Silk microtube assembly and sponge preparation into the bioreactor chamber and analysis of platelet production within this system. (A) Aqueous silk is dispensed into the chamber around the microtube and salt particles are added. After leaching out the salt, the resulting porous silk sponge is trimmed and sterilized. After seeding into the silk sponge, Mks migrate toward the microtube, adhere, and extend proplatelets through the microtube wall to release platelets into the microtube lumen. (Bi) SEM image showing a silk microtube embedded into the silk sponge (scale bar = 100 μm). (Bii) SEM image showing the porous morphology of silk sponge (scale bar = 100 μm). (C) Confocal microscopy analysis of different steps of Mks behavior within the silk microtube-sponge tissue system. (Ci) Silk sponge before Mks seeding (scale bar = 100 μm). (Cii) Mature Mks immediately after seeding into the silk sponge (green = CD61; blue = nuclei; scale bar = 100 μm). (Ciii) Migrated Mks in close contact with the microtube wall (green = CD61; blue = nuclei; scale bar = 50 μm). (Civ) Mks forming proplatelet on adhesion to the microtube wall (green = CD61; blue = nuclei; scale bar = 50 μm). (Cv) Mk extending proplatelets through the silk microtube wall. Arrow indicates proplatelet branch elongation through microtube wall with proplatelet tip protruding into the microtube lumen (green = CD61; blue = nuclei; scale bar = 50 μm). (Cvi) Boxes highlight proplatelet branches detectable along the inner wall of the silk microtube and platelets released into the microtube lumen (green = CD61; blue = nuclei; scale bar = 50 μm). For all immunofluorescence analysis, silk fibroin 3D scaffolds were stained with Hoechst 33258 and visualized in blue. (D) SEM imaging of different steps of Mks behavior within the silk microtube-sponge tissue system. (Di) Mature Mks adhesion on silk microtube outer wall (scale bar = 2 μm). (Dii-Diii) Migrated Mks forming proplatelet on adhesion to the microtube wall (scale bar = 2 and 10 μm). Arrows indicate silk pores that allow proplatelet elongation inside microtube lumen. (Div) Longitudinal section of silk microtube-sponge tissue system (scale bar = 200 μm). (Dv) Mk extending proplatelets through silk microtube wall (scale bar = 10 μm). (Dvi) Proplatelet branch stemming inside microtube lumen and released platelets (scale bar = 10 μm).