Name: fragility assessment of bovine cortical bone using scratch tests
Uploaded: 2017-11-30T13:00:00
Description: Watch this Scientific Journal Video about Fragility Assessment of Bovine Cortical Bone Using Scratch Tests at JoVE.com

This work was supported by the Department of Civil and Environmental Engineering and the College of Engineering at University of Illinois at Urbana Champaign. We acknowledge the Ravindra Kinra and Kavita Kinra Fellowship for supporting the graduate studies of Kavya Mendu. Scanning Electron Microscopy investigation was carried out at the facilities of the Frederick Seitz Material Research Laboratory and Beckman Institute at the University of Illinois at Urbana Champaign.

NOTE: The protocol described here, follows the animal care guidelines of the Illinois Institutional Animal Care and Use Committee.
1. Specimen Procurement
<ol>
	<li>Collect freshly harvested bovine femurs from a United States Department of Agriculture (USDA)-certified slaughterhouse and transport them in plastic air tight bags in a cooler. 
	NOTE: For the study conducted here, femurs were collected from animals that were 24 - 30 months old, corn-fed, and weighed about 1,000 - 1,100 poun...

<ol>
	<li>Akono, A., Reis, P., Ulm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+as+a+fracture+process:+From+butter+to+steel.">Scratching as a fracture process: From butter to steel.</a> Phys Rev Lett. 106 (20), 204302-204304 (2011).</li><li>Akono, A. T., Randall, N. X., Ulm, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+determination+of+the+fracture+toughness+via+microscratch+tests:+application+to+polymers,+ceramics,+and+metals.">Experimental determination of the fracture toughness via microscratch tests: application to polymers, ceramics, and metals.</a> J of Mat Res. 27 (02), 485-493 (2012).</li><li>Akono, A. T., Ulm, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+technique+for+characterizing+the+fracture+toughness+via+scratch+test+experiments.">An improved technique for characterizing the fracture toughness via scratch test experiments.</a> Wear. 313 (1-2), (2014).</li><li>Akono, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+size+effect+law+at+the+microscopic+scale:+Application+to+progressive-load+scratch+testing.">Energetic size effect law at the microscopic scale: Application to progressive-load scratch testing.</a> J of Nanomech and Micromech. 6 (2), (2016).</li><li>Kataruka, A., Mendu, K., Okeoghene, O., Puthuvelil, J., Akono, A. -. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cracking+during+nanoindentation+and+its+use+in+the+measurement+of+fracture+toughness.">Cracking during nanoindentation and its use in the measurement of fracture toughness.</a> MRS Proceedings. 356, 663-668 (1994).</li><li>Islam, A., Dong, X. N., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+modeling+of+a+nanoscratch+test+for+determination+of+in+situ+toughness+of+bone.">Mechanistic modeling of a nanoscratch test for determination of in situ toughness of bone.</a> J of the Mech Bhvr of Biomed Mat. 5 (1), 156-164 (2012).</li><li>McAlden, R. W., McGeogh, J. A., Barker, M. B., Court-Brown, C. M. <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+changes+in+the+tensile+properties+of+cortical+bone:+the+relative+importance+of+changes+in+porosity,+mineralization+and+microstructure.">Age-related changes in the tensile properties of cortical bone: the relative importance of changes in porosity, mineralization and microstructure.</a> J. Bone Joint Surg. 75, 1193-1205 (1993).</li><li>Zioupos, P., Gresle, M., Winwood, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatigue+strength+of+human+cortical+bone:+age,+physical,+and+material+heterogeneity+effects.">Fatigue strength of human cortical bone: age, physical, and material heterogeneity effects.</a> J of Biomed Mat Res Part A. 86 (3), 627-636 (2008).</li><li>Linde, F., S&#248;rensen, H. 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=The+effect+of+different+storage+methods+on+the+mechanical+properties+of+trabecular+bone.">The effect of different storage methods on the mechanical properties of trabecular bone.</a> J of Biomech. 26 (10), 1249-1252 (1993).</li><li>Zioupos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+in-vivo+fatigue+microdamage+and+its+relation+to+biomechanical+properties+in+ageing+human+cortical+bone.">Accumulation of in-vivo fatigue microdamage and its relation to biomechanical properties in ageing human cortical bone.</a> J of Microscopy. 201 (2), 270-278 (2001).</li><li>Yan, J., Clifton, K. B., Mecholsky, J. J., Reep, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+toughness+of+manatee+rib+and+bovine+femur+using+a+chevron-notched+beam+test.">Fracture toughness of manatee rib and bovine femur using a chevron-notched beam test.</a> J of Biomech. 39 (6), 1066-1074 (2006).</li><li>Xu, J., Rho, J. Y., Mishra, S. R., Fan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+and+nanoindentation+characterization+of+human+lamellar+bone+prepared+by+microtome+sectioning+and+mechanical+polishing+technique.">Atomic force microscopy and nanoindentation characterization of human lamellar bone prepared by microtome sectioning and mechanical polishing technique.</a> J of Biomed Mat ResPart A. 67 (3), 719-726 (2003).</li><li>Yan, J., Mecholsky, J. J., Clifton, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+tough+is+bone?+Application+of+elastic–plastic+fracture+mechanics+to+bone.">How tough is bone? Application of elastic–plastic fracture mechanics to bone.</a> Bone. 40 (2), 479-484 (2007).</li><li>Ritchie, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conflicts+between+strength+and+toughness.">The conflicts between strength and toughness.</a> Nat Mater. 10 (11), 817-822 (2011).</li><li>Kim, K. T., Ba&#382;ant, Z. P., Yu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-uniqueness+of+cohesive-crack+stress-separation+law+of+human+and+bovine+bones+and+remedy+by+size+effect+tests.">Non-uniqueness of cohesive-crack stress-separation law of human and bovine bones and remedy by size effect tests.</a> Intrnl J of Frac. 181 (1), 67-81 (2013).</li><li>Bazant, Z. P., Planas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fracture and size effect in concrete and other quasibrittle materials. 16, (1997).</li></ol>

Micro scratch tests induce a mixed-mode fracture3. Furthermore, in the short longitudinal bovine cortical bone specimens, fracture processes are activated as the probe digs deeper. For a 3-mm long scratch, the prismatic volume probed is around 3,600 &#181;m long, 600 &#181;m wide, and 480 &#181;m deep. This large volume helped in predicting a homogenized response. A non-linear fracture mechanics model enabled us to extract the fracture resistance based on the J-integral calculation<sup cl...

In this study, we measure the fracture toughness of bovine compact bone from the mesoscale (osteons) to the microscale (lamellar level) using a novel micro scratch technique1,2,3,4,5. Fracture processes including crack initiation and crack propagation in bone are directly influenced by length scales owing to the different structural constituents and organization at different levels of hierarchy. Therefore, assessing bone fracture at smaller length scales is essential to yielding a fundamental understanding of bone fragility. On the one hand, conventional tests such as three-point bending, compact tension, and flexure tests are commonly conducted on bovine femur and tibia for fracture characterization at the macroscopic scale6,7,8. On the other hand, to measure the fracture toughness at the microscopic scale, Vicker&#39;s indentation fracture was proposed9. Micro indentation was performed using the Vicker&#39;s indenter to generate radial cracks. Furthermore, the Oliver Pharr nanoindentation fracture toughness method was performed using a sharp cube corner indenter10.
In the above nanoindentation based fracture toughness studies, the lengths of the cracks thus generated were measured by the observer and a semi-empirical model was used to calculate the fracture toughness. However, these methods are irreproducible, subjective, and the results are highly dependent on the observer&#39;s skill due to the need to measure the crack lengths using optical microscopy or scanning electron microscopy. Moreover, scratch tests were conducted at the nano-scale, but the underlying mathematical model is not physics-based as it does not account for the reduction in strength due to cracks and defects11. Thus, a gap of knowledge exists: a method for fracture assessment at the microscopic level based on a physics-based mechanistic model. This gap of knowledge motivated the application of micro scratch tests to compact bone by focusing first on porcine specimens5. The study has now been further extended to understand bovine cortical bone.
Two different orientations of the specimens are possible: longitudinal transverse and short longitudinal. Longitudinal transverse corresponds to fracture properties perpendicular to the longitudinal axis of the femur. Whereas, short longitudinal corresponds to the fracture properties along the longitudinal axis of femur5. In this study, we apply scratch testing to bovine cortical bones to characterize the bone&#39;s fracture resistance in the short longitudinal direction.

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		<tr>
			<td>Table Top Diamond Band Saw</td>
			<td>McMaster Carr, Elmhurst, IL</td>
			<td>Model&#160; C-40</td>
			<td>Blade speed of 40 mph; Blade dimensions: 37 inch in diameter, 0.02 inch wide and 0.14 inch deep</td>
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		<tr>
			<td>Buehler Isomet 5000 Precision Cutter</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>112780</td>
			<td>Blade speed in the range of 200-5000 rpm in 50 rpm incrments; 8 inch diamond wafering blade</td>
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			<td>Branson 5800 Ultrasonic Cleanser</td>
			<td>(Through) Grainger, Peoria, Illinois</td>
			<td>39J365</td>
			<td>Bransonic CPXH ultrasonic bath has a tank capacity of 2.5 gal</td>
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			<td>Buehler Ecomet 250 Grinder - Polisher</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>497250</td>
			<td>8 inch base plate with a speed range from 10-500 rpm</td>
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		<tr>
			<td>Anton Paar, CSM Instruments Micro scratch tester</td>
			<td>Anton Paar Switzerland AG</td>
			<td>163251</td>
			<td>Compact Platform, Acoutstic Emission Sensor</td>
		</tr>
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			<td>JEOL 6060LV general purpose scanning electron microscope</td>
			<td>JEOL USA, Inc., Peabody, MA</td>
			<td></td>
			<td>Environmental scanning electron microscope which enables imaging at low vacuum levels.</td>
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		<tr>
			<td>Philips XL30 ESEM FEG&#160;</td>
			<td>FEI Company</td>
			<td></td>
			<td>Wet mode working of the instrument enables imaging of non conductive samples without altering them&#160;</td>
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			<td>Name</td>
			<td>Company</td>
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			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Bovine Femur</td>
			<td>L&#38;M Slaughter house, Georgetown, IL</td>
			<td></td>
			<td>Corn fed, 24-30 month old mature bovine specimens.</td>
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			<td>Alconox Powdered Precision Cleaner</td>
			<td>Alconox, Inc., 30 Glenn St., Ste. 309, White Plains, NY, 10603</td>
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			<td>Biodegradable, Non caustic, Interfering-residue free</td>
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			<td>Acrylic Plastic Casting</td>
			<td>Electron Microscopy Sciences</td>
			<td>24210-02</td>
			<td>Polymethyl Methacrylate</td>
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			<td>CarbiMet SiC Abrasive Paper 400 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080400</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>CarbiMet SiC Abrasive Paper 600 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080600</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>MicroCut Discs 800 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080800</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>MicroCut Discs 800 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>16081200</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>Texmet P For 8&#39;&#39; Wheel PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407638</td>
			<td>Polishing Cloth</td>
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			<td>8&#39;&#39; Microcloth PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407518</td>
			<td>Polishing Cloth</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 3 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406631</td>
			<td>Polishing suspension</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 1 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406630</td>
			<td>Polishing suspension</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 0.25 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406629</td>
			<td>Polishing suspension</td>
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			<td>MasterPrep Polishing Suspension, 0.05&#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>40-6377-032</td>
			<td>Polishing suspension</td>
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			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025126</td>
			<td>Buffer Solution</td>
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fragility assessment of bovine cortical bone using scratch tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

Atomic force microscopy was used to measure the roughness of the polished surface. As a rule of thumb, the specimen qualifies as a well-polished one if the surface roughness is an order of magnitude smaller than the surface features of interest. In this case, the measured surface roughness of 60 nm over a 40 &#181;m x 40 &#181;m area clearly falls within this criterion.
Figure 4 shows the force vers...

Watch this Scientific Journal Video about Fragility Assessment of Bovine Cortical Bone Using Scratch Tests at JoVE.com

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

The overall goal of this study is to characterize the fractured behavior of cortical bone at the mesoscale using microscopic scratch testing. This method can help answer key questions from the fields of biomechanics and fracture mechanics such as the fragility of biological tissues or the remodeling of compact bone. The main advantage of this technique is that it is adequate for small specimens.It is rigorous, reproducible and it is semi-destructive. In addition, the method is objective because we rely solely on force and depth measurements. Demonstrating the procedure will be Kavya Mendu, a graduate student in my laboratory.To begin this procedure, thaw the frozen femurs in a container with water for about two hours at room temperature. Then, cut multiple disk about 10-15 millimeters thick from the mid diaphysis region using a table top diamond band saw to produce specimens with uniform cross-sectional area of the cortical bone. After that, use a dissection kit to remove any soft tissue or flesh attached to the cortical bone.Cut the cross-sections of the femurs using a diamond wafering blade at low speed along the longitudinal axis of the bone under wet conditions to obtain multiple, ruffly cuboidal sections. Next, clean the specimens in the solution of 1.5%anionic cleaner and 5%bleach for 20 minutes in an ultrasonic cleaner. Following that, embed the cortical bone specimens in acrylic resin for ease of handling and stability by first coating the walls of the mold with a release agent.Then, mix the acrylic resin and hardener in a beaker. Place one of the cut cortical bone specimens into each mold with the surface to be scratched facing downwards. Pour the acrylic resin mix into these prepared specimen holders.Remove air bubbles trapped during the mixing process. Let the specimens cure for us to four to five hours. Subsequently, cut the embedded specimens into five millimeter thick disks at low speed exposing the surface to be scratched.Then, mount the specimens onto the aluminum disks of diameter 34 millimeters and height five millimeters using cyanoacrylate adhesive. Then, wrap the specimens in a gage shocked in HBSS. And refrigerate at four degrees celsius until further use.In this procedure, grind the bovine cortical bone specimens at room temperature using 400 grit and 600 grit silicon carbide papers for one minute and five minutes respectively. Maintain the grinder polisher at the base speeds of 100 rpm and 150 rpm respectively. After that, machine grind the bovine cortical bone specimens at room temperature on the 800 and 1200 grit papers for a duration of 15 minutes at each step.Maintain the grinder polisher at the base speed of 150 rpm, head speed of 60 rpm and operating load of one pound. Polish the specimens using three micrometer, one micrometer and 0.25 micrometer diamond suspension solutions in the same order on a hard, perforated, non-woven cloth for a duration of 90 each at room temperature. Maintain the operating load for each step at one pound, with the base and head speeds of the polisher at 300 rpm and 60 rpm respectively.Subsequently, polish the specimen using 0.05 micrometer alumina suspension solution on a soft, synthetic rayon cloth for a duration of 90 minutes at one pound, with a base and head speed of 100 rpm and 60 rpm respectively also at room temperature. Specimen grinding and polishing is crucial to yield the flat structure without significantly altering the microstructure. In between each consecutive step of grinding and polishing, place the specimens in a beaker with diionized water and keep the beaker in an ultrasonic bath for two minutes to clean the residue and avoid cross contamination.Then, view the surface features using an optical microscope or SEM imaging. Prior to the testing of cortical bone specimens, calibrate the Rockwell indenter tip using polycarbonate. Next, place the cortical bone specimen on the stage and chose the sight of scratch test using the optical microscope set up integrated to the micro scratch tester module.Then, apply a linear progressive load with a start load of 30 millinewtons and an end load of 30 newtons. The loading rate should be set to 60 newtons per minute and the scratch length to three millimeters. It is imperative to select a high density region for compact bone.Areas close to the cancelous bone, enlarge micropores, must be avoided, as they result in low values of the fracture resistance. Following that, perform series of scratch tests on the short, longitudinal, bovine cortical bone specimens. Wet the specimen surface with HBSS after every set of three to four scratch tests to keep them hydrated.After that, analyze the scratch test data based on non-linear, fracture mechanic modeling. Shown here is an optical microscopy image of the panorama of the scratch grove. Here's the corresponding plot of the force versus depth along the length of the scratch grove.Horizontal force corresponds to the resistive force detected by the sensors attached to the micro scratch tester stage. And the vertical force corresponds to the progressive linear force applied onto the cortical bone specimen. The SEM images of the scratch grove here show the micro-mechanisms such as crack deflection, crack bridging, fiber bridging and chipping at 40X, 10, 000X, 2, 400X and 5, 000X.While attempting the specimen preparation procedure, it is important to monitor the relative humidity and to keep the specimens wet at all times to prevent local dehydration. Following this procedure, other methods like x-ray computer tomography experiments can be preformed in order to answer addition questions like fracture micro-mechanisms at meso-and microscopic scales. After watching this video, you should have a good understanding of how to carry out microscopic scratch fracture tests on hard biological tissues.

fragility-assessment-of-bovine-cortical-bone-using-scratch-tests

Research

JoVE Journal

Bioengineering

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Modified Drop Tower Impact Tests for American Football Helmets

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards is presented that would provide better assessment of a helmet's on-field impact performance by including a faceguard on the helmet. In this study, a merger of faceguard and helmet test standards is proposed. The need for a more robust systematic approach to football helmet testing procedures is emphasized by comparing representative results of the Head Injury Criterion (HIC), Severity Index (SI), and peak acceleration values for different helmets at different helmet locations under modified NOCSAE standard drop tower tests. Essentially, these comparative drop test results revealed that the faceguard adds a stiffening kinematic constraint to the shell that lessens total energy absorption. The current NOCSAE standard test methods can be improved to represent on-field helmet hits by attaching the faceguards to helmets and by including two new helmet impact locations (Front Top and Front Top Boss). The reported football helmet test method gives a more accurate representation of a helmet's performance and its ability to mitigate on-field impacts while promoting safer football helmets.

This article provides a novel technique to assess the performance characteristics of American football helmets by inclusion of faceguards during NOCSAE Standard drop tests. Additionally, two more impact locations are proposed to be added to the NOCSAE certification.

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards ...

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 ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

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 ...

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...