Assessment of angiogenic properties of biomaterials using the chicken embryo chorioallantoic membrane assay

The angiogenic potential of a biomaterial is a critical factor for successful graft intake in tissue engineering. We developed a modified, rapid and reproducible chicken embryo chorioallantoic membrane ( CAM) assay to evaluate the ability of biomaterials in inducing blood vessel density. Five biomaterials including one-layer porcine small intestinal submucosa ( SIS), two-layer SIS, four-layer vacuum pressed (VP) SIS, polyglycolic acid ( PGA) and PGA modified with poly( lactic-co-glycolic acid) ( PLGA) were analyzed. A circular section ( 1.2 mm diameter) of each biomaterial was placed near a group of blood vessels in the CAM. Blood vessels around the biomaterials were captured with black and white images at 96 h post implantation; and the images were subjected to densitometry evaluation. One-layer SIS induced a significant increase in blood vessel density as compared to the cellulose nitrate negative control, and had the greatest increase in blood vessel density as compared to four-layer VP SIS, PGA, or PLGA modified PGA. Although two-layer SIS has enhanced physical structure for surgical manipulation, its induction in blood vessel density was significantly lower than the one-layer SIS. Stripping the SIS proteins or incubating one-layer SIS with neutralizing antibodies against basic fibroblast growth factor ( bFGF) or vascular endothelial growth factor ( VEGF) resulted in decreased angiogenesis. Consistent with results obtained from bladder augmentation animal models, these results confirmed that angiogenic growth factors were present in SIS and affected the angiogenic potential of biomaterials. These data also demonstrated that the CAM assay can be used to ascertain methodically the angiogenic potential of biomaterials.