One potentially important bone quality characteristic is the response of bone to cyclic (repetitive) mechanical loading. In small animals, such as in rats and mice, cyclic loading experiments are particularly challenging to perform in a precise manner due to the small size of the bones and difficult-to-eliminate machine compliance. Addressing this issue, we developed a precise method for ex vivo cyclic compressive loading of isolated mouse vertebral bodies. The method has three key characteristics: 3D-printed support jigs for machining plano-parallel surfaces of the tiny vertebrae; pivotable loading platens to ensure uniform contact and loading of specimen surfaces; and specimen-specific micro-CT-based finite element analysis to measure stiffness to prescribe force levels that produce the same specified level of strain for all test specimens. To demonstrate utility, we measured fatigue life for three groups (n = 5–6 per group) of L5 vertebrae of C57BL/6J male mice, comparing our new method against two methods commonly used in the literature. We found reduced scatter of the mechanical behavior for this new method compared to the literature methods. In particular, for a controlled level of strain, the standard deviation of the measured fatigue life was up to 5-fold lower for the new method (F-ratio = 4.9; p < 0.01). The improved precision for this new method for biomechanical testing of small-animal vertebrae may help elucidate aspects of bone quality.

Clinical bone sonometers applied at the calcaneus measure broadband ultrasound attenuation and speed of sound. However, the relation of ultrasound measurements to bone strength is not well-characterized. Addressing this issue, we assessed the extent to which ultrasonic measurements convey in vitro mechanical properties in 25 human calcaneal cancellous bone specimens (approximately 2×4×2cm). Normalized broadband ultrasound attenuation, speed of sound, and broadband ultrasound backscatter were measured with 500kHz transducers. To assess mechanical properties, non-linear finite element analysis, based on micro-computed tomography images (34-micron cubic voxel), was used to estimate apparent elastic modulus, overall specimen stiffness, and apparent yield stress, with models typically having approximately 25-30 million elements. We found that ultrasound parameters were correlated with mechanical properties with R=0.70-0.82 (p<0.001). Multiple regression analysis indicated that ultrasound measurements provide additional information regarding mechanical properties beyond that provided by bone quantity alone (p≤0.05). Adding ultrasound variables to linear regression models based on bone quantity improved adjusted squared correlation coefficients from 0.65 to 0.77 (stiffness), 0.76 to 0.81 (apparent modulus), and 0.67 to 0.73 (yield stress). These results indicate that ultrasound can provide complementary (to bone quantity) information regarding mechanical behavior of cancellous bone.

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Although second-generation high-resolution peripheral quantitative computed tomography (XCTII) provides the highest-resolution in vivo bone microstructure assessment, the manufacturer's standard image processing protocol omits fine features in both trabecular and cortical compartments. To optimize fine structure segmentation, we implemented a binarization approach based on a Laplace-Hamming (LH) segmentation and documented the reproducibility and accuracy of XCTII structure segmentation using both the standard Gaussian-based binarization and the proposed LH segmentation approach. To evaluate reproducibility, 20 volunteers (9 women, 11 men; aged 23-75 years) were recruited, and three repeat scans of the radii and tibias were acquired using the manufacturer's standard in vivo protocol. To evaluate accuracy, cadaveric structure phantoms (14 radii, 6 tibias) were scanned on XCTII using the same standard in vivo protocol and on μCT at 24.5 μm resolution. XCTII images were analyzed twice-first, with the manufacturer's standard patient evaluation protocol and, second, with the proposed LH segmentation approach. The LH approach rescued fine features evident in the grayscale images but omitted or overrepresented (thickened) by the standard approach. The LH approach significantly reduced error in trabecular volume fraction (BV/TV) and thickness (Tb.Th) compared with the standard approach; however, higher error was introduced for trabecular separation (Tb.Sp). The LH approach improved the correlation between XCTII and μCT for cortical porosity (Ct.Po) and significantly reduced error in cortical pore diameter (Ct.Po.Dm) compared with the standard approach. The LH approach resulted in improved precision compared with the standard approach for BV/TV, Tb.Th, Ct. Po, and Ct.Po.Dm at the radius and for Ct.Po at the tibia. Our results suggest that the proposed LH approach produces substantially improved binary masks, reduces proportional bias, and provides greater accuracy and reproducibility in important outcome metrics, all due to more accurate segmentation of the fine features in both trabecular and cortical compartments.