Establishment of an Early Vascular Network Promotes the Formation of Ectopic Bone

Vascularization is crucial for the induction of bone formation. In this study, we investigated the application of two subtypes of peripheral blood-derived endothelial progenitor cells (EPCs) to stimulate vessel formation in ectopic bone constructs. Early and late outgrowth EPCs (E-EPC and L-EPC, respectively) were characterized for their ability to form network structures in vitro and perfused vessels subcutaneously in mice. Only L-EPCs showed the formation of fully connected networks on Matrigel two-dimensional (2D) angiogenesis assays. The presence of multipotent stromal cells (MSCs) inhibited network formation in 2D assays, but stimulated network formation in three-dimensional plugs. In vivo studies revealed that at 2 weeks, the highest incidence of formed perfused vessels was reached by implanted E-EPC/MSC constructs and this could be attributed to the presence of E-EPCs. L-EPCs displayed a significantly lower frequency of blood vessel formation than E-EPCs and this was accompanied by a lowering of total luminal area densities. Nevertheless, combined E-EPC/L-EPC application somewhat increased the percentage incidence of perfused vessels. After 6 weeks, differences in vascularization were still obvious as all three EPC-based constructs contained higher numbers of perfused vessels than constructs containing MSCs alone. Bone was formed in all constructs at an incidence that coincided with high density of perfused vessels after 2 weeks. Altogether, our findings suggest the differential establishment of vascular networks by E-EPCs and L-EPCs and suggest the importance of early vasculogenesis in ectopic bone formation.