Increasing cell viability of 3D scaffolds for tissue engineering by means of an
atmospheric pressure plasma jet
V. Colombo1,2, P. Favia3,4, M. Gherardi1, R. Gristina4, F. Intranuovo3,
R. Laurita1, A. Liguori1, A. Stancampiano1
Alma Mater Studiorum - Università di Bologna
1
Department of industrial engineering (DIN)
2
Industrial Research Center for Advanced Mechanics and Materials (C.I.R.I.-M.A.M.)
Via Saragozza 8, 40123 Bologna, Italy.
Università degli Studi di Bari
3
Deptartment of Chemistry,
4
Institute for Inorganic Methodologies and Plasmas (IMIP-CNR)
Via Orabona 3, Bari, Italy
Abstract: In this work a nanosecond pulsed dual gas plasma jet has been used to
functionalize 3D porous poly (Ɛ -caprolactone) scaffolds. The effect of the plasma treatment
has been evaluated both with a chemical analysis, by means of an X-Ray Photoelectron
Spectroscopy, and a cell viability and morphological assay, evaluating top surface and section
modification.
Keywords: non-thermal atmospheric plasma, jet, 3D porous PCL scaffold, XPS,
functionalization, cells
1. Introduction
Three-dimensional porous biodegradable scaffolds act
as temporary backbone for the regeneration or repair of a
living tissue in most of tissue engineering applications [1].
The optimization of surface properties of scaffolds is a
critical aspect, as they influence the interactions between
the cells and the material. By modifying both chemical
surface properties and morphological parameters of the
scaffolds, a better control over cell adhesion mechanism
can be achieved. Both low pressure [2] and atmospheric
[3] plasma processes represent a very powerful tool to
modify surface chemistry of materials without influencing
their bulk properties. In this contribution the positive
effect of atmospheric plasma jet, recently developed by
some of the the Authors [4], modification of 3D
poly(Ɛ -caprolactone) (PLC) porous scaffolds on cell
colonization will be shown.
1.
Materials and Methods
Dual gas nanosecond pulsed plasma jet
A single electrode dual gas plasma jet driven by an high
voltage nanopulsed power supply (peak voltage (PV)
between 7 and 20 kV, repetition rate (RR) between 50 and
1000 Hz, a pulse duration about 40 ns and rise time 3 ns)
has been used as plasma source for the treatment of the
3D PCL porous scaffolds. Each 3D porous scaffold has
been treated in three different points of the top surface
with the following operative conditions: PV= 20 kV, RR
1000Hz. Argon or nitrogen have been used as primary gas
(flow rate 3 slpm). No secondary gas has been used.
Synthesis of scaffolds
3D PCL (Mw ~ 65,000, pellets, Sigma Aldrich) porous
scaffolds were produced with a very simple and
inexpensive technique, the solvent-Casting/ ParticulateLeaching (SCPL) [5,6]. This method consists of the initial
dissolution of PCL pellets into chloroform (20/80 wt/wt,
Fluka) by stirring for about 3h at room temperature (RT).
Then, sodium chloride crystals (NaCl, Sigma Ultra),
employed as porogen, are sieved to a specific size range
of 300-500μm and then homogeneously mixed to the
polymer solution at a 5/95 ratio, in order to create pores
with specific size into the scaffold. The viscous polymer
solution is then cast in Teflon moulds (10mm diameter,
2mm thickness) and dipped in ethanol (Carlo Erba
Reagents) for about 2h at RT to separate the chloroform in
a phase inversion process. The polymer/salt composite is
then leached in bidistilled water for 5 days to remove the
porogens and create the voids into the scaffold. With this
technique, the scaffold porosity could be changed in a
controlled way by varying the amount of salt added, while
the pore size could be controlled controlling the size of
the salt crystals. The resulting porous scaffolds were dried
into an oven for 3 minutes and stored in a desiccator up to
plasma modifications and/or characterizations.
X-Ray Photoelectron Spectroscopy
X-Ray Photoelectron Spectroscopy (XPS) analyses of
untreated and plasma-treated 3D PCL scaffolds were
carried out using a Theta Probe Thermo VG instrument
(2*10-9 mbar base pressure), equipped with a
monochromatic Al Ka X-ray source (hm = 1486.6eV) and
100W set power. Photoelectrons were collected at a 53°
take-off angle, corresponding to a sampling depth of
about 10nm. All spectra were recorded using a 300µm
aperture slot diameter, with a pass energy of 150eV for
survey scans and 100eV for high-resolution core level
scans. The electrostatic charging of samples was
neutralized by means of a flood gun (model 822-06 FG
400 lA emission current, 40V extraction voltage at
pressure 2 orders of magnitude higher than the base
pressure). All spectra were recorded and processed using
the Thermo Avantage 3.28 software (Thermo Electron
Corporation). Binding energies were charge corrected by
setting the C1s aliphatic carbon signal at 285.0eV [7]. The
C1s peak fitting of PCL scaffolds was performed using
four components at (1) 285.0, (2) 285.6, (3) 286.5 and (4)
289.1eV, considering the chemical structure of PCL. After
plasma modification, two further components were added
to the C1s peak fitting: at 288.0 (O-C-O/C=O, C5) and
290.4eV (O-C(=O)-O, C6). Fitted components data are
presented as mean atomic percentages (n=3). In order to
gain better insight into the penetration depth of the plasma
processing, sections of the scaffolds (parallel to the XZ
plane) were cut with a scalpel blade and analyzed with
XPS.
Cell culture
Cell culture experiments on untreated and plasma
modified PCL scaffolds were performed with the human
Saos-2 osteoblast cell line (ICLC, Italy). Cells were
grown in Dulbecco’s Modified Eagle Medium (DMEM,
Sigma, Italy), supplemented with 10% heat-inactivated
fetal bovine serum (FBS), 50 IU/ml penicillin, 50 IU/ml
streptomycin and 200 mM glutamine and kept at 37°C in
a saturated humid atmosphere containing 95% air and 5%
CO2. Cells were detached with a trypsin/EDTA solution
(Sigma, Italy) and suspended in the correct medium.
Before cell seeding, the scaffolds were pre-wet into cell
medium and evacuated in a vacuum machine, in order to
eliminate air bubbles inside the scaffolds and make all
their interconnected pores available for the transit of cells
and nutrients. The scaffolds were then placed in 48 wells
plates and seeded with 5•104 Saos-2 osteoblast cells.
Cell viability assay.
The mitochondrial activity of Saos-2 cells was
determined with the MTT colorimetric assay. This test can
detect the conversion of 3-(4,5-dimethylthiazolyl-2)
-2,5-diphenyltetrazolium bromide (Sigma, USA) to
formazan. The cell growth was stopped at 15, 39 and 87h.
After each time point, the cells were incubated with a
tenth of the medium of the bromide in 5% CO 2 (37°C, 2
h) to allow the formation of water insoluble formazan
crystals. This product was then dissolved in 10% Triton
X-100 and acidic isopropanol (HCl 0.1 N). The optical
densities (O.D.) of the solutions were read with a
spectrophotometer (Jenway 6505), at the wavelength of
570 nm with respect to the reference wavelength of 690
nm. Data (n = 3) were presented as means of O.D. values
+ S.D.
Cell morphology assay
To observe cellular morphology, cells were fixed, after
15, 39 and 87h, in 4% formaldehyde/PBS, at RT for 20
min, permealized with PBS containing 0.1% Triton X-100
and incubated with Alexa Fluor®488 phalloidin (green
fluorescence, Molecular Probes) at RT and for 30 min.
After rinsing with PBS actin filaments were observed
with an epifluorescence microscope (Axiomat, Zeiss,
Germany).
3. Results
XPS results
The chemical composition of untreated and plasma
treated scaffolds (top and section surfaces) were evaluated
by XPS. The legend of chemical group and binding energies
are reported in Table 1. Before plasma modification, fitted
C1s components of PCL surface were very similar to the
PCL composition reported in the literature, as shown in
Table 2.
After every plasma process, the content of total Oxygen
is slightly higher than untreated PCL scaffolds, confirmed
by the O/C ratios reported in Table 2 and 3. Nonetheless,
the shape of the C1s peak changed after plasma
modifications, observing variations of components
content.
In particular, after argon plasma jet treatment, on both
top and section surfaces, two new peaks appeared, C5 at
288.0 eV (O-C-O/C=O) and C6 at 290.4 eV (O-C(=O)-O)
(Fig. 1). This chemical change could be ascribable to
oxygen functionalities implanted during plasma treatment.
On the contrary, after nitrogen plasma jet treatment the
contributions of C5 and C6 to the C1s XPS spectrum
were negligible, both on top and in the scaffold section,
with a similarity to the pristine PCL scaffold, as reported
in Fig. 2. Thus, these plasma treatments did not make
decisive change in the chemical composition of PCL
scaffolds.
Chemical group
Binding Energy [eV]
C1
C-H/ C-C
285±0.2
C2
C-C-C=O
285.6±0.2
C3
C-C2-C=O
286.5±0.2
C4
C(O)=O
289.1±0.2
C5
O-C-O/C=O
288±0.2
C6
O-C(=O)-O
286±0.2
Table 1: Legend of chemical group and binding energies
gas , (A)1 minute per point, (B) 2 minutes per point.
Literature
CNT
Top surface
Section
Data
C
[at%]
70
77±1
74±1
76±2
O
[at%]
30
23±2
26±1
24±1
C1
[at%]
52
47±3
35±5
33±3
C2
[at%]
17
19±2
9±2
15±2
C3
[at%]
17
18±1
28±3
26±2
C4
[at%]
14
16±1
10±0.4
7±1
C5
[at%]
/
/
11±2
12±1
C6
[at%]
/
/
7±0.5
7±1
O/C
0.43
0.35 ±0.01
0.32±0.01
0.30±0.01
Figure 1: High resolution C1s spectrum of top scaffold surface
after the Argon plasma jet treatment.
Table 2. XPS chemical composition (atomic percent, at%) for
untreated (CNT) and Argon plasma treated PCL scaffold top and
section surfaces. Plasma treatment: Jet with Ar as primary gas ,
1 minute per point
CNT
Top
Section A
Surface A
Top
Section B
Figure 2 High resolution C1s spectrum of top scaffold surface
after the Nitrogen plasma jet treatment (A).
Quantitative and qualitative biological characterization
In the light of the results obtained by XPS, the adhesion
and colonization behavior of the human Saos-2
osteoblasts cell line were studied on argon plasma
modified PCL scaffolds and compared to the untreated
ones. The biological study was performed in terms of cell
proliferation, morphology and distribution at three culture
times (15, 39 and 87h).
MTT results at 15, 39 and 87h clearly show a better
viability of cells on modified scaffolds (Fig. 3).
In fact at each time point it can be observed a higher
absorbance value for cells grown on modified scaffolds
with respect to the ones grown on native PCL scaffolds.
Because the MTT assay evaluates the metabolic
activity of the cells, not giving information about the
variation of their number or shape, optical microscopy
analysis has also been performed, after fixing and staining
cells actin cytoskeleton with phalloidin conjugated with
an Alexafluor 488 chromophore. Morphological analyses
were performed with the aim of analyzing the influence of
plasma treatment in terms of cell spreading and
interaction with the surroundings, cell shape, size and
clustering phenomena. Fluorescence images of untreated
Surface B
C
[at%]
77±1
76±1
76±1
74±1
76.0±0.2
O
[at%]
23±2
24±1
24±1
26±1
24.0±0.2
C1
[at%]
47±3
53±3
54±2
51±2
52±3
C2
[at%]
19±2
12±2
12±2
11±2
12±3
C3
[at%]
18±1
16±1
16.1±0.1
16±3
16.0±0.1
C4
[at%]
16±1
15±1
15.0±0.1
17±2
16±1
C5
[at%]
/
3±1
2.1±0.1
4.0±0.5
2.6±0.7
C6
[at%]
/
1±0.2
0.8±0.1
1.0±0.2
1.4±0.1
0.32
0.31
0.35
0.3
±0.01
±0.01
±0.02
1±0.01
O/C
0.30±0.01
Table 3. XPS chemical composition (atomic percent, at%) for
untreated (CNT) and nitrogen plasma treated PCL scaffold top
and section surfaces. Plasma Treatment: Jet with N2 as primary
and plasma treated scaffolds seeded with Saos-2cells and
cultured for 120h, are illustrated in Figure 4 and enable
the observation of cell actin cytoskeleton in green.
Figure : Metabolic activity of Saos2 cells on control and argon
plasma jet treated scaffolds.
modified PCL scaffolds appeared well spread, clustered
and polygonal-shaped with the presence of actin stress
fibers in the cell cytoskeleton (Figure 4d). The increased
cell-cell interactions present in the surface of treated
scaffolds suggest a positive effect of plasma treatment on
the attachment of Saos-2 cells. Both MTT and
morphological data clearly evidenced improved cell
activity on plasma treated scaffolds.
4. Conclusions
A dual gas nanopulsed plasma jet has been used to treat
both surface and internal sections of 3D PCL porous
scaffolds. Negligible results have been found if nitrogen
gas has been used, while argon plasma jet treatment can
modify the chemical structure of the scaffold: XPS results
highlights that after treatment, C5 and C6 groups are
created in the scaffold, as a result of the implant of
oxygen functionalities. This functionalization has been
found both in the top and section surfaces of the 3D
scaffold and, suggesting the uniform treatment of the
scaffold surface but also internal pores. Metabolic activity
and fluorescence microscopy images of Saos-2 cells
cultured on plasma modified scaffold evidence the
improvement of cell activity compared to the control PCL
scaffold.
References
Figure 4: Fluorescence microscopy images of Saos-2 cells
cultured for 39h, on untreated (a,b) and Argon plasma jet
modified (c,d) PCL scaffolds at low (a,c) and high
magnifications (b,d). The cells were fixed in 4%
formaldehyde/PBS solution and incubated with Alexa
Fluor®488 phalloidin, allowing the observation of the cell actin
in green.
Low magnification images evidence that the chemical
modifications of the scaffolds interfere with the green
signal resulting in a high green background. This effect
cannot allow us to observe a change in cell number or
distribution between control (Figure 4a) and treated
(Figure 4c) scaffolds. At higher magnification, significant
differences in cell morphology can be easily detected
between Saos-2 cells adhering onto untreated and plasma
treated scaffolds. In the first case (untreated scaffolds),
the cells appeared isolated, with circle-shaped small body
(Figure 4b), hence suggesting a low cell affinity with the
substrate. Contrarily, Saos-2 cells seeded onto plasma
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