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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C., Bonfield, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crack+velocity+dependence+of+longitudinal+fracture+in+bone.">Crack velocity dependence of longitudinal fracture in bone.</a> J of Mat Sc. 15 (7), 1841-1849 (1980).</li><li>Mullins, L. P., Bruzzi, M. S., McHugh, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+microstructural+fracture+toughness+of+cortical+bone+using+indentation+fracture.">Measurement of the microstructural fracture toughness of cortical bone using indentation fracture.</a> J of Biomech. 40 (14), 3285-3288 (2007).</li><li>Harding, D. S., Oliver, W. C., Pharr, G. 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.

<table>
	<tbody>
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			<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>
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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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			<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>Consumables</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>CarbiMet SiC Abrasive Paper 400 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</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>
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			<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>
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			<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>
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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>
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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>
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			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</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>
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			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</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.

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9.1K Views.  University of Illinois at Urbana Champaign. 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 objectiv...