Anat Akiva^1,2, Johanna Melke^2,3, Nalan Liv⁴, Job Fermie⁴, Merula Stout¹, Harm van Ruremonde¹, Cilia de Heus⁴, Paul H.H. Bomans, Anne Spoelstra^1,2, Judith Klumperman⁴, Keita Ito^2,3, Sandra Hofmann^2,3,5, Nico A.J.M. Sommerdijk^1,2,

¹Laboratory of Materials and Interface Chemistry and Center for Multiscale Electron Microscopy, Department of Chemical Engineering and Chemistry. Eindhoven University of Technology, Eindhoven, The Netherlands.

²Institute for Complex Molecular Systems. Eindhoven University of Technology, Eindhoven, The Netherlands.

³Department of Biomedical Engineering. Eindhoven University of Technology, Eindhoven, The Netherlands.

⁴Section of Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, The Netherlands

⁵Institute for Biomechanics, ETH, Zürich , Switzerland.

Bone tissue is an organic-inorganic composite material that provides the mechanical support and protection for our bodies. Its impressive mechanical properties arise from the hierarchical organization of the organic collagen matrix that is mineralized with ultrathin, aligned inorganic crystals of carbonated hydroxyapatite (cHAP).

Despite its importance to the human body, still relatively little is understood about the mechanisms by which collagen mineralization occurs, and what the respective roles are of the collagen and the osteoblast cells in directing this process.

The ultimate system to study collagen assembly and mineralization is the one that can emulate this process as it occurs in vivo and still can provide a high level of accessibility to monitor and control the system in vitro. To this end, we use human mesenchymal stem cells (hMSC) that are differentiated into osteoblasts and deposit collagen in a 3D silk scaffold. Using a large set of microscopic tools (micro CT, SEM, 3D FIB/SEM, TEM and fluorescence), combined with spectroscopic (FTIR) and molecular tools (qPCR) we show that our 3D model system develops the main features that construct bone. We show that the hMSC differentiated into osteocytes that are embedded within the collagen matrix. The mineral is characterized as carbonated hydroxyapatite by FTIR, and the TEM images show that the mineral is embedded within the collagen matrix. These results set the ground for the use of our 3D living in vitro system to study the process of collagen mineralization using electron and fluorescent microscopy in high resolution and cryogenic conditions.