Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Summary

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.

Abstract

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 – nano-, micro-, meso-, and macroscopic – 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.

Introduction

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 technique^1,2,3,4,5. 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 scale^6,7,8. On the other hand, to measure the fracture toughness at the microscopic scale, Vicker's indentation fracture was proposed⁹. Micro indentation was performed using the Vicker'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'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's fracture resistance in the short longitudinal direction.

Protocol

NOTE: The protocol described here, follows the animal care guidelines of the Illinois Institutional Animal Care and Use Committee.

1. Specimen Procurement

1. 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.
2. Freeze the femurs at -20 °C until the start of the specimen preparation procedure. This temperature keeps the femurs fresh^12,13,14.

2. Cutting, Cleaning, and Embedding the Specimens

1. Thaw the frozen femurs in a container with water for about 2 h at room temperature.
2. 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.
3. Use a dissection kit to remove any soft tissue or flesh attached to the cortical bone.
4. 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 – longitudinal specimens are discussed. However, except for the direction of cutting, the preparation procedure remains the same for the transverse orientation.
5. 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.
6. Embed the cortical bone specimens in acrylic resin (herein polymethyl methacrylate (PMMA)) for ease of handling and stability.
   1. 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.
   2. 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.
7. 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.
8. Wrap the specimens in a gauge soaked in Hanks Balanced Saline Solution (HBSS) and refrigerate at 4 °C until further use^15,16.

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 protocols^13,17 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 x 8 mm² surface.

1. 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.
2. 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.
3. Polish the specimens using 3 µm, 1 µm, and 0.25 µm 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.
4. Polish the specimen using 0.05 µm 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.
5. 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.
6. 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.

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 µm and apex angle of 120° 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.

1. Prior to the testing of cortical bone specimens, calibrate the Rockwell indenter tip using polycarbonate as reference material³.
2. 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.
3. 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.
4. Perform series of scratch tests on the short longitudinal (Figure 3b) bovine cortical bone specimens as illustrated in Figure 3.
5. Wet the specimen surface with HBSS after a set of every three to four scratch tests to keep them hydrated.
6. Analyze the scratch test data based on non-linear fracture mechanics modelling².

Results

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 µm x 40 µm area clearly falls within this criterion.

Figure 4 shows the force vers...

Discussion

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 µm long, 600 µm wide, and 480 µm 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

Materials

Name	Company	Catalog Number	Comments
Table Top Diamond Band Saw	McMaster Carr, Elmhurst, IL	Model  C-40	Blade speed of 40 mph; Blade dimensions: 37 inch in diameter, 0.02 inch wide and 0.14 inch deep
Buehler Isomet 5000 Precision Cutter	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	112780	Blade speed in the range of 200-5000 rpm in 50 rpm incrments; 8 inch diamond wafering blade
Branson 5800 Ultrasonic Cleanser	(Through) Grainger, Peoria, Illinois	39J365	Bransonic CPXH ultrasonic bath has a tank capacity of 2.5 gal
Buehler Ecomet 250 Grinder - Polisher	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	497250	8 inch base plate with a speed range from 10-500 rpm
Anton Paar, CSM Instruments Micro scratch tester	Anton Paar Switzerland AG	163251	Compact Platform, Acoutstic Emission Sensor
JEOL 6060LV general purpose scanning electron microscope	JEOL USA, Inc., Peabody, MA		Environmental scanning electron microscope which enables imaging at low vacuum levels.
Philips XL30 ESEM FEG	FEI Company		Wet mode working of the instrument enables imaging of non conductive samples without altering them
Name	Company	Catalog Number	Comments
Consumables
Bovine Femur	L&M Slaughter house, Georgetown, IL		Corn fed, 24-30 month old mature bovine specimens.
Alconox Powdered Precision Cleaner	Alconox, Inc., 30 Glenn St., Ste. 309, White Plains, NY, 10603	1104-1	Biodegradable, Non caustic, Interfering-residue free
Acrylic Plastic Casting	Electron Microscopy Sciences	24210-02	Polymethyl Methacrylate
CarbiMet SiC Abrasive Paper 400 grit, 8 inch, PSA backed	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	36080400	Grinding - Abrasive Papers
CarbiMet SiC Abrasive Paper 600 grit, 8 inch, PSA backed	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	36080600	Grinding - Abrasive Papers
MicroCut Discs 800 grit, 8 inch, PSA backed	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	36080800	Grinding - Abrasive Papers
MicroCut Discs 800 grit, 8 inch, PSA backed	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	16081200	Grinding - Abrasive Papers
Texmet P For 8'' Wheel PSA	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	407638	Polishing Cloth
8'' Microcloth PSA	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	407518	Polishing Cloth
Meta Di Supreme Polycrystalline Diamond Suspension, 3 µm	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	406631	Polishing suspension
Meta Di Supreme Polycrystalline Diamond Suspension, 1 µm	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	406630	Polishing suspension
Meta Di Supreme Polycrystalline Diamond Suspension, 0.25 µm	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	406629	Polishing suspension
MasterPrep Polishing Suspension, 0.05µm	Buehler,41 Waukegan Rd, Lake Bluff, IL 60044	40-6377-032	Polishing suspension
HBSS, calcium, magnesium, no phenol red	Thermo Fisher Scientific	14025126	Buffer Solution