About 20, 3-month-old female, Sprague-Dawley rats were obtained from Charles River. The protocol pertaining to all procedures and aspects of the study was approved by the University of Alberta animal care and ethics committee. Before arrival, to induce an osteoporosis (OP)-like increase in bone resorption, ten rats were ovariectomized (OP-OVX); the remaining ten rats were sham-operated as normal controls (OP-Sham). All rats were euthanized 12 weeks after OVX and/or sham surgery and lumbar vertebrae 4-6 (L4-L6) were dissected. The muscle and soft tissues connected to vertebrae were removed, and samples were wrapped in paper towels dampened with phosphate buffered saline and stored frozen at -30 C for subsequent Micro-CT scan.
The excised vertebrae, L4, L5, and L6, were thawed at room temperature for 2 h before scanning. All vertebrae were imaged using a benchtop Micro-CT imager (SkyScan 1076; Bruker-MicroCT, Kontich, Belgium) at 18 mum voxel image resolution with 70 kV, 100 muA, and a 1.0 mm aluminum filter. Projection images were reconstructed using bundled vendor software (Nrecon 1.6.1.5; Bruker-MicroCT, Kontich, Belgium). Dataviewer 1.4.3 software was employed to orient the cross sectional images parallel to the transaxial plane. A cylindrical region of interest (ROI) was segmented from trabecular bone within the vertebral body. The diameter of each cylinder was constrained by the endocortical bone margin enclosing the vertebra. The vertebral growth plates at each end were used to landmark the top and bottom segmentation boundaries. We utilized CTAn 1.11.6.0 (Bruker-MicroCT, Kontich, Belgium) to morphometrically analyze the trabecular bone cylinder. In order to calibrate BMD with Hounsfield units (HU), two hydroxyapatite [Ca10(PO4)6(OH)2] phantoms (Computerized Imaging Reference Systems Inc., Norfolk, VA, USA) with BMD 0.250 and 0.750 g/cm3 were included in the calculation based on the assumption that HUair = -1000 and HUwater = 0. The threshold, 70 ~ 255, was chosen to segment out bone on 8-bit (0 ~ 255 gray level) bitmap (BMP) images. The measured structural parameters were BV/TV, BMD, Tb.Th, Tb.Sp, FD, Tb.Pf, DA, and Conn.Dn. Those parameters were calculated three dimensionally (3D) based on the volume of the cylinder. Except for Conn.Dn, all structure parameters were also calculated two dimensionally (2D) from cross sectional images slice by slice, along the longitudinal direction from caudal to cranial (Figure 1), and graphed with respect to the normalized height of the cylinder. The results were compared between groups and different levels of vertebrae in the same group.
The results of the Micro-CT structural parameters indicated that the largest difference between groups were in the 40% portion of the cylindrical length from the caudal side. Thus, it was selected for FEA (Figure 1). The cross sectional images of this region were imported into Mimics 14.11 (Materialise, Leuven, Belgium) to build finite element meshes. Since the voxel of the Micro-CT data was 18 mum ( 17.156 mum), that dimension was used to build voxel mesh (hexahedral elements) for FEA to maintain the details of the microarchitecture. To avoid losing or distorting microarchitecture of trabecule, no resizing or smoothing was undertaken prior to meshing. After creating hexahedral elements (hex8), the meshes were exported as an input file (*.inp) format of ABAQUS (SIMULIA, Providence, RI, USA). The remaining pre-processing to include material properties and boundary conditions were done in ABAQUS/CAE 6.10-1. Since bone is expected to behave elastically and failure expected with small strain, the analysis was set as linear and elastic with Young's modulus (E) = 24.5 GPa and Poisson's ratio (nu) = 0.3. All vertebrae were assigned with the same material property, as no significant difference of material properties was measured between normal and OVX bone. The bottom of the cylinder was fixed and a displacement (Deltad), 5% of the height of the cylinder (strain = 0.05), was applied to the top of the cylinder. The stiffness (k) of the bone was calculated by dividing the summation of the vertical reaction forces (R) on the fixation to the applied displacement. Since the sizes of individual vertebrae varied, the stiffness (which comprises information of geometry and material property) would also vary with size. Therefore, values of k were further normalized with the theoretical stiffness (k'), which was calculated from the size of each cylinder by assuming that the cylinder was solid (Figure 2). The index of bone stiffness was represented as k/k'.
PASW  Statistics 17.0 was used for statistical evaluations. The two-independent-samples test, Mann-Whitney U test, was used to find differences between individual groups on the same level of vertebra. This method was applied to both the results of Micro-CT structure parameters and the stiffness calculated by the FEA. The difference among three levels (L4, 5, and 6) of the same group was tested by Kruskal-Wallis H test. The Mann-Whitney U test was done to understand the difference between two individual vertebral levels. Results were shown as mean +- SD. An asymptotic value (p-value) <0.05 was used to evaluate the significance of differences. The correlation between Micro-CT structural parameters and the index of stiffness was described with Pearson correlation coefficients (r). The significance level was set at 0.05 (Sig. <0.05) with the two tailed test.