Angiogenesis is required for endochondral ossification during development and fracture healing; however the exact mechanisms and temporal relationship between the two processes remains unclear. In this study, we utilize an in vivo model of endochondral ossification in mice by implanting demineralized bone matrix (DBM) proximal to the femur to induce ectopic bone formation. TNP-470, a drug known to be anti-angiogenic, was used to inhibit vascularization during the time course of de novo bone formation in order to define the role of angiogenesis during the chondrogenic phase of endochondral bone formation. Day 2, day 8, and day 16 post-surgery were selected time points to represent pre-chondrogenic, chondrogenic, and bone mineralization stages, respectively. Plain x-ray and micro-CT analysis showed that inhibition of angiogenesis led to decreased mineralized tissue formation. Inhibited angiogenesis was confirmed with qRT-PCR. Most striking, however, is that while stem cells are recruited and committed to the chondrogenic lineage, subsequent chondrogenesis failed to progress based on the failure of Sox5 and Sox6 expression, which directs chondrocyte commitment. This expands the role for angiogenesis to a much earlier stage than currently thought and places the necessity of angiogenesis very early in the endochondral ossification process
