Alignment of angiogenic microvessels and the surrounding collagen matrix under anchored boundary conditions

Abstract

Previously we have observed an alignment of newly formed microvessels when 3D collagen gel cultures were axially anchored to their culture chamber . In contrast, microvessels in free‐floating gels exhibit a random orientation. The objective of this study was to examine changes in collagen alignment that occur during microvessel outgrowth over time in free‐floating and anchored 3D cultures. Microvessel cultures were fixed after 1 to 6 days in culture and endothelial cells were stained with Isolectin IB4‐Alexa 488. 3D image stacks of microvessels and collagen fibrils were obtained using confocal and confocal reflection microscopy, respectively. Microvessel and collagen fibril organization were quantified using standard image processing algorithms. There was significant alignment of vessels in anchored cultures, starting at 3‐4 days in culture. Similarly, the organization of collagen went from a random organization (anisotropy < 0.1) to a progressively more aligned orientation (anisotropy > 0.25) in a direction consistent with the microvessels. These results suggest that the orientation of microvessel outgrowth and matrix realignment follow similar patterns during angiogenesis. Although these events were highly correlated, it is unclear which event leads in the process of alignment.