Tissue factor and thrombomodulin expression on endothelial cell-seeded collagen modules for tissue engineering

The creation of functional tissue engineering constructs to repair or replace diseased tissues requires a well-formed vasculature network within the construct and the endothelial cells lining that vascular bed must display a nonthrombogenic phenotype. A new approach to tissue engineering involves the assembly of smaller components (modules fabricated at the hundred micron scale) into larger constructs. The modules, collagen gel containing the particular tissue cell of interest, are covered with endothelial cells prior to assembly so that the interconnected channels that are formed are lined with endothelial cells, creating a mimic of a vascular network. Here, we confirmed (using confocal microscopy primarily) that the human umbilical vein endothelial cells, seeded on collagen gel modules without a second embedded cell and without flow, bore the molecular markers of low thrombogenicity. Two days, after seeding on the modules, endothelial cells displayed the typical cobblestone morphology, formed tight cell-cell junctions and covered the whole module surface. Immunofluorescence staining showed that at both 2 days and 7 days after seeding, only a few cells expressed tissue factor while this number was dramatically increased after TNFalpha stimulation. On the other hand, thrombomodulin was expressed by the majority of seeded cells and expression was reduced after TNFalpha stimulation.