Fig. 2.

Image analysis-based macroscopic characterization of sound patterned microcapillary networks. Local cell density enhancement after sound patterning was quantified by (a) measuring the GFP+ area of the ROIs placed on (i) the patterned line (“On pattern” ROIs) of the concentric circles pattern and compared with the areas adjacent to the pattern line or with (ii) the cells seeded in stationary condition (“Distal”, “Proximal”, and “Random” ROIs) (scale bars = 500 µm). The measurements (b) were conducted on the first and second rings of the concentric rings pattern and showed 438 ± 103 % and 386 ± 104 % enhancement of local cell density for the first and second rings, respectively, compared to the stationary condition (n = 3, mean ± SD, ****p-value <0.0001). The microcapillary network's local thickness analysis (c) was performed on representative images obtained from the concentric circles pattern. Specifically, on (i) the patterned line and on representative images of (ii) the stationary condition (random) at day 1, 3, and 5 of tissue maturation (scale bars = 200 µm). The local thickness results (d) for the patterned capillaries during the 5 days in culture were 13.1 ± 24.7 µm, 10.6 ± 18.9 µm, and 4.1 ± 9.5 µm, whereas 6.6 ± 7.8 µm, 9.3 ± 12.5 µm, and 4.5 ± 8.8 µm for the random distribution in stationary conditions, measured at day 1, 3, and 5, respectively (mean ± SD). The evolution of the microcapillaries pattern (e) was monitored over the 5 days in culture by measuring the radial intensity profile of the concentric rings pattern, with peaks corresponding to single circles on each of the imaging days. The valley-to-peak ratio (f) of the radial profile's peaks was measured over the culture time and showed a linear increase, suggesting capillary growth also in between the concentric rings. Conversely, the thickness of the rings remained relatively constant, ranging between 80 and 120 µm. Analysis of the general orientation (g) presented by capillary-like structures, performed on overview images of samples, revealed that different pattern geometries (round and square) induced different orientation of the capillary-like structures at the macroscopic level. Immunofluorescent staining (h) of the microcapillary networks showed signs of tissue maturation and functionality (representative image from concentric rings pattern). (i) Pericyte cells were found to support the capillary structures (green = GFP-HUVEC, yellow = F-actin, scale bar = 100 µm), and (ii) VE-cadherin was expressed at cell-cell junctions along the patterned capillaries (violet = VE-cadherin, blue = Nuclei, scale bar = 500 µm).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)