We would like to thank the Materials and Structural Testing Core Facility at Mayo Clinic for technical support. In addition we would like to thank Lawrence J. Berglund, Brant Newman, Jorn op den Buijs, Ph.D., for their help during the study. This study was financially supported by the Grainger Innovation Fund from the Grainger Foundation.

NOTE: All studies presented in this protocol were approved by the Institutional Review Board (IRB) at Mayo Clinic. The bones were obtained over a period of 6 years from various organizations. All specimens were collected within 72 hours of death, wrapped in saline saturated towels, and stored at -20 &#176;C until preparation.
1. Measuring Bone Mineral Density Using DXA
<ol>
	<li>Remove specimens kept at -20 &#176;C freezer to thaw at room temperature for about 24 h; specimens do not need to be removed from original packing if most soft tissue has been removed.</li>
	<li>Use two 5 lb bags of rice to account for soft tissue. Cover the two rice bags on the DXA table with plastic bags to prevent contamination. The rice bags will simulate surrounding soft tissue (in vivo) during scanning as shown in Figure 1.</li>
	<li>Protect the surface of the DXA scanner with plastic lined paper and place 2 plastic wrapped rice bags on the scanner table (Figure 1A).</li>
	<li>Lay 2 femora (right and left) on top of rice bags such that the proximal end (including the femoral head) is centered on bags and the posterior side is down (Figure 1B). This mimics a patient lying on their back.</li>
	<li>Cover anterior/exposed proximal femur end with additional two 5 lb rice bags (Figure 1C).</li>
	<li>Position machine head over proximal femur and scan the femur according to the standard institutional procedure for patient BMD measurement (Figure 1C). Follow the specific DXA manufacturer instructions.
	<ol>
		<li>From the DXA machine software interface perform a normal femur scan. Select the femur exam, position the DXA arm on top of the cadaveric femur by pressing on the left or right arrow accordingly on the DXA arm, and start the exam by clicking on the &#34;start&#34; button. Perform the BMD analysis by clicking on &#34;Analyze&#34;. 
		NOTE: The resulting automatic T-score from the scan classifies the bone as normal, osteopenic or osteoporotic (Figure 1D). Follow the specific DXA manufacturer instructions.</li>
	</ol>
	</li>
</ol>
2. Cleaning, Cutting and Drilling the Distal End of the Bone
<ol>
	<li>Clean the most proximal 300 mm of the femur by carefully removing any remaining soft tissue from the bone. This step is needed to allow PMMA to contact the bone during the potting process in preparation for mechanical testing. The bones need not be thawed to room temperature for this process.</li>
	<li>Sanitize the workspace with 70% isopropyl alcohol and cover the table with absorbent paper pads with plastic film on one side (Figure 2A). Set the whole femur on the table (Figure 2B) for the entire process starting with cleaning to cutting (Figure 2A to 2H). Wear personal protective equipment (PPE) including gloves and eye protection.</li>
	<li>Scrape off excess periosteum and cut away excess tissue using a scraper and scalpel (Figure 2C, 2H).</li>
	<li>Place the bone in the custom-made cutting fixture as shown in Figure 2D with the head of the femur against the acrylic plate of the fixture.</li>
	<li>Align and hold the diaphysis against the two pins on the cutting fixture (Figure 2D).</li>
	<li>Secure the bone to the fixture by tightening down the slotted plate on the diaphysis; if bone does not lie flat on fixture, hang distal end off table top and also optionally zip tie the femoral neck as needed to hold the specimen in place (Figure 2D).</li>
	<li>Cut the distal shaft of the femur using the cast cutter (Figure 2E) through slotted plate as a guide; hold bone with a dry rag/towel for better grip.</li>
	<li>Remove the bone from the fixture; normal bone length after cut is 255 mm (Figure 2F).</li>
	<li>Clean the medullary cavity of marrow using a curette approximately 25 mm deep. Then, insert a gauze sponge to help dry the interior surface. Remove the gauze just prior to placing the distal end of the femur in the mold. Grip the bone with a dry towel/rag and drill a 10 mm hole through the distal end at approximately 25 mm from the proximal cut end of the specimen. Note: This is to allow the PMMA to penetrate the canal and secure the bone firmly.</li>
</ol>
3. Potting the Bone
<ol>
	<li>Design and fabricate potting container. The potting containers are made of 5 mm thick acrylic sheets and have the following outer dimensions: 50 mm by 50 mm square cross section and 100 mm tall (Figure 3A).</li>
	<li>Label potting container with appropriate bone identification (Figure 3A and 3B &#8211; see label on acrylic box).</li>
	<li>Adjust the embedding fixture to the proper orientation (left leg or right leg; e.g. 15&#176; or 30&#176; internal rotation).</li>
	<li>Place potting container in base of embedding fixture, place bone in the potting container (Figure 3B) and align neck with pointer in fixture (Figure 3C) to adjust the internal rotation angle of the bone to the desired value.</li>
	<li>Measure 60 g of PMMA powder and mix with 30 g of liquid resin under fume hood until the powder has dissolved. The mixture should be pourable. Use a disposable paper cup for this process.</li>
	<li>Pour mixture into potting container with bone under a fume hood (Figure 3D), allow to cure for about 10 - 15 min until PMMA is clear and hard. This should only fill ~ 1/2 of the potting container with PMMA. Carefully wrap the bone in saline saturated towels to prevent tissue dryness due to heat generation during PMMA polymerization (Figure 3E).</li>
	<li>Periodically check the femur to ensure it remains aligned in the container during curing.</li>
	<li>Remove femur from potting fixture and wrap with a saline soaked paper towel (Figure 3E).</li>
	<li>Prepare 90 g of PMMA under a fume hood as explained in step 3.5 and fill the potting container completely. Cure the PMMA for about 10 - 15 min until it becomes hard.</li>
	<li>After resin has cured, tightly wrap/rewrap bone in saline soaked paper towels, cover with plastic bag and store specimens at -20 &#176;C in freezer.</li>
</ol>
4. Imaging the Bone with X-ray
(CAUTION! Operate with proper care for X-ray radiation when using the machine)
<ol>
	<li>If using films for X-rays, turn X-ray developer on at least 20 min (per manufacturer instructions) prior to scanning by turning the knob clockwise (in the developer room).</li>
	<li>Ensure there is unexposed film in the cassette before X-raying; cassette should only be opened in a dark room.</li>
	<li>Turn machine on to unlock and extend the machine head.</li>
	<li>Place a cart under the path of the beam and place a cassette on the cart under the beam (Figure 4A).</li>
	<li>Place and position the femur on the cassette (Figure 4B); two orientations will be captured: medial-lateral view and anterior-posterior view. Label the specimen images accordingly.</li>
	<li>After first exposure, swap the lead and bone locations.</li>
	<li>Cover the already exposed half with lead and expose the bone in the second orientation on the unexposed side. This allows the user to use one X-ray film for a single bone in two orientations (Figure 4C-D).</li>
	<li>Change the cassette and rotate each femur into the second orientation.</li>
	<li>In the case of a single femur, cover half of the cassette with a lead cover to avoid initial exposure of the entire film to X-rays.</li>
	<li>Move behind the lead-lined portable wall for personal protection and use the trigger to expose the bones.</li>
	<li>On completion, return X-ray head to lock and store position, turn key to OFF on X-ray machine and remove exposed X-ray film.</li>
	<li>Develop the film to obtain X-ray images (Figure 4E) using a regular film developer. Turn on the white light in the room and locate the film developer. Turn on the red light and turn off the white light before opening the cassette and proceed with the film developing process. Open the cassette and put the film through the developer. Turn on the white light and turn off the red light once the film has been developed.</li>
</ol>
5. CT Scanning of Bones
<ol>
	<li>Remove bones from freezer about 24 h prior to scanning. Bones must be completely thawed prior to scanning.</li>
	<li>Ensure the bones are wrapped in plastic bags for scanning to minimize clean-up at the end.</li>
	<li>Place and secure the femur and the calibration phantom in the CT scanning fixture (Figure 5A-B). The fixture holds the calibration phantom (Figure 5C) and also holds the femur in an orientation (Figure 5D-E) identical with the orientation desired for subsequent mechanical testing. This cross registration is required to use the data from CT scanning (Figure 5F) in the QCT/FEA modeling process (described in a different protocol). 
	NOTE: The fixture is designed such that it exposes the femur for CT scanning without obstructing the proximal femur (femoral head, neck, greater trochanter, and proximal shaft).</li>
	<li>Make sure the fixture along with the femur in the CT scanner is properly aligned using the system lasers as guides (Figure 5D-E). Re-check the alignment of the fixture with the CT long axis laser (by pressing laser on/off button). The phantom does NOT need to be aligned with the laser as it is secured in the fixture. Zero the table position from the control panel of the machine by pressing the zero button (&#8594; 0 &#8592;) on the control panel.</li>
	<li>Following CT standard operating procedure, operate the CT machine at 120 kVp, 216 mAs, 1 s rotation time, and pitch of 1 using ultra high resolution mode (zUHR). This gives a slice thickness of 0.4 mm, and a pixel size of 0.30 - 0.45 mm depending on the size of the field of view (FOV).</li>
	<li>Check CT-scan data prior to mechanical testing to ensure that images of interest are captured and saved. Refreeze the bone at -20 &#176;C until the day of the experiment.</li>
</ol>

<ol>
	<li>Cristofolini, L., Schileo, E., Juszczyk, M., Taddei, F., Martelli, S., Viceconti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+testing+of+bones:+the+positive+synergy+of+finite-element+models+and+in+vitro+experiments.">Mechanical testing of bones: the positive synergy of finite-element models and in vitro experiments.</a> Philos Trans A Math Phys Eng Sci. 368, 2725-2763 (2010).</li><li>Cartner, J. L., Hartsell, Z. M., Ricci, W. M., Tornetta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+trust+ex+vivo+mechanical+testing+of+fresh-frozen+cadaveric+specimens?+The+effect+of+postfreezing+delays.">Can we trust ex vivo mechanical testing of fresh-frozen cadaveric specimens? The effect of postfreezing delays.</a> J Orthop Trauma. 25 (8), 459-461 (2011).</li><li>An, Y. H., Draughn, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mechanical testing of bone and the bone-implant interface. , (1999).</li><li>van Haaren, E. H., van der Zwaard, B. C., van der Veen, A. J., Heyligers, I. C., Wuisman, P. I., Smit, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+long-term+preservation+on+the+mechanical+properties+of+cortical+bone+in+goats.">Effect of long-term preservation on the mechanical properties of cortical bone in goats.</a> Acta Orthop. 79, 708-716 (2008).</li><li>Shaw, J. M., Hunter, S. A., Gayton, J. C., Boivin, G. P., Prayson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+freeze-thaw+cycles+do+not+alter+the+biomechanical+properties+of+fibular+allograft+bone.">Repeated freeze-thaw cycles do not alter the biomechanical properties of fibular allograft bone.</a> Clin Orthop Relat Res. 470 (3), 937-943 (2012).</li><li>Topp, T., 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=Embalmed+and+fresh+frozen+human+bones+in+orthopedic+cadaveric+studies:+which+bone+is+authentic+and+feasible?.">Embalmed and fresh frozen human bones in orthopedic cadaveric studies: which bone is authentic and feasible?.</a> Acta Orthop. 83 (5), 543-547 (2012).</li><li>Manske, S., 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=Cortical+and+trabecular+bone+in+the+femoral+neck+both+contribute+to+proximal+femur+failure+load+prediction.">Cortical and trabecular bone in the femoral neck both contribute to proximal femur failure load prediction.</a> Osteoporos Int. 20 (3), 445-453 (2009).</li><li>Rezaei, A., Dragomir-Daescu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+Strength+Changes+Faster+With+Age+Than+BMD+in+Both+Women+and+Men:+A+Biomechanical+Study.">Femoral Strength Changes Faster With Age Than BMD in Both Women and Men: A Biomechanical Study.</a> J Bone Miner Res. 30, 2200-2206 (2015).</li><li>Cristofolini, L., McNamara, B., Freddi, A., Viceconti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+measured+strains+in+the+loaded+femur:+quantification+of+experimental+error.">In vitro measured strains in the loaded femur: quantification of experimental error.</a> J Strain Anal Eng Des. 32, 193-200 (1997).</li><li>Dragomir-Daescu, D., 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=Robust+QCT/FEA+models+of+proximal+femur+stiffness+and+fracture+load+during+a+sideways+fall+on+the+hip.">Robust QCT/FEA models of proximal femur stiffness and fracture load during a sideways fall on the hip.</a> Ann Biomed Eng. 39, 742-755 (2011).</li></ol>

The authors have no relevant disclosures.

We presented a robust bone preparation protocol for ensuring mechanical testing and QCT/FEA modeling of femoral strength in a sideways fall on the hip configuration. This method became our standard in-house protocol. Over the course of 6 years, with varying personnel, about 200 femora were successfully prepared following this protocol. The outcomes of the protocol includes classifying bone conditions using DXA, ruling out metastatic diseases, previous fractures, or implants using X-ray, and obtaining mineral distribution and 3D geometry using CT for subsequent QCT/FEA modeling. Cutting, potting, and scanning fixtures were designed to accommodate left and right femora as well as for different bone orientations required for future testing, modeling, and analysis. The in-house fixtures assured repeatability and reproducibility of test samples.
Due to the complexity of bone experiments and the need for the combination of BMD, X-ray, and CT scanning before mechanical testing, femora must undergo multiple freeze/thaw cycles. With a proper protocol that minimizes the exposure to room temperature, freezing bone specimens preserves the tissue for mechanical testing, even long term3,4. Previous studies showed that freezing bones at -20 &#176;C does not alter their mechanical properties and that a few freeze/thaw cycles before testing is considered a safe and feasible process5,6. In our study, all femora experienced three freeze/thaw sequences at -20 &#176;C and room temperature, respectively, for DXA scanning, CT scanning, and mechanical testing.
In line with several previous studies, standardized rice bags were used while measuring BMD values of specimens using DXA to mimic in vivo soft tissue around bone7. We compared BMD values of our cadaveric cohort with BMD values of different patient populations and found their distributions to be very similar, suggesting the robustness of our protocol for BMD measurements8.
Femoral samples lack flat surfaces to be easily and properly aligned to a desired orientation for testing. If not done properly, this may impact the repeatability of the procedure and limit the accuracy of the experimental results9. To address this issue, several fixtures were designed and fabricated and standard operating procedures were implemented to make tissue handling independent of the users&#39; skill throughout the sample preparation process. While the femora were acquired and tested over several years, the protocol and the hardware remained the same reducing the potential preparation errors.
One important step of our bone preparation process was to perform CT scanning for 3D modeling of bone fracture using QCT/FEA. Thus, registration between CT scanning and future fracture testing was a necessary step in our femoral sample preparation protocol10.
The current method for bone preparation has some limitations. Although careful planning was implemented during the acquisition of the cadavers, dissection, potting and CT scanning, scheduling the various phases of femur preparation could be challenging due to personnel and equipment availability. Our process requires the specimens to be frozen and thawed at multiple time points. Nevertheless, the freezing time never exceeded more than two weeks, and the bones were subjected to a total of three freeze/thaw cycles. Also the bone preparation process was designed to minimize the operator errors. We observed only one error in potting the distal end of the proximal femur. One right leg femur was potted at the wrong internal rotation angle which, was discovered only after CT imaging. Subsequently, this femur was discarded from further data analysis. Therefore, a second operator might be needed for this step to check on the orientation of the femur before pouring the PMMA for potting. No other errors were observed in any of the other steps. Thus, it is significant to note that our process was very robust allowing for only one error, with multiple operators, during the preparation of about 200 proximal femora in the course of several years.
To provide a good quality control system that will minimize the likelihood of operator errors, certain parts of the protocol need to be repeated or re-checked by a second operator. For example, care should be taken during potting to assure that the femoral shaft is drilled to allow bone cement to enter the femur cavity, guaranteeing that the femur is rigidly fixed and will not loosen during testing. Additionally, potting the femur at the internal rotation angle of interest is usually performed by one operator. Before the PMMA is poured for potting the distal end, a second operator might be required to check that the internal rotation angle was set at the required value. Finally, during CT scanning of the femur, the alignment of the fixture holding the bone on the CT scanner bed is critical. One operator should precisely align the fixture with the CT-laser beams and a second operator should confirm that the fixture is properly aligned.
While the current protocol was designed specifically for fracture testing and modeling of femoral specimens in a sideways fall on the hip configuration, it can be easily extended to other loading scenarios including non-destructive testing, or adopted to test other bone types with appropriate fixture redesign.

Determining the true cadaveric proximal femoral fracture strength with mechanical testing is a destructive method that requires a rigorous testing approach for accurate measurements. In particular, proper bone preparation methods are necessary to maintain near in vivo integrity of the bones prior to mechanical fracture testing1. This is achieved by proper bone storage and minimizing handling at room temperature. This test data is extensively used to validate QCT/FEA models of femoral fracture which have the potential to be used clinically to understand the fracture risk, especially in osteoporotic patients. Unfortunately, there is no current standard procedure to prepare proximal femur samples for mechanical testing. A good testing procedure should ensure repeatability and reproducibility of the preparation process. Therefore, fixtures required for sample preparation need to be carefully designed and fabricated to minimize the likelihood of various testing errors. We also need to minimize the preparation time for which bone tissue is at room temperature and thus in danger of degradation with irreversible changes in mechanical properties. 
To this end, we have developed a procedure that preserves bone tissue across multiple preparation steps. This is important to ensure minimal exposure time at room temperature while also minimizing the number of freeze/thaw cycles which can affect tissue physical properties2. The entire procedure is long and nontrivial as the steps occurred over multiple weeks and required scheduling for scanning procedures and personnel availability. The steps included thawing bone samples, screening the samples using DXA scanning to obtain bone mineral density (BMD) values, X-ray to rule out any diseased specimens, and finally CT scanning to estimate distribution of bone mineral and femoral geometry. All the specimens were prepared for testing by removing extraneous soft tissues from the bone surface, cutting the femur to a length required for testing, and potting the femur in a desired orientation for simulating a sideways fall on the hip during subsequent testing. It is essential to keep the time period for all these operations as short as possible. A robust protocol is thus mandatory for consistent specimen preparation, tissue preservation between steps, and for reducing the overall preparation time. 
The aim of this paper is to present in detail the procedures involved in the preparation of femoral samples for subsequent mechanical testing under various conditions. Preservation of the bone tissue is crucial in this process and we achieved it by keeping specimens frozen between steps and keeping them carefully wrapped in saline saturated towels at all times except when scanning and mechanically testing the bones. Femora were also kept wrapped in saline wet towels during the steps involving PMMA curing to prevent dryness of the bone tissue.

<table><tbody><tr><td>CT potting container and scanning fixture</td><td>Internally manufactured</td><td>N/A</td><td>Custom designed and manufactured</td></tr><tr><td>CT scanner</td><td>Siemens</td><td>Somatom Definition scanner (Siemens, Malvern, PA)</td><td>CT scanning equipment</td></tr><tr><td>Quantitative CT Phantom</td><td>Midways Inc, San Francisco, CA</td><td>Model 3 CT calibration Phantom</td><td>Used for obtaining BMD values from Hounsfield units in the CT image</td></tr><tr><td>Dual Energy X-ray Absorptiometry scanner</td><td>General Electric</td><td>N/A</td><td>GE Lunar iDXA scanner for bone health or any similar BMD scanners</td></tr><tr><td>Hygenic Orhodontic Resin (PMMA)</td><td>Patterson Dental Supply</td><td>H02252</td><td>Controlled substance and can be purchased with proper approval</td></tr><tr><td>Freezer</td><td>Kenmore</td><td>N/A</td><td>This is a -20 &amp;deg;C storage for bones</td></tr><tr><td>X-ray scanner</td><td>General Electric</td><td>&amp;nbsp;46-270615P1</td><td>X-ray imaging equipment.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>X-ray films</td><td>Kodak</td><td>N/A</td><td>Used to display x-ray images</td></tr><tr><td>X-ray developer</td><td>Kodak X-Omatic</td><td>M35A X-OMAT&amp;nbsp;</td><td>Used for developing X-ray images</td></tr><tr><td>X-ray Cassette</td><td>Kodak X-Omatic</td><td>N/A</td><td>Used for holding x-ray films</td></tr><tr><td>5-pound Rice Bags</td><td>Great Value</td><td>N/A&amp;nbsp;</td><td>Used for mimicking soft tissue during the DXA scanning process</td></tr><tr><td>Physiologic Saline (0.9% Sodium Chloride)</td><td>Baxter</td><td>NDC 0338-0048-04</td><td>Used for keeping samples hydrated</td></tr><tr><td>Scalpels and scrapers</td><td>Bard-Parker</td><td>N/A</td><td>Used to clean the bone from soft tissue</td></tr><tr><td>Cast cutter</td><td>Stryker</td><td>810-BD001</td><td>Used to cut femoral shaft</td></tr><tr><td>Drilling machine</td><td>Bosch</td><td>N/A</td><td>Used to drill the femoral shaft</td></tr><tr><td>Fume Hood</td><td>Hamilton</td><td>70532</td><td>Used for ventilation when using making PMMA</td></tr></tbody></table>

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.

The cadaveric femora were shipped frozen and maintained at -20 &#176;C until preparation began. BMD scanning was performed using a DXA scanner to measure total hip and neck BMD as well as T-score for each specimen (Figure 1). A T-score is the number of standard deviations of the measured BMD compared to average values for young healthy subjects. It can range from -2.5 or lower for osteoporotic bones, between -1 and -2.5 for osteopenic bones and higher than -1 for normal bones. Once finished, bones were cleaned of excess tissue, and cut to remove the distal end using an in-house designed and fabricated cutting fixture (Figure 2). The specimens were then potted distally using a fixture designed for holding the bones in the desired internal rotation orientation; after placing the distal end into the potting container, the PMMA in liquid form was poured to fill the container (Figure 3). The X-ray images were obtained for paired bones together and for single bones separately to discern the presence of fracture or diseases, such as cancer, that might affect femoral strength (Figure 4). In the presence of such abnormalities, the condition of the bone should be documented when considered for future analyses. Finally, the femora were CT-scanned, in order to obtain CT images, using an acrylic CT scanning fixture designed to hold the bone in appropriate predetermined orientations (adduction and internal rotation angles) (Figure 5). The CT images are used to obtain 3-dimensional bone geometry and volumetric bone mineral distribution to be used in quantitative CT-based finite element analysis. Prior to subsequent fracture testing, all relevant data characterizing each femur such as the BMD values, X-ray images, and CT images were checked to ensure that data of interest were recorded and saved.
<img alt="Figure 1" src="/files/ftp_upload/54925/54925fig1.jpg" /> 
Figure 1: BMD Measurement Using DXA Scanning. (A) Rice bags and plastic lined papers; (B) Two bone specimens in desired orientations in scanner bed; (C) proximal femur ends covered with 2 rice bags during scanning; (D) Neck and total hip BMD measurements with associated T-scores. DXA scanning is performed using a clinical scanner to measure bone mineral density and estimate T-score. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54925/54925fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54925/54925fig2.jpg" /> 
Figure 2: Cleaning and Cutting Bones. (A) Cleaning and cutting desk; (B) bone sample tools for cleaning; (C) cleaning the shaft of a femur; (D) securing a sample in the cutting fixture; (E) cast cutter; (F) completed sample after cutting. A special fixture and bone cleaning and cutting tools are used to prepare the most proximal 255 mm length for testing; (G) curette used for cleaning intramedullary canal of the femur; (H) tool for cleaning superficial tissue in samples. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54925/54925fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54925/54925fig3.jpg" /> 
Figure 3: Femur Cleaning and Potting Process. (A) Potting fixture; (B) potting a femur in the fixture; (C) adjusting internal rotation angle to desired value; (D) pouring PMMA in the container; (E) potted bone wrapped in a saline saturated towels. A special fixture is used to set the internal rotation angle to a specified value. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54925/54925fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54925/54925fig4.jpg" /> 
Figure 4: Bone X-ray Process. (A) X-ray machine; (B) bone sample on a cassette with unexposed film, a second half of the cassette is covered by lead to avoid exposure of the entire film; (C) placing unexposed film in loading tray of developer in dark room; (D) developed film; (E) resulting X-ray image of a healthy femur. X-ray equipment is used to scan the bones in two positions to rule out prior fractures, implants, bone metastasis, or any structural abnormalities. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54925/54925fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54925/54925fig5.jpg" /> 
Figure 5: CT Scanning Using an Acrylic Fixture to Hold Bones in a Desired Orientation. (A) CT scanner; (B) fiberglass femur mounted in an acrylic fixture designed to hold bones in a desired orientation; (C) mounting a cadaveric femur in the fixture; (D) vertical alignment of the fixture using the CT long axis laser; (E) Alignment of the femur. An in-house designed fixture is used to hold the bone in a position identical to the subsequent testing position; bone alignment is obtained with the aid of the CT built-in lasers; (F) CT imaging of the femur. Trabecular and cortical bone can be visualized in the CT image. Cortical bone is represented by bright voxels (large Hounsfield Unit (HU) values) surrounding the trabecular bone which is represented by smaller HU values. Note: Care has to be taken to include all five rods and the entire proximal femur in the CT images. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54925/54925fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis at JoVE.com

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

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 ...

proximal-cadaveric-femur-preparation-for-fracture-strength-testing

Nanyang Technological University

Research

JoVE Journal

Medicine

9.3K Views.  Mayo Clinic.  We presenteren een robuust protocol over hoe om zorgvuldig te bewaren en voor te bereiden cadaveric dijbenen voor fractuur testen en kwantitatieve computertomografie beeldvorming. De werkwijze biedt precieze controle over ingangscondities voor het bepalen van verhoudingen tussen botdichtheid, breuksterkte en definiëren eindige elementenmodel geometrie en eigenschappen. 

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

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

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 ...

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 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 ...

Engineering

Subject-specific Musculoskeletal Model for Studying Bone Strain During Dynamic Motion

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is essential to determine the effect of high ground impact forces on lower-body bone deformation to better understand the mechanisms of bone stress injuries. Conventional strain gauge measurement has been used to study in vivo tibia deformation. This method is associated with limitations including invasiveness of the procedure, involvement of few human subjects, and limited strain data from small bone surface areas. The current study intends to introduce a novel approach to study tibia bone strain under high impact loading conditions. A subject-specific musculoskeletal model was created to represent a healthy male (19 years, 80 kg, 1,800 mm). A flexible finite element tibia model was created based on a computed tomography (CT) scan of the subject&#39;s right tibia. Laboratory motion capture was performed to obtain kinematics and ground reaction forces of drop-landings from different heights (26, 39, 52 cm). Multibody dynamic computer simulations combined with a modal analysis of the flexible tibia were performed to quantify tibia strain during drop-landings. Calculated tibia strain data were in good agreement with previous in vivo studies. It is evident that this non-invasive approach can be applied to study tibia bone strain during high impact activities for a large cohort, which will lead to a better understanding of injury mechanism of tibia stress fractures.

During landing, lower-body bones experience large mechanical loads and are deformed. It is essential to measure bone deformation to better understand the mechanisms of bone stress injuries associated with impacts. A novel approach integrating subject-specific musculoskeletal modeling and finite element analysis is used to measure tibial strain during dynamic movements.

Bone stress injuries are common in sports and military trainings. Repetitive large ground impact forces during training could be the cause. It is ...

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

Principles and Protocols of Mechanical Testing for Bone Fracture Susceptibility

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 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&#39;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&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

Author Spotlight: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests 

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.

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 ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

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

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