Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite C. Lange a , C. Li a , I. Manjubala a,b , W. Wagermaier a , J. Kühnisch c,d , M. Kolanczyk c,d , S. Mundlos c,d , P. Knaus e , P. Fratzl a,⇑ a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, 14476 Potsdam, Germany b Biomedical Engineering Division, School of Bio-Sciences and Technology, VIT University, Vellore 623 014, Tamilnadu, India c Institute for Medical Genetics, Charité, Augustenburger Platz 1, 13353 Berlin, Germany d Max Planck Institute of Molecular Genetics, Ihnestraße 63-73, 14195 Berlin, Germany e Institute for Chemistry and Biochemistry, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany article info Article history: Received 31 March 2011 Received in revised form 2 August 2011 Accepted 3 August 2011 Available online 10 August 2011 Keywords: Bone mineralization Small-angle X-ray scattering Wide-angle X-ray diffraction Murine model abstract It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particu- larly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were charac- terized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmen- tal scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the lon- gitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that min- eral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The development of the vertebrate skeleton is achieved by intramembranous bone formation and endochondral bone forma- tion (Karsenty, 2003; Zoetis et al., 2003; Forriol and Shapiro, 2005; Shapiro, 2008). Bone development starts with a differentia- tion of cells to chondrocytes or osteoblasts which form cartilage and bone (Olsen et al., 2000). Cartilage is replaced by bone by the process of endochondral ossification, while cell differentiation into osteoblasts produces intramembranous bone directly. The full hierarchical structure of bone is very complex and variable, but its basic building block, the mineralized collagen fibril, is rather uni- versal (Fratzl et al., 2004). Thus, on the nanometer scale, bone can be considered as a collagen/mineral composite consisting of mineral particles embedded in the collagen matrix. The collagen I molecules building up the collagen fibrils in bone are staggered from each other (Miller, 1984; Hodge, 1989; Weiner and Wagner, 1998) with an axial spacing of 64–67 nm, depending on the state of hydration and mineralization (Bonar et al., 1985; Price et al., 1997). Derived from the axial spacing the ends of the collagen mol- ecules are separated by holes of about 35 nm in wet unmineralized collagen fibrils and about 20 nm in wet mineralized collagen. These gap-zones are supposed to be the nucleation sites for the mineral- ization (Landis, 1995). Mineral crystals in bone are known to be elongated platelets with their orientation predominantly parallel to the longitudinal direction of the bone (Fratzl et al., 2004). The amount of mineral, the properties of the organic matrix and the geometrical arrangement of the two components determine the mechanical properties of the material. Since decades a fundamen- tal question in understanding the mineralization process in verte- brates is the nature of the first-formed mineral phase (Betts et al., 1981; Boskey, 1997; Weiner, 2006; Grynpas, 2007). The formation of a transient precursor mineral phase has been reported for invertebrates (Lowenstam and Weiner, 1985; Weiss et al., 2002) but is still controversially discussed for vertebrates. 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.08.003 ⇑ Corresponding author. Fax: +49 331 5679402. E-mail addresses: Claudia.Lange@mpikg.mpg.de (C. Lange), Peter.Fratzl@ mpikg.mpg.de (P. Fratzl). Journal of Structural Biology 176 (2011) 159–167 Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi