Name: Mechanical Testing Procedure to Check Bone's Susceptibility to Fracture
Uploaded: 2023-09-01T13:50:00
Description: Watch this Scientific Journal Video about Mechanical Testing Procedure to Check Bone's Susceptibility to Fracture at JoVE.com

mechanical testing procedure to check bone&#39;s susceptibility to fracture

골절 감수성에 대한 기계적 검사의 원리와 프로토콜

Mechanical testing of bone is the primary method to extract functional information related to a bone&#39;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 freezing bones in saline-soaked gauze, freezing in saline alone, or dehydrating bones in ethanol 1. Bones stored in ethanol have been shown to have increased stiffness and elastic modulus and decreased deformation parameters versus those stored frozen1. Even rehydrating the bones prior to testing does not recover these properties back to normal levels 1. Storing submerged in saline could cause damage to the bone since pressure is exerted as the saline expands. In addition, a complete thaw of the solution would be required to remove the bones for microcomputed tomography (&#181;CT) scanning. Consequently, freezing freshly harvested bones in saline-soaked gauze has become the standard storage method and is recommended throughout this protocol.
Because the size and shape of a bone affect its bulk strength and many disease models significantly alter bone size and morphology, engineering principles are used to normalize away the effects of size to produce properties that estimate the behavior of the tissue2. This approach requires cross-sectional geometry of the failure location, which is most commonly acquired using &#181;CT to create scans of the bones prior to testing. &#181;CT is widely used due to its availability and high image resolution. Moreover, contributions of soft tissue are not included, and scanning does not require chemical fixation or other modifications to the bone3,4. In all forms of CT, an X-ray source is focused on an object while a detector on the other side of the object measures the resulting X-ray energy. This produces an X-ray shadow of the sample that can be converted into an image3,5. The object being scanned is rotated (or the X-ray source and detector are rotated around the sample), generating images that can be reconstructed into a three-dimensional data set representing the object5.
Scan resolution, or how close together two objects can be and still be resolved individually, is controlled by changing the nominal voxel size or the size of a pixel in the resulting image. It is generally accepted that objects must be at least two times the size of a single voxel to be identified3, but a higher ratio will allow for improved precision. Further, larger voxels are more prone to partial volume effects: when a single voxel contains tissues of varying densities, it is assigned the average of these densities, rather than the specific density of a single tissue, which may lead to an over- or under-estimation of tissue areas and mineral density3. While these issues can be mitigated by choosing smaller voxel sizes, using a higher resolution does not ensure the elimination of partial volume effects and may require longer scan times3. When scanning bones ex vivo, a voxel size of 6-10 &#181;m is generally recommended to accurately assess the trabecular architecture of mouse bones. A larger voxel size of 10-17 &#181;m can be used for cortical bone, although the smallest reasonable voxel size should be used. This protocol uses a 10 &#181;m voxel size, which is small enough to differentiate key trabecular properties and minimize partial volume effects without extensive scan time.
X-ray energy and energy filter settings must also be selected carefully, as the high mineral density and thickness of bone tissue greatly attenuates and alters the transmitted X-ray energy spectrum. It is generally assumed that because the emitted X-ray spectrum is equivalent to the spectrum that exits the object6, using low-energy X-rays on dense objects such as bone can lead to an artifact known as beam hardening7. A higher voltage of 50-70 kVp is recommended when scanning bone samples to reduce the incidence of these artifacts5. In addition, inserting an aluminum or copper energy filter creates a more concentrated energy beam, further minimizing artifacts4,7. A 0.5 mm aluminum filter will be used throughout this protocol.
Finally, the scan rotation step and rotation length (e.g., 180&#176;-360&#176;), together control the number of images captured, which determines the amount of noise in the final scan4. Averaging multiple frames in each step can reduce noise but may increase scan time4. This protocol uses a rotation step of 0.7 degrees and a frame averaging of 2.
One final note about scanning: hydroxyapatite calibration phantoms should be scanned using the same scan settings as the experimental bones to enable the conversion of attenuation coefficients to mineral density in g/cm35. This protocol uses phantoms of 0.25 g/cm3 and 0.75 g/cm3 of hydroxyapatite, although different phantoms are available. Note that some scanning systems use internal phantoms as part of daily system calibration.
Once scanning is complete, the angular projections are reconstructed into cross-sectional images of the object, typically using the manufacturer&#39;s accompanying software. Whatever system is used, it is important to ensure that the whole bone is captured in the reconstruction and that thresholding is set appropriately to allow for the recognition of bone versus non-bone. After reconstruction, it is critical to rotate all scans in three dimensions so that bones are oriented consistently and properly aligned with the transverse axis, again using the manufacturer&#39;s software.
Following rotation, regions of interest (ROI) for analysis may be selected based on whether cortical properties, trabecular properties, or fracture geometry for mechanical normalization are desired. For the latter, ROIs should be selected after testing by measuring the distance from the fracture site to one end of the bone and using voxel size to determine the corresponding slice location in the scan file. The selected region should be at least 100 &#181;m in length, with the fracture point at the approximate center of the ROI, to provide adequate estimation4.
With ROIs selected, two properties are needed for mechanical normalization (to calculate bending stress and strain): the maximum distance from the neutral bending axis to the surface where failure is initiated (assumed to be the surface loaded in tension, determined by the testing setup), and the area moment of inertia around the neutral axis, (also dependent on testing setup). This protocol recommends the use of a custom code to determine these values. For access to the code, contact the corresponding author directly or visit the lab website at&#160;https://bbml.et.iupui.edu/ for more information.
Once &#181;CT scanning has been completed, mechanical testing can begin. Bending tests can be performed in either four-point or three-point configurations. Four-point bending tests are preferred as they eliminate shear stress in the bone between loading points, allowing for pure bending to occur in this region3. The bone will then fracture due to tension, creating a failure that is more representative of the true bending properties of the bone3. However, the bone must be loaded in such a way as to deliver the same load at both loading points (this can be facilitated with a pivoting loading head). In three-point bending tests, there is a large change in shear stress where the load point meets the bone, which causes the bone to break at this point due to shear, not tension3. ASTM standards recommend materials undergoing bending should have a length-to-width ratio of 16:1, meaning the length of the support span should be 16 times larger than the width of the bone to minimize impacts of shear8,9. This is often impossible to achieve when testing small rodent bones, so the loading span is simply made as large as possible but with as small of a change in cross-sectional shape as possible. Moreover, when performing four-point bending, the ratio between the lengths of the lower and upper span should be ~3:18, which can usually be achieved in the tibia, but it is difficult in the shorter femur. In addition, the thinner cortical walls of femurs make them susceptible to ring-type deformation which changes the shape of the bone cross-section during the test (this can be accentuated in four-point tests as a greater force is required to induce the same bending moment compared to three-point bending). Therefore, three-point bending will be utilized for mouse femora while four-point bending will be used for tibiae throughout this protocol.
Finally, it is important to properly power the study for statistical analysis. A general recommendation for mechanical testing is to have a sample size of 10-12 bones per experimental group to be able to detect differences, as some mechanical properties, especially postyield parameters, can be highly variable. In some cases, this may mean starting with a higher animal sample size given attrition that could occur during the study. Sample size analysis using existing data should be completed prior to attempting a study.
There are numerous limitations and assumptions, but bending tests can provide quite accurate results, especially when relative differences between groups are of interest. These properties, together with the analysis of trabecular architecture and cortical morphology, can provide better insight into disease states and treatment regimens. If care is taken with those aspects of the experiment that are in our control (e.g., harvesting, storing, scanning, and testing), we can feel confident that accurate results have been generated.

저자 스포트라이트: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests(전체 뼈 굽힘 검사의 정확성 및 재현성 향상) 

설치류 뼈의 전체 뼈 굽힘 시험과 관련된 연구의 설계, 실행 및 해석에 대한 실용적인 고려 사항

The current experimental challenges of bending tests include:Consistent placing of the bones in the same orientation, documenting the break progress of the bone during the test, and interpretation of complex results. Our protocol provides a guide not only for the testing procedure and data analysis for whole-bone bending tests, but also for sample preparation and storage. And this is the first instance where the entire protocol has been presented.Whole-bone mechanical testing is a quick and simple method to obtain measures of bone fracture susceptibility when compared to other testing methods.

Watch this Scientific Journal Video about Mechanical Testing Procedure to Check Bone's Susceptibility to Fracture at JoVE.com

Mechanical Testing Procedure to Check Bone's Susceptibility to Fracture  

골절에 대한 뼈의 감수성을 확인하기 위한 기계적 검사 절차

안락사된 쥐에서 대퇴골과 경골을 채취한 후, 운동 기하학을 계산하기 위해 각 샘플에 대해 재구성한 6-10마이크로미터 해상도의 마이크로 CT 스캔을 얻습니다. 테스트를 위해 모든 뼈를 해동하고 한 세션에서 동일한 실험의 모든 뼈를 테스트해야 합니다. 이제 시편에 적합한 감도와 용량을 가진 로드셀을 찾으십시오.시편의 파손 범위를 고려하여, 감도를 최대화하면서 50% 더 많은 용량을 가진 로드셀을 선택하십시오. 하중 및 지지 스팬 고정 장치를 찾습니다. 로드셀을 테스터의 상부 지지대에 나사로 고정하고 상부 하중 고정구를 로드셀에 부착하고 하부 고정구를 테스터의 하단 지지대에 장착하여 로드셀과 고정구를 설치합니다.그런 다음 모든 샘플에 대해 일정하게 유지되는 지지 스팬 길이를 선택합니다. 샘플 세트에서 가장 짧은 골격을 사용하여 지지 범위 거리를 결정하고 픽스처 사이에서 골격의 방향을 적절하게 지정합니다. 대퇴골의 3점 굽힘 테스트를 수행합니다.뼈의 앞쪽 표면을 지지 스팬에 대고 위치가 샘플의 단골 내에 있는지 확인합니다. 근위 끝의 세 번째 trochanter와 뼈가 원위 끝의 metaphysis 및 condyles로 넓어지는 전이 지점을 제외합니다. 로딩 스팬이 지지 스팬 내의 중앙에 있고 골격의 균일한 영역을 포함하는지 확인합니다.그런 다음 지지 스팬 거리를 측정하고 기록합니다. 지지 스팬이 설정되고 기록되면 뼈를 놓을 수 있는 충분한 공간을 두고 아래쪽 고정구를 위쪽 고정구에 가깝게 가져옵니다. 뼈를 식염수로 다시 넣거나 식염수로 재수화하십시오.모든 고정 부품이 단단하고 움직이지 않는지 확인하십시오. 기계식 테스터 소프트웨어에서 느린 변위 램프 속도로 굽힘 테스트 프로파일을 설계하여 점탄성 효과를 방지하여 골격이 파손될 때까지 하중을 가합니다. 테스트 프로필을 만든 후 모든 스터디 그룹에 대해 고유한 폴더를 만듭니다.적절하게 해동된 뼈를 선택한 다음 캘리퍼스를 사용하여 전체 길이를 측정하고 기록합니다. 뼈 샘플을 올바른 방향으로 픽스처에 로드합니다. 테스트 중인 샘플의 세부 정보를 반영하도록 파일 이름을 변경하고 각 테스트를 해당 폴더에 저장합니다.하중을 영점화하고 시스템의 무버를 활성화하여 하중 또는 변위 제어에 있지 않도록 합니다. 뼈의 위치를 고정하기 위해 뼈에 약간의 예압을 조심스럽게 가합니다. 샘플을 손상시키지 마십시오.약 0.25뉴턴의 예압을 목표로 합니다. 앞으로 이동하기 전에 원하는 뼈 방향이 일관되게 유지되는지 확인하십시오. 식염수를 넉넉하게 적셔 샘플을 수화하십시오.소프트웨어에서 시작 또는 실행을 선택하여 굽힘 테스트를 시작합니다. 테스트 기간 동안 샘플을 정확하게 관찰하고 문제 발생을 기록하는 것이 중요합니다. 뼈가 골절되기 시작할 때 면밀히 관찰하십시오.테스트는 설정된 제한에 도달하면 자동으로 중지됩니다. 그러나 오류가 발생하면 로드셀이 손상되지 않도록 수동으로 테스트를 중지하십시오. 테스트가 완료되면 캘리퍼스를 사용하여 말단부에서 중단점까지의 길이를 측정하고 기록합니다.

Research

JoVE Journal

Bioengineering

Mechanical Testing Procedure to Check Bone&#39;s Susceptibility to Fracture

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

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 ...