Uso do selante de fibrina derivado do veneno de serpente como arcabouço tridimensional para células tronco mesenquimais da medula óssea de Lobo-Guará (Chrysocyon brachyurus)
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Data
2017-08-03
Autores
Sesso, Andréa Sebelim [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O lobo-guará (Chrysocyon brachyurus) é encontrado em grande parte do território da América do Sul, mas 90% de seus exemplares se distribui principalmente no cerrado brasileiro, sendo o maior canídeo sul americano, e como tal, demanda grandes áreas em suas atividades. Consequentemente, ele sofre severamente os efeitos da perda de habitat pelas ações antrópicas. Isto o faz estar na lista vermelha pela União Internacional de Conservação da Natureza, como espécie ameaçada de extinção da fauna brasileira. Tendo em vista a necessidade de desenvolver estratégias para a conservação do lobo-guará, o objetivo deste projeto foi caracterizar e padronizar o isolamento, o cultivo e a criopreservação de células tronco mesenquimais derivado da medula óssea do lobo-guará, além de utilizar o selante heterólogo de fibrina derivado de veneno de serpente como arcabouço tridimensional possibilitando procedimentos de terapia celular para a espécie. Também foi realizada uma revisão bibliográfica sobre a espécie, terapia celular e selantes de fibrina. Para tanto, realizou-se a coleta de medula óssea de animais provenientes de CETAS e zoológicos; o cultivo das células tronco mesenquimais (CTMs); a caracterização a partir do uso de marcadores de superfície (CDs) por citometria de fluxo; o potencial de diferenciação das CTMs em linhagens celulares osteogênica, adipogênica e condrogênica; a análise da viabilidade celular pré e após a criopreservação; e por fim a observação e avaliação do selante de fibrina como arcabouço celular mostrando sua interação com as CTMs pela microscopia de varredura e viabilidade por microscopia confocal. A técnica empregada para coleta de medula óssea e posterior cultivo foi eficiente, sendo possível sua expansão até a sexta passagem. Os marcadores de superfície celular utilizados foram capazes de reconhecer as células possibilitando a sua caracterização por citometria de fluxo. As CTMs apresentaram potencial de diferenciação em linhagens osteogênica, adipogênica e condrogênica. O processo de criopreservação foi eficaz pois manteve alta a viabilidade celular após o seu descongelamento. A interação com o selante de fibrina foi adequada e manteve as células viáveis como verificado por microscopia confocal, demonstrando a estrutura tridimensional do selante por microscopia eletrônica de varredura. A terapia celular pode proporcionar para a espécie, um tratamento alternativo e eficaz na agilidade de processos regenerativos, minimizando o estresse em tratamentos convencionais de longa duração. O sucesso na criopreservação das CTMs torna possível a criação de um banco de células tronco que certamente ajudará na conservação da espécie.
Maned wolf (Chrysocyon brachyurus) is found in South America, but 90% of its specimens are distributed mainly in the Brazilian Cerrado, being the largest South American canid, and as such, it demands large areas. Consequently, maned wolf severely undergoes the effects of habitat loss by human actions. This makes it on the red list by the International Union for Conservation of Nature, as a threatened species of extinction of Brazilian fauna. Objective of this project was to characterize and standardize the isolation, cultivation and cryopreservation of mesenchymal stem cells derived from the bone marrow of maned wolf. In addition to using the fibrin sealant derived from snake venom as a three-dimensional scaffold enabling cellular therapy procedures for the species. A literature review on the species, cell therapy and fibrin sealants were also carried out. For that, the bone marrow was collected from CETAS and zoological animals; culture of mesenchymal stem cells (MSCs); characterization by surface markers (CDs) using flow cytometry; differentiation potential of MSCs into osteogenic, adipogenic and chondrogenic cell lines; analysis of cell viability before and after cryopreservation; and finally, observation and evaluation of the fibrin sealant as cellular scaffold showing its interaction with the MSCs by scanning microscopy and viability by confocal microscopy. The technique used for collection of bone marrow and subsequent culture was efficient, being possible to expand until the sixth passage. Cell surface markers used were able to recognize the cells allowing their characterization by flow cytometry. CTMs presented differentiation potential in osteogenic, adipogenic and chondrogenic linages. Cryopreservation process was effective because it maintained high cell viability after thawing. The interaction with the fibrin sealant was adequate and kept the cells viable as verified by confocal microscopy, demonstrating their three-dimensional structure of the sealant by scanning electron microscopy. Cell therapy may provide for the species an alternative and effective treatment in the agility of regenerative processes, minimizing stress in conventional long-term treatments. Success in cryopreservation of MSCs makes it possible to create a stem cell bank that will certainly help in its conservation.
Maned wolf (Chrysocyon brachyurus) is found in South America, but 90% of its specimens are distributed mainly in the Brazilian Cerrado, being the largest South American canid, and as such, it demands large areas. Consequently, maned wolf severely undergoes the effects of habitat loss by human actions. This makes it on the red list by the International Union for Conservation of Nature, as a threatened species of extinction of Brazilian fauna. Objective of this project was to characterize and standardize the isolation, cultivation and cryopreservation of mesenchymal stem cells derived from the bone marrow of maned wolf. In addition to using the fibrin sealant derived from snake venom as a three-dimensional scaffold enabling cellular therapy procedures for the species. A literature review on the species, cell therapy and fibrin sealants were also carried out. For that, the bone marrow was collected from CETAS and zoological animals; culture of mesenchymal stem cells (MSCs); characterization by surface markers (CDs) using flow cytometry; differentiation potential of MSCs into osteogenic, adipogenic and chondrogenic cell lines; analysis of cell viability before and after cryopreservation; and finally, observation and evaluation of the fibrin sealant as cellular scaffold showing its interaction with the MSCs by scanning microscopy and viability by confocal microscopy. The technique used for collection of bone marrow and subsequent culture was efficient, being possible to expand until the sixth passage. Cell surface markers used were able to recognize the cells allowing their characterization by flow cytometry. CTMs presented differentiation potential in osteogenic, adipogenic and chondrogenic linages. Cryopreservation process was effective because it maintained high cell viability after thawing. The interaction with the fibrin sealant was adequate and kept the cells viable as verified by confocal microscopy, demonstrating their three-dimensional structure of the sealant by scanning electron microscopy. Cell therapy may provide for the species an alternative and effective treatment in the agility of regenerative processes, minimizing stress in conventional long-term treatments. Success in cryopreservation of MSCs makes it possible to create a stem cell bank that will certainly help in its conservation.
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Palavras-chave
Biomaterial, Canídeos, Citometro de fluxo, Biomaterial, Canids, Flow cytometer