Fig 2: Quantitative RT-PCR analysis of osteogenesis-related genes in human cancellous bone of aged and young individuals. RUNX2 (A), Osterix (B), OPN (C), OCN (D), BMP-2 (E), ALP (F), FGF-2 (G), Cyclin D1 (H) WNT5a (I), Noggin (J), MMP-9 (K), MMP-13 (L). Samples (aged n = 11; young n = 17) were normalized to the housekeeping gene 18S rRNA and data are presented as mean ± S.E.M.; Student's t-test. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 3: Immunohistochemical staining of osteogenesis- and osteoclastogenesis-related proteins in human cancellous bone of old and young individuals. Sample size for aged individuals was n = 11 (RUNX2), 8 (ALP), 11 (OCN) and 11 (RANKL); for young individuals n = 5 (RUNX2), 6 (ALP), 5 (OCN) and 5 (RANKL). Additionally, the isotype control for each type of antibody is shown. Data are presented as mean ± S.E.M.; Student's t-test. (*P < 0.05). Scale bar: 20 µm.

Fig 4: Regulation of osteogenesis-related genes in human scaphoid non-unions compared to control adjacent cancellous bone. Quantitative RT-PCR determination of osteogenesis-related genes revealed that the majority of genes were significantly up-regulated (ALP (B); cyclin D1 (H); WNT5A (I); MMP-9 (J); MMP-13 (K)) compared to controls. Expression levels of RUNX-2 (A), OPN (C), OCN (D), BMP-2 (E) and FGF-2 (G) were similar in both tissues. The BMP inhibitor noggin was significantly reduced in non-unions (F). Gene expression determinations were performed relative to 18S expression and data are presented as mean ± S.E.M.; Wilcoxon signed rank test (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 5: ALP and pSMAD2/3 activity. (A) Immunohistochemistry for ALP revealed higher levels in scaphoid non-unions as opposed to cancellous bone. Likewise, immunofluorescence for phosphorylated SMAD2/3 revealed increased activity in scaphoid non-unions. Scale bars (A and B): 50 µm.