Fig. 5: Mkx gene knockdown or knockout induces osteogenic differentiation and calvarial bone repair.
A, B siRNA mediated knockdown of Mkx within calvarial suture cells. A Confirmation of siRNA mediated Mkx knockdown efficiency by qPCR, 48 h after siRNA treatment. B Representative Alizarin red staining at d 10 of osteogenic differentiation (left) and quantification (right). C, D Ad-Cre mediated knockout of Mkx within Mkxfl/fl calvarial suture cells. C Confirmation of Ad-Cre mediated Mkx knockout efficiency by qPCR, 48 h after Ad-Cre treatment. D Representative Alizarin red staining at d 10 of osteogenic differentiation (left) and quantification (right). E Schematic of calvarial bone defect. Mkxflox mice were locally administered Ad-Cre or Ad-GFP directly overlying the skull, and thereafter subjected to calvarial defect creation. Samples were harvested at d 28 post-defect. F Mkx gene knockout efficiency tested by immunostaining (left) and quantification (right) within the sagittal suture of Mkxflox mice 28 d after viral injection. G μCT reconstructions of the defect site in a top-down view (above) and sagittal cross-sectional images (below). Analysis performed at d 28 post-defect. Margins of original defect are indicated by dashed red lines. H–K μCT quantification of bone healing among control and Ad-Cre treated mice, including H bone volume (BV), I bone volume/tissue volume (BV/TV), J residual defect diameter, and K bone fractional area (BFA). L H&E staining of coronal cross section of the healing defect site from control and Ad-Cre treated mice, d 28 after defect. Black arrowheads indicate healing bone edges. Scale bar: 100 μm. M Immunostaining of OCN at the defect edge (left) and quantification of OCN (right). Dots in scatterplots represent values from individual measurement, whereas mean and 1 SD are indicated by crosshairs and whiskers. *p < 0.05; **p < 0.01. A two-tailed Student t-test was used for all comparisons.
