Potencial Osteogénico das células do estroma derivadas do tecido adiposo de diferentes localizações anatómicas do rato
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2019-02-15
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As células estromais derivadas do tecido adiposo (ADSCs) possuem a capacidade de se autorenovarem e o potencial de se diferenciarem em diferentes linhagens, incluindo a linhagem osteoblástica. Além disso, estas células podem ser facilmente obtidas a partir de procedimentos cirúrgicos minimamente invasivos, quando comparadas com as células estromais provenientes de outras fontes, nomeadamente da medula óssea. Estas características tornas as ADSCs atrativas para serem aplicadas na área de engenharia de tecidos, com ênfase particular nas estratégias regenerativas do tecido ósseo. Embora existam estudos acerca do impacto dos procedimentos de recolha e isolamento das ADSCs, pouca atenção tem sido dada às diferenças de localização anatómicas verificadas dentro do tecido adiposo, bem como o impacto dessas diferenças na atividade funcional das ADSCs. Posto isto, o objetivo deste trabalho experimental foi o isolamento e a caracterização biológica, em particular do potencial de diferenciação osteogénica das ADSCs, de diferentes localizações anatómicas, nomeadamente, periabdominal, subcutânea, perirenal, pericoronária, mesentérica e do tecido adiposo castanho, usando a rato como modelo experimental. O tecido adiposo foi recolhido das diferentes localizações anatómicas sendo estabelecidas as culturas de células estromais, através de um processo de dissociação enzimática. As culturas obtidas foram mantidas em condições basais e induzidas na linhagem osteogénica, através da adição de indutores comumente utilizados, sendo as culturas caracterizadas, em diferentes períodos, tendo em conta o seu potencial de diferenciação através de técnicas bioquímicas, histoquímicas e expressão genética. Os resultados obtidos mostraram que as culturas estabelecidas a partir das diferentes localizações anatómicas revelaram um comportamento biológico distinto, relativamente à morfologia, proliferação e atividade funcional. Comparativamente, o tecido adiposo periabdominal e subcutâneo apresentam valores mais elevados de marcadores de diferenciação osteogénica expressos numa fase precoce, como demonstrado pela expressão mais elevada do Runx2 e Col1a1. Por outro lado, o tecido adiposo perirenal apresenta valores mais elevados de marcadores de diferenciação osteogénica expressos numa fase mais tardia/avançada (i.e., Osterix e osteoclacina). Em termos gerais, e dadas as diferenças funcionais observadas nas ADSCs cultivadas a partir de diferentes localizações anatómicas, parece-nos que será fundamental adequar as zonas anatómicas do tecido adiposo às necessidades terapêuticas de diferentes abordagens regenerativas, de forma a que as populações celulares com maior potencial sejam utilizadas na otimização do processo de regeneração óssea.
Adipose-derived stromal cells (ADSCs) have self-renewal capacity and the potential to differentiate in different lineages, including osteoblast lineages. Additionally, these cells can be easily harvested trough a minimal invasive surgical procedure, as comparing to other sources, such as the bone marrow. These characteristics make ADSCs appealing for application in tissue engineering, with special interest for bone tissue engineering. While widespread data is available on the impact of fat tissue harvest and cell isolation procedures for adequate ADSCs functionality, little attention has been given to the differences anatomical locations verified within the fat tissue, as well as the impact of these differences on the functional activity of ADSCs. Accordingly, this experimental work aimed the isolation and biological characterization, in special the osteogenic potential of rat ADSCs from different anatomical locations, i.e., periabdominal, subcutaneous, perirenal, peri/epicardial, mesenteric, brown adipose tissue. The adipose tissue was isolated and the stromal cell culture was established through an enzymatic dissociation process. The obtained cultures were maintained in either basal or osteogenic conditions. The cultures were characterized at different periods, to evaluate the differentiation potential by biochemical and histochemical techniques and genetic expression. The results obtained showed that, established culture from the different anatomical locations revealed a distinct biological behavior with respect to morphology, proliferation and functional activity. Periabdominal and subcutaneous adipose tissue expressed higher levels of osteoblastic differentiation markers in an early stage, as demonstrated by the higher expression of Runx2 and Col1a1. On the other hand, perirenal adipose tissue presented higher expression of osteogenic differentiation markers, in a later stage (i.e., Osterix e osteoclacin). Broadly speaking, functional differences were observed in the ADSCs cultured from different anatomical groups. Therefor it seems to be fundamental adequate the anatomical zones of the adipose tissue to the therapeutic needs of different regenerative approaches, in a way that the cellular population with the most potential can be applied in bone regeneration.
Adipose-derived stromal cells (ADSCs) have self-renewal capacity and the potential to differentiate in different lineages, including osteoblast lineages. Additionally, these cells can be easily harvested trough a minimal invasive surgical procedure, as comparing to other sources, such as the bone marrow. These characteristics make ADSCs appealing for application in tissue engineering, with special interest for bone tissue engineering. While widespread data is available on the impact of fat tissue harvest and cell isolation procedures for adequate ADSCs functionality, little attention has been given to the differences anatomical locations verified within the fat tissue, as well as the impact of these differences on the functional activity of ADSCs. Accordingly, this experimental work aimed the isolation and biological characterization, in special the osteogenic potential of rat ADSCs from different anatomical locations, i.e., periabdominal, subcutaneous, perirenal, peri/epicardial, mesenteric, brown adipose tissue. The adipose tissue was isolated and the stromal cell culture was established through an enzymatic dissociation process. The obtained cultures were maintained in either basal or osteogenic conditions. The cultures were characterized at different periods, to evaluate the differentiation potential by biochemical and histochemical techniques and genetic expression. The results obtained showed that, established culture from the different anatomical locations revealed a distinct biological behavior with respect to morphology, proliferation and functional activity. Periabdominal and subcutaneous adipose tissue expressed higher levels of osteoblastic differentiation markers in an early stage, as demonstrated by the higher expression of Runx2 and Col1a1. On the other hand, perirenal adipose tissue presented higher expression of osteogenic differentiation markers, in a later stage (i.e., Osterix e osteoclacin). Broadly speaking, functional differences were observed in the ADSCs cultured from different anatomical groups. Therefor it seems to be fundamental adequate the anatomical zones of the adipose tissue to the therapeutic needs of different regenerative approaches, in a way that the cellular population with the most potential can be applied in bone regeneration.
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Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde