FGFR3 deficient mice have accelerated fracture repair

Abstract

Bone fracture healing is processed through multiple biological stages that partly recapitulates the skeletal development process. FGFR3 is a negative regulator of chondrogenesis during embryonic stage and plays an important role in both chondrogenesis and osteogenesis. We have investigated the role of FGFR3 in fracture healing using unstabilized fracture model and found that gain-of-function mutation of FGFR3 inhibits the initiation of chondrogenesis during cartilage callus formation. Here, we created closed, stabilized proximal tibia fractures with an intramedullary pin in Fgfr3^-/-mice and their littermate wild-type mice. Fracture healing was evaluated by radiography, micro-CT, histology, and real-time polymerase chain reaction (RT-PCR) analysis. The fractured Fgfr3^-/- mice had increased formation of cartilaginous callus, more fracture callus, and more rapid endochondral ossification in fracture sites with up-regulated expressions of chondrogenesis related gene. The fractures of Fgfr3^-/- mice healed faster with accelerated fracture callus mineralization and up-regulated expression of osteoblastogenic genes. The healing of fractures in Fgfr3^-/- mice was accelerated in the stage of formation of cartilage and endochondral ossification. Downregulation of FGFR3 activity can be considered as a potential bio-therapeutic strategy for fracture treatment.

Keywords: FGFR3; fracture treatment.

Figure 1

FGFR3 deficiency mice exhibit accelerated fracture healing. Tibia fractures were created in 8-week-old Fgfr3^-/- mice and wild-type (WT) controls. Tibias with fractures were harvested at 7, 14, 21 and 28 days after fracture. (A) Representative radiographs of fractured tibias. (B) Longitudinal sections of micro-CT 3-dimensional reconstructed calluses from WT and Fgfr3^-/- mice. (C) The quantitative analysis for calcified callus volume and BMD. Data are presented as Means ± SD (N=6-8/group, *p<0.05, **p<0.01).

Figure 2

Biomechanical properties of the fracture calluses. (A) Stiffness, (B) ultimate force to failure, (C) work to failure, and (D) ultimate displacement results were assessed by three-point bending in tibias from 21 and 28 days post-fracture in Fgfr3^-/- mice and wild-type (WT) controls. Data are presented as Means±SD (N=6/group, *p<0.05, **p<0.01).

Figure 3

Fractures in Fgfr3^-/-mice undergo accelerated endochondral bone formation. (A) Representative histology of fractured tibias stained with Safranin O/Fast Green show cartilage (red) and bone (green) formation. (B-D) Histomorphometric measurements (total callus area, cartilage area, bony callus area) were made from 20 sections for each group (N=5-6 mice/group). (E) Real-time RT-PCR mRNA expression analyses were performed for evaluating expressions of genes involved in chondrogenesis in callus from WT and Fgfr3-/- mice after fracture and results were expressed as fold changes relative to expression level of WT callus at PFD3. The analysis was repeated for three times. Data are presented as Means±SD ( N=5-6 mice/group, *p<0.05, **p<0.01).

Figure 4

The expression of genes involved in osteogenesis is altered in callus from Fgfr3^{-/ -}mice. Total RNA was extracted from callus in Fgfr3^-/- mice and controls (N=5 mice/group) at various time points after fracture. The following primer sets were used: RUNX2 (A), Col1a1 (B), ALP (C), OCN (D), FGFR1 (E), and FGFR2 (F). Results were expressed as fold changes relative to expression level of WT callus at PFD3. The analysis was repeated for three times. Data are presented as Means±SD ( N=5-6 mice/group, *p<0.05, **p<0.01).

Figure 5

Fgfr3^-/- mice have accelerated bone remodeling in fracture healing. Representative micrographs of sections of calluses from control and Fgfr3^-/- mice at 14 and 21days post-fracture stained histochemically for TRAP. (B) Number of TRAP-positive osteoclasts related to tissue area (N.Oc/T.Ar, #/mm²) and osteoclast surface relative to bone surface (Oc.S/BS, %) were assessed by computer-assisted image analysis. RANKL (C) and OPG (D) RNA levels in the fracture callus were examined by RT-PCR. Results were expressed as fold changes relative to expression level of WT callus at PFD7. Data are presented as Means±SD ( N=5-6 mice/group, *p<0.05, **p<0.01).

Figure 6

Schematic illustration of the role of FGFR3 in bone repairing. FGFR3 is strongly expressed in prehypertrophic chondrocytes, osteoblasts and periosteum during fracture repair. FGFR3 plays a negative role in bone repairing which is similar with its role in bone development.