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M.M. Pagliara, D. Lepore, P. Netti, F. Molle; 3D model of angiogenesis: study on directionality of GFs effects . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1879.
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Purpose: the goal of this study is to use 3D spheroid model of endothelial cells cultures in order to further simulate in vivo conditions. Using this model we evaluate the ECs capillary sprouts with a controlled directionality under the stimulus of a localized reservoir of a single angiogenic GFs (a single–molecular strategy) or of a combination of GFs (a multi–molecular strategy). Materials and Methods: two types of endothelial cells are used for our study: Human Umbilical Vein Endothelial (HUVE) cells, isolated from human umbilical veins of newborn babies using collagenase digestion, and Pig Aortic Endothelial (PAE) cells cultured at 37° C (5% CO2 , 100% humidity) in HAM’s F12 Media with 10% fetal bovine serum and 1% pen–strep. Alginate beads are used as a localized reservoir of GF. VEGF, bFGF, and TGF–α have been tested. Cells are observed with a confocal microscope directly or with a fluorescent labeling Results: we observed that spheroids embedded in collagen gel with an alginate bead of VEGF sprouted after 18 hours, 6 hours faster than a uniform concentration of VEGF. To determine if the physical presence of the bead affected the sprouting, we suspended spheroids in a collagen well with and without the alginate bead containing BSA. Since no difference in sprouting was found, we concluded that the alginate bead itself does not affect the sprouting. We also observed no effect by doubling the number of beads. Spheroids exposed to a bead containing all three factors remained thriving for up to 15 days, while beads containing only VEGF showed signs of necrosis after 5 days. Wells containing bFGF, either alone or in combination, grew sprouts that branched away from the original direction, aspiring to form capillary networks. Any wells without bFGF contained sprouts that were long and thin, with little branching present. Conclusions: as regards to the single molecular strategy, the GF local gradient seems to be the cause for the increases rapidity of angiogenesis thus indicating the key role of spatial and temporal distribution of GFs. We can also hypothesize a "plateau" behaviour of GFs concentration. As regards to multimolecular strategy, our study confirm the positive influence on sprouting of GFs combination and the key role played by bFGF.
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