Download Correlation of Age, Degeneration, and Biomechanical Properties of

Correlation of Age, Degeneration, and Biomechanical Properties of Human Lumbar Discs
with mRNAs Encoding Numerous Extracellular Matrix Components
1,2
Fisher, G D; 1Hashemi, J; 2Gill J B; 2Graham S; 2Hutson J C; +2Hardy, D M
Texas Tech University, Lubbock, TX; +2Texas Tech University Health Sciences Center, Lubbock, TX
[email protected]
1
INTRODUCTION:
Intervertebral discs are essential elements of the spine that function to
transmit load and absorb shock while enabling multi-axial motion.
Mechanics of the disc and their proximity to the spinal cord and nerves
make pathologies of the disc potentially debilitating. Degeneration and
herniation of the disc can result in severe pain and lead to a considerable
decrease in quality of life. While such defects can occur at any level of
the spine, those occurring within the lumbar region are among the most
common and have been cited as a widespread cause of low-back pain.
Although the involvement of both mechanical and molecular factors in
disc function and pathology has been reported, no study has correlated
gene expression with mechanical properties of the lumbar disc. In this
study, we tested the hypothesis that mechanical properties of lumbar
discs correlate with levels of mRNAs encoding extracellular matrix and
cell adhesion/signaling components.
METHODS:
Nine full, frozen, cadaveric spines (4 male, 5 female; ages 28-76
years, mean 49.7 years) were dissected in a 4 °C cold room without
allowing tissues to thaw. A senior anatomic pathologist graded all L2-L3
discs according to the system described in Boos et al (Spine 2002; 27:
2631-44), and a single numerical value for degeneration was calculated
for each disc by summing the scores of the individual histological
categories. For mechanical testing L3-L4 discs were removed from the
spines as motion segments (vertebra-disc-vertebra), leaving intact all
anterior and posterior elements up to and including the vertebral canal.
For gene expression studies, tissue from the outer anterior annulus
fibrosus of the L4-L5 discs was removed and stored at -80 °C.
Prior to mechanical testing, motion segments were thawed at room
temperature, placed in an acrylic testing chamber containing a
circulating solution of 150 mM NaCl, and equilibrated 15 min. Porous
stainless steel platens (50% porosity, 50 µm pore size) were placed
between the motion segments and testing apparatus to allow the flow of
saline through the cartilage endplates. Each motion segment was tested
using a materials testing system in unconfined uniaxial compression at a
rate of 0.1 mm/s. Following ten preconditioning cycles at a 1-mm
amplitude, motion segments were compressed to and held at 1 mm for
500 seconds followed by load-to-failure testing, to generate
measurements for calculation of viscoelastic properties in the form of
hysteresis and stress relaxation as well as compressive moduli (from
linear and toe regions of the stress-strain curve), failure strain, failure
strength, and strain energy density.
Total RNA isolated from annular disc tissue was used for PCR-based
expression profiling for 84 genes encoding extracellular matrix and cell
adhesion gene products using real time reverse transcription polymerase
chain reaction (RT-PCR; SuperArray Bioscience).
Because Ct (threshold cycle) values from RT-PCR output give a
relative measure of initial steady-state mRNA amount present in a tissue,
the Ct values for each gene product were used as a relative quantification
of gene expression in linear regression analyses. Prior to calculating
correlations, each Ct value for a given gene product was first normalized
to the average Ct value of its tissue’s five housekeeping genes. Age,
height, disc degeneration, and mechanical properties were then tested for
correlations with the normalized Ct values for all 84 target genes.
Because Ct is inversely related to the steady-state amount of mRNA in
the tissue, for ease in interpretation of results the signs of the
correlations reported in Table 1 are with respect to mRNA amount, not
Ct .
RESULTS:
Relative amounts of mRNAs encoding several extracellular matrix
and cell membrane components correlated significantly (|r|≥0.666) with
donor age, donor height, degeneration, and disc mechanical properties
(Table 1). Age correlated negatively with 16 mRNAs including – among
others – those encoding subunits of collagen, integrins, and laminins, as
well as two different matrix metallopeptidases. The only mRNA that
correlated positively with age was fibronectin. However, donor height
correlated with the mRNA concentration of MMP9. Degeneration
correlated with linear modulus at a correlation coefficient of -0.687.
Finally, mRNAs encoding three MMPs, three integrin subunits, and
catenin correlated with the six biomechanical properties measured.
Table 1. Correlation coefficients for mRNA comparisons.
Property
Age
Height
Degeneration
Linear Modulus
Toe Modulus
Failure Strength
Failure Strain
Stress Relaxation
Hysteresis Area
Gene
COL14A1
CTNND1
ITGA3
ITGB3
LAMA3
MMP12
NCAM1
SELP
VCAM1
MMP9
COL14A1
SELL
TNC
VCAN
ITGB2
ITGA8
ITGAV
CTGF
SELE
r-Value.
-0.672
-0.769
-0.754
-0.747
-0.775
-0.744
-0.759
-0.808
-0.720
+0.873
-0.690
-0.679
-0.832
+0.701
+0.757
+0.666
-0.669
-0.688
+0.702
Gene
COL4A2
FN1
ITGA8
LAMA2
LAMC1
MMP16
SELE
SPG7
r-Value.
-0.672
+0.738
-0.736
-0.678
-0.740
-0.775
-0.703
-0.727
ITGB5
CLEC3B
-0.682
-0.681
MMP12
MMP9
+0.711
+0.718
MMP3
CTNNB1
CTNNB1
-0.694
-0.725
-0.751
COL=Collagen, CTNN=Catenin, FN=Fibronectin, ITG=Integrin, LAM=Laminin,
MMP=Matrix metallopeptidase, NCAM=Neural cell adhesion molecule,
SEL=Selectin, SPG=Spastic paraplegia, VCAM=Vascular cell adhesion molecule,
CLEC=C-type lectin, TNC=Tenascin C, VCAN=Versican, CTGF=Connective
tissue growth factor
DISCUSSION:
Here we showed that age, degeneration, and mechanical properties of
lumbar discs correlate with steady-state mRNA levels for many different
gene products that function in signaling as well as maintenance and
turnover of the extracellular matrix in connective tissues. Our most
striking finding was the negative correlation of age with numerous
mRNAs encoding extracellular matrix and cell membrane components.
The results suggest that the expression of all of the genes listed in Table
1 – with the exception of fibronectin – is diminished with aging.
Because these genes may be classified into different functional
categories, this could imply a general inactivity of annulus fibrosus cells
and decreased matrix turnover with aging that could contribute to disc
degeneration.
Two different genes correlated with both age and a mechanical
property: MMP12 and ITGA8. Interestingly, both genes correlated negatively with age and positively with their respective mechanical property
(linear modulus for MMP12; failure strength for ITGA8). Because linear
modulus is a measure of stiffness, its positive correlation with MMP12
indicates that stiffer discs express MMP12 at higher levels. For a given
strain, a disc with a larger modulus experiences more load, so this
finding could result from a biological response of the cell to modify its
surrounding matrix more rapidly in response to the increased load.
Several of the genes in Tables 1 have not previously been
investigated for any potential role they may play in disc pathology. The
results of this study indicate that further research involving these genes
could prove worthwhile in uncovering meaningful conclusions related to
disc mechanics, biology, and pathology.
One limitation of this study is that mRNAs were quantified only in
the outer anterior region of each annulus fibrosus. Investigating other
radial and circumferential regions of the annulus would likely lead to
different results. Second, care should be taken when interpreting
significant correlations involving gene expression because the
significant correlation of a gene’s mRNA levels does not necessarily
imply a high level of expression of that gene in the tissue.
This work was supported in part by AR049767 and a joint
TTU/TTUHSC collaboration grant.
Poster No. 696 • ORS 2011 Annual Meeting