An optimized protocol for assessment of sputum macrorheology in health and muco-obstructive lung disease

Abstract

Background: Airway mucus provides important protective functions in health and abnormal viscoelasticity is a hallmark of muco-obstructive lung diseases such as cystic fibrosis (CF). However, previous studies of sputum macrorheology from healthy individuals and patients with CF using different experimental protocols yielded in part discrepant results and data on a systematic assessment across measurement settings and conditions remain limited.Objectives: The aim of this study was to develop an optimized and reliable protocol for standardized macrorheological measurements of airway mucus model systems and native human sputum from healthy individuals and patients with muco-obstructive lung disease.Methods: Oscillatory rheological shear measurements were performed using bovine submaxillary mucin (BSM) at different concentrations (2% and 10% solids) and sputum samples from healthy controls (n = 10) and patients with CF (n = 10). Viscoelastic properties were determined by amplitude and frequency sweeps at 25°C and 37°C with or without solvent trap using a cone-plate geometry.Results: Under saturated atmosphere, we did not observe any temperature-dependent differences in 2% and 10% BSM macrorheology, whereas in the absence of evaporation control 10% BSM demonstrated a significantly higher viscoelasticity at 37°C. Similarly, during the measurements without evaporation control at 37°C we observed a substantial increase in the storage modulus G′ and the loss modulus G″ of the highly viscoelastic CF sputum but not in the healthy sputum.Conclusion: Our data show systematically higher viscoelasticity of CF compared to healthy sputum at 25°C and 37°C. For measurements at the higher temperature using a solvent trap to prevent evaporation is essential for macrorheological analysis of mucus model systems and native human sputum. Another interesting finding is that the viscoelastic properties are not much sensitive to the applied experimental deformation and yield robust results despite their delicate consistency. The optimized protocol resulting from this work will facilitate standardized quantitative assessment of abnormalities in viscoelastic properties of airway mucus and response to muco-active therapies in patients with CF and other muco-obstructive lung diseases.

Ileal mucus viscoelastic properties differ in Crohn’s disease

“…To our knowledge rheological data on human intestinal mucus are absent; therefore, we compared our findings on human ileal mucus from controls (n=11) with macrorheological data on human airway mucus from controls (n=10; Figure 1B ). Some of the airway mucus samples from controls were previously included in a publication 33 . The demographic and basic clinical characteristics of the control group for airway mucus are presented in Table S1 .…”

“…We used the previously described macrorheological protocol for human airway mucus 33 . Details are provided in the Supplementary Information .…”

Modeling Cystic Fibrosis Chronic Infection Using Engineered Mucus-like Hydrogels

“…Additionally, the viscoelastic properties of the CF and healthy mucus-like hydrogels developed herein were within the same order of magnitude as the viscoelastic properties of CF and healthy airway mucus that have been analyzed and reported in literature. Recent sources have found that healthy and CF airway mucus have storage moduli within the range of 0.2 Pa to 93 Pa and loss moduli within the range of 0.1 Pa to 19 Pa 6,7 . The hydrogels developed here recapitulate the key differences in viscoelastic properties, mucin concentration, and solids concentration in physiological CF and healthy airway mucus.…”

“…Airway mucus acts as a protective barrier for the respiratory system by collecting and clearing harmful pathogens from the airways 1,2 . In cystic fibrosis (CF), mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the airway epithelial surface to be dehydrated, leading to increased solids and mucin protein concentrations and altered mucus viscoelasticity [3][4][5][6][7] . These altered airway mucus properties impair mucus clearance mechanisms such as mucociliary and cough mediated clearance [8][9][10] .…”

Effects of Elexacaftor/Tezacaftor/Ivacaftor on Sputum Viscoelastic Properties, Airway Infection and Inflammation in Patients with Cystic Fibrosis

“…Sputum rheology was measured with a cone and plate rheometer (Kinexus Pro+, Netzsch GmbH, Selb, Germany) as previously described [23]. The elastic modulus (storage modulus, G') and viscous modulus (loss modulus, G′′) were directly extracted from the linear viscoelastic region of the amplitude sweep.…”