Microdissection--elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick

Abstract

The concentrations of elements in avian growth cartilage were studied by electron probe x-ray emission microanalysis (EDX). The cartilage was prepared for analysis by freezing, freeze-fracturing, freeze-drying, and carbon coating techniques. Cells and matrix fragments were removed from the tissue by microdissection with a tungsten needle in a scanning electron microscope (SEM) equipped with a real-time stereoscopic viewing facility. The samples were analyzed in the same SEM by EDX. Elemental analyses were performed on each fragment at a distance from the tissue sample, and hence background radiation due to the sample was eliminated. An important finding was that the intracellular potassium concentration of chondrocytes in calcified cartilage was similar to the levels in the premineralized zones. This observation supports the view that chondrocytes do not die in the process of, or as a consequence of, mineralization of the surrounding matrix. Calcium peaks were seen in the matrix at all levels and in chondrocytes immediately prior to mineralization. In contrast, phosphorus levels were always high in cells and low or absent from the premineralized matrix. At the mineralization front the appearance of a phosphorus peak in the matrix just preceded the deposition of mineral. We propose that the transfer of phosphorus from cell to matrix is a rate-limiting step in mineralization. Finally, when rachitic and normal cartilage were compared, little difference was seen in the profile of either intracellular or extracellular elements. However, in rickets the mineralized matrix remained soft in consistency. We suggest that this may reflect a phosphorus-related calcification defect that prevents growth and interlocking of the apatite crystallites.