Name: imaging of the microstructural failure mechanism in the human hip
Uploaded: 2023-09-29T13:50:00
Description: Watch this Scientific Journal Video about Imaging of the Microstructural Failure Mechanism in the Human Hip at JoVE.com

Funding from the Australian Research Council (FT180100338; IC190100020) is gratefully acknowledged.

The protocol was developed and tested with 12 femur specimens received from a body donation program. The specimens were obtained fresh and stored at &#8722;20 &#176;C at the Biomechanics and Implants Laboratory of Flinders University (Tonsley, South Australia, Australia). Bone moisture was maintained throughout the experiment. The donors were Caucasian women (66-80 years of age). Ethics clearance was obtained from the Social and Behavioural Research Ethics Committee (SBREC) of Flinders University (Project # 6380).
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<ol>
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D., M&#252;ller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+microstructure+and+volume+fraction+in+predicting+failure+in+cancellous+bone.">The interaction of microstructure and volume fraction in predicting failure in cancellous bone.</a> Bone. 39 (6), 1196-1202 (2006).</li><li>Schileo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+what+extent+can+linear+finite+element+models+of+human+femora+predict+failure+under+stance+and+fall+loading+configurations.">To what extent can linear finite element models of human femora predict failure under stance and fall loading configurations.</a> Journal of Biomechanics. 47 (14), 3531-3538 (2014).</li><li>Schileo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accurate+estimation+of+bone+density+improves+the+accuracy+of+subject-specific+finite+element+models.">An accurate estimation of bone density improves the accuracy of subject-specific finite element models.</a> Journal of Biomechanics. 41 (11), 2483-2491 (2008).</li><li>Dall'ara, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonlinear+QCT-based+finite+element+model+validation+study+for+the+human+femur+tested+in+two+configurations+in+vitro.">A nonlinear QCT-based finite element model validation study for the human femur tested in two configurations in vitro.</a> Bone. 52 (1), 27-38 (2013).</li><li>Perilli, E., Parkinson, I. H., Reynolds, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+examination+of+human+bone:+from+biopsies+towards+the+entire+organ.">Micro-CT examination of human bone: from biopsies towards the entire organ.</a> Annali dell'Istituto Superiore di Sanit&#224;. 48 (1), 75-82 (2012).</li><li>Wearne, L. S., Rapagna, S., Taylor, M., Perilli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+scan+optimisation+for+mechanical+loading+of+tibia+with+titanium+tibial+tray:+A+digital+volume+correlation+zero+strain+error+analysis.">Micro-CT scan optimisation for mechanical loading of tibia with titanium tibial tray: A digital volume correlation zero strain error analysis.</a> Journal of the Mechanical Behavior of Biomedical Materials. 134, 105336 (2022).</li><li>Bennett, K. 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The present protocol allows for studying the time-elapsed micromechanics of hip fractures in three dimensions ex vivo. A radiotransparent (aluminium) compressive stage&#160;capable of applying a progressive deformation to the proximal half of the human femur and measuring the reaction force has been custom-designed, manufactured, and tested. A large-volume micro-CT scanner is employed in this protocol to provide a temporal sequence of image volumes displaying the entire proximal femur with progressive loading at...

Fractures of the femoral neck are a major burden to the aging population. Micro-computed tomography (micro-CT) imaging and concomitant mechanical testing&#160;allow for observing the bone microstructure and studying its relationship to bone strength, its age-related changes, and displacements under load1,2. However, until recently, micro-CT studies of bone under load were limited to excised bone cores3, small animals4, and human spine units5. The present protocol can quantify the displacement of the microstructure of the entire proximal human femur under load and after a fracture.
Several studies have been conducted to investigate the failure of the human femur, and at times, these have reached contrasting conclusions. For example, the age-related thinning of the cortical and trabecular structures is thought to determine the age-related susceptibility to fracture by causing elastic instability of the bone6,7, which is in apparent contrast with the high coefficient of determination of cortical strain and femoral strength predictions assuming no elastic instability (R2 = 0.80-0.97)8,9. Nevertheless, such studies have systematically underestimated the femoral strength (by 21%-29%), thus bringing into question the brittle and quasi-brittle bone responses implemented in the models8,10. One possible explanation for these apparently contrasting findings may reside in a different fracture behavior of entire bones compared to isolated bone cores. Therefore, observing the deformation and fracture responses of the bone microstructure in entire proximal femurs may advance knowledge of hip fracture mechanics and related applications.
Current methods for imaging entire human bones with micrometric resolution are limited. The gantry and the detector size must provide a suitable working volume to host the human proximal femur (approximately 13 cm x 10 cm, width x length) and possibly a pixel size in the order of 0.02-0.03 mm to ensure that relevant micro-architectural features can be captured11. These specifications can currently be met by some synchrotron facilities1 and some commercially available large-volume micro-CT scanners12,13. The compressive stage has to be radio-transparent in order to minimize X-ray attenuation while generating a force sufficient for causing a fracture to the human femur (e.g., between 0.9 kN and 14.3 kN for elderly white women)14. This large fracture load variation complicates the planning of the number of load steps to fracture, the overall experiment time, and the corresponding amount of data produced. To address this problem, the fracture load and location can be estimated via finite-element modeling by using the bone density distribution of the specimen from clinical computed tomography (CT) images1,2. Finally, after the experiment, the large volume of data generated needs to be processed for studying the failure mechanisms and energy dissipation capacity in the entire human femur.
Here, we describe a protocol for obtaining a sequence of three-dimensional microstructural images of the entire proximal femur under progressively increasing deformation, which causes clinically relevant fractures of the femoral neck2. The protocol includes planning the stepwise increment&#160;of the specimen compression, loading via a custom radio-transparent compressive stage, imaging via a large-volume micro-CT scanner, and processing the images and the load profiles.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbent tissue</td>
			<td>N/A</td>
			<td></td>
			<td>Maintain the bone moisture throughout the experiment</td>
		</tr>
		<tr>
			<td>Alignment rig</td>
			<td>Custom-made</td>
			<td></td>
			<td>Rig for positioning the specimen in the potting cup</td>
		</tr>
		<tr>
			<td>Aluminium potting cup</td>
			<td>Custom-made</td>
			<td></td>
			<td>Potting cup</td>
		</tr>
		<tr>
			<td>Bone saw</td>
			<td>N/A</td>
			<td></td>
			<td>Cut the specimen to size</td>
		</tr>
		<tr>
			<td>Calibration phantom QCT Pro</td>
			<td>Mindways Software, Inc., Austin, USA</td>
			<td>CT Calibration 13002</td>
			<td>Calibrate grey levels in the images into equivalent bone mineral (ash) density levels</td>
		</tr>
		<tr>
			<td>Clinical Computed-Tmography scanner</td>
			<td>General Electric Medical Systems Co., Wisconsin, USA</td>
			<td>Optima CT660</td>
			<td>Preliminary imaging for the prediction of the load step to fracture</td>
		</tr>
		<tr>
			<td>Compressive stage</td>
			<td>Custom-made</td>
			<td></td>
			<td>A 10 kg, radiotransparent compressive stage for applying and maintaining throught imaging a prescribed deformation to the specimen.</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Soesterberg, The Netherlands</td>
			<td>Vertex RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Femur specimen</td>
			<td>Science Care, Phoenix, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finite-element analysis software</td>
			<td>ANSYS Inc., Canonsburg, USA</td>
			<td>ANSYS Mechanical APDL</td>
			<td>Finite-element software package</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>N/A</td>
			<td></td>
			<td>Store specimens at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hard Drive</td>
			<td>Dell</td>
			<td></td>
			<td>Disk space: 500 GB per volume</td>
		</tr>
		<tr>
			<td>Image bnarization and segmentation software</td>
			<td>Skyscan-Bruker, Kontich, Belgium</td>
			<td>CT analyzer</td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Image elastic segmentation</td>
			<td>The University of Sheffield</td>
			<td>Bone DVC</td>
			<td>https://bonedvc.insigneo.org/dvc/</td>
		</tr>
		<tr>
			<td>Image processing and automation software</td>
			<td>The MathWork Inc.</td>
			<td>Matlab</td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Image registration software</td>
			<td>Skyscan-Bruker, Kontich, Belgium</td>
			<td>DataViewer</td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Image segmentation and FE modelling software</td>
			<td>Simpleware, Exeter, UK</td>
			<td>Scan IP</td>
			<td>Bone egmentation software</td>
		</tr>
		<tr>
			<td>Image stiching script</td>
			<td>Australian syncrotron, Clayton, VIC, AU</td>
			<td></td>
			<td>The script is available at IMBL</td>
		</tr>
		<tr>
			<td>Image visualization</td>
			<td>Kitware, Clifton Park, NY, USA</td>
			<td>Paraview</td>
			<td>Image visualization</td>
		</tr>
		<tr>
			<td>Image visualization</td>
			<td>Australian National University</td>
			<td>Dristhi</td>
			<td>Image visualization: doi:10.1117/12.935640</td>
		</tr>
		<tr>
			<td>Imaging and Medical beamline</td>
			<td>Australian syncrotron, Clayton, VIC, AU</td>
			<td></td>
			<td>Large object micro-CT beamline at the Australian Synchrotron</td>
		</tr>
		<tr>
			<td>Laptop</td>
			<td>Dell Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low-friction x-y table</td>
			<td>THK Co., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NI signal acquisition software</td>
			<td>National Instruments, Austin, TX</td>
			<td>NI-DAQmx</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline solution</td>
			<td>Custom-made</td>
			<td></td>
			<td>Maintain the bone moisture throughout the experiment</td>
		</tr>
		<tr>
			<td>Plastic bag</td>
			<td>N/A</td>
			<td></td>
			<td>Maintain the bone moisture throughout the experiment</td>
		</tr>
		<tr>
			<td>Rail</td>
			<td>SKF Inc., Lansdale, PA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-jack mechanism</td>
			<td>&#160;Benzlers, &#214;rebro, Sweden</td>
			<td>Serie BD (warm gear unit)</td>
			<td>stroke: 150 mm, maximal load: 10,000 N, gear ratio: 27:1, a displacement per revolution: 0.148 mm</td>
		</tr>
		<tr>
			<td>Single pco.edge sensor, lens coupled scintillator</td>
			<td>Australian syncrotron, Clayton, VIC, AU</td>
			<td></td>
			<td>Detector Ruby FOV: 141 x 119 mm; 2560 x 2160 px; 55 &#181;m/px; 50 fps</td>
		</tr>
		<tr>
			<td>Six axis load cell</td>
			<td>ME-Me&#223;systeme GmbH, Hennigsdorf, GE</td>
			<td>K6D6</td>
			<td>Maximal measurement error: 0.005%; maximal force: 10000 N; maximal torque: 500 Nm</td>
		</tr>
		<tr>
			<td>Strain amplifier</td>
			<td>ME-Me&#223;systeme GmbH, Hennigsdorf, GE</td>
			<td>GSV-1A8USB K6D/M16</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging of the microstructural failure mechanism in the human hip

Imaging the bone microstructure under progressively increasing loads allows for observing the microstructural failure behavior of bone. Here, we describe a protocol for obtaining a sequence of three-dimensional microstructural images of the entire proximal femur under progressively increasing deformation, causing clinically relevant fractures of the femoral neck. The protocol is demonstrated using four femora from female donors aged 66-80 years at the lower end of bone mineral density in the population (T-score range = &#8722;2.09 to &#8722;4.75). A radio-transparent compressive stage was designed for loading the specimens replicating a one-leg stance, while recording the applied load during&#160;micro-computed tomography (micro-CT) imaging. The field of view was 146 mm wide and 132 mm high, and the isotropic pixel size was 0.03 mm. The force increment was based on finite-element predictions of the fracture load. The compressive stage was used to apply the displacement to the specimen and enact the prescribed force increments. Sub-capital fractures due to opening and shear of the femoral neck occurred after four to five load increments. The micro-CT&#160;images and the reaction force measurements were processed to study the bone strain and energy absorption capacity. Instability of the cortex appeared at the early loading steps. The subchondral bone in the femoral head displayed large deformations reaching 16% before&#160;fracture, and a progressive increase in the support capacity up to&#160;fracture. The deformation energy linearly increased with the displacement up to&#160;fracture, while the stiffness decreased to near-zero values immediately before&#160;fracture. Three-fourths of the fracture energy was taken by the specimen during the final 25% force increment. In conclusion, the protocol developed revealed a remarkable energy absorption capacity, or damage tolerance, and a synergic interaction between the cortical and trabecular bone at an advanced donor age.

The images display the entire proximal femur, the pressure socket, the dental cement, the aluminum cup, and the wrapping tissue. The bone micro-architecture can be seen progressively deforming as the load increases before fracture and after fracture (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64947/64947fig04.jpg" /> 
Figure 4: The compressive stage con...

Watch this Scientific Journal Video about Imaging of the Microstructural Failure Mechanism in the Human Hip at JoVE.com

Imaging of the Microstructural Failure Mechanism in the Human Hip

The protocol enables the measurement of the deformation of the bone microstructure in the entire proximal human femur and its toughness by combining large-volume micro-CT scanning, a custom-made compressive stage, and advanced image processing tools.

Imaging the bone microstructure under progressively increasing loads allows for observing the microstructural failure behavior of bone. Here, we describe ...

This protocol enables the observation of the microstructure of the whole human femur as it deforms under load and fracture. By imaging the whole femur with microstructural resolution, this protocol makes it possible to study how the different trabecular cortical compartment of the bone, synergically determine its capacity to withstand the load. Advancing our understanding of the bone fracture mechanism can inform advanced diagnostic methods for osteoporosis.This protocol has been developed at the Australian Synchrotron, adapted for use in a commercial, large volume micro-CT scanner at University and applied to study different anatomical region, including the knee and shoulder. In principle, it can be extended to materials of similar size to whole human bones and joints. This study necessitates expertise in several disciplines, including imaging, solid mechanics, and computational modeling in biomechanics.To begin, using a clinical CT scanner, scan the femur specimen with a slice thickness and an in-plane pixel size of approximately 0.5 to 0.7 millimeters. Alongside the specimen, scan a CT densitometry calibration phantom, with five known concentrations of dye potassium hydrogen phosphate. Next, segment the bone geometry from the clinical CT images, calibrate gray levels in the images to corresponding bone density values, and Young's modulus, using the density to elastic modulus relationship.After creating a mesh of the segmented geometry, import the mesh into the finite element software. Fully constrained three to six millimeter deep, the distal end of the model, then replicate a single-leg stance loading configuration by applying a nominal force of 1000 Newton, abducted by eight degrees from the femoral shaft axis in the coronal plane and passing through the center of the femoral head. Solve the finite element model using the built-in PCG solver.Then by executing the indicated commands, generate an element table containing the first and third principle strain components at the element's centroid. Next, execute the indicated command to calculate the strain ratio between the first and third principle strain components in the model and the bone yield strain, intention and compression. Scale the nominal force by the peak strain ratio, intention and compression, and discard the biggest of the two to estimate the fracture load.Position a testing rig on the micro-CT rotation stage with the specimen in the reference unloaded condition and start the micro-CT scan. Repeat imaging twice for the unloaded condition and unwind the cable after scanning. Apply the force increment by manually actuating the screw jack mechanism at a constant rate of approximately one second per round, and perform the micro-CT imaging.Repeat the force increment until the specimen fractures, as indicated by a sudden drop in the reaction force. Perform micro-CT imaging of the fractured specimen. Then visualize the sequence of projection images at various load steps.Subsample the micro-CT images by four, to reduce the computation time. Rigidly co-register, in space, the images of the specimen under load, with those in the unloaded reference condition, using the distal diaphysis as the target of the co-registration. Create surface three dimensional models for visualization after binarizing the micro-CT images.Determine the displacement in the images over a grid 50 pixels in size, using bone DVC. Then determine the strain tensor, by converting the grid into a finite element model. Apply the calculated displacement at the nodes and solve the model.Next, using cubic interpolation, with the inter P three function in Mac-Lab, re-sample the displacement and strain volumes to match the size of the original size of micro-CT images. Visualize displacements, strain and microstructural images for large volume visualization and animation. For bone deformation analysis, display the permanent deformation of the bone by overlaying the images obtained in unloaded conditions and after the fracture.Then display the progressive micro structural deformation of the bone by overlaying the three dimensional models in unloaded conditions, at increasing load levels, and post fracture. Display the strain of the bone at the fracture location. Finally, using descriptive statistics and regression methods, analyze the deformation energy, stiffness, and displacement.Micro-CT imaging and concomitant mechanical tests allow for observing femoral neck fractures. An animation showed that the femoral head progressively rotated medially, while moving distally up to fracture. The head curvature flattened under the socket, where local cortical instability, but no instability of the underlying trabecular volume, was observed.Fracture onset occurs by bending the cortex, either progressing along the main compressive trabecular group, or by shear at about 45 degrees from the main principle compressive, trabecular axis. Strain exceeded bone yield, once the four succeeded 50%of the expected fracture load, reaching eight to 16%compression before fracture. Permanent deformation was observed in the head region under peak compression.Failure occurred under a complex strain state, showing compression, tension and shear strain. The deformation energy was a linear function of the displacement up to fracture, showing a stable fracture behavior. A critical aspect for replicating the protocol is obtaining the load step increment, which is important for controlling the number of load steps required to cause a fracture to displacement and planning of the experiment.Obtaining good quality images is also important for a meaningful analysis of the data. The belief that elastic instability that demands the steep increase of fracture incidents above 60 years of age focused the case of research in fragility prevention on cortical thickness. The elastically stable fracture behavior demonstrated by this protocol, in lightly osteoporotic bones, shifts the current focus to cortical and trabecular interactions.This procedure can advance considerative models of bone mechanics, inform the diagnosis of fragility and the design of implantable devices.

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