Identification of brushite in newly deposited bone mineral from embryonic chicks

Crystalline brushite, CaHPO 4·2H 2O, has been identified in embryonic chick bone by X-ray and electron diffraction. This was accomplished by independent analyses of bone powder fractions of varying density and therefore of varying degrees of mineralization. Although it was not detected in samples of whole bone powder by X-ray diffraction, brushite was found to be the major crystalline solid phase of calcium phosphate in the lowest density fractions corresponding to the least mineralized bone. Thus brushite appears to be a component of the earliest solid mineral phase deposited in bone tissue. Both brushite and hydroxyapatite were detected in fractions of bone powder of somewhat higher density, but only hydroxyapatite was present in the highest density fractions corresponding to the most highly mineralized portions of bone tissue. Electron diffraction of the unembedded, lowest density fractions of bone powder confirmed the presence of brushite. The relative Ca/P ratio of these early mineral deposits determined by electron probe microanalysis was also consistent with that of brushite. Most importantly, when either synthetic crystalline brushite or the lowest density bone powder was embedded in epoxy and thin-sectioned, no electron diffraction pattern of brushite or monetite was obtained. This result may explain why brushite has not been previously detected by electron diffraction of thin sections containing the mineral phase of bone even though such sections contain the newly deposited solid phase of Ca-P.