Name: method and instrumented fixture for femoral fracture testing in a sideways fall-on-the-hip position
Uploaded: 2017-08-17T12:30:00
Description: Watch this Scientific Journal Video about Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position at JoVE.com

We would like to thank the Materials and Structural Testing Core Facility and Division of Engineering at Mayo Clinic for technical support. In addition we would like to thank Lawrence J. Berglund, James Bronk, 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.

1. Large Fixture Attachment
<ol>
	<li>Remove standard fixtures from the machine.</li>
	<li>Move crosshead apart to accommodate the in-house fixture.</li>
	<li>Place the aluminum block (part No. 1 in Figure 1A) on the machine and securely fasten on the machine using two bolts; the hole in the center accommodates the machine load cell.</li>
	<li>Place the main fixture structure (part No. 2 in Figure 1B) on the aluminum block and securely attach it to the block u...

<ol>
	<li>Bouxsein, M. L., Szulc, P., Munoz, F., Thrall, E., Sornay-Rendu, E., Delmas, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+trochanteric+soft+tissues+to+fall+force+estimates,+the+factor+of+risk,+and+prediction+of+hip+fracture+risk.">Contribution of trochanteric soft tissues to fall force estimates, the factor of risk, and prediction of hip fracture risk.</a> J Bone Miner Res. 22, 825-831 (2007).</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>Zani, L., Erani, P., Grassi, L., Taddei, F., Cristofolini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+distribution+in+the+proximal+Human+femur+during+in+vitro+simulated+sideways+fall.">Strain distribution in the proximal Human femur during in vitro simulated sideways fall.</a> J Biomech. 48, 2130-2143 (2015).</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><li>Schileo, E., Balistreri, L., Grassi, L., Cristofolini, L., Taddei, F. <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> J Biomech. 47, 3531-3538 (2014).</li><li>Koivumaki, J. 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=Ct-based+finite+element+models+can+be+used+to+estimate+experimentally+measured+failure+loads+in+the+proximal+femur.">Ct-based finite element models can be used to estimate experimentally measured failure loads in the proximal femur.</a> Bone. 50, 824-829 (2012).</li><li>Pottecher, P., 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=Prediction+of+Hip+Failure+Load:+In+Vitro+Study+of+80+Femurs+Using+Three+Imaging+Methods+and+Finite+Element+Models—The+European+Fracture+Study+(EFFECT).">Prediction of Hip Failure Load: In Vitro Study of 80 Femurs Using Three Imaging Methods and Finite Element Models—The European Fracture Study (EFFECT).</a> Radiology. , 142796 (2016).</li><li>Rivadeneira, F., 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=Femoral+neck+BMD+is+a+strong+predictor+of+hip+fracture+susceptibility+in+elderly+men+and+women+because+it+detects+cortical+bone+instability:+the+Rotterdam+Study.">Femoral neck BMD is a strong predictor of hip fracture susceptibility in elderly men and women because it detects cortical bone instability: the Rotterdam Study.</a> J Bone Miner Res. 22, 1781-1790 (2007).</li><li>de Bakker, P. M., Manske, S. L., Ebacher, V., Oxland, T. R., Cripton, P. A., Guy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=During+sideways+falls+proximal+femur+fractures+initiate+in+the+superolateral+cortex:+evidence+from+high-speed+video+of+simulated+fractures.">During sideways falls proximal femur fractures initiate in the superolateral cortex: evidence from high-speed video of simulated fractures.</a> J Biomech. 42, 1917-1925 (2009).</li><li>Courtney, A. C., Wachtel, E. F., Myers, E. R., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+reductions+in+the+strength+of+the+femur+tested+in+a+fall-loading+configuration.">Age-related reductions in the strength of the femur tested in a fall-loading configuration.</a> J Bone Joint Surg Am. 77, 387-395 (1995).</li><li>Cheng, X. G., 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=Assessment+of+the+strength+of+proximal+femur+in+vitro:+relationship+to+femoral+bone+mineral+density+and+femoral.">Assessment of the strength of proximal femur in vitro: relationship to femoral bone mineral density and femoral.</a> Bone. 20, 213-218 (1997).</li><li>Courtney, A. C., Wachtel, E. F., Myers, E. R., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+loading+rate+on+strength+of+the+proximal+femur.">Effects of loading rate on strength of the proximal femur.</a> Calcif Tissue Int. 55, 53-58 (1994).</li><li>Keyak, J., Rossi, S., Jones, K., Les, C., Skinner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+fracture+location+in+the+proximal+femur+using+finite+element+models.">Prediction of fracture location in the proximal femur using finite element models.</a> Medical engineering &amp; physics. 23, 657-664 (2001).</li><li>Nishiyama, K. K., Gilchrist, S., Guy, P., Cripton, P., Boyd, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximal+femur+bone+strength+estimated+by+a+computationally+fast+finite+element+analysis+in+a+sideways+fall+configuration.">Proximal femur bone strength estimated by a computationally fast finite element analysis in a sideways fall configuration.</a> J Biomech. 46, 1231-1236 (2013).</li><li>Langton, C. M., Njeh, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physical measurement of bone. , (2016).</li><li>Ariza, O., 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=Comparison+of+explicit+finite+element+and+mechanical+simulation+of+the+proximal+femur+during+dynamic+drop-tower+testing.">Comparison of explicit finite element and mechanical simulation of the proximal femur during dynamic drop-tower testing.</a> J Biomech. 48, 224-232 (2015).</li></ol>

We proposed a protocol to fracture test proximal cadaveric femora in a fall on the hip configuration with which we have successfully tested about 200 samples. The protocol includes several in-house designed fixtures for femoral strength testing under different loading conditions. The fixture allows for testing of both right and left femora at different testing speeds and bone orientations. After mounting the fixture and the measuring instruments, a fiberglass femur is tested to fracture to assure that all the hardware an...

Development of novel methods for femoral fracture risk assessment and fracture prevention for a fall on the hip require a comprehensive understanding of the biomechanical processes involved during fracture. Cadaveric proximal femur strength testing has proven to be effective in determining the relation between femoral strength and factors affecting the structural capacity of the femur providing important insights in this process1,2,3. Experimentally measured femoral strength is also used for validation of Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA) which enables a non-invasive estimate of fracture strength4,5,6,7.
To date, there is no accepted standard procedure to test whole femoral specimens to fracture. To isolate clinically-measureable variables (such as bone mineral density and geometry) and their influence on femoral strength, it is imperative for experimental testing to be carried out in a controlled and repeatable manner. Cadaveric femora have irregular shapes and range in sizes8 and can be obtained from either male or female cadavers of different ages, making it impossible to test using built-in fixtures of standard testing machines. In a sideways fall on the hip event, the greater trochanter undergoes compressive loading, while the proximal femur may experience complex loading including compression, tension, bending moment, and torsion. Testing such loading scenarios adds complexity to the experimental design. Therefore, a fixture, as one important component of the testing protocol, must be specifically designed, fabricated, and installed to accommodate femoral samples of different shapes and sizes, and different testing speeds. This fixture must also hold the specimens for testing in a range of desired orientations to simulate possible impact loads from a fall on the hip. To meet such a variety of conditions, the fixture needs to have multiple stationary and moving components connected in a manner to minimize play in the system and to obtain a smooth load-displacement response.
Reliable data acquisition is also critical during testing. The experimental design must incorporate the necessary load cells, displacement transducers, signal amplifiers and conditioners to accurately measure forces and moments at all supports. Additionally, high speed videos of both the anterior and posterior views of the femur obtained synchronously with the acquisition of forces are necessary to help understand the sequence of events leading to fracture, characterize fracture types, and precisely define femoral strength4,9.
While there are valuable experimental studies in the literature on whole femur testing, published protocols either lack details on how the testing was performed or are very different from one study to another to truly make them reproducible10,11. The goal of the current work was to introduce a protocol for mechanical testing of femoral samples that can be used as the starting point for an effort to standardize bone tissue testing which can be repeatable and reproducible. To this end, we designed and fabricated a testing fixture which was used to test about 200 cadaveric femora. The testing fixture included a bottom fixture and a crosshead fixture. The bottom fixture (Figure 1A-E) holds the femur at a desired orientation during testing and includes a trochanter load cell and a 6-channel load cell connected to the femoral shaft. It also accommodates three independent translations to allow for positioning of the bone for fracture testing. A rotation point is added to mimic the knee joint. The major parts of the bottom fixture were made up of thick pieces of stainless steel and aluminum to make a very stiff fixture. A load cell is attached to the bottom fixture to measure compressive forces on the greater trochanter during testing. The crosshead fixture (Figure 2A-2E) includes two aluminum base plates and two very stiff slide ball bearings (attached together by an aluminum plate), to account for the movement of the femoral head during testing and also to accommodate for right and left femora. A load cell included in the crosshead fixture measures compressive forces. An aluminum cup attached to the load cell is used to apply the compressive loads to the femoral head. Our method was used for left and right femora of both sexes, with various sizes, neck-shaft angles, bone mineral density, and loading conditions mimicking a sideways fall on the hip. The testing speeds in our experiments were set at 5, 100, and 700 mm/s, but they can be set to any value available on the testing machine. The designed fixture had two main components, one connected to the crosshead of the testing machine and the other connected to the testing frame. Both parts were instrumented with load cells sufficient to measure force and moment boundary conditions at all supports. Additionally, two high-speed video cameras were used to record the fracture events during testing. After fracture, a set of X-rays and Computed Tomography (CT) scans were obtained for post experimental fracture analyses. Results obtained from these experiments including fracture strength and energy are currently used for additional research in diagnostic tools to eventually improve the assessment of proximal fracture strength in osteoporotic patients.

<table>
	<tbody>
		<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>Hygenic Orthodontic 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 -20oC storage for bones</td>
		</tr>
		<tr>
			<td>X-ray scanner</td>
			<td>General Electric</td>
			<td>46-270615P1</td>
			<td>X-ray imaging equipment.</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</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>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>Fume Hood</td>
			<td>Hamilton</td>
			<td>70532</td>
			<td>Used for ventilation when preparing PMMA for potting of specimens</td>
		</tr>
		<tr>
			<td>Single axis load cell</td>
			<td>Transducer Techniques, Temecula, CA, USA</td>
			<td>LPU-3K; S/N 219627</td>
			<td>Capacity 3000 LBS</td>
		</tr>
		<tr>
			<td>Six channel load cell</td>
			<td>JR3,Woodland, CA</td>
			<td>45E15A4</td>
			<td>Mechanical load rating 1000N</td>
		</tr>
		<tr>
			<td>Linear potentiometer</td>
			<td>Novotechnik, Southborough, MA, USA</td>
			<td></td>
			<td>Used to acquire linear displacements during testing</td>
		</tr>
		<tr>
			<td>Slide ball bearing</td>
			<td>Schneeberger</td>
			<td>Type NK</td>
			<td>Part of the testing fixture</td>
		</tr>
		<tr>
			<td>Mechanical testing machine</td>
			<td>MTS, Minneapolis, MN</td>
			<td>858 Mini Bionix II</td>
			<td>Used for compression of femur</td>
		</tr>
		<tr>
			<td>Lighting unit</td>
			<td>ARRI</td>
			<td></td>
			<td>Needed for high speed video recordings</td>
		</tr>
		<tr>
			<td>high-speed video camera</td>
			<td>Photron Inc., San Diego, CA, USA</td>
			<td>Photron Fastcam APX-RS</td>
			<td>Used to capture the high speed video recordings of the fracture events</td>
		</tr>
		<tr>
			<td>Photron FASTCAM Viewer</td>
			<td>Photron Inc., San Diego, CA, USA</td>
			<td>Ver.3392(x64)</td>
			<td>Used to view the high speed video recordings</td>
		</tr>
		<tr>
			<td>Camera lens</td>
			<td>Zeiss</td>
			<td>Zeiss Planar L4/50 ZF Lens</td>
			<td>Needed to high image resolution</td>
		</tr>
		<tr>
			<td>Signal conditioner board (DAQ)</td>
			<td>National Instruments</td>
			<td></td>
			<td>Input/output signal connector</td>
		</tr>
		<tr>
			<td>Signal Express</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td>Data acquisition software</td>
		</tr>
		<tr>
			<td>Laptop Computer</td>
			<td>Dell</td>
			<td>N/A</td>
			<td>Used to monitor and acquire all signals from the testing procedure</td>
		</tr>
	</tbody>
</table>

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-house fixtures are mounted after the standard fixtures are removed from the testing machine. First, the bottom heavy fixture is mounted and secured (Figure 1). This includes an extended arm to hold the 6-channel load cell which also enables the femoral shaft to be aligned at a desired adduction angle. Next, the crosshead fixture including two frictionless slide bearings is mounted to accommodate the application of load and femoral head movement during frac...

Watch this Scientific Journal Video about Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position at JoVE.com

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

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 ...

The overall goal of this experimental cadaveric femoral fracture testing protocol is to obtain consistent reproducible fracture strength measurements. This method can help answer key questions in osteoporosis research, by measuring the differences between osteoporotic and normal femoral strength. The main advantage of this technique is that, the testing apparatus mimics a physiological site based fall on the hip.For attachment of the large fixture, first, securely fasten the aluminum block onto the machine. Next, use bolts to secure the main fixture structure onto the aluminum block and support the fixture with the placement of a four ton jack, under the part of fixture that does not rest on the aluminum block. Then use more bolts to secure a six channel load cell fixture onto the main fixture.For attachment of the crosshead fixture, attach the first base plate with the machine crosshead set to absolute zero and the curved edges of the base plate, facing the front of the testing machine. Use one pivoting screw to attach the second base plate. Then, attach the assembly of the two slide bearings, to the second base plate.Rotate the second base plate in a such a way, that the second set of screws can be accessed from the top of the first base plate. Then set the machine crosshead to a relative position of 65 degrees, to allow manual rotation of the slides, orthogonal to the six channel load cell. Once the instrumented fixture has been assembled on the standard server hydraulic machine, attach a high speed camera and lighting equipment to tripods.Position the camera facing the testing machine and connect camera to data acquisition unit. After connecting the data acquisition system, manually push the load cell and observe the data signal traces in the view panel of the DAQ software. To confirm the proper connection and synchronization of the trigger signal, tricanteric load cell, head load cell, linear potentiometer and six channel load cell.Calibrate the linear potentiometer, secure the linear potentiometer fixture to the crosshead and attach the potentiometer to the fixture. Tighten the screws to lock the potentiometer body and plug the connector into the DAQ unit. Next, manually move the actuator 25 millimeters on a load frame, so that the potentiometer position translate from the maximum compression to the maximum extension and record the displacements and the corresponding voltage for at least three data points.Plot the displacement versus the voltage and fit a linear function to the data. Then input the slope of the linear equation as the calibration factor into the scanning parameter box of the DAQ software. To prepare the bone for testing, place a clean 24 hour thawed bone in the acrylic scanning fixture and mix 60 grams of poly or PMMA powder, with 30 grams of liquid resin in a disposable cup under a fume hood, until the powder has dissolved.When the PMMA is pourable, align an aluminum cup below the tricanter and decant the PMMA cement to half the height of the cup. Raise the fixture platform to fit the bone into the cup and allow the PMMA to polymerise for 10 to 15 minutes. When the PMMA is clear and hard, move the bone to the test fixture and center the aluminum cup on the plate, attached to the tricanteric load cell.Adjust the six channel load cell linear stages, so that the aluminum cup lightly touches the load cell and remove the pin from the fixture to a lateral rotation of the fixture. Then center and lower the crosshead for contact with the femoral head and image the femur from two sides. After verifying that the servo mechanical load frame is appropriately programmed, click start to initiate the test sequence for fracturing the test femur.After the test is complete, manually retract the actuator. Remove the bone from the fixture and tape the proximal broken end of the bone to the shaft. Then wrap the bone Wet towels and plastic bags and return the bone to 20 degree celsius storage.After fracture, the force recorded at the greater tricanter and the displacement recorded at the femoral head, using the six channel load cell can be plotted. The broken parts of the bone can then be taped together for further fracture type imaging and classification. While attempting this procedure, it's important to remember to verify that all of the data acquisition systems, function properly with fiberglass femurs before testing the cadaveric specimens.After its development, this technique paved the way for researchers in the field of computational biomechanics, to validate quantitative computer tomography based finite element predictions of strength in osteoporotic femurs. After watching this video, you should have a good understanding on how to test cadaveric femurs in a fall on a hip configuration.

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A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

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 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. ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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 ...