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Authors
Advisor(s)
Abstract(s)
As membranas fetais têm vindo a ganhar destaque na medicina regenerativa
devido à sua hipo-imunogenicidade, funções anti-inflamatórias e biocompatibilidade.
Estes interessantes materiais naturais, podem ser utilizados como matriz extracelular, uma
vez que são ricos em fatores de crescimento e proteínas. A membrana fetal é composta
pela membrana coriónica, a camada externa, e pela membrana amniótica, a camada
interna, ambas derivadas de tecido extraembrionário. Existe uma terceira camada entre
essas duas, a camada esponjosa, cujas características ainda não foram significativamente
exploradas.
A descelularização de tecidos e órgãos é uma plataforma tecnológica de sucesso
para a criação de materiais de suporte (scaffolding). Tem sido sugerido que o sucesso
desses materiais após a implantação, é devido aos sinais moleculares fornecidos pelos
componentes restantes da matriz extracelular (ECM) apresentados às células in vivo, à
medida que estas repovoam a superfície dos scaffolds descelularizados. Existem vários
protocolos descritos na literatura para descelularização de tecidos, sendo que alguns usam
compostos tóxicos e de tal forma agressivos que eliminam componentes da membrana
que são essenciais para a engenharia de tecidos e aplicações regenerativas.
No decorrer deste trabalho, foi testado um protocolo de descelularização
optimizado a partir de um outro já utilizado no grupo. As membranas fetais resultantes
foram caracterizadas morfológica e quimicamente através de diferentes técnicas,
nomeadamente histologia, espectroscopia de infravermelho com transformada de Fourier
(FTIR), calorimetria diferencial de varrimento e termogravimetria (DSC/TGA). A
possibilidade de usar a camada esponjosa das membranas fetais como um scaffold à base
de hidrogel também foi explorada. Para isso, as suas características foram avaliadas
usando FTIR, microscopia confocal Raman, microscopia de força atómica (AFM) e testes
reológico. O potencial zeta foi também determinado bem como a taxa de hidratação desta
membrana.
Os resultados obtidos mostraram que embora o novo protocolo de
descelularização tenha eliminado completamente as células da membrana amniótica,
algumas células permaneceram na membrana coriónica. Verificou-se também tratar-se de um protocolo menos agressivo, mantendo a estrutura e composição das membranas
descelularizadas mais próxima das membranas nativas.
O presente trabalho permitiu também um avanço na caracterização da camada
esponjosa, ainda pouco explorada, tendo em vista a sua possível utilização como um novo
hidrogel potencialmente muito interessante para aplicação em medicina regenerativa
Fetal membranes have been gaining attention for regenerative medicine due to their hypoimmunogenicity, anti-inflammatory functions and biocompatibility. These interesting natural materials can be used as an extracellular matrix once they are rich in growth factors and proteins. The fetal membrane is composed of the chorionic membrane, the outer layer, and the amniotic membrane, the inner layer, both deriving from extra embryonic tissue. There is a third layer between these two, the spongy layer, which characteristics have not been significantly explored yet. Decellularization of tissues and organs is a successful platform technology for creating scaffolding materials. It has been suggested that the success of these materials upon implantation is due to the molecular signals provided by the remaining extracellular matrix (ECM) components presented to probing cells in vivo as they repopulate the surface. There are several protocols described in the literature for tissue decellularization but some use toxic compounds and are so aggressive, that eliminate membrane components that are essential for tissue engineering and regenerative applications. In the course of this work, an improved decellularization protocol was tested. The resultant fetal membranes, were morphologically and chemically characterized using different techniques namely histology, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry and thermogravimetry (DSC/TGA). The possibility of using the spongy layer as an hydrogel based scaffold was also explored. For this, its characteristics were determined using FTIR, confocal Raman microscopy, atomic force microscopy (AFM), zeta potential and rheological tests. Also, the water swelling ratio of this layer was determined. The results obtained showed that although the new decellularization protocol completely eliminated cells of the amniotic membrane, some cells remained in the chorionic membrane. It was also verified to be a less aggressive protocol, keeping the structure and composition of the decellularized membranes closer to the native membranes. The present work also allowed for a breakthrough in the characterization of the spongy layer, still little explored, in view of its possible use as a potentially very interesting new hydrogel/scaffold for application in regenerative medicine
Fetal membranes have been gaining attention for regenerative medicine due to their hypoimmunogenicity, anti-inflammatory functions and biocompatibility. These interesting natural materials can be used as an extracellular matrix once they are rich in growth factors and proteins. The fetal membrane is composed of the chorionic membrane, the outer layer, and the amniotic membrane, the inner layer, both deriving from extra embryonic tissue. There is a third layer between these two, the spongy layer, which characteristics have not been significantly explored yet. Decellularization of tissues and organs is a successful platform technology for creating scaffolding materials. It has been suggested that the success of these materials upon implantation is due to the molecular signals provided by the remaining extracellular matrix (ECM) components presented to probing cells in vivo as they repopulate the surface. There are several protocols described in the literature for tissue decellularization but some use toxic compounds and are so aggressive, that eliminate membrane components that are essential for tissue engineering and regenerative applications. In the course of this work, an improved decellularization protocol was tested. The resultant fetal membranes, were morphologically and chemically characterized using different techniques namely histology, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry and thermogravimetry (DSC/TGA). The possibility of using the spongy layer as an hydrogel based scaffold was also explored. For this, its characteristics were determined using FTIR, confocal Raman microscopy, atomic force microscopy (AFM), zeta potential and rheological tests. Also, the water swelling ratio of this layer was determined. The results obtained showed that although the new decellularization protocol completely eliminated cells of the amniotic membrane, some cells remained in the chorionic membrane. It was also verified to be a less aggressive protocol, keeping the structure and composition of the decellularized membranes closer to the native membranes. The present work also allowed for a breakthrough in the characterization of the spongy layer, still little explored, in view of its possible use as a potentially very interesting new hydrogel/scaffold for application in regenerative medicine
Description
Keywords
Membranas fetais Membrana amniótica Membrana coriónica Camada esponjosa Descelularização Scaffolds Hidrogéis Fetal membranes Amniotic membrane Chorionic membrane Spongy layer Decellularization Scaffolds Hydrogels