Fig. 6. BMP9-induced ectopic bone formation from iSCAP cells is potentiated by Wnt3A but attenuated by β-catenin knockdown.

Subconfluent iSCAP-KD and iSCAP-Ctrl cells were infected with AdBMP9, AdWnt3A, AdBMP9+AdWnt3A, or AdGFP for 16h. Cells were collected for subcutaneous injections (3x10⁶ cells/site in 100μl PBS) into the flanks of athymic nude mice (n=5 each group). (A) Gross images and μCT analysis. At 4 weeks after injection, the animals were sacrificed. Masses formed at the injection sites were retrieved (a), fixed in formalin, and subjected to μCT imaging (b). No masses were detected the animals injected with GFP- transduced iSCAPs. The 3-D reconstruction was performed for all scanned samples (b), and the average total bone volume (c) were determined using the Amira 5.3 software. “**”, p<0.001 when compared with that of the BMP9-transduced iSCAP-Ctrl cells. (B) and (C) Histologic evaluation and Trichrome staining. After μCT imaging was completed, the samples were decalcified and subjected to paraffin-embedded sectioning for histologic evaluation, including H & E staining (B) and Trichrome staining (C). Representative results are shown. TB, trabecular bone; MBM, mineralized bone matrix; OM, osteoid matrix.