Precise Quantification of Angiogenesis in 3D Biomaterials Using in Vitro CAM Models: Enhancing 3Rs in Research and Minimizing in Vivo Dependency

Abstract The efficacy of tissue engineering applications depends on selecting a scaffold with angiogenic properties, as angiogenesis plays a pivotal role in delivering nutrients and oxygen to cells for proper function. This study employed the chorioallantoic membrane (CAM) assay as a 3R‐compliant model to investigate angiogenesis within an elastin‐fibrin scaffold. Angiogenesis was evaluated in 2D using ImageJ software and in 3D using Micro‐CT and confocal microscopy, respectively, following the intravascular injection of the contrast agents Indocyanine Green (ICG) and Microfil to quantify the vascular volume. Furthermore, we compared CAM analysis with in vivo vascularization using Optiray 350, an iodine‐based contrast agent. Our findings highlight the limitations of 2D quantification and identify significant differences in vascular volume quantification between Microfil and ICG ( p < 0.05). Specifically, ICG is distributed uniformly throughout the vascular network, unlike Microfil and Optiray 350, which have limited penetration in the microvessels within scaffolds. Our findings present a refined method for high‐resolution, 3D visualization and quantification of vascular networks within biomaterials by CAM. This approach enables rapid assessment of angiogenic potential, thereby significantly reducing reliance on animal testing. By supporting the principles of the 3Rs, this strategy offers a more ethical and scalable framework for preclinical biomaterial evaluation.