Effect of fibrin on osteogenic differentiation and VEGF expression of bone marrow stromal cells in mineralised scaffolds: a three-dimensional analysis.

The aim of the present study was to test the hypothesis that a fibrin matrix enhances the osteogenic differentiation and expression of vascular endothelial growth factor (VEGF) by human bone marrow stromal cells (hBMSCs) seeded into mineralised scaffolds. Porous calcium carbonate scaffolds were droplet seeded with hBMSCs using a matrix containing 3 % fibrinogen and cultured for 3 weeks. Seeded scaffolds without the fibrin matrix served as controls. The scaffolds were evaluated, using undecalcified thick sections, for fluorescence staining for nuclei, osteocalcin (OC) and VEGF. The sections were systematically scanned using optical sectioning and three dimensional distributions of cells and positive staining indicating expression of OC and VEGF were reconstructed from the z-stacks. The fibrin matrix maintained a significantly higher level of cell numbers after 2 d and 1 week and delayed the onset of osteogenic differentiation while sustaining a significantly higher level of OC and VEGF expression after 2 and 3 weeks, starting from the periphery of the scaffolds. There was a decrease in cell density from the periphery to the centre of the scaffolds in both groups The percentage of cells expressing OC and VEGF was significantly different between the centre and the periphery of the scaffolds in the fibrin(+) group but not in the controls. It is concluded that the fibrin matrix used appears to be a useful adjunct for supporting and sustaining osteogenic and angiogenic activity of hBMSCs in tissue engineered constructs. This could help to improve their performance in a clinical setting.