Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: A cryo-electron microscopy study Julia Mahamid a,⇑ , Amnon Sharir b,c , Dvir Gur a , Elazar Zelzer b , Lia Addadi a , Steve Weiner a a Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel b Department of Molecular Genetics, Weizmann Institute of Science, 76100 Rehovot, Israel c Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, 76100 Rehovot, Israel article info Article history: Received 4 January 2011 Received in revised form 18 March 2011 Accepted 18 March 2011 Available online 2 April 2011 Keywords: Biomineralization Mouse model Osteoblast Calvaria Carbonated hydroxyapatite Transient precursor abstract Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by spe- cialized cells – the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of devel- oping mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular min- eral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precur- sor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the ves- icles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The vertebrate skeleton is a unique organic–inorganic tissue that forms during embryonic development and is dynamically shaped and maintained throughout the animal’s life (Currey, 2002). Bones are complex structures, with a hierarchical organiza- tion encompassing a range from nanometers to centimeters (Wei- ner and Traub, 1992; Weiner and Wagner, 1998). At the fundamental structural level, all bones are comprised of type-I col- lagen fibrils reinforced by co-aligned crystalline platelets of car- bonated hydroxyapatite, a calcium phosphate mineral (Landis et al., 1993; Traub et al., 1989). Various cell types are responsible for the formation as well as the maintenance of the bone integrity and function (Peck and Woods, 1988). Bones of the vertebrate skeleton may develop via two distinct processes: intramembranous bone formation that proceeds through direct mineralization of a newly formed type-I collagen matrix and endochondral bone, involving the formation of a miner- alized cartilaginous precursor that is later replaced by a mineral- ized bone matrix (Caplan, 1987; Streeter, 1949). In both modes osteoblasts orchestrate the bone formation process, initiated by the synthesis of an organic extracellular matrix – the osteoid (Ca- plan, 1987), mainly composed of type-I collagen, but also contain- ing up to 10% non-collagenous proteins (Lowenstam and Weiner, 1989). Mineralization of the collagen fibrils thereafter takes place. Blood serum is the main source of ions in the vertebrate body, and contains significant concentrations of calcium and phosphate, sufficient for the deposition of carbonated hydroxyapatite (Jahnen- Dechent, 2000; Posner et al., 1978). However, the pathways through which the ions are translocated from the serum to the site of deposition within the extracellular matrix, and the precise involvement of cellular processes in mineral deposition are not clear (Gay et al., 2000). Two principal modes have been suggested to describe mineral deposition into collagen matrices: (i) crystals are actively nucleated from solution by charged non-collagenous proteins associated to the collagen gap zones, without intervention of intracellular processes (Glimcher, 1984; Veis and Perry, 1967); 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.03.014 ⇑ Corresponding author. Present address: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martins- ried, Germany. Fax: +49 89 8578 2641. E-mail address: mahamid@biochem.mpg.de (J. Mahamid). Journal of Structural Biology 174 (2011) 527–535 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi