Viscoelastic Properties of Hyaluronan in Physiological Conditions

Abstract

Hyaluronan (HA) is a high molecular weight glycosaminoglycan of the extracellular matrix (ECM), which is particularly abundant in soft connective tissues. Solutions of HA can be highly viscous with non-Newtonian flow properties. These properties affect the movement of HA-containing fluid layers within and underlying the deep fascia. Changes in the concentration, molecular weight, or even covalent modification of HA in inflammatory conditions, as well as changes in binding interactions with other macromolecules, can have dramatic effects on the sliding movement of fascia. The high molecular weight and the semi-flexible chain of HA are key factors leading to the high viscosity of dilute solutions, and real HA solutions show additional nonideality and greatly increased viscosity due to mutual macromolecular crowding. The shear rate dependence of the viscosity, and the viscoelasticity of HA solutions, depend on the relaxation time of the molecule, which in turn depends on the HA concentration and molecular weight. Temperature can also have an effect on these properties. High viscosity can additionally affect the lubricating function of HA solutions. Immobility can increase the concentration of HA, increase the viscosity, and reduce lubrication and gliding of the layers of connective tissue and muscle. Over time, these changes can alter both muscle structure and function. Inflammation can further increase the viscosity of HA-containing fluids if the HA is modified via covalent attachment of heavy chains derived from Inter-α-Inhibitor. Hyaluronidase hydrolyzes HA, thus reducing its molecular weight, lowering the viscosity of the extracellular matrix fluid and making outflow easier. It can also disrupt any aggregates or gel-like structures that result from HA being modified. Hyaluronidase is used medically primarily as a dispersion agent, but may also be useful in conditions where altered viscosity of the fascia is desired, such as in the treatment of muscle stiffness.

Keywords: fascia; hyaluronan; lubrication; viscoelasticity; viscosity.

Conflict of interest statement

Competing interests: No competing interests were disclosed.

Figures

Figure 1.. The hydrodynamic size of a hyaluronan chain depends on its molecular weight.

Hyaluronan chains with molecular weight of (from left to right) 0.1, 0.5, 1, 3 and 6 million have hydrodynamic diameters of approximately 50, 140, 210, 400, and 600 nm, respectively in physiological saline solution. The diameter of a small globular protein would be on the order of a few nm. Adapted from Cowman and Matsuoka .

Figure 2.. Specific viscosity of hyaluronan solutions, as a function of the concentration and intrinsic viscosity [η].

Experimental data for hyaluronan in physiological saline, plotted using the fitted equationη sp =c[η] + 0.42(c[η])2 + 7.77 x 10 -3(c[η])4.18 reported by Berriaud and coworkers , shows a marked increase in viscosity with increasing concentration and intrinsic viscosity. (Note that this data represents low shear conditions, where hyaluronan chains are not distorted or aligned with flow.) The experimental data can be compared with predictions based on theory. For an ideal case in which the hyaluronan molecules act independently, the specific viscosity would simply be equal to the productc[η]. When the molecules become crowded, the effective concentration increases, leading to a significant nonideality contribution, predicted by the last three terms of the mutual macromolecular crowding equation, ( Equation 2 in text).

Figure 3.. Model for steric exclusion of a globular protein by a hyaluronan molecule.

A illustrates the ability of small globular proteins to penetrate most of the hydrodynamic domain of the hyaluronan polymer.B shows the size of the excluded volume for a globular protein in the presence of a segment of a linear polymer as a crowding agent. The cross section of the cylindrical excluded volume has a radius equal to the sum of the radius of the crowding polymer and the thickness of a cylindrical shell determined by the radius of the globular protein. This figure has been reproduced with permission from Cowmanet al. (2012) in Structure and Function of Biomatrix. Control of Cell Behavior and Gene Expression. Ed. E.A. Balazs, pp.45–66. Copyright 2012 Matrix Biology Institute.

Figure 4.. Model for mutual macromolecular crowding of hyaluronan molecules.

The effective hydrodynamic domain of each chain is modeled as a sphere, the volume of which is dependent on the molecular weight to the 1.8 power. This figure has been reproduced with permission from Cowmanet al. (2012) in Structure and Function of Biomatrix. Control of Cell behavior and Gene Expression. Ed. E.A. Balazs, pp.45–66. Copyright 2012 Matrix Biology Institute.

Figure 5.. Shear rate dependence of the viscosity of a polydisperse hyaluronan sample (viscosity-average molecular weight = 1.7 million) at a concentration of 5 mg/ml in PBS at 25°C.

Data from three consecutive runs are shown. This figure has been reproduced with permission from Cowmanet al. (2011) Anal. Biochem., 417, 50–56. Copyright 2011 Elsevier Inc.

Figure 6.. Master curves for the elastic modulus (G') and the viscous modulus (G") of a solution of HA with a molecular weight of 2.8 x 10 6 as a function of the frequency of displacement.

This figure has been reproduced with permission from Gibbset al. Biopolymers1968, 6, 777–791. Copyright 1968 John Wiley & Sons, Inc.

Figure 7.. Friction coefficient plotted as a function of fluid viscosity and shear velocity divided by load (Stribeck curve) with corresponding lubrication film thickness.

The schematic shows boundary, mixed, and hydrodynamic lubrication regimes. This figure has been reproduced with permission from Coleset al. (2010) Curr. Opin. Colloid Interface Sci. 15, 406–416. Copyright 2010 Elsevier Ltd.

Figure 8.. Dependence of the cartilage boundary lubricating properties of hyaluronan alone at 3.33 mg/ml on molecular weight.

Regression lines are shown for (A) mean static µ static, Neq friction values at pre-sliding duration, T ps = 1,200 s, and (B) mean kinetic <µ kinetic, Neq> friction values at T ps = 1.2 s obtained vs log molecular weight of hyaluronan. Mean values in phosphate-buffered saline (PBS) and synovial fluid (SF) are shown for reference. This figure has been reproduced with permission from Kwiecinskiet al. (2011) Osteoarthritis Cartilage 19, 1356–1362. Copyright 2011 Osteoarthritis Research Society International.

Figure 9.. Lubricin (rhPRG4) can reduce the viscosity of HA (weight-average molecular weight of 1.5 million) solution when both are present at concentrations found in normal human synovial fluid.

Shear rate dependent specific viscosity at 25°C of hyaluronan at 3.3 mg/ml alone and with 450 μg/ml rhPRG4, shown in black. Predicted specific viscosity (experimental hyaluronan + experimental rhPRG4) shown in red. This figure has been reproduced with permission from Ludwiget al. (2014) Biorheology 51, 409–422. Copyright 2014 IOS Press and the authors.