Smooth muscle cells (SMCs) and collagen scaffolds are widely used in vascular tissue engineering but their interactions in remodeling at the microscale level remained unclear. We characterized microscale morphologic alterations of collagen remodeled by SMCs in six dimensions: three spatial, time, multi-channel and multi-position dimensions. In live imaging assays, computer-assisted cell tracking showed locomotion characteristics of SMCs; reflection and fluorescent confocal microscopy and spatial reconstruction images of each time point showed detailed morphologic changes of collagen fibers and spatial collagen-SMC interactions. The density of the collagen around the SMCs was changed dynamically by the leading edges of the cells. The density of the collagen following 24 h of cell-induced remodeling increased 51.61 ± 9.73% compared to unremodeled collagen containing cells for 1 h (P < 0.0001, n = 40) (NS vs. collagen without cells). Fast Fourier transform analysis showed that the collagen fibers' orientation changed from random (alignment index = 0.047 ± 0.029, n = 40) after 1 h into concordant with that of the SMCs (alignment index = 0.379 ± 0.098, P < 0.0001, n = 40) after 24 h. Mosaic imaging extended the visual field from a single cell to a group of cells in one image without loss of optical resolution. Direct visualization of alignment of actin fibers and collagen fibers showed the molecular machinery of the process of scaffold remodeling. This is a new approach to better understanding the mechanism of scaffold remodeling and our techniques represent effective tools to investigate the interactions between cells and scaffold in detail at the microscale level.

The development of a functional microvasculature is critical to the long-term survival of implanted tissue-engineered constructs. Dynamic culture conditions have shown to significantly modulate phenotypic characteristics and stimulate proliferation of cells within hydrogel-based tissue engineered blood vessels. Although prior work has described the effects uniaxial or equibiaxial mechanical stimulation has on endothelial cells, no work has outlined effects of threedimensional mechanical stimulation on endothelial cells within tubular vessel analogs. We demonstrate here that 7 days of 10% cyclic volumetric distension has a deleterious effect on the average length and density of angiogenic sprouts derived from pellets of bovine aortic endothelial cells. Although both groups demonstrated lumen formation, the sprouts grown under dynamic culture conditions typically had wider, less-branching sprout patterns. These results suggest that prolonged mechanical stimulation could represent a cue for angiogenic sprouts to preferentially develop larger lumens over cellular migration and subsequent sprout length.

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