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Desenvolvimento de modelo biomimético de córnea para avaliação da toxicidade ocular de produtos farmacêuticos: perfil inflamatório, caracterização e aplicabilidade / Biomimetic corneal model for assessment of pharmaceutical products eye toxicity: inflammatory profile, characterization and applicabilitySilva, Artur Christian Garcia da 27 March 2018 (has links)
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Previous issue date: 2018-03-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Cosmetic products eye toxicity assessment has been performed worldwide using alternative methods since animal testing has been banished for this purpose in many countries. In this context, reconstructed epithelial models have been used internationally for these evaluations, which are not available in Brazil due bureaucratic issues that limit their acquisition and use in national territory. Objective: In this work, an evaluation platform based on cellular inflammatory changes caused by exposure to eye irritants was proposed, as well as the development of a biomimetic corneal model from human keratinocytes, which was characterized and evaluated for applicability in ocular toxicity studies. Methods: Inflammatory profile of HaCat keratinocytes was determined after exposure to 13 different chemicals with different classification for ocular irritation (irritants and non-irritants) using Cytometric Bead Array (CBA) and ELISA methods. 3D models were obtained by cultivating HaCat cell line onto a collagen type I matrix, in air-liquid interface for five days. Morphological characterization was carried out through Hematoxylin-eosin staining and corneal epithelial biomarkers expression was assessed using indirect immunofluorescence. Predictive capacity assessment was performed by exposure of model to test substances with subsequently tissue viability assessment. Finally, applicability of system was verified by testing five marketed eyebrow Henna samples which were considered non-irritants due the lack of adequate labelling. Results and Discussion: A qualitative and quantitative correlation was established regarding the HaCat cells inflammatory profile and eye irritation degree of evaluated substances. The epithelial model presented similar morphology and biomarkers expression patterns similar to the found in human corneal epithelium, so that tissue viability after exposure to test substances reduced proportionately to the irritant potential of them. Furthermore, 3D model classified four of the five evaluated Henna samples as moderate or mild irritants. Conclusion: The developed epithelial model presented morphological and functional properties similar to those founded in human corneal epithelium, which enabled the distinction between irritants and non-irritants and it is also applicable to the investigation of botanical mixtures ocular toxicity potential. / Introdução: A avaliação da toxicidade ocular de produtos cosméticos tem sido mundialmente realizada mediante a utilização de métodos alternativos desde que a experimentação animal foi proibida para esse fim em muitos países. Nesse contexto, os modelos epiteliais reconstruídos têm sido internacionalmente utilizados para essa finalidade, os quais não encontram-se disponíveis no Brasil por questões burocráticas que limitam a importação e utilização dos mesmos no território nacional. Objetivo: Neste estudo, foram propostos uma plataforma de avaliação baseada em alterações celulares inflamatórias ocasionadas pela exposição a agentes irritantes oculares, bem como o desenvolvimento de um modelo biomimético de córnea a partir de queratinócitos humanos, o qual foi caracterizado e avaliado quanto à aplicabilidade para aplicação em estudos de toxicidade ocular. Metodologia: O perfil inflamatório dos queratinócitos HaCat foi determinado após exposição à 13 diferentes substâncias químicas, com diferentes perfis de irritação ocular (irritantes e não irritantes), por meio das técnicas de Cytometric Bead Array (CBA) e ELISA. Os modelos 3D foram obtidos mediante o cultivo da linhagem HaCat sobre uma matriz de colágeno tipo I, em interface ar-líquido por cinco dias. A caracterização morfológica do modelo foi realizada por meio de coloração com Hematoxilina-eosina e a expressão de biomarcadores do tecido epitelial da córnea utilizando-se imunofluorescência indireta. A avaliação da capacidade preditiva do modelo foi realizada mediante exposição deste às substâncias supracitadas, com posterior avaliação da viabilidade tecidual. Por fim, a aplicabilidade do sistema para avaliação de misturas foi verificada avaliando-se cinco amostras de Hena de sobrancelha comercializadas, sendo consideradas não irritantes pela ausência de rotulagem adequada. Resultados e discussão: Foi estabelecida correlação qualitativa e quantitativa em relação ao perfil inflamatório e à severidade das substâncias teste avaliadas. O modelo desenvolvido apresentou morfologia e expressão de biomarcadores semelhantes aos padrões encontrados na córnea humana, de modo que a viabilidade tecidual após a exposição às substâncias reduziu de maneira diretamente proporcional ao potencial irritante das mesmas. Ademais, das cinco amostras de Hena avaliadas, o modelo classificou quatro como irritantes moderados ou leves. Conclusão: O modelo epitelial desenvolvido apresentou características morfológicas e funcionais semelhantes às da córnea humana, permitindo a distinção entre substâncias irritantes e não irritantes, sendo também aplicável à investigação da toxicidade ocular de misturas botânicas.
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Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic ProductsMcCanna, David January 2009 (has links)
The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes.
The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.
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Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic ProductsMcCanna, David January 2009 (has links)
The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes.
The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.
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