Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vesicles and their role in calcification

Abstract

The onset of mineralization in embryonic chick femurs was studied as a model for the initiation of biological calcification. Electron microscopy confirmed the presence of calcifying matrix vesicles within newly formed bone, and showed that these vesicles were the initial site of crystal deposition. Matrix vesicles were first seen on day 6 of embryonic development, and already were present in considerable numbers on day 7, at which time mineral deposition was just beginning. As a reflection of initial mineralization the uptake of 45Ca and 40Ca into 7-day-old bones was studied during 2 days in organ culture. A control level of uptake was established using a defined culture medium, P-6. Addition of inorganic pyrophosphate (PP i) to this medium caused a marked increase in calcium uptake into areas of matrix which normally calcify in vivo. The maximal 45Ca uptake, greater than 4-fold, was achieved with 4 μg of P per milliliter of PP i and was partially heat-inhibitable. Since the matrix vesicles are known to be rich in inorganic pyrophosphatase, it is proposed that mineralization is promoted in vesicles by the enzymatic hydrolysis of pyrophosphate. The membrane-bounded matrix vesicles appear to provide the necessary enzymes and environment to concentrate calcium and phosphate for initiating crystal formation.