Download water as a third structural component in the composite architecture

WATER AS A THIRD STRUCTURAL COMPONENT IN THE COMPOSITE ARCHITECTURE OF BONE
Proton Signal
+*Wilson, EW; **Awonusi, A; *Kohn, DH; *Morris, MD; **Tecklenburg, MMJ; *Beck, LW
+*University of Michigan, Ann Arbor, MI
[email protected]
INTRODUCTION
layer was also lost upon dehydration of bone in air (Figure 1). Crystal
The remarkable mechanical properties of bone have long been
water, however, was not lost upon dehydration of bone in air or heating
recognized as a consequence of the composite nature of this material.
of deproteinated bone to 225˚C.
The combination of hard mineral crystallites in a flexible collagen
matrix produces a material of extraordinary strength and toughness for
ordered water layer
its weight. However, we have yet to fully account for these properties
on a molecular level using two-component mechanical models. Water is
crystal defect water
an abundant component of bone, up to 25% by weight. Despite that,
channel water
there have been few attempts to define the role that water plays in the
A
architecture of bone. It remains a daunting challenge to distinguish
6
relatively small amounts of structurally bound water molecules amidst
300x10
the bulk water in bone. Solid-state nuclear magnetic resonance (NMR)
techniques are useful for studying heterogeneous materials due to their
200
ability to selectively observe one component of a composite material and
their ability to chemically distinguish between molecular environments.
100
We have used solid-state NMR to investigate specific structural roles for
water in the composite architecture of bone.
Synthetic B-type carbonated apatite and powdered deproteinated
bone were characterized by proton NMR from 25-225˚C. Abundant
bulk water and hydrated collagen interference in powdered bone made it
necessary to use special measures to selectively observe structural water.
Phosphorus-filtered proton NMR1 allowed us to selectively observe
hydrogen-bearing species present within one nanometer of mineral
phosphate. Unmodified bone samples were characterized by NMR at
different hydration states. Hydration states were produced by drying wet
powdered bone in air (10-40% humidity) and monitored by observing
changes in the proton NMR spectrum of the bone.
RESULTS
Proton NMR spectra of synthetic B-type carbonated apatite and
deproteinated bone and phosphorus-filtered proton NMR spectra of bone
revealed the presence of two types of structural water within the mineral
crystallites of bone, at 2.3 and 5.2 ppm respectively (Figure 1). An
analysis of the chemical shifts of these water environments suggests
roles for these water molecules in stabilizing crystal defect sites. The
2.3 ppm water signal corresponds to water in an isolated environment
consistent with the hydroxide channel of the mineral, while the 5.2 ppm
water exists in a much more acidic environment consistent with
surrounding ionic contacts. Differences in NMR linewidths in spectra of
bone vs. synthetic carbonated apatite also indicated that the biological
material is a less-ordered, more heterogeneous mineral environment.
An organized water layer was observed on the surface of bone
mineral crystallites using a variety of solid-state NMR techniques. We
have previously measured an average water proton-phosphate
phosphorus distance of 2.4 Å,2 which defines the location of this water
layer. Phosphorus-filtered proton NMR spectra showed a strong water
signal at 6.0 ppm (Figure 1B), downfield of bulk water and crystal water
previously observed. This chemical shift indicates an increased local
acidity, and shows that these water molecules participate in close
hydrogen-bonding interactions not found in the mineral-only model
materials. Thus this water is proposed to directly participate in the bone
mineral-organic matrix interface.
The lability of the various structural waters of bone was investigated
through variable temperature (deproteinated bone) and variable
hydration state (bone) solid-state NMR measurements. The organized
water layer on the surface of bone mineral crystallites was found to be
exchangeable with abundant bulk water present in the bone tissue.2 This
20
10
ppm
0
-10
20
10
ppm
0
-10
B
300x10
6
Proton Signal
METHODS
Solid-state NMR techniques were employed to identify and
characterize distinct locations of water in synthetic B-type carbonated
apatite, powdered deproteinated bone, and powdered bone produced by
cryogenic milling of bone. Procedures used to obtain bone samples were
in accordance with the relevant state and federal laws and the policies of
the University of Michigan committee on the use and care of animals
and OSEH standards.
250
200
150
100
50
Figure 1. Phosphorus-filtered proton NMR spectra of A) hydrated and
B) dehydrated bone. Ordered water layer is lost upon dehydration to
reveal crystal water.
DISCUSSION
In this work, we have presented evidence that water occupies three
distinct structural locations in bone. Crystal-bound structural water
occupies vacancies in the imperfect carbonated apatite crystal lattice,
providing structural stability by forming hydrogen-bonding bridges
between neighboring ions. The presence of this water may explain why
these crystallites do not collapse or spontaneously rearrange into more
perfect (and less biologically appropriate) mineral crystals. The water
layer observed at the surface of bone mineral crystallites mechanically
couples the mineral and the collagen in bone. This water may serve as a
cushion against mechanical stress. Two possible mechanisms for this
are that 1) water movement allows bone to withstand stress with less
deformation and 2) water acts as a sacrificial layer, protecting collagen
from shear under uniaxial stress. We have added water to the list of
fundamental building blocks that define the molecular level structure of
bone. With a complete model of how these building blocks fit together,
it will be possible to accurately model the material and understand the
molecular origins of its unique mechanical properties. These results
have implications for tissue engineering as well, suggesting the design of
scaffold materials that maximize hydrogen-bonding interactions between
hydroxyapatite crystals and a surrounding flexible matrix.
REFERENCES
1. Crosby, RC, et al. 1988. J. Am. Chem. Soc. 110, 8550.
2. Wilson, EE, et al. 2005. J. Bone Miner. Res. 20:, 625.
ACKNOWLEDGEMENTS
This work was supported by University of Michigan Core Centers for
Musculoskeletal Research through NIH grant P30 AR46024 and by NIH
R01 AR047969 and NIH T90 DK070071.
AFFILIATED INSTITUTIONS FOR CO-AUTHORS
**Central Michigan University, Mt. Pleasant, MI
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 1586