The role of mucus sol phase in clearance by simulated cough

Using a simulated cough machine, we analyzed the effect of adding tensio-active liquids as sol phase simulant on the clearance of gel mucus simulant by cough. Polysaccharides crosslinked with sodium tetraborate were used at different concentration as gel mucus simulant. A drop of gel mucus simulant was deposited either directly on the model trachea or on a sol phase layer simulant (2% sodium dodecyl sulfate in water). The clearance of the mucus simulants was quantified by observing the movement of marker particles in the gel layer. The viscoelastic properties of gel mucus simulants were determined by using a viscoelastometer (SEFAM). The adhesive properties were analyzed by means of the platinum ring technique. The wettability of the mucus simulants was quantified by the automatic measurement of the contact angle of the drop of gel on the model trachea. We found that the addition of a sol phase significantly decreased by about 50% the adhesivity and wettability of the gel mucus simulants. This decrease was associated with a marked enhancement of cough clearance, whatever the viscoelastic properties of the gel mucus simulants. These results suggest that the sol phase is essential in bronchial respiratory mucus clearance by the cough mechanism.     

The role of mucus viscoelasticity in cough clearance

The relationships between mucus rheology, depth of mucus layer and clearance by simulated cough were examined in a study employing a model plexiglass trachea lined with gels formed from locust bean gum crosslinked with sodium tetraborate. The viscoelastic properties of the mucus simulants were determined by magnetic rheometry at 100 rad/s and expressed as mechanical impedance (dynamic stress/strain ratio) and loss tangent. Cough was simulated by opening a solenoid valve connecting the model trachea to a pressurized tank, using an upstream flow-constrictive element to shape the flow profile to approximate the pattern seen in a normal adult. Mucus clearance was quantitated by observing the movement of contrasting marker particles placed in the mucus layer. The median particle displacement was defined as the clearance index, Cl. For any initial depth of mucus, Cl increased with driving pressure in the tank, and for a given driving pressure, Cl increased linearly with increasing mucus depth. For a given driving pressure and depth, Cl decreased with increasing mechanical impedance of the mucus. At constant mechanical impedance, Cl increased with increasing loss tangent, in other words, cough clearance was impeded more by elasticity than viscosity. Mucus clearance was associated with transient wave formation in the lining layer. Thus dependence on viscoelasticity is consistent with observations that airflow-mucus interaction and wave formation are impeded by elasticity. The clearance vs. loss tangent relationship for cough is opposite to that found for ciliary clearance (Biorheology 1980, 17, 249), suggesting a natural balance in viscosity and elasticity for mucus to be cleared by both mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clearance of mucus by simulated cough

We examined the relationship between mucus rheology, depth of mucus layer, and clearance by simulated cough. A model trachea was constructed of rigid Plexiglas of rectangular cross section (1 X 2 X 35 cm). The bottom of the trachea was lined with mucus simulants, gels prepared from locust bean gum cross-linked with sodium borate. Cough was simulated by opening a solenoid valve connecting the model trachea to a pressurized tank. An upstream flow-constrictive element was used to shape the flow profile of the simulated cough to approximate the pattern seen in a normal adult. Clearance of mucus was quantitated by observing the movement of contrasting marker particles floating in the mucus layer. The median particle displacement per cough maneuver was defined as the clearance index (CI). We found that CI for any initial depth of mucus increased with the driving pressure in the tank. For a given driving pressure, CI increased linearly with increasing mucus depth. For a given driving pressure and depth, CI decreased with increasing mucus cross-link density. For mucus samples with comparable levels of dynamic viscosity, samples with higher elasticity cleared less well. Mucus clearance was associated with transient wave formation in the lining layer.     

Effect of airway wall flexibility on clearance by simulated cough

In our previous study, we investigated the relationship between mucus rheology, depth of mucus layer, and clearance by simulated cough. The purpose of the present study was to examine the effect of airway wall flexibility on the clearance of mucuslike gels. Transient airflows similar to cough were generated by both positive and negative pressure, the latter to mimic the dynamic compression that occurs during real cough. As in the previous study, the trachea was modeled as a trough of rectangular cross section with only the bottom lined with the mucus simulant. Clearance was followed by observing the displacement of marker particles. Since cough clearance is intimately related to wave formation in the mucus blanket, we hypothesized that clearance might be impeded if the wave formation occurred simultaneously in the wall and its lining layer. Thus, in one set of experiments the bottom rigid surface of the model trachea was replaced with a frame over which a flexible membrane could be drawn, whereas in the other set the rigid top was replaced by the frame. We also examined the effect of negative-pressure cough in excised canine tracheae, comparing the case where the tracheal membrane was free to deform vs. the case where it was secured. For the rigid-walled model, clearance by positive or negative pressure, with matched flow pattern, was the same. With the mucus simulant lining the flexible bottom surface, clearance increased with increasing membrane flexibility for negative-pressure cough and decreased for positive-pressure cough.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tracheal mucus rheology and epithelial potential difference in two day old puppies

The transepithelial potential difference (PD) value represents an integral of ion fluxes across the epithelium, and relates to the regulation of airway fluid. We studied six healthy two day old husky puppies for their tracheal mucus rheology and bioelectric properties, since this data in newborns are still unknown. PD (-mV, epithelium vs. subcutaneous space) was measured using the agar bridge technique in two locations - lower trachea and subglottic region. For the rheological analysis, the magnetic microrheometer was employed; data are presented as mechanical impedance log G* and loss tangent tan delta (1 rad/s). The mucus collection rate (mg/min) and the solid content (%) were determined by gravimetry. Mucociliary clearability, normalized to frog mucus, (MCFP) was determined directly by the frog palate method; a cough clearability index (CCI) was computed from simulated cough machine data obtained with mucus-like gels. The mucus collection rates and PD values were considerably lower than those observed in adult dogs; the mechanical impedance values were also reduced in comparison with adult data. The PD profile within the trachea (-13.9 +/- 1.2 mV lower trachea vs. -18.4 +/- 1.4 mV subglottical, i.e. more negative subglottically), however, is similar to that observed in adult dogs. Intratracheal profiles in mucus collection rate and mucus rheology were also comparable between puppies and adult dogs. The low collection rates in puppies, particularly in lower trachea, could indicate either reduced mucus volume or slower clearance. PD and collection rate correlated very strongly (r = 0.82, p = 0.0003). PD also correlated negatively with log G* (r = 0.73, p = 0.003) and positively with tan delta (r = 0.58, p = 0.03). MCFP and % solids correlated positively (r = 0.84, p = 0.0012), in contradistinction to the usual relationship, perhaps due to the presence of non-glycoprotein components that do not contribute to crosslink formation. The apparent maturation of airway bioelectric properties, mucus collection rate and mucus viscoelasticity are all consistent with the maturation of mucociliary clearance, which has previously been reported.     

Formulating gels for decreased mucociliary transport using rheologic properties: Polyacrylic acids

The purpose of these studies was to identify the rheologic properties of polyacrylic acid gels necessary for optimal reductions in mucociliary clearance. The mucociliary transport of 2 bioadhesive polyacrylic acid polymers, polycarbophil and carbopol, was assessed in vitro by measuring their clerance rates across explants of ciliated bovine tracheal tissue. The viscoelastic properties of polymer gels were measured in the presence of mucus using controlled stress rheometry. Combinations of apparent viscosity (η) and complex modulus (G^*) were found to be the most useful parameters in the identification of polyacrylic acid formulations capable of decreasing mucociliary transport rate (MTR). A narrow range of η and G^* values suitable for reducing mucociliary clearance, while remaining sufficiently fluid for intranasal administration, were identified. The correlations between the rheologic parameters of the polycarbophil gels and their mucociliary transport rates were used to identify other polyacrylic acid gels that also had suitable mucociliary clearance properties, demonstrating that these parameters can be used to direct the optimization of formulations using simple in vitro rheologic testing. Published: April 20, 2007     

Mucus transport in a miniaturized simulated cough machine: effect of constriction and serous layer simulant

The transport of mucus gel simulant (MGS) in a constricted simulated cough machine, using blood plasma as a serous layer simulant (SLS), was investigated. MGS was prepared from locust bean gum solutions crosslinked with varying amounts of added borate to produce gels of varying spinnability (filance). The model trachea was a plexiglass channel of rectangular cross-section with the plane bottom surface. The upper surface included a sinusoidal protrusion which provided a flow convergence with minimum gaps of 6, 4 and 2 mm. Experiments for mucus transport were conducted for these minimum gaps, as well as for the non-convergent case (12 mm gap). Miniaturization of sample quantity was achieved by keeping the MGS layer depth constant (0.5 or 1 mm) but reducing the zone of loading from 13.4 cm to 1 cm, thus reducing the sample requirement to as little as 0.2 ml. MGS transport was determined as the minimum displacement of a line of marker dye placed in the MGS layer at the point of minimum constriction gap. It was shown that in all cases (dry as well as with SLS), MGS transport increased as the minimum constriction gap between the plane and the convergent top surface decreased. This increase was further enhanced if an SLS of lesser viscosity was used. It was also found that the transport of MGS increased as the depth of MGS layer increased or as the filance decreased in both non-constricted and constricted cases. The relationship between MGS transport and filance was maintained even in the presence of an SLS layer.     

Analysis of the volume of fluid (VOF) method for the simulation of the mucus clearance process with CFD

The clearance of mucus through coughing is a complex, multiphase process, which is affected principally by mucus viscosity and airflow velocity; however, it is also critically affected by the thickness of the two layers of mucus—the serous and gel layers—and oscillation level. The present study examines the effects of the latter parameters more closely. To do so, the mucus clearance process is simulated with a transient 3D volume of fluid (VOF) multiphase model in ANSYS Fluent. The model includes mucus’ bilayer properties and a wide range of boundary conditions were tested. The model was analysed in both a straight tube and a realistic trachea. Ultimately, the model was able to both capture air-mucus interface wave evolution and predict the overall behaviour of the clearance process. The results were consistent with experimental clearance data and numerical airflow simulations, which indicates our methodology is appropriate for future studies. Ultimately, the mere presence of the serous layer was found to increase mucus clearance by more than 15 percent. An oscillating flow enhanced clearance by up to 5 percent. Interestingly, interface wave steepness was found to be inversely correlated with mucus thickness, but directly with mucus velocity, which suggests it will be an interesting parameter for further study.     

Comparative rheological profile of rat gastric and duodenal gel mucus

Methods for measuring the adhesiveness, plasticity, viscoelasticity and spinnability of mucus microsamples have been developed. The rheological properties of the rat gastric and duodenal gel mucus have been analyzed and compared. Using a controlled stress rheometer (Carri-Med), flow and creep experiments showed that gastroduodenal mucus exhibits a typically non-newtonian, pseudoplastic and viscoelastic behaviour. The apparent viscosity (7,800 +/- 11,000 Pa.s) and yield stress (24.9 +/- 8.5 Pa) of gastric mucus were significantly higher than the duodenal mucus viscosity (39 +/- 160 Pa.s) and yield stress (12.9 +/- 2 Pa). Spinnability of gastric mucus, measured with a Filancemeter (SEFAM), was significantly lower (4.9 +/- 2.5 mm) in comparison to duodenal mucus (6.9 +/- 1.5 mm). Adhesive properties of gastric mucosa (analyzed with the platinum ring method) were not significantly different in comparison to duodenal mucus (99.9 +/- 31.5 mN/m and 92.8 +/- 11.2 mN/m, respectively).     

Role of phospholipid lining on respiratory mucus clearance by cough.

Phospholipid lining, present at the respiratory mucus-mucosa interface, may have an important role in the protective function of the airways by its abhesive properties and may also facilitate mucus transport. To mimic respiratory mucus-mucosa interface, monolayers of three different forms of phosphatidylglycerol (PG) have been deposited on glass slides by the Langmuir-Blodgett technique. Mucus adhesion and clearance by cough of mucus on these PG-coated or noncoated surfaces have been analyzed and compared, using frog respiratory mucus as "normal" mucus. Among the three PG types studied, the phosphatidylglycerol distearoyl, which is the phospholipid with the longest saturated fatty acid chain, was found to significantly improve the mucus cough clearance by decreasing the mucus work of adhesion compared with the noncoated surfaces. On the other hand, phosphatidylglycerol dipalmitoyl did not improve mucus cough clearance although it decreased mucus adhesion, and phosphatidylglycerol dioleyl did not improve either mucus cough clearance or mucus adhesion.     

Mechanical characteristics of synthetic polyelectrolyte gel as a physical model of the cytoskeleton

A physical model of the cytoskeleton based on synthetic polyelectrolyte hydrogel of polymethacrylic acid has been proposed. From the physicochemical point of view, the structures of polyelectrolyte gel and the cytoskeleton show a high degree of similarity. It has been shown that polyelectrolyte gel can shorten and produce mechanical stress in response to changes in the composition of the surrounding solution. The mechanical properties of the model gel have been evaluated: Young modulus (2–6 kPa), stress relaxation time (0.1–1 s), and apparent viscosity (0.3–3 kPa s). The viscoelastic properties of the gel depend on the degree of its swelling. It has been demonstrated that the mechanical properties of gels of polymethacrylic acid are close to those of biological objects.     

Adhesive thermosensitive gels for local delivery of viral vectors

Local delivery of viral vectors can enhance the efficacy of therapies by selectively affecting necessary tissues and reducing the required vector dose. Pluronic F127 is a thermosensitive polymer that undergoes a solution–gelation (sol–gel) transition as temperature increases and can deliver vectors without damaging them. While pluronics can be spread over large areas, such as the surface of an organ, before gelation, they lack sufficient adhesivity to remain attached to some tissues, such as the surface of the heart or mucosal surfaces. Here, we utilized blends of pluronic F127 and polycarbophil (PCB), a mucoadhesive agent, to provide the necessary adhesivity for local delivery of viral vectors to the cardiac muscle. The effects of PCB concentration on adhesive properties, sol–gel temperature transition and cytocompatibility were evaluated. Rheological studies showed that PCB decreased the sol–gel transition temperature at concentrations >1% and increased the adhesive properties of the gel. Furthermore, these gels were able to deliver viral vectors and transduce cells in vitro and in vivo in a neonatal mouse apical resection model. These gels could be a useful platform for delivering viral vectors over the surface of organs where increased adhesivity is required.     

A new paradigm in respiratory hygiene: increasing the cohesivity of airway secretions to improve cough interaction and reduce aerosol dispersion

Background

Infectious respiratory diseases are transmitted to non-infected subjects when an infected person expels pathogenic microorganisms to the surrounding environment when coughing or sneezing. When the airway mucus layer interacts with high-speed airflow, droplets are expelled as aerosol; their concentration and size distribution may each play an important role in disease transmission. Our goal is to reduce the aerosolizability of respiratory secretions while interfering only minimally with normal mucus clearance using agents capable of increasing crosslinking in the mucin glycoprotein network.

Methods

We exposed mucus simulants (MS) to airflow in a simulated cough machine (SCM). The MS ranged from non-viscous, non-elastic substances (water) to MS of varying degrees of viscosity and elasticity. Mucociliary clearance of the MS was assessed on the frog palate, elasticity in the Filancemeter and the aerosol pattern in a "bulls-eye" target. The sample loaded was weighed before and after each cough maneuver. We tested two mucomodulators: sodium tetraborate (XL"B") and calcium chloride (XL "C").

Results

Mucociliary transport was close to normal speed in viscoelastic samples compared to non-elastic, non-viscous or viscous-only samples. Spinnability ranged from 2.5 ± 0.6 to 50.9 ± 6.9 cm, and the amount of MS expelled from the SCM increased from 47 % to 96 % adding 1.5 μL to 150 μL of XL "B". Concurrently, particles were inversely reduced to almost disappear from the aerosolization pattern.

Conclusion

The aerosolizability of MS was modified by increasing its cohesivity, thereby reducing the number of particles expelled from the SCM while interfering minimally with its clearance on the frog palate. An unexpected finding is that MS crosslinking increased "expectoration".     

Effect of pericellular matrix formation by chondrocytes cultured in agarose gel on the viscoelastic properties of agarose gel matrix

The viscoelasticity of chondrocyte-seeded agarose gel (AGC0) and that of chondrocyte-seeded agarose gel after 21 days of cultivation (AGC3) were investigated. In AGC3, pericellular matrix (PCM)-like material around each chondrocyte was found to be constructed, which was confirmed by an optical micrograph in conjunction with toluidine blue staining. The relaxation modulus of each of the chondrocyte-agarose gel composite systems was measured by a non-constrained indentation method. Stress-strain curves for all of the specimens examined had a toe region followed by a linear region terminated by specimen fracture. The slope of the linear region of AGC0 was smaller than that of AG, while the SS curve of AGC0 was indistinguishable from that of AGC3. All of the relaxation curves studied were typical of gels, having a fast relaxation process up to 10³ s followed by a plateau. The relaxation modulus of AGC0 was smaller than that of agarose gel (AG), the decrement in relaxation modulus from AG to AGC0 being attributed to the seeding of chondrocytes that have a smaller modulus than that of agarose gels. However, the relaxation modulus of AGC3 was increased at the early viscoelastic region in particular, as compared with that of AGC0. The increments in the relaxation modulus in AGC3 were attributed to the PCM-like material produced by chondrocytes, where the produced material may provide crosslink points and reinforce the agarose gel.     

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Rheological characterization of neutral and anionic polysaccharides with reduced mucociliary transport rates

The purpose of this research was to compare the viscoelastic properties of several neutral and anionic polysaccharide polymers with their mucociliary transport rates (MTR) across explants of ciliated bovine tracheal tissue to identify rheologic parameters capable of predicting the extent of reduction in mucociliary transport. The viscoelastic properties of the polymer gels and gels mixed with mucus were quantified using controlled stress rheometry. In general, the anionic polysaccharides were more efficient at decreasing the mucociliary transport rate than were the neutral polymers, and a concentration threshold, where no further decreases in mucociliary transport occurred with increasing polymer concentration, was observed for several of the neutral polysaccharides. No single rheologic parameter (ν, G′, G″, tan δ, G^*) was a good predictor of the extent of mucociliary transport reduction, but a combination of the apparent viscosity (ν), tangent to the phase angle (tan δ), and complex modulus (G^*) was found to be useful in the identification of formulations capable of decreasing MTR. The relative values of each of the rheologic parameters were unique for each polymer, yet once the relationships between the rheologic parameters and mucociliary transport rate reduction were determined, formulations capable of resisting mucociliary clearance could be rapidly optimized. Published: April 20, 2007     

Functional interactions of gastric mucus glycoprotein

The concentration-dependence of viscosity in solutions of purified glycoprotein from pig gastric mucus is of the form expected for simple polymer entanglement. At higher concentrations, however, a weak viscoelastic gel is formed, whose mechanical spectrum (over the frequency range 10⁻²---10² rad s⁻¹) indicates a more stable mechanism of interchain association, and is closely similar to that of native mucus. On prolonged exposure to solvent, reconstituted gels redissolve, while native mucus retains its structural integrity (as characterized by the storage modulus, G′) but releases a significant, variable amount of glycoprotein. On proteolytic digestion or disulphide reduction of the glycoprotein to its component subunits, network structure is lost, but the mechanical spectra of the resulting solutions show interactions beyond simple entanglement. From this evidence we suggest that in the sub-micrometre-sized ‘domains’ in which native mucus is secreted, the carbohydrate side chains of component glycoprotein molecules are interdigitated in a comparatively stable arrangement, with the polymeric subunit structure of the glycoprotein conferring the branching required for development of a three-dimensional network, and with a substantial, variable sol-fraction of free glycoprotein within the interstices of the gel. On solubilization of native mucus, the ‘domain’ structure is destroyed irreversibly. Interaction between domains, and between individual molecules in gels reconstituted from the component glycoprotein after extraction and purification, is by more transient, non-specific interdigitation and entanglement, to confer the overall flow and spreading characteristics of the gel.     

Viscoelastic properties of f-actin, microtubules, f-actin/alpha-actinin, and f-actin/hexokinase determined in microliter volumes with a novel nondestructive method

A nondestructive method to determine viscoelastic properties of gels and fluids involves an oscillating glass fiber serving as a sensor for the viscosity of the surrounding fluid. Extremely small displacements (typically 1-100 nm) are caused by the glass rod oscillating at its resonance frequency. These displacements are analyzed using a phase-sensitive acoustic microscope. Alterations of the elastic modulus of a fluid or gel change the propagation speed of a longitudinal acoustic wave. The system allows to study quantities as small as 10 microliters with temporal resolution >1 Hz. For 2-100 microM f-actin gels a final viscosity of 1.3-9.4 mPa s and a final elastic modulus of 2.229-2.254 GPa (corresponding to 1493-1501 m/s sound velocity) have been determined. For 10- to 100-microM microtubule gels (native, without stabilization by taxol), a final viscosity of 1.5-124 mPa s and a final elastic modulus of 2.288-2. 547 GPa (approximately 1513-1596 m/s) have been determined. During polymerization the sound velocity in low-concentration actin solutions increased up to +1.3 m/s (approximately 1.69 kPa) and decreased up to -7 m/s (approximately 49 kPa) at high actin concentrations. On polymerization of tubulin a concentration-dependent decrease of sound velocity was observed, too (+48 to -12 m/s approximately 2.3-0.1 MPa, for 10- to 100-microM tubulin). This decrease was interpreted by a nematic phase transition of the actin filaments and microtubules with increasing concentration. 2 mM ATP (when compared to 0.2 mM ATP) increased polymerization rate, final viscosity and elastic modulus of f-actin (17 microM). The actin-binding glycolytic enzyme hexokinase also accelerated the polymerization rate and final viscosity but elastic modulus (2.26 GPa) was less than for f-actin polymerized in presence of 0.2 mM ATP (2.28 GPa).     

Viscoelastic properties and dynamics of porcine gastric mucin

Gastric mucin is a glycoprotein known to undergo a pH-dependent sol-gel transition that is crucial to the protective function of the gastric mucus layer in mammalian stomachs. We present microscope-based dynamic light scattering data on porcine gastric mucin at pH 6 (solution) and pH 2 (gel) with and without the presence of tracer particles. The data provide a measurement of the microscale viscosity and the shear elastic modulus as well as an estimate of the mesh size of the gel formed at pH 2. We observe that the microscale viscosity in the gel is about 100-fold lower than its macroscopic viscosity, suggesting that large pores open up in the gel reducing frictional effects. The data presented here help to characterize physiologically relevant viscoelastic properties of an important biological macromolecule and may also serve to shed light on diffusive motion of small particles in the complex heterogeneous environment of a polymer gel network.     

Glottis effects on the cough clearance process simulated with a CFD dynamic mesh and Eulerian wall film model

In this study, we have reproduced the cough clearance process with an Eulerian wall film model. The simulated domain is based on realistic geometry from the literature, which has been improved by adding the glottis and epiglottis. The vocal fold movement has been included due to the dynamic mesh method, considering different abduction and adduction angles and velocities. The proposed methodology captures the deformation of the flexible tissue, considers non-Newtonian properties for the mucus, and enables us to reproduce a single cough or a cough epoch. The cough efficiency (CE) has been used to quantify the overall performance of the cough, considering many different boundary conditions, for the analysis of the glottis effect. It was observed that a viscous shear force is the main mechanism in the cough clearance process, while the glottis closure time and the epiglottis position do not have a significant effect on the CE. The cough assistance devices improve the CE, and the enhancement rate grows logarithmically with the operating pressure. The cough can achieve an effective mucus clearance process, even with a fixed glottis. Nevertheless, the glottis closure substantially improves the CE results.