Material Properties of the Extrafibrillar Matrix of Annulus Fibrosus in Tension and Compression
+Cortes, DH; Gerasimowicz, KM; Smith, LJ; Elliott, DM
Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA
+ [email protected]
INTRODUCTION
The annulus fibrosus (AF) of the disc is a highly nonlinear and
anisotropic biphasic material that undergoes a complex combination of
loads in multiple orientations. AF tensile mechanical behavior in the
lamellar plane is dominated by collagen fibers and has been accurately
modeled using exponential and crimp functions [1]. On the other hand,
AF mechanics perpendicular to the lamella, in the radial direction,
depend on the properties of the extrafibrillar matrix (EFM) with little
collagen fiber contribution. Other tissue parameters, such as
permeability and diffusion coefficients, also depend on the EFM
properties [2]. The elastic response of the EFM can be divided in the
contribution of solid (non-ionic) and osmotic (ionic) effects. The AF
EFM mechanical properties of the solid phase have not been measured.
These properties are essential for constitutive and finite element models
of the AF and disc. Additionally, degeneration may also alter both the
ionic and the non-ionic mechanical contributions of the EFM. The
objective of this study was to measure AF nonlinear mechanics of the
EFM in tension and compression using a combination of osmotic
swelling and confined compression. We hypothesized that the EFM, in
the absence of reinforcing collagen fibers, will exhibit a linear response
across all applied compression and tension loading.
METHODS
To test the mechanical properties of the EFM a confined compression
test was applied perpendicular to the lamellar plane containing collagen
fibers. Consequently, the collagen fibers will not be stretched and the
measured material properties will correspond to the EFM only. For an
unloaded sample, the osmotic pressure is balanced by a residual tension
of EFM. Therefore, the mechanical properties of measured in an isotonic
(0.15M) and hypotonic (0.06M)
solutions of NaCl can be considered
as tension properties (Fig. 1). For a
hypertonic (2M) solution of NaCl,
the osmotic pressure and the residual
tension in the EFM are small.
Figure 1. Definition of
Consequently,
the
properties
tension and compression
obtained
from
a
confined
relative to the reference
compression test in this solution
configuration
(hypertonic
correspond to the compression
bath solution)
properties (Fig. 1).
Cylinders 4mm in diameter and 2mm thick were prepared from
bovine tail outer AF. The sample was allowed to swell in sterile
solutions of 0.06, 0.15 or 2M NaCl and protease inhibitors for 4 hours.
After this swelling, the thickness of the sample was measured in the
confining chamber by applying a constant stress of 1 kPa (first preload)
until equilibrium was reached. After this preload, three ramps of 5%
strain were applied at a rate of 0.005%/s followed by a stress relaxation
periods of 160, 200, 250 min, respectively. To measure the reference
thickness of the sample, 1 kPa was applied in a 2M solution of NaCl (2nd
preload). The difference between the initial and reference thicknesses
was used to determine the swelling deformation. To estimate the osmotic
pressure, the fixed charge density (FCD) was calculated from the GAG
content [3]. The EFM solid phase stress was calculated by subtracting
the osmotic pressure from the applied stress and the aggregate modulus
was calculated as the slope of the equilibrium stress-stretch curve.
RESULTS
A negligible difference of the
fixed charge density (Fig. 2)
was measured from tissue
adjacent to sample (before
testing) and the samples (after
test). Higher compressive stress
and aggregate moduli were
obtained for the hypotonic
solution (0.06M) (Fig. 3). The
Figure 2. Comparison of the fixed
stress of the solid phase of the
charge density before (adjacent
EFM (after subtracting the
tissue) and after (sample) the test
osmotic pressure) was linear
(Median and Interquartile range, n
(Fig. 4A) and the linear
= 15).
aggregate modulus was 7 kPa.
b)
a)
Figure 3. Mechanical behavior of AF in confined compression for
several bath concentrations of NaCl: a) Stress, b) Aggregate modulus.
(Median and Interquartile range, n = 5)
a)
b)
Figure 4. Mechanical behavior of the solid phase of the EFM in tension
and compression: a) Stress-Stretch curve, b) Aggregate modulus as a
function of stretch. (Median and Interquartile range, n = 5)
DISCUSSION
The objective of this study was to measure the mechanical properties of
the solid phase of the EFM in tension and compression and determine
whether this material exhibits nonlinearity. As hypothesized, the EFM is
linear in tension and compression with an aggregate modulus of 7 kPa.
The advantage of this testing protocol is that the EFM can be isolated
from the fiber contribution. Additionally, the small sample size will
allow quantifying mechanical properties in several locations (e.g., inner
and outer AF). Previous attempts to measure EFM properties using
uniaxial tensile tests [4] have reported higher moduli. However, those
studies do not consider the solid and osmotic contributions separately
and they do not isolate the potential contribution of collagen fibers and
of in-plane fiber reorientation, which may have contributed to
overestimate the EFM properties. In addition, the sample dimensions
spanned from the inner to the outer annulus, therefore, the properties are
an average of these regions.
Several studies have analyzed the contribution of solid and osmotic
effects in disc tissues [5,6,7]. The contribution of the non-ionic
components of bovine nucleus pulposus is approximately 30% [5],
which is in accordance with the values reported in this study. The
contribution of the non-ionic components of AF has been analyzed using
a combination of enzymatic digestions and confined compression [6].
However, tensile properties cannot be obtained using this approach.
The dependence of the osmotic effects on the ion concentration of
the bath has been exploited as a loading mechanism for cartilage [8,9].
The difference between hypertonic and isotonic solutions in combination
with optical measurement of strain has been used to determine a
variation of strains and properties through the thickness of the sample
[8]. A recent study in cartilage [9] showed that the enzymatic removal of
GAGs produce a higher decrease in compressive modulus compared to
using hypertonic solutions. This suggests that GAGs may also have a
non-ionic mechanical contribution in compression. Future studies will
quantify human inner and outer AF EFM properties and the effect of
degeneration.
ACKNOWLEDGEMENTS: NIH NIAMS and NIBIB.
REFERENCES
[1] Guerin and Elliott, J. Ortho. Res., 2007 [2] Jackson et al., Spine,
2008 [3] Chahine et al., J. Biomech, 2004. [4] O’Connell et al., J. Bio.
Eng. 2009 [5] Heneghan and Riches, J. Biomech. 2008. [6] Perie et al.,
J. Bio. Eng. 2006. [7] Drost et al., J. Bio. Eng. 1995 [8] Narmoneva et
al., J. Biomech., 1999 [9] Canal Guterl et al., J. Biomech. 2010.
Poster No. 807 • ORS 2011 Annual Meeting