Changes to the Disordered Phase and Apatite Crystallite Morphology during Mineralization by an Acidic Mineral Binding Peptide from Osteonectin

Abstract

Noncollagenous proteins regulate the formation of the mineral constituent in hard tissue. The mineral formed contains apatite crystals coated by a functional disordered calcium phosphate phase. Although the crystalline phase of bone mineral was extensively investigated, little is known about the disordered layer's composition and structure, and less is known regarding the function of noncollagenous proteins in the context of this layer. In the current study, apatite was prepared with an acidic peptide (ON29) derived from the bone/dentin protein osteonectin. The mineral formed comprises needle-shaped hydroxyapatite crystals like in dentin and a stable disordered phase coating the apatitic crystals as shown using X-ray diffraction, transmission electron microscopy, and solid-state NMR techniques. The peptide, embedded between the mineral particles, reduces the overall phosphate content in the mineral formed as inferred from inductively coupled plasma and elemental analysis results. Magnetization transfers between disordered phase species and apatitic phase species are observed for the first time using 2D (1)H-(31)P heteronuclear correlation NMR measurements. The dynamics of phosphate magnetization transfers reveal that ON29 decreases significantly the amount of water molecules in the disordered phase and increases slightly their content at the ordered-disordered interface. The peptide decreases hydroxyl to disordered phosphate transfers within the surface layer but does not influence transfer within the bulk crystalline mineral. Overall, these results indicate that control of crystallite morphology and properties of the inorganic component in hard tissue by biomolecules is more involved than just direct interaction between protein functional groups and mineral crystal faces. Subtler mechanisms such as modulation of the disordered phase composition and structural changes at the ordered-disordered interface may be involved.

SPARC/osteonectin in mineralized tissue

“…In a recent study, a peptide (ON29) derived from the mineral-binding region of SPARC induced changes to the disordered phase and apatite crystallite morphology in an in vitro assay of matrix mineralization [43]. Apatite embedded with ON29 revealed significant decreases in the amount of water molecules in the disordered phase of crystal mineralization and reductions in phosphate content, however calcium content remain unchanged [43]. Decreases in phosphate levels altered the disordered phase of apatite crystal growth by inducing needle-like crystal morphology in culture as opposed to plate-like morphology.…”

“…Decreases in phosphate levels altered the disordered phase of apatite crystal growth by inducing needle-like crystal morphology in culture as opposed to plate-like morphology. Decreased hydroxyl transfer to the disordered phase was observed with peptide treatment, but overall mineral transfer was unaffected [43]. Hence, some aspects of the altered phenotype of SPARC-null bones might arise from deficiencies in crystal mineralization, specifically in the disordered interface, that in turn might influence turnover rates of bone.…”

Identification of Specific Hydroxyapatite {001} Binding Heptapeptide by Phage Display and Its Nucleation Effect

“…Understanding interaction of biomolecules and amorphous calcium phosphate clusters existing in the mineralizing solution is critical to their applications in tissue engineering and biomimetic material synthesis. Compared to Glu (E) in ELBP, acidic residues, such as Asp (D) and Glu (E), are not found in EHBP, which is somewhat surprising because acidic amino acid-rich sequences were presumed to be binding sites of osteonectin (a natural HAp-binding protein) to HAp crystal [52,53,54]. In general, it is believed that negatively charged amino acids, containing carboxylate and phosphorylated residues, play a key role in controlling HAp mineralization.…”

Bone biology: insights from osteogenesis imperfecta and related rare fragility syndromes

“…In a recent study, a peptide (ON29) derived from the mineral-binding region of SPARC induced changes to the disordered phase and apatite crystallite morphology in an in vitro assay of matrix mineralization, supporting a role of the protein in bone formation through its capacity to bind mineral [145]. Interestingly, during in vitro differentiation of WT osteoblasts, SPARC mRNA level remains relatively constant [144], but the protein level is higher at the initial stages of differentiation and subsequently decreases as cells mature and begin to express late osteoblastic markers [144].…”