O chá-verde apresenta efeitos terapêuticos na saúde geral, principalmente em função da epigalocatequina-3-galato (EGCG), componente com comprovada ação antioxidante, antiinflamatória, antimicrobiana, inativadora de endotoxina bacteriana e mineralizadora. Nesse sentido, a EGCG poderia apresentar papel promissor como medicação intracanal, durante o tratamento endodôntico de dentes portadores de necrose pulpar e lesão periapical. Assim, o objetivo deste estudo foi desenvolver um sistema de liberação tópica de EGCG, para uso no interior do sistema de canais radiculares, caracterizar suas propriedades físico-químicas bem como avaliar a possível alteração de cor dental após sua aplicação no interior dos canais radiculares de dentes decíduos e permanentes. Inicialmente, foi realizada a caracterização físico-química da EGCG por meio de espectrofotometria em Ultravioleta/Visível (UV/Vis), fluorescência e titulação potenciométrica. A seguir, foi desenvolvida uma formulação para liberação tópica prolongada de EGCG, utilizando um veículo viscoso (PEG 400). O comportamento espectral desse novo sistema de liberação de EGCG foi avaliado em função do tempo (1, 2, 3, 4, 5, 6, 24 e 27 horas) e na presença de dentes (decíduos e permanentes). Para a análise da possível alteração de cor dental, foram utilizados 20 dentes decíduos e 20 dentes permanentes, uniradiculares, recém-extraídos, provenientes do Banco de Dentes da FORP-USP. A cor da coroa dos dentes foi determinada antes (dia 0) e 21, 42 e 56 dias após a aplicação intracanal de EGCG, veiculada em água ou PEG 400, com auxílio do espectrofotômetro digital VITA Easyshade. De acordo com os resultados físico-químicos, o espectro de absorção da EGCG na região do UV/Vis apresentou um pico máximo em 274 nm e na fluorescência um pico máximo de absorção em 393 nm. A EGCG apresentou estabilidade em função do tempo e em meio ácido, tendo comportamento espectral alterado em contato com dentes decíduos e permanentes. Em meio básico o espectro de absorção da EGCG em luz UV/Vis foi alterado para 323 nm. O sistema de liberação tópica intracanal de EGCG não alterou suas propriedades físico-químicas, mantendo os valores de absorção em UV/Vis (278 nm) e fluorescência (377 nm), sua estabilidade em função do tempo e não alterou seu comportamento espectral em contato com dentes decíduos e permanentes. A aplicação do sistema proposto de liberação tópica da EGCG, no interior dos canais radiculares de dentes decíduos e permanentes, não causou alteração de cor dental significativa, após o período de 21, 42 e 56 dias. Com base nas metodologias e nos resultados obtidos no presente estudo pode-se concluir que a EGCG apresenta fluorescência e estabilidade em função do tempo e em meio ácido, enquanto em meio básico e na presença de elementos dentais seu comportamento espectral sofre alteração. O sistema desenvolvido para liberação controlada da EGCG manteve suas propriedades físico-químicas, apresentou estabilidade em função do tempo e não foi alterou seu comportamento espectral na presença dos elementos dentais. Ainda, a aplicação intracanal deste sistema não causou alteração de cor da coroa dental. / Green tea has therapeutic effects on general health, mainly due to epigallocatechin-3-gallate (EGCG), a component with proven antioxidant, anti-inflammatory, antimicrobial, inactivating bacterial endotoxin and mineralization action. In this sense, EGCG could present promising role as an intracanal medication during endodontic treatment of teeth presenting pulp necrosis and periapical lesion. Thus, the objective of this study was to develop a system for topical delivery of EGCG, for use inside the root canal system, characterize their physical and chemical properties as well as to evaluate the possible color change after its application in root canals of deciduous and permanent teeth. Initially, was performed physicochemical characterization of EGCG by spectrophotometry Ultraviolet/Visible (UV/Vis), fluorescence, and potentiometric titulation. Next, a formulation for topical extended release of EGCG using a viscous vehicle (PEG 400) was developed. The spectral characteristic of this new delivery system of EGCG was assessed in function of the medium (acidic, basic or neutral), the time (1, 2, 3, 4, 5, 6, 24 and 27 hours) and in the presence of teeth (deciduous and permanent). To the analysis of possible tooth color changes, 20 primary teeth and 20 permanent teeth, single-rooted, freshly extracted, from the Bank of Teeth FORP-USP, were used. The color of the tooth crown was determined before (day 0) and 21, 42 and 56 days after intracanal application of EGCG diluted in water or PEG 400, using a digital VITA Easyshade spectrophotometer. According to results the physicochemical spectrum of absorption of the EGCG showed maximum absorption peak at 274 nm in the UV/Vis and at 393 nm in the fluorescence. The EGCG presented stability as a function of time and in acid medium, and its spectral behavior was changed in contact with deciduous and permanent teeth. In basic medium the EGCG absorption spectrum in UV/Vis was changed to 323 nm. The topical delivery system of EGCG intracanal did not change its physicochemical properties, maintaining the values of absorption in UV/Vis (278 nm) and fluorescence (377 nm), its stability as a function of time, and does not change its spectral contact with deciduous and permanent teeth. The application of the proposed system for topical delivery of EGCG in the root canals of deciduous and permanent teeth, did not cause significant tooth color change after the period of 21, 42 and 56 days. Based on the methodologies and the results obtained in this study it can be concluded that EGCG presents fluorescence and stability as a function of time and in an acidic medium, whereas in basic medium and in the presence of dental elements its spectral behavior is changed. The system developed for EGCG controlled release maintains its physicochemical properties, presents stability with time and does not change its spectral behavior in the presence of dental elements. Yet, the intracanal application of this system did not change the color of the teeth\'s crown.

O uso de agentes químicos coadjuvantes da higienização bucal pode ser necessário para o controle da microbiota cariogênica de indivíduos com alto risco e atividade da doença cárie. Atualmente o agente antimicrobiano mais recomendado é o digluconato de clorexidina (CHX) devido ao seu amplo espectro de ação e efeito residual. Contudo, quando utilizado por longos períodos, este agente químico apresenta efeitos colaterais. Neste contexto, os polifenóis naturais, como a Epigalocatequina3galato (EGCG), derivada do chá verde, vêm sendo propostos como alternativa aos agentes antimicrobianos sintéticos. Entretanto, os polifenóis não apresentam estabilidade ao longo do tempo, podendo se oxidar rapidamente. Desta forma, o encapsulamento da EGCG em nanopartículas poderia aumentar a sua biodisponibilidade e estabilidade física e química, manter o efeito deste polifenol no tecido alvo e potencializar sua eficácia farmacológica. Assim, o presente estudo teve como objetivo desenvolver e caracterizar sistemas de encapsulamento de EGCG e avaliar, in vitro, sua atividade antimicrobiana frente a microorganismos cariogênicos. Inicialmente, foram preparadas nanopartículas poliméricas (NPP) e carreadores lipídicos nanoestruturados (CLN), que foram caracterizados e avaliados quanto à sua atividade antimicrobiana in vitro frente aos microorganismos Streptococcus mutans, Streptococcus sobrinus e Lactobacillus casei. Após a análise dos resultados microbiológicos, o CLN foi selecionado para o encapsulamento da EGCG (CLNEGCG), por apresentar atividade antimicrobiana frente à todos os microorganismos avaliados. O CLNEGCG foi preparado pelo método de emulsão e sonicação e caracterizado quanto ao diâmetro, índice de polidispersão (PdI), potencial zeta (PZ), eficiência de encapsulamento (EE), cristalinidade, capacidade de mucoadesão e morfologia. A atividade antimicrobiana in vitro da EGCG livre e encapsulada foi avaliada por meio da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM). O diâmetro, PdI e o PZ dos CLN foram 228nm, 0,216 e 36,53mV, respectivamente, sendo que o encapsulamento da EGCG não alterou significativamente estes parâmetros. O CLN apresentou forma esférica, estabilidade por 330 dias e propriedade mucoadesiva devido a presença de quitosana na superfície do CLN. Além disso, a quitosana favoreceu o encapsulamento da EGCG obtendose uma EE de ~96%. As concentrações inibitórias e bactericidas mínimas do CLNEGCG (33,75 a 67,5 µg/mL) foram menores do que as verificadas para a EGCG livre (250 a 2.000 µg/mL), comprovando o aumento do potencial antimicrobiano com o encapsulamento da EGCG em nanocarreadores híbridos. De acordo com esses resultados, o CLNEGCG, desenvolvido no presente trabalho, constitui um sistema com potencial para o uso tópico bucal, pois além de ser estável e apresentar propriedade de mucoadesão e morfologia adequada, apresentaram alta atividade antimicrobiana frente aos principais microorganismos envolvidos no processo carioso. / The use of oral hygiene adjuvants may be necessary to control the cariogenic microbiota of individuals with high risk and caries disease activity. Currently the most recommended antimicrobial agent is chlorhexidine digluconate (CHX) because of its broad spectrum of action and residual effect. However, this chemical has side effects. In this context, as an Epigallocatechin3gallate (EGCG), a derivative of green tea, as an alternative to synthetic antimicrobial agents. However, there is no stability over time and can oxidize rapidly. Thus, the encapsulation of EGCG in nanoparticles can increase their bioavailability and physical and chemical stability, maintain the effect of this polyphenol on the target tissue and potentiate its pharmacological efficacy. Thus, the present study aimed to develop and characterize EGCG encapsulation systems and to evaluate, in vitro, its antimicrobial activity against cariogenic microorganisms. Initially, polymer nanoparticles (NPP) and nanostructured lipid carriers (CLN) were prepared and evaluated for their in vitro antimicrobial activity against Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei microorganisms. After the analysis of the microbiological results, the CLN was selected for the encapsulation of EGCG (CLNEGCG), due to its higher antimicrobial activity. The CLNEGCG was developed by emulsion and sonication method and was characterized in relation to diameter, polydispersity index (PdI), zeta potential (PZ), encapsulation efficiency (EE), crystallinity, mucoadhesion capacity and morphology. The in vitro antimicrobial activity of EGCG and its ability to evaluate minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The diameter, PdI and PZ of the CLNs were 228nm, 0.216 and 36.53mV, respectively, and the EGCG encapsulation did not significantly alter these parameters. The CLN showed a spherical structure, stability for 330 days and a mucoadhesive property due to a presence of chitosan on the CLN surface. In addition, a chitosan favored EGCG encapsulation resulting in an EE of ~ 96%. The minimum inhibitory and bactericidal concentrations of CLGEGCG (33.75 to 67.5 µg / mL) were lower than those for free EGCG (250 to 2,000 µg / mL), with increased antimicrobial potential with EGCG encapsulation in hybrid nanocarriers. According to the results, the CLNEGCG, developed in the present study, constitutes a system with potential for oral topical use, besides being stable and possessing mucoadhesion properties, presented high antimicrobial activity against the main microorganisms involved in the carious process.

O chá verde, obtido da Camellia sinensis, é uma das bebidas mais populares em todo o mundo e, recentemente, tem sido foco de pesquisas científicas por apresentar efeitos benéficos na saúde geral. Estudos laboratoriais e epidemiológicos sugerem que, dentre os polifenóis que compõem o chá verde, a Epigalocatequina-3-galato (EGCG) é o mais bioativo e responsável por sua ação antioxidante, anti-inflamatória, antimicrobiana, inativadora de LPS bacteriano, anticarcinogênica, antitumoral, anti-angiogênica, anti-hipertensiva e reparadora tecidual, podendo atuar na prevenção e tratamento do câncer, doenças cardiovasculares, neurodegenerativas, hepáticas e renais. Em diversas doenças ósseas, a EGCG também desempenha um importante papel protetor, atuando na indução da mineralização e inibição da osteoclastogênese. No entanto, seus possíveis efeitos na inflamação e na reabsorção óssea associadas à lesão periapical ainda não foram avaliados. Portanto, o objetivo do presente estudo foi desenvolver uma formulação para uso endodôntico à base de EGCG com propriedades físico-químicas e biológicas que permitam seu uso como curativo de demora entre sessões. As formulações testadas incluíram: EGCG diluída em água e diferentes concentrações de EGCG (1,25; 5; 10 e 20 mg/mL) veiculadas em polietilenoglicol 400 (PEG). O óxido de zinco foi utilizado como agente radiopacificador. Em função da possível degradação dos produtos fenólicos em função do ambiente e do tempo, uma solução obtida por meio do contato prolongado da EGCG com o dente também foi avaliada (produto da degradação). Inicialmente, foi realizada a caracterização físico-química da formulação de EGCG por meio de espectrofotometria em Ultravioleta/Visível (UV/Vis), em contato com soluções contendo zinco, óxido de zinco e acetato de zinco. Os estudos biológicos foram realizados para avaliar sua compatibilidade tecidual no tecido subcutâneo de camundongos, por meio da avaliação do extravasamento plasmático após 24 horas e da análise de aspectos macroscópicos e microscópicos aos 7, 21 e 63 dias após a inserção de tubos de polietileno contendo as formulações. Neste estudo, uma pasta a base de hidróxido de cálcio (Calen®) foi utilizada como controle. Na análise macroscópica, realizada por meio de fotografias, foram atribuídos escores aos parâmetros de ulceração epitelial, vascularização, necrose e edema. Na análise microscópica, realizada em microscopia de luz convencional e de fluorescência, foram avaliados os parâmetros de infiltrado inflamatório, vascularização, focos de abscessos, cápsula fibrosa, edema e necrose. Os escores dos resultados biológicos foram submetidos à análise estatística utilizando o teste do Qui-quadrado ou Exato de Fisher e para análise dos resultados quantitativos do extravasamento plasmático foi utilizada análise de variância (ANOVA), seguida pelo pós-teste de Tukey. O nível de significância adotado foi de 5%. Os estudos físico-químicos mostraram que a EGCG manteve seu comprimento de onda original em 274 nm quando associada ao óxido de zinco, tendendo à formação de uma banda de absorção em 325 nm. A associação da EGCG com acetato de zinco ocasionou diminuição da banda de absorção em 274 nm e formação de uma nova banda em 323 nm. Os estudos biológicos mostraram, nos parâmetros macroscópicos que, em todos os períodos avaliados, todas as formulações apresentaram compatibilidade tecidual, com ausência de ulceração epitelial, presença de leve necrose tecidual superficial, edema e vascularização, não havendo diferença significante entre os grupos avaliados (p>0,05). Na análise microscópica, em todos os períodos foi verificada ausência de focos de abscesso, edema e necrose e presença de suave ou moderado infiltrado inflamatório, cápsula fibrosa e neovascularização, semelhante entre todos os grupos (p>0,05). A avaliação dos espécimes corados com HE em microscopia de fluorescência favoreceu a visualização dos vasos sanguíneos e de fibras colágenas, constituindo metodologia adicional para esta finalidade. Na avaliação do extravasamento plasmático, o produto da degradação da ECGG apresentou extravasamento de corante azul de Evans por grama de tecido inferior (0,0514 ±0,0220 mg/mL) às demais formulações testadas (p<0,05). Conclui-se que a formulação tópica à base de EGCG, desenvolvida para uso endodôntico, apresenta propriedades físico-químicas estáveis e compatibilidade tecidual. / The green tea, obtained from the Camellia sinensis, is one of the most popular drinks in the world and has recently been in the focus of scientific research due its beneficial effects on general health. Laboratory and epidemiological studies suggest that, among the polyphenols found on green tea, the epigallocatechin-3-gallate (EGCG) is the most bioactive and responsible for its antioxidant, anti-inflammatory, anti-microbial, bacterial LPS inactivation, anticarcinogenic, anti-tumoral, anti-angiogenic, anti-hypertensive and tissue repair and can act in the prevention and treatment of cancer, cardiovascular disease, neurodegenerative, liver and kidney diseases. In several bone diseases, EGCG also plays a major protective role, acting in inhibition of osteoclastogenesis and induction of mineralization. However, their possible effects on inflammation and bone resorption associated to apical periodontitis have not been evaluated. Therefore, the aim of this study was to develop a EGCG based formulation for endodontic use with physicochemical and biological properties that allow its use as intracanal dressing between sessions. The formulations tested included: EGCG diluted in water and different concentrations of EGCG (1.25, 5, 10 and 20 mg/ml) diluted in polyethylene glycol 400 (PEG). Zinc oxide was used as radiopacifier agent. Due to the possible degradation of phenolic compounds as a function of the environment and time, a solution obtained through prolonged contact with the tooth EGCG was also evaluated (degradation product). Initially, physicochemical characterization of EGCG formulation was performed by ultraviolet spectrophotometry/Visible (UV/Vis) in contact with solutions containing zinc, zinc oxide and zinc acetate. Biological studies were conducted to evaluate its tissue compatibility with subcutaneous tissue of mice by means of the evaluation of plasma leakage after 24 hours and the examination of macroscopic and microscopic features at 7, 21 and 63 days after insertion of polyethylene tubes containing the formulations. In this study, a calcium hydroxide pastebase (Calen®) was used as control. The macroscopic analysis performed by means of photographs, assigned scores to epithelial ulceration, vascularization, necrosis and edema parameters. Microscopic examination performed in conventional and fluorescence microscopy assessed the inflammatory infiltrate, vascularization, abscesses spots, fibrous capsule, edema and necrosis parameters. The scores of biological results were statistically analyzed by the chisquare test or Fisher\'s exact and for the quantitative analysis of the results of plasma extravasation the analysis of variance (ANOVA) followed by Tukey\'s post-test were used. The significance level was 5%. The physico-chemical studies showed that EGCG maintained its original wavelength at 274 nm when added to zinc oxide, tending to the formation of an absorption band at 325 nm. The combination of EGCG with zinc acetate led to decreased absorption band at 274 nm and formation of a new band at 323 nm. Biological studies showed, in the macroscopic parameters in all periods, that all formulations showed tissue compatibility, with no epithelial ulceration, presence of mild superficial tissue necrosis, edema and vascularization, with no significant difference between the groups (p > 0.05). On microscopic examination, in all periods was verified absence of abscess foci, edema and necrosis and the presence of mild or moderate inflammatory infiltrate, fibrous capsule and neovascularization, similar among all groups (p> 0.05). The evaluation of the specimens stained with HE under fluorescence microscopy enhancve the visualization of the blood vessels and collagen fibers, constituting an additional methodology for this purpose. In the evaluation of plasma extravasation, the product of degradation of ECGG presented values of Evans blue dye extravasation by gram of tissue (0.0514 ± 0.0220 mg / mL) lower to the other tested formulations (p <0.05). It is concluded that the topical formulation based on EGCG developed for endodontic use, presented stable physical and chemical properties and tissue compatibility.

O uso de agentes químicos coadjuvantes da higienização bucal pode ser necessário para o controle da microbiota cariogênica de indivíduos com alto risco e atividade da doença cárie. Atualmente o agente antimicrobiano mais recomendado é o digluconato de clorexidina (CHX) devido ao seu amplo espectro de ação e efeito residual. Contudo, quando utilizado por longos períodos, este agente químico apresenta efeitos colaterais. Neste contexto, os polifenóis naturais, como a Epigalocatequina3galato (EGCG), derivada do chá verde, vêm sendo propostos como alternativa aos agentes antimicrobianos sintéticos. Entretanto, os polifenóis não apresentam estabilidade ao longo do tempo, podendo se oxidar rapidamente. Desta forma, o encapsulamento da EGCG em nanopartículas poderia aumentar a sua biodisponibilidade e estabilidade física e química, manter o efeito deste polifenol no tecido alvo e potencializar sua eficácia farmacológica. Assim, o presente estudo teve como objetivo desenvolver e caracterizar sistemas de encapsulamento de EGCG e avaliar, in vitro, sua atividade antimicrobiana frente a microorganismos cariogênicos. Inicialmente, foram preparadas nanopartículas poliméricas (NPP) e carreadores lipídicos nanoestruturados (CLN), que foram caracterizados e avaliados quanto à sua atividade antimicrobiana in vitro frente aos microorganismos Streptococcus mutans, Streptococcus sobrinus e Lactobacillus casei. Após a análise dos resultados microbiológicos, o CLN foi selecionado para o encapsulamento da EGCG (CLNEGCG), por apresentar atividade antimicrobiana frente à todos os microorganismos avaliados. O CLNEGCG foi preparado pelo método de emulsão e sonicação e caracterizado quanto ao diâmetro, índice de polidispersão (PdI), potencial zeta (PZ), eficiência de encapsulamento (EE), cristalinidade, capacidade de mucoadesão e morfologia. A atividade antimicrobiana in vitro da EGCG livre e encapsulada foi avaliada por meio da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM). O diâmetro, PdI e o PZ dos CLN foram 228nm, 0,216 e 36,53mV, respectivamente, sendo que o encapsulamento da EGCG não alterou significativamente estes parâmetros. O CLN apresentou forma esférica, estabilidade por 330 dias e propriedade mucoadesiva devido a presença de quitosana na superfície do CLN. Além disso, a quitosana favoreceu o encapsulamento da EGCG obtendose uma EE de ~96%. As concentrações inibitórias e bactericidas mínimas do CLNEGCG (33,75 a 67,5 &micro;g/mL) foram menores do que as verificadas para a EGCG livre (250 a 2.000 &micro;g/mL), comprovando o aumento do potencial antimicrobiano com o encapsulamento da EGCG em nanocarreadores híbridos. De acordo com esses resultados, o CLNEGCG, desenvolvido no presente trabalho, constitui um sistema com potencial para o uso tópico bucal, pois além de ser estável e apresentar propriedade de mucoadesão e morfologia adequada, apresentaram alta atividade antimicrobiana frente aos principais microorganismos envolvidos no processo carioso. / The use of oral hygiene adjuvants may be necessary to control the cariogenic microbiota of individuals with high risk and caries disease activity. Currently the most recommended antimicrobial agent is chlorhexidine digluconate (CHX) because of its broad spectrum of action and residual effect. However, this chemical has side effects. In this context, as an Epigallocatechin3gallate (EGCG), a derivative of green tea, as an alternative to synthetic antimicrobial agents. However, there is no stability over time and can oxidize rapidly. Thus, the encapsulation of EGCG in nanoparticles can increase their bioavailability and physical and chemical stability, maintain the effect of this polyphenol on the target tissue and potentiate its pharmacological efficacy. Thus, the present study aimed to develop and characterize EGCG encapsulation systems and to evaluate, in vitro, its antimicrobial activity against cariogenic microorganisms. Initially, polymer nanoparticles (NPP) and nanostructured lipid carriers (CLN) were prepared and evaluated for their in vitro antimicrobial activity against Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei microorganisms. After the analysis of the microbiological results, the CLN was selected for the encapsulation of EGCG (CLNEGCG), due to its higher antimicrobial activity. The CLNEGCG was developed by emulsion and sonication method and was characterized in relation to diameter, polydispersity index (PdI), zeta potential (PZ), encapsulation efficiency (EE), crystallinity, mucoadhesion capacity and morphology. The in vitro antimicrobial activity of EGCG and its ability to evaluate minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The diameter, PdI and PZ of the CLNs were 228nm, 0.216 and 36.53mV, respectively, and the EGCG encapsulation did not significantly alter these parameters. The CLN showed a spherical structure, stability for 330 days and a mucoadhesive property due to a presence of chitosan on the CLN surface. In addition, a chitosan favored EGCG encapsulation resulting in an EE of ~ 96%. The minimum inhibitory and bactericidal concentrations of CLGEGCG (33.75 to 67.5 &micro;g / mL) were lower than those for free EGCG (250 to 2,000 &micro;g / mL), with increased antimicrobial potential with EGCG encapsulation in hybrid nanocarriers. According to the results, the CLNEGCG, developed in the present study, constitutes a system with potential for oral topical use, besides being stable and possessing mucoadhesion properties, presented high antimicrobial activity against the main microorganisms involved in the carious process.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 70-84). Also available online.

O tratamento radioterápico para pacientes com neoplasias de cabeça e pescoço pode trazer consequências secundárias graves como alterações da estrutura dental, com conseguinte prejuízo da função oral, a qual influencia negativamente a qualidade de vida. Recentemente trabalhos de pesquisa tem demonstrado que a radiação induz a expressão e ativação das metaloproteinases da matriz (MMPs), consideradas as principais enzimas responsáveis pela remodelação da matriz orgânica, incluindo os componentes e estruturas da junção amelodentinária (JAD). Questiona-se então se as alterações dentais observadas em pacientes pós-radioterapia poderiam ser causadas também pela ativação das MMPs que se encontram na JAD. O presente estudo apresentou três avaliações: a ativação e expressão das MMPs, a implementação de inibidores de proteases como método de inibição das MMPs e a ativação das MMPs devido a um desafio ácido. Para as medições foram utilizados 178 fragmentos dentais de molares, divididos aleatoriamente em 2 grupos (decíduos e permanentes) / 4 subgrupos experimentais (irradiados e não-irradiados). Os fragmentos foram expostos à radiacao com Co-60, com fracao de dose de 2 Gy, 5 dias consecutivos, ate atingirem a dose total de 60 Gy, com um total de 30 ciclos, durante 6 semanas. Com o objetivo de determinar a expressão e atividade das MMPs, foram realizados os ensaios de imunofluorescência e zimografia in situ, nos fragmentos dentais de 0,6mm, analisando os tecidos duros do esmalte, dentina e JAD. Para avaliar se produtos odontológicos inativam as MMPs, os dentes foram imersos em 0,5ml de digluconato de clorexidina a 0,12%, fluoreto de sódio a 0,05%, polifenol epigalocatequina 3-galato 400&mu;M e água destilada (grupo controle), por 1 hora. Assim também com objetivo de avaliar se em um ambiente ácido, as MMPs apresentariam maior atividade, os dentes foram colocados em contato com 20&mu;l de solucao desmineralizadora com pH de 4,8, por um minuto, e posteriormente lavados com 20&mu;l de água deionizada, por um minuto. De maneira geral pudemos observar que a irradiação ativa as MMPs na JAD e estes efeitos foram mais evidentes nos dentes permanentes que nos decíduos. Com relação à expressão das diferentes MMPs, foi observada uma maior expressão das MMPs-9 e -20 para dentes decíduos, e para dentes permanentes as MMPs-2, -9 e -20 apresentaram expressão semelhante. Tendo em vista que a irradiação foi capaz de ativar as MMPs expressas na JAD de dentes permanentes, e em busca de soluções capazes de inibi-las, observamos que o Digluconato de Clorexidina, o Fluoreto de Sódio e o Polifenol Epigalocatequina 3-galato inibiram a atividade das MMPs na JAD em dentes permanentes. Por último ao investigar o efeito de um desafio ácido, na atividade das MMPs, observamos que a desmineralização não aumentou a atividade das MMPs em dentes não irradiados, porém aumentou a atividade das MMPs em dentes irradiados. Comparando dentes irradiados submetidos ou não à desmineralização, observou-se que a desmineralização incrementou a atividade das MMPs, já induzida pela irradiação. / Radiotherapy for patients with head and neck cancer can have serious secondary consequences such as changes in tooth structure, with consequent loss of oral function which negatively influences an individual\'s quality of life. Recently research work has shown that radiation induces the expression and activation of matrix metalloproteinases (MMPs) which are considered the major enzymes responsible for the remodeling of the organic matrix, including the components and structures of the dentinoenamel junction (DEJ). It is questionable if the dental changes observed in post-radiotherapy patients could also be caused by the activation of MMPs that are in the DEJ. The present study has three assessments: the activation and expression of MMPs, the implementation of protease inhibitors such as method of inactivating MMPs and the activation of MMPs due to an acid challenge. The measurements that were used were 178 molar dental fragments randomly divided into 2 groups (deciduous and permanent) / 4 experimental subgroups (irradiated and non-irradiated). The samples were exposed to radiation using Co-60 at a cumulative dose of 2 Gy fraction, 5 consecutive days, until they reached a total dose of 60 Gy, with a total of 30 cycles for 6 weeks. In order to determine the expression and activity of MMPs immunofluorescence assays were performed and in situ zymography, the dental fragments of 0.6mm, analyzing the DEJ in three areas of the tooth (cervical, cuspal and groove of pit). To assess whether MMPs inactivate dental products, the teeth were immersed in 0.5 ml of chlorhexidine digluconate at 0.12%, sodium fluoride 0.05%, polyphenol epigallocatechin-3 gallate 400&mu;M and distilled water (control group) for 1 hour. To evaluate effects in an acidic environment, MMPs have higher activity, the teeth were put in contact with 20&mu;l of demineralizing solution with pH 4.8, for a minute, and then washed with 20&mu;l of deionized water, one minute. In general we observed that the radiation active MMPs in DEJ and these effects were more evident in the permanent teeth than in the primary teeth. Regarding the expression of different MMPs, showed the greatest expression of MMP-9 and -20 for deciduous teeth, and permanent teeth MMPs-2, -9 and -20 showed similar expression. Given that the irradiation was able to activate MMPs expressed in the DEJ permanent teeth, and looking for solutions that inactive them, we observed that the digluconate Chlorhexidine, the Sodium Fluoride and Polyphenol Epigallocatechin-3-gallate inhibited activity of MMPs in the DEJ in permanent teeth. Finally, to investigate the effect of an acid challenge, in the activity of MMPs, we observed that the demineralization did not increase the activity of MMPs in non-irradiated teeth but increased the activity of MMPs in irradiated teeth. Comparing irradiated whether subjected to demineralization teeth or not, it was found that demineralization increased activity of MMPs, induced by the radiation.

The polymeric gel-forming mucins provide the structural framework of saliva and the mucus barriers that cover the mucosal surfaces of the alimentary canal. Dietary compounds may influence the barrier properties of these protective layers. The effects of green tea polyphenols, which have many health benefits but have low bioavailability and contribute to the astringency of green tea, on the structural properties of the mucins in the alimentary canal are investigated here. Using well characterised, highly purified salivary mucins MUC5B and MUC7, and porcine gastric mucins, the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) on mucins were studied here. Using rate-zonal centrifugation coupled to agarose gel electrophoresis, atomic force microscopy and particle tracking microrheology, EGCG, at concentrations found in a cup of green tea, caused increased aggregation of MUC5B in human whole saliva, and increased aggregation and viscosity of purified MUC5B. It was revealed using recombinant proteins of the N- and C-terminal regions of MUC5B that EGCG had these effects by aggregating the terminal globular protein domains of MUC5B. In contrast, MUC5B trypsin-resistant high molecular weight glycopeptides were not aggregated by EGCG, demonstrating that the oligosaccharide-rich, highly-glycosylated regions of mucins are not involved in the EGCG-induced aggregation of mucins. EGCG also caused the majority of MUC7 in human whole saliva to aggregate, and purified MUC7 also showed substantial aggregation in the presence of EGCG.Porcine gastric mucins were also used in order to model human gastric mucins. First, the identity of the porcine gastric mucins was explored using tandem mass spectrometry and immunohistochemistry. This revealed that Muc5ac was expressed by the surface epithelium and was the prominent mucin in porcine gastric mucus. Muc6 was expressed by gastric submucosal glands, but was not a major component of the secreted mucus barrier. Porcine Muc5ac and Muc6 were shown to be aggregated by EGCG. These data demonstrate that mucins from both saliva and the stomach are substantially altered by EGCG. This may contribute to the astringency and low bioavailability of EGCG. In contrast, the green tea polyphenol epicatechin (EC) did not cause aggregation of salivary mucins or porcine gastric mucins, suggesting that the galloyl ring of EGCG (which is absent in EC) is important for its aggregation of mucins, and that EC has different mechanisms of astringency. The structure of the mucins in the alimentary canal was studied using Raman spectroscopy, Raman optical activity (ROA) and Tip-enhanced Raman spectroscopy (TERS). The secondary structure of the oligosaccharide-rich regions of mucins was shown to be largely disordered, with some contribution of poly-proline II helix. The N- and C-terminal regions of MUC5B were largely β-sheet in structure, with some disordered structure also present in the C-terminal region. Raman spectroscopy could reliably distinguish between MUC5B glycoforms, demonstrating the sensitivity of this technique to mucin glycosylation and secondary structure. The first TERS spectra along the length of a MUC5B chain are reported, and suggest that patterns may exist in the glycosylation of MUC5B. Therefore, Raman spectroscopies are novel tools that shed new light on mucin structure and in future may be useful for studying the changes to mucin structure during interactions, such as those with polyphenols.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and occurs in ~1 of every 700 live births. Individuals with DS present craniofacial abnormalities, specifically an undersized, dysmorphic mandible which may lead to difficulty with eating, breathing, and speech. Using the Ts65Dn DS mouse model, which mirrors these phenotypes and contains three copies of ~50% Hsa21 homologues, our lab has traced the mandibular deficit to a neural crest cell (NCC) deficiency in the first pharyngeal arch (PA1 or mandibular precursor) at embryonic day 9.5 (E9.5). At E9.5, the PA1 is reduced in size and contains fewer cells due to fewer NCC populating the PA1 from the neural tube (NT) as well as reduced cellular proliferation in the PA1. We hypothesize that both the deficits in NCC migration and proliferation may cause the reduction in size of the PA1. To identify potential genetic mechanisms responsible for trisomic PA1 deficits, we generated RNA-sequence (RNA-seq) data from euploid and trisomic E9.25 NT and E9.5 PA1 (time points occurring before and after observed deficits) using a next-generation sequencing platform. Analysis of RNA-seq data revealed differential trisomic expression of 53 genes from E9.25 NT and 364 genes from E9.5 PA1, five of which are present in three copies in Ts65Dn. We also further analyzed the data to find that fewer alternative splicing events occur in trisomic tissues compared to euploid tissues and in PA1 tissue compared to NT tissue. In a subsequent study, to test gene-specific treatments to rescue PA1 deficits, we targeted Dyrk1A, an overexpressed DS candidate gene implicated in many DS phenotypes and predicted to cause the NCC and PA1 deficiencies. We hypothesize that treatment of pregnant Ts65Dn mothers with Epigallocatechin gallate (EGCG), a known Dyrk1A inhibitor, will correct NCC deficits and rescue the undersized PA1 in trisomic E9.5 embryos. To test our hypothesis, we treated pregnant Ts65Dn mothers with EGCG from either gestational day 7 (G7) to G8 or G0 to G9.5. Our study found an increase in PA1 volume and NCC number in trisomic E9.5 embryos after treatment on G7 and G8, but observed no significant improvements in NCC deficits following G0-G9.5 treatment. We also observed a developmental delay of embryos from trisomic mothers treated with EGCG from G0-G9.5. Together, these data show that timing and sufficient dosage of EGCG treatment is most effective during the developmental window the few days before NCC deficits arise, during G7 and G8, and may be ineffective or harmful when administered at earlier developmental time points. Together, the findings of both studies offer a better understanding of potential mechanisms altered by trisomy as well as preclinical evidence for EGCG as a potential prenatal therapy for craniofacial disorders linked to DS.

Introduction: This thesis investigates the effect of isolated epigallocatechin-3-gallate on weight reduction in the adult population. An overview of Czech and foreign literature was provided on topics of obesity, the possibility of using green tea in its treatment and the positive effects of green tea on human health, followed by a systematic review and meta-analysis. Methodology: Using a three-phase systematic search strategy, published studies were searched in the Scopus database using predefined keywords. The aim of the search was to find randomized placebo-controlled studies lasting at least 8 weeks, which examined the effect of isolated EGCG in the absence of other active substances on the change of body parameters. The extracted data from the primary studies were then evaluated using meta-analysis and forest plot. As a result, a standardized mean difference with a 95 % confidence interval was calculated using a random effect model. Results: Six1 studies met the inclusion criteria, one2 of which was excluded due to insufficient evaluation in the critical appraisal of methodological quality. The remaining 5 studies provided a total sample of 325 participants who were diagnosed with a change in BMI induced by EGCG consumption. The result was a standardized average difference of -0.01, 95 % CI:...

Osteoarthritis is one of the most common causes of disability in adults in America. It is a progressive and degenerative disease where the articular cartilage is broken down and lost from the surfaces of bones causing chronic pain and swelling in the joints, and currently has no cure. The most commonly osteoarthritis starts from a focal lesion on the cartilage surface, which will expand on the surface and downwards through the thickness of the tissue. The current gold standard for correcting cartilage focal lesions is the osteochondral autograft/allograft transplantation (OAT), which replaces the defect with a fresh osteochondral graft. The main limiting factor for using the OAT comes from the limited number of autograft and allografts that are available for implantation. To address the concern of graft availability, this study will look at the development of a porcine osteochondral xenograft (OCXG). The first aim of this research is to establish a decellularization protocol that will remove the antigens and cellular debris, which are the leading causes of graft rejection when implanting animal tissue in humans. The second aim of this study is restoring the mechanical strength of the OCXG that was lost during the decellularization process through crosslinking the tissue using genipin and epigallocatechin gallate (EGCG). The third aim is comparing the performance of the complete crosslinked OCXG at different degrees of crosslinking in a long-term goat animal model. The final aim is an alternative way to correct focal lesions through the development of an injectable collagen stabilizing treatment with genipin and punicalagin that will slow or stop the growth of a lesion and prevent osteoarthritis.