Coculture of peripheral blood-derived mesenchymal stem cells and endothelial progenitor cells on strontium-doped calcium polyphosphate scaffolds to generate vascularized engineered bone.

Abstract

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.