Electron-microscopic microprobe analysis on the initial stages of mineral formation in the epiphyseal growth plate

Dry thin sections (300-500 nm thick) of shock-frozen, freeze-dried and embedded epiphyseal growth plates from the proximal tibia of guinea pigs were cut longitudinally from the plate. Dark round bodies (phi less than 0.5 micron) were observed using the scanning transmission mode of the electron microscope initially directly in the vicinity of the chondrocytes. They gradually spread out in the direction of the metaphysis to the center of the longitudinal septum and represent most probably the matrix vesicles. By use of a microscan of 0.25 X 0.25 micron the element-concentrations of these bodies were measured. The measurements started on those bodies that could be clearly recognized and were extended to a length of 30-40 micron in the metaphyseal direction. To obtain approximate quantitative results the registered CaK alpha and PK alpha x-ray counts were directly compared with counts of fully mineralized regions, the Ca and P contents of which are known. Ca as well as p could be detected in the first visible vesicle-like structures (Ca approximately 0.2%, P approximately 0.4%) and increased steeply in the metaphyseal direction, amounting to approximately 6% Ca and 3% P. These results may lead to the conclusion that Pi becomes split from phosphate esters and transformed into the matrix vesicles already in a very early stage of enrichment. Incorporation of Ca may be coupled with this process.