Fig. 6

TM-25659 promotes in vivo bone formation of ADSCs. a Schematic description of experimental procedure for in vivo transplantation. Adipose-derived stem cells (ASCs) were initially treated with osteoinductive medium and TM-25659 for consecutive 7 days, then harvested, seeded on porous β-TCP blocks as a carrier, and subcutaneously transplanted into nude mice (six animals per experimental group). Six weeks later all transplants were harvested for further analysis. b Hematoxylin and eosin (H&E) and Masson trichrome staining revealed markedly enhanced bone formation in samples from ADSCs treated with TM-25659 compared with vehicle-treated samples. Scale bar = 100 μm. c Quantification of bone formation in samples indicated significantly more bone formation in ADSCs treated with TM-25659. Ten images of Masson staining (400×) were randomly selected in the slides from two experimental groups and captured under microscopy. The area of new bone in each image was marked using ImageJ and the percentage of new bone over total area was calculated. d Immunohistochemical staining of osteopontin (OPN) and osteocalcin (OCN) in samples revealed elevated OPN and OCN abundance in samples from ADSCs treated with TM-25659 compared with vehicle-treated samples. Scale bar = 100 μm. Data are shown as fold-change compared with vehicle-treated samples which were defined as 1.0. Data shown here are mean ± SD from two independent experiments; **P < 0.01, by Student’s t test. NB new bone, S scaffold