Research ArticleAPPLIED PHYSICS

Nanostructure, osteopontin, and mechanical properties of calcitic avian eggshell

See allHide authors and affiliations

Science Advances  30 Mar 2018:
Vol. 4, no. 3, eaar3219
DOI: 10.1126/sciadv.aar3219
  • Fig. 1 The nanostructure of chicken eggshell (G. gallus).

    (A) SEM microstructure at low magnification and regional nomenclature of avian eggshell. (B to F) AFM (top; with a scanning area of 800 nm × 800 nm) and SEM (bottom) images of eggshell nanostructure as observed from the regions indicated in (A). (G) Histogram of nanostructure size distribution (Feret diameter) in the VCL (B), upper PL (C), middle PL (D), lower PL (E), and ML (F) layers. Significant difference is indicated by brackets (*P < 0.05, **P < 0.01, and ***P < 0.001). No significant difference (NS) (P > 0.05) between bars E and F. Values were compared by a two-tailed Student’s t test.

  • Fig. 2 Optical microscopy, EBSD, and 2D XRD of chicken eggshell.

    (A) Thin eggshell cross section viewed by conventional bright-field light microscopy showing nonhomogeneous distribution of organic and inorganic material throughout the eggshell layers. (B) Thin eggshell cross section [same as in (A)] viewed under cross-polarized light showing multiple, closely packed and well-defined columnar calcite units. (C) Crystal orientation map obtained by EBSD of a polished eggshell cross section showing slight internal crystalline misalignments (up to 4°) within the columns as depicted by different pseudocolor shades [see selected boxed areas in (D) and (E)]. (F) 2D XRD of a cross section of an eggshell showing an elongated single-crystal diffraction spot (arrows) and the associated intensity profile of a 104 calcite reflection as a function of the γ angle. Note the widening of the 104 peak due to varying crystallographic orientation within a columnar calcite unit. a.u., arbitrary units. (G) 2D XRD of a control powdered Iceland spar calcite crystal showing single-crystal diffraction spots (arrows) and the associated intensity profile of a 104 calcite reflection as a function of the γ angle.

  • Fig. 3 Electron microscopy of a nanodomain substructure in the PL.

    (A) Bright-field unstained TEM image of chicken eggshell after FIB sectioning of the PL showing mineral nanodomains with a diameter of 5 to 7 nm. (B) HRTEM lattice imaging of the nanodomains, with SAED (inset) showing both a single-crystal diffraction spot pattern and polycrystalline-derived diffraction rings. Evidence for further mineral suborientations within the nanodomains is indicated by the dashed lines. Contrast differences in (A) and (B) presumably result from the relative distributions of a mixture of organic and inorganic (amorphous and crystalline) components. (C) Single bright-field TEM image from a tilt series of the nanodomains in the PL. (D and E) 3D tomographic reconstructions of the same nanodomain region indicated by a box in (C) [solid rendering in (D) and surface rendering in (E)]. The surface rendering in (E) used a threshold that shows only the high-density regions indicated by the dashed lines in (D).

  • Fig. 4 OPN in chicken eggshell.

    (A) Immunoblotting for chicken eggshell OPN. Lane 1, total soluble protein extract from decalcified eggshell (1 M HCl); lane 2, acidic eggshell matrix proteins, not retained on CM Sephadex (see Methods). (B) Immunohistochemistry (pink) for OPN protein showing its incorporation and distribution throughout the full thickness of the eggshell (decalcified here). Brackets indicate areas in outermost PL with concentrated OPN amounts. (C) Immunogold labeling for OPN (arrows) and TEM showing the association of OPN with a lacy network of organic matrix dispersed throughout the PL of the eggshell. (B) and (C) are from demineralized shell samples, resulting in the artifactual partial collapse of an organic matrix structure.

  • Fig. 5 Nanostructure induced by OPN and protein occlusion within calcite.

    (A to C) AFM images of the interior of microtome-cut calcite crystals showing no nanostructure in the absence of OPN (A) but visible nanostructure after growth in the presence of 0.9 μM OPN (B) or 5.9 μM OPN (C) (scanning area, 800 nm × 800 nm). Insets in (A) to (C) show typical SEM images of calcite crystals from which AFM images were obtained after microtoming to expose the interior structure. (D) Immunoblotting after gel electrophoresis of dissolved crystals showing retrieved OPN and degraded OPN fragments (lane 1, OPN protein alone; lane 2, dissolved crystals growing in the presence of 5.9 μM). (E) Micro-Raman spectra from grown crystals, demonstrating a C–H protein peak. (F) Computationally simulated (RosettaSurface) conformer docking of the polyaspartate domain (99DDDDDDDND107) of chicken OPN on the obtuse and acute step of calcite (binding energy, approximately −14 kcal/mol for both cases). Calcite atoms: Ca, green; C, gray/white; O, red.

  • Fig. 6 Mechanical testing by nanoindentation of eggshell and synthetic calcite crystals and effects of physiologic eggshell dissolution.

    (A) Hardness distribution across the eggshell layers. (B) Elastic modulus distribution across the eggshell layers. (C) Hall-Petch plot of average hardness versus nanostructure size distribution in the eggshell layers. (D) Hardness values from synthetic calcite crystals grown in the absence (control) and presence of OPN (5.9 μM). Insets show typical images of residual indents on the specimen surface. Significant difference is indicated by a bracket (**P < 0.01). (E to G) AFM images of nanostructured VCL (E), middle PL (F), and ML (G) eggshell layers from a fertilized egg incubated for 15 days (scanning area, 1.2 μm × 1.2 μm). Insets show nanostructure size distribution of the different eggshell layers, comparing eggs that were not incubated to incubated eggs. No significant difference (P > 0.05) is observed between VCLs, whereas a significant difference (***P < 0.001) in size exists between the two groups for the PL and the ML. Values were compared by a two-paired Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/3/eaar3219/DC1

    fig. S1. Distribution of measured eggshell nanostructures by AFM.

    fig. S2. Eggshell nanostructure area measurements.

    fig. S3. Internal nanocrystal misalignments in the PL of chicken eggshell.

    fig. S4. TEM showing eggshell nanostructure.

    fig. S5. Semiquantification of OPN immunostaining across the eggshell thickness.

    fig. S6. Effect of OPN on nanostructure size in synthetic calcite crystals.

    fig. S7. OPN induces nanostructure in synthetic calcite crystals.

    fig. S8. Absence of nanostructure in synthetic control calcite crystal (no added OPN).

    fig. S9. Electron microscopy of a FIB section showing nanostructure in a synthetic calcite crystal grown with OPN (5.9 μM).

    fig. S10. Nanoindentation displacement curves for eggshell and synthetic calcite crystals grown in OPN (5.9 μM).

    movie S1. 3D reconstruction from a tilt series of the upper PL of avian chicken eggshell G. gallus.

    movie S2. 3D reconstruction of nanodomains found in the upper PL of the eggshell.

    movie S3. 3D reconstruction from a tilt series of the synthetic calcite crystal grown with 5.9 μM OPN.

    movie S4. 3D reconstruction of a nanostructured region found in the synthetic calcite crystal grown with 5.9 μM OPN.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Distribution of measured eggshell nanostructures by AFM.
    • fig. S2. Eggshell nanostructure area measurements.
    • fig. S3. Internal nanocrystal misalignments in the PL of chicken eggshell.
    • fig. S4. TEM showing eggshell nanostructure.
    • fig. S5. Semiquantification of OPN immunostaining across the eggshell thickness.
    • fig. S6. Effect of OPN on nanostructure size in synthetic calcite crystals.
    • fig. S7. OPN induces nanostructure in synthetic calcite crystals.
    • fig. S8. Absence of nanostructure in synthetic control calcite crystal (no added OPN).
    • fig. S9. Electron microscopy of a FIB section showing nanostructure in a synthetic calcite crystal grown with OPN (5.9 μM).
    • fig. S10. Nanoindentation displacement curves for eggshell and synthetic calcite crystals grown in OPN (5.9 μM).

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). 3D reconstruction from a tilt series of the upper PL of avian chicken eggshell G. gallus.
    • movie S2 (.mov format). 3D reconstruction of nanodomains found in the upper PL of the eggshell.
    • movie S3 (.mov format). 3D reconstruction from a tilt series of the synthetic calcite crystal grown with 5.9 μM OPN.
    • movie S4 (.mov format). 3D reconstruction of a nanostructured region found in the synthetic calcite crystal grown with 5.9 μM OPN.

    Files in this Data Supplement:

Navigate This Article