Simultaneous Cultivation of Human Endothelial-like Differentiated Precursor Cells and Human Marrow Stromal Cells on β-Tricalcium Phosphate

Aim: The size of a bone defect limits the ingrowth of bone-forming cells. Endothelial cell-like differentiated precursor cells (endothelial progenitor cells, EPC) enhance the neovascularization, while marrow stromal cells (MSC) promote the repair of bone defects. Our aim was to evaluate if both types of cells can be cocultivated on a beta-tricalcium phosphate (beta-TCP) matrix and maintain their differentiation capacity as well as to analyze the biologic activity of these cell constructs in vivo. Methods: MSC from human bone marrow and EPC from buffy coat were used. EPC and MSC, alone or in combination, were seeded on fibronectin-coated beta-TCP. After 2, 6, and 10 days the metabolic activity and the endothelial differentiation were tested. On day 10 real-time RT-PCRs for endothelial genes (von Willebrandt factor, vascular endothelial growth factor, and vascular endothelial growth factor-receptor 2), osteogenic genes (osteocalcin, cbfa-1, and collagen-1 alpha), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were performed. Cell-containing constructs were implanted into the critical-size defect of the femur of the nude rat. Bone formation and vascularization was determined after 1 week. Results: MSC and EPC on beta-TCP remain metabolically active over 10 days. They maintain their differentiation as measured by means of Dil-ac-LDL uptake (EPC) and gene expression of lineage typical genes (EPC+MSC). Although a potential osteogenic differentiation of MSC was maybe affected negatively, constructs loaded with MSC resulted in an increase of new bone mass. Constructs containing EPC resulted in an improved vasculogenesis in vivo. Discussion: MSC and EPC can be cultivated in combination on a fibronectin-coated beta-TCP, thereby partly maintaining their lineage typical gene expression. The results of the in vivo examinations suggest that beta-TCP combined with EPC and MSC can used as a suitable tool to foster bone healing.