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Anti-DKK1 Antibody Promotes Bone Fracture Healing Through Activation of β-Catenin Signaling

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Anti-DKK1 Antibody Promotes Bone Fracture Healing Through Activation of β-Catenin Signaling

Hongting Jin et al. Bone.

Abstract

In this study we investigated if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of β-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in β-catenin(Prx1ER) conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (μCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the β-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through β-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in β-catenin(Prx1ER) conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.

Keywords: Conditional knockout; Dkk1-Ab; Fracture healing; Mesenchymal progenitor cells; β-Catenin.

Conflict of interest statement

Disclosures

Ke and Babij are Amgen employees and have received Amgen stock or stock options, and Chen received research contract from Amgen. All other co-authors have no conflict of interest.

Figures

Fig. 1
Fig. 1
DKK1-Ab promotes bone fracture healing in CD-1 mice. Fracture procedure was conducted in 10-week-old mice treated with DKK1-Ab or Vehicle. X-ray radiographic analysis was performed in mice 1, 7, 10, 14, and 21 days after fracture. Radiographs show resolution of fracture lines at day 14 and 21 in mice receiving DKK1-Ab treatment, suggesting that fracture healing process is accelerated. Red arrows indicate the fracture lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
DKK1-Ab increases bone volume and mechanical strength of fracture callus. Micro-CT and mechanical testing were performed on bone samples derived from mice treated with DKK1-Ab or Vehicle at 7, 10, 14 and 21 days after fracture. Micro-CT analysis showed that bone volume and BV/TV were increased in callus tissues 10, 14 and 21 days after fracture in the mice treated with DKK1-Ab. Mechanical testing showed that maximal torque and stiffness were increased by DKK1-Ab treatment 14, 21 and 28 days after fracture. Data are presented as means ± SD. *p < 0.05, one-way ANOVA followed by Tukey’s test, n = 6.
Fig. 3
Fig. 3
DKK1-Ab increases cartilage and bone volume of fracture callus. Histological analysis showed that fracture healing process was accelerated by DKK1-Ab treatment. On day 7, cartilage area of callus tissues (Alcian blue staining positive area) was increased after DKK1-Ab treatment (A, B). On day 10–21, new woven bone formation in callus tissues was enhanced by DKK1-Ab treated (A, C). Black arrows: cartilage area; blue arrows: woven bone area. Data are presented as means ± SD. *p < 0.05, one-way ANOVA followed by Tukey’s test, n = 6.
Fig. 4
Fig. 4
DKK1-Ab enhanced chondrocyte- and osteoblast-specific marker gene expression in callus tissues of mice. Total RNA was extracted from callus tissues 7, 10, 14, 21 and28 days after fracture in the mice treated with or without DKK1-Ab. Expression of chondrocyte-specific genes was examined. Expression of Sox9 was increased at day 7 (A); expression of Col2a1 was increased at days 7, 10, and 14 (B); and expression of Col10a1 was increased at day 14, 21, and 28 (C). Expression of Runx2 and Osterix was increased at days 14, 21, and 28 (D and E) and expression of osteocalcin(OC) was increased at days 21 and 28 (F). Expression of Dkk1 and β-catenin was examined. Expression of DKK1 was decreased at days 7 and 10 (G) and expression of β-catenin was increased at days 7 and 10 (H). Data are presented as means ± SD. *p < 0.05, one-way ANOVA followed by Tukey’s test, n = 3.
Fig.5
Fig.5
Bone mass was decreased in β-cateninPrx1ER conditional KO mice. Tamoxifen was administered to 2-week-old mice and μCT and histological analyses were performed in 3-month-old mice. Significant bone loss was observed in μCT 3D images and histological sections (A and B). Quantitative μCT analysis showed that bone volume (BV/TV), bone mineral density (BMD), trabecular numbers (Tb.N.) and connectivity density (Conn D) were significantly decreased in β-cateninPrx1ER mice (C–E and H). In contrast, trabecular separation (Tb.Sp.) was significantly increased (F). *p < 0.05, unpaired Student t-test, n = 4.
Fig. 6
Fig. 6
DKK1-Ab had no significant effect on fracture healing in β-cateninPrx1ER mice. Fracture procedure was performed in 10-week-old β-cateninPrx1ER and Cre-negative control mice. Tamoxifen was injected for 5 days immediately following fracture followed by treatment with DKK1-Ab. Radiographs showed that fracture healing was accelerated by DKK1-Ab treatment in Cre-negative control mice. In contrast, DKK1-Ab had no significant effect on fracture healing in β-cateninPrx1ER mice. Fracture lines are still clear at day 21 and 28 in β-cateninPrx1ER mice with or without DKK1-Ab. Red arrows: fracture lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7
DKK1-Ab had no significant effect on bone structure and mechanical strength in β-cateninPrx1ER mice. μCT and mechanic testing were performed on bone samples collected from mice 7, 10, 14, 21 and 28 days after fracture. Results of μCT analysis and mechanical testing showed that there was a significant increase in bone volume at days 10 and 14 (A–C) and significant increases in maximal torque at day 10–28 and significant increases in the stiffness day 10, 14 and 28 in Cre-negative control mice treated with DKK1-Ab. In contrast, there were no significant changes in bone volume, maximal torque, and stiffness in β-cateninPrx1ER mice treated with DKK1-Ab (B–E). Significant reductions in maximal torque (at day 21 and 28) and the stiffness (at day 14–28) were observed in β-cateninPrx1ER mice compared to Cre-negative control mice. Data are presented as means ± SD. *p < 0.05, between DKK1-Ab and PBS groups (Cre-negative control mice); #p < 0.05, between Cre-negative and β-cateninPrx1ER groups (without DKK1-Ab); two-way ANOVA followed by Tukey’s test, n = 6.
Fig. 8
Fig. 8
DKK1-Ab had no significant effect on cartilage and bone volume of fracture callus in β-cateninPrx1ER mice. Histological analysis was performed on day 7, 10, 14 and 21 samples and showed that cartilage volume was increased at day 7 and woven bone was increased at days 14 and 21 in DKK1-Ab treated Cre-negative control mice (A–C). In contrast, DKK1-Ab had no significant effect on cartilage and woven bone volume in fracture callus of β-cateninPrx1ER mice (A–C). Black arrows: cartilage area and blue arrows: woven bone area. Data are presented as means ± SD. *p < 0.05, between DKK1-Ab and PBS groups (Cre-negative control mice);#p < 0.05, between Cre-negative and β-cateninPrx1ER groups (without DKK1-Ab); two-way ANOVA followed by Tukey’s test, n = 6.
Fig. 9
Fig. 9
DKK1-Ab had no significant effect on gene expression of fracture callus in β-cateninPrx1ER mice. Total RNA was extracted from callus tissues 7, 10, 14, 21 and 28 days after fracture. In Cre-negative control mice treated with DKK1-Ab, the expression of Sox9 and Col2a1 was increased at day 7 and 10 (A and B), Aggrecan (Agc1) expression was increased at day 14 (C); expression of Col10a1 was increased at days 14, 21, and 28 (D); and expression of Mmp9 and Mmp13 was increased at days 14 and 21 (E and F). InCre-negative control mice treated with Dkk1-Ab, the expression of Runx2 and Osterix was increased at days 14, 21, and 28 (G and H) and expression of osteocalcin (OC) was increased at days 21 and 28 (I). Expression of DKK1 was decreased at days 7 and 10 (J); and expression of β-catenin was increased at days 7 and 10 (K) in Cre-negative control mice treated with Dkk1-Ab. There was no significantly difference between DKK1-Ab and PBS treated groups in β-catenin cKO mice (A–K). Data are presented as means ± SD. *p < 0.05, between DKK1-Ab and PBS groups (Cre-negative control mice); #p < 0.05, between Cre-negative and β-cateninPrx1ER groups (without DKK1-Ab); two-way ANOVA followed by Tukey’s test, n = 3.
Fig. 10
Fig. 10
DKK1-Ab had no significant effect on osteoclast formation. TRAP staining was performed on day 10 and 14 samples and showed no significant difference in osteoclast numbers after DKK1-Ab treatment in Cre-negative control and β-cateninPrx1ER conditional KO mice (A). Expression of osteoclast-specific genes was examined. Expression of Opg and Rankl was increased at days 14 and 21 after DKK1-Ab treatment in Cre-negative control mice (B and C).

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