In this study, we aimed at generating osteogenic and vasculogenic constructs starting from the stromal vascular fraction (SVF) of human adipose tissue as a single cell source. SVF cells from human lipoaspirates were seeded and cultured for 5 days in porous hydroxyapatite scaffolds by alternate perfusion through the scaffold pores, eliminating standard monolayer (two-dimensional [2D]) culture. The resulting cell-scaffold constructs were either enzymatically treated to extract and characterize the cells or subcutaneously implanted in nude mice for 8 weeks to assess the capacity to form bone tissue and blood vessels. SVF cells were also expanded in 2D culture for 5 days and statically loaded in the scaffolds. The SVF yielded 5.9 +/- 3.5 x 10(5) cells per milliliter of lipoaspirate containing both mesenchymal progenitors (5.2% +/- 0.9% fibroblastic colony forming units) and endothelial-lineage cells (54% +/- 6% CD34+/CD31+ cells). After 5 days, the total cell number was 1.8-fold higher in 2D than in three-dimensional (3D) cultures, but the percentage of mesenchymal- and endothelial-lineage cells was similar (i.e., 65%-72% of CD90+ cells and 7%-9% of CD34+/CD31+ cells). After implantation, constructs from both conditions contained blood vessels stained for human CD31 and CD34, functionally connected to the host vasculature. Importantly, constructs generated under 3D perfusion, and not those based on 2D-expanded cells, reproducibly formed bone tissue. In conclusion, direct perfusion of human adipose-derived cells through ceramic scaffolds establishes a 3D culture system for osteoprogenitor and endothelial cells and generates osteogenic-vasculogenic constructs. It remains to be tested whether the presence of endothelial cells accelerates construct vascularization and could thereby enhance implanted cell survival in larger size implants. Disclosure of potential conflicts of interest is found at the end of this article.