Material compatibility and cell pluripotency of different bioinks. (A) Bright-field and fluorescent images at day 2 of the iPSCs being in contact with the three bioink compositions: (1) NFC/A 60/40 crosslinked with 100 mM CaCl2 solution, (2) NFC/A 80/20 crosslinked with 100 mM CaCl2 solution, and (3) NFC/HA crosslinked with 0.001% H2O2 solution (the scale bars represent 50 μm). Cell morphology and Oct4-positive staining (orange) indicated the compatibility and inertness of both NFC/A treatments. However, NFC/HA treatment changed the cell morphology to be spherical with less Oct4 staining and less cells. (B–D) Encapsulation of iPSCs in bioinks for a three-week differentiation period resulted in dissimilar cell distribution and differentiation phenotypes. (B) The NFC/A 60/40 bioink had a greater amount of clusters with larger diameters (each black arrow points to one cluster) compared to the 80/20 bioink (each gray arrow points to a cluster). By contrast, the NFC/HA bioink had minimal cell clusters (each white arrow points to one of the low-density cell clusters) (the scale bar represents 500 μm). (C) Alcian blue-van Gieson-stained histological sections revealed more rounded clusters in the NFC/A 60/40 bioink compared to the elongated clusters in the NFC/A 80/20 bioink and the lack of cells and clusters in the NFC/HA bioink. (D) Furthermore, the three bioinks supported the phenotypic expression of SOX9, aggrecan, and collagen type 2A1. (NFC/HA samples at week 3 were lacking due to the small amount of RNA). (E) At day 5 after printing, the NFC/A 60/40 bioink composition was superior for cell viability compared to the NFC/A 80/20 bioink, as shown in the wide-field fluorescence images with live staining (green, live) (the scale bars represent 200 μm). Confocal images (upper right corners) show signs of cell proliferation in the NFC/A 60/40 bioink with multiple cells in a cluster (DAPI, blue; actin, green) (the scale bar represents 50 μm).