To begin, dehydrate the osteochondral bone explant collected from sheep in a 40 milliliter glass vial containing 25 milliliters of dehydration solution. Place the vials on a rotating wheel for one hour at room temperature. After the last wash, replace the dehydration solution with 25 milliliters of xylene.
Place the glass vials on the rotating wheel for one hour at room temperature. Next, add 10%benzoyl peroxide, 10%dibutyl phthalate, and 450 microliters of N, N-Dimethylaniline diluted 1 to 20 in propanol, and place the glass vial at minus 20 degrees Celsius overnight. Now, place the explant into a medium-sized embedding mold in a plastic box.
Pour the methylmethacrylate benzoyl peroxide dibutyl phthalate N, N-aniline solution into the mold. Ventilate the mold with nitrogen flow for five minutes. Close the box hermetically and place it at four degrees Celsius for 48 hours for MMA polymerization and hardening.
After two days, remove the resin containing the explant from the mold. Cut the polymethylmethacrylate block containing the explant using a diamond saw along its long axis to generate a 1.5 millimeter thick section at 3000 RPM with a speed of three millimeters per minute. Grind the section with silicon carbide paper with ascending numbers.
Carbon coat the thick section with a 10 nanometer carbon film. Mount the section on an aluminum stub. Then, create a silver paint bridge between the top of the section and the stub to allow for electron charging evacuation to the ground.
Place the stub on the scanning electron microscopy, or SEM, stage. Close the chamber and initiate a vacuum. Turn on the electron beam and adjust the SEM settings to operate in back-scattered electron mode.
Set the gray level standards to 25 for carbon, 225 for aluminum, and 253 for silicon. After calibrating the SEM with the standards, acquire images of the specimen in back-scattered electron mode. Use the back-scattered electron image of the specimen to convert the gray levels into calcium content.
Then, plot the calcium distribution content of the image. Place the section on the stage of the Raman microspectrometer. Calibrate wave number for accuracy, and align the laser prior to measurement.
To collect the Raman spectra, use a 785 nanometer laser at 30 milliwatts. Set the time of integration to 20 seconds, repeat three times, and use a spectral range of 350 to 1, 800 per centimeter with a grading of 1, 200 lines per millimeter. Once the nanoindentation system is calibrated, place the sample in the optical system and identify the location for the nanoindentation.
Then, move the sample under the indentation device. Set the indentation depth to 900 nanometers with a loading-unloading speed of 40 millimeters per minute and a 15 second pause between loading and unloading. Set the cosine coefficient for bone tissue to 0.3 and start the nanoindentation.
