This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Jeonbuk National University Laboratory Animal Center, Jeonbuk National University, Korea. All animals were cared for in accordance with the regulations of the IACUC under the supervision of licensed veterinarians. The study was conducted according to the ARRIVE guidelines for the reporting of animal experiments. The surgical procedures and assessment of the unions were performed according to the methods of Liao et al .
Forty rats were divided into the following four groups, and ten rats were assigned to each group:
Group I: absorbable collagen sponge (ACS) (Gelfoam; Mascia Brunelli Spa, Milano, Italy) alone group; group II: DFDB graft group-cancellous bone chips (Community Tissue Services, Dayton, OH, USA); group III: autogenous bone graft group (iliac cortico-cancellous bone); group IV: rhBMP-2 implant group (Daewoong CG Bio Pharmaceutical Co. Ltd., Seoul, Korea) with ACS.
Pathogen-free 12-week-old female Lewis rats (230 – 290 g) were purchased from Orient Bio (Seoul, Korea). They were housed in a laminar flow cabinet with a 12 h light/dark cycle and maintained on standard laboratory chow ad libitum. Each rat was given an intraperitoneal injection of 0.1 mg/kg zoledronic acid (Zometa) once a week for 10 weeks. After that, femoral bone defect surgery was performed on all rats. All rats underwent radiographic studies every two weeks after the surgical procedure. Sixteen weeks after the operation, the bone volume in the defect site of the femur was assessed by micro-computed tomography. All animals were euthanized at 16 weeks after surgery and their right femurs were harvested. Union rate determination, manual palpation, and histologic studies were performed for the defect site of the femur.
All rats were anesthetized using ZoletilⓇ (Virbac, France) intraperitoneally (7 mg/kg). The right hind limb was shaved and disinfected with alcohol and povidone-iodine. A longitudinal 3-cm skin incision was made in the lateral aspect of the femur and the entire length of the right femur was exposed. The periosteum was stripped from the shaft. A 5-mm mid-diaphyseal full-thickness defect was created using a bone saw. A polyethylene plate (23 mm × 4 mm × 4 mm) was secured with four 0.99-mm Kirschner wires and two 0.53-mm steel cerclage wires on the lateral side of the femur, as previously described . Each testing material was inserted into the defect in groups II, III, and IV. ACS only was inserted into group I. Iliac cortico-cancellous bone was used for the autograft group. We performed an oblique 2-cm skin incision over the posterior superior iliac spine (PSIS) to retract the soft tissue and expose the PSIS. After cortical osteotomy, cortico-cancellous bone grafts were taken using a small-size curette. Autobone and DFDB were grafted in 0.5 cc amounts. For accurate grafting, we measured 0.5 cc using a 2-cc syringe. rhBMP was implanted at 10 µg/0.5cc. In the femoral defect model, 10 μg BMP was most effective in inducing bone formation [19,20].
All animals were anesthetized and radiographs were taken every two weeks. The rats were imaged with a high-resolution digital mammographic imager (Mammomat Novation, Siemens AG Medical Solutions, Erlangen, Germany). All images were obtained with exposure settings of 34 kVp and 110 mA at a magnification of 1.5×. Radiographic union was assessed by bony bridge formation of the cortex. A complete union was defined as the bony bridge formation of both cortexes in radiography while a partial union was defined as the bony bridge formation of one cortex. Nonunion was defined as no bony bridge formation.
Sixteen weeks after surgery, all rats underwent micro-CT scanning (NFR Polaris-G90, NanoFocusRay Co., LTF., Jeonju, Korea) to evaluate bone volume and healing of the defect site. The scanner was set at an X-ray voltage of 70 kVp with an X-ray current of 100 µA. The scans were completed over 360° of rotation of the X-ray tube. All micro-CT image data were acquired using live, free-breathing rats anesthetized by intraperitoneal ZoletilⓇ injections. We measured the bone volumes around the defect site using a micro-CT scanner (NFR Polaris-G90). A cylindrically shaped region of interest (ROI) was established with the middle of the bone defect site as the center, which had a diameter of 14.25 mm and a height of 6.65 m. The bone volume (BV), tissue volume (TV), and bone volume (BV/TV) percentage were measured in the ROI.
Manual Palpation and Manipulation
Manual palpation is a sensitive and specific method of assessing bone fusion [13,21-23]. Manual palpation with varus/valgus and anterior/posterior angulated force was performed for all harvested specimens with a particular focus at the defect region. All specimens were manipulated with a force high enough to evaluate the gross motion. When no motion was present, it was considered a complete union, as previously described .
We performed bony biopsies of the resected femurs to evaluate the bone healing potential. The resected femurs and jaws were fixed in 10% neutral buffered formalin and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 10 days or in rapid decalcifying solution (Calci-Clear Rapid, National Diagnostics, Atlanta, GA, USA) for 12 to 24 h. To evaluate the histologic changes, paraffin-embedded tissue sections were stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA).
Statistical analysis was carried out with Statistical Package for Social Sciences software (SPSS Inc., Seoul, Korea). The union rates detected by radiological evaluation (union rate), the manual test, and the bone volume in micro-computed tomography were compared using one-way analysis of variance (ANOVA) with a post-hoc test (Scheffe test). P-values of less than 0.05 were considered statistically significant.