| Summary: | 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.
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