Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Abstract

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.

FIGURE 1

Direct membrane osmometer. (a) Side view of the stainless steel cylindrical device with microchip pressure transducer. (b) Polymeric solutions are injected into the fluid chamber, and the resulting pressure is measured at the pressure port. Ion and fluid exchange occurs through the buffer portal located in the lid of the device. Dialysis membrane and stainless steel wire-mesh backing for the membrane are not shown.

FIGURE 2

Typical pressure response as a function of time for representative CS and PEG solutions (150 mg/ml).

FIGURE 3

Osmotic pressure of 20 kDa PEG solutions as a function of concentration. Current study (25°C) with corresponding polynomial fit; Wachtel and Maroudas (16) pressure measured at 25°C; Ehrlich et al. (12) pressure measured at 4°C.

FIGURE 4

Equilibrium osmotic pressure of CS-C solutions measured using the DMO as a function of fixed charge density (25°C), in various NaCl concentrations. Solid lines indicate the corresponding virial expansion polynomial fits.

FIGURE 5

Osmotic pressure of CS-A solutions measured at 0.15 and 2 M NaCl. The corresponding CS-C data points are plotted for comparison between the two mixtures of isoforms. Solid lines indicate the corresponding polynomial fits.

FIGURE 6

Osmotic pressure π measured in CS-C solutions of 150 mg/ml, at various NaCl concentrations. The breakdown of into its contributing components is hypothetical and illustrates the possibility that the contribution from configurational entropy decreases with decreasing salt concentration.

FIGURE 7

Osmotic pressure of proteoglycans in bovine articular cartilage at 25°C, in 0.015, 0.15, and 2 M NaCl from the study of Chahine et al. (15), with comparison to CS solutions from this study.

FIGURE 8

Compressive aggregate modulus, of bovine articular cartilage and osmotic modulus (Π, obtained from Eq. 7 using the virial coefficients of Table 2) and intrinsic modulus of the solid matrix ().