A method for measuring intra-tissue swelling pressure using a needle micro-osmometer

Abstract

The intervertebral disc's ability to resist load and facilitate motion arises largely from osmotic swelling pressures that develop within the tissue. Changes in the disc's osmotic environment, diurnally and with disease, have been suggested to regulate cellular activity, yet knowledge of in vivo osmotic environments is limited. Therefore, the first objective of this study was to demonstrate proof-of-concept for a method to measure intra-tissue swelling pressure and osmolality, modeling micro-osmometer fluid flux using Darcy's law. The second objective was to compare flux-based measurements of the swelling pressure within nucleus pulposus (NP) tissue against ionic swelling pressures predicted by Gibbs-Donnan theory. Pressures (0.03- 0.57 MPa) were applied to NP tissue (n = 25) using equilibrium dialysis, and intra-tissue swelling pressures were measured using flux. Ionic swelling pressures were determined from inductively coupled plasma optical emission spectrometry measurements of intra-tissue sodium using Gibbs-Donnan calculations of fixed charge density and intra-tissue chloride. Concordance of 0.93 was observed between applied pressures and flux- based measurements of swelling pressure. Equilibrium bounds for effective tissue osmolalities engendered by a simulated diurnal loading cycle (0.2-0.6 MPa) were 376 and 522 mOsm/kg H2O. Significant differences between flux and Gibbs-Donnan measures of swelling pressure indicated that total tissue water normalization and non-ionic contributions to swelling pressure were significant, which suggested that standard constitutive models may underestimate intra-tissue swelling pressure. Overall, this micro-osmometer technique may facilitate future validations for constitutive models and measurements of variation in the diurnal osmotic cycle, which may inform studies to identify diurnal- and disease-associated changes in mechanotransduction.

Fig. 1.. System schematic.

Depiction of probe-tissue microdialysis, with variables defined for application of Darcy’s law for radial flow.

Fig. 2.. Probe membrane permeability.

Schematic of setup for determining probe membrane permeabilities under known applied pressures, with variables defined for application of Darcy’s law for radial flow.

Fig. 3.. Membrane, tissue, and effective total permeabilities.

(a) Probe membrane permeabilities measured under known osmotic pressures, applied using polyethylene glycol (PEG). Gray bars denote 95 % confidence intervals. (b) Radial stretch ratios used to approximate tissue permeability, given changes in porosity with applied pressure. (c) Membrane, tissue, and effective total permeabilities calculated at each applied pressure (averages with SD).

Fig. 4.. Tissue composition from ICP-OES and Gibbs-Donnan.

(a) Tissue hydration following equilibration under osmotic pressure. (b) Intra-tissue sodium concentrations determined from ICP-OES and tissue hydration. (c) Tissue fixed charge densities for bovine NP in this study, calculated from Gibbs-Donnan equations. (d) Overlay of calculated fixed charge densities with those measured for human NP tissue. Black line represents FCD in mEq/EFW as presented by Urban and McMullin (1985). Figure reprinted from Urban JPG, McMullin JF (1985), with permission from IOS Press.

Fig. 5.. Tissue swelling pressures and osmolalities.

(a) Concordance between total tissue swelling pressures calculated from flux measurements and osmotic pressures applied using PEG during equilibrium dialysis. (b) Comparison of swelling pressures from flux and Gibbs-Donnan based measurements, as a function of FCD. (c) Concentration of tissue osmolytes calculated from flux measurements and Gibbs-Donnan equations. (*) indicates significant effect of method for Two-Way ANOVA, p < 0.001 (flux-based vs. Gibbs-Donnan ionic). (d) Comparison of swelling pressures from each method. Groups that do not share a letter are significantly different. Percentages indicate the portion of the flux swelling pressures accounted for by Donnan swelling.