Mouse polyclonal antibody against human cd31

The co-cultured prevascularized constructs were washed with PBS, fixed and stained with mouse polyclonal antibody against human CD31 (Abcam, Cambridge, MA), and followed by DyLight 488 horse anti-mouse IgG secondary antibody (Vector laboratories, Burlingame, CA). The samples were washed and incubated in 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) solution to label the cell nuclei. Images were captured under an Olympus FV-1000 confocal microscope. The vascular networks were quantified by the ImageJ software, using methods previously described 43 . We firstly chose one vessel that aligned along the main direction of the nanogratings and set the angle of it as 0 degree, the angles of other vessels were measured and defined as Δ Angle. Experiments were triplicated, and 5 non-overlapping panels were randomly analyzed from each individual sample for unbiased statistical analysis. The same immunofluorescent staining was performed with anti-CD146 and anti-CD166 primary antibody staining. CD146 is known as the melanoma cell adhesion molecule (MCAM), which is regarded as a pericyte marker 44 (link). CD166 stains activated leukocyte cell adhesion molecule, and it was used to detect hMSC-EC interaction 45 (link). Images were captured under an Olympus FV-1000 confocal microscope by depth scan every 1 µm. The obtained images were reconstructed into 3-D views by a Fiji software.

The MMP inhibition was performed to determine MMP inhibitor's influence on the structural properties of microvessels. A MMP inhibitor, APR 100 (Tocris Bioscience, Bristol, UK), was dissolved in dimethyl sulfoxide (DMSO) (Corning, NY) to prepare a 1mM stock solution. APR 100 is a selective inhibitor of MMP-2 (IC₅₀ = 12 nM), which also displays selectivity over MMP-9, MMP-3, MMP-1 and MMP-7 (IC₅₀ = 200, 4500, > 50000 and > 50000 nM respectively). Three different working concentrations (1 µM, 5 µM, 10 µM) of APR 100 were prepared in endothelial cell growth medium (EGM), which was used during the 7-day co-culture. No APR 100 was added in the control group. After 7 days of co-culture, prevascularized constructs were washed with PBS, fixed and stained with mouse polyclonal antibody against human CD31 (Abcam), followed by DyLight 488 horse anti-mouse IgG secondary antibody (Vector laboratories). Samples were mounted, and images were captured under an Olympus FV-1000 confocal microscope. Experiments were triplicated, and at least 5 non-overlapping areas from each sample were randomly observed.