OSTEOPONTIN-HYDROXYAPATITE INTERACTIONS IN-VITRO - INHIBITION OF HYDROXYAPATITE FORMATION AND GROWTH IN A GELATIN-GEL

Osteopontin is a phosphorylated bone matrix sialoprotein, postulated t o play a regulatory role in biomineralization. The effects of a crude preparation of rat bone osteopontin and a more highly purified bovine bone osteopontin were evaluated using a gel diffusion system to measur e effects of 0.1-100 mug/ml of this matrix protein on hydroxyapatite f ormation and crystal proliferation. Bovine osteopontin at concentratio ns greater than 25 mug/ml inhibited both hydroxyapatite formation and growth in a dose-dependent manner. Osteopontin at concentrations lower than 25 mug/ml had no detectable effect on the amount of mineral accu mulated in experiments with and without pre-formed hydroxyapatite seed crystals either when initial mineral deposition was assessed at 3.5 d ays, or when mineral formation and growth were assessed at 5 days. The re was a statistically significant dose-dependent decrease in crystal length at all concentrations tested. The rat osteopontin preparation h ad similar inhibitory abilities. Partial dephosphorylation of bovine o steopontin with alkaline phosphatase removed its inhibitory ability, a nd reduced its ability to bind calcium. The affinity of bovine osteopo ntin for hydroxyapatite was determined based on a Langmuir adsorption isotherm, with values of K (binding affinity) and N (number of binding sites) being 0.026 ml/mug and 1084 mug/M2, respectively. The data sug gest that, in this system, osteopontin is an effective inhibitor of hy droxyapatite formation and growth due to its affinity for the hydroxya patite crystals, In this system, osteopontin, distinct from other phos phoproteins which both promote and inhibit hydroxyapatite deposition, did not enhance mineral formation at any concentration tested.