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Advisor(s)
Abstract(s)
Durante a gravidez, várias estruturas são formadas para o correto desenvolvimento fetal, nomeadamente,
as membranas fetais. As membranas fetais são constituídas pela membrana amniótica e a membrana
coriónica que fornecem suporte mecânico, endócrino e imunitário para a proteção do feto. A membrana
amniótica consiste na camada mais interna que contém o líquido amniótico. É formada por uma camada
de células epiteliais, uma membrana basal e pelo estroma que é composto pelas camadas compacta,
fibroblástica e esponjosa. Por outro lado, a membrana coriónica é a camada mais externa e constituída
pela camada reticular, a membrana basal e pela camada de trofoblastos. Após o parto, as membranas são
descartadas uma vez que são consideradas lixo biológico. Contudo, possuem propriedades bastante
interessantes que se devem à presença de excelentes fatores de crescimento, citoquinas, células e outros
componentes, podendo ser utilizadas em diversas aplicações. De modo a evitar uma resposta
inflamatória e imunitária no hospedeiro, é necessário realizar o tratamento das membranas através de
certos processos para remover componentes celulares, preservando a estrutura e elementos da matriz
extracelular. Graças aos componentes estruturais, as membranas fetais têm vindo a ser utilizadas em
inúmeras aplicações na engenharia de tecidos e medicina regenerativa como scaffolds para o tratamento e
regeneração de vários tecidos danificados.
Assim, o principal objetivo deste trabalho foi avaliar o papel proliferativo e adesivo das membranas
amniótica e coriónica em dois tipos de linhas celulares, particularmente, a MC3T3-E1 e a RAW 264.7,
tendo em vista explorar a possível aplicação destas membranas em regeneração óssea. A MC3T3-E1 é
uma linha celular precursora de osteoblastos, comumente utilizada para avaliação da osteogénese. Já as
células RAW 264.7 são uma linha celular de macrófagos, passíveis de diferenciação em osteoclastos, os
quais têm um papel central na remodelação óssea. Inicialmente, começou-se por realizar a caracterização
das membranas através de técnicas de espectroscopia vibracional: espectroscopia e microscopia de
infravermelho com transformada de Fourier e microscopia confocal Raman, para adquirir informações
quanto à composição química e estrutura molecular das mesmas. Foi também possível identificar a
presença de bandas indicativas da existência de glicosaminoglicanos, o que indica que o procedimento
de descelularização, preservou, pelo menos em parte, estes constituintes da matriz. Posteriormente,
realizou-se a cultura das células MC3T3-E1 e as RAW 264.7 durante 1, 3 e 7 dias juntamente com as
membranas amniótica e coriónica. Após incubação, foram utilizados três tipos de testes para avaliar a
viabilidade e citotoxicidade das células, nomeadamente, o PrestoBlue, Live/Dead e o CytoTox 96.
Por fim, a microscopia confocal, a microscopia eletrónica de varrimento e a microanálise por raios-X,
foram utilizadas para analisar a morfologia das células MC3T3-E1 na presença das membranas e a
composição elementar da membrana amniótica e membrana coriónica. Observou-se uma alteração na
morfologia das células em ambas as membranas, bem como uma maior concentração de células na
presença da membrana coriónica relativamente à membrana amniótica. Além disso, a adesão das células
à superfície das membranas sugere que componentes importantes em ambas as membranas foram
preservados nas matrizes extracelulares. Assim, verificou-se que as membranas fetais demonstram ter
características únicas para serem utilizadas em várias aplicações da engenharia de tecidos, nomeadamente
para regeneração óssea, graças à preservação de componentes estruturais e integridade da matriz
extracelular após os procedimentos de descelularização e liofilização.
Throughout pregnancy, different structures are formed for the correct fetal development, namely, fetal membranes. Fetal membranes are comprised of the amniotic membrane and chorionic membrane, which protect the fetus by providing mechanical, endocrine, and immune support. The amniotic membrane is the innermost layer that contains the amniotic fluid. It is formed by a layer of epithelial cells, a basement membrane, and the stroma, which is composed of compact, fibroblast, and spongy layers. On the other hand, the chorionic membrane is the outermost layer and is essentially composed of a reticular layer, a basement membrane, and a trophoblast layer. After parturition, membranes are discarded since they are considered biological waste. Nonetheless, they have interesting properties due to the presence of excellent growth factors, cytokines, cells, and other components, so they can be used in a wide range of applications. To avoid an inflammatory and immune response in the host, it is necessary to perform a treatment to remove cellular components, preserving the structure and elements of the extracellular matrix. Thanks to the structural components, fetal membranes have been used in numerous applications as scaffolds for the treatment and regeneration of damaged tissue. Therefore, this work aimed to evaluate the proliferative and adhesive roles of amniotic and chorionic membranes in two types of cell lines, particularly MC3T3-E1 and RAW 264.7, to explore the potential application of these membranes in bone regeneration. MC3T3-E1 is an osteoblast precursor cell line, commonly used to evaluate osteogenesis. RAW 264.7 are macrophages cell lines, capable of differentiating into osteoclasts, which have a crucial role in bone remodeling. Initially, the characterization of the fetal membranes was performed by using vibrational spectroscopy techniques: Fourier-transform infrared spectroscopy and microscopy and confocal Raman microscopy, to acquire information regarding membranes’ chemical composition and molecular structure. It was also possible to identify the presence of bands related to the glycosaminoglycans, which reveals that the decellularization procedure preserved, at least partially, these components of the matrix. Posteriorly, cell culture of MC3T3-E1 and RAW 264.7 was done during 1, 3, and 7 days jointly with amniotic and chorionic membranes. After the incubation, three types of assays were used to evaluate cell viability and cytotoxicity, specifically PrestoBlue, Live/Dead, and CytoTox 96. Finally, confocal microscopy, scanning laser microscopy, and X-ray microanalyses were used to analyze MC3T3-E1 cell morphology when in contact with membranes and elementary composition of amniotic and chorionic membranes. It was observed a modification in cell morphology in both membranes, as well as a higher cell concentration in the presence of the chorionic membrane when compared to the amniotic membrane. Besides that, cell adhesion to the surface of the membranes suggested that important components on both membranes were preserved in their extracellular matrices. Thereby, fetal membranes demonstrated to have unique characteristics to be used in various applications in tissue engineering, specifically in bone regeneration, due to the preservation of structural components and extracellular matrix’s integrity, after the procedures of decellularization and lyophilization.
Throughout pregnancy, different structures are formed for the correct fetal development, namely, fetal membranes. Fetal membranes are comprised of the amniotic membrane and chorionic membrane, which protect the fetus by providing mechanical, endocrine, and immune support. The amniotic membrane is the innermost layer that contains the amniotic fluid. It is formed by a layer of epithelial cells, a basement membrane, and the stroma, which is composed of compact, fibroblast, and spongy layers. On the other hand, the chorionic membrane is the outermost layer and is essentially composed of a reticular layer, a basement membrane, and a trophoblast layer. After parturition, membranes are discarded since they are considered biological waste. Nonetheless, they have interesting properties due to the presence of excellent growth factors, cytokines, cells, and other components, so they can be used in a wide range of applications. To avoid an inflammatory and immune response in the host, it is necessary to perform a treatment to remove cellular components, preserving the structure and elements of the extracellular matrix. Thanks to the structural components, fetal membranes have been used in numerous applications as scaffolds for the treatment and regeneration of damaged tissue. Therefore, this work aimed to evaluate the proliferative and adhesive roles of amniotic and chorionic membranes in two types of cell lines, particularly MC3T3-E1 and RAW 264.7, to explore the potential application of these membranes in bone regeneration. MC3T3-E1 is an osteoblast precursor cell line, commonly used to evaluate osteogenesis. RAW 264.7 are macrophages cell lines, capable of differentiating into osteoclasts, which have a crucial role in bone remodeling. Initially, the characterization of the fetal membranes was performed by using vibrational spectroscopy techniques: Fourier-transform infrared spectroscopy and microscopy and confocal Raman microscopy, to acquire information regarding membranes’ chemical composition and molecular structure. It was also possible to identify the presence of bands related to the glycosaminoglycans, which reveals that the decellularization procedure preserved, at least partially, these components of the matrix. Posteriorly, cell culture of MC3T3-E1 and RAW 264.7 was done during 1, 3, and 7 days jointly with amniotic and chorionic membranes. After the incubation, three types of assays were used to evaluate cell viability and cytotoxicity, specifically PrestoBlue, Live/Dead, and CytoTox 96. Finally, confocal microscopy, scanning laser microscopy, and X-ray microanalyses were used to analyze MC3T3-E1 cell morphology when in contact with membranes and elementary composition of amniotic and chorionic membranes. It was observed a modification in cell morphology in both membranes, as well as a higher cell concentration in the presence of the chorionic membrane when compared to the amniotic membrane. Besides that, cell adhesion to the surface of the membranes suggested that important components on both membranes were preserved in their extracellular matrices. Thereby, fetal membranes demonstrated to have unique characteristics to be used in various applications in tissue engineering, specifically in bone regeneration, due to the preservation of structural components and extracellular matrix’s integrity, after the procedures of decellularization and lyophilization.
Description
Keywords
Amniotic membrane bioactive factors bone regeneration cell proliferation chorionic membrane tissue engineering