Made available in DSpace on 2014-12-17T14:05:20Z (GMT). No. of bitstreams: 1 DeborahAC_TESE.pdf: 2567069 bytes, checksum: 17be3c292d241e19b0a9d1323613899f (MD5) Previous issue date: 2012-04-13 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren?t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient / C?lulas mesenquimais estromais multipotentes, tamb?m conhecidas como c?lulas-tronco mesenquimais humanas (CTMH), s?o c?lulas multipotentes utilizadas em v?rias pesquisas de terapia celular, pois apresentam a capacidade de se diferenciar em m?ltiplas e diferentes linhagens, t?m grande capacidade de autorrenova??o e de expans?o in vitro, excelentes propriedades imunossupressoras e s?o capazes de secretar mol?culas bioativas que exercem efeitos tr?ficos. O cord?o umbilical ? uma fonte de CTMH cuja extra??o n?o necessita de um procedimento invasivo, al?m de n?o envolver controv?rsias ?ticas, pol?ticas e religiosas. Um dos problemas que envolvem linhagens de CTMH de diferentes fontes ? a possibilidade de ocorr?ncia de altera??es cromoss?micas e instabilidade gen?tica, que podem aparecer durante a expans?o in vitro. Al?m disso, as CTMH de um dos cord?es apresentaram uma altera??o cromoss?mica constitucional: invers?o parac?ntrica no bra?o curto do cromossomo 3, cari?tipo: 46,XY,inv(3)(p13p25~26). Em 3p25-26, est?o localizados v?rios genes de grande import?ncia biol?gica, como genes envolvidos com o reparo de DNA e outros respons?veis pelo desenvolvimento de tumores. O tit?nio ? um dos materiais mais utilizado para fabrica??o de implantes ortop?dicos e dent?rios, e ? considerado um excelente biomaterial, entretanto, as part?culas derivadas de pr?teses acumulam-se nos tecidos periprost?ticos e na medula ?ssea local, disseminam-se para linfonodos, f?gado e ba?o. As implica??es biol?gicas em longo prazo da dissemina??o sist?mica de part?culas de metais e seus efeitos cito e genot?xicos n?o est?o bem caracterizados. Neste trabalho investigamos a estabilidade gen?tica de CTMH isoladas da veia do cord?o umbilical durante a expans?o in vitro, ap?s a criopreserva??o, e em diferentes condi??es de cultivo, na presen?a e na aus?ncia de tit?nio, antes e ap?s o aparecimento de c?lulas senescentes no cultivo. As c?lulas foram isoladas, expandidas, diferenciadas em osteoblastos, adip?citos e condroblastos e analisadas com citometria de fluxo para comprovar que s?o c?lulas-tronco mesenquimais. As CTMH foram tratadas com diferentes doses/ tempo de exposi??o ? micropart?culas de tit?nio. A avalia??o da estabilidade gen?tica das CTMH foi realizada atrav?s da an?lise do cari?tipo, de hibrida??o in situ por fluoresc?ncia (FISH) e da an?lise do micron?cleo e outras altera??es nucleares (CBMN). Ficou estabelecido que as CTMH foram capazes de internalizar as micropart?culas de tit?nio, mas as c?lulas mant?m sua capacidade de prolifera??o, diferencia??o e preservam os mesmos marcadores de membrana. Al?m disso, demonstrou-se que existe um aumento na instabilidade gen?tica com o decorrer do tempo de expans?o in vitro, e esta instabilidade foi maior na presen?a de grande concentra??o de micropart?culas de tit?nio que induzem estresse oxidativo. ? necess?rio sempre avaliar os riscos/ benef?cios da utiliza??o do tit?nio na terapia tecidual envolvendo CTMH, considerando a biosseguran?a da utiliza??o da regenera??o ?ssea guiada que utiliza CTMH e tit?nio. Mesmo n?o se utilizando o tit?nio, ? importante que o uso terap?utico de tais c?lulas seja baseado em an?lises que garantam a qualidade, seguran?a e estabilidade celular, com a padroniza??o de programas de controle de qualidade adequados. Como conclus?o, sugere-se que a an?lise citogen?tica, FISH e a an?lise do micron?cleo e outras altera??es nucleares sejam realizadas nas CTMH antes de implantar num paciente, sejam elas cultivadas por longo tempo ou n?o