SUMMARY:Fetal mouse metatarsals are well-known models to study cartilage differentiation and osteoclastic resorption. We show here the outgrowth of PECAM-1 positive tubelike structures from the bone rudiments. This feature can be used to study angiogenesis in vitro. The area of outgrowth significantly increased with culture time, as shown by computerized image analysis of PECAM-1 positive tubelike structures. Treatment with recombinant human vascular endothelial growth factor (rhVEGF-A) stimulated the formation of tubelike structures. Treatment of explants with the angiogenesis inhibitor endostatin, the chemokine IP-10, and the thalidomide derivative phatolyl glutamic acid (PG-acid) resulted in an inhibition of the formation of PECAM-1 positive tubelike structures of 48.8% (Ϯ 4%), 50.2% (Ϯ12%), and 80.8% (Ϯ3%), respectively. Outgrowth of tubelike structures was partly dependent on endogenous VEGF-A because treatment with anti-mVEGF-A and truncated VEGF receptor 1 (soluble fms-like tyrosine kinase 1, sFlt1) strongly inhibited the formation of tubelike structures 74% (Ϯ 4%) and 38% (Ϯ 5%), respectively. Neither onset of tube formation nor total area of tubelike structures were changed when metatarsals were cultured on a fibrin gel or collagen type I gel. Tube formation required activation of matrix metalloproteinases because treatment of the bones with an inhibitor of matrix metalloproteinases completely inhibited migration and tube formation, whereas treatment with an inhibitor of plasmin had no effect. In conclusion, we describe a new in vitro model to study angiogenesis that can be used to test the angiogenic or antiangiogenic potential of novel test compounds that also combines the multicellularity of in vivo assays with the accessibility and flexibility of in vitro assays. (Lab Invest 2001, 81:5-15).