The nanometer-scale physiology of bone: Steric modeling and scanning
transmission electron microscopy of collagen-mineral structure
- SUPPLEMENTAL DOCUMENT Benjamin Alexander* 1
Tyrone L. Daulton* 2, 3
Guy M. Genin 1, 3
Justin Lipner 4, 5
Jill D. Pasteris 3, 6
Brigitte Wopenka 3, 6
Stavros Thomopoulos 3, 4, 5
* Ben Alexander and Tyrone Daulton contributed equally to this work
1
Department of Mechanical, Aerospace, and Structural Engineering, Washington
University, St. Louis, MO 63130
2
Department of Physics, Washington University, St. Louis, MO 63130
3
Center for Materials Innovation, Washington University, St. Louis, MO 63130
4
Department of Orthopaedic Surgery, Washington University, St. Louis, MO 63110
5
Department of Biomedical Engineering, Washington University, St. Louis, MO 63130
6
Dept. of Earth and Planetary Sciences, Washington University, St. Louis, MO 63130
Corresponding Author:
Stavros Thomopoulos, Ph.D.
Washington University
Department of Orthopaedic Surgery
660 South Euclid, Campus Box 8233
St. Louis, MO 63110
Phone: 314-362-8605
Fax: 314-362-0334
Email: ThomopoulosS@wudosis.wustl.edu
GLOSSARY
Hierarchical collagen structures in bone
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Collagen molecule: A triple-helix tropocollagen molecule approximately 300 nm in
length and 1.5 nm in diameter.
Microfibril: If microfibrils are not separable from fibrils as individual structural
entities, their definition is somewhat arbitrary since they can be defined by any
repeating sub-structure in a fibril. Microfibrils can be defined as an assemblage of
five strands of end-to-end stacked collagen molecules. The collagen within a
microfibril is stacked with a ~36 nm gap space between the N- terminus of one
collagen molecule and the C- terminus of the succeeding molecule. The five strands
are staggered (along their stacking direction) by ≈ 67 nm (approximately ¼ the length
of a collagen molecule, hence termed quarter-staggered). Alternatively, microfibrils
can be defined as five individual collagen molecules in a quarter-staggered
arrangement.
Fibril: A 50-500 nm diameter assemblage of ordered microfibrils.
Fiber: A 3-7 μm diameter close-packed bundle of fibrils.
Periodicity- and space-related terms
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Gaps regions: Periodically recurring regions within a microfibril in which axially
abutting collagen molecules leave open spaces between the termini of the collagen
molecules.
Gap channels: Open channels in fibrils formed when the gap spaces of their
constituent microfibrils are aligned.
Overlap region: Periodically recurring regions along the length of a fibril that have no
gap channels.
Intermolecular space: The volume between adjacent collagen molecules within fibrils,
excluding gap channels.
Bioapatite-related terms
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Bioapatite: The carbonated, compositionally complex, and substituted form of the
mineral hydroxylapatite (Ca5(PO4)3(OH)) that is present in bone.
Intrafibrillar bioapatite: Bioapatite within gap channels and within intermolecular
space in a microfibril; note we observed bioapatite predominantly in the former.
Extrafibrillar bioapatite: Bioapatite exterior to fibrils.
Mineralized: Regions in bone with abundant mineral internal (i.e., in their gap
channels) and external to the fibrils.
Mineral-deficient: Regions in bone with low or no concentrations of mineral internal
and external to the fibrils.
ANALYSIS OF EELS SPECTRA IMAGES
Plural scattering affects the absorption edge shape directly as well as the Bremsstrahlung
background. Close proximity of core-loss edges further complicate appropriate
background fitting. Plural scattering can be taken into account to a certain extent by
Fourier deconvolution with the low loss spectra. While the latest version of the Gatan
GIF (Quantum model) can collect low loss and core loss spectra maps simultaneously
(for Fourier deconvolution), this is not possible with GIF Tridiem, used in the current
study. Acquisition of simultaneous maps would require both large energy dispersions
(reducing energy resolution) and relatively low primary beam intensity in order to
prevent detector saturation by the zero-loss peak (severely limiting the signal from the
weaker core-loss edges). Collecting low loss and core loss spectral maps in serial is
problematic for two reasons. First, there may be differences in potential hydrocarbon
deposition by the beam, which would change the level of plural scattering in the two
maps. Second, spatial alignment of the two spectra image maps is required because of
specimen drift and potential tilt in orientation of thin film upon prolonged electron beam
exposure during map acquisitions. The acquisition of the core loss maps requires up to
12 hours and low loss maps require up to 2 hours.
We are presently experimenting with methodologies to serially collect and align core loss
and low loss spectra maps. Initial results have been consistent with (and reproduce the
same abundance trends) as displayed in our maps generated from spectra which have not
been Fourier de-convolved. For the measurements we report in the current study, thin
areas of the specimen were sought out for analysis to reduce possible plural scattering
effects. This is evident by the fact that we only analyzed mineralized regions in which
fibrils were visible and not obscured by extrafibrilar mineral. Analyses of low loss
spectra maps indicate that the typical thickness of regions examined were in the range ≈
0.05 - 0.30 λ (where λ is the mean free path for inelastic scattering).
SUPPLEMENTAL FIGURE CAPTION
Figure S.1: Representative electron energy core-loss spectra of mineralized bone over
the energy loss range 100 - 640 eV. The integration windows for each core-loss edge and
the measured core-loss signal used to quantify the relative elemental abundances are
shown by the vertical dashed lines and shaded regions of the spectra, respectively. The
background for the edges (arising from scattering events not associated with the
adsorption edge) was approximated by fitting a power law to the pre-edge regions
(schematically represented by the red curves). These background fits were then
subtracted from their respective spectral regions.
SUPPLEMENTAL VIDEO CAPTION
Steric Model Video:
Collagen molecules were approximated by 1.5 nm diameter, 300 nm length
cylinders. The basic unit used to construct the steric structural model consisted of five
individual collagen molecules in a quarter-staggered arrangement (this unit can be
viewed as an alternative definition of a microfibril). Fibrils were formed by three
dimensional stacking and packing of these units. Fibrils were constructed by first
stacking the five-collagen model units such that the C-terminus of the collagen molecules
from one unit abutted to N-terminus ends of the collagen molecules in the next unit with
a 36 nm gap between the abutting molecules. Secondly, the stacked units were packed
normal to the axis of the fibril in an idealized (and simplified) monoclinic lattice. The
packing was such that the gap spaces in each stack of units were aligned to those of the
other stacks to form gap channels.
Bioapatite crystals (2 x 30 x 40 nm) were inserted through the gap channels,
producing a banded pattern of intrafibrillar bioapatite and overlapping collagen molecules
along the length of the fibrils. The steric model predicts that for any gap channel, there
are five different orientations (in the plane normal to the axis of the fibril) in which
bioapatite crystals can be inserted.