Synchrotron SAXS/WAXS and TEM studies of zinc doped natural hydroxyapatite nanoparticles and their evaluation on osteogenic differentiation of human mesenchymal stem cells

In this study, hydroxyapatite (HA) nanoparticles were prepared from natural bone and doped with zinc (Zn-HA) by solid state reaction method. Structural characteristics of both samples were analyzed using transmission electron microscopy, synchrotron small- and wide-angle X-ray scattering techniques, which showed flake-like shape for HA and Zn-HA nanoparticles with an average diameter of 82 nm, and hexagonal crystal structure whose crystallinity increased with Zn doping. The osteogenic differentiation impact of nanoparticles was investigated on mesenchymal stem cells using alkaline phosphatase (ALP) activity, alizarin red staining (ARS) and real time-quantitative polymerase chain reaction (RT-PCR) analyses. The alkaline phosphatase activity of the stem cells in the presence of HA and Zn-HA nanoparticles in culture media increased by 2.6 and 4.3 times after two weeks, respectively. The results of ARS analysis demonstrated a significant increase in calcification of the culture media due to Zn doping. The results of gene expression from RT-PCR analysis revealed that the expression level of ALP, RUNX2, OPN, and OCN increased due to the presence of the nanoparticles, especially for the Zn-HA nanoparticles. The cellular analysis clearly revealed that the stem cell osteogenic differentiation of HA nanoparticles substantially increased by Zn doping. In particular, the Zn-HA nanoparticles significantly improved the osteogenic differentiation, making it a viable option for bone tissue engineering.