Synchrotron X-ray studies were conducted on native intramuscular shad and herring bones from the region consisting of newly secreted, unmineralized collagen fibrils, progressively toward the mineralization front, to the region where collagen fibrils are heavily mineralized. Two-dimensional (2D) X-ray diffraction/scattering patterns containing comprehensive 3D ultrastructural information over several levels of bone hierarchical structures were collected by adjusting the sample-to-detector distance.

Study on the native, unmineralized region revealed a new molecular packing scheme, which is significantly different from early postulates based on the quasi-hexagonal arrangement for tendon collagen, and is particularly suited to the biomineralization of bone. The deduced cross-sectional structure in bone collagen fibrils indicates the presence of spatially discrete microfibrils and an arrangement of intrafibrillar space to form “channels”, which could accommodate crystals with dimensions typically found in bone apatite.

Two analytical schemes were developed to extract the structure information contained in the 2D small-angle X-ray scattering (SAXS) patterns. The first scheme involved analyzing the equidistant meridional reflections resulting from the periodic structure of collagen fibrils in their axial direction. Conventional 2D analysis is difficult because of the large discrepancy between longitudinal and lateral length scales for individual fibrils, as well as their preferred orientation. The new approach represents an unapproximated analysis of the equidistant meridional reflections, which takes the exact separation of preferred orientation and fibril size effects into account. The analytical results (e.g. axial period, fibril diameter etc.) agree well with the parameters obtained from transmission electron microscopy.

The second scheme involved analyzing the fan-shaped near-equatorial diffuse scattering originating from the lateral arrangement of the mineral phase in the collagen matrix. TEM studies of intramuscular herring bone indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, also taking preferred orientation effects into account. Using this model, the diffuse intensity profile that dominates the small-angle equatorial region of the scattering pattern, could be quantitatively analyzed. The analysis leads to the thickness distribution of mineral crystals and that of the intercalated organic layers, and quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c-axis.

Analysis of the XRD patterns from regions at various stages of development revealed the 3D ultrastructural evolution of collagen fibrils during maturation and mineralization. In regions containing newly formed collagen fibrils, the molecular packing in fibrils and the fibril organization in bone were less ordered, as indicated by the reduced number of Bragg reflections and the poorer orientation of collagen fibrils. As it approached the mineralization front, each stage of the bone hierarchical structure became more organized. At the initial site of mineralization, the lateral packing order of collagen molecules in fibrils was significantly improved, suggesting a strong correlation between the collagen matrix structure and the mineralization process. The high degree of lateral order was destroyed by the onset of mineralization owing to the perturbation of the matrix structure by the deposition of apatite crystals.

The mineral packing in the collagen matrix was also evaluated for regions at different degrees of mineralization. The mineral platelets were found to be very thin and showed little growth in thickness during mineralization (from ∼1.8 nm at the initial site of mineralization to ∼2.1 nm at the heavily mineralized region). At the same time, the intercalated organic layers shrank from an average thickness of ∼7 nm to ∼5 nm. The average height of crystals along the crystallographic c-axis increased from 30 nm to 50 nm during mineralization but the orientation distribution of the mineral stacks remained unchanged. The information about the 3D ultrastructural evolution is critical for the correct understanding of the mechanical properties, biological and physiological functions of bone during aging and in pathological conditions.