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1 School of Fundamental Science and Technology, Keio University, Yokohama, Kanagawa, Japan
2 Department of System Desgin Engineering, Keio University, Yokohama, Kanagawa, Japan
3 Keio Leading-edge Laboratory of Science and Technology, Keio University, Yokohama, Kagnagawa, Japan
4 Department of Mechanical Engineering, Shibaura Institute of Technology, Minato-ku, Tokyo, Japan
* To whom correspondence should be addressed. E-mail: akinori{at}tani.sd.keio.ac.jp.
Shear stress stimulus is expected to enhance angiogenesis, the formation of micro-vessels. We determined the effect of shear stress stimulus on three-dimensional micro-vessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor (bFGF) to make a micro-vessel formation model. We observed this model in detail using phase contrast microscopy, confocal laser scanning microscopy and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 hours. Promotion of micro-vessel network formation was detectable after approximately 10 hours in the flow chamber. After 48 hours, the length of networks exposed to shear stress was 6.17 (± 0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (± 0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of micro-vessel network formation in the gel and expands the network due to repeated bifurcation and elongation.
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