Hindawi

International Journal of Polymer Science

Volume 2017, Article ID 3728485, 10 pages
https://doi.org/10.1155/2017/3728485

Research Article
Bioactive 3D-Shaped Wound Dressings Synthesized from
Bacterial Cellulose: Effect on Cell Adhesion of Polyvinyl Alcohol
Integrated In Situ

Marlon Osorio,1 Jorge Velásquez-Cock,1 Luz Marina Restrepo,2 Robín Zuluaga,3

Piedad Gañán,1 Orlando J. Rojas,4 Isabel Ortiz-Trujillo,5 and Cristina Castro6
1
Facultad de Ingenierı́a Quı́mica, Universidad Pontificia Bolivariana, Circular 1, No. 70-01, Medellı́n, Colombia
2
School of Health Sciences, Universidad de Antioquia, Calle 51D 62-29, Medellı́n, Colombia
3
Facultad de Ingenierı́a Agroindustrial, Universidad Pontificia Bolivariana, Circular 1, No. 70-01, Medellı́n, Colombia
4
Biobased Colloids and Materials Group (BiCMat), Department of Forest Product Technology, Aalto University,
School of Chemical Technology, Espoo, Finland
5
Facultad de Medicina, Universidad Pontificia Bolivariana, Calle 78B No. 72A-109, Medellı́n, Colombia
6
Facultad de Ingenierı́a Textil, Universidad Pontificia Bolivariana, Circular 1, No. 70-01, Medellı́n, Colombia

Correspondence should be addressed to Cristina Castro; cristina.castro@upb.edu.co

Received 5 March 2017; Accepted 1 August 2017; Published 18 September 2017

Academic Editor: Miriam H. Rafailovich

Copyright © 2017 Marlon Osorio et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We investigated wound dressing composites comprising fibrils of bacterial cellulose (BC) grown by fermentation in the presence
of polyvinyl alcohol (PVA) followed by physical crosslinking. The reference biointerface, neat BC, favoured adhesion of fibroblasts
owing to size exclusion effects. Furthermore, it resisted migration across the biomaterial. Such effects were minimized in the case of
PVA/BC membranes. Therefore, the latter are suggested in cases where cell adhesion is to be avoided, for instance, in the design of
interactive wound dressings with facile exudate control. The bioactivity and other properties of the membranes were related to their
morphology and structure and considered those of collagen fibres. Bioactive materials were produced by simple 3D templating of
BC during growth and proposed for burn and skin ulcer treatment.

1. Introduction reduce inflammatory and immune responses [7–12]. Modern

Skin ulcers and burns often result in extensive damage to
the skin layers [1]. Significantly, burns induce anatomical,
physiological, endocrinological, and immunological alter-
ations [2]. In the United States alone, over two million
burn injuries per year are brought to medical intervention.
Most of these injuries are minor; however, approximately
20,000 patients sustain severe burns requiring admission to
specialized units [3]. Most burn wounds are treated with
sterile gauze, saline solution, and topical prophylaxis [4].
These treatments, however, are not fully effective for pain
relief, hydration, and protection [5].
Modern wound dressings have been developed to facili-
tate functions at the wound area. These dressings are designed
to prevent wound dehydration, promote healing [6], and
wound dressings are classified as passive, interactive, or
bioactive [6]. The sole role of passive dressings is protection
while interactive dressings promote healing through a moist
wound environment. Interactive dressings, however, not only
maintain a moist environment but also interact with the
components of the wound bed to further enhance healing
[6, 13–15].
In wound dressing applications, there is a pressing need
to develop biocompatible hydrogel biomaterials [16]. In this
context, biocompatibility refers to the ability to perform with
an appropriate host response in the given application [17–19].
For this purpose, hydrogels are ideally suited since they are
able to absorb more than 70 wt% of their dry weight in water
[8].
2 International Journal of Polymer Science
A number of studies have highlighted the use of polyvinyl
alcohol (PVA), bacterial cellulose (BC), and their composites
in biomedical applications, including scaffolds for tissue engi-
neering, heart valves, articular cartilage, and artificial corneas
[20–22]. In fact, PVA/BC nanocomposites have been tailored
to suit the properties of skin, where BC and PVA mimic
collagen and elastin for their roles in natural soft tissues [23].
In addition, PVA/BC biomaterials can be shaped into gloves,
masks, and wearable webs due to the conformability of BC
during its fermentation [1]. Shaped wound dressings provide
a high degree of adherence to the tissues surrounding a lesion,
preventing their detachment in areas that are subjected to
movement (e.g., torso, hands, and face) [11].
Two primary approaches that are used toward PVA/BC
nanocomposites include in situ and ex situ processing [23,
24]. In contrast to ex situ synthesis, nanocomposite bio-
materials developed via in situ fermentation favour homo-
geneous material distribution, which promotes a number
of synergistic properties [25]. In this case, crosslinking is
required to limit the solubilization of the matrix. Recently, an
in situ PVA/BC nanocomposite was developed via physical
crosslinking (Castro et al.). This type of crosslinking avoids
the use of toxic compounds, including chemical crosslinkers
[26], some of which are known to be carcinogenic and neu-
rotoxic [27]. The in situ PVA/BC nanocomposites developed
so far present a highly porous, percolated morphology; their
chemical, mechanical, and thermal properties make them
appropriate as biomaterials [20, 26, 28–31]. Any biomedical
application, however, demands in vitro assessments, which
are available for BC biomaterials for wound dressings [32–
35]; however, to the best of our knowledge, no reports exist in
this context for PVA/BC nanocomposites obtained via in situ
fermentation.
Therefore, this study evaluates the morphological,
mechanical, water adsorption, and in vitro performance
(adherence and cell migration) of PVA, BC, and PVA/BC
nanocomposites for wound dressing. Our efforts included
the classification of the developed materials into currently
accepted wound dressings. The results indicate possibilities
for exudate management and the use of BC as bioactive
wound dressing that facilitates skin cell regeneration.
2. Materials and Methods
2.1. Materials. Glucose (PubChem CID: 64689), yeast
extract, peptone, disodium hydrogen phosphate (PubChem
CID: 24203), citric acid (PubChem CID: 311), potassium
hydroxide (PubChem CID 14797), silicon rubber, and PVA
(molecular weight 89000–98000 and 99+% hydrolysis,
PubChem CID 11199) were all acquired from Sigma-Aldrich.
Bacterial cellulose was produced by Komagataeibacter
medellinensis [36, 37]. Its elemental analysis included carbon
(44.2 ± 1.6%), hydrogen (6.3 ± 0.25%), and nitrogen (0.39
± 0.04%) [37]. The nitrogen content relates to the amino
acids from the residual bacteria after purification. The BC
crystallinity index is ∼0.65, with a I𝛼/I𝛽 ratio of ∼0.74, indi-
cating the enrichment of the I𝛼 polymorph [38]. Dulbecco’s
Modified Eagle Medium, foetal calf serum, and fibroblasts
in primary culture were employed for cell culturing. All
reagents were analytical grade. Fibroblasts were kindly
donated by the Grupo de Ingenierı́a de Tejidos y Terapias
Celulares, Universidad de Antioquia, Medellı́n, Colombia.
2.2. Bacterial Cellulose. In order to produce the BC biomate-
rial, 90 mL of Hestrin & Schramm culture medium (HS) [39]
was adjusted to pH = 3.6 with citric acid and then sterilized
in autoclave at 15 psi for 15 min. After reaching room tem-
perature, the culture medium was inoculated with 10 v/v%
of Komagataeibacter medellinensis [37] and distributed in
a six-well polystyrene plate. The fermentation was carried
out for 7 days at 28∘ C, after which the BC biomaterial was
extracted and purified with 5 wt% of KOH solution at room
temperature for 14 h followed by continuous rinsing with
deionized water until pH = 7.0. The resultant material was
sterilized in autoclave (121∘ C for 15 minutes) for later use.
2.3. PVA and PVA/BC Nanocomposites. The method to pro-
duce the PVA/BC nanocomposite included PVA (5 wt% con-
centration) that was solubilized in aqueous culture medium
(90∘ C and 1000 rpm) for 20 min, sterilized, and then inoc-
ulated with 10 v/v% of Komagataeibacter medellinensis, dis-
tributed in Petri dishes, and incubated for 15 days at 28∘ C.
These conditions were optimized in our previous efforts [26].
The obtained biomaterial was physically crosslinked in a
Labconco FreeZone Bulk Tray Dryer at atmospheric pressure
using six freezing/thawing cycles between 20∘ C and −20∘ C
using a heating/cooling rate of 0.1∘ C/min and a holding time
of 6 h. The relative amount of PVA/BC in the nanocomposite
was ca. 80/20 wt%. Purification of all biomaterials was per-
formed as described for BC. After achieving pH = 7.0, and
in order to sterilize the PVA and PVA/BC biomaterials for
cell culture, they were oven-dried overnight at 50∘ C, cut into
discs to fit the wells in the polystyrene plate, and sterilized
with ethylene oxide.
2.4. Composite Morphology. Scanning electron microscopy
(SEM) was used to examine the microstructure of the
biomaterials. The samples were swelled in distilled water
to equilibrium and then dehydrated by lyophilisation; once
dried, they were coated with gold (12 nm thick) using an ion
sputter coater and observed with a Jeol JSM 5910 LV electron
microscope operated at 10 kV at 200, 1000, and 5000x.
2.5. Wet Mechanical Strength. Young’s modulus and tensile
strength of the biomaterials (BC, PVA & PVA/BC) in wet
condition were evaluated in air using an Instron 5582 Uni-
versal Testing Machine, equipped with a 50 N load cell with
a cross head speed of 12.5 mm/min. For testing, dumbbell
shaped strips were used (dowel: width 5 mm × length 18 mm).
The mechanical tensile data were collected for 7 different
specimens per biomaterial type. Young’s modulus and the
tensile strength were determined according to ASTM D882
Standard Test Method for Tensile Properties of Thin Plastic
Sheeting in the initial slope.
2.6. Swelling. The swelling ratio of biomaterial is relevant in
characterizing the amount of nutrients available to cells and
International Journal of Polymer Science
cell migration products [8]. The swelling measurements were
carried out following the methodology reported by Chang
et al. [40]. To reach swelling equilibrium, PVA and PVA/BC
biomaterials were hydrated in distilled water for over 2 weeks
at room temperature. The equilibrium-swelling ratio was
experimentally determined as percentage of swelling (S%)
using
𝑊𝑠
𝑆% = ∗ 100, (1)
𝑊𝑑
where 𝑊𝑠 and 𝑊𝑑 are the weight of the swollen gel and dried
material (drying at 70∘ C and 0.7 bar of vacuum) measured
at room temperature. All measurements were recorded in
triplicate. The kinetics of water uptake was determined with
samples that were removed from water at regular time
intervals. The weight of each sample was gravimetrically
recorded. Before the measurements, the biomaterial surfaces
were wiped with filter paper to remove superficial water. The
sample weight was recorded as the average value of three
measurements. The results are expressed as water uptake (𝑊𝑢 )
and calculated following
(𝑊𝑡 − 𝑊𝑑 )
𝑊𝑢 = ∗ 100, (2)
𝑊𝑠
where 𝑊𝑡 is the weight of the swollen gel in distilled water at
time 𝑡 at room temperature.
2.7. Cell Adhesion. Sterile BC, PVA, and PVA/BC biomateri-
als were placed in a six-well plate for cell culture and hydrated
for 4 days with Dulbecco’s Modified Eagle Medium (DMEM)
and foetal calf serum (FCS) at 10 v/v%. After the fourth
day, the medium was exchanged and 50,000 fibroblasts were
seeded per well on the biomaterial surface. The incubation
time was 8 days, with medium changes every fourth day.
Fibroblasts, being present in great amounts on the skin, were
chosen to conduct the test. They are responsible for skin
cell regeneration and are used for testing biomaterials for
skin substitutes [11, 33, 41, 42]. To monitor cell adhesion,
the biomaterials were stained with haematoxylin-eosin and
imaged with a Leica microscope and micrometrics camera.
Furthermore, the BC-based biomaterial was stained with
ethidium bromide, and fluorescence images were recorded
using a Leica microscope at 40x and 100x and a Moticam
(Motic) of 10 megapixels.
2.8. Cell Viability. The numbers of viable cells were quan-
tified throughout the incubation time via the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay. This assay is based on the fact that only live cells can
reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to formazan [43]. The protocol was
performed as described by Weniger et al. [44]. Briefly, at 4
and 8 days’ incubation time, the BC biomaterial was cut into
small cubes, to fit the dimensions of a 96-well microtiter
plate. To each well, 90 𝜇L of DMEM and 10 𝜇L of MTT
0.5 w/v% were added. Next, the plate was incubated at
30∘ C and 70 rpm for 3 h. The insoluble formazan crystals
3
formed were dissolved in 100 𝜇L of isopropanol for 20 h
and measured spectrophotometrically in an enzyme-linked
immunosorbent assay reader (MultiskanGO, Thermo
Scientific) at 570 nm wavelength. The cell number per
biomaterial was established by totalizing the number of cells
per piece of biomaterial. Before the assay, a calibration curve
was developed by using 8 successive dilutions to half the
initial concentration, starting from 50,000 viable cells and
following the procedure described above. The results were
compared with Corning polystyrene well (control), which is
the reference substrate for cell maintenance. The tests were
carried out in triplicate at two independent times.
2.9. Histology. The histology procedure was performed to
evaluate the cross adhesion of human fibroblasts within the
BC-based biomaterial. Samples obtained after 16 days’ cell
culture were placed in a tissue processor (Thermo Scientific)
for 12 h at room temperature. To dehydrate the samples,
the following solvents were used: neutral formaldehyde,
isopropanol between 50 and 80 v/v%, pure isopropanol, pure
xylols, and paraffin at 60∘ C (1 : 3 : 3 : 3 : 2). After 12 h, the bio-
materials were embedded in a paraffin block at 56∘ C and were
cut using a microtome (Leica) to produce 1 𝜇m thick cross
sections. The sections were stained with haematoxylin-eosin
and images were recorded using an Olympus microscope
(100x).
2.10. 3D Moulding for Wound Dressing. Gloves were devel-
oped from BC. For this, an oxygen-permeable mould com-
prising silicon rubber was used. A nitrile glove was coated
with catalyzed silicon rubber and left for 24 h to vulcanize the
silicon; next, the silicon rubber glove was unmoulded. The
silicon mould was sterilized, filled with 500 mL of HS culture
medium, and inoculated at 10 v/v% with Komagataeibacter
medellinensis, and the system was incubated at room temper-
ature for 10 days; then the gloved shaped BC dressing was
unmoulded and purified as explained in previous sections for
BC.
3. Results and Discussion
3.1. Biomaterial Morphology. The morphology of the bio-
materials relates to their suitability for cell adhesion and
biocompatibility. BC presents a fibrillar structure while pore
features are typical in PVA and PVA/BC. The BC microstruc-
ture shows an open porous network (Figure 1 BC at 5000x),
comprising interconnected nanoribbons, 50–70 nm in width,
similar to collagen fibrils in the extracellular matrix (ECM)
[17, 45]. In the case of PVA/BC composites, the fibril structure
of BC is covered by PVA (Figure 1), which results from the
fact that cellulose acts as nucleation element for the PVA
biopolymer. In this way, PVA crystals grow from the BC
nanoribbons into the free spaces [46] (Figure 1, PVA/BC).
The morphology, including the texture and pore size,
affects the adsorption and adhesion of proteins and, conse-
quently, cell anchorage [47]. The dried PVA/BC nanocom-
posites presented large, closed pores, ca. 2 𝜇m in size, while
PVA exhibited open pores (1 𝜇m in size, see Figure 1). These
4 International Journal of Polymer Science
200x
PVA
PVA/BC
BC
Figure 1: Microstructure of the dry biomaterials obtained from for PVA, PVA/BC nanocomposite, and BC as assessed by scanning electron
micrographs at different magnifications (200 and 5000x).
results are related to phase separation during freeze-thawing
[30]. Open porosity is desirable because it allows nutrient
migration within the biomaterial and its supplementation to
cells. Thus, compared to the PVA/BC nanocomposites, the
single component BC and PVA are expected to exhibit higher
cell adhesion.
3.2. Wet Mechanical Properties. The wet mechanical prop-
erties of BC, PVA, and PVA/BC biomaterials are presented
as strain-stress profiles (Figure 2(a)). Hydrogels with vis-
coelastic behaviours were identified, similar to those of
human tissue, skin, cartilage, and so on [23]. This was more
distinctive for BC (Figure 2(b)), which showed a nonlinear
response (toe-in region) as has been described elsewhere
[48, 49]. This region results from the gradual stretching
of cellulose fibres upon loading, which is analogous to the
function of collagen fibrils [48].
5000x
This response was not evident for PVA or PVA/BC,
due to the highly entangled morphology that was generated
during the physical crosslinking [25]. The incorporation of
BC induced changes to the mechanical properties of PVA:
the slope of the linear zone, related to the stiffness, increased
due to the presence of the reinforcing BC. The skin elastic
modulus is ca. 1–3 MPa [50]. Compared to this value, the
results in Table 1 indicate that the tested biomaterials have
similar stiffness, which is expected to facilitate the comfort of
patients, avoiding stress concentration between the dressing
and the wounded area.
The values of tensile strength and Young modulus of
the biomaterials are summarized in Table 1. The inclusion
of BC in PVA increased Young’s modulus, which indicates
a stiffening of the matrix. This behaviour resulted from
good stress transfer upon the incorporation of the three-
dimensional BC nanoribbon network in PVA [25, 26, 51]. The
International Journal of Polymer Science 5

7 75

6 PVA
60
5

Water uptake (%)

45
Stress (MPa)

PVA/BC
3
30

2
BC 15
1

0 0
0 100 200 300 400 500 0 25 50 75 100 125 150 175
Strain (%) Time (h)

PVA
PVA/BC
(a) (b)
Figure 2: (a) Biomaterial strain-stress curves under uniaxial tension in the wet state for BC, PVA, and PVA/BC nanocomposites, as indicated.
(b) Kinetics of water uptake for PVA and PVA/BC nanocomposite (biomaterial rehydration profiles). The water content was measured
gravimetrically.

Table 1: Mechanical properties of biomaterials in the wet state: Young’s modulus and tensile strength under uniaxial stress (the test was
performed in air). Included is also the biomaterial swelling, swelling (%), water content (wt.%), time to reach equilibrium (h), and wet
thickness (mm). Biomaterial swelling, swelling as percentage, was calculated according to (1). The time to reach swelling equilibrium was
determined when mass changes of less than 1 wt.% were recorded. The wet thickness is reported in millimetres.

Time to reach
Young’s modulus Tensile strength Water content Wet thickness
Biomaterial Swelling (%) equilibrium
(MPa) (MPa) (wt.%) (mm)
(h)
PVA 1.13 ± 0.25 7.16 ± 1.46 298.93 ± 4.91 62.64 ± 3.08 5.5 0.42 ± 0.28
PVA/BC 2.29 ± 0.46 2.55 ± 0.29 305.65 ± 7.06 66.98 ± 3.03 1 0.17 ± 0.04
BC 3.30 ± 0.97 0.92 ± 0.17 6951.37 ± 200.92 98.56 ± 0.04 N/A 1.20 ± 0.32

maximum tensile strength of the biomaterial is influenced by
the presence of water, which interferes in the interaction of
polymer chains [52]. The tensile strength of PVA was reduced
by the incorporation of BC. This is explained by the fact that,
in the wet state, BC acts as a stress concentrator, because of
the interference of water located between the matrix and the
reinforcement, at the interfaces in the composite [52].
A Young’s modulus of ca. 3 MPa was measured for the
BC hydrogels, in agreement with other reports [53, 54].
The nanoribbons are linked to bulk water [8]; moreover
according to Konidari et al., water induces changes in the
mechanical properties of semicrystalline polymers due to
the plasticization of the amorphous regions and the possible
partial destruction of the crystalline ones [55]. Despite the
loss of mechanical performance (tensile strength) in the
presence of water, the hydrogel morphology is desirable for
cell regeneration since it mimics the morphology of collagen
[11] and, according to Bäckdahl et al. and Fu et al., this type
of effect is beneficial for the formation of cell layers on the
biomaterial [53, 54].
3.3. Swelling. Figure 2(b) illustrates the swelling behaviour of
PVA and PVA/BC nanocomposite, including their swelling
percentage, water content, time to reach swelling equilibrium,
and wet thickness. PVA and PVA/BC presented ca. 300%
swelling, indicating that the reinforcement of PVA with BC
did not affect its ability to absorb water. BC has a large
capacity to hold water (99 ± 0.04 wt.%), equivalent to a
relative swelling of ca. 7000%. The water holding capacity in
biomaterials is important because it determines the amount
of nutrients available to the cells [8]. Accordingly, these
results indicate the possibility of an extensive cell adhesion.
Regarding the kinetics of water uptake, swelling equi-
librium for PVA and PVA/BC biomaterials is not affected
by the inclusion of BC (ca. 67 wt% water content) (Table 1).
PVA/BC reached equilibrium in 1 h, while PVA took 5.5 h.
However, compared with PVA, the rate of water uptake is
higher for the PVA/BC nanocomposites. This behaviour is
related to the thickness of the biomaterial (Table 1), which
affects the diffusion of water, as described by Fick’s second
law [56]. The thickness of the nanocomposite is difficult
6 International Journal of Polymer Science

(a) (b) (c)

(d) (e)

Figure 3: In vitro cell adhesion on biomaterials, haematoxylin-eosin staining incubation time of 8 days. (a) PVA, (b) PVA/BC, and (c) BC.
Images taken at 40x. White arrows indicate the presence of cells. Cell densities: PVA and PVA/BC not detectable; BC 56934 ± 4657. Fluorescent
images of fibroblast on BC, (d) 4th day and (e) 8th day. Dye: ethidium bromide, cell nucleus is brighter than cytoplasm. Images token at 40x.
Fibroblasts increase their number on BC. At the 4th day cells are spread on the surface of BC surface. At the 8th day, cells display a confluent
monolayer on the surface of the biomaterial. Scale bar is 100 𝜇m.

to control in the in situ methodology because it depends
on the number of viable bacteria at the air-liquid interface.
We note that BC swelling occurs during fermentation, and
rehydration is limited after the material has been dried, due
to the “hornification” phenomenon [57].
3.4. In Vitro Cell Adhesion. The results for cell adhesion
on the biomaterials are shown in Figure 3. Adhesion was
observed for PVA, PVA/BC, and BC. For the PVA/BC
nanocomposite the closed pores affected the adhesion of
fibroblasts on the biomaterial. This is likely because adsorp-
tion and adhesion of proteins are hindered by the reduced
availability of nutrients [47]. Moreover, cells anchored
scarcely on PVA and PVA/BC, as observed in Figure 3. In
both cases, the number of cells was low compared to BC; from
50000 cells seeded only a few were detected. MTT assay did
not reveal any significant changes.
According to fluorescence imaging (see Figures 3(d)
and 3(e)) and MTT assessment of human fibroblasts on
BC (Table 2), cells adhered to the BC biomaterial in great
numbers. Cells anchored to BC from the fourth day. At
the 8th day, a confluent fibroblast monolayer was evident
wherein the cells displayed a fusiform morphology. This
type of morphology indicates healthy cells and appropriate
anchorage on BC [58, 59]. This is explained by the BC
microstructure, similar to that of collagen, and its chemical
configuration with three hydroxyl groups per monomer that
interact with polar sites in the cell through hydrogen bonds
[60]. Likewise, the high swelling of BC and its open porosity
Table 2: Cell density on BC with time. Plates for cell culture of
polystyrene were used as control.
Time (d)
Biomaterial
4 8
BC 15349 ± 10227 56934 ± 4657
Control 35265 ± 11064 60940 ± 17062
facilitated the sorption of cell culture medium; these factors
promoted cell anchorage.
Despite the presence of abundant hydroxyl groups in
PVA, protein adsorption and cell adhesion were prevented
[61]. In BC, fibroblasts adhered, a phenomenon that did
not occur in PVA/BC (see Figure 3). Thus, PVA/BC can be
better used as interactive dressings where cell adhesion is not
required, or even is unwanted [14]. Nonetheless according
to the swelling results these biomaterials control moisture,
that is, control exudate, without interacting directly with the
wound bed (cells). Others possible biomedical applications
of the PVA/BC nanocomposite include the development of
dura mater implants, drug delivery systems, and protein
immobilization [62–64]. In these applications cell adhesion
is not required.
3.5. Cell Viability. Cell viability is a critical test for wound
dressings, since they interact with cells and promote cell
movement to the injury site [65]. Table 2 shows the results
of the MTT viability test.
International Journal of Polymer Science
Cell culture
Bacterial cellulose
100x
(a)
Figure 4: BC wound dressing: (a) side view of BC histology cut, cells adhere on the biomaterial surface (image taken at 100x). (b) BC glove-
shaped wound dressing (hydrogel state) for treatment of hand lesions, especially those involving fingers.
The number of viable cells on BC at day 8 was similar
to that on the control. However, cells on BC increase in
their number faster than in the control; from day 4 to 8,
cells on BC increased fourfold in number. This effect can be
attributed to the BC microstructure and its surface chemistry
[60], as explained previously. The fact that fibroblasts are
viable on BC means that this biomaterial does not interfere
in the cell cycle, which make BC suitable for skin cell
regeneration as a bioactive wound dressing [6]. This result
is in agreement with fluorescent imaging (see Figures 3(d)
and 3(e)): good adhesion was observed on BC from day 4;
at day 8, a fibroblast monolayer was observed as a fusiform
and confluent morphology [32].
3.6. Histology. Figure 4(a) shows the side view of a monolayer
of fibroblasts on BC. It indicates that they did not migrate
across the biomaterial, as the pore size of the BC is c.a.
1 𝜇m in size and cells have an average size of 40 𝜇m [66].
However, it has been indicated in the literature that cells can
push BC nanoribbons and migrate transversely [67, 68]. This
phenomenon seemed not to occur in this case because of the
highly entangled BC network, which excluded cells by size.
Currently, research focuses on creating microporosities
in BC in order to enhance cell migration; some remark-
able strategies include the creation of sponges, inclusion of
paraffin spheres, and development of cocoon-like structures
[12, 66, 69, 70]. For wound dressing the fact that cells do not
migrate transversally in BC may be beneficial, for example,
in creating monolayers of bioengineered skin substitutes.
According to the results above, BC wound dressings can
be classified as bioactive; BC is able to control moisture
while interacting with skin cells. One interesting application
is the development of tissue engineering skin substitutes,
where BC can be covered by skin cells and then used for
wound treatments [6]. The addition of PVA will generate
an interactive wound dressing for the exudates management,
where interactions with cells are not required.
3.7. Glove-Shaped Wound Dressing. According to the results
presented for BC, a glove-shaped wound dressing was
7
(b)
designed for treating burns and skin ulcers (Figure 4(b)).
For these treatments, the high water holding capacity of
BC and its biocompatibility with skin cells are important to
keep the wound moist and to promote fibroblast adhesion,
respectively. All the above promote epithelialization and
wound healing [1].
The benefits of wound dressings can be expanded if
they are made from BC. Furthermore, the production of
related wound dressings is possible by using microorganism
bioengineering, using a silicone rubber membrane template
(permeable to oxygen). Only bacteria (Komagataeibacter
medellinensis) next to the inner mould wall have access to
oxygen and divide and produce cellulose. Therefore, BC is
produced only adjacent to the silicone rubber and it will
conform to the shape of the mould, a glove in this case. Such
wound dressings can be used as skin substitutes for difficult
areas such as hands and fingers.
4. Conclusions
A facile, robust, and novel method toward nanocomposites
with biocompatible bacterial cellulose (BC) and polyvinyl
alcohol (PVA) was applied by in situ fermentation and
physical crosslinking upon cellulose biosynthesized from
Komagataeibacter medellinensis. BC-based hydrogels were
found to be beneficial for the formation of a cell layer on
the biomaterial. BC presented a high degree of swelling,
which is vital for nutrient and adhesion protein adsorption.
The microstructure of PVA and PVA/BC scarcely allowed
cell adhesion. In contrast, fibroblasts cultured on BC were
identified in large number, forming fusiform and confluent
morphology. This observation is explained by the similarity
of BC with collagen. According to viability tests, fibroblasts
multiplied rapidly on BC, and the fibroblast monolayer
did not migrate significantly across the biomaterial. This is
mainly due to size exclusion effects; this is advantageous
for bioengineering monolayers of skin substitutes. According
to the properties presented by BC and taking advantage of
microorganism bioengineering, it was possible to develop
BC shaped as a glove for wound dressing. This material may
have application in the treatment of skin burns and ulcers. In
8 International Journal of Polymer Science
conclusion, PVA and PVA/BC nanocomposites can be used
if cell adhesion is not required, for instance, as interactive
wound dressing for exudate management. BC is ideally
suited as a bioactive wound dressing for development of
bioengineered skin substitutes; thus the biomaterials can be
used as wound dressings depending on the clinical situation.
Further investigation will be focused on in vivo studies to
validate the systems under real conditions, to determine their
safety in use and their pharmacokinetics.
Conflicts of Interest
The authors declare that there are no conflicts of inter-
est regarding the publication of this paper nor financial
interest in the subject matter or materials discussed in this
manuscript.
Acknowledgments
The authors acknowledge the Universidad Pontificia Boli-
variana, CIDI-UPB, Colombia, and Colciencias, Colombia,
for the funding support that allowed the completion of this
research.
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BioMed Research International
Volume 2015, Article ID 506059, 17 pages
http://dx.doi.org/10.1155/2015/506059

Research Article
Proteomic Changes of Tissue-Tolerable Plasma Treated Airway
Epithelial Cells and Their Relation to Wound Healing

Derik Lendeckel,1 Christine Eymann,1 Philipp Emicke,1 Georg Daeschlein,2

Katrin Darm,1 Serena O’Neil,1 Achim G. Beule,1 Thomas von Woedtke,3 Uwe Völker,4
Klaus-Dieter Weltmann,3 Michael Jünger,2 Werner Hosemann,1 and Christian Scharf1
1
Department of Otorhinolaryngology, Head and Neck Surgery, University Medicine Greifswald, 17475 Greifswald, Germany
2
Department of Dermatology, University Medicine Greifswald, 17475 Greifswald, Germany
3
Leibniz Institute for Plasma Science and Technology (INP), 17489 Greifswald, Germany
4
Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, 17475 Greifswald, Germany

Correspondence should be addressed to Christian Scharf; scharf@uni-greifswald.de

Received 10 April 2015; Revised 16 July 2015; Accepted 27 July 2015

Academic Editor: Isabel Sá-Correia

Copyright © 2015 Derik Lendeckel et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new
safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration
of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the
macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis
(2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of
beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound
closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound
healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and
120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative
stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the
adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or
downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma
effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma
application, for example, in the surgery field of otorhinolaryngology or internal medicine.

1. Background recently, possible applications of artificial plasma in diverse

Wound healing is an important necessity for survival. A
worldwide increasing number of patients suffering from
nonhealing and chronic infected wounds require the devel-
opment of new strategies for wound repair. One of these
promising strategies is the application of nonthermal plasma.
Plasma is defined as the fourth state of matter and is
generated by almost completely or partly ionisation of gas.
Naturally occurring thermal plasmas are found, for example,
as lightning in thunderstorms and in the magnetosphere of
astronomical objects. Plasma can also be created artificially
and is commonly used in the industrial sector [1–4]. But only
medical fields have begun to be investigated.
Besides thermal artificial plasma, which has become an
innovative tool in, for example, microsurgery for the ablation
and cauterisation of biological tissue [5–8], it is also possible
to create low-temperature artificial plasmas (“nonthermal” or
“cold”).
Due to many observed beneficial effects, the therapeutic
potential of cold plasma has emerged as a promising new
option in several clinical settings. In contrast to the thermal
plasma equivalent, cold plasma mediated effects have been
proposed to include modulation of the immune system [9–
11], arrest of tumour proliferation [12–16], and improvement
of wound healing [17–20].
2 BioMed Research International
Thus, the application of cold plasma to the skin is of
particular importance in dermatology, where conventional
therapies of chronic wounds are mostly insufficient. It has
been shown that positive effects of nonthermal plasma on
wound healing clearly result from its antiseptic properties
[18, 21–24]. But also the increase of cell proliferation and
migration as significant effects of particular low plasma doses
might be also responsible for the support of wound healing
[24].
Clinical studies have focused on wounds of the epidermis
like venous ulcers [18, 21, 25] or skin graft donor sites [20,
26], whereas molecular research has predominantly been
performed on common bacteria of the skin [23, 27, 28] or
keratinocytes [29, 30].
Recent studies and clinical trials have demonstrated the
efficacy of tissue-tolerable plasma (TTP) in wound healing
[13, 17]. It may have the potential to improve wound healing
in all clinical settings with healing in secondary intention.
However, understanding of the underlying molecular mech-
anisms is limited owing to the complexity of both the plasma,
which consists of charged particles, reactive oxygen species
(ROS), and several types of radiation (VUV, UVA, and UVB)
[28], and the tissue or cell. Available studies on cellular
mechanisms are limited to targeted molecular research based
on key mediators and their hypothetical downstream effects
[19].
To our knowledge, only two global transcriptomic studies
have been performed to analyse the entirety of the molecular
mechanisms in human epithelial cell lines influenced by indi-
rect plasma treatment [31, 32]. These recent transcriptomic
analyses revealed an influence on several biological pathways,
such as oxidative stress response, repair, and inflammation
signalling. Oxidative stress might be a result of the existence
of reactive oxygen or nitrogen species (ROS, RNS) such as
ozone, superoxide, peroxide, and hydroxyl radicals which are
generated by different plasma sources [14]. However, no gel-
based proteomic studies have been reported to date.
It is interesting to note that some recent clinical trials and
molecular research have already revealed dose-dependent
cellular effects in response to cold plasma treatment [29, 31,
33]. These studies indicate that increasing plasma doses could
heighten the proportion of cells which are forced to undergo
apoptosis, leading consequently to a progressive negation
of positive effects. The optimal dosage of tissue-tolerable
plasma (TTP) treatment based on evaluation of molecular
results has not been ascertained yet. Therefore, the aim of
the present study was to identify the effects of varying TTP
doses on molecular pathways in upper airway epithelial S9
cells at several time points. An in vitro wound healing model
was established to determine the macroscopically optimal
dosage of TTP treatment for wound regeneration, while a
2D-difference gel electrophoresis (2D-DIGE) approach was
used to quantify the proteomic changes in a hypothesis-
free manner and to evaluate the balance of beneficial and
adverse effects due to TTP application. Human S9 upper
airway epithelial cells serve as a model for mucosa cells of
the throat in a broader sense and plasma application could
be of particular interest, for example, for the field of surgery
in otorhinolaryngology.
2. Methods
2.1. Cultivation of S9 Epithelial Cells. Upper airway S9
epithelial cells [34] were incubated with 10 mL standard cell
cultivation medium MEM (Promocell) in a cell culture plate
at atmospheric conditions (at 37∘ C, 5% CO2 , and >85%
humidity) and were grown up to an approximate cell density
of 1 × 107 cells/cm2 . Cell counting was performed with CASY
(Roche Life Sciences). Then subcultivation was performed
by removing the cell cultivation medium, washing cells
with 5.0 mL PBS, and overlaying them with 1.0 mL trypsin-
solution (PAN Biotech, 10 ng/mL) for 5 to 15 minutes to
detach cells from the surface [35, 36]. Then, 3 mL standard
cell cultivation medium was added, and cells were repeatedly
pipetted, divided into aliquots, and transferred into new cell
culture plates, each containing 9 mL fresh cell cultivation
medium with a subcultivation ratio of 1 : 4, which corre-
sponded to approximately 1 × 107 viable cells per cell culture
plate. These subcultures were incubated as described above
until 95% confluency of cells was reached. Substitution of
medium against fresh medium was performed after 60 h.
2.2. Plasma Treatment of S9 Epithelial Cells. For plasma
treatment the kINPen08, an atmospheric pressure plasma jet
developed by the INP in Greifswald, was used ([24]; Figure
S1-A). To generate the plasma, argon gas was used and a high-
frequency voltage of 2–6 kVpp, 1.1 MHz, was applied to the
electrode. At the tip of the plasma jet, temperatures measured
ranged between 37∘ C and 42∘ C. Different plasma doses which
varied between 30 s and 360 s were applied to cell cultures. For
plasma application, the cell cultures covered with 1 mL stan-
dard cell medium were moved in a grid pattern underneath
the visible tip of the fixed plasma jet for the requested time
(supplementary Figure S1 (B-D) in the Supplementary Mate-
rial available online at http://dx.doi.org/10.1155/2015/506059).
After plasma treatment, untreated and treated cell cultures
were incubated for several time periods enabling assessment
of wound healing and proteome analyses (incubation for 24 h,
48 h, and 72 h before cell harvesting).
2.3. In Vitro Wound Model. In order to study effects of
plasma treatment on wound healing, we used cell cultures
of S9 upper airway epithelial cells in a cell culture plate
of 10 cm diameter. Cell cultures were treated according to
an established wound model, previously described by Beule
et al. and Roth et al. [35, 36]. In confluent cell cultures
21 circular wounds per plate were created using a 4 mm
sterile biopsy punch (pfm AG, Cologne, Germany) (Figure
S1-C). All wounds were documented photographically and
subsequently treated with the kINPen08, following a grid
pattern for a duration of 30 s, 60 s, 120 s, 240 s, and 360 s
(Figure S1-D). Untreated wounds (controls) were covered
with a light-proof lid for the same duration. Wound areas
were documented at a 4-fold magnification on an inverted
microscope (Nikon Eclipse) 24 h, 48 h, 72 h, 90 h, and 120 h
after appropriate incubation. The size of six wounds was mea-
sured on the photographs by using an area-calculating tool
of Photoshop CS5 (Adobe, San Jose, Calif., USA) (Figure 1).
BioMed Research International
Figure 1: Schematic description of wound measurement. The blue
line marks the initially punched wound border. The red line defines
the wound border of the current moment. 𝑟𝑤 = radius of the minimal
diameter of the wound; 𝑟𝑠 = radius of punched wound; 𝐴 𝑤 = wound
area related to the defined punched area 𝐴 𝑠 .
The average of each condition was calculated. Each of the
different treatment groups was tested against the untreated
control group. Effects were considered statistically significant
for p < 0.05 using ANOVA. Additionally, the nonparametric
Mann-Whitney U test was used to compare two groups (96 h,
120 h values, p < 0.05). Sphericity of wounds was assumed.
2.4. Sample Preparation for Proteomic Analyses. For cell
harvesting, the medium was removed and cells were washed
with PBS. After PBS removal, cells were incubated with 1.8 mL
sample buffer (8 M urea, 2 M thiourea) and detached with a
cell scraper (Greiner BioOne). For disruption, cells were ini-
tially shock-frozen in liquid nitrogen and then defrosted in a
thermomixer (Eppendorf, Hamburg, Germany) at 1400 rpm
at 30∘ C for 10 minutes. After five freeze and thaw cycles,
samples were centrifuged to remove cell debris. Supernatants
were transferred into new tubes and stored at −70∘ C prior
to further processing. Protein concentrations were estimated
using a Bradford assay (Bio-Rad, Munich, Germany) as
previously described [37].
2.5. Two-Dimensional Difference in Gel Electrophoresis (2D-
DIGE). Proteome analysis was performed for two separate
experimental series of four conditions (control, 30, 60, and
120 s of plasma treatment) and three time points (24, 48, and
72 h). Protein lysates were labelled with Cy-dyes (Cy3 or Cy5)
according to the manufacturer’s instructions (GE Healthcare,
Munich, Germany). In order to reduce intergel variations
an internal standard pool consisting of 50 𝜇g aliquots of
all samples was generated and labelled with the Cy2-dye.
50 𝜇g protein of each sample or internal standard was labelled
with 400 pmol of corresponding dye on ice in the dark for
30 min. Reaction was quenched with 10 mM L-lysine for
10 min under the same conditions. From all samples, four
technical replicates labelled either with Cy3-dye or with Cy5-
dye were analysed. For a two-dimensional-difference gel elec-
trophoresis (2D-DIGE) approach two labelled samples (Cy3
3
and Cy5, each 50 𝜇g) were mixed with the internal standard
(Cy2, 50 𝜇g) in rehydration buffer [38] and applied to IPG
strips (pH 4–7, 24 cm, GE Healthcare, Munich, Germany)
by in-gel rehydration. After rehydration overnight, isoelectric
focusing (IEF) was carried out as described earlier [38]. Sub-
sequently, the second dimension (SDS-PAGE) was carried
out using low-fluorescent glass plates (GE Healthcare) in
the PROTEAN plus Dodeca Cell system (Bio-Rad, Munich,
Germany). After 2D-PAGE, the Cy2 (internal standard), Cy3,
and Cy5 labelled proteins in each gel were visualized by using
a Typhoon 9400 laser scanner (GE Healthcare) at 100 microns
by using the following excitation/emission wavelengths:
488/520 nm (Cy2), 532/670 nm (Cy3), and 633/670 nm (Cy5).
The resulting images (3 per gel) were processed with dedi-
cated software as described below. Dose- and time-resolved
proteome analyses (dose-dependent: control versus 30 s, 60 s,
and 120 s of TTP treatment, time-dependent: the 24 h sample
of each dose versus 48 h, 72 h) were performed for controls
and samples treated with TTP.
2.6. Statistical Analyses. Analyses of the scanned gel images
and data analyses were performed with the software package
Delta2D (Version 4.2, Decodon GmbH, Greifswald, Ger-
many). After aligning all image sets in position by using the
internal standard (Cy2), all samples of a set were merged in
order to create a fused image (2D proteome map) in union
fusion mode [39]. Spot detection and editing were done with
the Delta2D software on the union fused image and then
spots and spot labels were transferred onto all other images
included in the analyses. TMeV software (implemented in
Delta2D) was used for statistical analyses and graphical dis-
play of expression profiles. Acquired spot expression values
were automatically normalized, first by correcting differences
between intensities of all gel images and then by calibrating
spot volumes (Cy3 or Cy5) to the internal standard (Cy2).
In order to identify differences in spot intensity, the values
of TTP treated samples were divided by the corresponding
baseline values of the untreated control. Stringent selection
criteria were employed in order to reduce false positive
results and changes were only considered significant when
the following three criteria were fulfilled: (1) the change of
the intensity ratio had to exceed a factor of 1.5, (2) the p value
of the corresponding analysis of variance test had to be lower
than 0.05 (one-way ANOVA), and (3) raw values of each spot
had to exceed 0.3, avoiding calculation of ratios of spots close
to background intensities. After TMeV analyses, principle
component analysis (PCA) [40] was performed to validate
reproducibility of experimental conditions and identify pos-
sible outliers. Each subset of sample (4 technical replicates per
group) was defined as point in an n-dimensional Cartesian
coordinate system, described by 1582 vectors, which represent
the amount of expression values of every detected protein
spot. Those samples were transferred into 2-dimensional
space by focusing on the principal components with the
largest variances. After removal of an obvious outlier in two
groups (60 s/24 h and 120 s/24 h), caused by a hardware failure
of the scanning device, regulated spots were identified with
MALDI-TOF-MS as described below.
4 BioMed Research International
2.7. Preparative 2D Gel Electrophoresis and Sample Prepa-
ration for Mass Spectrometry. Preparative two-dimensional
gel electrophoresis was performed as previously described
[38]. Briefly, 450 𝜇g of protein was pooled from treated and
untreated samples of each condition (75 𝜇g each) and added
to the rehydration buffer. The resulting 2D-PAGE gels were
stained with colloidal Coomassie brilliant blue according
to the manufacturer’s instructions (GE Healthcare). Digital
documentation of the gel images was performed by a trans-
mission light scan. Gel image analyses were performed with
the Delta2D software package (Decodon GmbH) as described
above. Spots of interest were processed for identification as
described by Eymann et al. [41]. Briefly, spots were excised
manually with a 2 mm picking head and transferred into 96-
well microplates, which were loaded with 100 𝜇L of Lichro-
solv water in each well. Tryptic digestion was performed auto-
matically in an Ettan Spot Handling Workstation (Amersham
Biosciences) as well as the subsequent spotting of peptide
solution onto MALDI targets. For peptide extraction, gel
pieces were covered with 60 𝜇L 50% v/v ACN/0.1% w/v TFA
and incubated for 30 min at 37∘ C. Supernatants containing
peptides were transferred into new microtiter plates. Peptide
extraction was performed again with 40 𝜇L of the same solu-
tion. Joined supernatants were now completely dried at 40∘ C
for 220 min. Peptides were dissolved in 2.2 𝜇L of 0.5% w/v
TFA/50% v/v ACN and 0.7 𝜇L of each solution was spotted
directly onto MALDI targets. Then, this sample solution was
mixed with 0.4 𝜇L of matrix solution by aspirating five times.
After drying for 10–15 min, samples were measured using the
MALDI-TOF-TOF instrument.
2.8. Matrix-Assisted Laser Desorption-Ionization Time-of-
Flight Mass Spectrometry (MALDI-TOF-MS). MALDI-TOF-
MS measurements were performed with a Proteome-
Analyzer 4800 (Applied Biosystems, Foster City, CA, USA).
Reflector mode was used in order to record the spec-
tra in a mass range from 900 to 3700 Da with a mass
focus to 2000 Da. Twenty-five subspectra with 100 shots
per subspectrum were accumulated for one main spectrum
using a random search pattern. The standard peptide search
tolerance was set to 50 ppm. The peak lists were created
and searched automatically by using GPS-Explorer software
package (Applied Biosystems, Foster City, CA, USA). These
peak lists were compared to a UniProt-SwissProt database
(Rel. 51.5 restricted to human taxonomy) by the MASCOT
search engine (Version 2.1). Positive identifications had to
reach the following specifications: sequence coverage of at
least 30% and a MOWSE-score of at least 49. Proteins and
peptides, which failed to meet the 30% sequence coverage
requirement, were reanalysed with more accurate MALDI-
TOF-MS. The MALDI-TOF-MS/MS analyses were used for
the five strongest peaks of the previous MS-spectrum. Here,
20 subspectra with 125 shots per subspectrum were accu-
mulated using a random search pattern. The same tools
were used for peak list interpretation. Results reaching a
MOWSE-score of at least 49 in reflector mode (MALDI-
TOF-MS) and being confirmed by subsequent measurement
of the strongest peaks (MS/MS) were regarded as posi-
tively identified proteins. The confirmation by subsequent
measurements (MS/MS) was particularly useful for protein
identification in spots, which contained multiple proteins.
Protein identifications and statistically relevant data were
combined via unique spot-IDs using the MSRepo database
software (Decodon, Greifswald, Germany). Categorization of
the identified proteins was achieved by using the PANTHER
Classification System (http://www.PANTHERdb.org/) [42].
2.9. Network and Protein Functional Analyses Using Special-
ized Software. Expression profiles of statistically significant
changing spots were exported. TMeV data were combined
with identification lists from mass spectrometry and then
imported into Ingenuity Pathway Analysis (Ingenuity Sys-
tems). Using this program, it was possible to create protein
networks and pathways, which contained the proteins dis-
playing changes in intensity and thereby placing individual
protein data into physiological context.
3. Results
3.1. Dose-Dependent Effects of Plasma Treatment on Wound
Healing of Upper Airway S9 Epithelial Cells. The effect of
various plasma doses on the rate of wound healing of S9
airway epithelial cells was examined over 120 hours in an in
vitro wound model. In comparison to the control cell cultures,
the application of low plasma doses (30, 60, and 120 s)
led to successive accelerated wound regeneration after 96 h.
Higher doses either resulted in significantly lower regener-
ated wound area (240 s) or even blocked the ability for regen-
eration and to close the wound area (360 s) (Figure 2(a)).
The 120 s dose provided the most intense stimulation of
wound healing after 48 h and at subsequent time points.
Statistical analysis ensured a significantly increased wound
healing rate after 96 h and 120 h (p < 0.05). No significance
could be determined for cells which were treated for 30
and 60 s, although there is a trend for increasing wound
healing potential over 120 h (Figure 2(a)). The difference in
the wound healing area between control cells and 120 s of
plasma treated cells is clearly visible as the regenerated wound
area comprised approximately 72% of the initial wound area
after 96 h and 86% after 120 h, whereas control samples
provided 57% and 72% for the same time points (Figure 2(b)).
The application of low plasma doses (≤120 s), in the following
termed tissue-tolerable plasma (TTP), seems to improve cell
proliferation and migration, thereby increasing the rate of
wound healing of S9 airway epithelial cells. Higher doses
seem to repress or stop cell proliferation/migration and could
have therefore cell damaging or lethal effects.
3.2. The Protein Expression Is Influenced by Tissue-Tolerable
Plasma (TTP) Doses as Revealed by 2D-DIGE and MALDI-
MS Analysis. To analyse possible changes in protein pat-
tern caused by the TTP treatment, a 2D-DIGE approach
comprising four conditions (untreated control, 30, 60, and
120 s TTP) and three time points after the TTP application
(24 h, 48 h, and 72 h) was used. These time points should be
most importantly for the evaluation of molecular changes
on proteomic level if the doubling time of cells is 36 h
BioMed Research International 5

100
∗ ∗
90

80

Regenerated wound area (%)

70

60

50

40

30

20

10

0
0 24 48 72 96 120
Time after TTP treatment (h)
Control TTP 120 s
TTP 30 s TTP 240 s
TTP 60 s TTP 360 s
(a)
Control
TTP 120 s

0 24 48 72 96 120
Time after TTP treatment (h)
(b)

Figure 2: Time course of wound healing processes of S9 epithelial cells. (a) The regenerated wound area was calculated by taking the average
of six representative wounds per culture plate/dose and time point. Significant differences (see ∗ ) were observed after 96 h and 120 h between
120 s plasma treated and untreated control samples. Plasma application for 30 s and 60 s provided no significant effect on wound healing, ∗ 𝑝 <
0.05 (Student’s 𝑡-test, 𝑛 = 6). (b) Cell cultures of plasma treated and untreated samples were cut in order to create circular artificial wounds.
The outer blue line represents the primary wound border. The red line represents the current healing border after 24 h, 48 h, 72 h, 96 h, and
120 h. For every time point, the effect of 120 s TTP treatment on wound healing is shown in comparison to untreated samples “control.”

and the macroscopic effects should happen after respective
proteomic changes. Across the 72 resulting 2D-DIGE gel
images 2094 different protein spots were clearly detected
on the fusion gel and analysed using Delta2D statistical
software tool for differential protein expression. The average
abundances of spots were quantified and 1582 protein spots
passed the one-way ANOVA test and could be determined
as significantly regulated (supplementary Table S1). Those
with relative changes in abundance greater than 1.5 times
between control and plasma samples (up and down) at 95%
confidence level (p ≤ 0.05) and identification by MALDI-MS
were further considered (778 protein spots) (supplementary
Table S2). In total, 553 unique proteins could be assigned
to all samples with many proteins represented in multiple
spots. Alterations in protein amounts between untreated and
treated samples could be also demonstrated in dual view
mode by Delta2D software (Decodon) as shown for one
example in Figure 3(a). In several spots the same protein was
identified by MALDI-MS, probably due to posttranslational
modification whose relevance was not further examined
in this analysis (Figure 3(b)). Further bioinformatical and
statistical analyses of the identified regulated proteins of S9
epithelial cells highlighted the proteins of interest that are
affected by TTP.
6 BioMed Research International

TRXR1
2
1
2

PCNA
3

GSTO1
3
4 7 6 5
5

PSME2
7

Control Plasma Plasma Plasma

30 s 60 s 120 s
Dual view control_24 h versus plasma 120 s_24 h Dual view versus control (24 h)
(a) (b)

Figure 3: Dual channel images of whole proteome separated by 2D-DIGE (a) or selected protein spots as sections from 2D-DIGE gels (b). (a)
Dual channel image of false coloured separated proteins of untreated (green) and 120 s TTP treated (red) S9 cells after 24 h. Protein spots with
increased amounts induced by TTP exposure appear as red or orange spots, and those with decreased amounts as green spots. Spots, which are
expressed with the same intensity on both gels, appear in yellow. Selected spots are numbered (1–7) and enlarged in (b). (b) Sections of selected
protein spots 1–7 under different dose conditions underlining the phenomenon of multiple spots of several proteins. TRXR1 = thioredoxin-
reductase 1; PCNA = proliferating cell nuclear antigen; GSTO1 = glutathione S-transferase omega 1; PSME2 = proteasome activator complex
subunit 2.

3.3. Principal Component Analysis (PCA) Uncovers Significant
Treatment Effects. PCA was used to determine the variation
within the proteomes and the significant effects of TTP
treatment on the S9 epithelial cells proteome. The first
and second principal components represented 47.7% of data
variance (PC1: 33.3%, PC2: 14.4%) and distinguished all
analysed samples in a very impressive way (Figure 4). After
removing two outliers, it could be observed that all four
technical replicates were tightly grouped, thus supporting the
experimental reproducibility.
The proteomes of S9 cells which had undergone TTP
treatment showed diverse distribution patterns depending
on treatment dose and time point. The cellular proteomes
of the control groups are closely clustered on the upper
left side, with limited movement across the various time
points. In contrast, 120 s TTP treated S9 cells are positioned
on the right side of the coordinate system with long shifts
among untreated and treated 24 h samples (arrow 3a) and
between different time points (24 h versus 48 h, arrow 3b).
The samples treated for 30 and 60 s are grouped between
the others, as their proteomes differed in fact from those of
untreated samples, but did not reach the distance of 120 s
versus control samples. A comparison of the longest spatial
distances between untreated and different treated samples
after 24 h (arrows 1a, 2a, and 3a) or between 24 h and 48 h
of each similar TTP treatment (1b, 2b, and 3b) clearly shows
that the directions of the spatial shifts are similar, respectively,
but the degree of shifts varied between the different doses
of TTP (1a, 2a, and 3a) and time points (1b, 2b, and 3b)
applied.
The PCA highlights that the highest dose of TTP treat-
ment (120 s) after 24 h resulted in the greatest shifts between
untreated and treated samples demonstrating the most signif-
icant effect on the proteome of S9 airway epithelial cells under
these conditions.
3.4. TTP-Dose- and Time-Dependent Effects on the S9
Cell Proteome. TTP-dose-dependent effects: The TTP-dose-
dependent alterations in S9 cell protein expression with
respect to up- and downregulated protein spots were exam-
ined and are shown in Figure 5(a). A total of 723 protein
spots satisfied specific criteria (p < 0.05; fold change cut-
off ±1.5; MS-identification) and could therefore be regarded
as significantly regulated during at least one TTP-dose-
time condition compared to untreated control samples. They
correspond to 369 unique identified proteins.
As shown in Figure 5(a), the amount of up- and downreg-
ulated protein spots increased with raising TTP doses at each
time point (one exception: 60 s/72 h). This dose-dependent
correlation is developed most strongly after 24 h of TTP
treatment. Thus, the strongest effect of regulation could be
observed for 120 s TTP treatment after 24 h with 537 regulated
protein spots (433 down- and 104 upregulated). This observed
distinct dose-dependent effect of TTP on S9 airway epithelial
cells supports the results of PCA analysis (see above). Among
the top 10 of upregulated proteins after 120 s TTP treatment
at 24 h are, for instance, GRP78 (Ca2+-binding, chaperone
function, 5.7-fold), DCTN1 (axonal transport of vesicles
and organelles, 4.4-fold), 2AAB (serine/threonine protein
BioMed Research International 7

1,235.33
57 showed a reduced protein level after 72 h (e.g., DP13B).
Contrarily, from 433 repressed protein spots after 24 h, 97
2a 988.26
showed an increased protein level after 72 h (e.g., PRDX3).
3a 741.2 This behaviour pointed out that a certain effect of recreation
1a from TTP treatment has to exist.

2b
494.13
Comparison of TTP-dose- and time-dependently induced
247.07 unique proteins: A possible overlap of unique proteins reg-
ulated by more than one TTP dose or time point was not
−2,100.34 −1,680.27 −1,260.2 −840.13 −420.07 420.07 840.13 1,260.2 1,680.27 2,100.34 considered in Figure 5. To exemplify that there are really
−247.07
1b
distinct TTP-dose- and time-specific protein alterations, we

3b
−494.13 compared the upregulated unique proteins by means of
VENN diagrams. Providing that mainly the function of
−741.2
upregulated proteins contributes to the observed macro-
−988.26 scopic effect, we wanted to answer the following questions:
−1,235.33
(1) How many (and which) proteins are only induced by
a special TTP dose, particularly by 120 s? (2) How many
X-axis = 1,Y-axis = 2 proteins are only induced by one time span? (3) How many
proteins are only induced over time without any impact of
Control_24 h Plasma 60 s_24 h
Control_48 h Plasma 60 s_48 h
TTP treatment? Figure 6(a) demonstrates that there is a TTP-
Control_72 h Plasma 60 s_72 h dose-specific induction of proteins. Two proteins are only
Plasma 30 s_24 h Plasma 120 s_24 h induced by the 30 s dose, four proteins are only upregulated in
Plasma 30 s_48 h Plasma 120 s_48 h response to 60 s TTP, and fourteen proteins showed increased
Plasma 30 s_72 h Plasma 120s_72 h amounts only after the 120 s TTP treatment (e.g., PSME1 and
2, proteasome activator complex). The comparison of time-
Figure 4: Principal component analyses (PCA) of proteomes of
untreated and TTP treated S9 cells. The first and second principal dependently induced proteins revealed that fourteen proteins
components for untreated and TTP treated (30 s, 60 s, and 120 s) S9 are only induced after 48 h and twenty-four proteins are
epithelial cells after different points of time (24 h, 48 h, and 72 h) only upregulated later (72 h) (Figure 6(b)). To exclude the
are shown. Each group consists of four technical replicates, except time-dependent effect, a comparison of TTP-dose-dependent
for the 24 h time point of 60 s and 120 s TTP treatment (only 3). induced proteins and time-dependently upregulated proteins
The distances of shifts in space are illustrated by arrows. Arrows 1a, was made (Figure 6(c)). Seventy proteins are solely induced
2a, and 3a display the deviation of untreated samples and different by the TTP treatment and twenty-five proteins showed both
treated samples after 24 h. Arrows 1b, 2b, and 3b represent the shift increased amounts by the impact of tissue-tolerable plasma
of treated samples after 24 h compared to their corresponding 48 h and by time period of 48 h or 72 h after the TTP treatment
time point. (e.g., CATB, catalase).

3.5. PANTHER Analysis of TTP-Dose-Dependently Regulated

phosphatase, 4.2-fold), and the 4-fold induced DP13B protein
Proteins. The gene ontology (GO) terms associated with
which is involved in the regulation of cell proliferation.
the TTP-dose-dependently regulated proteins were examined
Time-dependent effects: To analyse time-dependent alter-
using PANTHER. For 354 of the 369 identified proteins,
ations in S9 cell protein expression with respect to up- and
412 hits for molecular functions and 747 hits for bio-
downregulated protein spots, the proteomes of a specified
logical processes could be found. Binding, catalytic, and
dose of TTP treatment after 48 and 72 h were compared with
structural molecule activity appear to be the most affected
those after 24 hours. A total of 323 protein spots fulfilled all
molecular functions (Figure 7(a)), while metabolic and cel-
mentioned criteria which could be allocated to 182 unique
lular processes are the most influenced biological processes
proteins. As shown in Figure 5(b), there is a time-dependent
(Figure 7(b)). Consequently, PANTHER analyses revealed
effect under all conditions, in untreated cells as well as in TTP
that observed dose-dependent proteome alterations applied
treated samples. But whereas only a few proteins had changed
mainly proteins which are involved in common cellular pro-
their abundance time-dependently in untreated and 30 s
cesses of binding and catalytic activity. The time-dependently
TTP treated cells, significantly more proteins showed time-
affected proteins mainly function in protein expression and
dependent alterations after the 60 and 120 s TTP treatment
cell balance (data not shown).
(Figure 5(b)). Hence, the highest TTP dose (120 s) resulted
in the highest number of time-dependently regulated protein
spots after 72 h. 3.6. Cellular Adaptation due to Different Doses of TTP Treat-
A comparison of these time-dependently regulated ment as Revealed by IPA (Top Tox List and Top Molecular
protein spots (120 s, 72 h versus 120 s, 24 h) with dose- and Cellular Function List). Pathways and networks, where
dependently regulated protein spots (120 s, 24 h) revealed TTP-dose-dependently regulated proteins are involved, were
a large overlay of protein spots that are repressed time- further analysed using Ingenuity Pathway Analysis (IPA) to
dependently after 72 h and induced by 120 s TTP dose after determine the precise affected cellular pathways in response
24 h. Hence, from the 104 induced protein spots after 24 h, to TTP treatment. A ranking of the most determining stress
8 BioMed Research International

600 200

180
500
104 160
Number of regulated protein spots

Number of regulated protein spots

99
140
400 74 67
120
87
300 100

80
200 79 433
56 359 355 60 80
42
52 40 15 99
100 27 69 31
173 140 49
36 151 20 34
106 16 27 23 23
36 39 6
0 0 4 5
30 s_72 h
60 s_72 h
120 s_72 h
30 s_24 h
60 s_24 h
120 s_24 h

30 s_48 h
60 s_48 h
120 s_48 h

Control_48 h

30 s_48 h

60 s_48 h

120 s_48 h

Control_72 h

30 s_72 h

60 s_72 h

120 s_72 h
versus versus versus
control_24 h control_48 h control_72 h versus_24 h versus_24 h
TTP treatment dose versus control TTP treatment time versus 24 h
Upregulated Upregulated
Downregulated Downregulated
(a) (b)

Figure 5: Number of TTP-dose- and time-dependent up- and downregulated protein spots in S9 epithelial cells. (a) In the TTP-dose-
dependent approach, protein spots of all TTP doses (30 s, 60 s, and 120 s) were compared with untreated samples (control) after each time
point (24 h, 48 h, and 72 h). The number of significantly up- and downregulated protein spots (±1.5-fold, p ≤ 0.05) is shown. Note that a
TTP-dose-dependent increase of the number of regulated protein spots could be observed for each time with the greatest effect after 24 h. (b)
In the time-dependent approach, protein spots of each of 48 h and 72 h time points were compared to the corresponding 24 h time points.
The number of significantly up- and downregulated protein spots (±1.5-fold, p ≤ 0.05) is shown.

factors “Top Tox List” and the most affected molecular func- most affected response after 24 h for all TTP doses (rank
tions “top molecular and cellular function” was established. 1) and “oxidative stress” seems to be the most affected
The “Top Tox List” (Table 1(a)) provides an overview response after 48 h and 72 h (ranks 1 and 2). Apart from these
of the adjustment of cell balance, displaying the five most oxidative stress responses, “Decreases permeability transition
affected stress response mechanisms for each condition, of mitochondria and mitochondrial membrane”, which is
respectively. affected by low TTP doses (30 and/or 60 s) at any time
At first sight, there is a significant change of influenced is a further marker for the presence of oxidative stress, as
toxicity responses between 24 h after TTP treatment and an increase of mitochondrial membrane permeability is an
the 48 h and 72 h time points, independently of applied important initial step for apoptosis triggered by reactive
doses, as several response mechanisms on one hand are oxygen species (ROS) [43]. The same is true for “increases
strongly regulated after 24 h but not after 48 h and 72 h (e.g., transmembrane potential of mitochondria,” which is affected
“mitochondrial dysfunction,” “renal necrosis/cell death,” and to rank four or five after 60 and 120 s TTP treatment [44, 45].
“aryl hydrocarbon receptor signalling”) and on the other Additionally, the 30 s TTP dose regulated the “proteinuria-
hand, other response mechanisms are strongly regulated induced oxidative stress response.”
after 48 h and 72 h but not after 24 h (e.g., “mechanisms Besides oxidative stress responses, the regulation of “aryl
of gene regulation by peroxisome proliferators via PPAR𝛼,” hydrocarbon receptor signalling” at 24 h by 60 and 120 s doses
“oxidative stress,” and “cell cycle : G2/M DNA checkpoint indicated that cells furthermore had to prepare for xenobiotic
regulation”). “Nrf2-mediated oxidative stress” seems to be and carcinogen detoxification due to higher doses of TTP
affected independently of TTP dose and time point but treatment [46].
ranked first after 24 h. Proteins involved in the categories “mechanisms of
The major part of toxicity responses is obviously linked gene regulation by peroxisome proliferators via PPAR𝛼,”
to oxidative stress if “Nrf2-mediated oxidative stress” is the “PPAR𝛼/RXR𝛼 activation,” and “PXR/RXR activation” were
BioMed Research International 9

30 s_all versus co_24 h (55)

PHP14
TXNL5

3 0 1433E
6PGL
50 CATB, ERP29,
FETA, HYOU1
4 14 14 73 24
22
KBP, STK3 PDIA6, PSME1, 2
TCTP SPRC, TSNAX
STMN1 VATB2, GELS
MRE11
60 s_all versus co_24 h (79) 120 s_all versus co_24 h (86) 48 h_all versus 24 h (87) 72 h_all versus 24 h (97)
(a) (b)

For example,
For example,
MRE11
For PPT1, VINC
example,
70 86
CATB only time-
only dose-
dependent 25 dependent

All TTP dose-dependent (95) All time-dependent (111)

(c)

Figure 6: Comparison of upregulated proteins (TTP-dose and time-dependent) by VENN diagrams. (a) VENN diagram of all TTP-dose-
dependently upregulated single proteins visualizing the overlapping results between upregulated proteins found in 30 s, 60 s, or 120 s TTP
treated cells versus untreated cells (co). Proteins given in bold are only TTP-dose-dependently induced, and those given in light are induced
also time-dependently. (b) VENN diagram of all time-dependently upregulated single proteins visualizing the overlapping results between
upregulated proteins found in 48 h or 72 h incubated (TTP treated) cells versus 24 h incubated (TTP treated) cells. (c) VENN diagram of all
TTP-dose- and time-dependently induced proteins indicating a fraction of proteins that are induced by both TTP dose and time span, as well
as proteins that are solely regulated only by tissue-tolerable plasma or only over time.

regulated by not exact referable TTP doses. Peroxisome
proliferator-activated receptors (PPARs) have been discussed
in context of initial wound healing phases [47].
In general, the analyses give a global overview of the
major mediators of TTP treatment effects on cell balance.
Oxidative stress is thereby one of the most determining
factors.
The “top cellular and molecular function table”
(Table 1(b)) provides an overview of cell adaption mecha-
nisms, displaying the five most affected cellular pathways for
each parameter. “Posttranslational modification,” “protein
folding,” and “cell death” are the most regulated cellular
functions, showing an increasing number of up- or downreg-
ulated proteins with increasing doses of TTP which was
shown before in Figure 5(a).
The “cellular growth and proliferation” was mainly
affected by the 120 s dose, whereas no regulation could be
observed in response to the 30 s application. The sporadic
affected expression of “DNA replication, recombination, and
repair” indicated that TTP could also cause DNA damage.
3.7. Oxidative Stress Response and UV Damage Repair Pro-
teins. The oxidative stress response is one of the most deter-
mining toxicity factors. Besides reactive oxygen or nitrogen
species (ROS or RNS), plasma contains also a certain fraction
of UV radiation which could also induce the oxidative stress
response as well as DNA damage. The sporadic affected
expression of “DNA replication, recombination, and repair”
could be one indication for DNA damage caused by TTP
treatment. Therefore, a detailed view on single regulated
proteins participating in oxidative stress response (focus on
detoxification of reactive oxygen species) and UV damage
repair was accomplished in order to support the IPA results.
As shown in Figure 8 (for the whole protein list see supple-
mentary Table S3), there is a TTP-dose-dependent regulation
of both pathways with the highest number of regulated
protein spots observed after the 120 s dose of TTP treatment
during all time points. The largest TTP-dose-dependent
regulating effect with respect to the number of affected
protein spots could be found for oxidative stress response
proteins after 24 h indicating an early, direct consequence
10 BioMed Research International

Molecular functions Biological processes

9 6 64 25
15 34
17 43

27 213
150 60

61
73

61
136

148
Number of mapped genes: 354
Binding (GO:0005488)
Catalytic activity (GO:0003824)
Structural molecule activity (GO:0005198)
Enzyme regulator activity (GO:0030234)
Transcription regulator activity (GO:0030528)
Translation regulator activity (GO:0045182)
Transporter activity (GO:00052515)
Motor activity (GO:00037774)
Receptor activity (GO:0004872)
Antioxidant activity (GO:0016209)
(a)
63
68
Number of mapped genes: 354
Metabolic process (GO:0008152)
Cellular process (GO:0009987)
Cell communication (GO:0007154)
Transport (GO:0006810)
Cellular component organization (GO:0016043)
Developmental process (GO:0032502)
Cell cycle (GO:0007049)
Immune system response (GO:0002376)
System process (GO:0003008)
Apoptosis (GO:0006915)
(b)

Figure 7: Pie charts showing assignments of regulated proteins of the dose-dependent approach to molecular functions (a) and biological
processes (b). The charts were established by using PANTHER gene ontology. The number of genes that could be allocated to the
corresponding function or process is marked. For the 369 significant regulated proteins (p < 0.05) 354 hits with allocations could be found.
The standardized GO term is listed in brackets behind the function or process.

of TTP treatment (Figure 8(a)). The impact of TTP on the
regulation of UV damage repair proteins seems to be more
lasting if the number of regulated protein spots was increased
further after 48 h (Figure 8(b)).
As shown in supplementary Table S3, even a mod-
erate dose of TTP (30 s) induced the expression of only
two early oxidative stress response proteins, thioredoxin-
reductase (TXRX1) and thioredoxin domain-containing pro-
tein 5 (TXND5). The 60 and 120 s TTP treatment significantly
induced in a similar manner the expression of thioredoxin
(THIO) itself. Additionally, reactive oxygen species- (ROS-)
detoxification mechanisms seem to be affected, since super-
oxide dismutase (SODC), glutathione S-transferase (GSTO1),
and peroxiredoxin (PRDX3, 2) were observed to be decreased
in expression by 60 and 120 s TTP treatment although they are
needed for detoxification of increased free radicals.
The UV excision repair protein RD23 homolog B
(RD23B) showed a TTP-dose-dependent increasing induc-
tion after 24 h. An additional repair protein, the DNA double-
strand break repair protein MRE11, was upregulated only
by the 120 s dose (Table S3). As shown above, MRE11 was
solely upregulated by the impact of TTP, not over time (see
Figure 6(c)). In summary, this detailed analysis revealed a
strong dose-dependent increase of the number of regulated
oxidative stress proteins after 24 h indicating a direct impact
on molecular adaptation mechanisms. The impact of UV
radiation on cellular pathways could be observed as more
lasting compared to oxidative stress. Furthermore, a strong
dose-dependent increase of average fold changes is shown for
both adaptation pathways in Figure 9.
3.8. Proteins Associated with Cell Death/Apoptosis and Cell
Proliferation. “Cell death” and “cellular growth and prolifer-
ation” were under the top of molecular and cellular functions
affected by TTP. In order to get a closer view on single
regulated proteins participating in these pathways, a more
detailed analysis of the subgroups “antiapoptotic factors,”
“cell death/apoptotic factors,” and “cell proliferation/cell
division” was accomplished and should support the present
IPA results.
An increasing number of cell death/apoptotic factors and
protein spots functioning in cell proliferation/cell division
BioMed Research International 11

Oxidative stress response UV damage repair

16 16

Numbers of regulated protein spots

Numbers of regulated protein spots

14 14

12 12

10 10

8 8

6 6

4 4

2 2

0 0
30 60 120 30 60 120
(s) (s)

24 h 24 h
48 h 48 h
72 h 72 h
(a) (b)

Figure 8: Progressive increase of the number of regulated protein spots involved in oxidative stress response and UV damage repair. Number
of identified regulated protein spots involved in oxidative stress response (focus on detoxification of reactive oxygen species) (a) or UV
damage repair (b) is displayed for different TTP doses after several time points. Note that increasing TTP doses progressively influences both
adaptation pathways.

showed reduced protein levels in S9 cells correlating with
increasing TTP doses at any time point. The average fold
changes of respective normalized values of each condition
are shown in Figure 10. For the whole protein list see sup-
plementary Table S4. There is a clear TTP-dose-dependent
suppression after 24 h if the reduction of protein amount
was highest after 120 s TTP treatment at this time point.
In particular, the amount of one protein, the proliferation-
associated protein 2G4 (PA2G4), was strongly decreased
leading to an average expression value of about 10 for the
whole group of “cell proliferation/cell division” proteins. If
this effect could be observed only after 24 h, then it should
be more directly linked to the given TTP stimulus.
In contrast, protein levels of antiapoptotic factors are
increased up to 2.5-fold in a dose-dependent manner during
all time points indicating lasting impact of TTP on regulation
of respective proteins.
Taking both observations into account, it can be assumed
that TTP treatment has regulating effects on cell death and
apoptosis mechanisms, thus leading to longer cell survivabil-
ity.
4. Discussion
Many recent papers have highlighted the promising potential
of plasma treatment as a new therapeutic option for chronic
infected wound care. In several in vitro studies, different types
of wound relating cells in monolayer, such as keratinocytes,
fibroblasts, breast epithelial, and endothelial cells, were used
to analyse the different effects of plasma application [19,
22, 24, 25]. Despite these in vitro studies, the fundamental
nature of the interaction between plasma and human cells
is to a large extent unknown. But a detailed knowledge of
these interactions is essential for the evaluation of plasma
effects in relation to wound healing and the establishment
of new therapeutic plasma tools. In this study we analysed
for the first time plasma effects on cellular adaptation of
upper airway epithelial S9 cells. The standardized wound
model employed in this study simulates in vitro wound
healing in secondary intention, for example, after surgery
of the paranasal sinus, the larynx, or the trachea. As a
consequence, results obtained here have a direct clinical
impact for the medical application of cold plasma in internal
medicine (e.g., during bronchoscopy), thoracic surgery, and
otorhinolaryngology.
Furthermore, molecular changes due to plasma treatment
were studied for the first time at the proteome level in order
to complement the known macroscopic effects observed in
the wound model. First, tissue-tolerable plasma (TTP) doses
were determined in an in vitro wound healing model with
S9 epithelial cells. Plasma doses from 30 s up to 360 s were
tested in relation to wound regeneration after 24 h, 48 h, 72 h,
96 h, and 120 h, in which lower doses (30, 60, and 120 s)
resulted in dose-dependent improved wound healing rate
compared to untreated cells. Thereby, the 120 s dose caused
significantly the best wound healing properties after 96 and
120 h. Because higher doses (240, 360 s) resulted in lowered
or even blocked regeneration, only the lower plasma doses
were termed as tissue-tolerable plasma (TTP) and used for
the investigation of proteome alterations in the following.
Thus, the proteomes of 30, 60, and 120 s TTP treated S9 cells
were analysed and compared with untreated cells. Assuming
that proteome alterations in the following experiments and
molecular adaptation should happen before the development
of the observed macroscopic effect, the analysis focused on
cells harvested after 24, 48, and 72 h.
12 BioMed Research International

Table 1: Ranking of response mechanisms and adaptation of cellular functions due to TTP treatment as revealed by IPA. The charts were
established by using Ingenuity Pathway Analyses (IPA) to relate regulated proteins to their corresponding molecular mechanism and cellular
function.
(a) The “Top Tox List” displays the response mechanisms, which are most affected by several stress factors emerging due to TTP treatment. The top five response
mechanisms are listed for every dose (30 s, 60 s, and 120 s) after every time point (24 h, 48 h, and 72 h). Numbers (1–5) represent the ranking of the response
mechanisms for every parameter

24 h 48 h 72 h
Top Tox List
30 s 60 s 120 s 30 s 60 s 120 s 30 s 60 s 120 s
Nrf2-mediated oxidative stress 1 1 1 4 3 3 3
Decreases permeability transition of 2 1 5 1
mitochondrial membrane
Mitochondrial dysfunction 3
Response to proteinuria-induced
oxidative stress in renal proximal tubulus 4 2
cells
Increases bradycardia 5 5
Renal necrosis/cell death 2 3
Aryl hydrocarbon receptor signaling 3 2
PPAR𝛼/RXR𝛼 activation 4 3 4
Increases transmembrane potential of 5 5 4 5
mitochondrial membrane
Xenobiotic metabolism signaling 4
Mechanisms of gene regulation by 3 1 2 4
peroxisome proliferators via PPAR𝛼
Increases renal proliferation 4
Oxidative stress 2 1 1 2 2
PXR/RXR activation 5 3
Cell cycle: G2/M DNA checkpoint 5 1
regulation
TR/RXR activation 2 5
Acute renal failure panel 4
(b) The chart displays the most influenced molecular and cellular functions after 30 s, 60 s, and 120 s of TTP treatment and incubation time of 24 h, 48 h, and
72 h. Numbers represent the ranking of the top five cellular functions for every parameter

24 h 48 h 72 h
Top molecular and cellular functions
30 s 60 s 120 s 30 s 60 s 120 s 30 s 60 s 120 s
Posttranslational modification 1 1 1 1 1 2 1 5 1
Protein folding 2 3 2 2 3 4 3 4 3
Cell death 3 2 3 4 1 2 1 2
Cellular assembly and organization 4 4 4 2
Cellular movement 5 3 5
DNA replication, recombination, and repair 4 5
Cellular growth and proliferation 5 4 3 4
Cellular compromise 5 2 5
Cellular function and maintenance 5 3 3

In accordance with the PCA, the detailed statistical
proteome analysis highlights that the highest dose of TTP
treatment (120 s) after 24 h resulted in the greatest proteome
alterations between untreated and treated samples demon-
strating the most significant effect on the proteome of S9
airway epithelial cells under these conditions. If the 120 s
dose caused the best wound healing properties and the most
significant changes at the proteome level, then the question
arises if these molecular changes could be the reason for the
observed macroscopic effect. Which cellular and molecular
mechanisms could be responsible for the improved wound
healing properties?
As shown by IPA, various toxic responses or cellular and
molecular functions are affected by TTP treatment in terms of
up- or downregulation of their associated proteins. Undoubt-
edly, the most toxicity responses are linked to oxidative stress
BioMed Research International 13

response emphasizing oxidative stress as a possible key event

12
in the regeneration process of epithelial cells as well as in the
adaptation to plasma exposure. Oxidative stress might occur
10
Average fold change of expression

as a direct outcome of the existence of reactive oxygen or

nitrogen species (ROS, RNS) which are detectable in the gas
8 phase over the cells as well as in the culture medium after
the argon plasma treatment. These species can also enter the
6 cells possibly by diffusion or can induce new species within
the cells [24]. It might be that the impact of ROS or RNS
and the initiation of oxidative stress response are a necessary
4
prerequisite for initiation of wound healing.
The significant influence of several oxidative stress
2 response pathways is further supported by our single protein
analyses, which revealed partial progressive regulation of
0 downstream proteins involved in oxidative stress response,
Plasma 30 s Plasma 60 s Plasma 120 s
particularly in the detoxification of reactive oxygen species.
versus control_24 h The upregulation of thioredoxin-reductase and thioredoxin
Dose of TTP treatment itself might be a clear evidence for the already existing
defence against oxidative damage or at least for the signalling
Oxidative stress response
UV damage repair
of molecules like hydrogen peroxide or nitric oxide. Even
though the amounts of further detoxification enzymes like
Figure 9: Effect of TTP treatment on the average fold change of
superoxide dismutase and peroxiredoxin were decreased
protein spots involved in oxidative stress response and UV damage relatively, an involvement of these enzymes in the active
repair. The influence of different doses of TTP on oxidative stress defence against oxidative stress can be assumed. We suppose
response (focus on detoxification of reactive oxygen species) and that the oxidative stress elucidated by our TTP treatment is
UV damage repair is shown as average fold changes of all selected dosed as much as required for initiation of wound healing but
protein spots regardless of the fact that the single ratios have low enough to prevent profound cell damage. The induction
positive or negative values. For single ratios and their calculation of oxidative stress due to plasma treatment has also been
see supplemental Table S3. described for other cell types, both human [31, 48–50] and
animal cells [51, 52]. In contrast to many other studies (e.g.,
50), our proteomic approach was designed not to detect ROS
or RNS but to detect many downstream regulated proteins,
4 providing a new perspective on plasma treatment effects. The
pivotal role of oxidative stress in the wound healing process
2 can be regarded as independently of organism or cell type, as
Fold change of expression

0 revealed by both in vitro and in vivo settings [53–55].

The wound healing process of acute wounds is an intricate
−2 interplay between several cell types involving various forms
−4 of intracellular signalling and classical wound healing phases
like haemostasis, inflammation, and new-tissue formation.
−6
The last one includes, for example, keratinocytes migrating
−8 and proliferating into the wound [56]. Apoptosis of immune
cells may be the major key to end inflammation and to initiate
−10
the healing [57]. Our proteomic study analysing only a single
−12 epithelial cell type could demonstrate the induction of anti-
30 s 60 s 120 s 30 s 60 s 120 s 30 s 60 s 120 s apoptotic factors as well as the repression of proteins involved
versus control_24 h versus control_48 h versus control_72 h in apoptosis indicating that apoptosis seems to be inhibited
Dose of TTP treatment
by our TTP treatment in S9 cells. However, TTP treatment
caused contrary effects on cells, because it inhibits apoptotic
Antiapoptotic factors mechanisms on one hand and cell proliferation on the other
Cell death/apoptotic factors
hand. Although the majority of protein hits associated with
Cell proliferation/cell division
cell proliferation and cell division showed reduced protein
levels mainly 24 h after the 120 s TTP treatment (e.g., PA2G4),
Figure 10: Effects of TTP treatment on cell death and cell prolif-
eration pathways of S9 epithelial cells. The influence of different
we cannot exclude a certain proliferation and migration
TTP doses on cell death/apoptosis and cell proliferation pathways activity leading to the observed wound regeneration after 96
is shown as average fold changes of all selected protein spots taking and 120 h. In accordance with this assumption, the proteomic
into account the fact that single ratios have positive or negative approach revealed that the degree of repression of these
values. For single ratios and their calculation see supplemental Table proteins is highest after 24 h but is clearly reduced after 48
S4. and 72 h, indicated by a decreased number of affected proteins
14
and decreased repression factors. Furthermore, the DP13B
(APPL2) protein, involved in regulation of cell proliferation,
belongs to the top 10 of upregulated proteins in response to
120 s plasma dose.
The apparently concurrent repression of apoptosis and
cell proliferation might be an indication for the coexistence of
both successful and failed cell adaptation in response to TTP
treatment. Here, we have to underline that our proteomic
approach could only detect the sum of all cell events of
each culture plate including intact as well as injured cells
representing all molecular mechanisms that are responsible
for the final concentration of each protein. It might be that
there are some cells, immediately bordered on the wound,
which activate the cell proliferation/cell division at least to a
certain extent.
Positive effects of plasma treatment on wound healing
have also been shown several times in many studies [17, 18,
21, 25]. However, the positive effects were mostly ascribed
to a decrease of bacterial load in chronic wounds [18, 21].
Only recently, different studies focusing on epithelial cells
demonstrated that certain plasma doses increased cell prolif-
eration in treated cells but also activated apoptotic pathways
[24, 29–31]. In another very recently published study with
keratinocytes, a significant role of apoptosis after plasma
treatment was excluded [50]. Further studies showed that
plasma can support wound healing not only by its antiseptic
effects but also by the stimulation of proliferation and
migration of wound relating skin cells or by its proangiogenic
effect (reviewed in [24]).
In the current proteome study we analysed for the first
time a possible correlation between detected proteome alter-
ations, due to treatment with three different TTP doses, and
wound healing process without an involvement of infection.
To some extent, these first detected proteomic effects in
S9 epithelial cells are comparable with the sole transcriptomic
studies carried out with indirectly plasma treated HaCaT
keratinocyte cells [31, 32, 50]. Similar biological pathways,
such as oxidative stress response, repair, cell, death, and
proliferation, have been found as effected by both approaches.
Both proteomic and transcriptomic studies revealed that,
for the present, plasma treatment has to be considered as
double-edged sword yet. For example, our results indicate
that TTP treatment is probably not just affecting molecular
mechanisms during the time of application or rather over
one period of cell division but has in fact lasting impacts on
protein expression and cell balance. A more detailed time-
resolved view including also shorter times immediately after
the application of TTP should apparently demonstrate that
(1) the TTP dose affects the degree of molecular response
and that (2) posttranslational modifications of proteins may
appear as a consequence of oxidative stress (Emicke et al., in
preparation).
We were able to show that the effects of TTP treatment
on cells are highly dependent on the dose applied. The in
vitro wound model revealed an increasing wound healing
rate with increasing doses of TTP treatment. Our proteome
analyses uncovered a TTP-dose-dependent increase of the
number of regulated proteins functioning in different toxicity
responses and biological pathways. Particularly after 24 h,
BioMed Research International
the repression rate of proteins associated with apoptosis and
cell proliferation is increased. This highlights the need of
finding the optimal treatment dose to provide a balance
between cell proliferation and death before it could be safely
and routinely introduced in clinical use. In the current
experimental setup, the 120 s dose of TTP treatment may
be the optimal dose, if apoptotic pathways do not at least
counteract with cell proliferation. Precaution should be used,
as long term effects were not analysed so far and therefore
the risk of cumulative cell damage could not be evaluated
surely. In recent clinical trials, a dose of 120 s cold plasma
treatment was set as standard, since beneficial macroscopic
effects could be observed [21, 26, 48, 58–60]. Both studies
reported an enhanced wound healing rate on skin graft
donor sites or chronic ulcers without adverse effects for
patients; however, long term effects were not evaluated.
Lademann et al. [61] and Kramer et al. [33] focused on the
suitability and risk assessment of plasma on human skin.
Both concluded that plasma treatment entails no risk for
humans. Nevertheless, our current study showed at proteome
level that cell damaging effects of TTP treatment on airway
epithelial cells should not be disregarded. Future studies
should combine clinical trials with a molecular approach in
order to achieve a comprehensive understanding of tissue-
tolerable plasma.
The possible cell damaging effects of TTP treatment not
only may be a result of oxidative stress, but also may be
attributed to UV damage. Although no hints were obtained
from top toxicity lists and associated molecular functions
and biological processes, single protein analyses showed
a TTP-dose-dependent regulation of UV damage repair
mechanisms for all time points (e.g., upregulation of RD23B
and MRE11). This could be an underestimated source of long
term cell damage due to TTP treatment. The possible role of
UV radiation due to plasma treatment on cells was recently
discussed. Irradiation of cell cultures can lead to severe DNA
damage and apoptosis, but moderate doses are able to activate
UV damage response and trigger adaptation pathways which
lead to enhanced cell proliferation [62, 63]. Stoffels [63]
reasoned that cells appear to be fairly resistant against UV
radiation, if activated response mechanisms are sufficient to
cope with the imposed challenges. The possible UV damage
caused by plasma treatment is still persisting as a critical and
highly discussed point for plasma researchers. The current
study indicates that UV damage is probably subordinate to
oxidative stress regarding the effects of TTP treatment, at least
under the investigated conditions. But long term research
should be performed in order to evaluate the enduring effects
of both UV damage and oxidative stress. Additionally, the
more detailed time resolved view in the accompanying study
of Emicke et al. (in preparation) should highlight the role of
NRF2 in oxidative stress response and the identification of
Nrf2 targets if “Nrf2 mediated oxidative stress” was found as
a top of toxicity responses by our proteomic approach.
Last but not least, one limitation of this study has to
be taken into consideration. Cell cultures were manually
treated with TTP from the plasma jet “kINPen” by following
a grid pattern for the required time (30 s, 60 s, and 120 s). In
order to achieve equalization of treatment time for all cell
BioMed Research International
cultures, six time checkpoints were set along the grid pattern.
Nevertheless, absolute exactness of the manual procedure
cannot be guaranteed. Now, a new plasma source generation
has been developed by the INP Greifswald, which is able
to treat cell cultures automatically for a specified time,
following a defined pattern. For future studies, the automated
application would be recommended to avoid any variation
in application of treatment. Furthermore, it is necessary to
note that the results gathered from cell culture analyses with
only one single cell type cannot simply be transferred into
interacting physiology or extrapolated for other cell types
or complex organs. For example, the S9 airway epithelial
cells may be more resistant to TTP treatment and may show
different adaptation mechanisms than other human cells or
even animal cells. In the future, the aim of comprehensive
molecular research should be to combine in vitro and in
vivo macroscopic and molecular analysis [64]. However, this
first proteomic analysis indicates dose-dependent cellular
adaptation in response to TTP treatment and therefore
supports the results of the in vitro wound model.
5. Conclusion
This proteomic study provides an overall insight to cell
adaptation pathways of S9 epithelial cells induced by TTP
treatment. At the proteome level, oxidative stress could be
shown to be the most affected response of TTP treatment
and the possible key mediator for initiation of wound healing.
Although this study gives reason to assume that oxidative
stress is the greatest challenge for cells to cope with, the role
of radiation should not be disregarded. Therefore, further
analyses have to be performed in order to receive more
detailed knowledge. Furthermore, results of an in vitro wound
model highlight the dose-dependent wound healing acceler-
ating effects of TTP treatment. Treatment of cells for 30 s and
60 s showed marginal beneficial effects on cell regeneration,
whereas 120 s improved the wound healing rate by nearly
15% compared to untreated cells. Additionally, analyses on
protein level supported the wound model, as alterations in
protein expression profiles increased with the applied dose
and cell proliferation pathways were TTP-dose-dependently
regulated. However, higher doses of TTP seem to be also a
more potent cause for cellular imbalance, leading to increased
repression rate of proteins functioning in cell death/apoptosis
and cell proliferation. Hence, in vivo wound models should be
performed in order to evaluate the toxicity of TTP treatment
for animal or human physiology. This study confirms that
proteome analyses are a powerful tool to achieve molecular
understanding about well-known macroscopic TTP treat-
ment effects and should be implemented in clinical trials to
learn about strengths and weaknesses of plasma in human
medicine.
Abbreviations
TTP: Tissue-tolerable plasma
ROS: Reactive oxygen species
RNS: Reactive nitrogen species
15
2D-DIGE: Two-dimensional-difference gel
electrophoresis
Plasma dose: Treatment time.
Conflict of Interests
The authors declare that they have no competing interests.
Authors’ Contribution
Derik Lendeckel contributed to the analysis and interpre-
tation of data and drafting the paper. Christine Eymann
contributed to interpretation of the data and writing of
paper. Philipp Emicke contributed to gel image and mass
spectrometry analyses, statistical analyses, and drafting of
paper. Georg Daeschlein contributed to study and concept
design, laboratory support, revision of paper, and cofinancing
by provision of assistant technician Katrin Darm. Katrin
Darm contributed to cell cultivation, plasma treatment, and
DIGE experiment. Serena O’Neil contributed to data analysis
and drafting of paper. Achim G. Beule contributed to devel-
opment of wound model and revision of paper. Thomas von
Woedtke contributed to provision of kINPen plasma source,
acquisition of funding, and revision of paper. Uwe Völker
contributed to provision of mass spectrometry instruments
and IPA data analysis software. Klaus-Dieter Weltmann con-
tributed to provision of kINPen plasma source, acquisition of
funding, and revision of paper. Michael Jünger contributed to
laboratory support and cofinancing by provision of assistant
technician Katrin Darm. Werner Hosemann contributed to
acquisition of funding and revision of paper. Christian Scharf
contributed to design of study and experimental planning,
data analysis, conception, drafting and revision of paper, and
acquisition of funds.
Acknowledgment
This work was realized within the joint research project
“Campus Plasma-Med II”, funded by BMBF 13N11181.
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