PREPARATION OF HARD TISSUES – BONE AND TEETH – 2
DECALCIFICATION
To obtain thin sections of bone and teeth with a regular microtome, these tissues may be submitted to a decalcification procedure, meaning the removal of calcium salts deposited in their extracellular matrix.
This can be done in several ways. One of the most common is to immerse teeth or bone fragments for appropriate periods of time in solutions that remove calcium salts. Acid solutions solubilize the calcium crystals that leave the tissue and enter the sorrounding medium. However, over a long period of time these acid solutions can damage and distort the tissue structure, therefore weak (diluted) acid solutions must be used.
A better way is to place the tissues in solutions of substances that have a chelating action, such as ethylenediaminetetraacetic acid (EDTA). Chelators are molecules that have a high affinity for certain ions such as calcium and lead and bind to them. In consequence the ions of the tissues are gradually displaced into the chelating solution.
The action of chelating agents is more delicate than that of acids and provides images that are closer to that of the tissues in vivo.
After decalcification, the hardness of the tissues decreases and they can be subjected to routine histological preparation procedures, embedded in paraffin and sectioned with regular microtomes. The sections can then be stained using routine staining mixtures.
To obtain thin sections of bone and teeth with a regular microtome, these tissues may be submitted to a decalcification procedure, meaning the removal of calcium salts deposited in their extracellular matrix.
This can be done in several ways. One of the most common is to immerse teeth or bone fragments for appropriate periods of time in solutions that remove calcium salts. Acid solutions solubilize the calcium crystals that leave the tissue and enter the sorrounding medium. However, over a long period of time these acid solutions can damage and distort the tissue structure, therefore weak (diluted) acid solutions must be used.
A better way is to place the tissues in solutions of substances that have a chelating action, such as ethylenediaminetetraacetic acid (EDTA). Chelators are molecules that have a high affinity for certain ions such as calcium and lead and bind to them. In consequence the ions of the tissues are gradually displaced into the chelating solution.
The action of chelating agents is more delicate than that of acids and provides images that are closer to that of the tissues in vivo.
After decalcification, the hardness of the tissues decreases and they can be subjected to routine histological preparation procedures, embedded in paraffin and sectioned with regular microtomes. The sections can then be stained using routine staining mixtures.
The image is of a histological section of a decalcified bone fragment. As can be seen, the histological structure of the tissue is very well preserved and details of the cells and of the organic extracellular matrix are clearly observed.
Decalcified bone. Hematoxylin and eosin staining.
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