# Pneumatically Actuated Microfluidic Platform for Reconstituting 3D Vascular Tissue Compression
## 2 Results and Discussion
### The Effect of Solid Stress on Reactive Oxygen Species Generation  
Reactive oxygen species (ROS) are highly reactive oxygen-containing molecules generated as a byproduct of cellular metabolism [18]. Healthy cells regulate ROS levels and prevent oxidative stress by managing homeostatic antioxidant mechanisms, such as the catalysis of peroxides into water by  
Figure 5: Microvascular network viability according to six different pressures. Cell viability was examined after a 1-min duration each at six different pressures and representative fluorescence images from each sample are shown. Scale bar: \(80\)\(\upmu\)m.  
superoxide dismutase. Under unfavorable conditions, cells may exhibit diminished ROS removal capabilities, leading to oxidative-stress-induced damage and even death [19]. Common diseases caused by elevated ROS levels include cardiovascular diseases, pulmonary disorders, and diabetes, which are all related to endothelial dysfunction. In the case of diabetes, increased ROS production with reduced oxygen consumption results in damage to endothelial cells [20]. A microfluidic device introduced by [PERSON] et al. investigated the endothelial monolayer under pulsatile shear stress and showed that the effects of shear stress on endothelial cells and ROS levels are critical for mimicking the functionalities of endothelial cells in vitro [21]. To assess the ROS generation rates as a cellular response to biomechanical compressive stress, vascular tissues were observed at six levels of compressive stress. A significant increase of ROS levels were detected from 1.5 psi to 2.5 psi compared to readings below 1.5 psi. ROS production levels increased by 6.75-fold, 12.1-fold, and 28.63-fold at 1.5 psi, 2.0 psi, and 2.5 psi, respectively, compared to the baseline 0 psi condition (Figure 6A,B), indicating that ROS levels increased exponentially with mechanical stress. ABAQUS modeling corroborated the observed correlation between ROS generation and compression, also indicating that an abrupt increase of von Mises stress occurred from 1.5 psi to 2.5 psi conditions (Figure 3C,D). The results of both the experiment and simulation indicates the importance of investigating the effect of biomechanical stress on the fully perfusable 3D vascular networks under physiological and pathological conditions since they may reflect the various blood vessel damage patterns in vivo [22]. It can be extrapolated that higher levels of biomechanical stress may substantially increase the intracellular level of ROS beyond the tested parameters. These results suggest that applying a mechanical stimulus for 3D vascular tissue compression could generate mechanotransduction, which refers to the conversion of a mechanical stimulus into electrochemical activity [21]. The Effect of Compressive Stress on Vascular Permeability and Vascular Endothelial (VE)-Cadherin Expression  
Abnormal vascular permeability caused by barrier defects are often related to various pathological conditions, such as biomechanical stress and high concentrations of histamine or growth factors [23; 24]. To investigate the vascular permeability in response to biomechanical compressive stress, perfusable vascular networks were treated to 0 psi and 1.5 psi conditions for one minute each. Permeability measurements using FITC-dextran are commonly used for the visualization of blood vessel barrier function. Cultivated vascular networks were perfused with 20 kDa FITC-dextran and imaged in 5 s increments to show the gradual FITC-dextran diffusion from vascular to perivascular regions (Figure 7A). The permeability coefficient value of the 1.5-psi-conditioned vascular tissues was  
Figure 6: The reactive oxygen species (ROS) level in a microvascular network. (**A**) ROS levels assayed using H\({}_{2}\)DCFDA fluorescence dye (green). Scale bar: 80 \(\upmu\)m. (**B**) Quantitative analysis of the fluorescence intensity. Bars represent the mean \(\pm\) SEM from 4–5 devices per condition. \({}^{***}\)_p_\(<\) 0.001 in an unpaired two-tailed Student’s \(t\)-test.