Diffusion of MRI and CT Contrast Agents in Articular Cartilage under Static Compression

Abstract

Cartilage has a limited capacity for self-repair and focal damage can eventually lead to complete degradation of the tissue. Early diagnosis of degenerative changes in cartilage is therefore essential. Contrast agent-based computed tomography and magnetic resonance imaging provide promising tools for this purpose. However, the common assumption in clinical applications that contrast agents reach steady-state distributions within the tissue has been of questionable validity. Characterization of nonequilibrium diffusion of contrast agents rather than their equilibrium distributions may therefore be more effective for image-based cartilage assessment. Transport of contrast agent through the extracellular matrix of cartilage can be affected by tissue compression due to matrix structural and compositional changes including reduced pore size and fluid content. We therefore investigate the effects of static compression on diffusion of three common contrast agents: sodium iodide, sodium diatrizoate, and gadolinium diethylenetriamine-pentaacid (Gd-DTPA). Results showed that static compression was associated with significant decreases in diffusivities for sodium iodide and Gd-DTPA, with similar (but not significant) trends for sodium diatrizoate. Molecular mass of contrast agents affected diffusivities as the smallest one tested, sodium iodide, showed higher diffusivity than sodium diatrizoate and Gd-DTPA. Compression-associated cartilage matrix alterations such as glycosaminoglycan and fluid contents were found to correspond with variations in contrast agent diffusivities. Although decreased diffusivity was significantly correlated with increasing glycosaminoglycan content for sodium iodide and Gd-DTPA only, diffusivity significantly increased for all contrast agents by increasing fluid fraction. Because compounds based on iodine and gadolinium are commonly used for computed tomography and magnetic resonance imaging, present findings can be valuable for more accurate image-based assessment of variations in cartilage composition associated with focal injuries.

Introduction

Articular cartilage is a highly specialized connective tissue that covers ends of bones in synovial joints. It functions in load bearing and lubrication of joints by providing a nearly frictionless, gliding surface that evenly distributes mechanical loads resulting from physical activities (1–3). The major constituents of cartilage extracellular matrix (ECM) include primarily proteoglycans and type II collagen, together with interstitial fluid (4). Because articular cartilage is avascular and interstitial fluid comprises 70–85% of the tissue, solute transport throughout the ECM plays a crucial role for biological activities of cartilage (5). Furthermore, clinical imaging protocols developed for functional assessment of cartilage often depend on contrast agent partitioning through the ECM, mainly mediated by diffusion (6–8). Improved observation and knowledge of solute transport rates are pivotal considerations for precise application of clinical imaging techniques to assess tissue function and integrity (9).

Solute transport in articular cartilage is strongly affected by static compression due to changes in matrix structure. Studies of static compression effects on solute transport indicate reductions in the diffusivity and partitioning of larger molecules such as relatively large molecular mass polysaccharides (10). Diffusivities of several different solutes have been reported to decrease with increasing compressive strains (11–13). Perturbations to diffusion of solutes through compressed articular cartilage have been correlated with changes in mechanical properties and matrix density (14). However, each solute interacts with cartilage in specific ways, unique to its molecular mass, conformation, charge, and binding characteristics (10,15). Therefore, clear correlations between mechanical compression and solute transport parameters for a solute of interest is required to be established for a better understanding of ECM interactions with solute in vivo.

Cartilage has a limited intrinsic capability for regeneration, therefore early detection of degenerative changes is essential for effective treatment. X-ray computed tomography (CT) imaging or delayed gadolinium-enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) have been developed for this purpose (16–19). In these contrast agent-based imaging techniques, solute transport properties are used to assess tissue composition and function. Contrast agents, generally anionic, partition through the ECM in inverse proportion to the spatial distribution of fixed charge density (FCD) of the proteoglycans in cartilage matrix (20). According to the Donnan-Gibbs theory, the distribution of fixed charges and fluid content determines the equilibrium distribution of anionic diffusing molecules. However, because prohibitively long times are required to attain equilibrium, the use of contrast agents for determination of matrix composition by solute partitioning is limited. Long equilibration periods may result in inaccurate evaluation of FCD distribution by standard methods due to the difficulty of achieving a true steady-state distribution in vivo (which these methods assume). In addition, constant physiological clearance and uptake by other tissues may avoid true equilibrium even if enough time is given. Nonequilibrium transport, such as diffusion of contrast agents through cartilage therefore presents new possibilities for more accurate imaging protocols (6,9). Because anionic gadolinium-based and iodinated compounds are commonly used for studies of the CT and MRI contrast agent (9,20–23), elucidate correlations between changes in diffusion behavior of these contrast agents and matrix composition could provide a potential tool for cartilage integrity assessment.

Static compression provides a means for nondestructive adjustment of cartilage ECM composition and density, which are also altered in injury and disease. However, the sensitivity of contrast agent diffusivities to cartilage compression has not been well characterized. Because cartilage injury is accompanied with changes in glycosaminoglycan (GAG) density and water content, study of induced changes in these parameters due to the imposed strain originating from tissue compression can provide indirect means to evaluate the sensitivity of contrast agents to the biochemical variations involved in cartilage damage. Acquiring this information is essential for development of cartilage integrity assessment techniques based on diffusion rates. We therefore aimed to study the effects of static compression on diffusion of CT and MRI contrast agents through cartilage in vitro. Furthermore, alterations of ECM due to compression were characterized by determination of GAG and fluid contents. Insights obtained by investigation of diffusion of these contrast agents in cartilage under static compression may indicate means for evaluating cartilage properties in vivo by considering mechanical compression as an adjustable parameter that could be used to assess the interactions of contrast agents and ECM. Findings may also contribute to the development of improved techniques for assessing mechanical and biochemical properties of cartilage in the laboratory and in the clinic.

Materials and Methods

Cartilage explants preparation

Fresh adult bovine femurs were acquired from a local slaughterhouse. Visually intact osteochondral plugs (8 mm in diameter) were drilled perpendicular to the distal femur from the patellar groove by a power drill and coring bit (Snug-Plug Cutters, Veritas Tools, Ottawa, Canada). Plugs were kept at −20°C in phosphate buffered saline (PBS) with 0.1 mg/mL sodium azide and protease inhibitors (Sigma-Aldrich, St. Louis, MO) until further use. Osteochondral plugs were thawed to reach room temperature before sectioning. Samples were mounted on a microtome (RM2235, Leica Microsystems, Wetzlar, Germany) using a custom-built holder and superficial cartilage (thickness of 50–150 μm) was removed to obtain a more homogenous specimen with uniform properties. Disks were then sliced from the cartilage specimen with a variety of thickness ranging from 400 to 1000 μm using the microtome.

Solutes

Three contrast agents applicable in CT or MRI were studied. Sodium iodide (150 Da, 383112, Sigma-Aldrich, St. Louis, MO) and sodium diatrizoate hydrate (636 Da, S4506, Sigma-Aldrich, St. Louis, MO) are CT contrast agents, and gadolinium diethylenetriamine-pentaacid hydrate (Gd-DPTA, 548 Da, 381667, Sigma-Aldrich, St. Louis, MO) is a MRI contrast agent. Iodine-containing solutes are typical CT contrast agents; hence, negatively charged monovalent iodide and diatrizoate represent this broad category of contrast agents with different molecular masses, although the latter is a commercially available CT contrast agent (24). Gd-DTPA is the negatively charged divalent gadolinium-based MRI contrast agent commonly used in the dGEMRIC technique whose equilibrium distribution is known to be inversely proportional to the negative FCD of cartilage (18).

Absorption bath

For each contrast agent, 28 cartilage explants obtained from 10 different joints were used for diffusivity measurements. Explant disks (5 mm in diameter) were punched from the dissected cartilage using a biopsy punch (33–35, Miltex, York, PA). For each experiment, six sliced cartilage explants were immersed in 10 mL of solute absorption bath and equilibrated for a period of 20–24 h. Absorption baths were prepared by dissolving the contrast agent in PBS to obtain nominal concentrations of 200, 90, and 100 mM for sodium iodide, sodium diatrizoate, and Gd-DTPA, respectively. Adjustment of pH to 7.4 for Gd-DTPA absorption bath was achieved by use of hydrochloric acid before introducing cartilage samples to the bath.

Diffusivity Measurement

After equilibration, disks were removed from absorption baths and smaller explant disks (3 mm in diameter) were punched from them to reduce the surface adsorption effect (Fig. 1 A) (25). Explant disk dimensions, including thickness and diameter, were measured by a dissecting microscope (Stemi 2000-C, Zeiss, Jena, Germany). Two explants of the same thickness were then mounted within the two circular depressions in a Plexiglas chamber and compressed when a Plexiglas disk was placed on top and the entire sandwich configuration was sealed by means of a screw (Fig. 1, B and C). Hence, radially unconfined static compression was imposed to explant disks between impermeable Plexiglas platens. Design of the custom-built Plexiglas chamber and disk satisfy the boundary conditions and minimize the source of errors; however, possible bulging during the compression may have perturbed the idealized explant geometry.

Figure 1.

(A) Sequence of events for preparing cartilage explants for the desorption bath; (B) cartilage disks were mounted on the custom-built compression chamber and fluid (PBS) was circulated to induce radial solute transport, as shown by arrows; (C) schematic of custom-built compression apparatus.

The height between the Plexiglas chamber and disk was set to be fixed at 300, 450, or 600 μm so that different compressive strains were achievable depending on the free-swelling thicknesses of cartilage explant disks. The sandwich configuration was then transferred to a custom-made diffusion apparatus in which 7 mL PBS (initially free of contrast agent) was circulated around the statically compressed explants using a peristaltic pump. Aliquots of 400 μL were taken from the diffusion apparatus periodically at regular time intervals up to 12 h and were immediately replaced by the same amount of PBS to keep the bath volume constant at 7 mL. Explant disks were then allowed to equilibrate in desorption bath overnight so that the final desorption bath concentration was achieved.

Desorption bath solute concentration was measured for each of the contrast agents by different techniques. Iodide concentration in aliquots taken from the sodium iodine desorption bath was measured with a colorimetric iodide assay using a microplate reader (Berthold Mithras LB940, Bad Wildbad, Germany) by absorbance measurement at 610 nm for aliquots mixed with a starch-based solution (26). Sodium diatrizoate concentration was measured by a spectrophotometric procedure to determine the absorbance of aliquots at 230 nm in a UV-visible spectrophotometer (BioPhotometer plus, Eppendorf, Hamburg, Germany) (27). Detection of gadolinium concentration in desorption bath containing Gd-DTPA was performed by inductively coupled plasma atomic emission spectroscopy (ICP-AES); aliquots were acidified by addition of 4% nitric acid (HNO₃) and then injected into the ICP-AES apparatus (iCAP 6500, Thermo Scientific, Cambridge, England) via a peristaltic pump. The resulting signatures for iodine, sodium diatrizoate, and gadolinium were compared to a dilution series to quantify concentrations after establishing linear calibrations between absorbance and solute concentrations for sodium iodide and sodium diatrizoate.

The effective diffusivity, D, for transport across the cartilage surface is defined as the coefficient relating the flux to the gradient of solute concentration. Considering conservation of solute molecules, D can be determined from the equation governing the transient distribution of concentration (c) in an explant disk for one-dimensional radial transport from a block of tissue (14):

$$\frac{\partial c(r,t)}{\partial t}=\frac{D}{\varphi }\left[\frac{1}{r}\frac{\partial }{\partial r}\left(r\frac{\partial c}{\partial r}\right)\right],$$ (1)

where ϕ represents explant fluid volume fraction in the compressed state. Boundary conditions are ∂c/∂t(r = 0,t) = 0 by symmetry, and c(r = R,t) = 0 for the well-mixed (and relatively very low concentration) bath, where R is the radius of the compressed explant disk. Poisson’s ratio (radial-to-axial strain ratio, ν = 0.215), as a measure of volume change of cartilage under loading, was used to determine the compressed radius of each cartilage disc (14). The initial condition is c(r,0) = c₀. Solving Eq. 1.,

$$c\left(r,t\right)=\sum _{n=1}^{\infty }\frac{2c_0}{P_n{J}_1\left(P_n\right)}{J}_0\left(\frac{P_n}{R}r\right)\text{exp}\left(-\frac{D{\lambda }_n^{2}t}{\varphi }\right),$$ (2)

where J₀ and J₁ are the zero- and first-order Bessel functions of the first kind, P_n is the nth zero of J₀ and λ_n = P_n/R.

The average desorption bath solute concentration (c_d) for two samples in the desorption bath at any given time is determined by integration of Eq. 2. over the explant fluid volume followed by subtraction of the result from the total molecules of solute presented initially within the explants; therefore, c_d for two cartilage disks present in the bath would be (28):

$$c_d\left(t\right)=c_{d0}+\frac{c_0\pi h}{V_d}\left(R_1^2{\varphi }_1\left[1-\sum _{n=1}^{\infty }\frac{4}{P_n^2}\text{exp}\left(-\frac{D{\lambda }_{n1}^{2}t}{{\varphi }_1}\right)\right]+R_2^2{\varphi }_2\left[1-\sum _{n=1}^{\infty }\frac{4}{P_n^2}\text{exp}\left(-\frac{D{\lambda }_{n2}^{2}t}{{\varphi }_2}\right)\right]\right),$$ (3)

where c_d0, V_d, and h represent desorption bath solute concentration at t = 0, desorption bath volume and explant disk thickness in compressed state, respectively. The indices 1 and 2 denote cartilage samples 1 and 2, respectively. Initial solute concentration within the cartilage samples (c₀) is determined from conservation of solute molecules (28):

$$c_0=\frac{\left(c_{d\infty }-c_{d0}\right)V_d}{\varphi \pi h\left(R_1^2+R_2^2\right)}+K{c}_{d\infty },$$ (4)

where c_d∞ and K are the equilibrium solute concentration in the desorption bath and solute partition coefficient, respectively.

Diffusion coefficients were calculated by fitting the theoretical model to the experimental data for desorption bath solute concentration versus time (Fig. 2) (15). Aliquots acquired from the desorption bath were compensated by taking into account the aliquot removal and subsequent replacement of the corresponding amount of PBS during concentration measurements and a least squares method implemented in MATLAB (The MathWorks, Natick, MA) was employed to fit Eq. 3 to experimental data. Average of coefficients of determination (R²) for fitted curves was 0.941, 0.996, and 0.988 for sodium iodide, sodium diatrizoate, and Gd-DTPA, respectively, showing that the theoretical model accurately approximated the experimental data.

Figure 2.

Diffusivity was determined by least squares best fitting of Eq. 3. to experimental data for desorption bath concentration versus time. Aliquot removal during desorption bath experiments was compensated. Representative graph for determination of diffusivity of Gd-DTPA in an explant under 20% compression.

Glycosaminoglycan content and fluid fraction measurements

Per each experimental setup for diffusivity measurement, each of the individual explant disks in the desorption bath was used to determine fluid volume fraction and GAG content. To retrieve the free-swelling state, explant disks were left for 3 h in PBS after diffusion measurements. Explant wet weights were then measured by an analytical balance (AL204, Mettler Toledo, Mississauga, Canada). Explant disks were then lyophilized for 24 h by a freeze dry system (7670520, Labconco, Kansas City, MO) and dry weight was measured. The total explant fluid volume fraction under compression was calculated as the ratio between the explant fluid volume under compression (V_f) and the sum of the fluid volume and the solid volume (V_s) under compression (total volume):

$$\varphi =V_f/\left(V_f+V_s\right).$$ (5)

The explant fluid weight in the free-swelling state (W_f,fs), solid weight of the compressed cartilage sample (W_s), and explant volumetric strain (ε_v) were used to assay V_f assuming fluid matrix density (ρ_f) and solid matrix density (ρ_s) of 1 and 1.4 g/mL (29), respectively:

$$V_f=W_{f,fs}(1-{\epsilon }_v)/{\rho }_f-(W_s{\epsilon }_v/{\rho }_s).$$ (6)

W_f,fs and W_s were measured independently and ε_v was determined as the ratio between difference of explant total volume in the free-swelling state and explant total volume in the compressed state to the explant total volume in the free-swelling state:

$${\epsilon }_v=\frac{d_{fs}^2{h}_{fs}-{(d_{fs}+d_{fs}\nu \epsilon )}^{2}h}{d_{fs}^2{h}_{fs}},$$ (7)

where d_fs, h_fs, and ε are the explant diameter in the free-swelling state, explant thickness in the free-swelling state and axial strain, respectively.

To determine GAG content, lyophilized explants were digested overnight at 60°C in 1 mL of PBS solution containing 0.01% sodium azide, 5m M L-Cysteine hydrochloride monohydrate (C7880, Sigma-Aldrich, St. Louis, MO), and 125 μg/mL papain (P4762, Sigma-Aldrich, St. Louis, MO). Colorimetric assay using the dimethylmethylene blue spectrophotometric technique was then employed to estimate GAG weight fraction of explant disks (30). GAG concentration was expressed as GAG content per total weight of a compressed cartilage disc.

Partition coefficient

Effective partition coefficients for each of the contrast agents were evaluated using 7–8 explant disks, 5 mm in diameter, obtained randomly from three different joints. Explants were left to equilibrate in an absorption bath of sodium iodide, sodium diatrizoate and Gd-DTPA with nominal concentrations of 30, 10, and 60 mM, respectively, for a period of 24 h. Explants were then transferred to 250 μL blank PBS baths and incubated for 24 h. Equilibrium concentration of absorption and desorption baths for each of the contrast agents were measured by the techniques described previously. Wet and dry weights of cartilage explants were also measured as described previously.

The ratio of solute concentration within cartilage to that within surrounding bath at equilibrium is considered as the solute partition coefficient, K. Using conservation of solute in the desorption bath, K is determined as

$$K=\frac{c_d{V}_d}{V_0\varphi (c_a-c_d)},$$ (8)

where c_a represents the equilibrium adsorption bath solute concentration.

Statistical Analysis

Statistical analysis of the significance of differences was assessed using analysis of variance. The data for partition coefficients are reported as mean ± standard error of the mean. Differences were considered significant for p < 0.05. The data for diffusivity and GAG measurements are reported using least squares best-fit straight lines to data points. The 95% confidence and prediction intervals for the least square best-fits were calculated by SigmaPlot (Version 12.5, Systat Software, Chicago, IL). A multiple regression analysis of diffusivity versus GAG content and fluid fraction was performed using Minitab software (Release 16, Minitab, State College, PA) to investigate the relative importance of each of these parameters on diffusivity.

Results

Partition coefficients of contrast agents remained approximately constant in a range of measured GAG content of the cartilage matrix (Fig. 3 A). K, as measured under free-swelling conditions, had a tendency to decrease with a monotonic trend as the molecular mass of contrast agents increased (Fig. 3 B). Sodium iodide, with somewhat smaller molecular mass compared to the two other contrast agents, had a K of 0.740 ± 0.0415. This indicated that sodium iodide could relatively freely diffuse into the tissue fluid. For Gd-DTPA and sodium diatrizoate, partition coefficients were 0.469 ± 0.0142 and 0.385 ± 0.229, respectively, indicating that factors like steric interactions and electrostatic repulsion come into play and result in low partition coefficients.

Figure 3.

(A) Partition coefficients of contrast agents: sodium iodide, sodium diatrizoate, and Gd-DTPA in cartilage versus GAG content (g GAG per g explant). (B) Contrast agent partition coefficients within cartilage explants. Data are shown as mean ± SE (n = 7–8).

Although diffusivity coefficients for sodium diatrizoate and Gd-DTPA (with similar molecular mass but different anionic charge) were about the same, sodium iodide exhibited higher values for diffusivity. Diffusivities of sodium iodide and Gd-DTPA significantly decreased with increasing compressive strains (Fig. 4, A and C). The tendency for static compression to reduce the diffusion rate of contrast agent was stronger for iodide versus sodium diatrizoate and Gd-DTPA. Increasing static compression from 0% to 50% was associated with a decrease in the diffusivity of sodium iodide from 838 ± 111 μm²/s to 412 ± 95 μm²/s (Fig. 4 A). Diffusivity of Gd-DTPA decreased from 187 ± 31 to 133 ± 32 μm²/s in response to change of compression from 0% to 60% (Fig. 4 C). Although sodium diatrizoate approached a trend of decreasing diffusivity from 145 ± 19 to 116 ± 19 μm²/s when compression changed from 0% to 52% (Fig. 4 B), this was not statistically significant (p = 0.061).

Figure 4.

Contrast agent diffusivities versus static compression for: (A) sodium iodide, (B) sodium diatrizoate, and (C) Gd-DTPA. Solid lines represent linear least squares best-fit straight lines to data; dashed and dotted lines are the 95% confidence and prediction intervals on best-fit straight line parameters, respectively. In each of the A, B, and C graphs, each filled marker represents two samples present in the desorption bath at the specific static compression.

Altered matrix density induced by static compression was evident by GAG content and fluid volume fraction measurements. Although correlation of decreasing diffusivity with increasing GAG content (expressed as GAG weight per total explant weight) was found to be significant for sodium iodide and Gd-DTPA (stronger for sodium iodide than Gd-DTPA) (Fig. 5, A and C), trends for sodium diatrizoate was insignificant (Fig. 5 B). Altering GAG content from 0.0177 to 0.0.949, 0.0140 to 0.0629, and 0.0161 to 0.0648 due to compression resulted in decreased diffusivity from 772 ± 99 to 329 ± 143, 144 ± 16 to 125 ± 21, and 178 ± 20 to 128 ± 33 μm²/s for sodium iodide, sodium diatrizoate, and Gd-DTPA, respectively. To achieve unbiased design, explants were randomly obtained from a group of mixed explants from 10 different joints for each contrast agent; nevertheless, GAG content variation was relatively more noticeable for Gd-DTPA and sodium iodide than sodium diatrizoate. However, statistical analysis showed that GAG content is a less influential factor to dominate diffusivity of sodium diatrizoate than fluid volume fraction.

Figure 5.

GAG content (g GAG per g explant) dependences of diffusivity of cartilage explants for (A) sodium iodide, (B) sodium diatrizoate, and (C) Gd-DTPA. Solid lines represent linear least squares best-fit straight lines to data; dashed and dotted lines are the 95% confidence and prediction intervals on best-fit straight line parameters, respectively. Each of the A, B, and C graphs represents the same data set shown in Fig. 4, but plotted with different x axes.

Increased diffusivity significantly correlated with increasing fluid volume fraction for all three contrast agents. Diffusivity increased from 400 ± 123 to 778 ± 111, 110 ± 19 to 155 ± 14, and 124 ± 28 to 195 ± 26 μm²/s when fluid volume fraction increased from 0.66 to 0.97, 0.78 to 0.94, and 0.80 to 0.95 for sodium iodide, sodium diatrizoate, and Gd-DTPA, respectively (Fig. 6). Increasing trends were found to be significant as confirmed by analysis of variance and p value was equal or<0.005 for all contrast agents. The multiple regression analysis revealed that both GAG content and fluid volume fraction have substantial effects on diffusivity of sodium iodide, whereas fluid volume fraction is more influential than GAG content to affect diffusivity of sodium diatrizoate and Gd-DTPA.

Figure 6.

Fluid fraction dependences of diffusivity of cartilage explants for (A) sodium iodide, (B) sodium diatrizoate, and (C) Gd-DTPA. Solid lines represent linear least squares best-fit straight lines to data; dashed and dotted lines are the 95% confidence and prediction intervals on best-fit straight line parameters, respectively. Each of the A, B, and C graphs represents the same data set shown in Fig. 4, but plotted with different x axes.

Discussion

Clinical imaging techniques developed for early detection of cartilage degeneration rely upon contrast agent diffusion. Cartilage experiences a range of static and dynamic compressive forces on a daily basis due to its physiological loading environment (11,31). Characterization of contrast agent nonequilibrium diffusion in the presence of mechanical loading can therefore mimic these in vivo conditions and improve interpretation of CT or MR images. This study confirmed that contrast agent diffusivities in statically compressed cartilage were altered by changes in matrix density due to axial explant strain. Consistent with previous reports for relatively small solutes (11,13,14), diffusivity significantly decreased for sodium iodide and Gd-DTPA, and a strong trend for decreasing diffusivity was evident for sodium diatrizoate with increasing static mechanical compression; these changes in diffusivity also correlated with matrix density variations. Although experiments were performed using a simple geometry and controlled boundary conditions to quantify diffusion rates, diffusivity values obtained are not limited to those controlled conditions as diffusion is an intrinsic property and is independent of parameters such as tissue geometry, uptake by other tissues, and physiological clearance.

Equilibrium distributions of contrast agents determined by partition coefficient measurements at the free-swelling state were in good agreement with theoretical predictions and previous studies (13,32). Presently studied contrast agents are relatively small solutes, hence steric interactions may be of secondary importance in partitioning of solutes as compared to electrostatic interactions. As these molecules carry electric charges (iodide: −1, diatrizoate: −1, and Gd-DTPA: −2) identical to the negatively charged proteoglycans of the matrix solid network, partition coefficients of less than unity were expected due to electrostatic repulsion (33). Distributions of contrast agents at equilibrium were also influenced by solute molecular mass. The partition coefficient of sodium diatrizoate with its molecular mass of 636 Da deviates from unity more significantly than that for sodium iodide with its molecular mass of 150 Da though they contain identical charge. Sodium diatrizoate also showed a lower partition coefficient than Gd-DTPA, which has a greater electric charge, presumably because the greater molecular mass of sodium diatrizoate contributes more dominantly to its partitioning behavior in cartilage and hinders its penetration into cartilage ECM compared to the more negatively charged Gd-DPTA.

In clinical applications of CT and MRI under currently standard protocols, contrast agents are assumed to attain equilibrium distributions within a timeframe that is generally < 2 h (9,18,34–36). However, diffusivity measurements in this study demonstrated that periods required for near-equilibrium distribution of common contrast agents may vary from 4 to 8 h. Of course, the geometry and direction of solute transport involved in this study do not replicate in vivo conditions. However, previous studies of contrast agent diffusion through cartilage, which employ a peripheral quantitative CT instrument or the dGEMRIC technique to acquire distribution maps and determine diffusivities of similar contrast agents have also reported longer periods for equilibrium distribution (5–29 h) (6,9,37). Failure to achieve steady-state distribution of Gd-DTPA in cartilage samples during the conventional imaging timeframe has also been reported to possibly result in a fallaciously too high dGEMRIC index for bulk regions (38). These observations together imply that achieving a steady-state distribution under clinical conditions may not be feasible, hence investigating nonequilibrium diffusion characteristics of contrast agents rather than their equilibrium distributions could offer opportunities for more accurate assessment of cartilage integrity (22,37,39).

Activities such as standing, kneeling, or imitating physical activities are regular practices to facilitate transport of contrast agents through cartilage in a clinical context (9,18,40). These activities may influence the matrix density of cartilage by inducing mechanical strains. Effects of these preparation steps on diffusivity of contrast agents are usually considered to be beneficial, though present findings indicate that static mechanical compression can contribute strongly to reduced penetration rates of contrast agents. It is worth noting that activities involving dynamic compression are not simple to interpret as introduction of associated oscillatory fluid flows would need determination of advection coefficient, which governs the effects of dynamic compression. In a wide range of compressive strains applied to cartilage samples in this study, static compression was associated with significant decreases in diffusivity versus uncompressed explants (0% static compression) for sodium iodide and Gd-DTPA, whereas a near-significant trend for sodium diatrizoate was also observed. These findings were reasonably consistent with results of previous studies investigating compression effects on diffusion of small solute molecules such as tetramethylrhodamine (430 Da) and fluorophores (∼500 Da) (13,14).

Differences in solute molecular mass, charge, matrix GAG content, and fluid volume fraction in compressed explants largely explain the changes in diffusivities observed (6,32). The smaller molecular mass of sodium iodide versus sodium diatrizoate and Gd-DTPA, and the lower anionic charge versus Gd-DTPA were evidently influential factors for sodium iodide to exhibit the highest diffusivity. Sodium iodide also showed the strongest trend for changes in diffusivity over compression among the contrast agents studied. Sodium diatrizoate and Gd-DTPA have a similar molecular mass and also exhibited similar ranges of diffusivities. Nevertheless, Gd-DTPA showed a significant tendency for a decrease in diffusivity with compressive strain, whereas sodium diatrizoate appeared to be less affected. In addition to differences in anionic charge due to the lower anionic charge of sodium diatrizoate compared to Gd-DTPA that may affect solute diffusivity, alteration of GAG content in the statically compressed explants used was (unfortunately) more evident during experiments with Gd-DTPA than for those involving sodium diatrizoate.

Conclusion

Contrast agent diffusion through statically compressed articular cartilage was examined to determine the effects of matrix structure and composition on nonequilibrium transport phenomena. Results show that direct correlations exist between altered matrix density due to compression and contrast agent diffusivity, which can influence the distribution of contrast agent in clinically significant ways. Solute molecular mass and matrix GAG content and fluid volume fraction were found to be important determining factors of the diffusivities of these solutes of interest. Present findings can improve the understanding of interactions between cartilage and contrast agents used for CT and MRI. Furthermore, findings may indicate possibilities for more accurate clinical assessment of cartilage structure, composition, and function by including quantification of nonequilibrium solute transport in CT and MR clinical imaging.