocos de esmalte bovino (4 X 4 mm). Para cada um dos veículos a ser testado (solução ou gel) foram constituídos 5 grupos, sendo um grupo controle, constituído por água deionizada ou gel placebo e 4 grupos experimentais. Para as soluções, os grupos experimentais foram constituídos de: Mucina 0,27% + Caseína 0,5%, CaneCPI-5 0,025 g/L, CaneCPI-5 0,1 g/L e CaneCPI-5 1,0 g/L. Para os géis, os grupos experimentais foram: Mucina 0,27% + Caseína 0,5%, CaneCPI-5 0,1 g/L, CaneCPI-5 1,0 g/L e CaneCPI-5 2,0 g/L O tratamento dos espécimes com as soluções foi feito por 2 h a 30°C, sob agitação, enquanto com os géis foi feito por 1 min. Saliva estimulada foi coletada de 3 voluntários para formação da película adquirida (durante 2 h) sobre os espécimes. Os espécimes foram então incubados em solução de ácido cítrico 0,65% (pH = 3,4) por 1 min a 30ºC sob agitação constante. Cada espécime foi assim tratado uma vez ao dia durante 3 dias. As análises de microdureza de superfície (SHM) foram feitas e as alterações na SMH (SHM baseline SMH pós-erosão; %SHC) foram calculadas nos dias 1 e 3. Os dados foram analisados pelos teste de Kruskal-Wallis, seguido pelo teste de Dunn (p<0,05). As soluções contendo CaneCPI-5 a 0,1 e 1,0 g/l reduziram significativamente %SHC em comparação aos demais tratamentos, para ambos os períodos experimentais, sem diferença entre si. Para os géis resultados semelhantes aos das soluções foram obtidos apenas no primeiro dia de tratamento. Após 3 dias, entretanto, o efeito protetor foi perdido. Em conclusão, o tratamento com solução contendo CaneCPI-5 a 0,1 g/L parece ser uma boa alternativa para prevenir a erosão inicial do esmalte, o que deve ser confirmado em estudos utilizando condições experimentais mais similares às situações clínicas. / Dental erosion has a multifactorial etiology, thus, there are several preventive and therapeutic possibilities. Saliva is one of the most important biological factors involved, having a protective role against erosive challenges and contributing to the formation of the acquired enamel pellicle. The incorporation of proteins in the acquired enamel pellicle, by their addition to dental products such as mouthwashes or gels, for example, can affect its ability to protect against erosion. Preliminary experiments from our group have shown that a sugar cane-derived protein that was recently cloned, known as CaneCPI-5 has a strong interaction force with the enamel and is capable of protecting it against initial erosion. The aim of this study was to evaluate the effect of solutions or gels containing CaneCPI-5, in different concentrations, on the protection against initial enamel erosion in vitro. Bovine enamel blocks (n=150) (4X4 mm) were prepared. For each of the vehicles tested (solution or gel) 5 groups (one control, constituted by deionized water or placebo gel and four experimental) were constituted. For the solutions, the experimental groups were constituted of: 0.27% mucin + 0.5% casein, 0.025 g/L CaneCPI-5, 0.1 g/L CaneCPI-5 or 1.0 g/L CaneCPI-5. For the gels, they were: 0.27% mucin + 0.5% casein, 0.1 g/L CaneCPI-5, 1.0 g/L CaneCPI-5 or 2.0 g/L CaneCPI-5. The specimens were treated with the solutions for 2 h at 30°C under stirring or with the gels for 1 min. Stimulated saliva was collected from three volunteers and the acquired enamel pellicle was formed for 2 h. Then the specimens were incubated in a solution of 0.65% citric acid (pH = 3.4) for 1 min at 30°C under stirring. Each specimen was treated once a day for 3 days. Surface hardness analysis (SHM) was made and changes in SMH (SHM baseline - posterosion SMH; %SHC) was calculated on days 1 and 3. Data were analyzed by Kruskall-Wallis and Dunn´s tests (p<0.05). The solutions containing 0.1 and 1.0 g/L CaneCPI-5 significantly reduced %SHC when compared with the other treatments, for both experimental periods, without significant difference between them. As for the gels, similar results were obtained but only at the first day of treatment. After 3 days, the protective effect was lost. In conclusion, the treatment with solution containing 0.1g/L CaneCPI-5 seems to be a good alternative to prevent initial enamel erosion, what needs to be confirmed using experimental conditions that more closely resemble the clinical situation.

Syfte: Syftet med denna studie var att studera huruvida algen Ascophyllum Nodosum (AN) utövar någon effekt på proteasaktivitet i oralt plack, samt om effekten finns i algen från början eller om man måste inta den oralt för att få en systemisk effekt.En förhöjd proteasaktivitet har förknippats med gingivit och parodontit. Vi förväntar oss en minskad proteasaktivitet hos försökspersonerna, och därmed en minskad risk för gingivit och parodontit, efter intag av AN under en månads tid. Material och metod: Ett in vitro med en pilotstudie, och ett in vivo försök utfördes. I in vitro försöket användes pulveriserat och upplöst Ascophyllum Nodosum. I in vitro studien deltog 5 personer i åldersgruppen 20 till 30 år. Plackprover på försökspersonerna togs före och efter intag av algen i 4 veckor. Under båda försökstillfällena fick försökspersonerna inte ha borstat tänderna på 12h innan försöket. Resultat: Vid kombinering av pilotstudien och in vitro studien ses ingen signifikant skillnad gällande Ascophyllum Nodosums proteasaktivitet i pulveriserad form. Våra resultat erhållna från in vivo studien visar att det har skett en ökad proteasaktivitet i placket hos försökspersonerna efter en månads intag av Ascophyllum Nodosum. Konklusion: Denna studie visar på en tendens till en ökad proteasaktivitet orsakad av Ascophyllum nodosum. Studien har inte undersökt vilka proteaser som påverkats. På grund av komplexiteten i den orala miljön och de många olika typerna av proteaser, behöver fler studier utföras för att studera de exakta effekterna på den orala miljön. / Aims: The purpose of this study was to investigate if the alga Ascophyllum Nodosum (AN) exerts any effect on protease activity in plaque, if such an effect is present in the algae from the beginning or if it has to be taken orally to exert a systemic effect.Increased protease activity has been associated with gingivitis and periodontitis. We expected a reduced protease activity, and thus a potentially reduced risk for gingivitis and periodontitis, after ingestion of AN for a month.Materials and methods: One in vitro trial with a pilot study, and one in vivo trial was carried out. In the in vitro trial pulverized and dissolved AN was used to make a solution, tested for protease activity.In the in vitro study 5 subjects, aged 20 to 30 years, participated. Plaque samples were taken before and after ingestion of the algae for 4 weeks. Subjects were instructed not to brush their teeth 12 h before sampling.Results: When combining the results from the pilot and in vitro studies, no AN protease activity could be detected. Our in vivo results showed an increased protease activity in the plaque after a month of AN intake.Conclusion: This study indicates a tendency to an increased protease activity caused by Ascophyllum Nodosum. However, the study did not examine which protease was affected. Because of the complexity in the oral environment and the many different types of protease, more studies need to be executed to study the exact effects of AN on the oral environment.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Indiana University-Purdue University Indianapolis (IUPUI)School of dentistry / BACKGROUND: Fluoride and stannous ions have been reported to be relevant for dental erosion prevention. However, their interaction with the acquired dental pellicle (ADP), a clinically relevant erosion protective factor, is not well known and needs to be investigated. OBJECTIVES: To investigate the anti-erosive properties of fluoride-containing solutions and stannous solutions on enamel and dentin surfaces with a previously formed ADP. To characterize the protein profile of the ADP treated with the test solutions. METHODS: Phase I tested four solutions: SnCl2/NaF, NaF, SnCl2 and deionized water (DIW) (as negative control). Forty bovine enamel and dentin specimens 104 (4x4x2 mm3) were prepared and randomly distributed into 4 groups (n = 10). The specimens were incubated in clarified human saliva (CHS) for 24 h for pellicle formation and then they were subjected to a cycling procedure that included a 5-min erosive challenge (0.3-percent citric acid, pH 2.6); a 2-min treatment with the solution (between 1st, 3rd and 6th cycles); a 2-h immersion in CHS, and overnight immersion in CHS. Cycles were repeated 6x/day for 5 days. The outcome measure was surface loss (SL) using profilometry. Phase II: Thirty-two (32) bovine enamel specimens (882 mm3) (n = 8) were similarly prepared and incubated in saliva for 24 h and then treated with the solutions for 2 min followed by CHS immersion for 2 h. This cycle was repeated 3x for one day. The pellicles formed and treated with the test rinse solutions were collected, digested, and analyzed for specific protein content using liquid chromatography electrospray ionization tandem mass spectrometry (LCESI-MS/MS). RESULTS: Phase I: for enamel, SnCl2/NaF, SnCl2, NaF solutions provided 89 percent, 67 percent, and 42 percent SL reduction respectively compared with the control, while in dentin they provided 60 percent, 23 percent, and 36 percent, respectively, all significant at p < 0.05. Phase II: Seventy-two (72) common proteins were identified in all groups, 30 exclusive to DIW, 20 to SnCl2/NaF, 19 to NaF, and 13 to SnCl2. SnCl2/NaF increased the abundance of pellicle proteins than each one alone. CONCLUSION: SnCl2/NaF showed the best anti-erosive effect on both enamel and dentin. The findings suggest that the composition of acquired pellicle changes with different solutions, which may be related to their anti-erosive effect.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Bacterial adherence to the acquired dental pellicle, important in caries, is mediated by receptor-adhesin interactions such as Streptococcus mutans antigen I/II (I/II). Ten I/II epitopes from the A, V, P and C regions were chosen to determine their reactivity in human saliva. Underlying the body’s ability to immunologically respond to bacteria that lead to caries are the human leukocyte antigen (HLA) genes, specifically HLA class II (HLA-II) genes that control antigen presentation. Previous studies suggested that a specific HLA biomarker group (HLA-DRB1*04) may have differential control of immune responses to I/II. However, it was not known whether secretory IgA (SIgA) responses to the selected epitopes from HLA-DRB1*04 positive subjects were different compared to their non-biomarker counterparts (negative), or across other caries factors, since no study to date had thus assessed these questions. Methods: Per IRB approval, the study population was divided into age, sex and race matched DRB1*04 positive (n=16) and negative groups (n=16). SIgA-epitope (and whole cell) reactivity was determined using ELISA. Other caries factors were measured. Subjects received a clinical exam by a trained examiner. ix Differences between DRB1*04 positive and negative groups were examined using a two-sided, two-sample t-test. Results: DRB1*04 positive subjects had numerically, but not statistically, higher reactivity to 9 out of 10 epitopes, the exception being residues 834-853 from the V and P regions of I/II across multiple measures. Though statistically insignificant, DRB1*04 positive subjects also exhibited 25-30 μg mL-1 less total IgA (TIgA) than negative counterparts. All clinical caries data proved inconclusive when comparing groups, likely due to exogenous factors and sample size. Conclusion: DRB1*04 positive subjects showed a trend toward lower TIgA. Moreover, they also showed a lower SIgA response across multiple measures to 834-853, the I/II V and P region epitope. This region forms a sort of functional epicenter involved in collaboration between domains along the entire I/II antigen, and governs the region involved in initial attachment to the acquired dental pellicle. This region may be involved in an in vivo discontinuous conformationally specific immunogenic epitope that serves as an HLA-II binding motif which remains elusive.