Study On Construction Of Osteogenic And Vascular Endothelial Cell Sheets From Rat Adipose-derived Mesenchymal Stem Cells

Objective Two kinds of cell sheets with the potential abilities of osteogenesis and blood vessels formation derived from ADSCs were obtained by induced culture in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defect.Methods ADSCs were isolated from epididymis and dorsal adipose tissues of Sprague Dawley(SD) rats. Flow cytometry identificated the expression of surface marker, such as CD29, CD44, CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by oil red o staining, alkaline phosphatase(ALP) staining, alizarin red, vonkossa and alcian blue staining, respectively. ADSCs with high density(1x10~6/cm~2) were cultured in the osteogenic medium(contain 50?g/ml ascorbic acid). The forming process of cell sheet was observed and cell sheet was characterized by SEM, TEM and vonkossa staining. ADSCs were cultured in endothelial cell growth medium(EGM-2) containing 50ng/ml VEGF. After 14-21 d, flow cytometry was used to identify the expression of surface marker, such as CD29, CD44, CD31 and CD45. Matrigel matrix analyzed the function of blood vessel formation in vitro. ADSCs with high density(1x10~6/cm~2) were cultured in EGM-2 medium containing 50?g/ml ascorbic acid and 50ng/ml VEGF. The forming process of cell sheet was observed and cell sheet was characterized by H&E staining?TEM and immunofluorescence staining.Results The primary generation of ADSCs was short spindle and polygon, and the cells became uniform long spindle shapes after three generations. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD34. Adipogenic, osteogenic and chondrogenic differentiation were confirmed by the results of positive oil red o staining, alkaline phosphatase staining, alizarin red, vonkossa and alcian blue staining, respectively. ADSCs with high density(1x10~6/cm~2) were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 days. H&E staining image showed that the osteogenic cell sheet consisted of multilayer cells and abundant extracellular matrix. The cell sheet was positive with vonkossa staining. The TEM image showed that needle-like calcium salt crystals were deposited on the extracellular matrix. SEM image confirmed that spindle-shaped extracellular matrix and plenty of mineralized nodules were formed on surface of the cell sheet. These results suggested that osteogenic cell sheet had the potential osteogenesis ability. ADSCs with high density(1x10~6/cm~2) were cultured in the endothelial cell growth medium-2(EGM-2) containing 50?g/ml ascorbic acid and 50ng/ml VEGF. The cell sheet could be obtained after 16 days. The H&E staining showed that the endothelial cell sheet consisted of multilayer cells and a large number of ECM. The TEM image confirmed that the Weibel-Palade corpuscle which was the special structure of endothelial cell was seen in the cell. The expression of the specific endothelial marker CD31 was positive. These results suggested the endothelial cell sheet had a potential ability to form blood vessels.