Background Angiogenesis, the process of new vessel formation from a pre-existing vascular network, is essential for bone development and repair. New vessel formation and microvascular functions are crucial during bone repair, not only for sufficient nutrient supply, transport of macromolecules and invading cells, but also because they govern the metabolic microenvironment. Despite its central role, very little is known about the initial processes of vessel formation and microvascular function during bone repair.Methods To visualize and quantify the process of vessel formation and microvascular function during bone repair, we transplanted neonatal femora with a substantial defect into dorsal skin-fold chambers in severe combined immunodeficient (SCID) mice for continuous noninvasive in-vivo evaluation. We employed intravital microscopic techniques to monitor effective microvascular permeability, functional vascular density, blood flow rate and leukocyte flux repeatedly over 16 days. Oxytetracyclin and v. Kossa/v. Giesson staining was performed to quantify the calcification process in vivo and in vitro.Results Development of a hematoma surrounding the defect area was the initial event, which was accompanied by a significant increase in microvascular permeability and blood flow rate. With absorption of the hematoma and vessel maturation, permeability decreased continuously, while vascular density and tissue perfusion increased. Histological evaluation revealed that the remodeling of the substantial defect prolonged the invivo monitored calcification process.

Angiogenesis is essential for wound healing and proliferative processes such as bone formation and repair. Since increased expression of the vascular endothelial growth factor (VEGF) stimulates bone formation, it can be hypothesized that surgical procedures leading to a systemic increase of VEGF for instance during wound healing, influence enchondral ossification processes and might be responsible for observed growth phenomena during callus distraction. To study the mechanisms of angiogenesis in soft tissue during unilateral callus distraction, lengthening of the right tibia was performed in 12 beagles. After osteotomy, application of a ring fixator and after five latency days, distraction was started for 25 days. A control group of four additional beagles underwent no surgical procedure. Subsequent to the distraction period (Group A), muscle samples from six beagles were taken from the distracted side (ds) and the contralateral non-distracted side (n-ds), six beagles underwent an additional consolidation period of 25 days (Group B). Samples were analyzed for VEGF, VEGFR-1 and VEGFR-2 mRNA expression using real-time PCR and protein expression using Western Blot analysis. Muscles from both extremities showed significantly increased expression of VEGF and its cognate receptors VEGFR-112. Expression decreased significantly after the consolidation period, whereby the level at the nondistracted side decreased more than the level at the distracted side. Interestingly VEGF and VEGFR-I levels at the non-distracted side were significantly higher than at the distracted side. In contrast VEGFR-2, the receptor that mediates endothelial cell proliferation, showed higher levels at the distracted than at the non-distracted side. These findings indicate that callus distraction results not only in locally increased expression of VEGF and its receptors, but leads also to increased VEGF and VEGFR-112 levels at distant sides and might therefore be responsible for the observed growth phenomena during callus distraction.

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