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 pounds.</li>
	<li>Freeze the femurs at -20 &#176;C until the start of the specimen preparation procedure. This temperature keeps the femurs fresh12,13,14.</li>
</ol>
2. Cutting, Cleaning, and Embedding the Specimens
<ol>
	<li>Thaw the frozen femurs in a container with water for about 2 h at room temperature.</li>
	<li>Cut multiple discs about 10 - 15 mm 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.</li>
	<li>Use a dissection kit to remove any soft tissue or flesh attached to the cortical bone.</li>
	<li>Cut the cross sections of the femurs obtained in step 2.2 using a diamond-wafering blade on a low speed saw under wet conditions along the longitudinal axis of the bone to obtain multiple roughly cuboidal sections. 
	NOTE: Here, only specimen preparation and scratch tests performed on the short &#8211; longitudinal specimens are discussed. However, except for the direction of cutting, the preparation procedure remains the same for the transverse orientation.</li>
	<li>Clean the specimens in a solution prepared using 1.5% anionic cleaner and 5% bleach for a duration of 20 min in an ultrasonic cleaner.</li>
	<li>Embed the cortical bone specimens in acrylic resin (herein polymethyl methacrylate (PMMA)) for ease of handling and stability.
	<ol>
		<li>To embed the specimens, first coat the walls of the mold with a release agent. Then mix the acrylic resin and hardener in a beaker, as per instructions given by the PMMA manufacturer.</li>
		<li>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. Let the specimens cure for a duration of up to 4 - 5 h.</li>
	</ol>
	</li>
	<li>Cut the embedded specimens into 5-mm thick discs, exposing the surface to be scratched, using the low speed saw and mount the specimens on to metal (aluminum) discs of diameter 34 mm and height 5 mm using cyanoacrylate adhesive.</li>
	<li>Wrap the specimens in a gauge soaked in Hanks Balanced Saline Solution (HBSS) and refrigerate at 4 &#176;C until further use15,16.</li>
</ol>
3. Grinding and Polishing Protocols
NOTE: A pre-requisite to high-precision testing at small-length scales is a smooth and levelled surface of specimens. Previous polishing protocols13,17 result in a large surface roughness, leading to substantial inaccuracy in measurement. The challenge lies in achieving low average surface roughness, less than 100 nm, over a large area 3&#160;x 8 mm2 surface.
<ol>
	<li>Grind the bovine cortical bone specimens at room temperature using 400 grit and 600 grit Silicon Carbide papers for 1 min and 5 min, respectively. Maintain the grinder-polisher at base speeds of 100 rpm and 150 rpm, respectively.</li>
	<li>Machine grind the bovine cortical bone specimens at room temperature on the 800 and 1,200 grit papers for a duration of 15 min for each step. Maintain the grinder-polisher at a base speed of 150 rpm, head speed of 60 rpm, and operating load of 1 lb.</li>
	<li>Polish the specimens using 3 &#181;m, 1 &#181;m, and 0.25 &#181;m diamond suspension solutions in the same order on a hard, perforated, non-woven cloth for a duration of 90 min each at room temperature. Maintain the operating load for each step at 1 lb with the base and head speeds of the polisher at 300 rpm and 60 rpm, respectively.</li>
	<li>Polish the specimen using 0.05 &#181;m alumina suspension solution on a soft, synthetic rayon cloth for a duration of 90 min at 1 lb with base and head speed of 100 rpm and 60 rpm, respectively, also at room temperature.</li>
	<li>Put the specimens in a beaker with de-ionized water and put the beaker in an ultrasonic bath for 2 min in between each consecutive step of grinding and polishing to clean the residue and avoid cross contamination.</li>
	<li>View the surface features using optical microscopy and SEM imaging. 
	NOTE: As shown in Figure 1, osteons, Haversian canals, cement lines, interstitial regions, and lacunae were observed on the bovine cortical bone specimens. These imaging methods reveal the porous, heterogeneous, and anisotropic nature of cortical bone specimens. Additionally, advanced surface examination of the specimens was performed to assess the quality of the polished surface. A representative polished surface is shown in Figure 2.</li>
</ol>
4. Micro Scratch Test
NOTE: Micro scratch tests are performed on the polished bovine cortical bone specimens using a micro scratch tester (Figure 3). A diamond Rockwell indenter with a tip radius of 200 &#181;m and apex angle of 120&#176; is used for the study. The instrument allows the application of a linear progressive load up to 30 N. Furthermore, the instrument is equipped with high-accuracy sensors to measure the horizontal load, penetration depth, and acoustic emissions generated due to scratching. The instrument can capture the panoramas of scratch grooves.
<ol>
	<li>Prior to the testing of cortical bone specimens, calibrate the Rockwell indenter tip using polycarbonate as reference material3.</li>
	<li>Place the cortical bone specimen on the stage and choose the site of scratch test using the optical microscope set up integrated to the micro scratch tester module.</li>
	<li>Apply a linear progressive load with a start load of 30 mN and end load of 30 N. The loading rate should be set to 60 N/min and the scratch length to 3 mm.</li>
	<li>Perform series of scratch tests on the short longitudinal (Figure 3b) bovine cortical bone specimens as illustrated in Figure 3.</li>
	<li>Wet the specimen surface with HBSS after a set of every three to four scratch tests to keep them hydrated.</li>
	<li>Analyze the scratch test data based on non-linear fracture mechanics modelling2.</li>
</ol>

<ol><li>Akono, A., Reis, P., Ulm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scratching+as+a+fracture+process%3A+From+butter+to+steel%5BTitle%5D)+AND+%22Phys+Rev+Lett%22%5BJournal%5D)">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="https://www.cambridge.org/core/journals/journal-of-materials-research/article/experimental-determination-of-the-fracture-toughness-via-microscratch-tests-application-to-polymers-ceramics-and-metals/77F2DBD39131579AD83C981C8BCB7C0B">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.sciencedirect.com/science/article/pii/S0043164814000593">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://ascelibrary.org/doi/abs/10.1061/(ASCE)NM.2153-5477.0000105">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. -T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscopic+assessment+of+bone+toughness+using+scratch+tests%5BTitle%5D)+AND+%22Bone+Reports%22%5BJournal%5D)">Microscopic assessment of bone toughness using scratch tests.</a> Bone Reports. 6, 17-25 (2017).</li>
<li>Melvin, J. W., Evans, F. G. Crack propagation in bone. ASME Biomech Symp. , New York. (1973).</li>
<li>Norman, T. L., Vashishth, D., Burr, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+groove+on+bone+fracture+toughness%5BTitle%5D)+AND+%22J+of+Biomech%22%5BJournal%5D)">Effect of groove on bone fracture toughness.</a> J of Biomech. 25 (12), 1489-1492 (1992).</li>
<li>Behiri, J. C., Bonfield, W. <a target="_blank" href="https://link.springer.com/article/10.1007/BF00550605">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/pubmed?term=((Measurement+of+the+microstructural+fracture+toughness+of+cortical+bone+using+indentation+fracture%5BTitle%5D)+AND+%22J+of+Biomech%22%5BJournal%5D)">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="https://www.cambridge.org/core/journals/mrs-online-proceedings-library-archive/article/cracking-during-nanoindentation-and-its-use-in-the-measurement-of-fracture-toughness/B98631CB666CA3CD3D554135BEC2D845">Cracking during nanoindentation and its use in the measurement of fracture toughness.</a> MRS Proceedings. 356, Cambridge University Press. 663-668 (1994).</li>
<li>Islam, A., Dong, X. N., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanistic+modeling+of+a+nanoscratch+test+for+determination+of+in+situ+toughness+of+bone%5BTitle%5D)+AND+%22J+of+the+Mech+Bhvr+of+Biomed+Mat%22%5BJournal%5D)">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/pubmed?term=((Age-related+changes+in+the+tensile+properties+of+cortical+bone%3A+the+relative+importance+of+changes+in+porosity%2C+mineralization+and+microstructure%5BTitle%5D)+AND+%22J.+Bone+Joint+Surg%22%5BJournal%5D)">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/pubmed?term=((Fatigue+strength+of+human+cortical+bone%3A+age%2C+physical%2C+and+material+heterogeneity+effects%5BTitle%5D)+AND+%22J+of+Biomed+Mat+Res+Part+A%22%5BJournal%5D)">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., Sørensen, H. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+different+storage+methods+on+the+mechanical+properties+of+trabecular+bone%5BTitle%5D)+AND+%22J+of+Biomech%22%5BJournal%5D)">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/pubmed?term=((Accumulation+of+in-vivo+fatigue+microdamage+and+its+relation+to+biomechanical+properties+in+ageing+human+cortical+bone%5BTitle%5D)+AND+%22J+of+Microscopy%22%5BJournal%5D)">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/pubmed?term=((Fracture+toughness+of+manatee+rib+and+bovine+femur+using+a+chevron-notched+beam+test%5BTitle%5D)+AND+%22J+of+Biomech%22%5BJournal%5D)">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/pubmed?term=((Atomic+force+microscopy+and+nanoindentation+characterization+of+human+lamellar+bone+prepared+by+microtome+sectioning+and+mechanical+polishing+technique%5BTitle%5D)+AND+%22J+of+Biomed+Mat+ResPart+A%22%5BJournal%5D)">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/pubmed?term=((How+tough+is+bone%3F+Application+of+elastic%E2%80%93plastic+fracture+mechanics+to+bone%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">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/pubmed?term=((The+conflicts+between+strength+and+toughness%5BTitle%5D)+AND+%22Nat+Mater%22%5BJournal%5D)">The conflicts between strength and toughness.</a> Nat Mater. 10 (11), 817-822 (2011).</li>
<li>Kim, K. T., Bažant, Z. P., Yu, Q. <a target="_blank" href="https://link.springer.com/article/10.1007/s10704-013-9821-8">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBazant%2C+ZP+%22Fracture+and+size+effect+in+concrete+and+other+quasibrittle+materials%22">Fracture and size effect in concrete and other quasibrittle materials</a>. 16, CRC press. (1997).</li>
</ol>

The authors have nothing to disclose.

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><tr><td>Table Top Diamond Band Saw</td><td>McMaster Carr, Elmhurst, IL</td><td>Model&amp;nbsp; 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></tr><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></tr><tr><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></tr><tr><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></tr><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><tr><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></tr><tr><td>Philips XL30 ESEM FEG&amp;nbsp;</td><td>FEI Company</td><td></td><td>Wet mode working of the instrument enables imaging of non conductive samples without altering them&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Femur</td><td>L&amp;amp;M Slaughter house, Georgetown, IL</td><td></td><td>Corn fed, 24-30 month old mature bovine specimens.</td></tr><tr><td>Alconox Powdered Precision Cleaner</td><td>Alconox, Inc., 30 Glenn St., Ste. 309, White Plains, NY, 10603</td><td>1104-1</td><td>Biodegradable, Non caustic, Interfering-residue free</td></tr><tr><td>Acrylic Plastic Casting</td><td>Electron Microscopy Sciences</td><td>24210-02</td><td>Polymethyl Methacrylate</td></tr><tr><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></tr><tr><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></tr><tr><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></tr><tr><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></tr><tr><td>Texmet P For 8&#039;&#039; Wheel PSA</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>407638</td><td>Polishing Cloth</td></tr><tr><td>8&#039;&#039; Microcloth PSA</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>407518</td><td>Polishing Cloth</td></tr><tr><td>Meta Di Supreme Polycrystalline Diamond Suspension, 3 &amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>406631</td><td>Polishing suspension</td></tr><tr><td>Meta Di Supreme Polycrystalline Diamond Suspension, 1 &amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>406630</td><td>Polishing suspension</td></tr><tr><td>Meta Di Supreme Polycrystalline Diamond Suspension, 0.25 &amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>406629</td><td>Polishing suspension</td></tr><tr><td>MasterPrep Polishing Suspension, 0.05&amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>40-6377-032</td><td>Polishing suspension</td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>14025126</td><td>Buffer Solution</td></tr></tbody></table>

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...

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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 ...

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

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Bioengineering

9.4K Views.  University of Illinois at Urbana Champaign. Deze studie analyseert de breuk-taaiheid van boviene corticaal bot op de sub-meso-niveaus met behulp van microscopisch kleine kras tests. Dit is een originele, objectieve, rigoureus, en reproduceerbare methode voorgesteld om sonde breuk-taaiheid beneden de macroscopische schaal. Mogelijke toepassingen zijn wijzigingen studeren in bot kwetsbaarheid als gevolg van ziekten zoals osteoporose.

Video: Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Developmental Biology

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 ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Medicine

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 ...

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 ...

Cortical Bone Assessment Using the Ultrasonic BDAT Device

Cortical Bone Assessment Using Ultrasonic Guided Waves: A Reproducibility Study in a Healthy Population

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected to double between 2020 and 2050. Therefore, it is essential to detect patients at risk of fragility fracture at the population scale. The current golden standard is dual X-ray absorptiometry (DXA), providing the areal bone mineral density (aBMD). Ultrasonic devices, usually more portable and cheaper than X-ray devices, represent interesting DXA alternatives as screening tools. However, operator dependency is usually recognized as their main drawback. In this study, the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device is presented in detail. The dedicated ultrasonic probe is placed at the one-third distal radius of the non-dominant forearm using conventional coupling gel. The guided interface provides in quasi-real time (about 2 Hz) four parameters of interest: velocities of the first arriving signal (vFAS) and the A0 mode (vA0),&#160;cortical thickness (Ct.Th) and porosity (Ct.Po), as well as four quality parameters. The operator moves the probe slowly at the measurement site, carefully observing the feedback provided by the interface until finding a stable position and starting a series of 10 acquisitions. When at least four consistent series are obtained, the measurement ends, and an automatic report is generated. The measurement usually takes about 5 min to complete. To determine the robustness of this protocol, a reproducibility study was conducted among 3 operators (one expert and two novices) and 14 healthy participants (6 women, 8 men, 21-53 years old). The intraclass correlation coefficients (ICC) were found good for vA0 (0.76), Ct.Po (0.80) or excellent for Ct.Th (0.87) and vFAS (0.91). The standard deviations were found to be less than 10% of the total ranges in clinical practice.

Here, we present the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device in detail and test it in a reproducibility study, considering 14 healthy participants and 3 operators. Reliability, measured with the intraclass correlation coefficients (ICC), was good to excellent for four parameters of interest.

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

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

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

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

Biology

Quasistatic Load-to-Fracture Test of CAD/CAM Occlusal Veneers on Dentin Analogs

Quasistatic Mechanical Testing for Computer-Aided Design and Manufacturing Occlusal Veneers Cemented to Milled Dentin Analog Material

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored tooth&#39;s vitality, aesthetics, and function. New computer-aided design and computer-aided manufacturing (CAD/CAM) ceramic-like material are&#160;now available. To guarantee longevity, a dental clinician must know these&#160;newly launched product&#39;s mechanical strength compared to the relatively brittle glass-matrix ceramic. Furthermore, a tooth substitute has been promoted for laboratory investigation, especially after the pandemic, and more evidentiary support is required for its application.
This study developed a laboratory protocol for a monotonic load-to-fracture test to determine the fracture strength of 1 mm-thick CAD/CAM occlusal veneers. Master dies were milled from high-pressure fiberglass laminate, which has&#160;similar elastic modulus and bond strength as hydrated dentin. They were mounted into polyvinyl chloride (PVC) end caps with cold-curing epoxy resin. Occlusal veneers, also called tabletop restorations, were milled from lithium disilicate (LD) and resin nanoceramic blocks (RNC) and cemented to prepared master dies using dual-cured adhesive resin cement. They were&#160;allowed to cure fully by storing in distilled water for 48 h at 37 &#176;C.
All samples were then placed in a universal testing machine and loaded via a non-fixed 5.5 mm stainless-steel ball that allows lateral movement as would occur against the antagonist&#160;teeth. Compression was applied at a 1 mm/min rate, and the load-displacement graph was generated. The average maximum load-bearing capacity of restorations in the RNC group (3,212.80 &#177; 558.67 N) was significantly higher than in the LD group (2727.10 &#177; 472.41 N) (p &#60; 0.05). No debonding was found during the test.&#160;Both CAD/CAM materials may have a similar flaw distribution. Hertzian cone crack was found at the loading site, whereas radial cracks propagating from the cementation surface were found close to the margin in both groups.

A quasistatic load-to-fracture test with a non-fixed stainless steel ball was developed to determine the fracture strength of minimally invasive posterior computer-aided design and manufacturing restorations cemented to dentin analog materials. This test models the typical loading regime responsible for the fracture of dental restorations.

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

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