Control of angiogenesis is a major challenge to promotion of vascularization in the field of tissue engineering. In particular, shear stress is recognized as an important mechanical factor controlling new vessel formation. However, the effects of steady and pulsatile shear stress on endothelial cell (EC) network formation remain unclear. Here, we systematically investigated their effects. Compared with pulsatile shear stress, steady shear stress at 1.0 Pa increased cell numbers in EC networks as well as the distribution of networks and pseudopodia in the deep range after 48 h. To further investigate the process of EC network growth, we focused on the effect of flow frequency on network elongation dynamics. Pulsatile shear stress at 1.0 Pa increased the extension and retraction velocities and separation of networks, resulting in the formation of unstable EC networks. In contrast, steady shear stress application resulted in the formation of extended and stable EC networks composed of many cells. Thus, two types of three-dimensional network growth were observed, depending on flow pulsatility. A combination of the type of ECs, such as aortic and microvascular ECs, and flow characteristics, such as flow magnitude and frequency, may have important implications for the construction of well-developed three-dimensional EC networks.
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