cystic fibrosis (CF)

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this concept was established using micro-rheology but was later applied to shear rheology [4] without considering inertia effects. We show that inertia highly ...
Böni, L.‡, Radtke, T., Bohnacker, P., Maggi-Bebba, M., Kriemler, S., Benden, C., Dressel, H., and Fischer, P.

cystic fibrosis (CF) – a genetic disease causing sputum accumulation in the airways Cystic fibrosis (CF) is the most common genetic life-limiting disease. In CF highly viscous mucus secretions accumulate in the lungs (Fig. 1) and in the pancreas, causing tissue damage, chronic infection, and inflammation. Regular clearance of the accumulated mucus, called sputum in the airways, is therefore of critical importance to lung health [1]. Generally, mucus clearance is known to be influenced by viscoelasticity [2]. Earlier works established a link between in vitro clearance and rheological properties of mucus by mimicking the two major clearance processes: mucociliary clearance (MC) and cough clearance (CC). MC was simulated using low frequency deformations (1 rad/s) – similar to the beating frequency of epithelial cilia - whereas CC was simulated using 100 rad/s, being similar to speeds occurring during coughing [2,3]. However, this concept was established using micro-rheology but was later applied to shear rheology [4] without considering inertia effects. We show that inertia highly affects the torque response of the soft CF sputum at high frequencies and suggest to limit frequencies to < 10 rad/s in shear rheology [5].

normal airway: lined with thin layer of mucus

left lung

airway with CF: thick mucus blocks airways alveoli bacterial infection

Fig. 1: Sputum in CF lungs (adapted form Jay Smith/Discover)

so far: rheology was linked to in vitro sputum clearance coughing transport

AIRWAYS

amplitude

similar to high frequency deformations (100 rad/s)

time

MUCOCILIARY CLEARANCE beating cilia (1 rad/s) mucociliar transport

mucus layer EPITHELIAL CELLS

sputum clearance and micro-rheology empirically correlated [2]

good clearance

1 0.8

CCI

0.6 0.4

bad clearance

0.2

0.4 0.6 0.8

amplitude

derived [3]

good clearance

1 0.8

MCI

0.6 0.4

Cough Clearance Index (CCI) CCI = 3.44 - (1.07 x log G*) + (0.89 x tan δ) à used to simulated cough clearance in rheology (100 rad/s) Mucociliary Clearance Index (MCI) MCI = 1.62 - (0.22 x log G*) - (0.77 x tan δ) à used to simulate mucociliary clearance in rheology (1rad/s)

bad clearance

0.2

0.4 0.6 0.8

similar to low frequency deformations (1 rad/s)

time

1

tan δ ciliary clearance

mucus layer

cough clearance

COUGH CLEARANCE shearing air current (100 rad/s)

tan δ

CCI and MCI are frequently used parameters in medical studies to rheologically assess sputum clearance performance [3,4]

1

clearance of mucus/viscoelastic fluids at const. G* determined by simulated cough (adapted from [2])

watch out: sample modulus and inertia limit use of high frequency oscillations Fig.2: Frequency sweep of CF sputum (45 samples averaged) CF sputum is a very soft biomaterial (G0 = 9.6 ± 5.6 Pa) Soft gel-like behavior: G’ ∝ ω0.17 , phase angle (16° < δ < 25°) High within-subject variability; large sample size needed for statistics when testing effects of drugs, exercise, etc [8]

on ensati comp inertia heometer by r

a

Ms ≈ Me; Ma ≈ 0

a

δ

Ms

c

b

Ms > Me > Ma

Me > Ma > Ms Ma

Ma

Ma Me

Me

b

Ms

δ

c

Ms

Me

δ

δ

Ms sample torque Ma accelera"on or iner"a torque Me electric or total torque (Ms+Ma) sample phase angle

conclusions

materials & methods Sputum samples were obtained from 15 clinically stable CF patients at three points in time. The samples were snap frozen (-80°C) after collection and thawed in the fridge (4°C) 6 hours before measuring. For sample loading micropipette tips (1 ml) were cut in the front with a scalpel to increase the die size and thus to minimize preshear. Measurements were performed on a MCR 702 (Anton Paar) rheometer in parallel plate mode, using micro-roughened 25 mm plates (PP25 S sandblasted, Anton Paar) to avoid slip and a gap of 0.5 mm. Frequency sweeps (strain amplitude 1%) and amplitude sweeps (angular frequency 1 rad/s) (not shown) were performed at 20°C using a solvent trap. Calculated inertia boundaries are based on equations by Ewoldt et al. [7]

Fig. 3: Sample and electric torque of a representative CF sample Instrument inertia dominates torque response of CF sputum at high frequencies (> 10 rad/s) [6,7]

CF sputum is a very soft, viscoelastic biomaterial Limit frequencies to < 10 rad/s in shear rheology to avoid inertia Rethink the MCI / CCI concept: spinnability or large amplitude oscillatory shear (LAOS) – both large deformation measurements - could prove more effective to simulate clearance in vitro

acknowledgements and funding We thank Jörg Läuger for his help with the interpretation of the inertia effects and André Königs for his assistance in patients’ recruitment. This work was funded by the Swiss Cystic Fibrosis Society (CFCH)

[1] Cutting, GR., Nat Rev Gen, 2015;16:45-56 [2] King, M., Biorheology, 1987;24:589-597 [3] App, E., et al., CHEST, 1998;114,:171-7 [6] Läuger, J., Stettin, H., JoR, 2016;60:393-406 [4] Daviskas, E., Respirology, 2005;10:46-56 [7] Ewoldt, RH., et al., Complex Fluids in Biological Systems, Springer (2015) [5] Radtke,T., et al., BMC Pulm Med, 2018;18:99 [8] Radtke,T., et al., Respir Physiol Neurobiol, 2018;254:36-39

‡ Department of Health Science and Technology, ETH Zürich [email protected] Annual European Rheology Conference, 2018