NUCLEATION AND INHIBITION OF HYDROXYAPATITE FORMATION BY MINERALIZED TISSUE PROTEINS

Many proteins found in mineralized tissues have been proposed to funct ion 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 [osteoc alcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondro calcin (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 mu g/ml (9 nM) and 10 mu g/ml (67 nM) respective ly, OC, OPN, ON and CC all lacked nucleation activity at concentration s up to 100 mu g/ml. Inhibition of HA formation de novo was studied wi th calcium phosphate solutions buffered by autotitration. OPN was foun d to be a potent inhibitor of HA formation [IC50 = 0.32 mu g/ml (0.01 mu M)] whereas OC was of lower potency [IC50 = 6.1 mu g/ml (1.1 mu M)] ; BSP, ON and CC all lacked inhibitory activity at concentrations up t o 10 mu g/ml. The effect of OPN on HA formation de novo is mainly to i nhibit crystal growth, whereas OC delays nucleation. These findings ar e consistent with the view that BSP and DPP may play roles in the init iation of mineralization in bone and dentine respectively. OPN seems t o be the mineralized tissue protein most likely to function in the inh ibition of HA formation, possibly by preventing phase separation in ti ssue fluids of high supersaturation.