Experimental animals
Sixty-one female C57/BL/6 mice ~14 weeks of age (body weight of ~20 g) were used (Harlan Sprague-Dawley, Inc., Indianapolis, IN). Five animals were housed per cage at the Laboratory Animal Resource Center of Indiana University School of Medicine, and they were fed with standard laboratory chow and water ad libitum. The animals were allowed to acclimate for 2 weeks before the experiment. All procedures, performed in this study, were in accordance with the Institutional Animal Care and Use Committee guidelines at Indiana University School of Medicine and approved.
Mechanical loading
The mouse was placed in an anesthetic induction-chamber to induce sedation and mask-anesthetized using 2% isoflurane. Using the custom-made piezoelectric mechanical loader, mechanical loads were applied for 3 minutes per day for 3 consecutive days to the left knee through the lateral-medial direction (Fig. 1). The mice were randomly divided into four groups for four loading frequencies (5, 10, 15 or 20 Hz, N = 8), and the loads with a peak-to-peak force of 0.5 N were applied. Using the same procedure, the right knee was placed under the loader without oscillatory loading and used as the non-loading control. After loading, the mouse was allowed normal cage activity and any abnormal behavior, weight loss or a diminished food intake was monitored.
Piezoelectric joint loader
The loader consisted of four bimorph-type piezoelectric actuators (LPD 1260X; Megacera, Saitama, Japan). A voltage signal was sent through a 16-bit data-acquisition board (PCI-6052E; National Instruments, Austin, TX) and a piezo-driver (model PZD 700-L; Trek, New York, NY) (Fig. 1). Before the loading experiment, loads applied to the knee were calibrated using an anesthetized mouse. A strain gauge (CEA-06-062UW; Vishay Measurements Group, Raleigh, NC) was attached on the aluminum cantilever connected to the stator and known forces from 0 to 10 N were given. To position the knee properly, the lower end of the loading rod and the upper end of the supporter were designed to form a pair of semispherical cups. The lateral and medial epicondyles of the femur together with the lateral and medial condyles of the tibia were confined in the cups. Because of the complex anatomy of the knee, the lateral side of the femur is less projected outwards. Therefore, the femoral epicondyle apparently receives fewer loads than the tibial condyle. The tip of the loader had a contact area of 4 mm diameter. To avoid stress concentration between the knee and the loader, the loading surface and the supporter were covered with silicon rubber.
Measurement of strain
Five animals were used for strain measurements (Fig. 2). After euthanasia, the periosteal surface of a left femur was exposed. A strain gauge (Model EA-06-015DJ-120, Measurements Group Inc., NC) was trimmed into 0.5 mm in width and 2.2 mm in length, and attached with glue to the mid-diaphysis (50% along the length of the femur). Note that it was unavoidable to remove a part of surrounding tissue for attachment of the strain gauge but the loading site in the knee was kept intact. Bone histomorphometry was conducted using 0.5-N loads, and therefore the strain was also measured with the same loading conditions. The distal epiphysis was loaded at 5, 10, 15 and 20 Hz with 0.5 N. Voltage signals from the strain gauge were processed with a signal conditioning amplifier (2210, Measurement Group Inc., NC), and Fourier transform was conducted. The measurement was repeated 5 times, and the peak-to-peak voltage was converted to the actual strain value using the standard calibration line [15, 16]. Loads were confined with the loading rod, and no bending moment was presumably generated to the femur or the tibia.
Fluorescence labeling and sample preparation
Mice were given an intraperitoneal injection of calcein (Sigma, St. Louis, MO), a fluorochrome dye, at 30 μg/g body mass on days 2 and 6 after the last loading (Fig. 3). Animals were sacrificed 13 days after the last loading, and femurs were harvested for histomorphometric analyses. The isolated bones were cleaned of soft tissues, and the distal and proximal ends were cleaved to allow infiltration of the fixatives with 10% neutral buffered formalin. After 48 h in the fixatives, bones were transferred to 70% alcohol for storage. Specimens were dehydrated in a series of graded alcohols and embedded in methyl methacrylate (Aldrich Chemical Co., Milwaukee, WI). The transverse sections (~80 μm in thickness) were removed from the mid-diaphysis, ~8 mm distant from the distal end using a diamond-embedded wire saw (Delaware Diamond Knives, Wilmington, DE), and they were mounted on standard microscope slides.
Bone histomorphometry
Using a Nikon Optiphot microscope (Nikon, Inc., Garden City, NY) and a Bioquant digitizing system (R & M Biometrics, Nashville, TN), we measured total perimeter (B.Pm), endocortical perimeter, single-labeled perimeter (sL.Pm), double-labeled perimeter (dL.Pm), and double-labeled area (dL.Ar). From these measurements we derived mineralizing surface (MS/BS = [1/2 sL.Pm + dL.Pm]/B.Pm in %), mineral apposition rate (MAR = dL.Ar/dL.Pm/4 in μm/day), and bone formation rate (BFR/BS = MAR × MS/BS × 365 in μm3/μm2 per year). To evaluate the effects of the loading frequencies the relative parameters such as rMS/BS, rMAR, and rBFR/BS were derived as differences between the loaded and the control femurs. In drawing a fluorescent intensity curve, a MetaMorph Imaging System (version 3.6, Universal Imaging Co.) was used. Note that the fluorescent labeling data represent bone formation during the days 2 and 6 after the last loading.
The total bone cross-sectional area (mm2), bone medullary area (mm2), and cortical thickness (mm) were also measured. The cross-sectional cortical area was determined by subtracting the bone medullary area from the total bone cross-sectional area. The cortical thickness was defined as the mean distance between the endosteal and the periosteal surfaces on both anterior and posterior sides. The measurements were taken at the middle of each side, and the mean value was determined from two independent measurements. The relative alteration was calculated as differences between loaded (L) and control (C) femurs such as ([L - C]/C × 100 in %).
Bone porosity
Intracortical porosity was determined from the tibial and femoral transverse sections of the non-loaded limbs in the mid-diaphysis (~50 μm thickness; N = 24). Using a Nikon Optiphot microscope and a Bioquant digitizing system, we measured cross-sectional cortical area (mm2), total porous area (mm2), and the number of pores whose area was larger than an identifiable threshold of 11 μm2 with the optical system. From these measurements, we derived intracortical porosity (ratio of the porous area to the total bone area in %) and pore density (number/mm2). The mean value was calculated from 2 independent measurements.
Microcomputed tomography (μCT)
Micro-CT was performed using a desktop μCT-20 (Scanco Medical AG, Auenring, Switzerland). The hindlimb was harvested keeping the intact knee. The sample was placed in a plastic tube filled with 70% ethanol and centered in the gantry of the machine. A series of cross-sectional images were captured in an 8-mm segment including the knee at 30-μm resolution. Images were imported into Scion Image software (Scion Corp., Fredrick, MD, USA), and three-dimensional reconstruction was conducted.
Statistical analysis
The data were expressed as mean ± SEM. Statistical significance among groups was examined using ANOVA, and a post-hoc test was conducted using a Fisher's protected least significant difference (PLSD) for all the pairwise comparisons. A paired t- test was employed to evaluate statistical significance between the loaded and control femurs. All comparisons were two-tailed and statistical significance was evaluated with p < 0.05.