Analysis of Intracellular Zn-Hydroxyapatite Nucleation in the Early Stages of Human Osteogenic Differentiation Combining Synchrotron-Based and Diffraction Techniques

The core knowledge about biomineralization is provided by studies on the advanced phases of the process mainly occurring in the extracellular matrix. This PhD project is based on the analysis of the early stages of biomineralization by evaluating the chemical fingerprint of the initial mineral nuclei deposition in the intracellular milieu and their evolution toward hexagonal hydroxyapatite. The study is conducted on human bone mesenchymal stem cells (bMSC) exposed to an osteogenic cocktail for 4 and 10 days, exploiting laboratory X-ray diffraction techniques and cutting-edge developments of synchrotron based 2D and 3D cryo X-ray microscopy. The synchrotron-based phase contrast imaging revealed few small intracellular depositions @4th day of differentiation and a lot of extracellular and intracellular mineral nuclei @10th day. The 3D reconstructions of phase-contrast images of bMSC at 10 days allowed browsing the entire cell along its thickness to explore the three-dimensional localization of the mineral depositions. Several spots are localized in the cytoplasm of the cell, indicating that the biomineralization starts in the intracellular environment. Synchrotron X-ray Fluorescence Microscopy was carried out to determine the chemical composition of mineral nuclei formation at nanoscale, confirming that mineral accumulation starts early during the osteogenic differentiation of bMSC and providing experimental evidence about the Zn role in HA nucleation. The X-ray fluorescence tomography was performed to obtain the 3D visualization of Ca, P and Zn elemental distribution. Wide/Small Angle X-ray Scattering and X-ray Absorption Near-Edge Spectroscopy revealed the hexagonal HA as the unique crystalline structure present in differentiating bMSC. In conclusion, thanks to the combination of 3D high-resolution synchrotron-based X-ray techniques, it has been demonstrated that biomineralization starts with hydroxyapatite nucleation in the intracellular environment, rapidly evolving toward a hexagonal hydroxyapatite crystal very similar to the one present in human bone as detected after just ten days of osteogenic induction.