Static compression is associated with decreased diffusivity of dextrans in cartilage explants

The chondrocytes of adult articular cartilage rely upon transport phenomena within their avascular extracellular matrix for many biological activities. Therefore, changes in matrix structure which influence cytokine transport parameters may be an important mechanism involved in the chondrocyte response to tissue compression. With this hypothesis in mind, partitioning and diffusion of 3-, 10-, and 40-kDa dextrans conjugated to tetramethylrhodamine, and 430-Da tetramethylrhodamine itself, were measured within statically compressed bovine articular cartilage explants using a novel experimental apparatus and desorption fluorescence method. Partitioning and diffusion were examined as functions of solute molecular weight and matrix proteoglycan density, and diffusion was measured versus static compression up to 35% volumetric strain. In general, partition coefficients and diffusivities were found to decrease with increasing solute molecular weight. In addition, for a given solute, diffusivities decreased significantly with increasing static compression. Results therefore suggest a possible role for transport limitations of relatively large molecular weight solutes within the extracellular matrix in mediating the biological response of chondrocytes to cartilage compression.     

Dynamic compression augments interstitial transport of a glucose-like solute in articular cartilage

Solute transport through the extracellular matrix is essential for cellular activities in articular cartilage. Increased solute transport via fluid convection may be a mechanism by which dynamic compression stimulates chondrocyte metabolism. However, loading conditions that optimally augment transport likely vary for different solutes. To investigate effects of dynamic loading on transport of a bioactive solute, triangular mechanical loading waveforms were applied to cartilage explants disks while interstitial transport of a fluorescent glucose analog was monitored. Peak-to-peak compression amplitudes varied from 5-50% and frequencies varied from 0.0006-0.1 Hz to alter the spatial distribution and magnitude of oscillatory fluid flow. Solute transport was quantified by monitoring accumulation of fluorescence in a saline bath circulated around the explant. Individual explants were subjected to a series of compression protocols, so that effects of loading on solute desorption could be observed directly. Maximum increases in solute transport were obtained with 10-20% compression amplitudes at 0.1 Hz; similar loading protocols were previously found to stimulate chondrocyte metabolism in vitro. Results therefore support hypotheses relating to increased solute transport as a mediator of the cartilage biological response to dynamic compression, and may have application in mechanical conditioning of cartilage constructs for tissue engineering.     

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response.

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly (p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly (p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.     

Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis

The biomechanical functions of articular cartilage are governed largely by the composition and density of its specialized extracellular matrix. Relationships between matrix density and functional indices such as mechanical properties or interstitial solute diffusivities have been previously explored. However, direct correlations between mechanical properties and solute transport parameters have received less attention, despite potential application of this information for cartilage functional assessment both in vivo and in vitro. The objective of this study was therefore to examine relationships among solute diffusivities, mechanical properties, and matrix density of compressed articular cartilage. Matrix density varied due to natural variation among explants and due to applied static compression. Matrix density of statically compressed cartilage explants was characterized by glycoaminoglycan (GAG) weight fraction and fluid volume fraction, while diffusion coefficients of a wide range of solutes were measured to characterize the transport environment. Explant mechanical properties were characterized by a non-linear Young's modulus (axial stress-strain ratio) and a non-linear Poisson's ratio (radial-to-axial strain ratio). Solute diffusivities were consistently correlated with Young's modulus, as well as with explant GAG weight and fluid volume fractions. Therefore, in vitro mechanical tests may provide a means of assessing transport environments in cartilage-like materials, while in vivo measurements of solute transport (for example with magnetic resonance imaging) may be a useful complement in identifying localized differences in matrix density and mechanical properties.     

Modeling of neutral solute transport in a dynamically loaded porous permeable gel: implications for articular cartilage biosynthesis and tissue engineering

A primary mechanism of solute transport in articular cartilage is believed to occur through passive diffusion across the articular surface, but cyclical loading has been shown experimentally to enhance the transport of large solutes. The objective of this study is to examine the effect of dynamic loading within a theoretical context, and to investigate the circumstances under which convective transport induced by dynamic loading might supplement diffusive transport. The theory of incompressible mixtures was used to model the tissue (gel) as a mixture of a gel solid matrix (extracellular matrix/scaffold), and two fluid phases (interstitial fluid solvent and neutral solute), to solve the problem of solute transport through the lateral surface of a cylindrical sample loaded dynamically in unconfined compression with frictionless impermeable platens in a bathing solution containing an excess of solute. The resulting equations are governed by nondimensional parameters, the most significant of which are the ratio of the diffusive velocity of the interstitial fluid in the gel to the solute diffusivity in the gel (Rg), the ratio of actual to ideal solute diffusive velocities inside the gel (Rd), the ratio of loading frequency to the characteristic frequency of the gel (f), and the compressive strain amplitude (epsilon 0). Results show that when Rg > 1, Rd < 1, and f > 1, dynamic loading can significantly enhance solute transport into the gel, and that this effect is enhanced as epsilon 0 increases. Based on representative material properties of cartilage and agarose gels, and diffusivities of various solutes in these gels, it is found that the ranges Rg > 1, Rd < 1, correspond to large solutes, whereas f > 1 is in the range of physiological loading frequencies. These theoretical predictions are thus in agreement with the limited experimental data available in the literature. The results of this study apply to any porous hydrated tissue or material, and it is therefore plausible to hypothesize that dynamic loading may serve to enhance solute transport in a variety of physiological processes.     

Preservation and analysis of nonequilibrium solute concentration distributions within mechanically compressed cartilage explants

Solute transport within articular cartilage is of central importance to tissue physiology, and may mediate effects of mechanical compression on cell metabolism. We therefore developed and applied a freeze-substitution method for fixation of cartilage explant disks which had been compressed axially during radial solute desorption. Dextrans were used as model solutes. Explant morphology was well preserved and nonequilibrium solute concentration distributions were stable for several hours at room temperature. For desorption from explants compressed statically to 0-46% strain, analysis of laser confocal images and comparison to a theoretical model permitted measurement of effective diffusivities. Results were consistent with previous studies suggesting a role for transport limitations in mediating the decreases of chondrocyte metabolic rates associated with static compression. In explants compressed dynamically (23+/-5% strain at 0.001 Hz), evidence was obtained for the augmentation of effective transport rate of 3 kDa dextrans by oscillatory interstitial fluid flows. This suggests that augmented solute transport may play a role in mediating the increases of chondrocyte metabolic rates associated with dynamic compression. Methods appear suitable for quantitative studies of transport within mechanically compressed cartilage-like tissues, and may be valuable for identification of loading environments which optimize solute transport in tissue engineering applications.     

Effects of tension-compression nonlinearity on solute transport in charged hydrated fibrous tissues under dynamic unconfined compression

Cartilage is a charged hydrated fibrous tissue exhibiting a high degree of tension-compression nonlinearity (i.e., tissue anisotropy). The effect of tension-compression nonlinearity on solute transport has not been investigated in cartilaginous tissue under dynamic loading conditions. In this study, a new model was developed based on the mechano-electrochemical mixture model [Yao and Gu, 2007, J. Biomech. Model Mechanobiol., 6, pp. 63-72, Lai et al., 1991, J. Biomech. Eng., 113, pp. 245-258], and conewise linear elasticity model [Soltz and Ateshian, 2000, J. Biomech. Eng., 122, pp. 576-586; Curnier et al., 1995, J. Elasticity, 37, pp. 1-38]. The solute desorption in cartilage under unconfined dynamic compression was investigated numerically using this new model. Analyses and results demonstrated that a high degree of tissue tension-compression nonlinearity could enhance the transport of large solutes considerably in the cartilage sample under dynamic unconfined compression, whereas it had little effect on the transport of small solutes (at 5% dynamic strain level). The loading-induced convection is an important mechanism for enhancing the transport of large solutes in the cartilage sample with tension-compression nonlinearity. The dynamic compression also promoted diffusion of large solutes in both tissues with and without tension-compression nonlinearity. These findings provide a new insight into the mechanisms of solute transport in hydrated, fibrous soft tissues.     

Site-specific effects of compression on macromolecular diffusion in articular cartilage

Articular cartilage is the connective tissue that lines joints and provides a smooth surface for joint motion. Because cartilage is avascular, molecular transport occurs primarily via diffusion or convection, and cartilage matrix structure and composition may affect diffusive transport. Because of the inhomogeneous compressive properties of articular cartilage, we hypothesized that compression would decrease macromolecular diffusivity and increase diffusional anisotropy in a site-specific manner that depends on local tissue strain. We used two fluorescence photobleaching methods, scanning microphotolysis and fluorescence imaging of continuous point photobleaching, to measure diffusion coefficients and diffusional anisotropy of 70 kDa dextran in cartilage during compression, and measured local tissue strain using texture correlation. For every 10% increase in normal strain, the fractional change in diffusivity decreased by 0.16 in all zones, and diffusional anisotropy increased 1.1-fold in the surface zone and 1.04-fold in the middle zone, and did not change in the deep zone. These results indicate that inhomogeneity in matrix structure and composition may significantly affect local diffusive transport in cartilage, particularly in response to mechanical loading. Our findings suggest that high strains in the surface zone significantly decrease diffusivity and increase anisotropy, which may decrease transport between cartilage and synovial fluid during compression.     

Validation of theoretical framework explaining active solute uptake in dynamically loaded porous media

Solute transport in biological tissues is a fundamental process necessary for cell metabolism. In connective soft tissues, such as articular cartilage, cells are embedded within a dense extracellular matrix that hinders the transport of solutes. However, according to a recent theoretical study (Mauck et al., 2003, J. Biomech. Eng. 125, 602–614), the convective motion of a dynamically loaded porous solid matrix can also impart momentum to solutes, pumping them into the tissue and giving rise to concentrations which exceed those achived under passive diffusion alone. In this study, the theoretical predictions of this model are verified against experimental measurements. The mechanical and transport properties of an agarose–dextran model system were characterized from independent measurements and substituted into the theory to predict solute uptake or desorption under dynamic mechanical loading for various agarose concentrations and dextran molecular weights, as well as different boundary and initial conditions. In every tested case, agreement was observed between experiments and theoretical predictions as assessed by coefficients of determination ranging from R²=0.61 to 0.95. These results provide strong support for the hypothesis that dynamic loading of a deformable porous tissue can produce active transport of solutes via a pumping mechanisms mediated by momentum exchange between the solute and solid matrix.     

Convection and Diffusion in Charged Hydrated Soft Tissues: A Mixture Theory Approach

The extracellular matrix of cartilage is a charged porous fibrous material. Transport phenomena in such a medium are very complex. In this study, solute diffusive flux and convective flux in porous fibrous media were investigated using a continuum mixture theory approach. The intrinsic diffusion coefficient of solute in the mixture was defined and its relation to drag coefficients was presented. The effect of mechanical loading on solute diffusion in cartilage under unconfined compression with a frictionless boundary condition was analyzed numerically using the model developed. Both strain-dependent hydraulic permeability and diffusivity were considered. Analyses and results show that (1) In porous media, the convective velocity for each solute phase is different. (2) The solute convection in tissue is governed by the relative convective velocity (i.e., relative to solid velocity). (3) Under the assumption that all the frictional interactions among solutes are negligible, the relative convective velocity for α-solute phase is equal to the relative solvent velocity multiplied by its convective coefficient (H ^α) which is also known as the hindrance factor in the literature. The relationship between the convective coefficient and the relative diffusivity of solute is presented. (4) Solute concentration profile within the cartilage sample depends on the phase of dynamic compression.     

A Biomechanical Triphasic Approach to the Transport of Nondilute Solutions in Articular Cartilage

Biomechanical models for biological tissues such as articular cartilage generally contain an ideal, dilute solution assumption. In this article, a biomechanical triphasic model of cartilage is described that includes nondilute treatment of concentrated solutions such as those applied in vitrification of biological tissues. The chemical potential equations of the triphasic model are modified and the transport equations are adjusted for the volume fraction and frictional coefficients of the solutes that are not negligible in such solutions. Four transport parameters, i.e., water permeability, solute permeability, diffusion coefficient of solute in solvent within the cartilage, and the cartilage stiffness modulus, are defined as four degrees of freedom for the model. Water and solute transport in cartilage were simulated using the model and predictions of average concentration increase and cartilage weight were fit to experimental data to obtain the values of the four transport parameters. As far as we know, this is the first study to formulate the solvent and solute transport equations of nondilute solutions in the cartilage matrix. It is shown that the values obtained for the transport parameters are within the ranges reported in the available literature, which confirms the proposed model approach.     

Dynamic loading of immature epiphyseal cartilage pumps nutrients out of vascular canals

The potential influence of mechanical loading on transvascular transport in vascularized soft tissues has not been explored extensively. This experimental investigation introduced and explored the hypothesis that dynamic mechanical loading can pump solutes out of blood vessels and into the surrounding tissue, leading to faster uptake and higher solute concentrations than could otherwise be achieved under unloaded conditions. Immature epiphyseal cartilage was used as a model tissue system, with fluorescein (332 Da), dextran (3, 10, and 70 kDa) and transferrin (80 kDa) as model solutes. Cartilage disks were either dynamically loaded (± 10% compression over a 10% static offset strain, at 0.2 Hz) or maintained unloaded in solution for up to 20 h. Results demonstrated statistically significant solute uptake in dynamically loaded (DL) explants relative to passive diffusion (PD) controls for all solutes except unbound fluorescein, as evidenced by the DL:PD concentration ratios after 20 h (1.0 ± 0.2, 2.4 ± 1.1, 6.1 ± 3.3, 9.0 ± 4.0, and 5.5 ± 1.6 for fluorescein, 3, 10, and 70 kDa dextran, and transferrin). Significant uptake enhancements were also observed within the first 30s of loading. Termination of dynamic loading produced dissipation of enhanced solute uptake back to PD control values. Confocal images confirmed that solute uptake occurred from cartilage canals into their surrounding extracellular matrix. The incidence of this loading-induced transvascular solute pumping mechanism may significantly alter our understanding of the interaction of mechanical loading and tissue metabolism.     

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

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.     

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

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.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.     

Effects of dynamic loading on solute transport through the human cartilage endplate

Nutrient and metabolite transport through the cartilage endplate (CEP) is important for maintaining proper disc nutrition, but the mechanisms of solute transport remain unclear. One unresolved issue is the role of dynamic loading. In comparison to static loading, dynamic loading is thought to enhance transport by increasing convection. However, the CEP has a high resistance to fluid flow, which could limit solute convection. Here we measure solute transport through site-matched cadaveric human lumbar CEP tissues under static vs. dynamic loading, and we determine how the degree of transport enhancement from dynamic loading depends on CEP porosity and solute size. We found that dynamic loading significantly increased small and large solute transport through the CEP: on average, dynamic loading increased the transport of sodium fluorescein (376 Da) by a factor of 1.85 ± 0.64 and the transport of a large dextran (4000 Da) by a factor of 4.97 ± 3.05. Importantly, CEP porosity (0.65 ± 0.07; range: 0.47–0.76) strongly influenced the degree of transport enhancement. Specifically, for both solutes, transport enhancement was greater for CEPs with low porosity than for CEPs with high porosity. This is because the CEPs with low porosity were susceptible to larger improvements in fluid flow under dynamic loading. The CEP becomes less porous and less hydrated with aging and as disc degeneration progresses. Together, these findings suggest that as those changes occur, dynamic loading has a greater effect on solute transport through the CEP compared to static loading, and thus may play a larger role in disc nutrition.     

Temperature affects transport of polysaccharides and proteins in articular cartilage explants

Solute transport phenomena mediate many aspects of the physiology and contrast agent-based clinical imaging of articular cartilage. Temperatures up to 10°C below standard body temperature (37°C) are common in articulating joints during normal activities and clinically (e.g. cold treatment of injuries). Therefore it is of interest to characterize the effects of temperature changes on solute transport parameters in cartilage. A range of fluorescent solutes including fluorescein isothiocyanate, 4 and 40kDa dextrans, myoglobin, insulin and chondroitin sulfate were prepared and used in assays of solute effective partition coefficient and effective diffusivity in bovine intermediate zone articular cartilage explants maintained at 10, 22 or 37°C. Trends for increasing partition coefficient with increasing temperature were evident for all solutes except chondroitin sulfate, with significant changes between 22 and 37°C for 4kDa dextran, insulin and myoglobin. Diffusivities of most solutes tested also tended to increase with increasing temperature, with significant changes between 10 and 22°C for FITC, 40kDa dextran and myoglobin. Oddly, insulin diffusivity decreased significantly as temperature increased from 22 to 37°C while chondroitin sulfate diffusivity exhibited no clear temperature dependence. These results highlight solute-specific temperature dependences of transport phenomena which may depend upon molecular weight, chemical structure, molecular conformation, and solute-matrix and solute-solute interactions. The articular cartilage explants themselves exhibited small but significant changes in water and glycosaminoglycan contents during experiments, underscoring the importance of solute-matrix interactions. Solute transport parameters in cartilage and their temperature dependences are therefore not easily predicted, and case-by-case experimental determination may be essential.     

Tensorial electrokinetics in articular cartilage

Electrokinetic phenomena contribute to biomechanical functions of articular cartilage and underlie promising methods for early detection of osteoarthritic lesions. Although some transport properties, such as hydraulic permeability, are known to become anisotropic with compression, the direction-dependence of cartilage electrokinetic properties remains unknown. Electroosmosis experiments were therefore performed on adult bovine articular cartilage samples, whereby fluid flows were driven by electric currents in directions parallel and perpendicular to the articular surface of statically compressed explants. Magnitudes of electrokinetic coefficients decreased slightly with compression (from approximately -7.5 microL/As in the range of 0-20% compression to -6.0 microL/As in the 35-50% range) consistent with predictions of microstructure-based models of cartilage material properties. However, no significant dependence on direction of the electrokinetic coupling coefficient was detected, even for conditions where the hydraulic permeability tensor is known to be anisotropic. This contrast may also be interpreted using microstructure-based models, and provides insights into structure-function relationships in cartilage extracellular matrix and physical mediators of cell responses to tissue compression. Findings support the use of relatively simple isotropic modeling approaches for electrokinetic phenomena in cartilage and related materials, and indicate that measurement of electrokinetic properties may provide particularly robust means for clinical evaluation of cartilage matrix integrity.     

The distributions and diffusivities of small ions in chondroitin sulphate, hyaluronate and some proteoglycan solutions

The distributions and diffusivities of Na+, Ca2+ and Cl- in chondroitin sulphate (CS), hyaluronate (HA) and proteoglycan solutions were measured using equilibrium dialysis and a capillary tube method. Measurements were made for a range of glycosaminoglycan (GAG) concentrations up to those normally found in dense connective tissue (10% CS, 2.5% HA), ionic strengths up to normal physiological concentrations (0.15 M) and for different combinations of monovalent and divalent cations. The partition coefficients, Ki, of the positive ions increased with increasing matrix concentration and with decreasing ionic strength but with one exception the selectivity coefficient KCaNa = square root of KCa/KNa was close to unity, indicating nearly ideal Donnan distributions. The ionic diffusivities decreased very much like those of small neutral solutes with increasing matrix concentration and with one exception were relatively independent of ionic strength, The exception in both cases was low matrix concentrations and low ionic strengths for which the diffusivity of Ca2+ was an order of magnitude lower and selectivity coefficients were approximately 2. We conclude that at physiological ionic strengths and GAG concentrations the distributions of small ions are determined by simple electrostatic interactions, without binding or condensation, and the diffusivities are not affected by the electrostatic field.