Early stage mineralization in tissue engineering mapped by high resolution X-ray microdiffraction.

The specific routes of biomineralization in nature are here explored using a tissue engineering approach in which bone is formed in porous ceramic constructs seeded with bone marrow stromal cells and implanted in vivo. Unlike previous studies this model system reproduces mammalian bone formation, here investigated at high temporal resolution. Different mineralization stages were monitored at different distances from the scaffold interface so that their spatial analysis corresponded to temporal monitoring of the bone growth and mineralization processes. The micrometer spatial resolution achieved by our diffraction technique ensured highly accurate reconstruction of the different temporal mineralization steps and provided some hints to the challenging issue of the mineral deposit first formed at the organic-mineral interface. Our results indicated that in the first stage of biomineralization organic tissue provides bioavailable calcium and phosphate ions, ensuring a constant reservoir of amorphous calcium phosphate (ACP) during hydroxyapatite (HA) nanocrystal formation. In this regard we suggest a new role of ACP in HA formation, with a continuous organic-mineral transition assisted by a dynamic pool of ACP. After HA nanocrystals formed, the scaffold and collagen act as templates for nanocrystal arrangement on the microscopic and nanometric scales, respectively.