Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio

Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of both osteogenic and angiogenic outcome parameters upon two-dimensional and three-dimensional culture conditions. Cultures were performed in four different media: osteoblastic cell proliferation medium, osteogenic medium (OM), endothelial medium, and a 1:1 mixture of the latter two media. Mineralization within the cocultures was observed only in OM. Subsequent experiments in OM showed that alkaline phosphatase activity, mineralization, and CD31(+) staining were highest for cocultures at a 50:50 HMSC/HUVEC ratio. Therefore, the results from the present study show that a HMSC/HUVEC coculture ratio of 50:50 in OM is the best combination to obtain both osteogenic and angiogenic differentiation.