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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

Abstract

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone's susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone's susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

Introduction

Mechanical testing of bone is the primary method to extract functional information related to a bone's susceptibility to fracture. In preclinical studies, several testing modalities can be used but by far the most common is the bending of long bones. These tests are easy to perform and can be used on bones ranging in size from human to mouse. As mice are one of the most commonly studied animals in preclinical research, this protocol will focus on bending tests performed on the femora and tibiae of mice.

Prior to performing bending tests, bones must be properly harvested and stored. The most common storage methods have traditionally been....

Protocol

All procedures described throughout this protocol that involved animals have been approved by the Indiana University School of Science Institutional Animal and Use Committee (IACUC) prior to the procedure. Animals described in the procedure were euthanized via CO2inhalation followed by cervical dislocation as a secondary means of euthanasia.

1. Harvest, storage, and thawing of bones

  1. Harvest and storage
    1. Place the mouse ventral side up. Use a s.......

Representative Results

Upon completion of CT scanning, most inadequate scans can be caught in reconstruction. Often, poor scans will have a high misalignment compensation that is a clear indicator of an error during the scan. However, errors may occur in other steps and could also lead to inaccurate data. These errors can often be spotted as the individual calculated architectural properties are examined. If values fall far out of the range of others in a group, the scan, ROI, and method of calculating the properties should be re-examined.

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Discussion

Throughout the scanning and testing process, there are moments when troubleshooting and optimization are appropriate. The first of these occurs when scanning bones using µCT. While many systems come with a holder in which one object may be held and scanned, custom holders can be fabricated to scan multiple bones at the same time. Scanning multiple bones can be an excellent point for optimization, but caution should be taken throughout the scanning and analysis process to ensure artifacts are not being induced. As X-.......

Acknowledgements

The work done to develop this protocol has been supported by the National Institutes of Health [AR072609].

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Materials

NameCompanyCatalog NumberComments
CTAnBrukerNACT Scan Analysis Software
DataViewerBrukerNACT Scan Rotation Software
Matrix Laboratory (MATLAB) 2023aMathWorksNACoding platform used for data analysis
NReconBrukerNACT Scan Reconstruction software
SKYSCAN 1272-100 kV w/ 16 MP CCD detector, incl 3D Suite Software  Micro Photonics IncSKY-016814Micro-CT system that can non-destructively visualize up to 209 mPs in every virtual slice through an object 

References

  1. Vesper, E. O., Hammond, M. A., Allen, M. R., Wallace, J. M. Even with rehydration, preservation in ethanol influences the mechanical properties of bone and how bone responds to experimental manipulation. Bone. 97, 49-53 (2017).
  2. Jepsen, K. J., Silva, M. J., Vashishth, D., Guo, X. E., van der Meulen, M. C.

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