Endothelial cell attachment to porous substrates via focal adhesions is initially affected by pore spacing and not absolute porosity. Maturation of endothelial monolayers via cell-cell interactions occurred over the same time course regardless of substrate porosity, pore size and contact area. Cell-matrix interactions at maturity were dictated by substrate stiffness.
Data
Fig. 1 SEM images of 0.5 mm and 3.0 mm pore size membranes. Spacing between pores is one pore diameter. Porosity for both membranes is 22.7%.
Fig. 2 Cell attachment to substrates through focal adhesions at 24 hours. HUVEC were cultured on tissue culture plastic (TCP), non-porous, 3.0 µm high porosity, 0.5 µm high porosity SiO2 membranes, and stiff (E = 2 MPa), and soft PDMS (E = 5kPa). Cells were stained with DAPI (nuclei, blue), phalloidin (actin filaments, green), and anti-vinculin (focal adhesion vinculin, red).
Fig. 3 Quantification of focal adhesion formation. Formation of distinct focal adhesions was quantified for all substrates – number of focal adhesions per cell and percent of cells with distinct focal adhesions. Error bars are +/- SD. Sample size >50 for each substrate.
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Fig. 4 Endothelial Barrier Maturity. HUVEC were cultured to form a monolayer on tissue culture plastic (TCP), non-porous, 3.0 µm high porosity, 0.5 µm high porosity SiO2 membranes, and stiff (E = 2 MPa), and soft PDMS (E = 5kPa). Cells were stained with DAPI (nuclei, blue), anti-VE-cadherin (cell-cell junctions, green), and anti-vinculin (focal adhesions, red) to visualize formation of endothelial barriers.
Fig. 5 High-resolution fluorescence image of focal adhesion formation on 3.0 µm pore size SiO2 membranes upon reaching confluence. White circles are locations of pores. Solid circles are locations where focal adhesions (vinculin, red) are present over the pore or at the pore edge.
Supplemental Data
Fig. S1 Cell Spread Area at 24 hrs. HUVEC spread area was quantified 24 hrs post-seeding on TCP, non-porous SiO2 membranes, 3.0 µm pore size SiO2, 0.5 µm pore size SiO2, stiff PDMS (E = 2 MPa), and soft PDMS (E = 5kPa). The box plots represent median and IQR of spread area. Whiskers are +/- 1.5IQR.
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