PTH 1-34 Ameliorates the Osteopenia and Delayed Healing of Stabilized Tibia Fracture in Mice with Achondroplasia Resulting from Gain-Of-Function Mutation of FGFR3

Abstract

Bone fracture healing is processed through multiple stages including the cartilaginous callus formation and its transition to bony callus. FGFR3 negatively regulates chondrogenesis and enhances osteogenesis during skeleton development. We previously found in mice carrying gain-of-function mutation of FGFR3 that FGFR3 delays the healing of un-stabilized fracture that heals mainly through endochondral ossification. Since fracture is regularly treated in clinics with rigid fixation, and stabilized fracture is healed largely through intramembranous ossification, we asked whether FGFR3, a key regulator of osteogenesis, also affect the regeneration of stabilized fracture. We found that gain-of-function mutation of FGFR3 inhibits the initiation of chondrogenesis and the subsequent bone formation. We further studied whether PTH1-34 can improve the osteopenia and delayed healing of the stabilized tibia fracture in mice with achondroplasia. Fracture healing was evaluated by radiography, micro-CT, biomechanical tests, histology, and real-time polymerase chain reaction (RT-PCR) analysis. We found that PTH 1-34 can alleviate the decreased bone mass and compromised architecture in ACH mice. Histological analysis revealed that administration of PTH1-34 increased the size of both the total callus and cartilaginous callus at 14 days after the surgery in ACH mice. RT-PCR data suggested that systemic PTH1-34 accelerated the initiation of chondrogenesis and chondrocyte maturation (earlier and higher levels of expression of chondrogenesis related markers) and enhanced the osteogenic differentiation in the fracture callus in ACH mice. These results indicate that the PTH1-34 administration resulted in an enhanced callus formation during bone fracture healing in ACH mice, which is at least in part mediated by an increase of cartilaginous callus at early stage and the promotion of bone formation in bony callus. In summary, in this study we revealed that FGFR3 delays the regeneration of stabilized fracture by inhibiting both the chondrogenesis and osteogenesis, and PTH1-34 treatment can improve the dysregulated bone metabolism and delayed bone injury healing resulting from gain-of-function mutation of FGFR3.

Figure 1

PTH1-34 alleviates the osteopenia in ACH mice. (A) BMD of total femurs from ACH and WT mice measured by DEXA (n=8). (B) Quantitative micro-CT analyses of distal femoral metaphysis (BV/TV, Tb.N and Tb.Th) at 12 weeks (n=6-8). (C) Histology (H&E staining) of the metaphysis of proximal tibiae in wild-type mice and ACH mice treated by vehicle and PTH 1-34 at 12 weeks. (magnification ×200). (D) Double calcein labeling of the undecalcified femurs. (E) Quantitative data of MAR. Graphs show mean value ± SD (*, P <0.05; **, P <0.01; ***, P <0.001; compared with WT control mice. #, P <0.05; ##, P <0.01; compared with ACH control mice).

Figure 2

PTH1-34 treatment alleviates the impaired fracture healing of ACH mice. (A) Representative radiographs of fractured tibias. (B) The quantitative analysis for total callus volume. (C) The quantitative analysis of bony callus volume. (D) The quantitative analysis of BMD. Data are presented as Means ± SD (N=6-8/group; *, P <0.05; **, P <0.01; ***, P <0.001; compared with WT control mice at the same group. ##, P <0.01; compared with ACH control mice at the same group).

Figure 3

Biomechanical properties of the fracture calluses of ACH were improved by PTH 1-34 treatment. (A) Stiffness, (B) ultimate force to failure, and (C) work to failure were assessed by three-point bending in tibias at 28 days post-fracture in four groups. Data are presented as Means±SD (N=6/group, *p<0.05; **p<0.01; ***p<0.001, compared with WT control mice. #, P <0.05; ##, P <0.01; compared with ACH control mice).

Figure 4

Effects of exogenous PTH on cartilaginous callus formation and its transformation into bony callus. Representative micrographs of paraffin sections of calluses from mice at 1,2 and 3 weeks post fracture (A). Areas of the total callus (B, E), cartilaginous callus (C, F) and bony callus (D, G) were measured by computer-assisted image analysis. Each value is the Means±SD of determinations in 6-8 animals from each group (*p<0.05; **p<0.01; ***p<0.001, compared with WT control mice. #, P <0.05; ##, P <0.01; compared with ACH control mice).

Figure 5

Analysis of the PTH 1-34 effects on cellular differentiation during fracture healing in WT and ACH mice. Total RNA was extracted from callus in mice at various time points after fracture. Real-time RT-PCR mRNA expression analyses were performed for evaluating expressions of genes involved in chondrogenesis in callus from mice after fracture and results were expressed as fold changes relative to the expression level of WT callus at PFD7(A-D). The following primer sets were used: cbfa1 (E), alp (F), ocn (G) and col1 (H) to evaluate the osteogenic differentiation during fracture healing. Results were expressed as fold changes relative to the expression level of WT callus at PFD7. The analysis was repeated for three times. Data are presented as Means±SD (N=5-6 mice/group; *, P <0.05; **, P <0.01; ***, P <0.001; compared with WT control mice at the same group. #, P <0.05; ##, P <0.01; compared with ACH control mice at the same group).