A ZONAL ANALYSIS OF INORGANIC AND ORGANIC CONSTITUENTS OF EPIPHYSIS DURING ENDOCHONDRAL CALCIFICATION

A dissection procedure has been devised to permit zonal analysis of the epiphyseal plate of fetal calf leg bones. Samples of whole and washed tissue from the various zones were analyzed for their content of electrolyte and organic constituents, as well as for density, ash and moisture. Results showed that as ash content and density increased, water content decreased. Ash levels in calcifying cartilage zones were unexpectedly high. When expressed as a percentage of dry weight, washed calcified cartilage had the highest content of any zone. In the early stages of the mineralization of cartilage, Na content (mmoles/l of fresh tissue) decreased as Ca and inorganic P increased. Magnesium levels increased as calcification proceeded, but only at a fraction of the rate of Ca and P. Ratios of Ca/inorganic P were highest in resting cartilage (non-differentiated hyaline cartilage), suggesting an initial binding of Ca to chondromucoproteins. However, at the onset of calcification in proliferating cartilage, Ca/P ratios were much lower (ca. 1.50), but gradually increased with advancing mineralization. Marked changes occurred in the composition of the organic phase during endochondral calcification. As determined by hydroxyproline analysis, collagen content progressively decreased during cartilaginous calcification, but increased rapidly during bone formation. As determined by hexosamine and sulfur analysis, chondromucoproteins were at highest levels in proliferating cartilage and decreased steadily as calcification increased. However, although calcification was already well advanced in hypertrophic cartilage, large amounts of mucopolysaccharide still were present. Sulfur/hexosamine ratios showed a slight decline during the early stages of calcification, but increased markedly with further mineralization. Sialic acid levels were elevated in epiphyseal cartilage over those in resting cartilage or bone. Lipids increased rapidly during cartilaginous calcification, but were greatly reduced in fully-formed bone. The significance of these findings is discussed. © 1969 Springer-Verlag.