Department of Orthopaedic Surgery, Okayama University Medical School, Japan.

Epiphyseal growth cartilage of the femoral head obtained from Wistar rats was investigated after fixation by a rapid-freezing and freeze-substitution. Liquid helium was used in order to achieve a fast cooling rate without ice-crystal damage during the rapid freezing. Use of the rapid-freezing and freeze-substitution procedure provided better ultrastructural preservation of the chondrocyte than conventional chemical fixation methods. This procedure allowed a more reliable approach to electron probe analysis. X-ray microanalysis of the specimens confirmed that calcium is not detected in the initial matrix vesicles as a result of the freezing process. The results suggest that calcium release from precipitates occurs in the free state without any detectable formation of hydroxyapatite at the initial stage of calcification and that calcium is not tightly bound to the matrix vesicles.