Fig. 1

Assessment of the roles that fibrous tissues and fibroblasts played on osteogenesis. A Masson staining was performed on tissues harvested from patients with bone nonunion. Blue staining indicated the presence of fibrous tissues while the red staining indicated the presence of newly formed bone. Scale bars of the ×100 photos were 100 μm. B Tissues obtained from the experiments of BMP2 induced ectopic bone formation were subjected to Masson staining. Blue staining represented fibrous tissues and the red staining indicated the presence of newly formed bone. Scale bars of the ×100 photos were 100 μm. C After being labelled with 2.5 µM CFSE (green fluorescence), a total of 5 × 10³ MSCs were cultured either alone or with 1.5 × 10⁴ fibroblasts in a single well of a 24-well plate for 72 h. Subsequently, the cells were harvested and subjected to flow cytometry. Cells exhibiting green fluorescence signals lower than parental cells were indicative of proliferating cells. Three independent experiments were conducted, and the average percentages of proliferating MSCs were calculated and shown (*P < 0.05). M MSC, F fibroblast. D After being labelled with 2.5 µM CFSE (green fluorescence), a total of 5 × 10³ MSCs were cultured with an equal number of fibroblasts in free DMEM on the upper layer of Corning cell culture insert with 8.0 μm polycarbonate membrane. The complete culture media, supplemented either with a vehicle or with 200 ng/mL BMP2, was placed below the cell permeable membrane. Representative immunofluorescent staining images of the fibroblasts and MSCs that migrated through the Transwell membrane were presented. Green cells indicated MSCs, red cells indicated α-SMA⁺ cells (fibroblasts), and blue colour indicated DAPI, a marker of nucleus. Scale bars, 100 μm. E Quantitative analysis of the numbers of MSCs and fibroblasts in C was conducted. Cell numbers of at least three fields were counted and average cell numbers were calculated (*P < 0.05; **P < 0.01). F A total of 1 × 10⁴ MSCs were cultured either alone or with 3 × 10⁴ fibroblasts in a single well of a 12-well plate. The cells were treated either with a vehicle or with 200 ng/mL BMP2 for 7 days. Subsequently, the cells were harvested and subjected to ALP staining. Representative images of the wells and cells were presented. Scale bars, 100 μm. M MSC, F fibroblast. G The percentages of ALP⁺ cells in E were quantitatively analysed. The average percentages of ALP⁺ cells were calculated from at least three fields and shown (*P < 0.05; **P < 0.01). H 1 × 10⁴ MSCs were cultured either alone or with 3 × 10⁴ fibroblasts in a single well of a 12-well plate. The cells were treated either with a vehicle or with 200 ng/mL BMP2 for 14 days. Subsequently, the cells were harvested and subjected to Alizarin red staining. Representative images of the wells and cells were presented. Scale bars, 100 μm. I The Alizarin red⁺ area percentages in G were quantitatively analysed. The average percentages of Alizarin red⁺ areas were calculated from at least three fields and presented (*P < 0.05; **P < 0.01). M MSC, F fibroblast. J MSCs and fibroblasts were either cultured alone or cocultured as indicated in I. Subsequently, the 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)