Matrix vesicle calcification in rat epiphyseal growth plate cartilage prepared anhydrously for electron microscopy

The preparation of mineralizing tissues for electron microscopy by methods that involve the use of aqueous solvents may result in translocation and/or removal of mineral, leading to the possible erroneous interpretation of initial calcifying loci. The failure of a recent cryoultramicrotomy study to detect a mineral phase associated with matrix vesicles raised doubts about the role of the matrix vesicle in initial mineralization and prompted us to reexamine the ultrastructure of calcifying cartilage using three different anhydrous preparatory techniques. Matrix vesicles or structures similar in profile and location were identified in rat epiphyseal growth plate cartilages that had been subjected to freeze-substitution, cryoultramicrotomy, or ethylene glycol fixation. Furthermore, mineral deposits, as confirmed by energy dispersive x-ray analysis, were observed within or in close association with the matrix vesicles in the extracellular matrix of the growth plate cartilage by all three methods. No evidence of mineral deposition at other sites preceding matrix vesicle calcification was found. Our studies support the conclusion of many investigations that matrix vesicles are the initial site of growth plate calcification.