Gastropod Slime-Based Gel as an Adjustable Synthetic Model for Human Airway Mucus.

Airway mucus works as a protective barrier in the human body, as it entraps pathogens that will be later cleared from the airways by ciliary transport or by coughing, thus featuring the rheological properties of a highly stretchable gel. Nonetheless, the study of these physical barrier as well as transport properties remains limited due to the restricted and invasive access to lungs and bronchi to retrieve mucus and to the poor repeatability inherent to native mucus samples. To overcome these limits, we report on a biobased synthetic mucus prepared from snail slime and multibranched thiol cross-linker, which are able to establish disulfide bonds, in analogy with the disulfide bonding of mucins, and therefore build viscoelastoplastic hydrogels. The gel macroscopic properties are tuned by modifying the cross-linker and slime concentrations and can quantitatively match those of native sputum from donors with cystic fibrosis (CF) or non-cystic fibrosis bronchiectasis (NCFB) both in the small- and large-deformation regimes. Heterogeneous regimes were locally found in the mucus model by passive microrheology, in which both diffusive and non-diffusive motion are present, similar to what is observed in sputa. The biobased synthetic approach proposed in the present study thus allows to produce, with commercially available components, a promising model to native respiratory mucus regarding both mechanical and, to a lesser extent, physicochemical aspects.

Ex-vivo effects of intrapulmonary percussive ventilation on sputum rheological properties.

Intrapulmonary percussive ventilation (IPV) has been postulated to enhance mucociliary clearance by improving tracheobronchial sputum rheological properties. The IPV effects on linear (viscoelasticity) and non-linear (flowing) rheological properties of 40 sputum samples collected from 19 patients with muco-obstructive lung diseases were investigated ex-vivo. Each sputum sample was split into 4 aliquots. These aliquots were independently placed in a circuit connected on one side to an IPV device and on the other side to a lung model that simulated spontaneous adult breaths. IPV was superimposed on simulated breathing. Three aliquots were exposed to a different IPV setting, modifying either percussion frequency or amplitude (4 Hz-200 L/min, 10 Hz-200 L/min, 10 Hz-140 L/min). One aliquot was only exposed to breathing (IPV was switched off, control condition). Each aliquot underwent 5 min of the pre-fixed mechanical stimulation before being recollected to proceed to rheological analysis. Neither percussion frequencies nor amplitudes had a significant impact on any sputum rheological properties studied. These results need to be confirmed in vivo.

Sputum handling for rheology.

The rheology of sputum is viewed as a powerful emerging biophysical marker for monitoring muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). However, there is no unified practice to process sputa from collection to analysis, which can lead to highly variable, and sometimes inconsistent results. The main objective of this study is to bring light into the handling of sputum samples to establish a standardised and robust protocol before rheological measurements. Sputum collected from 22 CF and 10 NCFB adults, was divided into control (vortexed and fresh: non-heated and non-frozen) and three treated conditions (either non-vortexed, heated or frozen). In addition, 6 CF expectorations were used to study the dynamics of ageing over 24 h. Sputum's mechanical properties were measured with a rotational rheometer to obtain their properties at rest, elastic ([Formula: see text]) and viscous moduli ([Formula: see text]), and at the onset of flow, critical deformation ([Formula: see text]) and critical stress ([Formula: see text]). We demonstrate that heating sputum is completely destructive while freezing sputa at [Formula: see text] has no discernible effect on their rheology. We also show that the variability of rheological measurements largely resulted from the sample's macroscopic heterogeneity, and can be greatly reduced by non-destructive vortex homogenisation. Finally, we observed contrasted ageing effects as a fonction of purulence: while the viscoelasticity of purulent samples reduced by half within 6 h after collection, semi-purulent samples did not evolve. These results guide towards a robust unified protocol for simple sputum handling in rheometry. We therefore suggest to vortex and snap freeze sputum samples immediately after collection when direct testing is not possible.

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer.

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G', G", G*, and tan δ) and gel point characteristics (γc and σc) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is <10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more "liquid-like" behavior overall (e.g., higher tan δ). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

Rheological analysis of sputum from patients with chronic bronchial diseases.

Bronchial diseases are characterised by the weak efficiency of mucus transport through the lower airways, leading in some cases to the muco-obstruction of bronchi. It has been hypothesised that this loss of clearance results from alterations in the mucus rheology, which are reflected in sputum samples collected from patients, making sputum rheology a possible biophysical marker of these diseases and their evolution. However, previous rheological studies have focused on quasi-static viscoelastic (linear storage and loss moduli) properties only, which are not representative of the mucus mobilisation within the respiratory tract. In this paper, we extend this approach further, by analysing both quasi-static and some dynamic (flow point) properties, to assess their usability and relative performance in characterising several chronic bronchial diseases (asthma, chronic obstructive pulmonary disease, and cystic fibrosis) and distinguishing them from healthy subjects. We demonstrate that pathologies influence substantially the linear and flow properties. Linear moduli are weakly condition-specific and even though the corresponding ranges overlap, distinct levels can be identified. This directly relates to the specific mucus structure in each case. In contrast, the flow point is found to strongly increase in muco-obstructive diseases, which may reflect the complete failure of mucociliary clearance causing episodic obstructions. These results suggest that the analysis of quasi-static and dynamic regimes in sputum rheology is in fact useful as these regimes provide complementary markers of chronic bronchial diseases.

Dynamics of colloid accumulation under flow over porous obstacles.

The accumulation of colloidal particles to build dense structures from dilute suspensions may follow distinct routes. The mechanical, structural and geometrical properties of these structures depend on local hydrodynamics and colloidal interactions. Using model suspensions flowing into microfabricated porous obstacles, we investigate this interplay by tuning both the flow pattern and the ionic strength. We observe the formation of a large diversity of shapes, and demonstrate that growing structures in turn influence the local velocity pattern, favouring particle deposition either locally or over a wide front. We also show that these structures are labile, stabilised by the flow pushing on them, in low ionic strength conditions, or cohesive, in a gel-like state, at higher ionic strength. The interplay between aggregate cohesion and erosion thus selects preferential growth modes and therefore dictates the final shape of the structure.

Thermocapillary migration in small-scale temperature gradients: application to optofluidic drop dispensing.

We experimentally investigate the thermocapillary migration induced by local laser heating of the advancing front of a growing droplet confined in a microfluidic channel. When heating implies an effective increase in interfacial tension, the laser behaves as a "soft door" whose stiffness can be tuned via the optical parameters (beam power and waist). The light-driven thermocapillary velocity of a growing droplet, which opposes the basic flow, is characterized for different types of fluid injection, either pressure or flow rate driven, and various channel aspect ratios. Measurements are interpreted using a simplified model for the temperature gradient at the interface, based on a purely diffusive, three-layer system. Considering the mean temperature gradient, we demonstrate that the classical large-scale temperature gradient behavior is retrieved in the opposite case when the thermal gradient length scale is smaller than the droplet size. We also demonstrate that the thermocapillary velocity is proportional to the smallest droplet curvature imposed by the channel confinement. This suggests that the thermocapillary velocity is in fact proportional to the mean temperature gradient and to the largest interface curvature radius, which both coincide with the imposed one and the spherical droplet radius in large-scale and unconfined situations. Furthermore, as used surfactant concentrations are largely above the critical micelle concentration, we propose a possible explanation, relying on state-of-the-art considerations on high-concentration surfactant-covered interfaces for the observed effective increase in interfacial tension with temperature. We also propose a mechanism for explaining the blocking effect at the scaling-law level. This mechanism involves the temporal evolution of hydrodynamic and thermocapillary forces, based on experimental observations. We finally show that this optocapillary interaction with a microfluidic droplet generator allows for controlling either the flow rate (valve) or the droplet size (sampler), depending on the imposed fluid injection conditions.