Cell spreading is affected by the surface energy on elastomer substrates of different stiffness. (A) Morphology of hBMSCs on functionalized substrates seeded at 5,000 cells per square centimeter after 24-h culture on PDMS and PEO-PDMS substrates of different stiffness (soft, 0.07–0.10 kPa; stiff, 2.15–2.40 MPa). Cells were immunostained with an antibody against vinculin (red), Alexa-488 phalloidin (green), and DAPI (blue). (Upper row scale bar, 100 μm; Lower row scale bar, 20 μm.) Images of cells on stiff substrates were reported from our previous publication (12). (B) Attachment of hBMSCs on PDMS and PEO-PDMS substrates when seeded at 25,000 cells per square centimeter after 1-h culture (n = 3). Data on stiff substrates were reported from our previous publication (12). (C) hBMSCs spreading area on PDMS and PEO-PDMS when seeded at 5,000 cells per square centimeter after 24-h culture (n = 4; number of cells ≥2,400). Data on stiff substrates were taken from our previous publication (12). Data are represented as mean ± SD. Significance is indicated for P ≤ 0.05 (***P ≤ 0 0.001, ****P ≤ 0.0001).