Acceleration of callus formation during fracture healing using basic fibroblast growth factor-kidney disease domain-collagen-binding domain fusion protein combined with allogenic demineralized bone powder

Both femurs were harvested from 36 C3H/HeN (H-2k) mice, and bone lipids were removed by treatment with chloroform/methanol. The harvested femoral bones were broken into small fragments, which were then passed through a 1-mm filter to collect the bone powder. To prepare DBP, the bone powder was demineralized using 0.6 N HCl for 18 h at 4°C. The particle size distributions were determined by laser scattering using a LMS-30 Micron Sizer (Seishin Enterprise Co., Ltd., Tokyo, Japan) and cumulative size distribution limits of D10, D50, and D90, which correspond to the percentage of particles (10%, 50%, and 90%, respectively) in a sample that is below a certain size. The surfaces of the DBP were observed by scanning transmission electron microscopy (SEM; JSM-7400F; JEOL Ltd., Tokyo, Japan).

Recombinant human bFGF was purchased from Kaken Pharmaceuticals (Tokyo, Japan). The construction of the fusion protein of bFGF, PKD, and the CBD derived from C. histolyticum class II collagenase (ColH) was previously described [18]. The biological activities of purified bFGF-PKD-CBD were confirmed using a proliferation assay with cultured periosteal mesenchymal cells [19]. bFGF-PKD-CBD exhibited the same cell proliferation ability as bFGF in vitro. The affinity of bFGF-PKD-CBD for allogenic DBP was confirmed in vitro by ELISA, as previously described [18]. In the assay, 0.064 nmol of bFGF-PKD-CBD bound to 1 mg of allogenic DBP.

All procedures involving the handling of animals adhered to the guidelines of the animal ethics committee of Kitasato University. A specific pathogen-free colony of C57BL/6J mice was housed in a semi-barrier system under controlled conditions (temperature, 23°C ± 2°C; humidity, 55% ± 10%; and lighting, 12-h light/dark cycle) throughout the study at Nippon Charles River Laboratories (Kanagawa, Japan). Mice were allowed access to standard rodent chow (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. Fractures were generated in the femurs of 72 C57BL/6J mice by first making a 4-mm medial parapatellar incision in the left knee under sterile conditions to laterally dislocate the patella [20]. A 0.5-mm hole was then drilled into the intracondylar notch, into which a nail (0.5 mm in diameter) was inserted retrograde for the creation of fractures, which were generated with a wire saw (0.22 mm in diameter) using a small lateral approach. After performing the fracture generation procedure, the nail was implanted deeply to stabilize the fracture. Soft x-rays were used to confirm the successful creation of fractures [20]. As we previously demonstrated that the combination of 0.58 nmol bFGF-PKD-CBD and collagen powder accelerated bone formation in mice [19], in the present study, the C57BL/6J mice were divided into four groups (18 mice/group), 8 mg DBP mixed with either phosphate-buffered saline (PBS) (DBP/PBS, control group), 0.58 nmol bFGF (0.58 nmol DBP/bFGF group), 0.058 nmol bFGF-PKD-CBD (0.058 nmol DBP/bFGF-PKD-CBD group), or 0.58 nmol bFGF-PKD-CBD (0.58 nmol DBP/bFGF-PKD-CBD group) was grafted into fracture sites. The mice were allowed to use their fractured leg without restriction immediately after the grafting procedure until micro-CT analysis was performed at 2, 4, and 6 weeks (n = 6, each time point).

Femurs and the surrounding muscle were excised from sacrificed mice at 2, 4, and 6 weeks after fracture generation and treatment and were then stored in 4% paraformaldehyde for 48 h at 4°C. Micro-CT images of whole femurs in PBS were obtained using a microfocus X-ray CT system (inspeXio SMX-90CT; Shimadzu Co., Ltd., Tokyo, Japan) and the following settings: acceleration voltage, 90 kV; current, 110 mA; voxel size, 20 μm/pixel; and matrix size, 1,024 × 1,024. From the obtained images, 10-mm regions of interest (500 slices) in the midfemur were defined. Three-dimensional (3D) image analysis software (Tri-3D-Bon; Ratoc System Engineering Co., Ltd, Tokyo, Japan) was used to measure new bone volume and bone mineral content in the defined regions, as previously described [21]. For assessing new bone volume and bone mineral content, a hydroxyapatite (HA) calibration curve was generated from the data obtained from phantom images prepared with 200, 300, 400, 500, 600, 700, and 800 mg HA/cm³. The bone mineral content in each sample was determined by comparing the measured densities in the micro-CT images to the HA calibration curve, and new bone was defined using a threshold value of ≥300 mg/cm³.

The prepared allogenic DBP was morphologically characterized by SEM, which showed that the particles appeared irregular in shape and had a rough surface (Figure 1A, B). Based on measurements of the diameter of the bone particles, the values for D10, D50, and D90 were determined to be 154.32, 266.11, and 439.94 μm, respectively (Figure 1C).

Callus formation at fracture sites in mice femurs grafted with DBP in PBS (PBS/DBP) or DBP loaded with bFGF (bFGF/DBP) or bFGF-PKD-CBD (0.058 nmol and 0.58 nmol bFGF-PKD-CBD/DBP) was evaluated after 2, 4, and 6 weeks by micro-CT imaging (Figure 2A–F). Similar amounts of callus formation and bone mineral content were observed for femurs treated with PBS/DBP, bFGF/DBP, 0.058 nmol bFGF-PKD-CBD/DBP, and 0.58 nmol bFGF-PKD-CBD/DBP at 2 weeks (Figure 2E, F). However, after 4 weeks, fracture sites grafted with 0.58 nmol bFGF/DBP and 0.058 or 0.58 nmol bFGF-PKD-CBD/DBP had significantly higher levels of callus volume and bone mineral content compared to those grafted with PBS/DBP (Figure 2E, F). After 6 weeks, the callus volume and bone mineral content in fracture sites grafted with 0.58 nmol bFGF-PKD-CBD/DBP were significantly higher than those found in the other three groups of treated femurs. In addition, no differences in callus formation or bone mineral content in fracture sites treated with PBS/DBP, bFGF/DBP, or 0.058 nmol bFGF-PKD-CBD/DBP femur were observed at 6 weeks (Figure 2E, F).