Figure 5.

Expansion of MSCs contributes to different results in cartilage engineering. (a) Flow cytometric analysis was performed to characterize the phenotypes of the P3 BMSCs and uncultured BMMNCs. (b) Cell viability analysis was performed to detect live/dead cells. Viable cells appear green, whereas dead cells appear red. Scale bar: 500 μm. (c) Cytoskeletal morphology was assessed using phalloidin staining of the actin cytoskeleton and cell nuclei via Hoechst 33258 by confocal microscopy. Scale bar: 500 μm. (d) H&E staining. Scale bar: 500 μm. (e) Cell proliferation was measured based on the DNA content after 7, 14 and 21 days of culture in 3D collagen hydrogels and chondrogenic supplements; n=5. (f) The GAG production was normalized to the total DNA production to evaluate the biosynthetic activity; n=5. (g) Safranin-O staining. Scale bar: 500 μm. (h) Genes related with chondrogenesis (COL1A1, COL2A1, COL10, SOX9 and ACAN) were detected by real-time PCR after 7, 14 and 21 days of culture in 3D collagen hydrogels and chondrogenic supplements; n=5. Error bars, mean±S.D. *P<0.05; **P<0.01; ***P<0.001