Improved in vitro cultivation of endothelial progenitor cells as basis for dermal substitutes with enhanced angiogenic capabilities.

PURPOSE

Endothelial progenitor cells (EPCs) are a promising cell source for vascular tissue engineering approaches in surgery. Diverse biomaterials have been used as matrices for EPC cultivation. In this in vitro study, fibrin in combination with growth factors was examined as an optimized culturing scaffold for EPCs.

METHODS

EPCs were isolated from peripheral blood by density gradient centrifugation and positive selection using CD34-specific magnetic beads. Cells were seeded on fibrin for 3, 5, and 10 days or on fibronectin for control purposes. The growth factors erythropoietin (EPO), granulocyte-monocyte colony-stimulating factor (GM-CSF), and hepatocyte growth factor were added as indicated. Cell proliferation and integrity measurements were performed, and EPC differentiation was determined by fluorescence-activated cell sorting (FACS) analysis.

RESULTS

EPCs cultured on fibrin showed a significantly higher proliferation rate and suffered less from matrix-induced cell death compared to EPCs cultured on fibronectin. Additionally, fibrin was a stronger stimulus for the differentiation of EPCs into a mature endothelial phenotype, as revealed by the expression of adult endothelial markers by FACS analysis. Moreover, EPO and GM-CSF enhanced EPC proliferation and differentiation to a greater extent when EPCs were grown on fibrin.

CONCLUSIONS

We conclude that EPC cultivation on fibrin is superior compared to the commonly used fibronectin as a scaffold for tissue engineering of vascular structures. The addition of different growth factors, reported to stimulate EPC growth, further improves the beneficial effects of this matrix.