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, 8 (1), 13696

First Evidence of Octacalcium Phosphate@osteocalcin Nanocomplex as Skeletal Bone Component Directing Collagen Triple-Helix Nanofibril Mineralization

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First Evidence of Octacalcium Phosphate@osteocalcin Nanocomplex as Skeletal Bone Component Directing Collagen Triple-Helix Nanofibril Mineralization

Paul Simon et al. Sci Rep.

Abstract

Tibia trabeculae and vertebrae of rats as well as human femur were investigated by high-resolution TEM at the atomic scale in order to reveal snapshots of the morphogenetic processes of local bone ultrastructure formation. By taking into account reflections of hydroxyapatite for Fourier filtering the appearance of individual alpha-chains within the triple-helix clearly shows that bone bears the feature of an intergrowth composite structure extending from the atomic to the nanoscale, thus representing a molecular composite of collagen and apatite. Careful Fourier analysis reveals that the non-collagenous protein osteocalcin is present directly combined with octacalcium phosphate. Besides single spherical specimen of about 2 nm in diameter, osteocalcin is spread between and over collagen fibrils and is often observed as pearl necklace strings. In high-resolution TEM, the three binding sites of the γ-carboxylated glutamic acid groups of the mineralized osteocalcin were successfully imaged, which provide the chemical binding to octacalcium phosphate. Osteocalcin is attached to the collagen structure and interacts with the Ca-sites on the (100) dominated hydroxyapatite platelets with Ca-Ca distances of about 9.5 Å. Thus, osteocalcin takes on the functions of Ca-ion transport and suppression of hydroxyapatite expansion.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) High–resolution TEM from region B in Fig. S10b reveals a polycrystalline sample consisting of nanoplatelets of (mainly) HAP. (b) The presence of HAP is indicated by the respective (100) and (002) reflections of the FFT. (c) Fourier filtered image of (a). For filtering the (002) reflection of HAP was used. Nanoparticular HAP domains are marked by white frames. The striation within the platelets corresponds to 3.44 Å (002 reflection), thus the c–axis of HAP (see white arrows). White framed area is taken for further analysis in Fig. 2. (d) The mask used for Fourier filtering encompasses exclusively the (002) reflection of HAP.
Figure 2
Figure 2
(a) Zoomed image of Fig. 1c (white frame). Individual mineralized fibrils are observed by Fourier filtering using the HAP (002) reflections only. Three individual alpha-chains entangling each other and forming a triple–helix are seen close to the white arrow. (b) The triple–helices form bundles, which are mineralized by apatite. The model beneath the experimental observed triple–helix demonstrates the idealized arrangement of the individual chains. The fine striation of 3.44 Å along the long axis of the triple–helices corresponds to the (002) reflection and thus the c–axis of HAP as indicated at the left bottom. (c) Bundle of mineralized triple–helices with the typical width of 1.5 nm of an individual fibril. (d) At thinner areas and at higher magnification (digitally zoomed) the winding of the alpha–chains becomes even more evident. Within the triple–helix (borders marked with white lines) the steep angle of the alpha–chain with respect to the long–axis and the spiral nature is revealed. (e) Comparison of predicted superstructure along the triple–helix of 2.86 nm (left) and supercoiling as read out from the TEM high–resolution (right). (f) Indication of winding period of alpha–chains. The periodicity along the triple–helix amounts to 0.86 nm and is evoked by the alpha–chain helix indicated on the model at the right.
Figure 3
Figure 3
(a) The occurrence of mineralized triple–helices is not confined to rat bone only but is also found in human hard tissue: Assembly of mineralized collagen microfibrils (left arrow) and triple–helices (right arrow) of human bone. (b) Zoomed area of (a) displaying mineralized triple–helices situated at the right together with the model of a triple–helix. The winding of the alpha–chains within the fibril is clearly imaged.
Figure 4
Figure 4
(a) In overview images, pearl necklace-like strings were observed in the sample. The strings consist of beads, which are about 2 nm in size. (b) High–resolution of the strings: Small angle filtered TEM image at higher resolution showing only the large spacings caused by the organic components. Spherical structures with about 2 nm in diameter covering the fibril and forming a pearl necklace string (white arrows). (c) Fourier filtered image using the (002) and (100) reflections of HAP, and the (100) reflection of OCP. OCP with [0–12] orientation is present along the osteocalcin pearl necklace strings (see (a)) whereas HAP extends straight along the collagen fibril. (d) Fourier transform in the small angle region clearly reveals the presence of OCP besides HAP (see arrows).
Figure 5
Figure 5
(a) Filtered image of the white framed area in Fig. 1a. For filtering all crystal reflections of HAP and OCP together with the small angle region corresponding to organic components (collagen fibrils and osteocalcin structures) were considered. The image appears highly structured by collagen fibrils superimposed with pearl necklace-like arrangements (red arrows) along the collagen fibrils indicating the presence of osteocalcin. (b) Magnified view taken from the top left region marked by a white frame in (a). One of the osteocalcin chains is marked by arrows. (c) High-resolution image where only crystal reflections were taken into account for filtering. The osteocalcin beads are clearly visualized between the HAP crystals appearing as vertical bright-dark stripes. (d) Magnified area of the bottom left part of (b) (white frame) showing the osteocalcin chain and single osteocalcin individuals close by (white circles). The preferred binding sites of osteocalcin to the Ca sites of the OCP/HAP structures amount to about 9.5 Å (Ca…Ca distances along b-axis within the OCP (100) plane) as well as along b-axis within the HAP (100) plane. In addition, binding distances of 5.45 Å are observed. (e) FFT of (c) with osteocalcin chain revealing the presence of OCP and HAP. (f) Mask used for Fourier filtering including all crystal reflections and small angle region. In (c) only the crystal reflections were taken into account without the small angle area.
Figure 6
Figure 6
Rat vertebra prepared by conventional ultramicrotomy. (a) Overview image. (b) Magnified image displays necklace pearl strings and individual osteocalcin molecules (see arrows) in the sample. (c) At high–resolution, the OC protein is visualized as concentric sphere (see dotted areas). One OC molecule is situated directly on the HAP/OCP crystal on the left whereas the other is near the crystal. (d) The FFT clearly indicates the presence of octacalcium phosphate in connection with OC. (e) Single crystal platelet of OCP in (100) orientation from rat trabecular bone. (f) The small angle filtered image indicates an additional globular structure (nearly close packing) consisting of the non–collagenous protein osteocalcin. (g) The Fourier filtered HR–TEM image shows the OCP lattice ((100) plane) together with assumably osteocalcin or/and another NCP (one molecule indicated by a white circle together with the Ca binding sites of about 9.5 Å distance). The region of interest is marked by a white frame in (f). (h) Fourier transform of (g) showing the presence of OCP.
Figure 7
Figure 7
In vitro mineralization of OC. (a) Occasionally, pure OCP platelet formation (several 100 nm in size) was observed under the influence of OC. (b) High-resolution of the crystal shows the presence of OCP, [100] zone. (c–e) Zoom series of OCP/HAP plates. (e) At higher magnification, beads of OC are imaged covering the surface and the edges of the plates. Pearl necklace formation is dominating but also isolated individuals are revealed, see arrows. (f) Filtered high-resolution with corresponding FFT, [100] zone. At bottom left, the crystal lattice shows a discontinuation (arrow) due to the presence of OC. (g) Magnified view of red marked area in (f). In high-resolution the disruption zone amounts to 2 nm corresponding to the diameter of a pearl necklace structure. Individual binding sites (arrows) of the carboxylic groups are identified bearing strong resemblance to the binding sites identified for bone. (h) Non-filtered high-resolution of another disruption zone of crystal lattice. At right top, a 2 nm sized OC is imaged containing three binding sites marked with three arrows and a dotted white circle.
Figure 8
Figure 8
TEM of conventional ultrathin diamond cuts of human femur. (a) Overview image with pearl bead strings of 2 nm diameter indicating the presence of osteocalcin, see arrows. At the center bottom a 50 × 50 nm2 large HAP crystal plate is imaged with OC particles on its surface resembling to white pearl bead structures suppressing the HAP growth normal to the plate plane. (b) The Fourier filtered high–resolution image displays a massive HAP crystal at the top of the micrograph. At certain regions, the crystal lattice is disturbed due to the presence of OC (see orange circles at right). (c) At higher magnification a gap within the crystal plate is revealed corresponding to the size of OC proteins. Even the binding sites are resolved which appear as small circles with pronounced dots in the center (distances of 11 and 8 Å). (d) The FFT of the high–resolution image show reflections at small angle corresponding to 18.69 Å, thus proving the presence of OCP. (e) OC is also found between HAP crystals with different orientations serving as delimiter. The insertion of OC evokes mechanical stress and leads to remarkable lattice distortions of the upper crystal (green dotted line). The binding sites within OC show 10.7 Å distance from each other. (f) The massive crystal plate on the right is confined by a string of OC particles marked in orange. At least six binding sites (see arrows) are arranged in a row.
Figure 9
Figure 9
Possible scenario and schematic sketch of calcification steps in and at a collagen fibril derived from snapshots. (a) Part of a collagen fibril consisting of protein triple–helices in parallel arrangement. (b) Pre–orientation of Ca3–triangles (Δ) and phosphate groups (x) as precursor–scenario containing motifs of the hydroxyapatite (HAP) crystal structure, pH > 6.5. (c) Formation of HAP nanocrystals (red) with platy habit (100) and the c–axis direction in parallel orientation to the fibril extension. (d) Growth of HAP nanoplatelets. (e) Attachment of osteocalcin (OC) on the composite surface (collagen fibril and HAP nanoplatelets). Lowering the pH (<6.5). (f) Epitaxial growth ((100) HAP/(100) OCP) of octacalcium phosphate (OCP, blue) on HAP (red).
Figure 10
Figure 10
(a) Structural relations between octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4 ·5H2O, P-1, a = 19.69 Å, b = 9.52 Å, c = 6.84 Å, α = 90.2 °, β = 92.5 °, γ = 108.9 °) and hydroxyapatite (HAP, Ca10(PO4)6(OH)2, P63/m, a = b = 9.42 Å, c = 6.88 Å, α = β = 90.0 °, γ = 120.0 °). Projections along c* (OCP) and c (HAP). Atoms restricted to Ca (OCP: blue, HAP: red), H2O (OCP: green), OH (HAP: red cross). Apatite–like layers in OCP (“Pseudo-HAP”) indicated by “unit cells” (broken lines in yellow). The epitaxial relation (100, OCP)/(100, HAP) is close to ideal, see schematic sketch in (b): Distribution of Ca- sites (red/blue) on (100, OCP)/(100, HAP) as present (for instance) on (100, OCP ≙ “pseudo–HAP”) close to the H2O–containing layers of OCP (see yellow arrows in (a)). Green circles: Possible areas (diameter of about 2 nm) for attachment of folded osteocalcin molecules. See TEM images in Figs. 4–7. Ca atoms (HAP, OCP) in plane. (c) Schematic sketch of the epitaxial growth of OCP (blue) on HAP (red) by lowering the pH (<6.5) under the influence of osteocalcin (OC). Formation of H2O molecules by reaction of OH (from “surface channels” on (100) in the HAP crystal structure) with H+ and local transformation of HAP (red) to “pseudo-HAP” (orange). Restriction to OH positions in HAP (x, red) and water molecules in “pseudo-HAP” (o, green) as well as H2O–molecules (, green) within the water–rich layers in OCP. Projections along c (HAP) and c* (OCP), respectively. (d) The HAP-OCP interface model proposed by Fernandez et al. where [−12–10] HAP coincide with [010] OCP and where the c-HAP-axis is parallel to the c-OCP axis. The angle between a-HAP and a-OCP amounts to 131°.

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