Dexamethasone pretreatment and osteogenic induction with combined dexamethasone and BMP-2 treatment enhance the osteogenic differentiation of BMSCs and MuSCs.

<p>A: Schematic representation of the cell culture protocol. Gross images of ALP staining (ALP) and Von Kossa staining (VK) of BMSCs (B) and MuSCs (D). Quantitative analysis of the mRNA expression of ALP and osteocalcin in BMSCs (C) and MuSCs (E). The fold change in gene expression was normalized to that of BM-Dex-AG or Mu-Dex-AG. Bars show the mean and SEM. Statistical significance was confirmed between the BM and BM-Dex groups (ALP: p = 0.015, OCN: p = 0.023) and between the Mu and Mu-DEX groups (ALP: p = 0.019, OCN: p = 0.015). Effects of combinations of differentiation reagents were significant for ALP in BM-Dex (p = 0.032) and Mu-DEX (p = 0.019) and for OCN in Mu-DEX (p = 0.024).</p

Ectopic bone formation analyses.

<p>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 <i>P</i> < 0.05.</p

Colony formation assay of BMSCs and MuSCs.

<p>A: Schematic representation of the colony formation unit assay protocol. Cells seeded at P2 were allowed to form single-cell-derived colonies with or without 10^-7 M dexamethasone for 7 days before osteogenic induction. ND-ND indicates normal growth medium at P0, P1, and P2. ND-D indicates normal growth medium at P0 and P1 and dexamethasone-containing medium at P2. D-ND indicates dexamethasone-containing medium at P0 and P1 and normal growth medium at P2. D-D indicates dexamethasone-containing medium at P0, P1, and P2. Gross images of BMSCs (B) and MuSCs (D) in each dish stained by ALP and crystal violet. Quantification of the total colony number and fraction of ALP-positive colonies (%) among total colonies in BMSCs (C) and MuSCs (E). *** denotes P < 0.001 as determined by Student’s t-test.</p

Western blot analyses of the SMAD1/5/8 and phosphorylation of SMAD 1/5.

<p>Western blot analyses of P-SMAD 1/5, SMAD1/5/8 and α-tubulin expression in BMSCs (A) and MuSCs (B) under four different osteogenic induction conditions with or without dexamethasone.</p

Dexamethasone affects cell proliferation during osteogenic differentiation.

<p>The absorbance at 585 nm was measured for dye extracted from the wells, and ratios relative to the standard are presented in the graphs. A: BM, B: BM-Dex, C: Mu, and D: Mu-Dex</p

RT-PCR primers used in this study.

<p>RT-PCR primers used in this study.</p