Development of human osteogenic cell sheets co-cultured with endothelial and pericyte-like cells
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2015-12-03
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Recentemente, o uso de cell sheets tornou-se uma realidade na área da engenharia de tecidos, existindo alguns estudos que provam a viabilidade desta tecnologia como alternativa aos tradicionais métodos baseados no uso de scaffolds. O uso de cell sheets permite não só dispensar o uso de biomateriais de suporte mas também criar substitutos de tecidos, ex-vivo, com uma organização celular própria mantendo altamente coesivas as adesões célula-célula e as interacções com a matriz extracelular. Contudo, a evolução desta tecnologia tem sido limitado por algumas das barreiras que têm vindo a impedir o progresso das estratégias tradicionais de engenharia de tecidos, nomeadamente a ausência de uma vasculatura apropriada que permita a integração dos constructs in vivo. Deste modo, este trabalho propõe a criação de um construct baseado na tecnologia de cell sheets, obtido co-cultivando células osteogénicas, endoteliais e com marcadores pericíticos, com o objetivo de promover a vascularização de tecido ósseo novo e o grau de maturação e estabilidade da rede vascular formada.
Numa primeira fase deste trabalho começou-se por otimizar as condições de cultura celular para o fabrico de cell sheets osteogénicas a partir de células mesenquimais humanas derivadas da medula óssea (hBMSCs). Esta otimização permitiu evoluir em seguida para o design de um sistema de co-cultura de células endoteliais da veia do cordão umbilical (HUVECs) e células com potencial perivascular (CD146+), criando um modelo para a vascularização de constructs produzidos in vitro com potencial para aplicação em engenharia de tecido ósseo. A medula óssea foi anteriormente proposta como uma possível fonte de células com potencial perivascular, nomeadamente as que expressam o marcador celular CD146. Deste modo, as hBMSCs isoladas da medula óssea foram cultivadas em diferentes meios de cultura e caracterizadas tendo em conta a expressão de CD146 e dos marcadores associados a células mesenquimais, CD90, CD73 e CD105. Os resultados de citometria de fluxo demonstraram que a adição de TGF-β1 ao meio de cultura provoca um aumento de expressão do marcador CD146 nas hBMSCs, de aproximadamente 50% para um valor superior a 97%. Simultaneamente, verificou-se que alterações na expressão de CD146 estão associadas a alterações da morfologia celular. A análise das co-culturas por imunocitoquímica mostrou que as células induzidas a expressar CD146 e as HUVECs foram capazes de migrar naturalmente e de se organizar sobre uma superfície rica em colagénio composta por células osteogénicas confluentes, sugerindo a existência de um cross-talk eficiente entre todas as células que compõem o sistema de co-cultura. A capacidade do construct formar, in vivo, tecido osteo-comprometido, vascularizado e funcional foi avaliada após a transplantação subcutânea do construct em ratinhos. A análise de imunohistoquímica das cell sheets transplantadas revelou a integração das HUVECs com a rede vascular do hospedeiro assim como o potencial osteogénico do construct, tal como comprovado pela detecção da expressão da osteocalcina. Adicionalmente, a análise do diâmetro dos vasos positivos para o marcador CD146 revelou um aumento da média do diâmetro dos vasos na condição experimental, o que confirma a hipótese colocada e reforça a relevância do modelo proposto na maturação e estabilização dos vasos sanguíneos formados.
In recent years the use of cell sheets for tissue engineering (TE) purposes has become a reality, with some studies showing that it is a reliable alternative for the traditional scaffold-based approaches by avoiding their associated shortcomings. This technology permits not only to eliminate the exogenous scaffolding biomaterials but also to create ex vivo tissue-like substitutes with organized cellular entities and cohesive cell-to-cell and cell-extracellular matrix (ECM) interactions. However, the progression of this technology is being limited by some of the barriers which have been hampering the evolution of other TE strategies, such as the lack of appropriate vasculature to supply thicker constructs in vivo. It is herein proposed the creation of a cell sheet construct, by co-culturing osteogenic, endothelial and pericyte-like cells, with the purpose of enhancing the vascularization of newly formed bone tissue and also the degree of maturation and stability of the vascular network. The optimization of the culture conditions to fabricate osteogenic cell sheets derived from human bone marrow stromal cells (hBMSCs) was the first step of this work. This optimization allowed furthering evolving to the design of a co-culture system with human umbilical cord vein endothelial cells (HUVECs) and pericyte-like cells as a model for the vascularization of in vitro cultured constructs with potential for bone tissue engineering applications. Bone marrow was previously proposed as a source of perivascular-like cells, in particular those expressing CD146, therefore hBMSCs were isolated, cultured under different conditions and screened for the expression of CD146 as well as for the Mesenchymal Stem Cells (MSCs) markers CD90, CD73 and CD105. Flow cytometry results demonstrated that supplementation of standard hBMSCs culture medium with TGF-β1 promote an increase of CD146 expression in hBMSCs, from approximately 50% to more than 97%. Moreover, changes in CD146 expression are associated with different cellular morphologies. Immunocytochemistry assays performed on the co-cultures showed that induced CD146+ hBMSCs and HUVECs migrated and organized themselves over a thin, collagen-rich, osteogenic cell sheet, suggesting the existence of an efficient cross-talk involving all the co-cultured cell types. Further studies concerning the ability of these constructs to form functional, vascularized and osteo-committed tissue in vivo were performed using a well described protocol for subcutaneous cell sheets transplantation on mice. Immunohistochemistry analysis of transplanted cell sheets revealed the integration of HUVECs with host network vasculature as well as the osteogenic potential of the created construct, as shown by the expression of osteocalcin. Additionally, analysis of the diameter of CD146 positive blood vessels showed a higher mean vessel diameter for the experimental condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability.
In recent years the use of cell sheets for tissue engineering (TE) purposes has become a reality, with some studies showing that it is a reliable alternative for the traditional scaffold-based approaches by avoiding their associated shortcomings. This technology permits not only to eliminate the exogenous scaffolding biomaterials but also to create ex vivo tissue-like substitutes with organized cellular entities and cohesive cell-to-cell and cell-extracellular matrix (ECM) interactions. However, the progression of this technology is being limited by some of the barriers which have been hampering the evolution of other TE strategies, such as the lack of appropriate vasculature to supply thicker constructs in vivo. It is herein proposed the creation of a cell sheet construct, by co-culturing osteogenic, endothelial and pericyte-like cells, with the purpose of enhancing the vascularization of newly formed bone tissue and also the degree of maturation and stability of the vascular network. The optimization of the culture conditions to fabricate osteogenic cell sheets derived from human bone marrow stromal cells (hBMSCs) was the first step of this work. This optimization allowed furthering evolving to the design of a co-culture system with human umbilical cord vein endothelial cells (HUVECs) and pericyte-like cells as a model for the vascularization of in vitro cultured constructs with potential for bone tissue engineering applications. Bone marrow was previously proposed as a source of perivascular-like cells, in particular those expressing CD146, therefore hBMSCs were isolated, cultured under different conditions and screened for the expression of CD146 as well as for the Mesenchymal Stem Cells (MSCs) markers CD90, CD73 and CD105. Flow cytometry results demonstrated that supplementation of standard hBMSCs culture medium with TGF-β1 promote an increase of CD146 expression in hBMSCs, from approximately 50% to more than 97%. Moreover, changes in CD146 expression are associated with different cellular morphologies. Immunocytochemistry assays performed on the co-cultures showed that induced CD146+ hBMSCs and HUVECs migrated and organized themselves over a thin, collagen-rich, osteogenic cell sheet, suggesting the existence of an efficient cross-talk involving all the co-cultured cell types. Further studies concerning the ability of these constructs to form functional, vascularized and osteo-committed tissue in vivo were performed using a well described protocol for subcutaneous cell sheets transplantation on mice. Immunohistochemistry analysis of transplanted cell sheets revealed the integration of HUVECs with host network vasculature as well as the osteogenic potential of the created construct, as shown by the expression of osteocalcin. Additionally, analysis of the diameter of CD146 positive blood vessels showed a higher mean vessel diameter for the experimental condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Engenharia de tecidos , Regeneração óssea , Técnicas de cultura de células , Scaffolds