Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca]×[PO₄] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 μg/ml (9 nM) and 10 μg/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 μg/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC₅₀ = 0.32 μg/ml (0.01 μM)] whereas OC was of lower potency [IC₅₀ = 6.1 μg/ml (1.1 μM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 μg/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.