Protocols used to induce the differentiation of the iPSCs in the bioprinted materials were inspired by current strategies with the highest success rate, relying on co-culturing with irradiated mature chondrocytes (iChons)8. Growth factors combined with a 3D environment are essential for directing iPSCs towards the chondrogenic lineage. Factors such as TGFβ1, TGFβ3, GDF5, and BMP2 have been found to be crucial for the production of the important hyaline cartilage matrix components collagen type II, IX, and XI and aggrecan21,22,23.

To ensure the formation of a functional mimic of cartilage tissue, visualization of the 3D arrangement of the extracellular matrix (ECM) and living cells in native hydrated conditions is central. Hence, bright-field microscopy was complemented with nonlinear microscopy to simultaneously acquire second-harmonic generation (SHG) images of collagen and two-photon excited autofluorescence (TPEF) images of living cells in unlabeled, printed constructs24.

To ensure that the bioinks were compatible with the iPSC phenotypic properties, 2D monolayer culturing was conducted on the bioprinted materials with different dry weights or volume percent ratios of the structural (NFC) and cell-supporting (A or HA) components together with the crosslinking solutions (Fig. 1A and Supplementary Table 1). Brightfield microscopy images confirmed no morphological or proliferative changes for the NFC/A 60/40 wt% and NFC/A 80/20 wt% bioinks with the CaCl 2 crosslinking solution compared to unprinted controls. Positive staining for iPSC marker OCT4 was indicative of cellular pluripotency for the cells in culture with the NFC/A bioink treatment. When in contact with the NFC/HA bioinks, the cells were rounded, with low positive OCT4 staining; hence, this material and the crosslinking conditions induced phenotypic changes of the cells away from pluripotency (Fig. 1A). The crosslinking agent, H 2 O 2 , which was used for NFC/HA, was not the cause of the reduction of OCT4 since H 2 O 2 exposure alone did not reduce OCT4 staining (Supplementary Fig. 1).

Figure 1 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 CaCl 2 solution, (2) NFC/A 80/20 crosslinked with 100 mM CaCl 2 solution, and (3) NFC/HA crosslinked with 0.001% H 2 O 2 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). Full size image

The proliferation of iPSCs encapsulated in different bioink compositions and maintained in DEF-CS medium was examined for one week to ensure that the material was compatible with supporting 3D expansion. Observations made using bright-field microscopy demonstrated the differences in cell distribution and cluster sizes (Fig. 1B, Supplementary Table 1, and Supplementary Fig. 2). The NFC/HA bioink showed little to no proliferation of the limited cell population remaining in the construct after encapsulation. The NFC/A 80/20 wt% constructs contained an even distribution of cells that proliferated into smaller, elongated and more irregular-shaped cell clusters. The greatest amount of growth and largest spherical clusters were observed within the NFC/A 60/40 wt% constructs by bright-field microscopy as well as after sectioning and staining with Alcian blue-van Gieson (Fig. 1B and C and Supplementary Table 1).

After three weeks of growth factor-mediated differentiation (with prior maintenance of iPSCs in the hydrogels in DEF-CS for one week), RNA expression showed phenotypic increases in chondrogenic markers for all three types of bioinks (Fig. 1D). Hence, the NFC/HA bioink still seemed promising for use in further experiments if an increase in cell number could be achieved through improved proliferation and delivery. The iPSCs printed with the NFC/A 60/40 wt% bioink showed better survival than iPSCs printed in the NFC/A 80/20 wt% bioink (Fig. 1E; more live cells (stained green) are seen in the NFC/A 60/40 wt% bioink, and increased survival was observed with time, Supplementary Fig. 3). Hence, using a lower concentration (60 wt%) of NFC yielded a higher number of viable cells, although a higher concentration (80 wt%) of NFC yielded more stable and defined printed constructs (as can be seen in Fig. 1E, lower; the printed grid lines using NFC/A 60/40 did not retain the same thickness and homogeneity compared to those using 80/20, Supplementary Fig. 4).

To direct iPSCs towards chondrocytes, we used a combination of growth factors (in short, Wnt 3a and Activin A for 3 days followed by GDF5 + BMP2 + TGFβ1 for up to five weeks) printed in the NFC/HA (Supplementary Fig. 5) or NFC/A bioink or co-cultured with irradiated chondrocytes (iChons) using the growth factors GDF5, BMP2, TGFβ1 and TGFβ3. First, the iPSCs and the iChons were printed in separate strands in an overlapping grid structure (Supplementary Fig. 6A). The production of GAGs was moderately increased in the intersections, indicating that crosstalk between chondrocytes and iPSCs was beneficial (Supplementary Fig. 6B and C). Therefore, iPSCs and iChons were mixed at a ratio of 1:1 and printed together. Clones of the cells appeared 1 week after printing, suggesting cell proliferation and clonal expansion (Fig. 2A, confocal microscopy images). The co-cultured printed constructs intentionally had twice the cell density from the start compared to the control constructs with only iPSCs or iChons to maintain a comparable constant iPSC density due to the mortality of the iChons (Fig. 2B). The iChons control constructs showed a decreasing number of visible cells over time when analyzed by TPEF microscopy and were diminished after 3 weeks in culture (Fig. 2B, iChons, and Supplementary Fig. 6). In agreement with previous findings that the use of irradiation to make chondrocyte replication incompetent also causes apoptosis, all cells were dead within 25 days of irradiation12. Fluorescence in situ hybridization (FISH) analysis of the 3D-printed constructs after 5 weeks (Fig. 2C) further supported the disappearance of iChons over time. This observation implies that the iPSCs are the most likely source for cartilage matrix formation. The co-cultured printed protocol gave rise to the appearance of hyaline-like cartilaginous tissue at week 5, which was visualized by (i) Alcian blue-van Gieson staining of collagen, connective tissue and acidic polysaccharides, such as glycosaminoglycans in cartilage (blue), and (ii) Safranin-O staining, which stains cartilage red (Fig. 3B). Furthermore, areas with dark staining indicated hyaline cartilage tissue generation that was similar to stained native cartilage tissue and, in areas of increased Alcian blue-van Gieson staining, nuclei clusters with lacunae were seen (Fig. 3B, zoomed in, upper row). An increase in cell number was correlated with higher GAG production (Fig. 3B and C (TPEF, cells are yellow in 3C), corresponding areas in an unstained serial section are marked with red and green squares). While no significant difference in the SHG signal could be detected between the areas (Fig. 3C), the production of cartilage-specific collagen type II was evident by immunohistochemistry using a specific antibody for collagen type II (Fig. 3D, green; and Supplementary Fig. 7A) and was surprisingly more intense than the human control cartilage (Supplementary Fig. 7B), further suggesting that cartilage-like tissue had been generated after five weeks of differentiation. The immunohistochemistry results were important since the prints containing alginate, even without cells, gave a background staining with Alcian blue-van Gieson. The lack of significant difference in the SHG signal between the areas (Fig. 3C) could result from the fine collagen II fibrils generating such a weak SHG signal that their build-up disappears in the high background interference from cellulose. The sensitivity of this technique could be improved by using polarization sensitive SHG microscopy25. We next wanted to determine whether we could generate cartilage mimic tissue from iPSCs without using iChons. We succeeded in generating higher GAG production from iPSCs in an area (Fig. 4A and B) when the iPSC prints (60:40 NFC/A) were initially maintained in a conditioned DEF medium since single cell survival is dependent on factors produced from other iPSCs (Fig. 4C). Finally, we assessed the level of pluripotency protein OCT4 prior to and after printing (Fig. 5). OCT4 could be detected even in the presence of iChons, which have been shown to produce BMP212, after 1 week of printing when maintained in the conditioned DEF medium. OCT4 signals were not detected after differentiation for 3 or 5 weeks (Fig. 5).

Figure 2 Co-culture increased iPSC density in the NFC/A 60/40 bioink after printing. (A) Confocal microscopy images of the NFC/A 60/40 co-culture samples at week 0 and week 1 stained for actin (green) and nuclei (blue) show cells evenly distributed with cluster formation after week 1, thus indicating proliferation (the scale bar represents 50 μm). (B) Label-free nonlinear microscopy (two-photon excitation fluorescence, with the autofluorescence of cells shown in yellow) images show similar distributions in the co-cultures compared to the confocal images. However, a decrease in the cell number was seen over time for the irradiated chondrocyte (iChons)-only prints as expected (co-culture prints were conducted with a 1:1 ratio of iPSC to iChons) (the scale bar represents 50 μm). (C) Fluorescence in situ hybridization (FISH) stained only the X chromosomes (X chromosomes, green; Y chromosomes, red) in the co-cultured cartilage-like tissue. Female line, iPSCs; male, iChons (the scale bar represents 10 μm). Full size image

Figure 3 Three-dimensional-bioprinted cartilage-like tissue. (A and B) Histology sections of the 3D-bioprinted constructs. (A) At week 3 (blank - no cells), week 0, week 1, and week 2 of differentiation, which followed 2 weeks of proliferation in the iPSC maintenance medium (stained with Alcian blue-van Gieson for proteoglycans/glycosaminoglycans (GAGs) (blue) and nuclei (brown)) (the scale bar represents 100 μm). (B) The 3D-bioprinted chondrocyte-derived iPSCs (printed together with iChons, which had been diminished) at week 5 of differentiation, zoomed in (upper row) and whole section (lower row) images of sections stained for GAGs, Safranin O for cartilage (with nuclear counterstain), and hematoxylin and eosin (H&E) for extracellular matrix (with nuclear counterstain) (the scale bar represents 100 μm or 500 μm). (C) Label-free images of unstained sections (of areas corresponding to red and green boxes from the lower row of B) shows highly dense cell areas (cell autofluorescence, yellow) and collagen-like fibrils (second-harmonic generation, cyan). The highly dense cell area in the red box corresponded to higher GAG staining (the scale bar represents 50 μm). The number of cells per ml was calculated from the high-density (red square) and low-density (green square) areas. (D) Fluorescent image of an immunohistochemistry section (from the same 3D printed sample as B and C) stained for collagen type II (green) (with nuclear counterstain shown in blue), which shows the production of extracellular matrix collagen type II in a representative cell cluster (the scale bar represents 10 μm). Full size image

Figure 4 Three-dimensional-bioprinted cartilage-like tissue from iPSCs excluding iChons. (A and B) Histology sections of 3D-bioprinted constructs at week 6 (week 1 proliferation in conditioned DEF medium for the iPSCs plus 5 weeks of chondrogenic differentiation with TGFβ1, TGFβ3, GDF5 and BMP2) (stained with Alcian blue-van Gieson for proteoglycans/glycosaminoglycans (GAGs) (blue) and nuclei (brown)) (the scale bars represent 500 μm in A and 100 μm in B). (C) Cell survival of single iPSCs in the DEF medium or in the iPSC-conditioned DEF medium. Full size image