cell bodies as well as their arrangement in colonies [18, 22]. Here,

we used rhodamine conjugated phalloidin according to the manu-

facturer’s manual for phallotoxins to characterize the colonized

scaffolds with respect to potentially formed endothelial layers.

1. Prepare the rhodamine staining solution freshly (0.1% rhoda-

mine phalloidin supplied stock diluted in PBS containing 1%

BSA and one drop per ml NucBlue reagent). The staining

Fig. 3 Exemplary images of rabbit endothelial progenitor cells (rbEPC) and human saphenous vein endothelial

cells (HSVEC) cultivated for 96 h on fibronectin coated bacterial nanocellulose vascular grafts. By histological

examination of the cell constructs, we found highly colonized areas with apparently preserved endothelial

functionality as indicated by AcLDL uptake. (a) Confocal microscopical image of CD31 stained HSVEC

indicating tight cell-cell junctions. (b) Confocal microscopical image showing the merge of AcLDL uptake

assay (green), Phalloidin (red), and NucBlue (blue) of rbEPC, indicating that the cells were functional intact and

expressed a distinct cytoskeleton. (c) Conventional fluorescence microscopy image of rbEPC after subjection

to AcLDL uptake assay. The presence of intracellular green AcLDL particles reflects a functional state of the

endothelial cells. (d) 3D-reconstruction of confocal microscopical image stack, displayed is the merge of

AcLDL uptake assay (green), Phalloidin (red), NucBlue (blue) of rbEPC. By 3D reconstruction, the morphology of

the EC layer can be estimated i.e., by the thickness and arrangement of stress fibers in the cytoskeleton. (e, f)

Images of acridine orange staining of rbEPC. (e) Magnification of whole cross-section (see rectangular region

of interest in f). (f) Cross-section through fibronectin coated BNC vascular graft. The acridine orange staining

allows to detect an overall graphical impression of the cell distribution on the luminal side of the graft, showing

more and less confluent areas

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