Fig. 19. Stress gradients within hydrogels influence cell differentiation in three dimensions. (A) Schematics of the process for creating three-dimensional multicellular hydrogels and encapsulating human mesenchymal stem cells (hMSCs). Briefly, prepolymer type I collagen was added to PDMS molds and hMSCs were suspended, before polymerizing at 37 °C. Liquid agarose was added to the mold to encase the collagen hydrogel at 4 °C. (B) Phase image of hMSCs in three-dimensional structures at day 0. (C) The hydrogel constructs with encapsulated hMSCs were suspended in mixed media and after 14 days, the cells at the edge of the constructs differentiated down an osteogenic lineage (blue) and those at the center underwent adipogenesis (red) (oil droplets, alkaline phosphatase staining) (D) Longitudinal section and (E) cross-sections confirmed the patterning of lineage specification in a tension-dependent manner (scale bar, 250 μm). Reprinted from Ruiz et al. ³⁶⁸ with permission from John Wiley and Sons publishing. Copyright (2008).