Osteochondral microtissue design, fabrication, and validation. (A) Schematic diagram of one microtissue assembly within a single well platform, cross-sectional side view. cc, chondrocyte construct; ec, endothelial construct; hf, human fibroblasts; lc, lower medium conduit; mc, mesenchymal construct; oc, osteoblast construct; p, platform; sf, synovial fluid; uc, upper medium conduit; a1, lower adapter with porous screen to allow communication between the lc and oc and the uc and cc chamber; a2, upper adapter, to seal the upper conduit. Red arrows indicate direction of fluid flow. (B) Schematic of one microtissue assembly within a single well platform showing fluid flow (red arrows) of the lc and its relation to the endothelial layer (ec), oc and the porous barrier (a1). (C) Combination of multiple microtissue platform configurations, 96-well format, and single well format. (D) Three-dimensional model fabricated microtissue components. (E) Microtissue components fabricated with E-shell 300™ (EnvisionTec, Gladbeck, Germany) polymer resin using projection stereolithography. (F) Order of microtissue assembly before culture. (G) Histological view of interface between osteoblast and mesenchymal construct on a fully assembled microtissue construct after 6 weeks of culture. Arrows indicate a dense structure between the two layers. Bar = 100 μm. (H) Native bone plugs in collagen gel with or without endothelial cells (ECs) cultured for 4 weeks immunohistochemically stained for osteoprotegerin. B, bone plug; G, collagen gel. Bar = 100 μm. (I) Osteoprotegerin ELISA analysis of media samples conditioned by bone plugs coated in collagen gel with or without ECs cultured for 1, 4, or 6 weeks. *P = 0.0362.