Analysis of microstructure in mouse femur and decalcification effect on microstructure by electron microscopy

Microstructure and decalcification effect by ethylenediaminetetraacetic acid (EDTA) on microstructure were studied for the compact bone of mouse femur by optical and electron microscopy. Especially the (002) reflection plane on the selected area electron diffraction (SAED) of hydroxyapatite (HA) was analyzed in detail. Two types of HA crystals were observed by transmission electron microscopy (TEM). One was needle-like crystals known as general HA crystals, and the other was flake-like crystals. Major constituents of two types of crystals were calcium, phosphorus, and oxygen. The Ca/P ratios of two types of crystals were close to the ideal value of HA within experimental error. Intensity data obtained from each crystals were also very similar. These results indicated that two types of crystals were actually same HA crystals. It was noticed that the (002) reflection plane on SAED displayed ring, spot, or arc patterns in accordance with orientations of HA crystals. Decalcification by EDTA process obsecured outline of osteons and havarsian canals, and changed morphology of the bone section. As the results of decalcification it was observed by TEM-EDS (Energy Dispersive Spectroscopy) that all peaks of calcium and phosphorus disappeared, and intensity of oxygen peak was substantially reduced. Moreover, collagen appeared to be disaggreated.