Fractal-like hierarchical organization of bone begins at the nanoscale

Abstract

INTRODUCTION: The components of bone assemble hierarchically to provide stiffness and toughness. Deciphering the specific organization and relationship between bone’s principal components—mineral and collagen—requires answers to three main questions: whether the association of the mineral phase with collagen follows an intrafibrillar or extrafibrillar pattern, whether the morphology of the mineral building blocks is needle- or platelet-shaped, and how the mineral phase maintains continuity across an extensive network of cross-linked collagen fibrils. To address these questions, a nanoscale level of three-dimensional (3D) structural characterization is essential and has now been performed. RATIONALE: Because bone has multiple levels of 3D structural hierarchy, 2D imaging methods that do not detail the structural context of a sample are prone to interpretation bias. Site-specific focused ion beam preparation of lamellar bone with known orientation of the analyzed sample regions allowed us to obtain imaging data by 2D high-resolution transmission electron microscopy (HRTEM) and to identify individual crystal orientations. We studied higher-level bone mineral organization within the extracellular matrix by means of scanning TEM (STEM) tomography imaging and 3D reconstruction, as well as electron diffraction to determine crystal morphology and orientation patterns. Tomographic data allowed 3D visualization of the mineral phase as individual crystallites and/or aggregates that were correlated with atomic-resolution TEM images and corresponding diffraction patterns. Integration of STEM tomography with HRTEM and crystallographic data resulted in a model of 3D mineral morphology and its association with the organic matrix. RESULTS: To visualize and characterize the crystallites within the extracellular matrix, we recorded imaging data of the bone mineral in two orthogonal projections with respect to the arrays of mineralized collagen fibrils. Three motifs of mineral organization were observed: “filamentous” (longitudinal or in-plane) and “lacy” (out-of-plane) motifs, which have been reported previously, and a third “rosette” motif comprising hexagonal crystals. Tomographic reconstructions showed that these three motifs were projections of the same 3D assembly. Our data revealed that needle-shaped, curved nanocrystals merge laterally to form platelets, which further organize into stacks of roughly parallel platelets separated by gaps of approximately 2 nanometers. These stacks of platelets, single platelets, and single acicular crystals coalesce into larger polycrystalline aggregates exceeding the lateral dimensions of the collagen fibrils, and the aggregates span adjacent fibrils as continuous, cross-fibrillar mineralization. CONCLUSION: Our findings can be described by a model of mineral and collagen assembly in which the mineral organization is hierarchical at the nanoscale. First, the data reveal that mineral particles are neither exclusively needle- nor platelet-shaped, but indeed are a combination of both, because curved acicular elements merge laterally to form slightly twisted plates. This can only be detected when the organic extracellular matrix is preserved in the sample. Second, the mineral particles are neither exclusively intrafibrillar nor extrafibrillar, but rather form a continuous cross-fibrillar phase where curved and merging crystals splay beyond the typical dimensions of a single collagen fibril. Third, in the organization of the mineral phase of bone, a helical pattern can be identified. This 3D observation, integrated with previous studies of bone hierarchy and structure, illustrates that bone (as a material, as a tissue, and as an organ) follows a fractal-like organization that is self-affine. The assembly of bone components into nested, helix-like patterns helps to explain the paradoxical combination of enhanced stiffness and toughness of bone and results in an expansion of the previously known hierarchical structure of bone to at least 12 levels.