Three-Point Bending of Rat Femur in the Mediolateral Direction: Introduction and Validation of a Novel Biomechanical Testing Protocol

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Abstract
Mediolateral three‐point bending of the rat midfemur was developed to enable the assessment of the mechanical competence of the elliptic bone cross‐section in terms of its widest diameter, the apparent primary direction of bone adaptation to loading. Introduction: Today, the most commonly used method to characterize the biomechanical properties of appendicular long bones is the three‐point bending testing of the midfemur in the anteroposterior (AP) direction. However, as the diameter of the elliptic cross‐section of femoral diaphysis is widest in the orthogonal mediolateral (ML) direction, the femoral diaphysis should also show the highest resistance to bending along this direction. The objective of this study was thus to introduce and validate a mechanical testing protocol for femoral midshaft along the ML direction. Materials and Methods: To determine the repeatability of the novel testing protocol, 38 pairs of rat femora underwent a comprehensive structural analysis by pQCT followed by ML three‐point bending. For comparison of the repeatability, corresponding tests were performed on the femoral neck. To validate the novel testing direction, the left hindlimb of 24 rats was neurectomized for 6 months, whereas the right limb served as an intact control. After excision, one half of these pairs of femora were randomly subjected to three‐point bending test in the conventional AP direction and the remaining in the orthogonal ML direction. Results: The precision (CVrms) of breaking load, stiffness, and energy absorption of the femoral midshaft in the ML direction was 3.8%, 6.6%, and 14.5%, respectively. The corresponding values for femoral neck compression test were 7.6%, 17.9%, and 18.7%, respectively. The loading‐induced effect on the femoral midshaft (difference between the neurectomized [nonloaded] and contralateral intact [loaded] femur) was +2.2%, +1.9%, and +2.1% in the AP direction and −18.9%, −17.6%, and −20.3% in the ML direction (p < 0.01 for all comparisons), respectively. Conclusions: Our results show that testing of rat femoral midshaft in the ML direction is a precise and biologically valid method to determine the structural strength of this widely used skeletal site in experimental bone research.

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