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

Abstract

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.
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").
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 microL to 150 microL of XL "B". Concurrently, particles were inversely reduced to almost disappear from the aerosolization pattern.
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".

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BMC Pulmonary Medicine
Open Access
Research article
A new paradigm in respiratory hygiene: increasing the cohesivity of
airway secretions to improve cough interaction and reduce aerosol
dispersion
Gustavo Zayas*
1
, John Dimitry
2
, Ana Zayas
2
, Darryl O'Brien
1
and
Malcolm King
1
Address:
1
Mucobiology Research Unit, Pulmonary Research Group, University of Alberta, Edmonton, Canada and
2
Summer students, University
of Alberta, Edmonton, Canada
Email: Gustavo Zayas* - g.zayas@ualberta.ca; John Dimitry - john.dimitry@med.ucalgary.ca; Ana Zayas - ana.zayas@ualberta.ca;
Darryl O'Brien - do1@ualberta.ca; Malcolm King - malcolm.king@ualberta.ca
* Corresponding author
Abstract
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".
Published: 02 September 2005
BMC Pulmonary Medicine 2005, 5:11 doi:10.1186/1471-2466-5-11
Received: 01 February 2005
Accepted: 02 September 2005
This article is available from: http://www.biomedcentral.com/1471-2466/5/11
© 2005 Zayas et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Infectious respiratory diseases are a prime cause of mor-
bidity, mortality and health system utilization worldwide,
but have a greater impact on the developing and least
developed countries. Respiratory infections including
tuberculosis (TB) caused 5.5 million deaths during 2001.
Mortality rates are only part of the burden, since individ-
uals with infections are often unable to work, becoming
trapped in a vicious cycle of ill health and poverty [1].
Viruses and bacteria cause most of the infectious respira-
tory diseases. These pathogens can travel from country to
country in different continents in a matter of hours, as
exemplified by the recent outbreak of SARS [1,2]. Chil-
dren, the elderly, and those with defective immune sys-
tems or with underlying chronic diseases are at highest
risk of being affected by airborne infectious diseases.
Health care providers are at risk of acquiring transmissible
respiratory diseases while providing care or working in
contaminated environments. The SARS outbreak has vali-
dated these concerns in advance of the feared flu pan-
demic. SARS has also confirmed that infectious diseases
can cause great disruptions in all sectors – economic, edu-
cational, recreational, familial – and can bring health care
systems to near collapse. Several strategies and devices
have been designed to protect various sectors of society
whether at peacetime or during conflicts.
Different individual protective barriers, such as surgical
masks, face shields and respiratory protection equipment
have been used; however, they yield a much lower protec-
tion than the theory might indicate [3]. The individual
protective equipment must be chosen according to the
agent and to the activities performed by the user, and con-
sider the compliance of the user and the adequacy of the
fit and the effectiveness of the seal. Wearing well-fitted
protective equipment for long periods of time could
impair the satisfactory performance of duties of health
care providers. Hence adequate and cost-effective strate-
gies are still needed.
Vaccination is the main strategy to control outbreaks of a
number of infectious diseases including the flu pandemic,
but the WHO states that other advance control measures
will definitively be required since vaccines take time to
become available [4].
Tuberculosis (TB) caused by Mycobacterium tuberculosis is a
curable, transmissible malady known for at least 3,400
years. The WHO and other organizations estimate that
two billion people around the world are infected with M.
tuberculosis. Eight million new active cases of TB are
reported annually, mostly in age-productive adults. Two
million people died of TB in 2002, more than in any pre-
vious year in history, becoming the leading killer of adults
worldwide [5].
M. tuberculosis can develop resistance to multiple, first-line
anti-TB drugs (MDR-TB). This type of resistance spirals the
costs beyond 100 times per patient, bringing more suffer-
ing to countries afflicted with TB [6]. Involuntary quaran-
tine in TB patients with non-adherent attitudes is enforced
as a last resource measure [7] to control its spread.
In cystic fibrosis, recurrent and persistent respiratory
infection by Pseudomonas aeruginosa is a major cause of
morbidity and mortality in CF patients. Jones et al. [8]
concluded that cross-infection outbreaks of P. aeruginosa
were caused by respiratory aerosol dissemination.
Infectious respiratory diseases, whether viral or bacterial,
are transmitted to non-infected subjects when an infected
patient expels pathogenic microorganisms to the sur-
rounding environment when coughing, sneezing or even
possibly when talking. Duguid [9] determined that the
number of particles of 100 µm or less in diameter con-
tained in the aerosol formed during speaking were
between 0 – 200 particles, while during coughing there
were 0 – 3,500 particles and during sneezing there were
4,500 – 1, 000,000 particles. Fennelly [10], studying the
size of the infectious particles generated during cough,
estimated that close to 50% of them were fine particles,
less than 2 µm range, small enough to reach the alveoli
within the lungs.
For the transmission of any respiratory pathogen at least
three components are required: a) the transmissor or
source (infected person), b) the surrounding environ-
ment, and c) the recipient (non-infected person). It is
important to additionally consider the concentration of
infectious particles determined by the volume of the space
and its ventilation, the length of time of exposure, as well
as the status of defense mechanisms of the exposed
individual.
This prompted a research question: Is it possible to con-
trol the transmission of infectious respiratory diseases by
modulating the physical characteristics of viscoelasticity,
cohesivity and surface tension of the respiratory secretions
to minimize the aerosolization that facilitates transmis-
sion of airborne diseases – a transmission-blocking
approach? We found in our literature search no report in
regards to this type of approach or drugs. Before testing
the mucomodulators we established a fundamental crite-
rion: Can we modulate the physical characteristics of the
respiratory secretions to reduce aerosolization without
affecting normal clearance function, or even better by
improving it?

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Hence, we focused our attention on the mucus layer of the
airways. The mucin macromolecule (Figure 1) consists of
a protein core surrounded by short oligosaccharide side-
chains, held together by different links: O-glycosidic
bonds, disulphide bridges, hydrogen bonds and ionic
bonds, which are the targets of the existing mucolytic
agents [11].
The mucus along with serous fluid forms the airway sur-
face fluid (ASF) that provides a protective milieu for the
airways. The composition and physical characteristics of
ASF allow for normal ciliary activity and airway protection
[12,13]. When disruption of normal secretory or mucocil-
iary clearance processes occurs, respiratory secretions can
accumulate and impair pulmonary function, reduce lung
defenses and increase the risk for infection and possibly
neoplasia [14].
Ninety-five per cent (95%) of mucus is water and the
remaining 5% is made up of proteins, carbohydrates,
salts, etc. Therefore, when the airway mucus layer interacts
with high-speed airflow during coughing, there is forma-
tion of droplets of different sizes that are expelled to the
surrounding environment as an aerosol. The concentra-
tion of droplets and their size distribution may each play
an important role in the transmission of respiratory infec-
tious diseases.
Micron-sized droplets dry quickly, remain airborne for
long periods and reach the lower respiratory system when
Schematic diagram of the macromolecules of mucus with the different types of crosslinksFigure 1
Schematic diagram of the macromolecules of mucus with the different types of crosslinks. Sodium tetraborate would contrib-
ute to crosslink formation involving accessible galactose moieties in the oligosaccharide side-chains. Calcium chloride would
add crosslinking through divalent ionic interactions.
1. COVALENT BONDS
2. IONIC BONDS
• mucin macromolecules have both
positive and negative fixed charges,
which are capable of interacting
3. HYDROGEN BONDS
• H-bonds link the
oligosaccharide side-chains
4. VAN DER WAALS' FORCES
• interdigitation between
oligosaccharide moieties
may be important
5. INTERMINGLING
• physical entanglements
between mucin macromolecules
6. EXTRACELLULAR DNA
&F-ACTIN
• parallel network formation in infection
• glycoprotein subunits are linked
primarily by intramolecular S-S bonds
Each of these types of
bonds is subject to
Mucolysis
Types of Bonds Occurring in a Mucous Gel
1
4
6
3
5
1
|
O
O
H
H
S
S
2
S
S
SO
3
NH
3
+

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inhaled. These breathable particles might indirectly infect
many persons, e.g. via a common ventilation system. In
contrast, large droplets are propelled onto the nearest sur-
face – oral, respiratory, or ocular mucosa of a nearby per-
son – or settle readily to the floor. Regarding transmission,
larger particles may have an immediate effect – direct but
limited – while micron-size particles may have more indi-
rect effects with longer-lasting and widespread
consequences.
For this study, our basic goal was to assess a new genera-
tion of mucomodulators in a novel approach that has the
potential to modulate the physical characteristics of the
mucin glycoprotein gel network. This intervention would
increases the mucin crosslinking binding sites to reduce
the aerosolizability on mucus simulants (surrogate of
transmissibility), and/or forming poorly soluble mucin
complexes. We aimed to achieve the former while interfer-
ing only minimally with normal mucus clearance and
hopefully even improving clearability in patients with air-
way mucus retention.
Our hypothesis is that transmission of airborne diseases
can be controlled by implementing a co-adjuvant phar-
macological intervention, additional to the indicated
standard treatment, aimed at modulating the physical and
biochemical characteristics of the respiratory secretions of
both the transmissor who carries the infection to mini-
mize expectorated material that carries the pathogens, and
in the non-infected potential recipient.
Methods
For this phase we used mucus simulants for testing the
effect of the mucomodulators. We also used our ex vivo
frog palate exposure model in combination with our sim-
ulated cough machine to assess the clearability as well as
the aerosol formation capability of the artificial mucus
with or without mucomodulators.
Mucus simulants (MS)
The MS ranged from a non-viscous, non-elastic substance
(water) to MS of varying degrees of viscosity and elasticity.
To prepare viscoelastic mucus simulants, we followed the
procedure described by King et al. [15]. Mucus simulants
were formulated and developed to be transported within
the normal range of velocity in a ciliated epithelium and
cleared from a simulated cough machine (SCM) set to
have a close to normal human cough. Mucus simulants
viscous-only were prepared dissolving different concen-
trations (0.5 %, 0.75 % and 1 %) of locust bean gum
(LBG) in warm Ringer solution, and then adding activated
charcoal, bromophenol blue 1% or Coomassie blue to
dye the solution for better visualization.
Frog palate preparation
From a bullfrog, Rana catesbiana, the upper portion of the
head was removed following the procedures described in
previous work [16,17], by cutting with scissors through
from the junction of the posterior pharynx and esophagus
out to the skin of the back. This procedure was carried out
after lowering the body temperature of the frog for 30 – 60
minutes inside a refrigerator to abolish pain sensations.
The palate was examined for macroscopic lesions, such as
ulcers, petechia or redness as evidence of inflammation;
only palates free of inflammatory indicators were used.
Any blood remaining in the epithelial surface was care-
fully washed away, then the excised head was placed pal-
ate side facing upwards on a piece of gauze saturated with
frog Ringer solution (FRS) in a Petri dish.
The palate was placed inside the frog chamber, a wooden
box with a glass top and fitted glass front, and manipu-
lated through glove openings. The humidity inside the
box was maintained near 100% using a Pari jet nebulizer,
and the temperature was kept between 22° and 24°C by
a rheostat-controlled, externally mounted light source.
Before carrying out any measurement, the palate was
allowed to stabilize inside the box for 15 minutes before
testing.
The FRS was prepared by mixing standard Ringer injection
with sterile water (2:1). The composition of standard frog
Ringer (in mmol/L) is 90 NaCl, 3 KCl, 2 CaCl2, and 15
NaHCO3 (220 mosm/L). The Health Sciences Animal
Policy and Welfare Committee, University of Alberta
approved the experimental procedures involving animals
Simulated mucus transport velocity (MTV) determination
The palate was placed under a dissecting stereomicro-
scope provided with a reticulated eyepiece. Mucociliary
clearance was determined by observing the movement of
particles of charcoal powder gently deposited on a sample
of simulated mucus on the palate surface; its clearance
was visually monitored and MTV determined. The dis-
placement of 3 – 5 µL of endogenous frog mucus sample
was calculated by dividing the distance traveled by the
transit time across the 0.3-inch (7.62 mm) segment
marked between 0.1 and 0.4 inches in the graduated eye-
piece. To obtain control and simulated mucus transport
velocities, at least five measurements of the time required
for the mucus sample to travel the defined distance were
made.
Tissue and mucus sample collection
Samples of tissue and mucus from frog palates exposed to
topical application of mucomodulators, were immersed
in glutaraldehyde 2.5%, and stored at 4°C for later scan-
ning electron microscope (SEM) studies. The epithelial

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tissue was carefully dissected and separated from the pal-
ate musculature.
Aerosol formation assay
To assess the aerosol pattern formed after coughing, we
used a range of fluids of varied physical characteristics
from non-viscous, non-elastic substances such as water, to
highly viscoelastic substances such as simulated mucus
samples of varying degrees of viscosity and elasticity. To
better visualize the dispersion pattern of the fluids
exposed to high-speed airflow in the SCM, we stained all
the fluids.
Spinnability
The capacity of a fluid to form threads when stretched, an
expression of elasticity, was assessed in the Filancemeter
(SEFAM, France) prior to exposure to airflow in the SCM.
A volume of approximately 50 µL of the sample to be
studied is introduced in a reservoir that is then sucked in.
A low current is applied to the sample from a voltage sup-
ply, and when the sample is stretched vertically, the appa-
ratus electronically measures the maximum length of the
thread at the moment of the rupture of the thread. For
every mucus sample, four measurements of the length of
the thread at the moment of rupture were made, and used
to compute the spinnability in mm.
Cough aerosolization
The simulated cough machine was used to test the main
hypothesis that the modulated mucus will result in less
aerosolized (airborne) material following cough. Since
cough is the most critical process of mucus aerosolization,
the successful reduction of droplet production during
cough will likely predict reduced amounts emitted during
sneezing and speaking. All fluids were placed at different
distances from the "mouth" opening of the simulated air-
way to examine if distance from the target may be a factor
in determining the dispersion pattern. Later, we decided
on a fixed distance to place the sample of MS to be
exposed to high-speed airflow interaction. The target was
a "bullseye-type" (circles within circles). We also placed
paper sheets to lateral sides, on top, and below the target
sheet to account for any fluid that is dispersed in areas
other than on the target sheet.
Cough clearability assay
The modified simulated cough machine system comprises
the following elements: A 10-L tank with compressed air,
which serves as a pressure reservoir that generates airflow,
simulating the lungs during a cough maneuver. The pres-
sure generated prior to a normal adult cough is approxi-
mately 8 psi, and we used this pressure value to simulate
the airflow pattern of a human cough for each trial.
This modified machine has a model "airway or trachea"
consisting of a 28 cm long rigid acrylic tube with a circular
2.5 cm cross-section. It has an 8 cm detachable muzzle
end-piece (59.805 g) that enabled us to weigh the sample
of MS loaded before and immediately after each cough
maneuver and report the mass of MS expelled or retained.
A solenoid valve located between the pressure reservoir
and the model trachea control the gas released from the
tank.
An aliquot of sputum is layered on the bottom of the
model trachea and driven forward by the pressurized gas.
The distance traveled by sputum samples under standard-
ized cough-simulating airflow is used as a measure of
cough clearability. The target was placed at a working dis-
tance of 40 cm from the mouth of the SCM. The simulated
cough maneuver is repeated four times with each sputum
sample.
Airway clearance ("expectoration" of mucus simulant
from the "airways"): Measurements of the weight of the
MS sample loaded before and after each cough maneuver
were carried out in a Mettler AE 163 microbalance.
Scanning electron microscopy
Samples of mucus and tissue were placed in 2.5% glutar-
aldehyde solution immediately after collection and stored
at 4°C until processing. The samples were post-fixed in
1% osmium tetroxide in Millonig's buffer at room tem-
perature for one hour. They were then washed in a series
of ethanol (50 – 100%), ten minutes at each step, fol-
lowed by two additional periods of absolute ethanol (10
minutes each). The samples were further dehydrated by
critical point drying at 31°C for 5 – 10 minutes, then
mounted on a specimen holder for SEM and dried over-
night in vacuum desiccators. In the final stage of prepara-
tion for viewing, the samples were sputter coated with
gold (Edwards, model S150B Sputter Coater). Samples
were viewed using SEM (Hitachi S-2500). Images were
scanned directly to a computer and stored as image files
for subsequent viewing and analysis.
Mucomodulators
We explored four different approaches that have the
potential to accomplish our primary goals of reducing aer-
osolization of mucus while promoting airway clearance.
However, for the purpose of this study, we report only two
of them here. Sodium tetraborate, named also as XL"B" or
mucomodulator B, and calcium chloride, named also as
XL "C" or mucomodulator C. The other approaches, high
molecular weight dextran, named also XL "D" or muco-
modulator D, and polyarginine, named also XL "A" or
mucomodulator A will be reported independently in
other publications [18].

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Sodium tetraborate (XL "B")
Sodium tetraborate causes reversible crosslink formation
between galactose units [19], which are the major neutral
sugar components of mucin. Addition of borate to galac-
tose polymers preferentially raises elasticity relative to vis-
cosity. We added three different volumes of 0.1 M borate
(1.5 µL, 15 µL and 150 µL) to the MS to produce three lev-
els of crosslinking, and exposed each of the gels to four
cough maneuver measurements per trial.
Calcium chloride (XL "C")
Calcium chloride increases the concentration of divalent
cations in the mucus. Conceptually, this is the opposite of
what occurs in nature during mucin exocytosis, where
intracellular mucin granules, which were held tightly
through ion crosslinks, give way to much looser interac-
tions as ion exchange occurs during fusion with the apical
membrane [20]. A volume of 4 µL of frog Ringer and 4 µL
of three different concentrations of calcium chloride
(0.001 M, 0.01 M and 0.1 M) were directly applied
through a micropipette to a fresh frog palate in order to
assess their effect on mucus transport velocity and on the
morphology of the tissue and mucus layer.
Statistical analysis
Data are expressed as mean ± standard deviation unless
otherwise stated. A paired Student-T test was used for sim-
ple comparison. Neuman-Keuls analysis was applied for
repeated measurements. The level of significance was set
at 5 %.
Results
In this study we report the effect that the mucomodulator
XL"B" has on MS on spinnability, clearance by ciliary
action and by high-velocity airflow interaction (cough
maneuver), as well as some effects that XL "C" has on the
physical appearance of frog mucus and on native mucus
transport on the frog palate.
Effects of sodium tetraborate
Staining the samples
Bromophenol Blue made the MS solutions very watery
and it reduced viscosity of the MS; hence we rejected the
use of this dye from further testing. Activated charcoal did
not alter the viscoelastic properties of the MS sample and
helped to better visualize the gel sample on the frog pal-
ate; however charcoal adds mass to the sample that even-
tually might alter the dispersion pattern. Coomassie Blue
did not alter the viscoelastic properties of MS; hence it was
used to dye the samples.
Transportability
Samples of MS of non-viscous, non-elastic properties
(frog Ringer solution) of approximately 0.2 – 0.5 mL at
room temperature were placed on the frog palate; these
dispersed on the surface and were not transported by cili-
ary action.
Viscous-only samples in the low range of concentration
(LBG 0.5%) did not maintain their shape and dispersed
on the epithelial surface and were not propelled by ciliary
action. Viscous-only samples in a higher concentration
range (LBG 1.0%) maintained their shape and were trans-
ported very slowly by ciliary action, approximately five
times slower than control.
Thread formation
Non-viscous, non-elastic samples and the viscous-only
samples did not produce any measurable spinnability in
the Filancemeter. Working with samples of viscoelastic
MS, we attained repeatable lengths of the filament in the
low range (1 – 10 mm) and high range (30 – 50 mm) val-
ues in the Filancemeter, but found it difficult to consist-
ently obtain medium ranges (11 – 29 mm), even using
different combinations of viscosity and elasticity in MS.
Elasticity of the crosslinked MS (length of the filament)
increased in a direct proportion to the volume (1.5 µL, 15
µL and 150 µL) of 0.1 M added borate (Figure 2).
Paired T-testing showed significant differences in thread
formation when comparing the three added volumes. The
Newman-Keuls test also indicated a significant difference
(p < 0.05) in thread formation of the MS when comparing
medium added volume (15 µL) to high volume (150 µL)
of sodium tetraborate. Also, the Newman-Keuls test indi-
cated a very significant difference (p < 0.01) in thread for-
mation when comparing low added volume (1.5 µL) to
high volume (150 µL) of borate.
The average linear velocity transport rate by ciliary action
of crosslinked mucus simulant in the frog palate was
approximately 60 percent (46% – 76%) as effective as
native mucus transport used as control (100%) when dif-
ferent volumes (1.5 µL – 150 µL) of 0.1 M sodium tetrab-
orate were applied. When different volumes of lower
concentration borate (0.01 M) were applied, the transport
velocity rate was no more than 30 percent (13% – 30%)
as effective as control.
"Cough clearance"
Non-viscous, non-elastic MS samples (water) placed at
various distances from the mouth of the artificial airway
and exposed to a simulated cough maneuver had a high
degree of dispersion onto the bullseye target.
A sample of approximately 0.5 mL of crosslinked MS
stained with Coomassie Blue was loaded inside the
detachable portion of the artificial airway and then
weighed in a microbalance. The cough machine was trig-
gered and the detachable portion with the remaining

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aliquot of the sample not expelled by the cough maneuver
was immediately weighed again. The amount of MS
expelled from the artificial airway increased in direct pro-
portion to the volume of 0.1 M sodium tetraborate added
(Figure 3). Paired T-test analysis showed there exists a sig-
nificant difference comparing the volume of MS sample
expelled with increasing volume of 0.1 M added borate.
The Newman-Keuls test indicated a significant difference
(p < 0.01) in the weight of MS expelled from the SCM
when comparing low added volume (1.5 µL of XL "B") to
high volume (150 µL of XL "B").
MS loaded in the simulated airway and exposed to high-
speed airflow interaction (simulated cough) broke up into
multiple droplets of various sizes that were expelled in the
form of a cloud of aerosol that landed on the target. Some
portions of the MS expelled did not reach the target but
landed on pieces of paper placed below, above and along
the lateral sides of the corridor between the mouth and
the target. The amount of aerosol striking the target and
the amount of fine aerosol decreased progressively with
added volume of crosslinking agent (Figure 4).
The volume of XL "B" added to the MS sample was
inversely related to the number of droplets formed after a
cough maneuver and airflow interaction; the larger the
volume added, the fewer droplets were formed. It was also
observed that as the added volume of XL "B" (borate)
increased, the size of the droplets formed after the airflow
interaction increased; hence few, big cohesive droplets
were formed, which did not reach the target but did fall in
the paper placed immediately below the exit. The propor-
tion of MS sample remaining in the "airway" after a cough
maneuver correspondingly decreased with increasing
Effect of the crosslinking agent XL "B" (sodium tetraborate) on spinnability in artificial mucusFigure 2
Effect of the crosslinking agent XL "B" (sodium tetraborate) on spinnability in artificial mucus. * p < 0.05 with respect to 150
µL; ** p < 0.01 with respect to 150 µL.
Spinnability vs. Volume of
Crosslinkin
g
A
g
ent Added to Mucus
0
10
20
30
40
50
60
1.5 15 150
Volume (µL) 0.1 M XL "B
**
*

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added volume of crosslinking agent (from 53% for 1.5 µL
to 40% for 15 µL to just 4% for 150 µL added sodium
tetraborate).
Calcium chloride effects
XL "C" (calcium chloride 5 µL) applied topically to our ex
vivo frog palate model, causes mucus "clumping" without
modifying the normal physical appearance of the ciliated
surface when compared with ciliated surface exposed to
frog Ringer solution (Figure 5).
Direct application (5 µL) of three concentrations (0.001
M, 0.01 M, 0.1 M) of CaCl2 on the frog palate seems to
moderately slow the way that native frog mucus is trans-
ported by ciliary action in the epithelial model (Figure 6).
The Newman-Keuls analog procedure showed a signifi-
cant difference (p < 0.05) of MTV of frog mucus samples
only when the lowest concentration was applied,
compared to the other concentrations and to the Frog
Ringer control.
Discussion
In this study we have acquired preliminary evidence that
the use of mucomodulators yields acceptable mucociliary
clearance in an ex vivo epithelial model, increases "expec-
toration" in a model system, and yet achieves a desired
target of a significant reduction in fine aerosol formation.
We have also obtained graphic evidence that one of our
mucomodulator agents, calcium chloride, which
increases the concentration of divalent cations, in fact
increases the interaction of the ion crosslinks in the frog
mucus, resulting in modification of the physical structure
Cough clearance ("expectoration") of sodium tetraborate (XL :B") crosslinked mucous gel simulants, i.e. the percentage of ini-tial load clearing the mouth of the artificial trachea during the cough maneuverFigure 3
Cough clearance ("expectoration") of sodium tetraborate (XL :B") crosslinked mucous gel simulants, i.e. the percentage of ini-
tial load clearing the mouth of the artificial trachea during the cough maneuver. ** p < 0.01 with respect to 150 µL.
Percentage of Mucus Expelled from a
Simulated Cough Machine
vs. Volume
of Added Crosslinking Agent
0
20
40
60
80
100
120
1.5 15 150
Volume (uL) of 0.1M XL "B" added to mucus simulant
**
**

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of the mucous layer. This action suggests that the
aerosolizability of the mucus or the ability to form tiny
droplets may be decreased.
To enhance the validity of the obtained results, we formu-
lated an artificial mucus according to King [19] that dis-
played physical properties of viscosity, elasticity,
reproducible thread formation and transportability in a
ciliated epithelium within an acceptable range that most
closely simulates airway mucus. 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". Rubin & Van der Schans [21]
stated, "If an intervention is thought to have a directly meas-
urable effect on sputum properties or on sputum clearability and
if these changes cannot be demonstrated in vitro, it is unlikely
that they will be observed in clinical trials."
Results presented in this article and obtained thus far from
our model system – mucous gel simulant, artificial airway,
simulated cough function and ex vivo epithelial model –
are limited but striking, and clearly support our muco-
modulator approach. We were encouraged to continue
with an in vivo mammalian model that will be presented
independently (18). We expect that similar results will
eventually be observed in clinical trials. Results from this
study suggest that the mucomodulators tested appear not
to affect the morphology or function of the mucociliary
apparatus.
We have designed a new mucomodulator therapy – aiming
to change the physical properties of ASF to enhance the
clearance of mucus from the respiratory tract, as well as to
optimize aspects of lung defense that depend on the
mucous layer. Mucomodulators work differently than
mucolytics, since the latter were designed to disrupt the
structural macromolecules that give respiratory tract
mucus its physical characteristics, and from other agents
designed to increase mucus flow by stimulating ciliary
activity or improving periciliary fluid hydration. As used
here, mucomodulators act to increase the cohesivity of
airway mucus simulants. Mucomodulator therapy to
combat mucus retention may become a major considera-
tion in the treatment of cystic fibrosis and other chronic
lung diseases in which mucus hypersecretion and
impaired airway clearance produce symptoms [11], and
require adequate and novel secretion management. With
the mucomodulator approach, we intend to adjust or reg-
ulate the mucin macromolecules that give respiratory
mucus its physical characteristics in order to minimize the
aerosolization that facilitates transmission of airborne
diseases. It should be noted that it is not "normal" to
Aerosolization and dispersion pattern effects for sodium tetraborate crosslinked mucous gel simulantsFigure 4
Aerosolization and dispersion pattern effects for sodium tetraborate crosslinked mucous gel simulants. Aerosol pattern at 40
cm. following a standardized cough maneuver. Both the amount of aerosol striking the target and the amount of fine aerosol
decreased progressively with added crosslinking agent.
1.5 µl 15 µl 150µl

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cough and aerosolize particles that can be breathed in by
others, although it is common. Thus a potential therapy
that could reduce this tendency to aerosolize particles
when coughing should be considered a form of modula-
tion in the sense of restoring a balance or state of
homeostasis.
Cellular debris and other byproducts of infection contrib-
ute to make more difficult the clearance of infected mucus
in CF patients, requiring antibiotics and mucolytic ther-
apy. These approaches have helped to reduce the mortal-
ity rate in CF and other patients; however, there still
remains a long way to go to reduce transmission of path-
ogens in non-infected subjects.
We have invented a novel and non-intuitive approach to
improving airway mucus clearance by adding crosslinks in
a selective and controlled manner through a specialized
new generation of mucomodulators. If further testing
bears out our initial findings, it could lead to treatments
that would reduce the rate of morbidity and improve the
quality of life of individuals who have difficulties with air-
ways secretions management.
Essentially, we have found preliminary evidence that cer-
tain mucomodulators that increase cohesive interactions
between mucin macromolecules lead to a mucus that is
more easily clearable, most likely by airflow-dependent
mechanisms, as suggested by results obtained from our
Effect of the crosslinking agent calcium chloride (XL "C") on the physical changes of the fresh frog mucusFigure 5
Effect of the crosslinking agent calcium chloride (XL "C") on the physical changes of the fresh frog mucus. SE micrograph show-
ing the "clumping" effect of calcium chloride applied topically to the frog palate. The ciliated surface of the palate is seen below
the mucus and does not appear different from control views.

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SCM The latter is a preliminary answer to a recurring
question from lung experts: that using this approach
could "gum up" the airway secretions. Such an improve-
ment in mucus quality related to clearance would benefit
many, if not most, patients with chronic airway disease,
including those suffering from chronic obstructive pul-
monary disease, who depend in whole or in part on air-
flow clearance mechanisms to maintain airway hygiene
[13,22]. Further work is needed in clarifying the mecha-
nisms involved in the clearance of the airway secretions.
This novel approach might be of particular importance for
those in the ICU or those with spinal cord lesions who
require assisted ventilation. At the same time, increasing
the cohesiveness in this manner leads to a reduction in
fine aerosol formation during expectoration – a clear
advantage in helping to control the spread of airborne
infections. This new approach may be applicable to a
range of infectious disease threats occurring naturally like
the feared next flu pandemic or the possible deliberate use
of biological agents.
There are some additional critical advantages of this novel
technology: Health Canada and the US FDA and other
regulatory agencies have already approved the selected
compounds for pharmaceutical use in humans. If any of
these compounds (or a combination thereof) is judged
suitable for clinical trials, established safety data could be
utilized to fulfill some of the regulatory requirements for
pre-clinical studies.
Some areas of applicability would include transmission
prevention of clinically and epidemiologically important
pathogens like M. tuberculosis and MDR-TB, in the feared
flu pandemic and SARS-CoV, as well as P. aeruginosa in
cystic fibrosis. Our mucomodulators would most likely
represent containment for transmission of respiratory
pathogens especially during outbreaks, allowing strategic
sectors of society to be protected and remain functional,
like the health system, law enforcement, service providers,
as well as the work force.
Some other critical advantages of this novel technology
are: Our low cost mucomodulators will not need to
Effect of different concentrations of sodium tetraborate (XL "B'') on MTV of frog mucus. * p < 0.05 with respect to the other concentrationsFigure 6
Effect of different concentrations of sodium tetraborate (XL "B'') on MTV of frog mucus. * p < 0.05 with respect to the other
concentrations.
*
Effect of XL "C" (CaCl
2
) on Frog Palate
Mucus Transport Velocity
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
FR 0.001 0.01 0.1
Molar Concentration
Mucus Transport
Velocity (mm/sec)
*

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undergo constant development and reformulation due to
viral or bacteria mutation or resistance. The technology is
designed to act on the host rather than the causative
organism, which will provide more longevity to it. The
compounds under consideration are less perishable than
vaccines, amenable to distribution, particularly in warm
climates, and simple to manufacture to even allow for
rapid response in outbreak situations. In addition, some
of the concept testing could involve veterinary trials in air-
borne animal diseases, an aspect which we are also
exploring.
In model studies using vegetable polysaccharides, sodium
tetraborate preferentially raises elasticity relative to viscos-
ity, and current knowledge prior to the development of
mucomodulators indicated that tetraborate solutions
would favour mucociliary clearability at the expense of
cough clearability and aerosolizability [12] Now, it
appears that cough clearability and aerosolizability are
both linked to cohesivity, but in opposite senses, at least
within the range of the current study. Thus it appears that
sodium tetraborate solutions, by altering mucous gel
cohesivity, can decrease aerosolizability (fine aerosol for-
mation during coughing) while increasing cough cleara-
bility (bulk clearance of mucus).
In summary, the mucomodulator technology is innova-
tive in that it represents a reversal in current thinking
about respiratory disease management. The resulting
product will be an unprecedented type of tool in airway
hygiene, in secretion management as well as in prevention
of droplet-spread illnesses, close person-to-person-con-
tact and airborne transmission – a safe, efficacious, non-
vaccine preventative therapy.
Competing interests
The author(s) declare that they have no competing
interests.
Acknowledgements
This project was funded in part by the Canadian Cystic Fibrosis Foundation
and the Canadian Institutes of Health Research.
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