Quantitation of angiogenesis in the chick chorioallantoic membrane model using fractal analysis

Objective quantitation of new vascular growth in angiogenesis assays has been difficult to achieve. The aim of this study was to test the potential applicability of fractal geometry to objectively quantify angiogenesis in the chick chorioallantoic membrane (CAM). To accomplish this, vascular growth in normal (NC) and tumor grafted (TGC) CAM was compared. Samples of human tumors of various origins acquired from biopsy specimens were grafted to CAMs at Days 6-12 of incubation. Images of 6- to 16-day NC and 10- to 17-day TGC were acquired and the box-counting method was used to estimate fractal dimension (Df). Vascular density (rho v) was derived from box counts. For NC, Df and rho v increased steadily from Days 6 to 14, peaked, and began to decrease by Day 15. Results from TGC showed that Df and rho v surrounding tumor grafts varied from those of NC. Complexity and density indices were calculated as amount of change in Df and rho v between TGC and NC of the same age, respectively. Based on these indices, differences in the angiogenic response elicited by various tumors were readily detected. Our results suggest that differences in Df and rho v, in both the temporal and spatial senses, are accurate and repeatable measures of vascular growth and may be useful for objective quantitation of the angiogenic response.