1. J Mater Sci: Mater Med
DOI 10.1007/s10856-012-4807-z
Plasma surface modification of polylactic acid to promote
interaction with fibroblasts
Tinneke Jacobs • Heidi Declercq • Nathalie De Geyter • Ria Cornelissen
Peter Dubruel • Christophe Leys • Arnaud Beaurain • Edmond Payen •
Rino Morent
•
Ó Springer Science+Business Media New York 2012
Abstract In this work, medium pressure plasma treatment of polylactic acid (PLA) is investigated. PLA is a
biocompatible aliphatic polymer, which can be used for
bone fixation devices and tissue engineering scaffolds. Due
to inadequate surface properties, cell adhesion and proliferation are far less than optimal and a surface modification
is required for most biomedical applications. By using a
dielectric barrier discharge (DBD) operating at medium
pressure in different atmospheres, the surface properties of
a PLA foil are modified. After plasma treatment, water
contact angle measurements showed an increased hydrophilic character of the foil surface. X-ray photoelectron
spectroscopy (XPS) revealed an increased oxygen content.
Cell culture tests showed that plasma modification of PLA
films increased the initial cell attachment both quantitatively and qualitatively. After 1 day, cells on plasmatreated PLA showed a superior cell morphology in
T. Jacobs Á N. De Geyter Á C. Leys Á R. Morent (&)
Research Unit Plasma Technology (RUPT), Department
of Applied Physics, Faculty of Engineering, Ghent University,
Sint-Pietersnieuwstraat 41, 9000 Ghent, Belgium
e-mail: Rino.Morent@UGent.be
H. Declercq Á R. Cornelissen
Department of Basic Medical Science, Faculty of Medicine
and Health Science, Ghent Universtity, De Pintelaan 185 6B3,
9000 Ghent, Belgium
P. Dubruel
Polymer Chemistry and Biomaterials Group, Department
of Organic Chemistry, Faculty of Sciences, Ghent University,
Krijgslaan 281 S4, 9000 Ghent, Belgium
A. Beaurain Á E. Payen
´
Unite de Catalyse et Chimie du Solide, UMR CNRS 8181,
´
ˆ
Universite des Sciences et Technologies de Lille, Bat. C3,
´
Cite Scientifique, 59655 Villeneuve d’Ascq, France
comparison with unmodified PLA samples. However, after
7 days of culture, no significant differences were observed
between untreated and plasma-modified PLA samples.
While plasma treatment improves the initial cell attachment, it does not seem to influence cell proliferation. It has
also been observed that the difference between the 3 discharge gases is negligible when looking at the improved
cell-material interactions. From economical point of view,
plasma treatments in air are thus the best choice.
1 Introduction
Polylactic acid (PLA) is a biodegradable aliphatic polymer,
which is frequently used in biomedical applications. These
applications include sutures, stents, drug delivery systems,
bone fixation devices (plates, pins, screws, etc.) and tissue
engineering scaffolds [1–4]. This broad application range
stems from the fact that PLA is a biocompatible, non-toxic
and biodegradable polymer with a total degradation time of
several years. Although PLA is widely used, the surface
properties are far from optimal. Mainly the low wettability
and low surface energy remain an important issue, since
these properties adversely affect cell attachment and proliferation [5]. A poor cell adhesion and proliferation onto
the surface can lead to a poor incorporation of the implant,
infections, inflammations and even complete implant failure. Therefore, surface properties of PLA need to be altered
to enhance cell-material interactions.
Non-thermal plasmas have previously been proven to be
able to modify the wetting and adhesive properties of
various polymers [6–14]. The success of plasma treatment
in polymer surface modification can be attributed to the
fact that it is a genuine surface modification technique
which only affects the outermost atomic layers of a
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2. J Mater Sci: Mater Med
material. Moreover, it is a fast, versatile and environmentally friendly technology. Over the past decades, research
has been mainly focused on traditional polymers such as
polypropylene, polyethylene, polyethylene terephthalate,
etc. Recently however, a few authors have studied the
plasma surface modification of biodegradable polymers,
such as PLA [3, 4, 15–24]. Nevertheless, these studies
mainly use vacuum (0.2 kPa) plasmas as surface modification tools, since these plasmas offer a good control over
the plasma chemistry. For industrial applications, the use of
atmospheric plasmas is however preferred, since sample
handling and scalability for industrial in-line processing is
much easier for these type of plasmas [25, 26]. Moreover,
the high costs of the vacuum pumping equipment remain a
great drawback in low pressure plasma technology.
It is however also possible to combine the advantages of
both vacuum and atmospheric pressure plasma technologies by working in the medium pressure range. At medium
pressure, a large plasma volume can be easily obtained,
which can result in a higher overall productivity [8].
Recently, it has been shown that at low energy densities,
plasma treatment at medium pressure is more energy efficient than at atmospheric pressure [27]. Furthermore, different (toxic) chemicals and gases can be used, since
medium pressure plasma technology works in a closed
plasma chamber. Nevertheless, the pumping equipment is
relatively inexpensive [8].
Recognizing the above, in this paper, a medium pressure
dielectric barrier discharge (DBD) will be used to modify
the surface of a PLA foil. A DBD is generated between two
electrodes, of which at least one is covered with a dielectric
material. The function of the dielectric layer is to stabilize
the discharge and to prevent electric arc formation by
limiting the current. Due to the easy formation of a stable
regime and due to their scalability, DBDs are very convenient tools for surface modification [25]. Usually, DBDs
operate in the filamentary mode: the discharge consists of
many tiny current filaments, named microdischarges [28,
29]. These microdischarges are of nanosecond duration and
are randomly distributed over the dielectric surface. Under
special, quite restrictive conditions homogeneous, so-called
glow DBDs can be obtained. In these DBDs, there is no
microdischarge activity and the discharges are thus diffuse
and uniform.
After the description of the experimental medium
pressure DBD set-up, DBD surface treatments in 3 different atmospheres (dry air, argon and helium) will be
investigated. The differences in surface modifications
among the 3 discharges will be explored using contact
angle measurements and X-ray photoelectron spectroscopy
(XPS). Contact angle measurements will be used to study
the wettability of the plasma-treated PLA samples, while
the relative surface chemical composition of the samples
123
will be obtained using XPS. Besides surface characterization, also cell culture tests will be performed on PLA
samples to determine whether plasma treatment is capable
of improving the cell-material interactions.
2 Experimental set-up
2.1 Polymer film
Commercially available PLA film was purchased from
Goodfellow Cambridge Limited (England). The films have
a thickness of 50 lm and are not subjected to any pretreatment step before plasma modification. For the plasma
treatments, small rectangles were cut from the foil with
dimensions of approximately 3 9 4 cm, while for the cell
culture tests circular samples with a diameter of 14 mm
were used.
2.2 DBD set-up
The experimental set-up (see Fig. 1) consisted of two circular copper plates with a diameter of 7 cm, both covered
with a glass plate which acts as a dielectric barrier. The
distance between the glass plates was 7 mm and the electrodes were placed within a cylindrical enclosure made of
PVC (inner diameter: 25 cm, height: 25 cm). The upper
electrode was connected to an AC power source (frequency = 5 kHz), while the lower electrode was connected to earth, either by a resistor (R) or a capacitor (C).
After placing the polymer sample on the lower glass plate,
the chamber was evacuated by a rotary vane pump below a
pressure of 2 kPa. After that, different procedures were
applied depending on the discharge gas. For helium and
argon, the plasma chamber was subsequently flushed for
3 min with helium or argon at a rate of 5.2 slm (standard
liters per minute) while keeping on the pumping system.
This purging step was performed to avoid variations in
residual oxygen concentration in the vacuum chamber.
After 3 min of flushing, the rotary vane pump was turned
off and the chamber was filled with helium or argon (Air
liquide–Alphagaz 2) at a rate of 5.2 slm. When atmospheric
pressure (101 kPa) was reached, the plasma chamber was
pumped down to 5 kPa while the gas flow was decreased to
0.2 slm. Subsequently, the AC power source was turned on
while the gas flow was maintained at 0.2 slm and the
pressure in the discharge chamber was kept constant at
5 kPa by slightly pumping during plasma treatment. For
treatments in dry air, a different procedure was applied,
since the remaining oxygen concentration in the discharge chamber was not critical when dry air was used
as discharge gas. After the chamber was evacuated
below a pressure of 2 kPa, it was filled with dry air

3. J Mater Sci: Mater Med
¨
system (Kruss GmbH, Germany). This system is equipped
with a high precision liquid dispenser to precisely control
the drop size of the water. A drop of 2 ll of distilled water
was placed on the PLA substrate and via an interline CCD
video camera, the drop image was stored. Measurement of
the static contact angle was fully automatic by using the
computer software provided with the instrument. The values of the contact angle were obtained using LaplaceYoung curve fitting based on the imaged sessile water drop
profiles. For each sample, 5 contact angles were measured
and an average value was calculated. Standard deviations
varied from 0.5 to 2.5°.
2.4 X-ray photoelectron spectroscopy
Fig. 1 Schematic diagram of the experimental DBD set-up. (1 gas
cylinder, 2 mass flow controller, 3 plasma chamber, 4 pressure gauge,
5 valve, 6 pump)
(Air Liquide–Alphagaz 1) to 50 kPa. Afterwards, the
chamber was again pumped down to 5 kPa while a constant air flow of 0.2 slm was fed into the system between
the glass plates. By slightly pumping, the pressure in the
discharge chamber was maintained at 5 kPa.
To determine in which discharge mode the DBD was
operating for each discharge gas, current–voltage waveforms have been acquired. A high voltage probe (Tektronix
P6015A) was connected to the upper electrode to measure
the high voltage applied to the reactor, while at the same
time the discharge current was obtained by measuring the
voltage over a small resistor of 50 X, connected in series to
the ground. Both waveforms were then recorded using an
oscilloscope (Tektronix TDS210, 60 MHz). To calculate
the discharge power of the DBD, the resistor (R) was
replaced by a capacitor (C) of 10 nF and the voltage across
this capacitor is proportional to the charge stored on the
electrodes. When plotting this charge as a function of
applied voltage, a Lissajous figure can be constructed and
from this figure, the discharge power can be calculated [28,
29]. Since the discharge power was not the same for the
different discharge gases, the results in this work will be
presented as a function of energy density instead of treatment time to make an objective comparison between the
various plasma treatments. This energy density was calculated by multiplying the plasma exposure time by the
plasma power and by dividing this by the area of the
plasma electrodes.
2.3 Water contact angle measurements
Within 5 min after plasma treatment, water contact angles
¨
were obtained with a commercial Kruss Easy Drop optical
Besides contact angle measurements, polymer samples
were also studied by XPS to obtain the surface elemental
composition and to get an insight into the chemical
functions introduced on the surface by the plasma treatments. XPS measurements were performed on a Kratos
Axis Ultra DLD spectrometer equipped with a monochromatic Al Ka X-ray source (hm = 1,486 eV) at a
power of 150 W. The pass energy for survey spectra was
160 eV with 1.0 eV step size and for high-resolution C1s
spectra 20 eV with 0.1 eV step size. The pressure in the
main chamber during analyses was not higher than
2.1 9 10-9 Pa, the analysis area was approximately
400 9 700 lm and the spectra were acquired at an
emission angle of 90°. Elements present on the surface
were identified from survey spectra and quantified (in
at %) with CasaXPS software using a Shirley background
and applying the relative sensitivity factors supplied by
the manufacturer of the instrument. CasaXPS software
was also used for the curve fitting of the high-resolution
C1s peaks: the hydrocarbon component of the C1s spectrum (285.0 eV) was used to calibrate the energy scale
and a Shirley background was chosen. After this calibration step, the C1s peaks were deconvoluted using
Gaussian–Lorentzian peak shapes with a 30 % Lorentzian
component and the full-width at half maximum (FWHM)
of each line shape was constrained below 1.6 eV.
2.5 Cell culture tests
2.5.1 Cell culture and cell seeding onto the PLA films
HFF-1 cells (human foreskin fibroblasts, ATCC) were
cultured in DMEM glutamax medium (Gibco Invitrogen)
supplemented with 10 % foetal calf serum (FCS, Gibco
Invitrogen), 2 mM L-glutamine (Sigma-Aldrich, Belgium),
P/S (10 U/ml penicillin, 10 mg/ml streptomycin, Gibco
Invitrogen) and 100 mM sodium-pyruvate (Gibco Invitrogen). Cells were cultured at 37 °C in a humidified
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4. J Mater Sci: Mater Med
atmosphere containing 5 % CO2. PLA films (diameter
14 mm) were left untreated or subjected to plasma treatment (dry air, argon, helium) with the above mentioned
characteristics. Subsequently, the PLA films were sterilized
using ethylene oxide (Maria Middelares Hospital, Ghent,
Belgium). After the sterilization cycle of 4 days, cell culture experiments were started. Cells were seeded at a
density of 40,000 cells/100 ll medium per film in 24-well
culture dishes. The cells were allowed to adhere for 4 h
before additional medium (400 ll) was added. Cell adhesion and proliferation were evaluated after 1 and 7 days.
Cells cultured on tissue culture polystyrene (TCPS) were
taken as a positive control.
2.5.2 Phase contrast and fluorescence microscopy
To visualize cell attachment and distribution on the PLA
films, the cell cultures were evaluated using inverted
phase contrast microscopy (Olympus inverted Research
System Microscope, XCellence Pro software, Aartselaar,
Belgium).
In addition, a live/dead staining (Calcein AM/propidium
iodide) was performed to evaluate cell viability. After
rinsing, the supernatant was replaced by 1 ml phosphate
buffered saline (PBS) solution supplemented with 2 ll
(1 mg/ml) calcein AM (Anaspec, USA) and 2 ll (1 mg/ml)
propidium iodide (Sigma). Cultures were incubated for
10 min at room temperature, washed twice with PBS
solution and evaluated by fluorescence microscopy (Type
U-RFL-T, Olympus, Aartselaar, Belgium). Evaluations
were done 1 and 7 days post-seeding.
2.5.3 MTT viability
The colorimetric MTT assay, using a 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck
Promega) was performed to quantify cell viability and
proliferation on the PLA films. The tetrazolium component
is reduced in living cells by mitochondrial dehydrogenase
enzymes into a water- insoluble purple formazan product,
which can be solubilized by addition of lysis buffer and
measured using spectrophotometry.
The cell culture medium was replaced by 0.5 ml
(0.5 mg/ml) MTT reagent and cells were incubated for 4 h
at 37 °C. The MTT reagent was removed and replaced
by 0.5 ml lysis buffer (1 % Triton X-100 in isopropanol/
0.04 N HCl) for 30 min. 200 ll of the dissolved formazan
solution was transferred into a 96 well plate and measured
spectrophotometrically at 580 nm (Universal microplate
reader EL 800, Biotek Instruments). Triplicate measurements were performed at the same time-points as the
microscopic evaluation.
123
2.5.4 PrestoblueTM viability
Additionally, the PrestoblueTM assay (Invitrogen) was
applied to quantify cell viability and proliferation on the
PLA films. PrestoblueTM is a blue non-fluorescent, cell
permeable compound (resazurin-based solution), that is
reduced by living cells into a fluorescent compound (resorufin). The fluorescence intensity was performed on the
Wallac 1420 Viktor 3TM plate reader (PerkinElmer, Inc.) at
535 nm (excitation)/615 nm (emission). Triplicate measurements were performed at the same time-points as the
microscopic evaluation.
3 Results and discussion
3.1 Characterization of the discharge
A photograph of the discharge operating in dry air can be
seen in Fig. 2. Although the discharge looks uniform, the
DBD actually works in the filamentary mode. This mode
can be determined by measuring current–voltage waveforms, as shown in Fig. 3 for the discharge operating in
helium.
Similar figures have been found for the other discharge
gases and are therefore not shown in this paper. On the
current waveform, several peaks superimposed on the
capacitive current can be distinguished. These peaks indicate microdischarge activity and thus clearly show that the
discharge operates in the filamentary mode. The discharge
consists of numerous tiny discharge filaments called microdischarges, which are randomly distributed over the
dielectric surface and are of nanosecond duration. Despite
the non-uniform nature of the discharge, previous experiments have shown that the applied plasma treatment results
in a homogeneous surface modification [27].
To calculate the power during treatment, Lissajous figures were also recorded. An example of such a Lissajous
figure can be seen in Fig. 4 for the helium discharge and
similar figures have been obtained for the other discharge
gases. The discharge power during treatment, as calculated
from these Lissajous figures, together with the range of
Fig. 2 Photograph of the DBD operating in dry air

5. J Mater Sci: Mater Med
Fig. 3 Voltage current curve of the DBD discharge operating in
helium
treatment times used to treat the PLA surfaces and the
corresponding energy densities are shown in Table 1.
3.2 Contact angle measurements
Water contact angle measurements are a quick and
straightforward way to determine the wettability of the
PLA surface and Fig. 5 shows the results of these measurements as a function of energy density. Error bars are
not included in this figure for sake of clarity, however, as
mentioned in the experimental section, standard deviations
on these measurements vary between 0.5 and 2.5°. From
Fig. 5, it is clear that by applying a plasma treatment, the
PLA contact angle decreases, indicating an increased PLA
surface wettability. At a certain energy density, the contact
angle does not decrease any further suggesting a saturation
of the treatment effect. For PLA samples treated in dry air,
the lowest contact angle value (58°) is obtained at a very
low energy density (200 mJ/cm2), which corresponds to a
treatment time of approximately 4 s. For argon treatments,
a similar minimum contact angle value (57°) is obtained,
however, the energy density necessary to obtain this value
is higher (300 mJ/cm2). By a helium plasma treatment, a
Fig. 4 Lissajous figure of the DBD discharge operating in helium
slightly lower contact angle (50.5°) can be reached, however, a high energy density (550 mJ/cm2) should be applied
to obtain this contact angle. These differences between the
discharge gases could be explained by the chemical composition of the samples after plasma treatment. In the next
section, the chemical analysis of the samples will therefore
be discussed.
From literature, it is well-known that a plasma-modified
surface tends to recover to its original hydrophobic state
[30]. This so-called ageing phenomenon has also been
investigated in this work since the PLA samples need to be
subjected to a 4 day sterilization cycle before cell culture
tests can be started. Therefore, it is very important to know
how the surface properties will vary in the first few days
after plasma treatment. Within this context, plasma-treated
samples with maximum wettability were stored in ambient
air at room temperature, while the water contact angle
value was evaluated as a function of storage time, as shown
in Fig. 6. As shown in this figure, the treated PLA samples
undergo a hydrophobic recovery which could be attributed
to a reorientation of the implanted chemical groups towards
the bulk of the material to reach a more energetically
favorable position. Another effect which could cause this
ageing behavior is the reaction of plasma-treated surfaces
with atmospheric minorities (CO2, H2O), which could lead
to the neutralization of the polar groups responsible for the
increased wettability [31, 32]. Consequently, the contact
angle will increase after plasma treatment resulting in a
partial loss of the treatment effect.
During the first 4 days of ageing, the increase in contact
angle is the most pronounced, while after this first ageing
period the contact angle only slightly increases. However,
after 14 days of ageing, the contact angle values remain
well below the value of the untreated samples (72.5°)
indicating that at least a part of the treatment effect is
permanent. To quantify the differences in ageing between
the discharge gases, the loss in treatment efficiency L (%)
is calculated according to the following equation:
À
Á
L ¼ 100 Ã htreated À hageing = ðhtreated À huntreated Þ
where huntreated is the water contact angle of the untreated
PLA, htreated the saturated water contact angle after plasma
treatment and hageing the saturated water contact angle after
14 days of ageing. With this formula, one can find that
Ldry air = 52 %, Largon = 54 %, and Lhelium = 58 %, suggesting that the discharge gas does not significantly influence the ageing behavior of plasma-treated PLA samples.
Because of this ageing phenomenon and the fact that the
sterilization cycle prior to cell adhesion and proliferation
experiments lasts for 4 days, cell culture tests in this work
cannot be conducted on PLA samples exhibiting the
highest hydrophilicity. Nevertheless, the ageing experiments clearly show that even after 4 days of ageing, the
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6. J Mater Sci: Mater Med
Table 1 Experimental parameters for the DBDs sustained in dry air,
argon and helium
C
O
N
C–C/C–H
285 eV
C–O
287 eV
O–C=O
289.1 eV
No
75.0
25.0
0
57.0
23.0
20.0
65.7
34.3
0
36.6
32.0
31.2
65.6
34.3
0.1
37.1
32.0
30.9
Helium plasma
Discharge
power (W)
Treatment
Argon plasma
Treatment time
(s)
Energy density
(mJ/cm2)
Dry air plasma
Gas
Table 2 Surface atomic composition (%) and concentration (%) of
the different chemical bonds on untreated and plasma-treated PLA
samples. (air: treatment time = 4 s, energy density = 200 mJ/cm2;
argon: treatment time = 8 s, energy density = 300 mJ/cm2; helium:
treatment time = 20 s, energy density = 550 mJ/cm2)
65.2
34.6
0.2
37.0
32.1
30.9
Dry air
0–10
2.1
0–550
Argon
0–15
1.6
0–650
Helium
0–20
1.1
0–575
Fig. 5 Variation of the contact angle as a function of the energy
density for plasma-treated PLA samples in dry air, argon and helium
Fig. 6 Variation of the contact angle with ageing of PLA samples
after plasma treatment in dry air, argon and helium
PLA samples still have a considerably higher hydrophilicity than the untreated sample.
3.3 XPS measurements
To be able to explain the increased wettability of the PLA
surfaces after plasma treatment, XPS measurements were
performed. By using XPS, the chemical composition of the
PLA surface can be determined. In the framework of these
XPS measurements, PLA samples have been plasma-treated for a considerable time to ensure saturation of the
treatment effect. The obtained XPS results have been
123
summarized in Table 2 and Fig. 7. From Table 2, the
changes in chemical composition after plasma treatment
become evident. Upon plasma treatment, the oxygen content at the surface strongly increases: for treatments in dry
air, helium and argon, the oxygen content increases from
25 to 34 %. For argon and helium plasmas, also a very
small amount of nitrogen incorporation is detected (0.1 and
0.2 % respectively). This increased oxygen (and nitrogen)
content is responsible for the strong increase in wettability
of the PLA samples after plasma exposure. The incorporated oxygen and nitrogen during argon and helium plasma
treatment can be attributed to the presence of impurities in
the discharge chamber. In the used set-up it is very difficult, if not impossible, to complete remove the residual air
in the plasma chamber before filling it with the discharge
gas. Moreover, impurities in the discharge chamber can
also stem from working gas impurities or can be desorbed
from the reactor walls. Due to its high reactivity, mostly
oxygen will be incorporated in the PLA films by plasma
treatments in helium and argon. From Table 2, it can be
seen that there are no large differences in chemical composition between the air, helium and argon plasma-treated
samples although there is a significant difference in water
contact angle (see Fig. 5). This contradiction could be
attributed to a different surface roughness: in our previous
work, it has been found that air and argon plasma treatments result in PLA surfaces with a high roughness [23]
which can in turn lead to higher water contact angle values.
The surface roughness of helium plasma-treated samples is
lower, which could result in a lower water contact angle
value.
By curve-fitting the high-resolution C1s peaks, the
chemical bonds present on the surface of the untreated and
plasma-treated PLA samples can be determined. Figure 7
shows the C1s peaks of the PLA surfaces before and after
plasma treatment in dry air, argon and helium. The C1s
envelope of the untreated PLA can be decomposed into 3
components: a peak at 285.0 ± 0.1 eV corresponding to
C–C and C–H bonds, a peak at 287.0 ± 0.1 eV due to C–O
functional groups and a peak at 289.1 ± 0.1 eV which can

7. J Mater Sci: Mater Med
Fig. 7 High-resolution C1s
peaks of PLA before and after
plasma treatment in dry air,
argon and helium. (air:
treatment time = 4 s, energy
density = 200 mJ/cm2; argon:
treatment time = 8 s, energy
density = 300 mJ/cm2; helium:
treatment time = 20 s, energy
density = 550 mJ/cm2)
be attributed to O–C=O groups [33, 34]. As shown in Fig. 7,
following plasma treatment, considerable changes in the
C1s peaks can be seen. The peaks at 287.0 and 289.1 eV
increase relatively compared to the peak at 285.0 eV after
plasma treatment in dry air, argon and helium. For argon and
helium plasma treatments also nitrogen is incorporated and
the peak at 287.0 eV can therefore correspond to both C–O
and C–N functional groups [34].
Based on these deconvoluted C1s peaks, the concentrations of the different chemical bonds on the PLA samples can be obtained and the results are shown in Table 2.
The concentration of the C–C and C–H bonds clearly
decreases after plasma treatment, while the concentration
of C–O (C–N) and O–C=O groups increases. No significant
differences in PLA chemical bond concentration can be
observed between the plasma treatments in air, helium and
argon.
also obtained by the PrestoBlueTM assay, as shown in Fig. 8.
Both assays thus show an increased amount of viable
attached cells on the PLA plasma-treated films compared to
the pristine PLA, with no significant changes between the
different discharge gases.
After 7 days, the amount of viable cells as determined
by the MTT assay on the untreated samples is 53 %, while
on the plasma-treated samples it varies between 50.3 and
54.7 % relative to the control (100 %). Results obtained by
the PrestoBlueTM assay are higher and vary between 78.2
and 85.3 % relative to the control (100 %). Based on the
results after 1 and 7 days of cell seeding, one can thus
3.4 Cell culture tests
3.4.1 MTT and PrestoBlueTM analysis
Figure 8 illustrates the amount of viable cells adherent on
the PLA films as determined by MTT and PrestoBlueTM
assays after 1 and 7 days of incubation, relative to the data of
the tissue culture plate control on day 7. According to the
MTT assay, after 1 day, the amount of cells on the PLA films
without plasma treatment is 13.7 ± 1.3 %. This amount
increases to 18.2 ± 4.6, 21.8 ± 1.5 and 22.4 ± 2.0 %
respectively after plasma treatments in dry air, argon and
helium. At the same post-seeding time, the amount of viable
cells on the control is 40.4 ± 1.1 %. Similar results were
Fig. 8 Percentage of viable HFF cells cultured on PLA films (no
treatment, dry air, argon and helium plasma treatment) for 1 and
7 days relative to the control (tissue culture polystyrene) on day 7.
The amount of viable cells was quantified with the MTT assay and
Presto BlueTM (PB) assay. Error bars represent standard deviations of
triplicate measurements
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8. J Mater Sci: Mater Med
Fig. 9 Photographs of HFF
cells cultured on PLA films (no
treatment (a, b), dry air (c, d),
argon (e, f) and helium (g,
h) plasma treatment) for 1 (a, c,
e, g) and 7 (b, d, f, h) days in
comparison with the control (i,
j) (tissue culture polystyrene).
Phase-contrast micrographs and
fluorescent micrographs (inserts
of b, d, f, h, j) after live/dead
staining. Arrows are showing
well spread cells with an
elongated morphology.
Arrowheads are showing cells
with a round morphology. (scale
bar: 200 lm; scale bar inset:
100 lm)
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9. J Mater Sci: Mater Med
conclude that the applied plasma treatments improve the
initial cell attachment, but do not seem to affect cell
proliferation.
3.4.2 Phase contrast and fluorescent microscopy analysis
Adherent cells on the PLA films were examined by phase
contrast microscopy (Fig. 9) and fluorescent microscopy
after live/dead staining (Fig. 9b, d, f, h, j inserts). One day
after seeding, predominantly cells with a round morphology were observed on untreated PLA films (Fig. 9a
arrowhead). However, some cells with an elongated morphology were also observed (Fig. 9a arrow). Control cultures of HFF cells were well spread and have an elongated
morphology (Fig. 9i). Cells seeded on plasma-treated PLA
films (Fig. 9c, e, g) were also well spread out with an
elongated morphology (arrows) and only a limited number
of cells with a round morphology (arrowhead) could be
observed. Independent of the observed cell morphology,
cells were viable as detected with live/dead staining
(photographs not shown).
After 7 days of culture on the PLA films, independent of
the type of plasma modification, all cells were well spread
with an elongated morphology (Fig. 9d, f, h) similar to the
control (Fig. 9j). However, well spread cells with an
elongated morphology were also observed on the untreated
PLA film (Fig. 9b). All cells were viable as detected with
live/dead staining (Fig. 9b, d, f, h, j inserts). A confluent
monolayer of cells could be observed in the centre of the
PLA films. Control cultures were confluent throughout the
whole tissue culture dish. This explains the quantitative
difference in the MTT and PrestoBlueTM assay of PLA
films (MTT assay: 50.3–54.7 %; PrestoBlueTM assay:
78.2–85.3 %) in comparison with the control (100 %).
Evaluation of the cultures by phase contrast and fluorescent
microscopy after live/dead staining thus clearly supports
the MTT and PrestoBlueTM data.
4 Conclusion
This paper describes the effect of a medium pressure DBD
plasma operating in different atmospheres (dry air, argon
and helium) on the surface of PLA samples. It was shown
that the plasma source was capable of reducing the water
contact angle and thus increasing the wettability. Although
the samples are subjected to ageing, the contact angle after
14 days of ageing stays well below the value of the
untreated samples, indicating that at least a part of the
treatment effect is permanent. From XPS, it was found that
plasma treatments in air, helium and argon result in surfaces with a similar chemical composition: mainly oxygencontaining functionalities are incorporated after plasma
treatment. Cell culture tests showed both quantitatively as
well as qualitatively that plasma modification of PLA films
increased the initial cell attachment. After 1 day, cells on
plasma-treated PLA showed a superior cell morphology in
comparison with unmodified PLA samples. After 7 days of
culture, this superior cell morphology is however not
observed, suggesting that plasma treatment does not affect
cell proliferation. From cell culture tests, it was also found
that the differences between the discharge gases are negligible when looking at the improved cell-material interactions. This observation can be attributed to the similar
chemical composition of the PLA surfaces plasma-treated
in air, argon and helium. Nevertheless, from economical
point of view, treatments in air are the best choice.
Acknowledgments R. Morent acknowledges the support of the
Research Foundation Flanders (FWO) for a post-doctoral research
fellowship.
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