Name: cantilever bending of murine femoral necks
Uploaded: 2022-01-05T14:00:00
Description: Watch this Scientific Journal Video about Cantilever Bending of Murine Femoral Necks at JoVE.com

The study was supported by the NIH P30AR069655 and R01AR070613 (H. A. A.).

Animal studies were approved by The University of Rochester Committee of Animal Resources. The mice used in this study were C57BL/6 males and females ranging from age 24-29 weeks of age. Mice were housed in standard conditions with food and water ad libitum. Upon euthanasia via carbon dioxide inhalation, followed by cervical dislocation, 20 right femurs (10 male and 10 female) were harvested and frozen at -20 &#176;C until tested.
1. Creation of custom 3D printed mounting guides...

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This protocol outlines a reliable cantilever bending test for murine femoral necks. The natural cantilever flexure scenario that occurs at the femoral neck is typically not represented in standard 3- and 4-point bending tests5. This testing method is better and more reliably replicates the type of femoral neck fractures experienced by bone fragility patients. The main focus when performing this protocol is eliminating the variability due to inconsistent potting of the femoral shaft. Critically, cl...

Fracture risk is a serious medical concern associated with osteoporosis. Over 1.5 million fragility fractures are reported each year in the United States alone, with fractures occurring in the hip, specifically the femoral neck, as the leading fracture type1. It is estimated that 18% of women and 6% of men will experience a femoral neck fracture in their lifetime2, and the mortality rate at 1 year following the fracture is greater than 20%1. Therefore, mouse models that allow biomechanical testing of the femoral neck can be suitable for studying fragility fractures. Mouse models also offer powerful tools to elucidate translatable cellular and molecular events involved in osteoporosis potentially. This is due to the availability of genetic reporters, gain and loss of function models, and the expansive library of molecular techniques and reagents. Mechanical testing of mouse bones can provide necessary outcome measures to determine bone health, genotypic and phenotypic variations that could explain the etiology of the disease, and assess therapies based on outcome measures of the quality of the bone and the risk of fracture3.
The anatomy of the femoral neck creates unique mechanical loading scenarios, which typically lead to flexural (bending) fractures. The femoral head is loaded in the acetabular socket at the proximal end of the femur. This creates a cantilever bending scenario on the femoral neck, which is rigidly attached to the femoral shaft distally4. This differs from traditional 3- or 4-point bending tests on the femoral mid-diaphysis. While these tests are helpful, they do not replicate the loading that typically leads to fragility fractures in osteopenic and osteoporotic individuals in terms of fracture location or the loading scenario.
To better assess fragility fracture risk in mice, it was sought to improve the reproducibility of cantilever bending tests of murine femoral necks. As theoretically predicted, the loading angle on the femoral head relative to the femoral shaft has been shown to significantly affect the outcome measures5, thereby creating a challenge for reliability and reproducibility of reported outcomes. To ensure proper and consistent alignment of the femurs during sample preparation, guides were designed, and 3D printed based on anatomic measurements made on &#181;CT scans of C57BL/6 mouse femurs. The guides were designed to aid in consistently potting the samples such that the femoral shaft is maintained at ~20&#176; from the vertical loading direction. This angle was chosen because it maximizes the stiffness while minimizing the maximal bending moment along the femoral shaft, which increases the likelihood of femoral neck fractures and leads to more consistent and reproducible testing5. Guides were 3D printed in various sizes to accommodate anatomical differences between samples and used to hold samples in a stable position while potting in acrylic bone cement. The stiffness, maximum force, yield force, and maximum energy were calculated from the force-displacement graphs. This testing method showed consistent results for the aforementioned biomechanical outcome. With practice and the aid of the 3D printed guide, measurement errors due to misalignment can be minimized, resulting in reliable outcome measures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#188;&#8221; x &#188;&#8221; square aluminum tubing</td>
			<td>Grainger</td>
			<td>48KU67</td>
			<td>Cut to lengths of 1/2&#34; to 1&#34; lengths</td>
		</tr>
		<tr>
			<td>1 kN load cell</td>
			<td>Instron</td>
			<td>2527-130</td>
			<td>Any load cell with sub 1 N resolution can be used.</td>
		</tr>
		<tr>
			<td>3.5x-45x Zoom Stereo Boom Microscope</td>
			<td>Omano</td>
			<td>OM2300S-GX4</td>
			<td>Microscope used to precisely line up samples with loading platen.</td>
		</tr>
		<tr>
			<td>3D printed guides</td>
			<td>Custom made</td>
			<td></td>
			<td>Angled slots at 73.13&#176;, with diameters between 1.9 mm and 2.2 mm</td>
		</tr>
		<tr>
			<td>3D printed mount</td>
			<td>Custom made</td>
			<td></td>
			<td>Tapped with M10 threads to fit the mount attachment and with 2 M4 threaded holes adjacent sides to hold the aluminum tubing with sample in place.</td>
		</tr>
		<tr>
			<td>Acrylic Base Plate Material Kit</td>
			<td>Keystone Industries</td>
			<td>921392</td>
			<td>Mix 3.5 g of powder with 2 mL of liquid. This will be enough for approximately 8 samples, and will begin to harden quickly.</td>
		</tr>
		<tr>
			<td>Amira</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>Used to compile &#181;CT scans</td>
		</tr>
		<tr>
			<td>Biaxial stage</td>
			<td>Custom made</td>
			<td></td>
			<td>Used to center femoral head of sample under the loading platen.</td>
		</tr>
		<tr>
			<td>BioMed Amber Resin</td>
			<td>formlabs</td>
			<td>RS-F2-BMAM-01</td>
			<td>Any resin from formlabs could be used for this project.</td>
		</tr>
		<tr>
			<td>Bluehill 3</td>
			<td>Instron</td>
			<td>V3.66</td>
			<td>Software used to set up loading protocol and collect load, displacement and time data.</td>
		</tr>
		<tr>
			<td>ElectroPuls 10000</td>
			<td>Instron</td>
			<td>E10000</td>
			<td>Mechanical testing system</td>
		</tr>
		<tr>
			<td>Faxitron UltraFocus</td>
			<td>Faxitron BioOptics</td>
			<td>2327A40311</td>
			<td>X-ray imaging system</td>
		</tr>
		<tr>
			<td>Form 2</td>
			<td>formlabs</td>
			<td>F2</td>
			<td>Used to print the mount and guides</td>
		</tr>
		<tr>
			<td>Form 2 Resin Tank LT</td>
			<td>formlabs</td>
			<td>RT-F2-02</td>
			<td>LT Tank was used to be compatible with the BioMed Resin</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>ImageJ</td>
			<td>Used to assess &#181;CT and X-ray images</td>
		</tr>
		<tr>
			<td>Laxco iLED Series LED Light Source</td>
			<td>ThermoFisher Scientific</td>
			<td>AMPSILED30W</td>
			<td>Light source used in conjugtion with microscope.</td>
		</tr>
		<tr>
			<td>Loading platen</td>
			<td>Custom made</td>
			<td></td>
			<td>This can be any metal rod that is tapered to a diameter of approximately 2.5 mm. We used an M6 screw that was tapered on a lathe.</td>
		</tr>
		<tr>
			<td>Mount attachment</td>
			<td>Custom made</td>
			<td></td>
			<td>To secure the 3D printed mount to the load cell. We used a M10/M6 threaded rod</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010031</td>
			<td>Need to rehydrate the samples once acrylic base plate material has set.</td>
		</tr>
		<tr>
			<td>Plumber&#39;s putty</td>
			<td>Oatey</td>
			<td>31174</td>
			<td>Used to seal the end of the aluminum tubing when pouring acrylic base plate material in. Any clay or putty could be used.</td>
		</tr>
		<tr>
			<td>PreForm</td>
			<td>formlabs</td>
			<td>Preform 3.15.2</td>
			<td>Formlabs software</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td>Samples can be laid flat in culture dish and covered in PBS to rehydrate.</td>
		</tr>
		<tr>
			<td>vivaCT 40</td>
			<td>Scanco</td>
			<td>&#181;CT 40</td>
			<td>Representative set or actual samples can be scanned prior to printing of guides to calculate femoral shaft angle and diameter.</td>
		</tr>
	</tbody>
</table>

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.

When potted with the aid of the guide, the femoral shafts were aligned at 21.6&#176; &#177; 1.5&#176;. While this represents &#60;10% deviation from the intended angle of 20&#176;, the coefficients of variation (COV) of the potting angle across all samples tested were 7.6% and 6.5% for male and female mice, respectively (n = 10 per group) as verified by pre-test planar x-rays (Figure 5). Additionally, the post-testing X-rays should be used to assess the mode in which the samples failed. Fail...

Watch this Scientific Journal Video about Cantilever Bending of Murine Femoral Necks at JoVE.com

Cantilever Bending of Murine Femoral Necks

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 ...

The cantilever bending setup outlined in this protocol creates a reproducible approach for generating femoral neck fractures, which are more clinically relevant and can be utilized in murine studies to look at osteoporosis. This protocol creates a testing platform that improves reproducibility, leading to decreased coefficient of variation in outcome measures. Therefore, it minimizes the sample size needed for robust studies.Begin by cutting tubing sections of length 1/2-inch to 1-inch using 1/4-inch by 1/4-inch square aluminum tubing. Label each aluminum segment with the sample IDs with an etching tool. Fill half of the tubing segments with putty and place them into a fixture to hold them upright.Place the clean femurs flat on the benchtop so the anterior surface is facing up. Then, place the 3D-printed guide directly below the third trochanter, where the shaft diameter becomes consistent. Hold the proximal and distal ends with one hand.Firmly press the femur onto the workbench, and using the other hand, place the 3D-printed guide on the midshaft of the femur to prevent the femur from rotating to the lateral or medial side. Then, place them in front of the corresponding aluminum segments. Fill the aluminum segments with bone cement until almost full, leaving a little room for displacement.Place the femurs with guides in the correct aluminum segment and allow the bone cement to set. After the bone cement has hardened, place the samples in a Petri dish with room temperature PBS and allow them to rehydrate for two hours. Use a mechanical testing system to attach and calibrate a load cell with the resolution less than 1 Newton.Attach a fixture with a square slot to firmly hold the aluminum segments with the samples. Attach set screws to the two sides of the holding fixture to firmly hold samples in place. Then, attach a loading platen into the actuator.Place a stereo microscope on a surface directly in front of the MTS. Place light sources around the system if additional lighting is needed to see the setup through the microscope. In the MTS software, begin the creation of a new flexural protocol.Ensure that the protocol will operate in displacement control. Set the loading rate of the protocol to 0.5 millimeters per second. If the software has a setting for soft keys, add the soft keys Balance and Zero Extension to the protocol.Ensure that the software program will record the time in seconds, load in Newtons, and extension or displacement in millimeters at a minimum sampling rate of 100 hertz. Save the new protocol and return to the main screen of the software program to begin the testing of a new sample set. Obtain an X-ray image of the samples in the aluminum pots.Multiple samples can be imaged at once. Ensure that the anterior view of samples is captured to allow for verification measurements of potting angle. Place an aluminum segment with sample into the holding fixture and tighten the set screws.Lower the actuator until it is within a few millimeters of the femoral head. Use the stereo microscope to adjust the biaxial stage to align the position of the femoral head directly underneath the loading platen. Lock the biaxial stage in place.In the MTS software, zero the position of the actuator and balance the load cell using the added soft keys Balance and Zero Extension. Then, begin the loading protocol. Depending on how much space was left between the loading platen and the sample, testing will only take 10 to 30 seconds.After testing, capture another anterior X-ray of the sample. This will be used to discern and document the mode of fracture. The coefficients of variation, or COV, from measured flexural properties of mouse femoral necks are presented here.COV represents the ratio of the standard deviation and mean of a data set, and its decrease indicates a tighter grouping of the individual data points around the mean. This protocol decreased the COV for maximum load compared to other publications performing similar testing. A representative force displacement curve displaying a 0.2%offset linear fit is used to derive the stiffness and yield point.Selected outcome measures are plotted, displaying mean and standard deviation, including maximum load at failure, stiffness, maximum displacement at failure, and and work to failure. Asterisks indicate significant differences determined using a one-tailed unpaired t-test. Femoral necks for male mice were significantly stronger and stiffer than specimens from female mice.In addition, the female femoral necks experienced more significant deformations and work to failure compared to specimens from male mice. This is consistent with the lower bone mineral density in females and underscores the sensitivity of the test to detect physiologically-relevant differences. Biomechanical outcome measures, including maximum load, stiffness, maximum displacement, and work to failure, were plotted against the potting angle and correlated using a simple linear regression for the male cohort, female cohort, and all samples grouped together.Solid black lines show linear regression of group samples, and dotted lines indicate confidence intervals. Variability in the potting angle did not significantly affect the maximum load, maximum displacement, or failure work. However, as the potting angle increased, the stiffness increased.The most important thing to remember while attempting this protocol is to keep the potting procedure and loading rates the same to better compare your results to previously published data. If using an alternative to bone cement, such as a bismuth alloy that can be softened to remove the sample after testing, samples can undergo other procedures, such as Raman spectroscopy, DEXA, or three-point bending, to elucidate additional mechanical, chemical, and structural properties.

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