Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis

Background: A dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation. Methods: Early osteogenic activities of the co-cultures (+/- VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (mu CT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31. Results: ALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs. Conclusion: These studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.