Recent research by Amy Lerner has described "variations within a single growth plate (1)." It was found that "regional variations in bone growth are most strongly explained by the amount of matrix associated with each chondrocyte domain (2)." The growth rate of the peaks versus the valleys is twice as fast. In this project we hope to see these variations in the cell shape and their surrounding structures.
We obtained our samples from the distal femur and tibia of a 6 week old New Zealand White rabbit; this age was chosen to avoid mechanical deformation of the developing structure. The desired bone sections were dissected out. The sample was cut into longitudinal cross sections, 2-3 mm wide, with a diamond saw. The bone fragments were then fixed in formalin. The fixed sample was transferred into 3% Biz solution for 96 hours, then transferred into saline solution for 48 hours, which was changed daily and periodically agitated.
After preparation, the samples were placed in a dessicator for 24-48 hours. The cuts from the diamond saw smeared the topographical information on the sample, so it was necessary to put the samples in liquid nitrogen and fracture them with a razor blade. These fractured samples were mounted on sample stages with conductive carbon glue, then gold plated.
The gold plating is necessary to create a conductive environment in which the electrons from the beam can be transmitted off of the sample without causing a charge buildup. To effectively gold plate the samples we used the sputtering process. In this method, the sample stages were placed in a bell jar in which a vacuum was created to a pressure of 10-3 torr. Then argon gas was bled into the bell jar to create a plasma with the gold. This facilitates the acceleration of the gold toward the sample. The sputtering process continued for two minutes to allow for an even coating of gold on the samples. The bone fragments were then ready to be viewed under the scanning electron microscope.
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