Mice with a heterozygous Lrp6 deletion have impaired fracture healing

Abstract

Bone fracture non-unions, the failure of a fracture to heal, occur in 10%-20% of fractures and are a costly and debilitating clinical problem. The Wnt/β-catenin pathway is critical in bone development and fracture healing. Polymorphisms of linking low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt-binding receptor, have been associated with decreased bone mineral density and fragility fractures, although this remains controversial. Mice with a homozygous deletion of Lrp6 have severe skeletal abnormalities and are not viable, whereas mice with a heterozygous deletion have a combinatory effect with Lrp5 to decrease bone mineral density. As fracture healing closely models embryonic skeletal development, we investigated the process of fracture healing in mice heterozygous for Lrp6 (Lrp6 (+/-)) and hypothesized that the heterozygous deletion of Lrp6 would impair fracture healing. Mid-diaphyseal femur fractures were induced in Lrp6 (+/-) mice and wild-type controls (Lrp6 (+/+)). Fractures were analyzed using micro-computed tomography (μCT) scans, biomechanical testing, and histological analysis. Lrp6 (+/-) mice had significantly decreased stiffness and strength at 28 days post fracture (PF) and significantly decreased BV/TV, total density, immature bone density, and mature area within the callus on day-14 and -21 PF; they had significantly increased empty callus area at days 14 and 21 PF. Our results demonstrate that the heterozygous deletion of Lrp6 impairs fracture healing, which suggests that Lrp6 has a role in fracture healing.

Figure 1

Biomechanical measures of intact bone. There was no statistical difference in (a) stiffness and (b) maximum load in Lrp6 ^+/− and Lrp6 ^+/+ controls at any time point. Error bars indicate one s.d. above the Lrp6 ^+/+ group and one s.d. below the Lrp6 ^+/− group.

Figure 2

Biomechanical measures of fractured femurs. There was a significant decrease in the (a) stiffness and (b) maximum load at 28 days post fracture (PF) in the Lrp6 ^+/− group. Error bars indicate one s.d. above the Lrp6 ^+/+ group and one s.d. below the Lrp6 ^+/− group (*P<0.05, Lrp6 ^+/− and Lrp6 ^+/+ at the time point).

Figure 3

Stiffness and maximum load ratios of fractured to intact femurs. There was no statistical difference in the (a) stiffness load ratio at any time point. There was a significant decrease in the (b) maximum load ratio at 28 days post fracture (PF) in the Lrp6 ^+/− group. Error bars indicate one s.d. above the Lrp6 ^+/+ group and one s.d. below the Lrp6 ^+/− group (*P<0.05, Lrp6 ^+/− and Lrp6 ^+/+ at the time point).

Figure 4

Bone volume over tissue volume (BV/TV) and immature bone volume over tissue volume (IV/TV) ratio for fracture callus. There was a significant decrease in BV/TV and IV/TV at days 14 and 21 post fracture (PF) in the Lrp6 ^+/− group. Error bars indicate one s.d. above the Lrp6 ^+/+ group and one s.d. below the Lrp6 ^+/− group (*P<0.05, Lrp6 ^+/− and Lrp6 ^+/+ at the time point, and note that IV/TV significance is indicated below the data points).

Figure 5

Total callus density, mature callus density, and immature callus density in the fracture callus. There was a significant decrease in (a) total callus density at days 14 and 21 post fracture (PF) in the Lrp6 ^+/− group and in the (b) immature callus density at day 14 PF. There was no significant difference in the mature callus density at any time point. Error bars indicate one s.d. above the Lrp6 ^+/+ group and one s.d. below the Lrp6 ^+/− group (*P<0.05, Lrp6 ^+/− and Lrp6 ^+/+ at the time point).

Figure 6

Total callus area, mature callus area, immature callus area, and empty callus area in the fracture callus. There was no statistical difference in the (a) total area or immature area at any time point. There was a significant increase in the (b) empty area and a significant decrease in the (b) mature area at days 14 and 21 post fracture (PF) in the Lrp6 ^+/− group. There was no statistical difference in total area or immature area at any time point. Error bars indicate one s.d. above for the group with the larger area and one s.d. below for the group with the smaller area (*P<0.05, Lrp6 ^+/− and Lrp6 ^+/+ at the time point).

Figure 7

Representative β-catenin staining (for Wnt signaling activity, brown). There is similar β-catenin expression in the Lrp6 ^+/− group (left) compared with the Lrp6 ^+/+ controls (right) throughout healing (day 14 post fracture (PF), top; and day 28 PF, bottom, shown here).

Figure 8

Representative tartrate-resistant acid phosphatase (TRAP) staining (for osteoclasts, purple). There is similar TRAP expression in the Lrp6 ^+/− group (left) compared with the Lrp6 ^+/+ controls (right) throughout healing (day 14 post fracture (PF), top; and day 28 PF, bottom, shown here).