Fig. 5.

Cell viability, proliferation, adhesion, chondrogenic maintenance, and osteogenic potential of the printed upper- and lower-layer hydrogels in vitro. A, B) Viabilities of chondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs) were tested with CCK-8 (n = 3). The control group comprised cells that were cultured in an untreated medium. C) Images of live/dead staining were captured after culturing chondrocytes and BMSCs with various scaffolds for 7 d (live cells: green, dead cells: red. Scale bar: 100 μm. D) Schematic of chondrocytes and BMSCs were separately seeded on upper-layer (i) and lower-layer (ii) hydrogels. E) Image of chondrocyte adhesion morphology on scaffolds on day 7. Nucleic acids are depicted in blue, while F-actin is visualized in red. Scale bar: 40 μm. Immunofluorescence (IF) staining of collagen type II (COL II) and collagen type I (COL I) was utilized to evaluate chondrocyte phenotype. Scale bar: 200 μm. F) Images of BMSCs adhesion behavior in 7 d. Scale bar: 100 μm. Alizarin red staining was used to assess osteogenic potential. Scale bar: 400 μm. G) Chondrogenic maintenance of chondrocytes (i) and osteogenic differentiation of BMSCs (ii) were assessed at the mRNA level (qRT-PCR) (n = 3). All data were analyzed by one-way ANOVA, and error bars indicate mean ± SD, *p < 0.05, **p < 0.01, and ***p < 0.001.