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

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

<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