Modulation of Wnt signaling influences fracture repair

Abstract

While the importance of Wnt signaling in skeletal development and homeostasis is well documented, little is known regarding its function in fracture repair. We hypothesized that activation and inactivation of Wnt signaling would enhance and impair fracture repair, respectively. Femoral fractures were generated in Lrp5 knockout mice (Lrp5-/-) and wild-type littermates (Lrp5+/+), as well as C57BL/6 mice. Lrp5-/- and Lrp5+/+ mice were untreated, while C57BL/6 mice were treated 2x/week with vehicle or anti-Dkk1 antibodies (Dkk1 Ab) initiated immediately postoperatively (Day 0) or 4 days postoperatively (Day 4). Fractures were radiographed weekly until sacrifice at day 28, followed by DXA, pQCT, and biomechanical analyses. Lrp5-/- mice showed impaired repair compared to Lrp5+/+ mice, as evidenced by reduced callus area, BMC, BMD, and biomechanical properties. The effects of Dkk1 Ab treatment depended on the timing of initiation. Day 0 initiation enhanced repair, with significant gains seen for callus area, BMC, BMD, and biomechanical properties, whereas Day 4 initiation had no effect. These results validated our hypothesis that Wnt signaling influences fracture repair, with prompt activation enhancing repair and inactivation impairing it. Furthermore, these data suggest that activation of Wnt signaling during fracture repair may have clinical utility in facilitating fracture repair.

Figure 1

Longitudinal radiographs. Representative longitudinal radiographs from male and female Lrp5−/− and Lrp5+/+ mice, as well as male mice treated with vehicle, Dkk1 Ab Day 0, and Dkk1 Ab Day 4. Radiographs are arranged by group (columns) and by time-point (rows). Each longitudinal series depicts the same animal at each of the indicated time points.

Figure 2

Longitudinal analysis of callus area. Graphs depicting mean callus area, measured from digital radiographs, over weeks 2, 3, and 4 for: (A) Female Lrp5+/+ and Lrp5−/− mice; (B) male Lrp5+/+ and Lrp5−/− mice and; (C) vehicle and Dkk1 Ab-treated mice. Error bars are ±1 SEM. p-Values for tests of differences between genotypes (Lrp5+/+vs. Lrp5−/−) are p =0.0009 at week 2, p =0.0039 at week 3, and p =0.0048 at week 4. p-Values for tests of differences among treatments (vehicle vs. Dkk1 Ab Day 0 vs. Dkk1 Ab Day 4) are indicated on the graph.

Figure 3

Dual-energy X-ray absorptiometry (DXA). Graphs illustrating average femoral BMD, BMC, and area of isolated femora as determined by DXA analyses. All raw values were normalized to contralateral intact values for analysis of genotype and sex effects (A–C). Actual values are reported for both intact and fractured (callus) femurs from vehicle and Dkk1 Ab-treated mice (D–F). Error bars are ±1 SEM. p-Values for tests of differences between genotypes (Lrp5+/+vs. Lrp5−/−) and among treatments (vehicle vs. Dkk1 Ab Day 0 vs. Dkk1 Ab Day 4) are given.

Figure 4

Peripheral quantitative computed tomography (pQCT). Series of graphs showing average cross-sectional density and area obtained from pQCT scanning of intact and fractured femora. All raw values were normalized to contralateral intact values for analysis of genotype and sex effects (A–C). Actual values are reported for both intact and fractured (callus) femurs from vehicle and Dkk1 Ab-treated mice (E, F). Error bars are ±1 SEM. p-Values for tests of differences between genotypes (Lrp5+/+ vs. Lrp5−/−) and among treatments (vehicle vs. Dkk1 Ab Day 0 vs. Dkk1 Ab Day 4) are given.

Figure 5

Biomechanical properties. Results of 4-point bending tests used to determine biomechanical properties of energy to failure, strength and stiffness of intact and fractured femora. All raw values were normalized to contralateral intact values for analysis of genotype and sex effects (A–C). Actual values are reported for both intact and fractured (callus) femurs from vehicle and Dkk1 Ab-treated mice (D–F). Error bars are ±1 SEM. p-Values for tests of differences between genotypes (Lrp5+/+vs. Lrp5−/−) and among treatments (vehicle vs. Dkk1 Ab Day 0 vs. Dkk1 Ab Day 4) are given.