Earliest phases of chondrogenesis are dependent upon angiogenesis during ectopic bone formation in mice

Endochondral ossification is the process where cartilage forms prior to ossification and in which new bone forms during both fracture healing and ectopic bone formation. Transitioning to ossification is a highly coordinated process between hypertrophic chondrocytes, vascular endothelial cells, osteoblasts and osteoclasts. A critical biological process that is central to the interactions of these various cell types is angiogenesis. Although it is well established that angiogenesis is crucial for fracture repair, less is known pertaining to the role of angiogenesis in ectopic bone formation. Furthermore, fracture repair models are complicated by extensive trauma, subsequent inflammatory responses and concurrent repair processes in multiple tissues. In order to more definitively characterize the relationship between angiogenesis and postnatal endochondral ossification, a model of ectopic bone formation was used. Human demineralized bone matrix (DBM) was implanted in immune-deficient mice (rag null (B6.129S7-Rag1^tm1/MOM/J)) to induce ectopic bone. Inhibition of angiogenesis with either a small molecule (TNP-470) or a targeted biological (Vascular Endothelial Growth Factor Receptor type 2 [VEGFR2] blocking antibody) prevented ectopic bone formation by 83% and 77%, respectively. Most striking was that the progression of chondrogenesis was halted during very early phases of chondrocyte differentiation between condensation and prehypertrophy (TNP-470) or the proliferative phase (VEGFR2 blockade) prior to hypertrophy, while osteoclast recruitment and resorption were almost completely inhibited. Our results demonstrate angiogenesis plays a developmental role in endochondral bone formation at a much earlier phase of chondrogenesis than suggested by prior findings.