Fractal-like hierarchical organization of bone begins at the nanoscale

Abstract

The components of bone assemble hierarchically to provide stiffness and toughness. However, the organization and relationship between bone's principal components-mineral and collagen-has not been clearly elucidated. Using three-dimensional electron tomography imaging and high-resolution two-dimensional electron microscopy, we demonstrate that bone mineral is hierarchically assembled beginning at the nanoscale: Needle-shaped mineral units merge laterally to form platelets, and these are further organized into stacks of roughly parallel platelets. These stacks coalesce into aggregates that exceed the lateral dimensions of the collagen fibrils and span adjacent fibrils as continuous, cross-fibrillar mineralization. On the basis of these observations, we present a structural model of hierarchy and continuity for the mineral phase, which contributes to the structural integrity of bone.

Figure 1

Three projections of bone structure as observed by TEM and corresponding electron diffraction patterns. (A) The filamentous pattern shows curved, more than 100 nm-long crystals. (A’) diffraction pattern from (A) shows well-defined (002) plane-related reflections, which are oriented in the direction of the elongated crystals. (B) The lacey pattern comprises groups of parallel, slightly bent crystals surrounding electron-transparent voids. The absence of the (002) plane-related reflections in (B’) indicates that the crystals’ c-axes are oriented out of plane. (C) Nested rosettes, a previously unknown pattern, shows crystals of about 5 nm in size arranged into left-handed helices. (C’) Corresponding diffraction pattern with (002) plane-related reflections being absent, similar to that in (B’). Higher magnification of the same motifs, D-F. (D) The filamentous motif. (E) The lacey motif. (F) The rosette motif. Note a nearly hexagonal outline of the dark crystallite in (F); the inset shows a high-resolution transmission electron microscope (HRTEM) view of the area indicated by the small white square, and the white circles in A, B and C indicate the areas from which the selected-area electron diffraction patterns A’, B’ and C’ were recorded.

Figure 2

Reconstructed and rendered STEM tomogram in different projections of an FIB-milled specimen of mature human lamellar bone. Panel (A) shows mostly the filamentous pattern with a fragment of the lacey pattern in the bottom left corner – these patterns originate from two adjacent lamellae. Panel (B) shows the same volume slightly tilted around the horizontal axis. Panel (C) shows the same volume (A) tilted approximately 50-60° around the horizontal axis. Panel (D) shows the same volume (A) tilted approximately -30° around the horizontal axis. Note the angular offset of approximately 60° between the crystallites of the neighboring motifs apparent in (C). Colored arrows indicate the axes of the reconstructed 3D-volume orientation in space.

Figure 3

Reconstructed and rendered STEM tomogram in different projections of an FIB-milled specimen of mature human lamellar bone. Tomogram of a sample with the lacey motif at the 0°-tilt angle. Panels A, B and C show the same volume viewed vertically (A), tilted approximately 30° around the horizontal axis (B), and tilted approximately 60° around the horizontal axis (C). Note that in the right top corner of (C) the faint D-periodicity of collagen can be detected. Panel (D) shows a fragment of the same sample in such an orientation that acicular projections of the crystallites appear, resembling the rosette pattern in Fig. 1C. Colored arrows indicate the axes of the reconstructed 3D-volume orientation in space.

Figure 4

Labeling algorithm and comparison of the lacey and filamentous patterns in bone in reciprocally oriented projections. (A) Tomogram slice with superimposed labels of the lacey (blue) and filamentous (pink) patterns. (B) Labeled volumes show extensively aggregated and coalesced elongated entities of variable size and irregular shape. (C) Volumes cropped to an identical size in all 3 dimensions are transformed in such a way that former XY-planes now are XZ-planes, i.e. the in-plane labels are viewed in the out-of-plane orientation, and vice versa. (D) Digital manipulation of the filamentous label field (from left to right): original labels in a cubic stack are averaged in terms of pixel value to form a pseudo-2D image, which is similar to the corresponding area in (A). The resliced label field stack is averaged in terms of pixel value to form a pseudo-2D image in an orthogonal direction. (E) Digital manipulation of the lacey label field follows the same sequence of steps. Note the similarity of the resliced projected filamentous label field to the original lacey motif, and vice versa.

Figure 5

Evaluation of the tomogram shown in Fig. 2. Individual labels selected within the lacey pattern (A-E) and the filamentous pattern (F-H). Only the mineral aggregates that showed the least degree of confluence with each other were selected. Panels (A) and (B) show the same ten labels in situ, in two different projections; panels (C – E) show four out of ten labels in individually adjusted projections to highlight their 3D shape. (F) Ten individual labels from the filamentous pattern, of which two are shown in adjusted projections, (G, H). There are 3 levels of confluence of mineral formations with each other: i) lateral merging of needle-shaped entities into platelets, ii) planar merging of platelet-shaped entities into stacks of 2-4 with a uniform gap between them, iii) merging of adjacent stacks at an angle (like fan blades, especially obvious in C, E, H) with a wedge-shaped clearance between them. Almost all labels in three dimensions show a delicate twist, especially visible in (B). The overall label density to the total-volume ratio was the same in both samples (approximately 0.45-0.5) – not to be confused with bone mineral density.

Figure 6

Proposed model of crystal organization in bone, and comparison with 2D projections obtained from TEM and STEM (second column of panels) and the tomogram reconstructed from the STEM tilt series (amber colored, third column of panels). The first column of panels shows the orientation of a thin specimen with respect to the ordered array of mineralized collagen fibrils in lamellar bone. The last column of panels features the same simplified 3D model of bone apatite crystals viewed in three different projections, in-plane, out-of-plane and edge-on views. For the sake of clarity, note that the model drawing has fewer concentric tiers of curved filaments than it presumably would accommodate.

Figure 7

Discernible segment length of collagen fibrils in the extracellular matrix of bone. (A) Reconstructed volume of demineralized and stained collagen fibrils in bone, and (B) individual fibrils color-labeled where they can be continuously traced in the edge-on view. Each of 100 labels is shown in a different color visualizing the distribution of segment lengths.

Figure 8

A scheme of hierarchical organization in bone. For levels VII-XII (green) see Reznikov et al. (4, 29, 62). Both ordered and disordered motifs of lamellar bone comprise mineralized collagen fibrils (VI) that are 80-120 nm thick and form a continuous network. Collagen fibrils are composed of quasi-hexagonally packed microfibrils (V), each of which incorporates multiple staggered triple helices (IV) that in turn are formed from repetitive chains (III) of amino acids (II). Collagen levels V to II are discussed in detail by Orgel et al. (5, 35, 39, 63, 64). Collagen panels III and IV are courtesy of Dr. Joseph Orgel. The inorganic component of the mineralized collagen fibrils (VI) itself incorporates several nested structural motifs, listed as follows in decreasing order of complexity: mineral aggregates (V), stacks of platelets (IV), platelets (III) and acicular crystals (II).