Surgical technique used to construct the ovariectomies and the femoral fracture model
Approval was obtained from Oita University’s Animal Research Committee prior to animal experimentation (Oita University Institutional Animal Ethics Committee, no. 182402). Before the procedure, a total of 40 female Sprague–Dawley rats (24 weeks old; CLEA Japan, Inc., Tokyo, Japan) were anesthetized via intraperitoneal injection containing 0.3–0.4 ml of 0.15 mg/kg medetomidine + 2 mg/kg midazolam + 2.5 mg/kg butorphanol. All operations were conducted in a standard sterile environment. Rats underwent ovariectomy or sham surgery, raised for 8 weeks, and then underwent weight measurement to confirm weight gain. After 8 weeks, all rats underwent femoral osteotomy. The right hind limb was prepared for the operation. With the rats in the lateral position, the right femur was located using the posterolateral approach. The periosteum of the femur was circumferentially incised, elevated, and stripped. Subsequently, the femur at the osteotomy site was exposed. A transverse osteotomy was made with a sagittal saw (Stryker, Kalamazoo, MI, USA) without cooling at the midshaft of the femoral bone. Fracture fragments were contacted and stabilized, and the intramedullary was then fixed using a stainless-steel wire (diameter, 1.4 mm). The wire was cut on the surface of the intercondylar groove to avoid restriction of knee joint motion. The fascial and skin incisions were closed with a 3-0 nylon suture. During surgery, a mixture of medetomidine + midazolam + butorphanol was used for the anesthesia; medetomidine and butorphanol have strong analgesic effects. The rats were housed in separate cages, given food and water ad libitum, and their conditions were monitored daily.
A total of 40 operated Sprague–Dawley rats were divided into four groups: the control group (C group; n = 10; administered saline), the romosozumab group (R group; n = 10; administered romosozumab, 25 mg/kg), the vitamin D3 group (VD group; n = 10; administered active vitamin D3, 0.2 µg/kg), and the romosozumab plus vitamin D3 group (R + VD group; n = 10; administered romosozumab plus vitamin D3). Romosozumab (Amgen Inc, Japan, Tokyo) was administered once a month and active vitamin D3 (Rocaltrol, Kyowakirin, Inc, Japan, Tokyo) was administered twice a week. In addition, a sham-operated group (n = 10) was prepared, but the group was not analyzed statistically with the other four groups. The study design is shown in Fig. 1.
Harvesting of femurs and blood
Ten weeks after femoral osteotomy, 0.15 mg/kg medetomidine hydrochloride + 2.0 mg/kg midazolam + 2.5 mg/kg butorphanol was injected into the peritoneum, and cervical dislocation was performed. The operated right femoral bones were explanted, and the right bones were separated from the stainless-steel wire before analysis. We used the left femoral bones, which had not been operated upon, to evaluate the effect of the combined treatment of romosozumab with active vitamin D3 on the bones of OVX rats using microcomputed tomography (micro-CT) analysis.
Soft X-ray analysis
The right femoral bones obtained at the 10-week time point were photographed using Softex X-ray apparatus (Softex CSM-2; Softex, Tokyo, Japan) with HS Fuji Softex film (Fuji Film, Tokyo, Japan) at 45 cm, 30 kV, and 15 mA for 20 s. The fusion was quantified using the anteroposterior (A–P) and lateral radiographs. Three independent, blinded observers scored the bone formation in each rat using a four-point scale. Fracture union was judged by visual assessment of the mineralized callus bridging the fracture line on the A–P radiographs (right side: 1 point; left side: 1 point) and lateral radiographs (anterior side: 1 point; posterior side: 1 point). The bone fusion was considered to be >2 points with soft X-ray images on a four-point scale. In addition, the Radiographic Union Scale in Tibial Fractures (RUST) was used to assess all specimens. The RUST score is based on the presence or absence of a callus and of a visible fracture line at a total of four cortices visible on the A–P and lateral radiographs, and its four-point minimum corresponds to a fracture that is deemed not healed, whereas its 12-point maximum corresponds to a fracture that is deemed healed with all cortices bridged with a callus, without a fracture line. Kooistra et al. reported the reliability and validity of the RUST scale in human long bone.
After the specimens were harvested, they were dissected until they were free of soft tissue. They were then immersed in 70% ethanol for 1 day and in 99.5% ethanol for 1 day. The bones were then sequentially immersed in acetone for 1 day, in 99.5% ethanol for 1 day, and in 2-propanol for 1 day. The next day, the bones were embedded in glycolmethacrylate, without previous decalcification, and left to stand for 10 days. Using a fully automated rotary microtome (Leica RM2255, Leica, Nussloch, Germany), the femurs were cut into 3-µm thick sections, and the specimen was stained with toluidine blue.
Fracture bone micro-CT analysis (operated side)
The bone micro-CT imaging was performed according to the guidelines. The explanted femoral bones were scanned using a Sky-Scan 1172 tomograph (Bruker micro-CT, Kontich, Belgium) with a voxel size of 20 mm. Data were collected at 100 kV and 100 mA and reconstructed using the cone-beam algorithm. Each femoral bone was set on the object stage, and sample scanning was performed over 180° of rotation with an exposure time of 105 ms. A cylindrical volume of interest with a diameter of 20 mm and a height of 27 mm was selected, which displayed the microstructure of the rat femoral bones (comprising the cortical and trabecular bones). Data analysis was performed using CT Analyzer software (Bruker micro-CT). The region of interest was set as the area of fracture healing and was defined by the fracture area filled with new bone; the structural indices of the femoral fracture areas (9 × 9 × 8.4 mm; a fracture gap in the center) were calculated using this software. During the three-dimensional analysis, the bone volume/tissue volume (BV/TV), the BV of cortical bone, the TV, the trabecular thickness (Tb. Th), the trabecular number (Tb. N), and the trabecular separation (Tb. Sp) were measured.
Bone micro-CT analysis (nonoperated side)
The bone micro-CT imaging was performed according to the guidelines . The explanted femoral bones were scanned using a Sky-Scan 1172 tomograph (Bruker micro = CT, Kontich, Belgium) with a voxel size of 20 mm. Data were collected at 100 kV and 100 mA and reconstructed using the cone-beam algorithm. Each femoral bone was fixed in a sample holder and set with the vertical axis on the object stage, and sample scanning was performed over 180° of rotation with an exposure time of 105 ms. A cylindrical volume of interest with a diameter of 20 mm and a height of 27 mm was selected, which displayed the microstructure of the rat femoral bones (comprising the cortical and trabecular bones). Data analysis was performed using CT Analyzer software (Bruker micro-CT). The region of interest was set at the distal femoral area, and the structural indices of the femoral area of trabecular bone analysis (0.8–3.8 mm) from the growth-plate reference level and the area of cortical bone analysis (3.0–3.8 mm) from the growth-plate reference level were calculated using this software. During the three-dimensional analysis, the BV/TV, the BV of trabecular bone, the TV, the Tb. Th, the Tb. N, the Tb. Sp, the BV of cortical bone, the cortical bone area (Cr. Ar), and the cortical bone thickness (Cr. Th) were measured.
Nonfractured femurs were used to test the bone strength. Three-point bending tests were conducted using a universal material testing system (Instron 5865; Instron, Kanagawa, Japan). The femur was placed on the sample support with its anterior surface facing upward and the center of the femoral shaft located at the center of the support. The load was applied at a rate of 1 mm/s until the bone was fractured. A program prepared by Instron was used for data analysis; the measured parameters were the maximum load, maximum bending stress, stiffness, Young’s modulus, and toughness.
Measurement of serum levels of bone turnover markers
Blood samples (100 µL) were collected when the animals were euthanized at week 10. Serum levels of osteocalcin (OC), an osteogenesis marker, were measured with a Rat Osteocalcin enzyme-linked immunosorbent assay (ELISA) Kit (RK03858, Woburn, MA). Cross-linked C-telopeptides of type-1 collagen (CTX-1), a bone-resorption marker, were measured using the Rat Cross-linked C-telopeptide of Type-1 Collagen ELISA Kit (RK03603, Woburn, MA).
Statistical analysis of the four groups, but not the sham group, was performed. Normality was confirmed by the Shapiro–Wilk test. Variables that were normative were subjected to one-way analysis of variance to compare the differences between the four groups. One-way analysis of variance with the Bonferroni posthoc test was used for the four-point test results, RUST, morphometric analysis, biomechanical analysis, and bone turnover marker analysis. For the four-point test and RUST, kappa statistics were performed to check for interobserver variability. The kappa statistic corrects the observed agreement for a possible chance agreement among observers. Agreement was rated as follows: poor, κ = 0–0.20; fair, κ = 0.21–0.40; moderate, κ = 0.41–0.60; substantial, κ = 0.61–0.80; excellent, κ > 0.81. A value of 1 indicated absolute agreement, whereas a value of 0 indicated agreement no better than chance. All analyses were performed using Statistical Package for the Social Sciences software (SPSS V25.0; IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY). Statistical significance was set at p < 0.05.