High-precision method for cyclic loading of small-animal vertebrae to assess bone quality

Abstract

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.

Effects of ex vivo ionizing radiation on collagen structure and whole-bone mechanical properties of mouse vertebrae

“…The total bone volume of the vertebrae was measured on the L5 only (ImageJ 2.0, BoneJ2). After manual segmentation of the trabecular compartment, the following parameters were measured between the top and bottom surface of the L5 vertebra: trabecular bone volume fraction (Tb.BV/TV), number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) (Scanco Medical µCT Evaluation Program v6.5) [29]. The micro-CT analysis confirmed successful random sample distribution; there were no significant differences in bone quantity or microarchitecture between the groups.…”

“…Lumbar vertebrae (L3, L4, L5, S1) were excised and gently cleaned of soft tissue, wrapped in saline-soaked gauze (Gibco PBS 1X, pH 7.4), and stored at -20°C. In preparation for mechanical testing, the vertebral bodies of the L4 and L5 levels were isolated, endplates precisely planed using a diamond microtome (Leica SP1600 Saw Microtome, Wetzlar, Germany) and posterior elements removed [29].…”

Dietary countermeasure mitigates simulated spaceflight-induced osteopenia in mice

“…MicroCT analysis was also performed on the fourth lumbar (L4) vertebrae. Before scanning, vertebrae were processed to remove all transverse processes that project posteriorly in preparation for mechanical testing, as detailed below 76 . If a vertebral body was at all compromised during sample preparation, sample was removed from further analysis.…”

“…Mechanical testing -monotonic compression. The vertebral column was processed to isolate the L4 vertebra and further prepared for mechanical testing as described previously 76 . In brief, all soft tissue was carefully removed, and endplates and posterior elements were removed using a diamond-saw microtome (Leica SP1600 Microtome, Wetzlar, Germany) to isolate the vertebral body and ensure plano-parallel testing surfaces.…”

“…In brief, all soft tissue was carefully removed, and endplates and posterior elements were removed using a diamond-saw microtome (Leica SP1600 Microtome, Wetzlar, Germany) to isolate the vertebral body and ensure plano-parallel testing surfaces. Uniaxial compressive monotonic testing was performed as described previously 76 . In brief, after microCT was performed as detailed above, uniaxial compressive testing was performed at ambient temperature at a platen displacement rate of 0.01 mm/sec.…”