Effect of mechanical convection on the partitioning of an iodinated anionic contrast agent in a bovine patella model
+2Entezari, V; 1,2Bansal, P N; 1Grinstaff, MW; 1Stewart RC; 2Snyder, BD
+1Departments of Chemistry and Biomedical Engineering, Boston University, Boston, MA; 2Center for Advanced Orthopaedic Studies, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, MA;
[email protected]
INTRODUCTION:
Magnetic Resonance Imaging1 and Computed Tomography2,3 along with
anionic contrast agents have been utilized to quantify and monitor
changes in the glycosaminoglycan content of articular cartilage. The
delayed gadolinium enhanced magnetic resonance imaging of cartilage
(dGEMRIC) uses gadolinium (Gd-DTPA2-), and Contrast Enhanced CT
(CECT) uses the iodinated contrast agents (iothalamate or ioxaglate),
that act as a mobile anionic probe that partitions itself throughout the
ECM in inverse proportion to the fixed negative charge density of the
proteoglycans comprising the ECM. Various studies have confirmed that
dGEMRIC can differentiate between healthy and arthritic cartilage both
in vitro and in vivo. CECT is a promising new technique that, allows for
faster acquisition of high resolution data, is cheaper and can image
subchondral bone and cartilage simultaneously. Previous studies2,3,5 have
shown the capability of CECT to quantify changes in GAG content of
articular cartilage, however most of these studies have either used
excised osteochondral or chondral samples. In addition, the results from
these studies have demonstrated the extremely slow diffusion rate of the
contrast agent into the cartilage ECM. The aim of this study was to
develop an experimental method whereby an intact bovine patella could
be subjected to dynamic deformation when surrounded by the contrast
agent to simulate more closely the in vivo conditions in a joint.
Specifically our aims were; (1) To evaluate the effect of mechanical
convection on the mass transfer of an iodinated anionic contrast agent
into intact bovine patellar surface 2) To assess the ability of CECT of
cartilage to correlate with spatial changes in GAG content.
METHODS: Study Design: Three intact bovine patellae from
skeletally mature cows were used in this study. Each patellae were
mounted on a custom designed holder which allowed for accurate
positioning of the patella during scanning and mechanical loading and
subjected to the following sequence: 1) Passive Diffusion of the contrast
agent into and out of the cartilage ECM using an anionic contrast agent
(CystoConray-II) (2) Mechanical convection of the anionic contrast
agent into the tissue. Contrast Enhanced Computed Tomography
Imaging: three sequential, 100µm thick, transaxial pQCT (Stratec,
Germany) images were obtained at 70µm in plane resolution. Three
slices were placed across the patellar 10 mm apart from each other.
Three 1 mm diameter holes were driddeld thorugh the bone to be used as
physical marker during imaging. The CT data sets were imported into
Analyze™ (Analyze™, BIR, Mayo Clinic, MN) and the cartilage was
segmented using spline-based manual segmentation. The mean cartilage
x-ray attenuation values using the Hounsfield Scale were obtained by
averaging attenuation values for all cartilage tissue over the three
transaxial CT images Passive Diffusion: Before immersing the patellae
in the contrast agent for the passive diffusion study, each patella was
imaged to obtain baseline values for cartilage x-ray attenuation. Then
each patella was immersed in CystoConray-II for 0.5, 1, 1.5, 2, 3, 5, 16
and 24 hours and imaged using a pQCT (Stratec, Germany) scanner. The
patella was immersed in 200 mL of CystoConray-II (400 mOsm/kg) at
34˚C to simulate in vivo joint environment. After 24 hours of immersion,
the patella was immersed in 500 mL of 400 mOsm/kg saline solution for
14 hours to allow contrast agent to diffuse out of the cartilage matrix.
Mechanical Convection: A custom fixture was built to hold the patella
while being subjected to mechanical convection in a material testing
machine (Instron 8511, Norwood MA). The fixture was built out of
acrylic so that it did not interfere with the CT imaging and allowed for
easy fixing and removal between the scanner and the material testing
machine. In order to apply an evenly distributed force during
convection, a conformal plate was fabricated for each patella out of an
elastic dental impression material (Vinylpolysiloxane/putty, Virtual,
Italy). Then, the same patella was affixed in the mechanical testing
machine and poured over the cartilage surface. The average cartilage
thickness was measured perpendicular to the surface using line profile
module in Analyze in ten different locations. In order to simulate the
walking cycle, 10% deformation under displacement control was applied
at a frequency of 1 Hz to the cartilage surface while controlling the
temperature to 34 ˚C. Each patella was subjected to 7.5, 15, 22.5, 30, 45,
60, 90 and 150 minutes of mechanical loading and scanned using the
pQCT scanner. Biochemical assessment of GAG content: The patellar
surface was divided into a matrix of (5 rows and 3 columns) and the
cartilage was separated from the subchondral bone using a razor blade
and the wet mass of the cartilage was obtained. The total GAG weight
per mg wet weight of cartilage for each sample was calculated using the
1,9-dimethylmethylene blue (DMMB) colorimetric assay. Statistical
Analysis: Paired t test was used to compare the means CT attenuation of
each patella after simple diffusion (SD) and mechanical convection
(MC). Linear regression analysis (SPSS, Chicago) was used to express
the x-ray attenuation measured by CECT as a function of the GAG
content for each patellar region. Significance level was set at α error of
0.05.
RESULTS: The mean CT attenuation for whole cartilage was
significantly higher after mechanical convection compared to passive
diffusion at the same time point. At 30 minute of convection the CT
attenuation was 25.5% higher than passive diffusion and had reached
79.9% (p = 0.0001*) of CT attenuation after 24 hours (Fig.1a). Linear
regression analysis showed that the CECT attenuation accounted for
93% of the variation in spatial GAG content (R2 = 0.93, p = 0.001*,
Figure 1b)
B
A
Figure1: CECT attenuation as a function of time (A). The
dotted lines are for mechanical convection and the solid lines
are for passive diffusion. The X-axis is on a log scale. Linear
regression between CECT attenuation and GAG content (B).
DISCUSSION:
The present study is the first to quantify the time required to achieve
equilibrium concentration of an anionic CT contrast agent in an intact
bovine patellar surface. The results of this study demonstrate that
mechanical convection results in enhanced mass transfer of an anionic
contrast agent in an intact bovine patellar surface. The environmental
conditions of temperature, osmolality and deformation were controlled
in this experiment to mimic the joint environment; hence the results
represented here could be easily translated in vivo. Further, it was shown
that the CECT imaging of cartilage is able to correlate with the spatial
variation of GAG content. This result validates the previous findings of
CECT studies2,3, in a more realistic model. With mechanical convection,
at 30 minutes the cartilage had achieved ~80% of the maximum CT
attenuation reached during passive diffusion. This finding is encouraging
from a clinical standpoint for patients with knee pain. Studies, such as
this will help us further understand the physical behavior of these
contrast agents and help make CECT of articular cartilage, a feasible
clinical tool.
REFERENCES:
(1) Bashir, A, et al., Magn Reson Med 1996;36:665-73 (2) Bansal, PN, et al., Trans. of the ORS,
Paper1655 Vol.33, 2008. (3) Palmer, AW, et al., PNAS 2006; 103:19255-19260
Poster No. 856 • 56th Annual Meeting of the Orthopaedic Research Society