Differential activation of endogenous TGF-β and BMP signaling between FOB and POb cells.

<p>(A) Immunoblotting analysis using specific anti-phosphoSmad-2 and phoshoSmad-1/5 antibodies shows a more intense phosphorylation of Smad-2 in POb than FOb cells. In contrast, analysis with anti-phoshoSmad-1/5 antibody reveals a stronger staining in FOb. To assess for the total amount of endogenous Smad-2 and Smad-1/5 and to control for equal loading and transfer of the samples membranes were reprobed with anti-Smad-2, Smad-1/5 and anti-α-Tubulin antibodies. Histogram below represents quantification of phosphorylated Smad-2 and Smad-1/5 proteins obtained by Image J program. The relative intensity of each band was normalized to their respective α-Tubulin loading controls. (B) immunofluorescent staining using anti-phosphoSmads antibodies as above confirms the results obtained by immunoblotting analysis. Immunofluorescent staining using anti-phosphoBcl-2 antibody detects higher levels of the anti-apoptotic protein Bcl-2 in FOb compared to POb. Dapi nuclear counterstaining. (C) Immunoblotting analysis performed as above (A) showing that the differential activation of the two signaling pathways observed between FOb and POb cells is maintained throughout their osteogenic differentiation.</p

Enhanced activation of endogenous Wnt signaling in FOb and exogenous activation of Wnt signaling in POb cells protects from apoptosis.

<p>(A) Time-course Caspase-3 activity performed on FOb and POb cells undergoing osteogenic differentiation with or without Dkk and sFRP (150 ng/ml of each), as illustrated by histogram this treatment upregulates the apoptotic activity in treated FOb cells (B) The same treatment reduces dramatically mineralization of the extracellular matrix in both cell types. (C) Contrary, treatment with Wnt3a (50 ng/ml) robustly decreases apoptosis in POb cells compared to untreated cells. Wnt3a has a similar effect also on FOb cells. (D) Mineralization of the extracellular matrix detected by Alizarin red staining reveals a significant increase of osteogenesis in treated POb cells. (E) and (F) Immunodetection of active/unphosphorylated β-catenin showing the effective inhibition and/or activation of Wnt signaling upon specific treatments.</p

Apoptotic profile of FOb and POb cells undergoing to osteogenic differentiation.

<p>(A) Alizarin red staining of FOb and POb cells at differentiation day 21 shows striking differences between FOb and POb, with FOb cells having a more robust mineralization and larger bone nodules as seen in the lower panel (Magnification 10X). (B) Quantification of Alizarin red staining. (C) Time course of Caspase-3 activity performed during osteogenic differentiation of FOb and POb cells harvested from different mouse stages reveals significant higher apoptotic activity in POb cells with a peak at d12 of differentiation. Abbreviations: (E), embryonic; (P), postnatal.</p

Exogenous activation of BMP signaling decreases apoptosis in POb cells.

<p>(A) Histogram illustrating a time-course of Caspase-3 activity performed on FOb and POb cells undergoing osteogenic differentiation with or without exogenous BMP-2 (200 ng/ml). BMP-2 treatment decreases apoptosis markedly in POb cells compared to untreated POb, a lower apoptotic activity is also observed in BMP-2 treated FOb as compared to untreated FOb cells. (B) Alizarin red staining and its quantification (lower panel) showing higher levels of mineralization in BMP-2 treated cells. (C) Profile of Caspase-3 activity in FOb and POb cells undergoing osteogenic differentiation with or without 200 ng/ml of noggin, inhibitor of BMP signaling, reveals that this treatment upregulated apoptosis with major effect on P7 osteoblasts (D) Noggin treatment inhibits dramatically mineralization of the extracellular matrix on both FOb and POb treated cells, as assessed by Alizarin red staining. The lower panel represents quantification of Alizarin red staining. (E) and (F) immunoblotting analysis with specific anti-phosphoSmad-1/5 antibody to validate the effective inhibition and/or activation of BMP signaling upon specific treatments. To control for equal loading and transfer of the samples membranes were reprobed with anti-Smad-1/5 and anti-α-Tubulin antibodies.</p

Enhanced proliferative activity of <i>Axin2</i>^<i>-/-</i> FOb and POb.

<p>(<b>A</b>) <i>In vitro</i> BrdU assay performed on <i>Axin2</i>^<i>-/-</i> and wild type FOb and POb cells undergoing differentiation reveals increased proliferative activity in <i>Axin2</i>^<i>-/-</i> cells than corresponding wild type. (<b>B</b>) <i>in vivo</i> PCNA immunostaining performed on coronal sections derived from frontal and parietal bones of pN21 <i>Axin2</i>^<i>-/-</i> and wild type mice, also indicates an increase in proliferation of <i>Axin2</i>^<i>-/-</i> frontal and parietal bones compared wild type. (<b>C</b>) Quantification of PCNA staining obtained by calculating the percentage of PCNA positive cells over the total cell number counted at least in five equivalent areas of each bone, indicates the lowest cell proliferation activity in wild type parietal bone, whereas <i>Axin2</i>^<i>-/-</i> parietal bone displays activity similar to that of wild type frontal bone. Scale bar = 150 μm.</p

Enhanced Activation of Canonical Wnt Signaling Confers Mesoderm-Derived Parietal Bone with Similar Osteogenic and Skeletal Healing Capacity to Neural Crest-Derived Frontal Bone

<div><p>Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of <i>axin2</i>, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between <i>Axin2</i>^<i>-/-</i> and wild type calvarial bones, with <i>Axin2</i>^<i>-/-</i> showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in <i>Axin2</i>^<i>-/-</i> and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in <i>Axin2</i>^<i>-/-</i> mice as compared to wild type, with <i>Axin2</i>^<i>-/-</i> parietal bone and derived osteoblasts displaying a “neural crest-derived frontal bone-like” profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.</p></div

<i>In vivo</i> calvarial healing of <i>Axin2</i>^<i>-/-</i> and wild type frontal and parietal bones.

<p><b>(A</b>) Two-millimeter (2mm) defects were created in the frontal and parietal bones of 7 month-old <i>Axin2</i>^<i>-/-</i> and wild type mice (n = 3). Quantification of defect repair according to microCT-scan results. Statistical analysis was conducted utilizing the Mann-Whitney Test. P-values: *P≤ 0.05. (<b>B</b>) Pentachrome staining of coronal sections of skull at post-operative week 8 showing the repair of calvarial bone defects as determined by yellow color. Bone regeneration was higher in <i>Axin2</i>^<i>-/-</i> frontal and parietal bones as compared to wild type bones. <b>(C)</b> Histogram showing the distance between the osteogenic fronts (dashed) and marked by arrows (Objective magnification 5x).</p

<i>In vitro</i> differentiation and osteogenic-related gene expression analysis of <i>Axin2</i>^<i>-/-</i> and wild type FOb and POb cells.

<p>(<b>A)</b> quantitative PCR analysis performed on wt and <i>Axin2</i>^<i>-/-</i>FOb and POb showing a significant upregulation of cWnt signaling target genes in both, <i>Axin2</i>^<i>-/-</i>FOb and POb as compared to wt osteoblasts. (<b>B</b>) enhanced activation of cWnt signaling in <i>Axin2</i>^<i>-/-</i>FOb and POb is further confirmed by indirect immunofluorescence analysis showing larger number of cells with positive nuclear staining for active β-catenin. Positive nuclear staining is more dramatic in <i>Axin2</i>^<i>-/-</i>POb. Nuclear counterstaining was performed with DAPI (objective magnification 10x). Scale bars = 50μm. (<b>C</b>) Cells were cultured with differentiation medium for 18 days. Mineralization of extracellular matrix as assessed by alizarin red staining indicates a more robust mineralization in <i>Axin2</i>^<i>-/-</i> FOb and POb as compared to corresponding wild type FOb and POb. (<b>D</b>) Magnification of alizarin red stained bone nodules. (<b>E</b>) Quantification of alizarin red staining as showed above (panel A) confirms enhanced osteogenic capacity of FOb and POb cells derived from <i>Axin2</i>^<i>-/-</i> mice. (<b>F</b>) RT-PCR analysis of osteogenic markers showing significant higher up-regulation of the <i>Runx2</i>, <i>Alpl</i> and <i>Bglap</i> in <i>Axin2</i>^<i>-/-</i> FOb and POb. (<b>G</b>) Histograms representing quantification of each electrophoretic band obtained by Image J program. Each band was normalized to its <i>Gapdh</i> content. *P≤ 0.05.</p

Characterization of Cells Isolated from Genetic and Trauma-Induced Heterotopic Ossification

<div><p>Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common <i>in vitro</i> properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured <i>in vitro</i>, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for <i>in vitro</i> investigations for drug screening purposes.</p></div

Figure 3

<p>LOH and copy number changes on chromosomes 11, 3, 4, 1, 17, and 7. Global view of common areas of LOH (left) and copy number change (right) in 22 primary neuroblastomas. Each sample is depicted as a series of vertical bars in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel).</p