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 x PO4 product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 microgram/ml (9 nM) and 10 micrograms/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 micrograms/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 microgram/ml (0.01 microM) whereas OC was of lower potency IC50 = 6.1 micrograms/ml (1.1 microM); BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 micrograms/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.