The calcium-buffering phase of bone mineral: some clues to its form and formation.

Devitalized, neonatal, rat calvariae incubated in a buffered solution release Ca2+ and Pi during the first day into the medium until levels reach 1.2 mM and 2.6 mM, respectively. Thereafter levels gradually decrease to stabilize by the fifth day at 0.5 mM and 2 mM. Comparison of these solubility changes with those of known calcium phosphate salts suggests that calvarial solubility is controlled by a mixture of impure, soluble, apatite precursors, which at cell death transform to an impure apatite. Increasing concentrations of Mg2+ increased the rate of Ca2+ release and, at 3 mM, stabilized Ca2+ by the fifth day at 0.9 mM. Decreasing the pH from 7.4 to 7.2 produced similar solubility changes as did the addition of citrate at 0.3 mM, phosphocitrate at 0.3 mM, ATP at 0.1 mM, and the bisphosphonate, HEBP, at 0.1 mM. Osteocalcin did not increase calvarial solubility but was able to slow the rate of calcium phosphate phase transition if present at the time of in vitro crystal formation. Phosphocitrate and HEBP were also more effective when present during in vitro crystal formation. HEBP was most effective when present during biological crystal formation, as shown by the increased solubility of devitalized calvariae removed after in vivo administration of HEBP. In vivo manipulations of osteoclast activity produced changes in plasma calcium which correlated with the solubility of the corresponding calvariae after removal and devitalization. Low milk intake increased calvarial solubility. Increasing doses of 1,25(OH)2D3 increased plasma calcium and calvarial solubility, both of which were reversed by injection of acetazolamide. It was concluded from this survey of devitalized bone solubility that calcium exchange between bone and body fluids can buffer calcium homeostasis in the young rat. The exchange is passive. The active components appear to be osteoblastic formation of soluble apatite precursors and their stabilizers and, in reverse, osteoclastic transformation of apatite to precursors by H2CO3 secretion.