Figure 5.

Prealigned SCs can influence HMEVCs to form an oriented vessel network following the same direction. (A) Illustration of the experiment, where 100 × 10³ SCs were seeded on an aligned fibrous scaffold and cultured in SC proliferation medium for 7 DIV to form aligned cell bands. The scaffold was then covered with 300 μL of fibrin containing 1.5 M HMVECs/mL. The co-culture was maintained for 10 DIV in normal VM. Illustration made with biorender (https://biorender.com/). (B) Scanning electron microscopy (SEM) image of the scaffold showing the presence of aligned microsized polymeric fibers composed of PEOT/PBT. The scale bar represents 200 nm. (C) After 7 DIV in the scaffold, SCs were highly aligned. F-actin is shown in red and DAPI in blue. The scale bar represents 50 μm. (D) Presence of prealigned SCs promoted the formation of vessels (CD31, red) that follow an overall fiber direction (right). The mean length of these vessels is 550.9 ± 265.3 μm. In the absence of SCs within the scaffold/fibrin platform, HMVECs did not form vessels or follow any overall direction (left). (E) SCs immunostained by S100 (green) associated closely at the z-plane of the vessels (CD31, red). The vascular channels also displayed wide lumens as seen by the CD31 orthogonal projections of the xz- and yz-planes. (F) Quantification of HMVEC orientation via coherence measurement on equally sized ROIs from CD31+ micrographs (where 0 is full isotropy and 1 is full anisotropy). The presence of SCs promoted a statistically significant increase (p < 0.0001) in cell alignment. The bars represent the mean ± SD from five replicates per condition. Ten measurements were taken per sample. Statistics were performed using an unpaired t-test, where ****p < 0.0001.