Poster No. 1395 • 54th Annual Meeting of the Orthopaedic
Research Society
Microscopic MRI Analysis of Rat Intervertebral Disc – A Basis
for Disc Tissue Engineering Applications
Yoram Zilberman1, Galit Saar2, Hadassah Shinar2, Keren
Keinan-Adamsky2, Gadi Pelled1, Gil Navon2, Dan Gazit1,31Skeletal
Biotech Laboratory, Hebrew University–Hadassah Medical Campus,
Jerusalem, Israel; 2School of Chemistry, Tel Aviv University, Tel
Aviv,
Israel; 3Department of Surgery – International Stem Cell
Institute, Cedars Sinai Medical Center, Los Angeles,
[email protected]
Introduction: Low back pain is a common cause of disability and
has enor-mous socioeconomic consequences (1). In cases of severe
pain caused by the pres-sure of bulging nucleus pulposus (NP) on
surrounding nerves, disectomy or nucle-us ablation are used. These
surgical procedures do not repair the disc problem.Repair is an
ideal therapeutic approach, which restores the normal structure
andfunction of the disc (2). Tissue engineering could regenerate a
damaged disc by theintroduction of cells, like Mesenchymal Stem
Cells (MSCs). One of the majorgoals in tissue regeneration research
is the establishment of valid imaging modali-ties for quantitative
analytical evaluation of the repair processes. We have previ-ously
shown that Double Quantum Filtered (DQF) MRI can be used for
theanalysis of tendon repair using MSCs (3). In this study we
hypothesized thatadvanced MRI methods including DQF-MRI could be a
useful tool for the eval-uation of the collagen fibers in a rat
model of IVD degeneration after NP ablation.The ability of non
invasive monitoring of the IVD changes at the early stage
post-nucleus ablation will provide an important base line for
future attempt in discrepair.
Materials and Methods: Using Fluoroscopy and Micromanipulator
two nee-dles were inserted into the NP through the postero-lateral
aspect of WISTAR ratscoccyges spine (C3 or C4). NP was removed by
wash with PBS till the leakage ofNP substance through the opposite
needle was completed.
To analyze the early changes in the IVD after NP removal,
animals were sac-rificed on day 5, C3-C4 segments were immersed in
fluorinate oil for the MRImeasurements. T2 relaxation times were
acquired using the multi slice multi echo ssequence (128x128,
TR/TE=3000/3 ms, 128 echoes, 8.45T). The contribution ofthe
residual dipolar interaction to T2 was refocused by dipolar echo
refocusing: 90
o
– [τcp – 90o – τcp]n – Imaging (4,5), where τcp is short (50 μs)
and n = 20-5000. The
relaxation time obtained by this method is denoted TDE.1H and 2H
double quan-
tum filtered (DQF) images were acquired with the basic DQF pulse
sequence: 90�
– τ/2 – 180� – τ/2 – 90� – tDQ – 90� – Imaging (6), where tDQ is
the double quan-
tum evolution time and τ is the creation time of the 2nd rank
tensors.Results: T2-weighted images of intact and ablated IVDs are
given in Fig. 1.
For the intact disc there is a clear distinction between the
nucleus and the annulus.After removal of the NP the width of the
disc is significantly decreased, the imageis relatively dark, and
the distinction between the AF and the NP is lost T2 valuesof
intact NP and AF were 83.5±8.6 ms and 20.3±2.9 ms (n=11)
respectively andfor the ablated disc 15.0±1.5 ms (n=2). Dipolar
echo refocusing gave relaxationtimes, TDE, of 171.5±32.5 ms and
31.0±6.2 (n=11) for the intact NP and AF,respectively, and 32.6±5.6
ms (n=2) for the ablated disc. Magnetization transferexperiments
followed the same trend: high magnetization transfer ratio (MTR)
forthe intact AF, almost negligible MTR for the intact NP and a
relatively high MTRfor the ablated disk.
The intact AF is clearly depicted by both 1H and 2H DQF (Fig.
2). In contrastto other MRI modalities, this compartment is also
partially highlighted in theablated disc. In the 2H DQF image, most
of the area of the ablated disc is high-lighted.
Discussion: Our results revealed higher concentration and better
ordering ofthe collagen fibers in the AF of the control disc
relative to the NP. The shorteningof T2 and TDE, the larger MTR and
the
1H and 2H DQF results obtained in theablated disc suggest an
increase in the concentration of the collagen fibers in theinner
part of the disc. This indicates a collapse of the collagen fibers
of the annu-lus into the void left after the NP removal and the
decrease of the disc width. TDEresults are in accordance to T1ρ
obtained in human intact and degenerated sam-ples (7,8).
As was previously pointed out (9), 1H DQF signal is obtained
only when thereis a substantial residual dipolar interaction of
water molecules as a result of high
degree of order of the tissue. In contrast, 2H DQF can be seen
in relatively disor-dered systems (10). This is the reason why in
the ablated disc the 1H DQF of theinner part appears as a dark spot
while in the 2H DQF one can see the effect of therelatively
disordered collagen fibers. The conclusions concerning the
morphologi-cal changes of the ablated disc are highly supported by
the histology (Fig. 3), inwhich shortly after NP removal the
collagen fibers of the annulus spread into theinner part of the
disc. These results demonstrate our ability to monitor very
earlychanges in the IVD post-nucleus ablation. They also provide us
with a baseline anda detection method for disc regeneration.
References: 1. Biering, SRF. Dan Med Bull, 29, 1982; 2. Alini, M
et al, Spine,28, 2003; 3. Hoffmann et al, J Clin Invest, 116, 2006;
4. Muller, K et al, J. Magn.Reson, 90, 1990; 5. Eliav, U et al,
Proc. ISMRM 6th Mtg, 602, 1998; 6. Tsoref, Let al, Magn Reson Med,
40, 1998; 7. Johannessen, W et al, Spine, 31, 2006; 8.Majumdar, S,
NMR Biomed, 19, 2006; 9. Eliav, U et al, J Magn Reson, 137,
1998;10. Sharf, Y et al, J Magn Reson, 107, 1995.
Fig 1. T2-weighted images of intact (A,B) and ablated (A,C) IVD
5 days after NP removal. A– Sagittal sections, TE=9ms; B,C – Axial
sections, TE=21ms.
Fig 2. 1H and 2H DQF images of intact and ablated IVD 5 days
after NP removal.
Fig 3. Histology (Masson Trichrom) of intact and ablated IVD 5
days after NP removal. a -Intact; b -Ablated
