The ultrastructure of slam-frozen bone mineral

Bone salt may be altered by preparative procedures. 'Slam' freezing can usefully be applied to bone mineral because it minimizes preparation and preserves the tissue chemistry. The structure and composition of the mineral in 'slam'-frozen neonatal mouse calvaria (which require neither previous slicing nor manipulation) was examined by transmission electron microscopy and X-ray microanalysis in unstained sections, 0.25 micron thick (i.e. unusually thick). Comparison was made with fresh intact calvaria and with 'snap'-frozen histological sections of mature rat femora. Under the optical microscope, calcified microspheres, up to 1 micron in diameter, were evident within 'young' osteocytes and within the extracellular matrix of both immature and mature, unstained and von Kossa-stained bone. In the electron microscope, microspheres of similar dimension and distribution were observed after 'slam' freezing and were divided into two groups. One group, found inside and outside cells, had a substructure of closely packed, electron-dense, rounded bodies 30-40 nm in diameter; despite their unusual stability in EDTA, X-ray microanalysis indicated high levels of both calcium and phosphorus. The other group was found at the calcification front and, although similar to the first group in size and chemical composition, these microspheres had a substructure of clusters of 5-nm-thick electron-dense filaments containing mineral that was characteristically EDTA labile. The 30- to 40-nm dense bodies did not appear to be mitochondrial and were absent from customary fixed and resin-embedded, ultrathin, stained preparations. They were not observed singly and their aggregation into arrangements of microspheres, sometimes linked by bridges, may be an important preliminary step in the development of the filamentous clusters. Needle-shaped and plate-like crystals of bone mineral were absent. It was concluded that 'slam' freezing preserves both intracellular and extracellular bone salt in the form of microspheres within which the mineral may modulate from dense bodies into filamentous arrays of variable density with maturity.