Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate

Early mineral deposits within calcifying rat epiphyseal growth plates were studied by bright field and selected-area dark field electron microscopy, and X-ray microanalysis. These mineral deposits were prepared in situ by high-pressure freezing, freeze substitution, and low-temperature embedding, and were examined in unstained, stained, and ethyleneglycol tetraacetic acid (EGTA)-treated stained thin sections. On unstained sections mineral rods occur within an amorphous density of calcium and phosphorus (CaP). X-ray microanalysis of stained sections reveals that the location of electron-dense deposits does not always correspond to that of the CaP mineral deposits identified in electron microscopic images. Such an analysis showed a depletion of both Ca and P in stained sections at sites corresponding to high levels of these elements in unstained sections. Staining thus demineralizes early deposition sites of CaP; at the same time lead (Pb) and uranium (U) bind to the organic components of the extracellular matrix formerly associated with Ca and P. This substitution phenomenon alters the overall fine structure of mineral sites by depleting the amorphous density of Ca and P, and by creating isolated rodlike structures that have formerly been interpreted as representing hydroxyapatite (HAP) crystals. Selected-area dark field imaging shows nascent sites of HAP crystals to be associated with the limiting membrane of matrix vesicles, but such crystals were undetectable at these sites with conventional bright field images. Dark field imaging also showed that the typical 30-80 nm crystal rods found in calcified cartilage consist of aggregates of HAP crystals.