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

Objective To investigate if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a critical role in fracture healing process and if DKKl-Ab promotes fracture healing through activation of β-catenin signaling.Methods1. Experimental Animals:(1) 10-week-old mice were subjected to tibial open fracture. After surgery, mice were divided into two groups:DKKl-Ab treatment group (25 mg/kg, subcutaneous injection, twice a week for 28 days); and PBS control group. (2) To generate PrxlCreER;β-cateninfx/fx (β-catenin cKO) mice, p-cateninfx/fx mice were bred with PrxlCreER transgenic mice.10-week-old β-catenin cKO mice and Cre-negative mice were subjected to tibia fracture. Mice were treated with DKKl-Ab and PBS, respectively. Tamoxifen (TM, lmg/lOg body weight/day, i.p. injection for 5 days) was administered right after fracture surgery.2. Tibial Fracture Model: An intramedullary pin was inserted into the tibia at the knee and an open fracture in the proximal tibia diaphysis was performed using a No.11 scalpel blade. The fibula was left undamaged so as to produce a stabilized fracture. Specimens were harvested at days 7,10,14,21 and 28 for Micro-CT, histology, biomechanical testing and RNA analysis.3. Cre-recombination Efficiency:To determine if the PrxlCreER transgene could target floxed genes specifically in mesenchymal cell in fracture site, PrxlCreER transgenic mice were bred with Rosa26 reporter mice. Tamoxifen (TM,1 mg/10 g body weight/day, i.p. injection for 5 days) was administered right after fracture. The mice were sacrificed at day 10. Cre-recombination efficiency was evaluated by X-Gal staining.4. Radiographic and p.-CT Analyses: Weekly radiographs (Faxitron X-ray, Wheeling, IL) were obtained to monitor bone healing. Specimens were scanned at 10.5-micron isotropic resolution using a Scanco VivaCT 40 (Scanco Medical AQ Switzerland) at indicated time points. Callus total volume (TV), callus bone volume (BV) and callus bone mineral density (BMD) were determined.5. Biomechanical Torsion Testing: Fracture specimens were mounted on an EnduraTec TestBenchTM system with a 200 N.mm torque cell (Bose Corp., Minnetonka, MN) and tested in torsion at a rate of 10/sec until failure to determine the torsional stiffness, ultimate torque, ultimate rotation, and strain energy to failure.6. Quantitative PCR: The fracture callus and 1mm of adjacent bone was harvested and total RNA was extracted using the Qiagen Plus RNeasy kit. cDNA was synthesized from 1μg of RNA per callus using a iScript cDNA synthesis kit (Bio-Rad). Real-time RT-PCR analysis was performed using murine specific primers for chondrogenesis and osteogenesis related genes (Sox9, Co12al,ColX, Runx2, Osterix, Osteocalcin, Dkkl, β-catenin).7. Histology & Histomorphometry: Specimens were harvested at 7,10,14,21 and 28 days, and fixed in 10% NB-Formalin for 3 days and decalcified for 14 days in 14% EDTA and then paraffin embedded.3-μm sections were cut and prepared and Alcian blue/H&E staining was performed. Histomorphometric analysis was performed using Osteometrics software (Decatur, GA).8. Statistical Analysis:Results were presented as the mean ± standard deviation. Statistical analyses included Student’s t-tests and two-way ANOVA. p<0.05 was considered as significant.Results1. X-Gal staining showed that the Cre-recombination efficiency was 63% in the callus tissue. X-Gal positive cells include mesenchymal cells, osteoblasts, chondrocytes and bone marrow cells in the callus tissue.2. Radiographic and μ-CT analyses showed that administration of DKKl-Ab enhanced bone callus formation. The fracture line was more obscure in DKKl-Ab group at day 14 and 21. In Prx1CreER;β-cateninfx/fx (β-catenin cKO) mice, the fracture line was easy to see in both DKKl-Ab and PBS treated group at day 14 and day 21.μ-CT data showed a significant increase in bone volume of fracture callus treated with DKKl-Ab for 14,21 and 28 days compared to controls. The bone volume of β-catenin cKO mice was decreased compared with Cre-negative control group in day 14,21 and 28. Moreover, this phenotypic alteration can not be reversed by DKK1-Ab, suggesting that DKK1-Ab enhanced fracture healing through activation of β-catenin signaling.3. Histological data showed that the progression of fracture healing was accelerated in DKK1-Ab treated group. However, less and smaller callus tissues were found in β-catenin cKO mice. Histomorphometric data demonstrated that the cartilage area was increased treated with DKK1-Ab at day 7, and the woven bone area was increased at day 14 and 21 after DKK1-Ab treatment. There was less cartilage and woven bone areas in β-catenin cKO mice. In contrast, there was no significant difference between DKK1-Ab and PBS treated groups in β-catenin cKO mice.4. Results of biomechanical testing showed that treatment with DKK1-Ab enhanced bone formation. The maximum torque and stiffness were significantly increased at day 10,14,21 and 28 in DKK1-Ab treated group. The callus of β-catenin cKO mice was more fragile than control group at day 10.5. Gene expression data showed that the expression of cartilage marker genes (Sox9, Col2al, and ColX) was increased at day 7 in DKK1-Ab treated group. The expression of bone marker gene (Runx2, Osterix, and Osteocalcin) was increased at day 14,21 and 28 in DKK1-Ab treated group. The expression of dkkl was decreased at day 7 and the expression of P-catenin was up-regulated at day 7 and 10 in DKK1-Ab treated group. Treatment with DKK1-Ab did not alter the expression of the genes mentioned above in β-cateninPrxlERmice. Conclusion In the present studies, we demonstrated that treatment with DKK1-Ab increased bone callus formation and mechanical strength during fracture healing. DKK1-Ab enhances fracture healing by increasing mesenchymal cell proliferation and expansion and promoting osteoblast and chondrocyte differentiation. The fracture healing process is delayed when the β-catenin gene is deleted in mesenchymal progenitor cells during early fracture healing stage. DKK1-Ab seems acting through β-catenin signaling to enhance fracture healing since DKK1-Ab lost its activity to promote fracture healing in β-catenin cKO mice.