Crystallization of Bone-Like Mineral in the Presence of the Regulatory Bone Protein Osteocalcin

Alex Kulpanovich, Chemistry , Bar Ilan University , Givatayim, Israel
Taly Iline-Vul,, Chemistry , Bar Ilan University , Petah Tikva , Israel
Merav Nadav-Tsubery, Chemistry , Bar Ilan University , El'ad, Israel
Gil Goobes, Chemistry , Bar Ilan University , Rehovot, Israel

Apatite crystals form the sturdy inorganic part of human teeth and bones; hence the importance of understanding their crystallization processes inside the biological tissue. In the human body, the crystallization process is influenced and regulated by acidic proteins called biomineralization proteins. Osteocalcin (OC), a gamma-carboxyglutamic acid rich protein, is commonly found embedded in the apatite layers of bone. The high affinity of OC to bone mineral enables it to influence bone mineralization, though its regulatory function is not known yet. To examine the impact of OC protein on the mineral composition, crystal growth and the interactions of OC with bone apatite, we precipitated hydroxyapatite (HAP) in the presence of human OC. The resultant, biomaterial contained agglomerates of OC with HAP nanocrystals. This biomaterial was characterized using XRD, TEM, SEM, TGA, ICP and BET measurements. In addition, solid-state NMR experiments were employed to analyze in detail HAP surface and bulk structure, and to investigate OC-HAP interactions.

HAP crystals prepared alone are small platelet-shaped and markedly disordered whereas crystals co-precipitated with OC have a well-ordered hexagonal structure with slightly elongated morphology. Dynamic 1H to 31P magnetization transfer experiments, from which proton-phosphate proximity can be crudely inferred, show that OC modifies the content of the hydrated layers on the surface of HAP and in the bulk of the mineral. Proximity measurements from OC sidechains to faces of the formed HAP crystals, reveal that acidic residues in the protein take part in the interaction with the surface of the mineral.