Chemical Fingerprint of Zn–Hydroxyapatite in the Early Stages of Osteogenic Differentiation

[Image: see text] The core knowledge about biomineralization is provided by studies on the advanced phases of the process mainly occurring in the extracellular matrix. Here, we investigate 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 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. We demonstrate that biomineralization starts with Zn–hydroxyapatite nucleation within the cell, rapidly evolving toward hexagonal hydroxyapatite crystals, very similar in composition and structure to the one present in human bone. These results provide experimental evidence of the germinal role of Zn in hydroxyapatite nucleation and foster further studies on the intracellular molecular mechanisms governing the initial phases of bone tissue formation.