Figure 4: ADSCs can undergo osteogenic and adipogenic differentiation in 3D. (a, b) ADSCs were subjected to control or osteogenic differentiation treatments in 0.2% aNFC for 21 days and subsequently stained for calcium deposition with Alizarin Red S. Images of differentiated ADSCs (b) clearly show calcium deposition (red), whereas no differentiation was observed in control cells (a). Bars in (a) and (b): 500 μm. (c) Image analysis of calcium deposition showed significantly higher calcium deposition within differentiated cells compared to the control. (d–g) Immunocytochemical stainings with antiosteocalcin (d, e) and antiosteopontin antibodies (f, g) reveal strong immunopositivity in the osteogenically differentiated cells (e, g) compared to control cells (d–f). (h–j) ADSCs were subjected to adipogenic differentiation within 0.2% aNFC for 21 days. Following Oil Red O staining, lipid deposition was observed in treated cells (arrows in (i) and (j)) compared with the untreated control (h). Bars in (h) and (i): 200 μm; bar in (j): 50 μm.