Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro

Detalhes bibliográficos
Autor(a) principal: Kronemberger, Gabriela Soares
Data de Publicação: 2018
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da UNIGRANRIO
Texto Completo: http://localhost:8080/tede/handle/tede/354
Resumo: New bone formation is desirable in a variety of pathological settings, such as osteoporosis. Adipose derived stem/stromal cells (ASCs) are considered a promising cell source for the formation of this tissue in vitro through tissue engineering techniques. A new approach in the field of tissue engineering corresponds to the use of stem-cell spheroids as modular units for fabricate biological tissues in vitro. Thus, the aim of this work was to standardize an osteogenic induction model in vitro from spheroids of human ASCs produced by micromolded nonadhesive hydrogel. ASCs were isolated by mechanical dissociation from human lipoaspirate samples obtained according to the local research ethics committee. Two strategies for osteogenic differentiation induction were evaluated in ASCs spheroids produced by micromolded nonadhesive hydrogel. Spheroids maintained in culture medium without inducing factors were maintained as controls. Measurements of spheroid diameter were performed using phase contrast optical microscopy images. Spheroids induced by hrBMP-7 and by TGF-β3 presented a diameter mean of 370 μm and 450 μm respectively. The hrBMP-7 induced spheroids showed high cell viability (85%), quantified by 7-AAD exclusion detected by flow cytometry. The TGFβ3 induced spheroids also showed high cell viability throughout the weeks of culture, as assessed by commercial fluorescence kit. The TGF-β3-induced spheroids exhibited more calcium deposits than those induced by hrBMP-7, identified by alizarin red staining in histological sections. In both cases, X-ray microanalysis (EDX) showed the presence of calcium only in induced spheroids. The hrBMP-7 induced spheroids showed higher alkaline phosphatase mRNA expression (p = 0.0015) and lower TSP-1 expression (p = 0.00016) when compared to control spheroids. However, there was no difference in RUNX2 and Sox9 expression. The TGF-β3 induced spheroids had a higher composition of typical extracellular matrix components, such as collagen I, osteocalcin, osteopontin, biglycan and tenascin C by immunohistochemistry when compared to those induced by hrBMP-7. In addition, the mechanical resistance to compression of spheroids induced by TGF-β3 was higher when compared to spheroids induced by hrBMP-7 (mean of 360μN and 20μN, respectively). Therefore, it was possible to standardize an in vitro osteogenic induction model of human ASCs spheroids. It was also concluded that induction by TGF-β3 followed by induction with osteogenic medium is able to produce differentiated spheroids with greater efficiency of osteogenic differentiation than spheroids induced by hrBMP-7. In this work, it was possible to standardize a model of in vitro osteogenic induction of human ASCs spheroids, opening a new perspective for regenerative medicine.
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spelling Baptista, Leandra SantosPereira, Karina Ribeiro da SilvaBaptista, Leandra SantosPereira, Karina Ribeiro da SilvaPereira, Leonardo da Cunha BoldriniSouza, Kleber Luiz de Araujo eSant' anna Filho, Celso Barbosa deKronemberger, Gabriela Soares2019-10-01T18:25:13Z2018-10-31KRONEMBERGER, Gabriela Soares. Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro. 2018. 128 f. Dissertação (Programa de Pós-Graduação em Biomedicina Translacional) - Universidade do Grande Rio, Duque de Caxias, 2018.http://localhost:8080/tede/handle/tede/354New bone formation is desirable in a variety of pathological settings, such as osteoporosis. Adipose derived stem/stromal cells (ASCs) are considered a promising cell source for the formation of this tissue in vitro through tissue engineering techniques. A new approach in the field of tissue engineering corresponds to the use of stem-cell spheroids as modular units for fabricate biological tissues in vitro. Thus, the aim of this work was to standardize an osteogenic induction model in vitro from spheroids of human ASCs produced by micromolded nonadhesive hydrogel. ASCs were isolated by mechanical dissociation from human lipoaspirate samples obtained according to the local research ethics committee. Two strategies for osteogenic differentiation induction were evaluated in ASCs spheroids produced by micromolded nonadhesive hydrogel. Spheroids maintained in culture medium without inducing factors were maintained as controls. Measurements of spheroid diameter were performed using phase contrast optical microscopy images. Spheroids induced by hrBMP-7 and by TGF-β3 presented a diameter mean of 370 μm and 450 μm respectively. The hrBMP-7 induced spheroids showed high cell viability (85%), quantified by 7-AAD exclusion detected by flow cytometry. The TGFβ3 induced spheroids also showed high cell viability throughout the weeks of culture, as assessed by commercial fluorescence kit. The TGF-β3-induced spheroids exhibited more calcium deposits than those induced by hrBMP-7, identified by alizarin red staining in histological sections. In both cases, X-ray microanalysis (EDX) showed the presence of calcium only in induced spheroids. The hrBMP-7 induced spheroids showed higher alkaline phosphatase mRNA expression (p = 0.0015) and lower TSP-1 expression (p = 0.00016) when compared to control spheroids. However, there was no difference in RUNX2 and Sox9 expression. The TGF-β3 induced spheroids had a higher composition of typical extracellular matrix components, such as collagen I, osteocalcin, osteopontin, biglycan and tenascin C by immunohistochemistry when compared to those induced by hrBMP-7. In addition, the mechanical resistance to compression of spheroids induced by TGF-β3 was higher when compared to spheroids induced by hrBMP-7 (mean of 360μN and 20μN, respectively). Therefore, it was possible to standardize an in vitro osteogenic induction model of human ASCs spheroids. It was also concluded that induction by TGF-β3 followed by induction with osteogenic medium is able to produce differentiated spheroids with greater efficiency of osteogenic differentiation than spheroids induced by hrBMP-7. In this work, it was possible to standardize a model of in vitro osteogenic induction of human ASCs spheroids, opening a new perspective for regenerative medicine.A nova formação do tecido ósseo é desejável em uma variedade de cenários patológicos, como a osteoporose. Células-tronco/estromais derivadas do tecido adiposo (ASCs, do inglês adipose derived stem/stromal cells) são consideradas uma fonte celular promissora para a formação deste tecido in vitro através de técnicas de engenharia tecidual. Uma nova abordagem na área de engenharia de tecidos corresponde à utilização de esferoides de células-tronco como unidades modulares para fabricar tecidos biológicos in vitro. Dessa forma, o objetivo deste trabalho foi padronizar um modelo de indução osteogênica in vitro a partir de esferoides de ASCs humanas fabricados em hidrogel de agarose micromoldado. ASCs foram isoladas por dissociação mecânica de amostras de lipoaspirado humano obtidas de acordo com o comitê local de ética em pesquisa. Duas estratégias para a indução à diferenciação osteogênica foram testadas em esferoides de ASCs fabricados em hidrogel de agarose micromoldado. Esferoides mantidos em meio de cultivo sem fatores indutores foram mantidos como controles. Medidas do diâmetro dos esferoides foram realizadas a partir de imagens de microscopia óptica de contraste de fase. Esferoides induzidos por hrBMP-7 e por TGF-β3 apresentaram média de diâmetro 370µm e 450µm respectivamente. Esferoides induzidos por hrBMP-7 apresentaram alta viabilidade celular (85%), avaliada por exclusão de 7-AAD detectada por citometria de fluxo. Esferoides induzidos por TGF-β3 também apresentaram elevada viabilidade celular ao longo das semanas de cultivo, avaliada por kit comercial de fluorescência. Esferoides induzidos por TGF-β3 apresentaram mais depósitos de cálcio do que os induzidos por hrBMP-7, identificado por coloração de alizarina vermelha em cortes histológicos. Em ambos os casos, as análises por microanálise de raio-X (EDX) mostraram a presença de cálcio apenas em esferoides induzidos. Esferoides induzidos por hrBMP-7 apresentaram maior expressão de mRNA de fosfatase alcalina (p = 0,0015) e menor expressão de TSP1 (p = 0,00016) do que esferoides controles. No entanto, não houve diferença na expressão de RUNX2 e Sox9. Esferoides induzidos por TGF-β3 apresentaram maior composição de componentes típicos da matriz extracelular óssea, como colágeno I, osteocalcina, osteopontina, biglicana e tenascina C, avaliada por imunohistoquímica do que os induzidos por hrBMP-7. Além disso, a resistência mecânica à compressão de esferoides induzidos por TGF-β3 foi superior à de esferoides induzidos por hrBMP-7 (média de 360µN e 20µN, respectivamente). Portanto, foi possível padronizar um modelo de indução osteogênica in vitro de esferoides de ASCs humanas. Conclui-se ainda que a indução por TGF-β3 seguida de indução com meio osteogênico forma esferoides diferenciados com maior eficiência de diferenciação osteogênica do que esferoides induzidos por hrBMP-7. Neste trabalho, foi possível padronizar um modelo de indução osteogênica in vitro de esferoides de ASCs humanas, abrindo uma nova perspectiva para a medicina regenerativaSubmitted by Janser dos Santos Nascimento (janser.nascimento@unigranrio.com.br) on 2019-10-01T18:25:13Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Gabriela Soares Kronemberger.pdf: 4259312 bytes, checksum: c308c4a72fa3009dc4615977829ed522 (MD5)Made available in DSpace on 2019-10-01T18:25:13Z (GMT). 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dc.title.por.fl_str_mv Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
title Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
spellingShingle Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
Kronemberger, Gabriela Soares
Ciências Biomédicas
Ossos
Células-tronco
Ossos - Crescimento
CIÊNCIAS DA SAÚDE
title_short Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
title_full Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
title_fullStr Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
title_full_unstemmed Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
title_sort Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro
author Kronemberger, Gabriela Soares
author_facet Kronemberger, Gabriela Soares
author_role author
dc.contributor.advisor1.fl_str_mv Baptista, Leandra Santos
dc.contributor.advisor2.fl_str_mv Pereira, Karina Ribeiro da Silva
dc.contributor.referee1.fl_str_mv Baptista, Leandra Santos
dc.contributor.referee2.fl_str_mv Pereira, Karina Ribeiro da Silva
dc.contributor.referee3.fl_str_mv Pereira, Leonardo da Cunha Boldrini
dc.contributor.referee4.fl_str_mv Souza, Kleber Luiz de Araujo e
dc.contributor.referee5.fl_str_mv Sant' anna Filho, Celso Barbosa de
dc.contributor.author.fl_str_mv Kronemberger, Gabriela Soares
contributor_str_mv Baptista, Leandra Santos
Pereira, Karina Ribeiro da Silva
Baptista, Leandra Santos
Pereira, Karina Ribeiro da Silva
Pereira, Leonardo da Cunha Boldrini
Souza, Kleber Luiz de Araujo e
Sant' anna Filho, Celso Barbosa de
dc.subject.por.fl_str_mv Ciências Biomédicas
Ossos
Células-tronco
Ossos - Crescimento
topic Ciências Biomédicas
Ossos
Células-tronco
Ossos - Crescimento
CIÊNCIAS DA SAÚDE
dc.subject.cnpq.fl_str_mv CIÊNCIAS DA SAÚDE
description New bone formation is desirable in a variety of pathological settings, such as osteoporosis. Adipose derived stem/stromal cells (ASCs) are considered a promising cell source for the formation of this tissue in vitro through tissue engineering techniques. A new approach in the field of tissue engineering corresponds to the use of stem-cell spheroids as modular units for fabricate biological tissues in vitro. Thus, the aim of this work was to standardize an osteogenic induction model in vitro from spheroids of human ASCs produced by micromolded nonadhesive hydrogel. ASCs were isolated by mechanical dissociation from human lipoaspirate samples obtained according to the local research ethics committee. Two strategies for osteogenic differentiation induction were evaluated in ASCs spheroids produced by micromolded nonadhesive hydrogel. Spheroids maintained in culture medium without inducing factors were maintained as controls. Measurements of spheroid diameter were performed using phase contrast optical microscopy images. Spheroids induced by hrBMP-7 and by TGF-β3 presented a diameter mean of 370 μm and 450 μm respectively. The hrBMP-7 induced spheroids showed high cell viability (85%), quantified by 7-AAD exclusion detected by flow cytometry. The TGFβ3 induced spheroids also showed high cell viability throughout the weeks of culture, as assessed by commercial fluorescence kit. The TGF-β3-induced spheroids exhibited more calcium deposits than those induced by hrBMP-7, identified by alizarin red staining in histological sections. In both cases, X-ray microanalysis (EDX) showed the presence of calcium only in induced spheroids. The hrBMP-7 induced spheroids showed higher alkaline phosphatase mRNA expression (p = 0.0015) and lower TSP-1 expression (p = 0.00016) when compared to control spheroids. However, there was no difference in RUNX2 and Sox9 expression. The TGF-β3 induced spheroids had a higher composition of typical extracellular matrix components, such as collagen I, osteocalcin, osteopontin, biglycan and tenascin C by immunohistochemistry when compared to those induced by hrBMP-7. In addition, the mechanical resistance to compression of spheroids induced by TGF-β3 was higher when compared to spheroids induced by hrBMP-7 (mean of 360μN and 20μN, respectively). Therefore, it was possible to standardize an in vitro osteogenic induction model of human ASCs spheroids. It was also concluded that induction by TGF-β3 followed by induction with osteogenic medium is able to produce differentiated spheroids with greater efficiency of osteogenic differentiation than spheroids induced by hrBMP-7. In this work, it was possible to standardize a model of in vitro osteogenic induction of human ASCs spheroids, opening a new perspective for regenerative medicine.
publishDate 2018
dc.date.issued.fl_str_mv 2018-10-31
dc.date.accessioned.fl_str_mv 2019-10-01T18:25:13Z
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dc.identifier.citation.fl_str_mv KRONEMBERGER, Gabriela Soares. Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro. 2018. 128 f. Dissertação (Programa de Pós-Graduação em Biomedicina Translacional) - Universidade do Grande Rio, Duque de Caxias, 2018.
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identifier_str_mv KRONEMBERGER, Gabriela Soares. Produção de esferoides a partir de células-tronco/estromais de tecido adiposo humano para fabricação de tecido ósseo in vitro. 2018. 128 f. Dissertação (Programa de Pós-Graduação em Biomedicina Translacional) - Universidade do Grande Rio, Duque de Caxias, 2018.
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