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Abstract(s)
A engenharia dos tecidos utiliza diferentes métodos e materiais para tentar obter estruturas com propriedades semelhantes aos tecidos que existem no organismo. Para isso utiliza diferentes materiais que são designados por biomateriais, contribuindo significativamente para diversos avanços no campo da Medicina Regenerativa. Estes têm como função reparar, substituir, manter ou melhorar a função particular de um tecido ou órgão.
O presente projecto de investigação teve como objectivo avaliar a adesão celular e a viabilidade de hidrogéis tendo como base o dextrano oxidado, para posterior aplicação na medicina regenerativa, principalmente na produção de substitutos de vasos sanguíneos.
Para isso procedeu-se inicialmente ao isolamento de células de músculo liso humanas, para posterior realização de ensaios de biocompatibilidade de vários hidrogéis com diferentes formulações de dextrano oxidado ligado aos agentes reticulantes ácido adipidico dihidrazido (AAD) e/ou gelatina (Gel) dissolvidos em DMEM-F12 ou em PBS. Estes biomateriais foram fornecidos pelo Departamento de Engenharia Química da Universidade de Coimbra. A presença das células de músculo lisas humanas na cultura primária foi confirmada por imunofluorescência com sinal positivo para a proteína de alfa - actina.
Porém os primeiros ensaios de biocompatibilidade foram realizados com células de músculo liso de rato (A7r5) devido ao rápido crescimento destas células. E também de modo a proceder à optimização do método do MTT que foi realizado de modo a testar a citotoxicidade das várias formulações de dextrano.
Ao longo deste estudo adicionou-se as células A7r5 e as células de músculo liso humanas aos diferentes hidrogéis, observou-se para os dois tipos de células de músculo liso que existe uma maior adesão e crescimento celular para as formulações de grau de oxidação inferior de dextrano, sendo o dextrano oxidado 5% (DexOx 5%) onde se obtiveram os melhores resultados. Assim como se observou maior adesão quando os materiais foram dissolvidos em DMEM-F12 em comparação aos dissolvidos em PBS.
Os resultados obtidos fornecem novos dados importantes sobre a biocompatibilidade de hidrogéis tendo como base o dextrano oxidado para que, após futuros ensaios in vivo possam, num futuro próximo, ser utilizados na medicina regenerativa, na formação de substitutos de vasos sanguíneos
Tissue Engineering uses different methods and materials to try to obtain structures with properties similar to that in tissues of organism. For that, it uses different materials, designated by biomateriais, contributing significantly to several progresses in the field of the Regenerative Medicine. These have as function to repair, to substitute, to maintain or to improve the peculiar function of tissue or organ. This thesis focuses the cell adhesion assessment on oxidized dextran-based hydrogels, for further application in regenerative medicine, mainly in the production of substitutes for blood vessels. For that, we started by isolating smooth muscle cells, for subsequent accomplishment of biocompatibily assays of several oxidized-dextran (dexOx) based hydrogels, linked to the agents reticulates adipic acid dihydrazide (AAD) and/or gelatine (Gel) dissolved in DMEM-F12 or in PBS.These biomateriais were supplied by the Department of Chemical Engineering of the Coimbra University. By using indirect immunofluorescence the cells were identified as smooth muscle cells by the presence of alpha smooth muscle actin. However the first biocompatibily assays were accomplished with smooth muscle cells from rat (A7r5) due to the fast growth of these cells. It is also in way to proceed to the optimization of the method of MTT that was accomplished to test the cytotoxicity of the several dextran formulations. Along this study it was added the cells A7r5 and human smooth muscle cells to the hydrogels, it was observed for the two types of cells of smooth muscle that exist a larger adhesion and cellular growth for the formulations of smaller oxidation degree, being the oxidized-dextran 5% (dexOx 5%) where they were obtained the best results. As well as larger adhesion it was observed when the materials that were dissolved initially in the culture medium (DMEM-F12) in comparison with dissolved in PBS. The subsequent evaluation of the cytotoxicity (through the assay of reduction of MTT) has concordant results with what was observed by optical microscopy. They are verified the largest absorvâncias for the materials than they were dissolved in DMEM-F12. The results obtained gave new information about biocompatible of oxidized dextran-based hydrogels, after in vivo assays can, in a near future, be applied in regenerative medicine, in the production of substitutes for blood vessels.
Tissue Engineering uses different methods and materials to try to obtain structures with properties similar to that in tissues of organism. For that, it uses different materials, designated by biomateriais, contributing significantly to several progresses in the field of the Regenerative Medicine. These have as function to repair, to substitute, to maintain or to improve the peculiar function of tissue or organ. This thesis focuses the cell adhesion assessment on oxidized dextran-based hydrogels, for further application in regenerative medicine, mainly in the production of substitutes for blood vessels. For that, we started by isolating smooth muscle cells, for subsequent accomplishment of biocompatibily assays of several oxidized-dextran (dexOx) based hydrogels, linked to the agents reticulates adipic acid dihydrazide (AAD) and/or gelatine (Gel) dissolved in DMEM-F12 or in PBS.These biomateriais were supplied by the Department of Chemical Engineering of the Coimbra University. By using indirect immunofluorescence the cells were identified as smooth muscle cells by the presence of alpha smooth muscle actin. However the first biocompatibily assays were accomplished with smooth muscle cells from rat (A7r5) due to the fast growth of these cells. It is also in way to proceed to the optimization of the method of MTT that was accomplished to test the cytotoxicity of the several dextran formulations. Along this study it was added the cells A7r5 and human smooth muscle cells to the hydrogels, it was observed for the two types of cells of smooth muscle that exist a larger adhesion and cellular growth for the formulations of smaller oxidation degree, being the oxidized-dextran 5% (dexOx 5%) where they were obtained the best results. As well as larger adhesion it was observed when the materials that were dissolved initially in the culture medium (DMEM-F12) in comparison with dissolved in PBS. The subsequent evaluation of the cytotoxicity (through the assay of reduction of MTT) has concordant results with what was observed by optical microscopy. They are verified the largest absorvâncias for the materials than they were dissolved in DMEM-F12. The results obtained gave new information about biocompatible of oxidized dextran-based hydrogels, after in vivo assays can, in a near future, be applied in regenerative medicine, in the production of substitutes for blood vessels.
Description
Keywords
Artéria umbilical Células do músculo liso Engenharia de tecidos Biomateriais