Figure 1.
Histological evaluation of relative amounts of tissues in the fracture calli at different time points post fracture.
WT: white columns, Fzd9−/−: grey columns. All values are presented as median, interquartile ranges, minimum and maximum. n = 5–10; Mann-Whitney-U-test, *p<0.05. TOT: total osseous tissue, Cg: cartilage, FT: fibrous tissue. A: day 10, WT: n = 6, Fzd9−/−: n = 5, B: day 24, WT: n = 7, Fzd9−/−: n = 8, C: day 32, WT: n = 10, Fzd9−/−: n = 8.
Figure 2.
Representative histological sections of the fracture calli of WT and Fzd9−/− mice at different time points post fracture.
The upper panel shows sections of osteotomized WT femurs and the lower one femurs of Fzd9−/−. Decalcified sections 10 days post fracture were stained with Safranin O. Undecalcified femurs 24 and 32 days post fracture were stained with Giemsa.
Figure 3.
Immunostaining of β-catenin at day 10 and histochemical TRAP staining indicating osteoclasts at day 24 in the fracture calli.
WT: upper panel, Fdz9−/−: lower panel. β-catenin was expressed in osteoblasts (OB) and proliferating chondroblasts (CB) but to a lesser extend in hypertrophic chondrocytes (HC). C: cortex. There were no differences between both genotypes. Only TRAP positive cells with ≥2 nuclei were identified as osteoclasts (OC). TRAP-staining either revealed no significant differences between both genotypes.
Figure 4.
Immunostaining of osteoblast differentiation markers Runx2 and Osteocalcin at day 10.
WT: upper panel, Fdz9−/−: lower panel. Runx2 (left) was expressed mainly in the nucleus of preosteoblastic cells and osteoblasts (OB) located in the fracture callus. Osteocalcin (right) was mainly localized in the cytoplasma of osteoblasts near mineralized matrix and in the bone matrix. There were no differences in staining of Runx2 and Osteocalcin between both genotypes. C: cortex, OT: osteocytes.
Figure 5.
Immunostaining of chemokines Cxcl5 and Ccl2 at day 10.
WT: upper panel, Fdz9−/−: lower panel. C: cortex. Cxcl5 (left) and Ccl2 (right) were expressed by precursor cells, osteoblasts and chondrocytes in both genotypes, but staining was less intense in the absence of Fzd9. C: cortex, HC: hypertrophic chondrocytes, OB: osteoblasts, OT: osteocytes.
Figure 6.
µCT evaluation of the fracture calli of WT and Fzd9−/− mice at days 24 and 32 post fracture.
A: Bone volume/total volume (BV/TV). B: Bone mineral density (BMD) C: 3D-models of femurs at day 24 in anterior-posterior (left) and lateral view (right). The upper panel shows WT femurs and the lower panel Fzd9−/− femurs. Fzd9−/− mice exhibited a less mineralized callus at both investigated time points. At day 24, the geometry of the calli of Fzd9−/− mice was different from WT mice, resulting in an increased moment of inertia (Ix) (see Figure 7C).
Figure 7.
Mechanical characterization of the fracture calli of WT and Fzd9−/− mice at days 24 and 32 post fracture.
All values are presented as median, interquartile ranges, minimum and maximum. n = 5–10; Mann-Whitney-U-test, *p<0.05. A: Apparent Young's Modulus Eapp calculated as the ratio of flexural rigidity and moment of inertia, which describes the apparent material properties of the newly formed tissue. B: Flexural rigidity of the fracture calli measured by 3-point-bending test. C: Moment of inertia (Ix) of the fracture calli measured by micro-computed tomography (µCT).