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.
