Immobilized DPP and other proteins modify OCP formation

Abstract

Osteonectin, gamma-carboxyglutamic acid-containing (Gla) protein, and dentin phosphoprotein were covalently attached to sepharose beads and inoculated in solutions at two different degrees of supersaturation with respect to both octacalcium phosphate (OCP) and hydroxyapatite. In both solutions, the inhibitory activity towards de novo formation of calcium phosphate that these proteins display when freely dissolved in solution was completely eliminated when they were immobilized on the sepharose at concentrations of up to 5 micrograms/mg wet beads. In the solution that was more highly supersaturated with respect to OCP, the immobilized dentin phosphoprotein, moreover, was found to induce de novo formation of OCP in proportion to the concentration of the protein immobilized. For example, at 10 micrograms/ml of the immobilized dentin phosphoprotein, the induction period was reduced more than 50%. However, in the solution considerably less supersaturated with respect to OCP, none of the immobilized proteins were capable of inducing OCP or apatite deposition. These findings suggest that the immobilized dentin phosphoprotein could work as a nucleating substrate for the OCP phase in solutions where calcium and phosphate concentrations are sufficiently higher than equilibrium saturation levels for the OCP phase.

Dentin Mineralization and the Role of Odontoblasts in Calcium Transport

“…DPP, immobilized on agarose beads, has also been shown to induce an OCP-like phase. 33 Boskey and coworkers, using a gelatin gel system, showed that DPP does not necessarily need to be immobilized by covalent attachment to a solid substratum in order to induce mineral.34 In their system, DPP at concentrations < 1 pg/ml promoted HAP formation, whereas high DPP concentrations were inhibitory. In the steady-state agarose gel system of Hunter and Goldberg, the anionic, phosphorylated bone sialoprotein (BSP) induced formation of HAP.…”

Osteocalcin: Skeletal and extra‐skeletal effects

“…Moreover in the steady‐state gel system OC acts at the early stage of mineralization, by inhibiting the nucleation of hydroxyapatite (Hunter et al, 1996). In contrast OC had no effects when attached to agarose beads (Doi et al, 1993) and loss‐of‐function of the OC gene in mice demonstrated that OC did not affect bone mineralization. OC deficient bone did not show differences in mineral apposition rates and bone mineral content when compared with wild‐type mice (Ducy et al, 1996).…”

Bone mineralization

“…The role of osteocalcin in bone formation and calcification is still, however, uncertain. In vitro studies have shown that calcium phosphate precipitation is delayed by OC in solution, and that this effect disappears if OC is immobilized on sepharose beads (178). Inhibition by warfarin of vitamin K, which functions as a cofactor in the carboxylation of glutamate residues, induced a fall in bone OC content, but did not cause significant bone changes in rats (179) and did not alter bone mineral density or the markers of bone turnover in monkeys (180).…”

“…Secondly, most of the information on the properties and activities of single noncollagenous proteins has been obtained by means of in vitro research, when it is known that they change according to whether the proteins are free in solution or attached to a substrate. Thus, osteonectin, osteocalcin and dentin phosphoprotein in solution delay calcium phosphate precipitation, whereas this effect disappears when the proteins are immobilized on sepharose beads (178). Third, the action of the proteins on the induction of mineral formation changes with their concentration and this, in its turn, can change during the mineralization process.…”