Coculture of Peripheral Blood-Derived Mesenchymal Stem Cells and Endothelial Progenitor Cells on Strontium-Doped Calcium Polyphosphate Scaffolds to Generate Vascularized Engineered Bone Wei-Li Fu, MD, PhD, 1 Zhou Xiang, MD, PhD, 1 Fu-Guo Huang, MD, 1 Zhi-Peng Gu, PhD, 2 Xi-Xun Yu, PhD, 2 Shi-Qiang Cen, MD, PhD, 1 Gang Zhong, MD, PhD, 1 Xin Duan, MD, PhD, 1 and Ming Liu, MD, PhD 1 Vascularization of engineered bone tissue is critical for ensuring its survival after implantation and it is the primary factor limiting its clinical use. A promising approach is to prevascularize bone grafts in vitro using endothelial progenitor cells (EPC) derived from peripheral blood. Typically, EPC are added together with mesenchymal stem cells (MSC) that differentiate into osteoblasts. One problem with this approach is how to promote traditional tissue engineering bone survival with a minimally invasive method. In this study, we examined the effectiveness of administering to stimulate the release of peripheral blood stem cells and their co- culturing system for generating prevascularized engineered bone. Cells were isolated by Ficoll density gradient centrifugation and identified as EPC and MSC based on morphology, surface markers, and functional analysis. EPC and MSC were cocultured in several different ratios, and cell morphology and tube formation were assessed by microscopy. Expression of osteogenesis and vascularization markers was quantified by enzyme- linked immunosorbent assay (ELISA), polymerase chain reaction, and histochemical and immunofluorescence staining. Increasing the proportion of EPC in the coculture system led to greater tube formation and greater expression of the endothelial cell marker CD31. An EPC:MSC ratio of 75:25 gave the highest expression of osteogenesis and angiogenesis markers. Cocultures adhered to a three-dimensional scaffold of strontium-doped calcium polyphosphate and proliferated well. Our findings show that coculturing peripheral blood-derived EPC and MSC may prove useful for generating prevascularized bone tissue for clinical use. Introduction B iomedical engineering of bone holds promise for treating large bone defects or nonunion. 1,2 Perhaps the greatest challenge in the field of bone regeneration is en- gineering bone composites that survive in vivo for long periods and integrate well at the site of repair. Traditional composites, comprising a scaffold seeded with mesenchy- mal stem cells (MSC), often show poor survival after im- plantation. This is due, in part, to necrosis in interior regions due to lack of vascularization and, in part, to the rapid death of MSC, with up to 99% of cells dying within a few days. 3 Engineered bone tissue usually relies on passive oxygen and nutrient diffusion, for which the effective distance is limited to 100–200 mm, making it likely that the tissue will die because of lack of oxygen and nutrients and insufficient waste removal. 4 Native bone does not suffer this problem because it is a highly vascularized tissue, in which osteo- blasts interact with blood vessels to maintain the metabolism and homeostasis of the local microenvironment. 5 In fact, interaction of osteoclasts and endothelial cells is critical for osteogenesis during embryonic development as well as for bone repair and regeneration after injury. To mimic these processes of natural bone, researchers have begun to explore ways to engineer vascularized bone composites based on the coculture of osteogenic and endo- thelial cell lines. 6–9 These cells are typically adult stem or progenitor cells because they can be isolated from various sources and show greater proliferative capacity and differ- entiation potential than terminally differentiated cells. The osteogenic cells used in vascularized bone tissue are usually MSC derived from the mesoderm. These cells are particularly appealing in bone regeneration studies because of their properties of self-renewal, multipotent differentia- tion, homing, and immunomodulatory activity. 10–12 Of the numerous tissues that contain MSC, 13 peripheral blood may 1 Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu, P.R. China. 2 College of Polymer Science and Engineering, Sichuan University, Chengdu, P.R. China. TISSUE ENGINEERING: Part A Volume 21, Numbers 5 and 6, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2014.0267 948