Figure 3.

Schematic representation of the experimental approach. Human chondrocytes were isolated from nasal septum biopsy, expanded, and dedifferentiated up to the sixth culture passage (P6). Cells were then embedded in Af, AGf, and AUBMf and subjected to the indicated analysis at days 7 and 14. Dedifferentiation process from passage 0 (P0) to passage 6 (P6) has been monitored: protein expression of cartilage-related genes (Col2A1, Aggrecan, and Sox9), and Col1A1 was investigated by immunocytochemical analysis. Alcian Blue staining for sulfate glycosaminoglycans (GAGs) detection is also reported. Cell morphology was evaluated by hematoxylin staining. Representative optical photomicrographs are reported. Pictures of at least four random fields of three replicates were captured for densitometric analysis using ImageJ software. Data are presented as means of pixels per 100 cells ±SEM (*p ≤ 0.05). Cell viability of embedded cells has also been investigated, and optical and fluorescence photomicrographs after double staining with Calcein-AM/propidium iodide at days 7 and 14 of culture in basal medium are reported. The green fluorescence indicates the presence of calcein-labeled live cells, while propidium iodide-labeled dead cells are revealed by red fluorescence. Merged photomicrographs are reported.