Phosphorylated osteopontin peptides inhibit crystallization by resisting the aggregation of calcium phosphate nanoparticles

Under near-physiological pH and ionic strength, the role of a 14 amino acid segment of osteopontin (OPN) in inhibiting hydroxyapatite (HAP) nucleation and growth was kinetically examined by measuring the induction time via pH monitoring. The phosphorylated 14-mer OPN peptide segments (PP) significantly inhibit nucleation of HAP by markedly increasing induction times with an increase in the number of phosphorylation sites at their lower concentrations (<156 nM). The presence of phosphate groups in OPN peptides not only enhances the stability of the calcium phosphate (Ca-P) nanoparticles and limits their aggregation, but also inhibits the phase transformation from amorphous calcium phosphate (ACP) to the final crystalline phases. The extent of phosphorylation of OPN peptides is important to its protective roles in inhibiting crystallization by resisting the aggregation of Ca-P nanoparticles at a given concentration range. The nonphosphorylated peptide segment (NPP) had relatively little effect on induction times at concentrations lower than 156 nM, whereas at a higher concentration (234 nM), the effect of NPP in inhibiting HAP nucleation was enhanced. These results clearly showed that OPN inhibits HAP crystallization by prolonging induction times and delaying subsequent growth in a phosphorylation and/or concentration-dependent manner.