Assessing angiogenesis during fracture healing.

Angiogenesis, the sprouting of new capillaries from existing blood vessels, is crucial for normal fracture healing. Angiogenesis is a complex process involving a variety of growth factors and several cell types. The mechanism regulating angiogenesis during fracture repair is not well understood, and the relationships between angiogenesis, chondrogenesis, and osteogenesis are also undefined. In vivo animal models have been useful for determining angiogenic mechanisms. In particular, a murine model has been developed that offers the advantages of easy animal handling, low cost, reliable healing, and the availability of molecular and genetic techniques for research. However, the small size of mice provides challenges, including the inability to assess vascularization using techniques that have been employed in larger animals. Therefore, we developed and optimized techniques specifically for studying angiogenesis during mouse fracture repair. These techniques include blood vessel casting, micro-computed tomography (micro-CT), immunohistochemistry, in situ hybridization, and genetic labeling of endothelial cells. Blood vessel casting and micro-CT are useful for visualization of small blood vessels. Immunohistochemistry using anti-PECAM (platelet endothelial cell adhesion molecule) or CD34 antibodies and genetic approaches using Tie2-cre transgenic mice can be used to label endothelial cells, visualize blood vessels including capillaries, and provide structural information about the vascularization of the fracture callous. Lastly, expression patterns of important growth factors regulating angiogenesis could be assessed by molecular approaches such as in situ hybridization.