Fig. 2

The effects of fibroblasts and conditional media from fibroblasts on BMP2 induced osteoblast differentiation of MSCs. A Schematic illustration showed the experimental protocols. MSCs were directly co-cultured with conditional media from fibroblasts or indirect co-cultured with fibroblasts in Transwell. BMP2 was utilized to induce osteoblast differentiation of MSCs. B A total of 3 × 10⁴ fibroblasts were cultured on the upper layer of the Corning cell culture insert with 0.4 μm polyester membrane. 1 × 10⁴ MSCs were placed below the cell permeable membrane for 7 days, supplemented either with a vehicle or with 200 ng/mL BMP2. Subsequently, the cells were harvested and subjected to ALP staining. Representative images of the wells and cells were presented. Scale bars, 100 μm. C The percentages of ALP⁺ cells in B were quantitatively analysed. The average percentages of ALP⁺ cells were calculated from at least three fields and shown (**P < 0.01). D A total of 3 × 10⁴ fibroblasts were cultured on the upper layer of Corning cell culture insert with 0.4 μm polyester membrane. 1 × 10⁴ MSCs were placed below the cell permeable membrane for 14 days, supplemented either with a vehicle or with 200 ng/mL BMP2. Subsequently, the cells were harvested and subjected to Alizarin red staining. Representative images of wells and cells were presented. Scale bars, 100 μm. E The percentages of Alizarin red⁺ areas in D were quantitatively analysed. The average percentages of Alizarin red⁺ areas were calculated from at least three fields and presented (**P < 0.01). F MSCs were treated as indicated in D. Subsequently, the cells were harvested and total RNAs were extracted. cDNAs were synthesized and subjected to qPCR. Relative mRNA levels of Runx2, Ocn and Osx to Gapdh in MSCs were calculated and presented (*P < 0.05; **P < 0.01). G 1 × 10⁴ MSCs were cultured with conditional media from fibroblasts and treated either with 200 ng/mL BMP2 or the control vehicle for 14 days. Subsequently, the cells were harvested and subjected to Alizarin red staining. Representative images of wells and cells were presented. Scale bars, 100 μm. H The percentages of Alizarin red⁺ areas in G were quantitatively analysed. The average percentages of Alizarin red⁺ areas were calculated from at least three fields and presented (**P < 0.01). I MSCs were treated as indicated in G. Subsequently, cells were harvested and the total RNAs were extracted. cDNAs were synthesized and subjected to qPCR. Relative mRNA levels of Runx2, Ocn and Osx to Gapdh in MSCs were calculated and presented (*P < 0.05; **P < 0.01)