Rat Ovariectomy Model and Experimental Groups
12 weeks old female Sprague-Dawley rats (Experimental Animal Center, Chinese General Hospital of PLA, Beijing, China), body weight 350-400 g were used for this experiment. The rats were maintained on commercial rat chow with 0.95% calcium and 0.67% phosphate. Rats were housed in a room that was maintained at 70°F with a 12-h light/dark cycle. All animals were treated according to the animal care guidelines, Department of Health, PR China with the approval of the PLA General Hospital Ethical Committee on Animal Research. Bilateral ovariectomies were performed using dorsal approach as previously reported.
Fifty rats were divided into five groups with 10 animals in each group. Group 1: animals received sham surgery; ovaries were exteriorized but not removed. For Groups 2, 3, 4 and 5, all animals received bilateral ovariectomies and waited for 12 weeks, and then used in the following experiments (as detailed in Figure 1): Group 1 (Sham) and Group 2 (OVX) received subcutaneous injection of the vehicle buffer twice weekly for 12 weeks. The other 3 groups were treated as the following: Group 3, OVX + ALN, 28 μg/kg subcutaneous injection twice/week for 12 weeks; Group 4, OVX + rhOPG-Fc, animals received 5 mg/kg rhOPG-Fc subcutaneous injection per day for 2 weeks starting from 20 weeks after the OVX surgery; Group 5, OVX + rhOPG-Fc+ ALN, the animals were treated twice/week with 28 μg/kg ALN subcutaneous injection, and 5 mg/kg rhOPG-Fc was injected daily for 2 weeks starting from 20 weeks after the OVX surgery. All animals were killed at 24 weeks following OVX surgery and bone samples collected for further examinations.
Materials
Anti-RANKL antibody was purchased from Santa Cruz, USA. BCA Protein assay kit was from PIERCE, Rockford, IL, USA. Polyvinylidene difluoride membrane filter was from Millipore, Tokyo, Japan. The ECL system was from Amersham Biosciences, Co., Piscataway, NJ, USA. Densitometric analysis was done with an ATTO Densitograph (ATTO, Tokyo, Japan). ALN was obtained from Merck Company. Anti-β-actin mouse monoclonal antibody was from Sigma, USA. rhOPG-Fc (22-201 Amino acid) was from Fuchun Zhongnan Company, Shanghai, PR China and the details of the characterization of rhOPG-Fc was published previously [3].
Bone Protein Preparation and Western Blotting
The 2nd-5th caudal vertebrae were collected and prepared according to Miyazaki et al [4]. In brief, all connective tissues were removed and 100 mg of vertebral bone were crushed by surgical pliers, homogenized with BAP buffer (50 mM Tris-HCL buffer, pH 7.5, containing 0.3 mM phenylmethyl fluoride, 1.0 mM benzamidine, and 0.1% Triton X-100). The homogenate was centrifuged at 12,000 g for 20 min at 37°C. Resultant supernatant was used for Western blot analysis. Western blotting was performed using a previously described method [5]. β-actin was used as an internal standard; Equal amount of proteins separated by electrophoresis were transferred to 2 polyvinylidene difluoride membrane filter; the proteins on the membranes were incubated with antibodies of anti-RANKL and anti-β-actin overnight at 37°C. After washing, the filters were reacted with peroxidase-conjugated secondary antibodies for 60 minutes at room temperature. The reactive bands were detected with the ECL system, and the relative intensities of the bands were calculated and expressed by percentage change.
Urine and Serum Biochemical Assays
Rats were housed in individual metabolic cages, fasted for 24 h before the urine samples were collected at the day before surgery, 12 weeks and 24 weeks days following OVX surgery. Twenty four-hour urine samples were collected over sodium azide in metabolic cages. To minimize contamination with dietary (soybean) proteins, the rats were starved during the collection period.
For serum collection, at each time-point, 500-1000 μl peripheral blood was collected from the tail vein with a plastic 1-ml heparinized syringe and further heparin (at a final concentration of approximately 0.5 mg heparin per ml of blood) was added to the blood. The red blood cells were removed by centrifugation for 10 min at 4500 × g and the serum were collected. The sera were filtered through a 0.22-μm filter for sterilization as well as in order to remove any fragments of platelets.
Urinary levels of calcium (Ca2+), phosphonate (P), creatinine (Cr) and serum levels of Ca, P were measured by standard laboratory tests. Serum alkaline phosphatase (ALP) and osteocalcin (OSC) were measured using ELISA plates from R&D system according to the manufacturer's instructions.
Bone Mineral Density (BMD) Measurement
At day 0 (before surgery), 12 weeks (development of osteopenia), and 24 weeks (before killing) following OVX surgery, whole body BMD were measured using lunar-DXA IQ (Lunar company, USA) and the BMD at the 4th, 5th and 6th vertabrae. The subjects were placed on the scan table in the supine position. All scans were performed in slow mode and analyzed using Lunar smart scan version software with the slowest scan mode. The measurements of the hip and vertebrae were repeated three times, and the means were calculated.
Biomechanical Testing
The mechanical properties reflect the true quality of bone and they are used as primary outcome measurement parameter in this study. Three-point bending test described by Turner and Bur [6] was used to measure the mechanical strength of intact femurs. The femur was placed on custom-made struts, 9 mm apart, with a 100-N superior load cell delivered through a superior strut directed to the mid-diaphyseal region at a rate of 1 mm/min. Load-displacement curves were recorded using a servo-hydraulic materials testing machine (858 Mini Bionix, MTS Corp., Minneapolis, MN, USA). Data were collected concerning peak load to failure, and stiffness was calculated from load-displacement curves, and elastic load, elastic stress and Young's modulus (maximum slope of the stress-strain curve) were calculated and compared. L3 vertebrae were also measured by vertical compression at the center along the cephalocaudal axis; the deformation and volume changes were determined. Biomechanical parameters including ultimate load (Fmax), maximum stress (Fmax/cross-sectional area), elastic load, elastic stress and Young's modulus (maximum slope of the stress-strain curve) were calculated and analyzed.
Bone Histomorphometry
To determine bone formation rate, rats received subcutaneous injections of 10 mg/kg of Calcein at 12 and 2 days before termination. Tibiae were collected and embedded in methylmethacrylate. Serial longitudinal sections of 4-μm, 8-μm and 200-μm thickness were cut. 4-μm sections were stained with Von Kossa and toluidine blue; the 8-μm sections were mounted without staining for measuring the bone growth rate using the cacein labeling; the bone mineral apposition rate was demonstrated by the distance of the two calcein labeling lines divided by 10 days. The 200-μm thick sections were mounted without staining for taking digital photographs. Histomorphometry measurements were performed on the proximal metaphyseal region (between 2 and 4 mm distal to the growth plate/metaphyseal junction) using a digital image analysis system (Osteomeasure, Inc., Atlanta, GA, USA). Trabecular area, perimeter, single- and double-labeling surfaces, osteoclast number, osteoid surface were measured. Trabecular number, thickness, mineralizing surface, mineral appositional rate, BFR/surface volume (BFR/BS), and osteoclasts number per millimeter were calculated according to the methods reported by Parfitt et al [7].
Statistics
The mean and standard deviation (SD) were shown. All the data were analyzed with one-way ANOVA test and the inter-relationship function of the two agents, ALN and OPG was analyzed using SPSS software (SPSS Version 10; SPSS Inc., Chicago, IL, USA). Significant difference was considered at p < 0.05.