Ectopic bone formation analyses.

A: Bone formation capability of muscle-derived cells. Representative histological sections of a scaffold loaded with BMSCs or MuSCs cultured with both dexamethasone and BMP-2. Scale bar: 1 mm (left panels), 200 μm (middle panels) and 50 μm (right panels). Black arrows indicate new bone formation in the scaffold. Black arrow heads indicate osteocytes and green arrow heads indicate bone lining cells. B: Newly formed bone, T: β-TCP, P: Porous area. B: Recruitment of cells residing in muscle tissue to participate in BMP-2-induced ectopic bone formation. Cells labeled prior to local BMP-2 administration were detected in the newly formed woven bone area. Representative histological sections were stained with H&E and evaluated for i-QD fluorescence. The black arrows in the H&E image show the locations of fluorescently labeled cells indicated with white arrows in the fluorescent image. Scale bar: 200 μm. B: Newly formed bone, T: β-TCP, P: Porous area. C: Augmentation of ectopic bone formation by dexamethasone. C-1, 2: Representative histological sections of an excised scaffold that had been loaded with BMP-2 alone and a scaffold that had been loaded with dexamethasone and BMP-2. The sections were stained with H&E and immunostained for osteocalcin. Black arrows indicate new bone formation in the scaffold. Red arrows indicate osteocalcin positive staining area. Scale bar: 1 mm (top panels of C-1), 200 μm (bottom panels of C-1) and 50 μm (C-2). B: Newly formed bone, T: β-TCP, P: Porous area. C-3: Quantification of bone formation at 3 weeks after transplantation. The Y axis indicates the bone formation ratio calculated as total bone area/total scaffold area. Each bar represents the mean with the standard deviation (SD). *denotes P < 0.05.