The role of angiogenesis in a murine tibial model of distraction osteogenesis.

Distraction osteogenesis (DO) is one of the most dramatic in vivo applications of mechanical stimulation as a means of inducing bone regeneration. A simple and reproducible murine model of tibia distraction osteogenesis was developed using a monolateral fixator. Bone formation was assessed histologically over a 35-day time course. The steady state expression of a broad family of angiogenesis-associated genes was assessed by microarray hybridization analyses over the same time course, while the immediate gene response that was induced during each cycle of distraction was assessed at 30 min and 8 h after the first and last rounds of activation of the fixator. Distraction osteogenesis promoted new bone formation primarily through an intramembranous process with maximal osteogenesis during the active distraction period. Histological analysis also showed that dense cortical bone continued to be formed, during the consolidation phase, for 2 weeks after distraction ended. The analysis of steady state mRNA expression levels over the time course of DO showed that VEGF-A and neuropilin, an alternate receptor for VEGF-A, both angiopoietin (Ang) 1 and 2 factors, and the Ang receptor Tie2 were the critical angiogenic factors during DO. A key transcriptional regulator of many of the angiogenic factors, hypoxia-induced factor1alpha (Hif-1a), the FGF binding protein pleiotropin/OSF1, and multiple MMP(s), were also induced during the active distraction period. Examination of the expression of angiogenic factors that were induced after each cycle of activation, demonstrated that Hif-1a, Nrp1, and VEGF-A were all cyclically induced after each increment of distraction. These results suggest that these factors are early mediators that are produced by distraction and contribute toward the processes that promote bone formation. These experiments represent the first step in defining the molecular mechanisms that regulate skeletal regeneration and the functional relationship between angiogenesis and osteogenesis during distraction osteogenesis.