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).
1. Planning a specimen-specific load step increment
<ol>
	<li>Scan the femur specimen using a clinical CT scanner targeting a slice thickness and an in-plane pixel size of approximately 0.5-0.7 mm. This step can be completed by an expert radiographer at any public imaging facility using standard pre-recorded imaging protocols for bone visualization.</li>
	<li>Together with the specimen, scan a CT densitometry calibration phantom with five known concentrations of dipotassium hydrogen phosphate (K2HPO4, equivalent density range approximately between 59 mg&#8729;cm&#8722;3 and 375 mg&#8729;cm&#8722;3).</li>
	<li>Segment the bone geometry from the clinical CT images15, mesh the segmented geometry of the bone, and map the isotropic material properties element by element to the calibrated bone density values by using the density-to-elastic modulus relationship reported by Schileo et al.8. Save the mesh for further analysis in the finite-element software. Complete each step by following the relevant guidelines provided with the segmentation and finite-element software.</li>
	<li>Import the mesh into the finite-element software. Fully constrain the 3-6 mm distal end of the model. Apply a nominal force of 1,000 N, adducted by 8&#176; from the femoral shaft axis in the coronal plane and passing through the center of the femoral head. This loading condition mimics a static one-leg stance task (orthoload.com).</li>
	<li>Solve the finite-element model using the built-in PCG solver (convergence tolerance: 1 x 10&#8722;7). 
	&#8203;NOTE: Here the finite element software ANSYS was used.
	<ol>
		<li>Generate an element table containing the first and third principal strain components at the element centroid by executing the following commands: 
		/POST1 
		ETABLE,, EPTO1,1 
		&#8203;ETABLE,, EPTO3,3</li>
		<li>Calculate the strain ratio between the first and third principal strain components in the model and the bone yield strain in tension (0.73% strain) and compression (1.04% strain)8 (Figure 1) by executing the following commands: 
		SMULT,RFT,EPTO1, ,1/0.0074,1, 
		SMULT, RFT,EPTO3, ,1/0.0104,1,</li>
	</ol>
	</li>
	<li>Scale the nominal force by the peak strain ratio in both tension and compression, and discard the biggest of the two in order to obtain an estimate of the fracture load. Determine the load increment as 1/4 of the calculated fracture load1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64947/64947fig01.jpg" /> 
Figure 1: The calculation of the fracture load. The finite-element strain map, the equations used to convert the nominal force into the fracture load (left), and the loading scheme displaying the femur (center right), the distal aluminum (top right) cup, and the polyethylene pressure socket (bottom right). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64947/64947fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of the femur specimen assembly (Figure 2)
<ol>
	<li>Remove the specimen from the freezer (&#8722;20 &#176;C).</li>
	<li>Thaw at room temperature (RT) for 24 h while keeping the specimen in a waterproof plastic bag wrapped in absorbent material soaked in a physiological solution to maintain the bone moisture.</li>
	<li>Cut the femoral diaphysis at 180 mm from the proximal femoral head.</li>
	<li>Center the femoral head on the vertical axis of the alignment rig by aligning the concave-shaped polyethylene pressure socket (Figure 2D) and the femur head.</li>
	<li>Align the plane containing the femoral neck and the diaphysis axis with the frontal plane&#160;(Figure 2).</li>
	<li>Rotate the diaphyseal axis to 8&#176; adduction so that the vertical axis represents the orientation of the hip reaction force during a static single-leg stance (Figure 2).</li>
	<li>Prepare the dental cement by following&#160;manufacturer&#39;s instructions.</li>
	<li>Pot the distal end of the specimen in an aluminum potting cup that is 55 mm deep, filling up the aluminium cup with dental cement. Allow no less than 30 min for the cement to complete curing.</li>
	<li>Store the specimen assembly at &#8722;20 &#176;C.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64947/64947fig02.jpg" /> 
Figure 2: The alignment rig. A frontal (left) and lateral (right) photo of the alignment rig displaying (A) the frame, (B) the aluminum potting cup, (C) a synthetic femur model, and (D) the spherically shaped pressure socket. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64947/64947fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Compression stage assembly
NOTE: The compression stage&#39;s external dimensions are 245 mm diameter, 576 mm height, and 14 kg weight, excluding the sample. The compression stage consists of two main parts: the compression chamber and the actuator, which are assembled as follows:
<ol>
	<li>Compression chamber
	<ol>
		<li>Mount the polyethylene pressure socket (104 mm diameter, 60 mm height) at the bottom of the aluminum cylinder (203 mm diameter, 3 mm wall thickness), which is closed by a welded aluminum plate at one end (bottom).</li>
	</ol>
	</li>
	<li>Actuator
	<ol>
		<li>Assemble the top structure using the disk, the three rods, the triangular plate, and the vertical rail (Figure 3).</li>
		<li>Mount the screw-jack mechanism (stroke: 150 mm, maximal load: 10,000 N, gear ratio: 27:1, displacement per revolution: 0.148 mm) on the triangular plate.</li>
		<li>Mount the angular adaptor onto the linear rail.</li>
		<li>Mount the low-friction x-y table onto the angular adaptor.</li>
		<li>Mount the six degrees of freedom load cell (maximal measurement error: 0.005%; maximal force: 10,000 N; maximal torque: 500 Nm) onto the low-friction table by aligning the x-z plane of the load cell to the frontal plane of the top structure.</li>
		<li>Connect the actuator screw to the angular adaptor.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64947/64947fig03.jpg" /> 
Figure 3: The custom-made radiotransparent compression stage assembly. A photo (left) and a model (right) of the compressive stage. (A) The compression chamber, which is a 3 mm thick aluminium cylinder closed at the bottom; (B) the actuator assembly with the top structure; (C) the screw-jack mechanism; (D) the low-friction x-y table; and (E) the six-axis load cell are displayed and indicated on the model. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64947/64947fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Setting up the experiment
<ol>
	<li>Thaw the specimen at RT for 24 h while keeping it in a waterproof plastic bag wrapped in absorbent material soaked in a physiological solution to maintain the bone moisture.</li>
	<li>Mount the aluminium cup specimen assembly to the load cell by aligning the frontal plane of the specimen assembly with that of the actuator.</li>
	<li>Assemble the top structure, including the specimen, into the compression chamber. Take care to align the femoral head with the spherical concavity on the polyethylene pressure socket. Ensure that the femoral head is engaged but slack within the spherical cavity of the pressure socket.</li>
	<li>Place the compressive stage on the rotation stage of the micro-CT scanner at Imaging and Medical Beamline (IMBL).</li>
	<li>Connect the load cell (error &#60; 0.005%; maximum force: 10,000 N; maximum torque: 500 Nm) to the strain amplifier.</li>
	<li>Connect, via USB, the strain amplifier to a laptop computer equipped with the application software provided with the load cell.</li>
	<li>Actuate the screw mechanism in the compression stage by moving the specimen downward toward the pressure socket while monitoring the reaction force measured by the load cell in the laptop. Stop the screw mechanism once a compression force equal to 100 N is achieved. Unload the specimen to 50 N pre-load.</li>
	<li>Select the single pco.edge sensor lens-coupled scintillator &#34;Ruby&#34; (http://archive.synchrotron.org.au/31-australian-synchrotron/imbl/811-preparation-for-imaging-experiments).</li>
	<li>Set the field of view to 76.31 mm x 64.39 mm, which for the 2,560 pixels x 2,160 pixels array size provides a pixel size of 29.81 &#956;m.</li>
	<li>Set the axis of the rotating stage to 8 mm (horizontally) from the axis of the field of view (off-set scanning mode) to extend the field of view to 145.71 mm x 64.39 mm at a pixel size of 29.81 &#956;m.</li>
	<li>Set the scanning parameters to a beam energy of 60 keV, a rotational increment of 0.1&#176;, two batches of 180&#176; rotation (off-set scanning), an exposure time of 50 &#956;s, and a frame-averaging of two per rotational position.</li>
	<li>Set the scan to acquire five consecutive, vertically stacked scans, with a 26 mm vertical shift each, so that the total height of the scanned volume is 132.2 mm for a total scanning time of 30 min.</li>
</ol>
5. Mechanical testing with concomitant microstructural imaging
<ol>
	<li>Perform micro-CT (pixel size: 0.03 mm) imaging twice in the reference condition (taken as a zero-strain condition).</li>
	<li>Apply the force increment by manually actuating the screw-jack mechanism at a constant rate of approximately 1&#160;s per round (0.1-0.2 mm/s).</li>
	<li>Perform micro-CT imaging.</li>
	<li>Repeat step 5.2 and step 5.3 up to causing the fracture of the specimen, as indicated by a sudden drop in the reaction force.</li>
	<li>Perform micro-CT imaging of the fractured specimen.</li>
	<li>Stitch the 1,800 projection images (2,560 pixels x 896 pixels in size, 76.8 mm x 26.88 mm, width x height, 32-bit floating point images). The process stitches two projection images (taken in horizontal off-set scanning mode), and the five vertically shifted images, hence&#160;producing&#160;a single projection image.
	<ol>
		<li>Reconstruct the volume of the cross-section images (4,407 images, each image 4,888 x 4,888 pixels in size), and save them as 32-bit, floating point files in .TIFF format (occupying 392 GB of disk space).</li>
		<li>Apply a 3 x 3 Gaussian filter to reduce noise. Convert the images into 8-bit (256 greylevel images, saved in bitmap format, occupying approximately 100 GB per volume). 
		&#8203;NOTE: In this work, the processing of the images was conducted using software available at the Australian Synchrotron under the guidance of the operator of the IMBL.</li>
	</ol>
	</li>
</ol>
6. Calculation of the displacement and strain field
<ol>
	<li>Subsample the cross-section images by four (120 &#956;m/pixel) to reduce the computation time.</li>
	<li>Rigidly co-register in space the images of the specimen under load to those of the specimen in the unloaded reference condition. Use the distal diaphysis as the target of the co-registration (Supplementary File 1 and Supplementary File 2).</li>
	<li>Create surface three-dimensional models (.STL files) for visualization after binarizing the micro-CT images11.</li>
	<li>Elastically register the image volume to the reference volume using a grid size equal to 50 pixels (SDER = 0.076% strain error, BoneDVC,&#160;https://bonedvc.insigneo.org/dvc/) to determine the displacements at the nodes of the grid.</li>
	<li>Convert the grid into a finite-element model. Apply the nodal displacement calculated by BoneDVC to the model. Solve the model to determine the strain tensor over the entire bone volume.</li>
	<li>Repeat the analysis in the region showing the highest strain levels using the full-resolution images.</li>
	<li>Map the DVC strain maps to the full-resolution images using cubic interpolation with the interp3 function (Matlab)2.</li>
	<li>Visualize the displacements, strain, and microstructural images for large-volume visualization and animation (Matlab)2.</li>
</ol>
7. Analysis
<ol>
	<li>Display the permanent deformation of the bone (damage) by overlaying the images obtained in the unloaded conditions and after the fracture2.</li>
	<li>Display the progressive microstructural deformation of the bone by overlaying the three-dimensional models in unloaded conditions, at increasing load levels, and post-fracture2.</li>
	<li>Display the strain of the bone at the fracture location2.</li>
	<li>Analyze the deformation energy, stiffness, and displacement using descriptive statistics and regression methods2.</li>
</ol>

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	<li>Martelli, S., 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=Time-elapsed+synchrotron-light+microstructural+imaging+of+femoral+neck+fracture.">Time-elapsed synchrotron-light microstructural imaging of femoral neck fracture.</a> Journal of the Mechanical Behavior of Biomedical Materials. 84, 265-272 (2018).</li><li>Martelli, S., Giorgi, M., Dall' Ara, E., 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=Damage+tolerance+and+toughness+of+elderly+human+femora.">Damage tolerance and toughness of elderly human femora.</a> Acta Biomaterialia. 123, 167-177 (2021).</li><li>Perilli, 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=Dependence+of+mechanical+compressive+strength+on+local+variations+in+microarchitecture+in+cancellous+bone+of+proximal+human+femur.">Dependence of mechanical compressive strength on local variations in microarchitecture in cancellous bone of proximal human femur.</a> Journal of Biomechanics. 41 (2), 438-446 (2008).</li><li>Thurner, P. J., 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=Time-lapsed+investigation+of+three-dimensional+failure+and+damage+accumulation+in+trabecular+bone+using+synchrotron+light.">Time-lapsed investigation of three-dimensional failure and damage accumulation in trabecular bone using synchrotron light.</a> Bone. 39 (2), 289-299 (2006).</li><li>Jackman, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative,+3D+visualization+of+the+initiation+and+progression+of+vertebral+fractures+under+compression+and+anterior+flexion.">Quantitative, 3D visualization of the initiation and progression of vertebral fractures under compression and anterior flexion.</a> Journal of Bone and Mineral Research. 31 (4), 777-788 (2016).</li><li>Mayhew, P. 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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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All the authors declare no conflicts of interest.

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><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 &amp;deg;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>&amp;nbsp;Benzlers, &amp;Ouml;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 &amp;micro;m/px; 50 fps</td></tr><tr><td>Six axis load cell</td><td>ME-Me&amp;szlig;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&amp;szlig;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 ...

imaging-of-the-microstructural-failure-mechanism-in-the-human-hip

Universidad San Sebastian

Research

JoVE Journal

Engineering

729 Views.  Queensland University of Technology. 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. 

Video: Imaging of the Microstructural Failure Mechanism in the Human Hip

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

Bioengineering

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Medicine

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

In Vivo Quantification of Hip Arthrokinematics during Dynamic Weight-bearing Activities using Dual Fluoroscopy

Several hip pathologies have been attributed to abnormal morphology with an underlying assumption of aberrant biomechanics. However, structure-function relationships at the joint level remain challenging to quantify due to difficulties in accurately measuring dynamic joint motion. The soft tissue artifact errors inherent in optical skin marker motion capture are exacerbated by the depth of the hip joint within the body and the large mass of soft tissue surrounding the joint. Thus, the complex relationship between bone shape and hip joint kinematics is more difficult to study accurately than in other joints. Herein, a protocol incorporating computed tomography (CT) arthrography, three-dimensional (3D) reconstruction of volumetric images, dual fluoroscopy, and optical motion capture to accurately measure the dynamic motion of the hip joint is presented. The technical and clinical studies that have applied dual fluoroscopy to study form-function relationships of the hip using this protocol are summarized, and the specific steps and future considerations for data acquisition, processing, and analysis are described.

In Vivo Quantification of Hip Arthrokinematics during Dynamic Weight-bearing Activities using Dual Fluoroscopy

Dual fluoroscopy accurately captures in vivo dynamic motion of human joints, which can be visualized relative to reconstructed anatomy (e.g., arthrokinematics). Herein, a detailed protocol to quantify hip arthrokinematics during weight-bearing activities of daily living is presented, including the integration of dual fluoroscopy with traditional skin marker motion capture.

Several hip pathologies have been attributed to abnormal morphology with an underlying assumption of aberrant biomechanics. However, structure-function ...

The Use of Mixed Reality in Custom-Made Revision Hip Arthroplasty: A First Case Report

The technology of 3D printing and visualization of anatomical structures is rapidly growing in various fields of medicine. A custom-made implant and mixed reality were used to perform complex revision hip arthroplasty in January 2019. The use of mixed reality allowed for a very good visualization of the structures and resulted in precise implant fixation. According to the authors' knowledge, this is the first described case report of the combined use of these two innovations. The diagnosis preceding the qualification for the procedure was the loosening of the left hip's acetabular component. Mixed reality headset and holograms prepared by engineers were used during the surgery. The operation was successful, and it was followed by early verticalization and patient rehabilitation. The team sees opportunities for technology development in joint arthroplasty, trauma, and orthopedic oncology.

A complex revision hip arthroplasty was performed using a custom-made implant and mixed reality technology. According to the authors&#39; knowledge, this is the first report of such procedure described in the literature.

The technology of 3D printing and visualization of anatomical structures is rapidly growing in various fields of medicine. A custom-made implant and mixed ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

Biology

Clinical Application of Artificial Intelligence Preoperative Planning System Combined with Expert Database Retrieval in Complex Revision Hip Surgery

Accurate preoperative planning in revision hip arthroplasty is crucial for achieving successful outcomes. To enhance the intuitive evaluation of acetabular bone defect severity and leverage previous successful experience in revision hip arthroplasty, this study proposes a novel approach based on expert surgical case database retrieval and is initially implemented in clinical application.&#160;In this study, five patients who required revision hip arthroplasty were preoperatively planned to employ the expert case database surgical planning system.The patient&#39;s imaging data was entered into the system and matched with cases in the expert case database. Based on the expert&#39;s surgical experience, a revision surgery plan was recommended. If no suitable case was found, the model and position of the prosthesis were planned based on patient-specific reconstruction results. A total of five patients were enrolled in this study, four males and one female, with a mean age of 50.6 years. The diagnosis was aseptic prosthesis loosening after hip arthroplasty. The mean operative time was 123.2 min, and the mean intraoperative hemorrhage was 672 mL. No intraoperative complications, such as vascular or nerve injury, were observed. In Case 2, for instance, the application of this innovative planning scheme enabled the surgeon to delineate the revision surgery plan for this patient in the preoperative period, thereby reducing the operative time and intraoperative hemorrhage. Furthermore, patients could be apprised of the outcomes of analogous cases in advance. Leveraging a big data analysis approach through our comprehensive case database enables automated identification of matching expert treatment plans throughout the entire process. This particularly benefits inexperienced orthopedic surgeons by providing accurate guidance on surgical strategies to assist them in selecting appropriate prosthetic sizes and mounting positions. Additionally, the matching results can offer patients visualizations depicting predicted postoperative outcomes.

This study proposes a novel artificial intelligence preoperative planning approach based on expert surgical case database retrieval in revision hip arthroplasty. Additionally, the technique was initially employed in five patients, exhibiting a reduction in operative time and intraoperative hemorrhage.

Imaging of the Microstructural Failure Mechanism in the Human Hip

Summary

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Chapters in this video

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

A Simple Critical-sized Femoral Defect Model in Mice

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

In Vivo Quantification of Hip Arthrokinematics during Dynamic Weight-bearing Activities using Dual Fluoroscopy

The Use of Mixed Reality in Custom-Made Revision Hip Arthroplasty: A First Case Report

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Clinical Application of Artificial Intelligence Preoperative Planning System Combined with Expert Database Retrieval in Complex Revision Hip Surgery