Enhanced microscopic dynamics in mucus gels under a mechanical load in the linear viscoelastic regime

Mucus is a biological gel covering the surface of several tissues and ensuring key biological functions, including as a protective barrier against dehydration, pathogen penetration, or gastric acids. Mucus biological functioning requires a finely tuned balance between solid-like and fluid-like mechanical response, ensured by reversible bonds between mucins, the glycoproteins that form the gel. In living organisms, mucus is subject to various kinds of mechanical stresses, e.g., due to osmosis, bacterial penetration, coughing, and gastric peristalsis. However, our knowledge of the effects of stress on mucus is still rudimentary and mostly limited to macroscopic rheological measurements, with no insight into the relevant microscopic mechanisms. Here, we run mechanical tests simultaneously to measurements of the microscopic dynamics of pig gastric mucus. Strikingly, we find that a modest shear stress, within the macroscopic rheological linear regime, dramatically enhances mucus reorganization at the microscopic level, as signaled by a transient acceleration of the microscopic dynamics, by up to 2 orders of magnitude. We rationalize these findings by proposing a simple, yet general, model for the dynamics of physical gels under strain and validate its assumptions through numerical simulations of spring networks. These results shed light on the rearrangement dynamics of mucus at the microscopic scale, with potential implications in phenomena ranging from mucus clearance to bacterial and drug penetration.

Mucus is a biogel ubiquitous across both vertebrates and invertebrates (1–3). The main mucus macromolecular components are a family of glycosylated proteins called mucins (4–6). Hydrophobic, hydrogen-bonding, and Ca2+-mediated (7) interactions between mucins are responsible for macromolecular associations determining the viscoelastic properties of mucus, which, in turn, control its biological functions (2, 5). Alteration of the viscoelastic properties compromises mucus functionality, resulting in severe diseases (8, 9).Mucus viscoelasticity stems from the reversible nature of the bonds between its constituents, which ensure solid-like behavior on short time scales while allowing flow on longer time scales. Rheological studies on mucus reporting the frequency dependence of the storage, G′, and loss, G″, components of the dynamic modulus reveal G′>G″, with G′ only weakly dependent on angular frequency ω on time scales of 0.1 to 100 s (8–10), a behavior typical of soft solids (11). Stress-relaxation tests probe viscoelasticity on longer time scales, up to thousands of seconds. They reveal a power law or logarithmic decay of the shear stress with time (12–14), indicative of a wide distribution of relaxation times, ascribed to the variety of macromolecular association mechanisms and the mucus complex, multiscale structure (7, 14, 15).Alongside conventional rheology, microrheology has gained momentum since it investigates the mechanical response of mucus on the length scales relevant to its biological functions, from a fraction of a micrometer up to ∼10 μm (7, 8, 16–19). Microrheology infers the viscoelastic moduli from the microscopic dynamics of tracer particles embedded in the sample (20), either due to spontaneous thermal fluctuations or externally driven, e.g., by a magnetic field. Mucus viscoelasticity as measured by microrheology is found to depend on the size of the tracer particles, the local environment they probe, and the length scale over which their motion is tracked (7, 8, 16, 17, 19, 21). Below ≈1 μm, microrheology data are dominated by the diffusion of the probe particles within the mucus pores, as inferred from the analysis of the localization of the tracers’ trajectories (7, 17), their dependence on probe size (8, 16, 21), and on the amplitude of the external drive in active microrheology (16). On larger length scales, microrheology reports the local viscoelasticity, which converges toward the macroscopic one above ≈10 μm, as revealed by the probe-size and drive-amplitude dependence of active microrheology (16).In vivo, mucus is submitted to stresses of various origin, involving strain on the microscopic scale, as in cilia beating in muco-ciliary clearance (22) and bacterial penetration (23, 24), up to macroscopic scales, e.g., during coughing and peristalsis (3, 25). Stresses due to the osmotic pressure exerted by the environment (26) or resulting from changes in hydration (27, 28) can modify the structure of mucus and, e.g., impair mucus clearance. By contrast, little is known on the impact of stress on the dynamics of mucus, in particular, at the microscopic level. Conventional rheology indicates that mucus is fluidized upon applying a large stress (29, 30), beyond the linear regime. This behavior is typical of soft solids (31–33); in concentrated nanoemulsions and colloidal suspensions and in colloidal gels, fluidization in the nonlinear regime has been shown to stem from enhanced microscopic dynamics (34–40). However, for mucus, we still lack knowledge of the effect of an applied stress on the microscopic dynamics.Here, we couple rheology and light and X-ray photon correlation methods to investigate the microscopic dynamics of pig gastric mucus under an applied shear stress. Surprisingly, we find that small stresses, well within the macroscopic linear viscoelastic regime, transiently enhance the mucus dynamics by up to 2 orders of magnitude. We propose a simple, yet general, model for the dynamics of physical gels under strain that rationalizes these findings.