Tese de Doutorado
Avaliação da diferenciação osteogênica de células-tronco do tecido adiposo humano cultivadas em espuma de vidro bioativo e biorreator de perfusão para engenharia de tecido ósseo
Fecha
2012-03-02Autor
Alexandra Rodrigues Pereira da Silva
Institución
Resumen
Tissue Engineering is a multidisciplinary science whose goal is to combine a structural matrix and cells to form a construct able to promote regeneration of injured tissue. Bioactive glass foam (BG) produced by sol-gel is an osteoinductive material that provides a network of interconnected macropores necessary for cell colonization. The use of human adipose stem cells (hASC) present advantages such as easy collecting, large numbers of cells and rapid expansion in vitro, capable of differentiating into osteoblasts. The use of bioreactors (BR) in three-dimensional cell culture enables greater efficiency for cell nutrition and application of mechanical forces, important modulators of bone cell physiology. In this context, the objective of this study is to evaluate the osteogenic differentiation of hASC seeded on bioactive glass foam and cultured in three-dimensional perfusion bioreactor for the production of a functional construct for bone tissue engineering. The synthesis and characterization of BG was initially performed, resulting in an interconnected network with pore size range 100-500m and 88% porosity. Evaluation of static and dynamic degradation and bioactivity of BG foam showed that 0.1 ml/min flow rate was able to maintain the pH of the SBF solution and permeate the sample, and led to deposition of a carbonated hydroxyapatite layer on the sample surface and inner pores. The extraction and characterization of hASC were performed and a new protocol for hASC cultivation in Leibovitz CO2 independent medium (LEI) was developed for use in the perfusion BR. A comparison between hASC grown in standard DMEM and LEI was also performed, showing similar morphological and phenotypic caracteristics and ability to differentiate into osteogenic phenotype when cultured in LEI supplemented with osteogenic factors (LEI O). The static cultivation of hASC seeded on the BG foam in LEI for 7, 14 and 21 days showed a great adaptation of cells to the matrix at 7 days, a peak of alkaline phosphatase activity (ALP) at 14 days in LEI, evidencing the BG osteoinductive action on hASC, which was confirmed by immunofluorescence. Finally, the hASC cultivation on BG in the BR demonstrated a significant increase in cell proliferation and viability at 7 days of culture, ALP activity peak at 14 days, with increased production in LEI group. In addition, immunofluorescence assay revealed expression of osteopontin (OP), osteocalcin (OC) and collagen type I from 7 to 21 days culture, being more evident and strong at 21 days culture in LEI O . Moreover, the cells changed from a spindle form to a cuboidal shape. Immunofluorescence of the group cultivated in LEI at 21 days also showed the expression of these proteins, but with a less intense signal and lower change in cell morphology. The PCR assay confirmed the expression of OP, OC and FA genes expressed by hASC at all cultivation times in LEI O in the perfusion bioreactor. Therefore it can be concluded that hASC seeded in BG and cultivated in BR presented phenotypic change to osteoblastic cells, suggesting this is a promising strategy for cell culture to obtain a functional construct for bone tissue engineering.