Effect of silk film topography and stiffness on human megakaryocyte adhesion and proplatelet formation. (A) Silk films are prepared by dispensing a silk and PEO solution onto a PDMS mold. The surface of the mold may contain a grating pattern with defined depth and width. When the solution dries, a silk film is formed that contains a dispersion of PEO porogen. The film is finally soaked in phosphate-buffered saline to remove the PEO porogen. (B) Representative light microscopy image of silk film porosity (scale bar = 25 µm). (C-D) Analysis of Mk adhesion and proplatelet formation on silk film with different topography coated with fibrinogen (average ± standard deviatoin [SD], n = 3, *P < .05). Results are presented relative to silk film with no pattern. (E) Atomic force microscopy elastic modulus values obtained over hydrated low, medium, and high films. Distributions are displayed as percent of total sample points measured per bin. All samples had a minimum of 300 measurements. (F) There was no significant difference in Mk adhesion between the different stiffness samples (average ± SD, n = 4, P = not significant). (G) The low stiffness samples had similar proplatelet formation compared with the medium stiffness but significantly higher percentage compared with the high stiffness samples (average ± SD, n = 4, *P < .01). (H) Representative β1-tubulin staining of Mks cultured on silk films with different stiffness coated with fibrinogen, after a 16-hour incubation. The low stiffness silk films supported long proplatelet extensions and increased silk film stiffness appeared to decreased proplatelet branching (scale bar = 50 µm).