differences in contrast agent diffusion reflect changes in composition and structure of articular cartilage . however , in clinical application the contrast agent concentration in the joint capsule varies , which may affect the reliability of contrast enhanced cartilage tomography ( cect ) . in the present study , effects of concentration of x - ray contrast agents on their diffusion and equilibrium distribution in cartilage were investigated . full - thickness cartilage discs ( d = 4.0 mm , n = 120 ) were detached from bovine patellae ( n = 24 ) . the diffusion of various concentrations of ioxaglate ( 5 , 10 , 21 , 50 mm ) and iodide ( 30 , 60 , 126 , 300 mm ) was allowed only through the articular surface . samples were imaged with a clinical peripheral quantitative computed tomography scanner before immersion in contrast agent , and after 1 , 5 , 9 , 16 , 25 , and 29 hours in the bath . the diffusion coefficient of iodide ( 473  133 m / s ) was greater ( p  0.001 ) than that of ioxaglate ( 92  46 m / s ) . in full - thickness cartilage , the partition coefficient ( at 29 h ) of iodide ( 71  5% ) was greater ( p  0.02 with most concentrations ) than that of ioxaglate ( 62  6% ) . significant differences in partition and diffusion coefficient of two similarly charged ( 1 ) contrast agents were detected , which shows the effect of steric interactions . however , the increase in solute concentration did not increase its partition coefficient . in clinical application , it is important that contrast agent concentration does not affect the interpretation of cect imaging . articular cartilage is avascular , hence diffusion ( of , e.g. , nutrients , wastes , cytokines ) through the extracellular matrix is essential to tissue function . from a general viewpoint , diffusion flux is proportional to the gradient of the chemical potential , which is proportional to the concentration gradient in dilute systems . diffusion is substantially affected by the matrix fixed charge density ( fcd ) and size of the diffusing molecule . differences in diffusion of contrast agents have been reported to reflect changes in tissue composition . in general , diffusion is hindered when molecular size approaches the pore size of the matrix . in the deep zone of articular cartilage , negatively charged proteoglycans ( pgs ) bind water to the matrix and provide resistance to compression and reswelling when external pressure is released . in degenerated cartilage , these properties are altered . early signs of cartilage degeneration include pg loss , disruption of collagen network , and increase of water content . as differences in contrast agent diffusion can reflect changes in tissue composition , imaging of diffusion processes could enable noninvasive assessment of cartilage integrity . contrast enhanced cartilage tomography ( cect ) and delayed gadolinium enhanced magnetic resonance imaging of cartilage ( dgemric ) have been used for imaging of tissue pg content by means of quantification of cartilage fcd . in dgemric , it is assumed that an anionic contrast agent reaches an electrochemical equilibrium with cartilage fcd , enabling measurement of glycosaminoglycan ( gag ) concentration through a change in tissue t1 value . the dgemric technique is based on the donnan theory of membrane equilibrium , with two assumptions , that is , the existence of diffusion equilibrium and a constraint on free diffusion of the charged molecules through the fixed negatively charged pg network . when the pg concentration decreases the concentration of anionic contrast agent increases , and t1 value decreases . analogously in cect , increase in contrast agent concentration enhances x - ray attenuation in the tissue . it has been shown that diffusion equilibrium for common clinical contrast agents , gadopentetate and ioxaglate , may be reached in vitro only after 20 hours in human articular cartilage . long diffusion times are not feasible in clinical applications , since most of the contrast agent is excreted within a few hours . however , contrast agent penetration may be enhanced by joint loading that induces interstitial fluid flow and solute advection . in dgemric , the applied contrast agent concentration is typically 1 to 2 mm . in cect studies , 5 to 100 times higher or even stronger contrast agent concentrations , compared with dgemric concentrations , have been applied . high contrast agent concentrations ensure sufficient x - ray attenuation in cartilage , and thus produce higher image contrast . however , ct arthrography , in which full - strength contrast agent is used , needs to be discriminated from cect , in which diluted contrast agent may need to be used . the reason for this is that in cect imaging the x - ray attenuation values may not exceed the range measurable with clinical scanners . ct arthrography gives information on cartilage morphology , whereas cect has been proposed for evaluation of cartilage composition . a concentration dependence of solute diffusion exists in many systems , and in theory , concentration of a solute can affect also its partition coefficient . however , the concentration dependence is often minimal in diluted solutions . particularly in diffusion studies with tracer substance concentrations below a few parts per million , the effect of concentration for example , the diffusion coefficient of dextran has been shown to increase as solute concentration increases , but opposite findings on concentration dependence of diffusion coefficient of dextran have also been reported . it has been suggested that solute  solute and solute  matrix interactions may become important only with highly concentrated solutes , and in tightly packed matrix . at present , concentration dependence has not been studied with charged x - ray contrast agents . in clinical application of cect , the contrast agent concentration in joint capsule varies between individuals due to variable amount of synovial fluid , especially in inflamed joints . importantly , if x - ray contrast agent concentration in the joint capsule affects the equilibrium distribution and diffusion rate , it could significantly affect the interpretation of clinical cect examinations . in the present study , we investigate the effect of concentrations of ioxaglate and iodide solutions on their diffusion properties in bovine articular cartilage in vitro . the problem is simplified by assuming that fick s law can sufficiently describe the macroscopic diffusion of contrast agent , and all relevant factors are merged into one diffusion coefficient . samples are imaged with a clinical peripheral quantitative computed tomography ( pqct ) scanner before immersion in contrast agent and after 1 , 5 , 9 , 16 , 25 , and 29 hours in the bath , and diffusion coefficients and near - equilibrium distributions of ioxaglate and iodide solutions are determined . fresh and visually intact patellae ( n = 24 ) from 16- to 24-month - old bovines were dissected within 6 hours after slaughter . the tissue was obtained from a local slaughterhouse ( atria plc , kuopio , finland ) and , thus , no approval was needed from the committee on research ethics of the university of eastern finland . the preparation procedure and imaging protocol have been previously described . in brief , five full - thickness cartilage discs ( d = 4.0 mm ) were detached from each patella with a dermal biopsy punch and a razor blade ( fig . 1a ) . as one disc from each patella was used as a reference , the total number of samples in contrast agents was 96 . were used , and four discs from each patella were divided among four concentrations of one contrast agent . thus , for each concentration 12 discs from 12 different patellae were prepared . in the end , this was either because some discs were excluded due to image artifacts or because they were not stable in the sample holder during immersion . cartilage surrounding the detached discs , that is , the remainder of the lateral upper quadrant of patellae ( fig . ( a ) cartilage discs were detached from the lateral upper quadrant of a bovine patella within 6 hours after slaughtering . ( b ) a basin carried six sample holders with one cartilage disc in each and contained 50 ml contrast agent solution adjusted to have isotonic osmolarity . ( c ) a cotton wad soaked in physiological saline was placed inside the tube to keep the microenvironment fully moist . a rigid polytetrafluoroethylene tube inside the silicone tube supported the cartilage disc during the experiment . ( d ) the peripheral quantitative computed tomography ( pqct ) slice thickness was 2.3 mm , and the in - plane pixel size was 0.20  0.20 mm . each slice was imaged five times and averaged for each time point to improve the signal - to - noise ratio . discs were mounted in a custom - made sample holder where contrast agent penetration was allowed only through the articular surface ( fig . all solutions contained inhibitors for proteolytic enzymes ( 5 mm ethylenediamine - tetraacetic acid disodium salt [ edta ] ; vwr international s.a.s . , fontenay , france ) and bacterial growth ( 100 units / ml penicillin , 100 g / ml streptomycin ; euroclone spa , siziano , italy ) . the samples were imaged with a clinical pqct device ( xct 2000 , stratech medizintechnik gmbh , pforzheim , germany ) without contrast agent , and after 1 , 5 , 9 , 16 , 25 and 29 hours in the contrast agent bath ( fig . 1b and d ) . tube voltage of 58 kvp , slice thickness of 2.3 mm , and pixel size of 0.20  0.20 mm were applied . subsequently , the cartilage discs were fixed in 10% formalin before processing the samples for histological analyses . two contrast agents were used , each at four different concentrations : hexabrix ( mallinckrodt inc . , st . louis , mo ) and sodium iodide ( sigma - aldrich inc . , st . the hexabrix dissociates into two anionic ioxaglate ( m = 1,269 g / mol ) , one meglumine ( m = 195 g / mol ) , and one sodium ( na , m = 23 g / mol ) molecule . sodium iodide dissociates into sodium ( na ) and iodide ( i , m = 127 g / mol ) in physiological saline . the ioxaglate molecule contains six iodine atoms and produces six times greater x - ray attenuation than iodide . hence , the 50 ml volume of phosphate buffered saline contained 5 , 10 , 21 , or 50 mm ioxaglate or 30 , 60 , 126 , or 300 mm sodium iodide . solutions were adjusted to isotonic osmolarity ( 295 - 320 mosm ) and ph 7.4 . however , in sodium iodide at 300 mm the high contrast agent concentration induced hyperosmolarity ( 625 mosm ) . the mean x - ray attenuation profile of native cartilage was subtracted from the x - ray attenuation profiles acquired during immersion in contrast agent . after that the attenuation inside the cartilage disc was normalized with the contrast agent attenuation in the bath . subsequently , mean surface - to - deep - cartilage partitioning profiles as well as volume - averaged partitioning were calculated as described previously . however , there is no need to calculate the actual concentrations for the present analysis , but it can be done , for example , with equations found in silvast et al . contrast agent partition coefficient was calculated at every time point as the ratio of contrast agent  induced attenuation in the cartilage and bath solution , c / cbath  100% . note that , for simplicity , the term partition is used here to refer to normalized solute concentrations during immersion and not only at complete equilibrium . diffusion coefficients were calculated using a one - dimensional ( 1d ) finite element ( fe ) model based on the fick s law ( comsol inc . , the model was fitted to volume - averaged contrast agent concentrations at each time point to determine the diffusion coefficient ( fig . 3 ) . water , uronic acid , and hydroxyproline contents in the tissue were analyzed from fresh cartilage surrounding the detached discs , that is , the remainder of the lateral upper quadrant of the patella ( fig . water contents were determined from the difference between wet and dry weights after lyophilization for 17 hours . lyophilized samples were incubated with 1 mg / ml papain ( sigma ) in 150 mm sodium acetate including 50 mm cysteine hydrochloride ( cys - hcl , sigma ) and 5 mm ethylenediaminetetra - acetic acid ( edta , vwr ) , ph 6.5 , and 60 c for 3 hours . a spectrophotometric assay for hydroxyproline was performed after hydrolysis of the freeze - dried and papain - digested tissue . the amounts of uronic acid and hydroxyproline were normalized to wet weights to compensate for variation in sample sizes . thickness of cartilage discs were measured with an optical microscope ( smz-10 , nikon corporation , tokyo , japan ) . irvine , ca ) stained histological sections via optical density measurement using a light microscope ( ernst leitz gmbh , wetzlar , germany ) . for practical reasons , only the samples immersed in the highest contrast agent concentrations ( 50 mm ioxaglate and 300 mm iodide ) were analyzed . in the analysis , the width of the region of interest was 650 m and its height was the full cartilage thickness . the exact wilcoxon signed ranks test for two related samples was used to investigate significance of differences in contrast agent partition coefficients , partitioning profiles , and diffusion coefficients between the different contrast agent bath concentrations . wallis test was used to investigate the differences in contrast agent partition and diffusion coefficients , and in reference parameters between experimental sample groups . there were no significant differences in diffusion or partition coefficients ( at 29-hour time point ) between different contrast agent concentrations ( table 1 ) . the diffusion of iodide was significantly ( p  0.001 ) faster than that of ioxaglate with ten times higher mass ( table 1 , fig . the average diffusion coefficients at 20 c were 92  46 m / s ( mean  standard deviation ) and 473  133 m / s for ioxaglate and iodide , respectively . the volume averaged partition coefficient ( at 29-hour time point ) of iodide ( 71  5% ) was greater ( p < 0.001 ) than that of ioxaglate ( 62  6% ) ( table 1 ) . the difference in partition coefficient for ioxaglate and iodide was significant ( p  0.02 ) between all concentrations , except ioxaglate concentrations 21 mm and iodide concentration of 30 mm . diffusion coefficients ( d ) and contrast agent partition coefficients ( at 29 hours ) as a function of contrast agent bath concentration at 20 c note : there were no significant differences in diffusion coefficients or volume - averaged partition coefficients between different contrast agent concentrations . ( a , b ) volume - averaged x - ray attenuation ( mean  sd , arbitrary units , a.u . ) in full - thickness bovine articular cartilage discs as a function of immersion time in contrast agent solutions . ( c , d ) volume - averaged partition coefficients ( % ) of contrast agents as a function of immersion time . iodide reached diffusion equilibrium faster than ioxaglate , and its near - equilibrium partition coefficient ( at 29 hours ) was significantly higher than that of ioxaglate . representative experimental ( mean  standard deviation ) and simulated normalized contrast agent concentrations as function of time . the presented data are from iodide 126 mm and ioxaglate 21 mm measurements . the shapes and levels of the contrast agent concentration distributions were similar ( fig . 4 ) . in general , the partition coefficient of iodide was greater than that of ioxaglate at every time point , although the difference decreased with immersion time . furthermore , at low concentrations the contrast agent distributions were less uniform , as revealed by higher standard deviations , compared with those with higher contrast agent concentrations . mean contrast agent partition profiles (  sd ) in bovine articular cartilage at different time points ( 1 , 5 , and 29 hours ) . the partition coefficients ( c / cbath  100% ) of iodide were systematically greater than those of ioxaglate , albeit the difference reduced with time . as the samples were of different thicknesses the individual distributions were normalized , and only relative distances are presented . cartilage composition in the two sample groups was found to be similar except for small but significant difference in the uronic acid contents ( table 2 ) . composition and thickness of samples immersed in ioxaglate or iodide note : statistically significant difference ( exact kruskall wallis test ) between the sample groups was revealed only in the uronic acid content . the average optical density profiles , that is , pg distribution profiles , of native samples in different sample groups were similar ( fig . 5 ) . however , the average optical density of the sample group immersed in 300 mm iodide after 29-hour immersion in contrast agent bath was higher ( p = 0.04 ) than that of adjacent native samples . in samples immersed for 29 hours in 50 mm ioxaglate solution , the average optical density was similar to that of the native samples in the superficial and transitional zones but lower ( p = 0.001 ) in the deep cartilage . the light microscopic images of safranin o  stained sections showed no or only minor pg loss after 29-hour immersion ( fig . 6 ) . optical density ( mean  sd ) , that is , proteoglycan distribution in native safranin o  stained samples ( open circles ) and adjacent samples immersed for 29 hours ( closed triangles ) in either 50 mm ioxaglate ( left ) or 300 mm iodide ( right ) . on the x - axis  surface the sections are highly identical , and no significant proteoglycan loss after immersion of 29 hours can be detected . the primary aim of this study was to examine the effect of contrast agent concentration on its diffusion properties in articular cartilage . in clinical cect , the contrast agent concentration in the joint capsule will vary between patients . this is due to variation in the amount of synovial fluid and the state of inflammation . furthermore , contrast agents of various concentrations ( ranging from 1 to 400 mm ) have been used . in theory , concentration of a solute can affect its partition coefficient . however , the experimental data published on this issue are rather contradictory , and there is no general agreement on the effect of contrast agent concentration on its diffusive properties . to investigate this issue , the effects of ioxaglate and iodide concentrations on their diffusion and temporal distribution in bovine articular cartilage were studied in vitro . in contrast to earlier studies with gels , this study was unable to demonstrate that increase in solute concentration increases its partition coefficient in cartilage . furthermore , in the present study we did not see statistically significant differences in volume - averaged diffusion coefficients as contrast agent concentrations were varied . a possible explanation for this might be that although concentration dependence of diffusion exists in most systems , often in diluted solutions the dependence is only minor . clinically , it is crucial that contrast agent concentration does not influence the reliability of cect imaging . the osmolarities of the contrast agent solutions were adjusted to be physiological , that is , 300 mosm . as an exception possibly , this could drag some water out of the cartilage , and consequently change the cartilage volume . based on a previous study , cartilage shrinkage in hyperosmolar solution is rather small ( 4% to 7% ) . this , together with limited resolution of the applied clinical pqct scanner ( pixel size 0.20 mm ) , may have masked the possible changes in cartilage volumes . in clinical use , this could lead to dramatic consequences provided that the joint is extensively loaded immediately after the contrast agent enhanced imaging . the average diffusion coefficient of negatively charged iodide ( m = 127 g / mol ) was 473 m / s , which compares well with the one reported in a previous study , 475 m / s . the present value for average diffusion coefficient of ioxaglate ( charge = 1 , m = 1,269 g / mol ) was 92 m / s , which is lower than that reported previously ( 143 m / s ) . the differences between the diffusion coefficients of molecules with identical charge but with different sizes clearly reveal the effect of steric hindrance in cartilage matrix . as a consequence , the increasing size of a mobile molecule slows down diffusion and can delay the time window for optimal clinical contrast enhanced imaging . in this study , no systematic differences in volume - averaged partition coefficients at 1- or 29-hour time points between different contrast agent concentrations were seen . however , there were sporadic differences between ioxaglate concentrations at 5- , 16- , and 25-hour time points , and between iodide concentrations at 9- and 25-hour time points . the reason for this is not clear , but it may have arisen from limited sample number ( average n = 9/concentration ) and relatively poor signal - to - noise ratio . as expected , the partition coefficient of iodide was greater than that of the substantially larger ioxaglate . this result is consistent with the earlier observations , which indicate that the partition coefficient decreases with increasing macromolecular size . in addition , our measurements showed that the partition coefficients of ioxaglate and iodide decrease toward the deep cartilage . these findings are in line with the earlier reports , indicating that the partition coefficient decreases with increasing matrix concentration , and reflects variation in cartilage water content . this is also in accordance with the observation that increased collagen cross - linking decreases contrast agent penetration in cartilage . it is also interesting to compare figure 4 with observations of torzilli , who found distributions of similar shape and level with neutral mobile molecules . ioxaglate and iodide are both anionic molecules with the same charge . as mentioned in the literature , the donnan equilibrium is believed to satisfactorily explain the transport properties of ionic species in liquids . in the ideal donnan equilibrium , the partition coefficients of contrast agents of different size but with same charge should be equal , when the effects of molecule size and hydration on electrostatic interactions are excluded . in the present study , the partition coefficients of small iodide were systematically greater than those of ioxaglate , albeit the difference reduced with time . this indicates that tissue fcd and the electric charge of the solute molecule do not fully account for diffusion and distribution of the charged contrast agent in cartilage . furthermore , this shows the effect of steric hindrance in cartilage matrix . traditionally , pgs have been thought to form the small pore network and collagens to have smaller role in controlling the tissue permeability . however , based on numerical modeling , it has been suggested that collagens also could have a significant role in cartilage permeability . furthermore , in an earlier study almost complete pg cleavage was induced using trypsin . in that study , it was found that contrast agent diffusion in the pg - cleaved cartilage matrix was still almost as slow as in intact tissue . these findings support the idea that collagens also have significant contribution on diffusion of charged contrast agents in articular cartilage . consistent with earlier studies , diffusion of contrast agents took several hours to reach equilibrium . increasing attenuation even after 16 hours of diffusion could be seen with the highest ioxaglate concentrations . with the most concentrated iodide solutions the near - equilibrium was reached only after 5 hours , but the attenuation was still seen to slightly increase even after 9 hours of diffusion . fluid flow related to dynamic loading has been shown to enhance solute transport in articular cartilage , being especially important for large solutes . in clinical situations , it may be essential to induce advection in articular cartilage in order to accelerate contrast agent penetration , since contrast agents begin to be excreted within less than few hours . this is already the case in clinical dgemric protocols , in which 10-minute exercise is typically performed immediately after intravenous injection of contrast agent . although inhibitors of proteolytic enzymes were added to contrast agent solutions , the lengthy immersion time may jeopardize the structure and composition of the cartilage samples . however , optical density measurements showed only minor pg loss in the deep tissue of samples immersed in ioxaglate for 29 hours , and no pg loss after 29 hours in the iodide immersion . instead , the results suggest that iodide increased optical density after contrast agent immersion . this result may be explained by the fact that safranin o binds stoichiometrically to a negative charge in cartilage tissue . as a result , the remnants of negative contrast agent may bind safranin o and raise the apparent pg content determined by using the measurements of optical density . if this happens , the effect should be highlighted at the highest concentrations of the contrast agent . however , the natural spatial variation in gag content could also coincidentally contribute to this finding . brownian motion becomes quicker and viscosity of contrast agent solutions decrease at higher temperatures , experiments conducted at body temperature would likely show accelerated contrast agent diffusion . thus , the present results may not directly translate to the in vivo situation . in summary , in addition , the partition coefficient of iodide was greater than that of ioxaglate , and the difference was maximal at 1 hour after immersion . moreover , the averaged partitioning for full - thickness cartilage was independent of the bath solute concentration . clinically , it is important that variation in contrast agent concentration between patients and anatomical locations do not jeopardize the diagnostic reliability of cect imaging .
objective : differences in contrast agent diffusion reflect changes in composition and structure of articular cartilage . 
 however , in clinical application the contrast agent concentration in the joint capsule varies , which may affect the reliability of contrast enhanced cartilage tomography ( cect ) . in the present study , effects of concentration of x - ray contrast agents on their diffusion and equilibrium distribution in cartilage were investigated.design:full-thickness cartilage discs ( d = 4.0 mm , n = 120 ) were detached from bovine patellae ( n = 24 ) . 
 the diffusion of various concentrations of ioxaglate ( 5 , 10 , 21 , 50 mm ) and iodide ( 30 , 60 , 126 , 300 mm ) was allowed only through the articular surface . 
 samples were imaged with a clinical peripheral quantitative computed tomography scanner before immersion in contrast agent , and after 1 , 5 , 9 , 16 , 25 , and 29 hours in the bath.results:diffusion and partition coefficients were similar between different contrast agent concentrations . 
 the diffusion coefficient of iodide ( 473  133 m2/s ) was greater ( p  0.001 ) than that of ioxaglate ( 92  46 m2/s ) . 
 in full - thickness cartilage , the partition coefficient ( at 29 h ) of iodide ( 71  5% ) was greater ( p  0.02 with most concentrations ) than that of ioxaglate ( 62  6%).conclusions : significant differences in partition and diffusion coefficient of two similarly charged ( 1 ) contrast agents were detected , which shows the effect of steric interactions . 
 however , the increase in solute concentration did not increase its partition coefficient . in clinical application , it is important that contrast agent concentration does not affect the interpretation of cect imaging .