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Review
. 2021 Dec 9:6:100057.
doi: 10.1016/j.yjsbx.2021.100057. eCollection 2022.

Hierarchical organization of bone in three dimensions: A twist of twists

Daniel J Buss  1 , Roland Kröger  2 , Marc D McKee  1   3 , Natalie Reznikov  4
Affiliations
Review

Hierarchical organization of bone in three dimensions: A twist of twists

Daniel J Buss et al. J Struct Biol X. .

Abstract

Structural hierarchy of bone - observed across multiple scales and in three dimensions (3D) - is essential to its mechanical performance. While the mineralized extracellular matrix of bone consists predominantly of carbonate-substituted hydroxyapatite, type I collagen fibrils, water, and noncollagenous organic constituents (mainly proteins and small proteoglycans), it is largely the 3D arrangement of these inorganic and organic constituents at each length scale that endow bone with its exceptional mechanical properties. Focusing on recent volumetric imaging studies of bone at each of these scales - from the level of individual mineralized collagen fibrils to that of whole bones - this graphical review builds upon and re-emphasizes the original work of James Bell Pettigrew and D'Arcy Thompson who first described the ubiquity of spiral structure in Nature. Here we illustrate and discuss the omnipresence of twisted, curved, sinusoidal, coiled, spiraling, and braided motifs in bone in at least nine of its twelve hierarchical levels - a visualization undertaking that has not been possible until recently with advances in 3D imaging technologies (previous 2D imaging does not provide this information). From this perspective, we hypothesize that the twisting motif occurring across each hierarchical level of bone is directly linked to enhancement of function, rather than being simply an energetically favorable way to assemble mineralized matrix components. We propose that attentive consideration of twists in bone and the skeleton at different scales will likely develop, and will enhance our understanding of structure-function relationships in bone.

Keywords: Biomineralization; Bone; Electron Microscopy; Helicoidal Structures; Hierarchical Organization; Review; Skeleton; Volume Microscopy and Tomography.

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Conflict of interest statement

The authors declare that they have no competing financial interests or personal relationships that could appear to have influenced the work reported in this paper.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Hierarchical levels of the full skeleton and of whole bones. (A,B) A mouse skeleton imaged using microcomputed tomography (µCT). The same structural plan can be observed in the skeletons of mammals: here, the murine spine shows the same principle curves as the human spine – cervical and lumbar lordosis (convexity oriented forwards) and thoracic kyphosis (concavity oriented forwards). (C, D, E) Individual bones (imaged by µCT) scaled to the same figure height: sheep femur (C) with its shallow c-shaped curvature, partridge humerus (D) with an s-shaped curvature, and squirrel humerus (E) showing a screw-shape geometry.
Fig. 2
Fig. 2
Cortical-to-trabecular bone transition (hierarchical Level 3) in the metaphyses of the sheep femur (A, B) and the squirrel humerus (C, D). Note that both proximal metaphyses show geometric “vorticity” of trabecular buttresses (A, C), but this is not that obvious in the distal metaphyses (B, D).
Fig. 3
Fig. 3
Vascular canals in a sheep femur (hierarchical Level 4), and winding of lamellar layers around the canals (osteons, hierarchical Level 5). (A) Resorption cavities and incomplete osteons within a juvenile sheep. (B) Less-abundant resorption canals and incomplete osteons in a mature sheep femur. Note the similarity between B and the “hatching” of osteonal canals in the human femur (from Hert et al. (Heřt et al., 1994) , with permission). (D) Spiral winding of co-oriented mineralized collagen fibrils around the central capillary of an osteon, from Wagermaier et al. (Wagermaier et al., 2006), with permission. The lamellar assembly in the whole osteon can be viewed as a series of concentric nested coils. Note the switch in handedness in the outermost layer. (E) Preceding work by Gebhardt (Gebhardt, 1906) in 1906, who empirically illustrated the stiffening effect of multi-layered coiling assemblies with varying pitch and handedness: either in tension, compression or torsion, there will always be a subset of lamellae that resist such deformation axially.
Fig. 4
Fig. 4
Hierarchical Level 6 (ordered and disordered repeats in the lamellar structure), and Level 7 (packing of co-aligned collagen fibrils into quasi-cylindrical bundles). These images were acquired using the FIB-SEM slice-and-view method on demineralized and stained lamellar bone. (A) Grey-scale 3D volume in which collagen fibrils appear dark grey, interfibrillar ground matter appears light grey, the lamina limitans lining canaliculi is white, and osteocyte processes are also white. (B) The same canaliculi as in A, surface-rendered and shaded to illustrate their meandering course across the ordered bundles, and their screw-shaped axial twist. (C) Segmentation of the ordered arrays of collagen, 3D rendered and superimposed on a semi-transparent grey-scale volume (same sample and same orientation as in A). Note the braided and gently twisted appearance of splitting and merging bundles of collagen fibrils. (D) Disordered collagen fibrils alternate with the ordered bundles and house the cellular processes. Here, the disordered phase has been segmented using deep learning-aided segmentation, and its content is about 30% with respect to the total volume of the extracellular matrix (original nonquantitative work by Reznikov et al. (Reznikov et al., 2013) underestimated the proportion of the disordered array by volume).
Fig. 5
Fig. 5
Hierarchical Level 8a – prolate ellipsoidal mineral tessellations that populate bundles of ordered collagen fibrils in lamellar bone. (A) 2D view from Fig. 4A and 4C. (B) An orientation- and magnification-matched 2D view of undemineralized bone from Buss et al. (Buss et al., 2020). (C) A further magnified 2D image of a typical mineral aggregate – a “tesselle”. (D) A 3D rendering of a typical single tesselle, as outlined by dashed red ellipses in panels B and C (the tesselle in panel C is one 2D cross-sectional view of its subsequent 3D volume shown in panel D). The rectangular box in panel C roughly corresponds to the complete volume of interest presented in the following Fig. 6. (E) Hierarchical Level 8b showing a supertwisted model of a mineralized collagen fibril with a characteristic stagger of triple helices and a 5° molecular tilt with respect to the fibril axis, reminiscent of the seed arrangement in the sunflower, as suggested by Charvolin and Sadoc (Charvolin and Sadoc, 2011, Charvolin and Sadoc, 2012). Gap regions between collagen triple helices are depicted in transparent red with the hydroxyapatite crystals represented in yellow. (F, G) The same 3D supertwisted collagen model rotated around the vertical axis (“yaw”) to accentuate the resultant spiraling alignment of the gaps and the mineral crystallites that would be confined to the gap zones. Panels H and I show the hierarchical structure of collagen only at the levels of triple helices, α-chains and amino acids (images adapted from the RCSB Protein Data Bank (Bella et al., 1994, Kramer et al., 1998, Sehnal, 2021); https://doi.org/10.2210/pdb1CAG/pdb). The corresponding organizational levels for bone mineral are shown in Fig. 6, Fig. 7.
Fig. 6
Fig. 6
Hierarchical Levels 9, 10 and 11 of bone mineral organization: stacks, plates and needles. (A) STEM tomography of a FIB-milled foil of lamellar bone. The contrast originates from the crystallites’ electron density, and all the crystallites are segmented (using a deep neural network), volume-rendered, and color-coded following a watershed transformation. (B) Twenty digitally separable crystallite aggregates are shown in situ (in unmodified orientations) within the same STEM tomographic volume as in A. The remaining tomogram volume is rendered transparent. The yellow inset in the bottom right corner of panel B schematically illustrates the orientation of collagen fibrils within the tomogram rendered in panels A and B. (C) A gallery of 20 digitally separable crystallite aggregates ranging in size between 106 nm3 and 105 nm3 (in a descending order). Individual aggregates are intentionally shown in re-orientations different from those orientations observed in situ.
Fig. 7
Fig. 7
Hierarchical Level 12, here pertaining only to the mineral component of bone, is represented by the unit cells and atoms themselves comprising bone mineral crystallites (A). Panel B shows a Fourier transform of the area outlined in (A) by the white square.
Fig. 8
Fig. 8
Contemporary understanding of the hierarchical structure of bone. According to the recent inventory, there are approximately twelve levels: 1) skeletons, made of 2) bones, made of 3) cortical and trabecular tissue, made of 4) cortical osteons, fibrolamellar bone packets, and trabecular lamellar packets, all of which contain 5) lamellar bone, made of 6) lamellae, made of 7) ordered collagen motifs that form 8) bundles, surrounded by the disordered collagen motif. The bundles are made of 9) collagen fibrils, made of 10) triple helices, made of 11) alpha-helices, made of 12) amino acids. The mineral organization in 3D shows its own hierarchical organization starting at level 8) of mineralized collagen bundles, that contain 9) tessellated prolate ellipsoids of mineral, made of 10) mineral platelets, made of 11) laterally merging acicular crystals, made of 12) unit cells. Because the cascade of hierarchical levels splits at the micrometer level for organic and inorganic matter, and because same-level mineral and collagen units have different shapes and even scale (for example, the tesselles, and the collagen fibrils, which are both level 9), and also for visual flow and continuity between levels, we intentionally did not number the levels in the figure.
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