Atmospheric non-thermal plasma surface modification of poly(L-lactic acid)
electrospun nanofibers affects scaffold hydrophilicity and fibroblast
morphological response
L. S. Dolci1, S. D. Quiroga1, M. Gherardi2, R. Laurita2, A. Liguori2, E Ghedini2,3, P. Sanibondi3, A. Fiorani4, L. Calzà1,
V. Colombo2,3, M.L Focarete1,4
Alma Mater Studiorum – Università di Bologna
Health Sciences and Technologies - Interdepartmental Center for Industrial Research (HST-ICIR), Via Tolara di Sopra
50, 40064 - Ozzano dell'Emilia, Italy
2
Department of Industrial Engineering (DIN), Via Saragozza 8, 40123 Bologna, Italy
3
Advanced Mechanics and Materials - Interdepartmental Center for Industrial Research (AMM-ICIR) , Via Saragozza 8,
40123 Bologna, Italy
4
Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, RU
Bologna), Via Selmi 2, 40126 Bologna, Italy
1
Abstract: In the present work atmospheric pressure non-thermal plasma surface modification
of poly(L-lactic acid) electrospun scaffold for biomedical applications is investigated. Plasma
treatment improves scaffold hydrophilicy and increases the concentration of carboxyl groups
(–COOH) on the surface of the scaffold. The morphology of cells cultured on pristine and
plasma treated electrospun scaffolds is compared with that of cells cultured on cultrex
substrates.
Keywords: Electrospun scaffold, non-thermal plasma, hydrophilicity, functionalization, cells
1. Introduction
Surface
modification
of
artificial
polymeric
biomaterials has been extensively studied as an effective
strategy
to
improve
material
biocompatibility,
biointegration and to promote cell adhesion, proliferation
and differentiation, without altering material bulk
properties [1, 2]. An ideal biomaterial should display
proper surface properties to promote biointegration, in
terms
of
chemical
composition,
biochemical
functionalization and surface charge.
Electrospun
nanofibrous
scaffolds
made
of
poly(L-lactic acid) (PLLA) have been widely studied as
artificial scaffolds to mimic the fibrillar arrangement of
the extracellular matrix (ECM) both from a
morphological and a mechanical point of view. Given the
possibility to easily modulate the structure of the scaffolds
during the electrospinning fabrication processes [3],
electrospun PLLA scaffolds have been proven successful
as tissue-engineering implants [4], wound dressings [5],
in neural tissue engineering [3], peripheral nerve studies
[6] and cardiac repair [7]. PLLA, and polyesters in
general, are hydrophobic materials. Hydrophobicity is
often responsible for some undesired effects related to
practical use of biomaterials, such as unspecific protein
adsorption, adhesion of bacterial agents and reduced
water wetting. Among the various techniques available to
increase hydrophilicity, plasma treatment is a solvent-free
process that can provide a uniform physical and chemical
modification of the scaffold surface without altering its
bulk properties. This versatile process has been already
proven viable for introducing specific functional groups
on scaffold surface to increase cell viability and
proliferation [8, 9], increasing adhesion of biomolecules
[10] and enhancing cellular acceptance through
subsequent functionalization of the scaffold surface [11].
In this study, we investigate plasma surface
modification of PLLA scaffolds fabricated by
electrospinning technique using a linear corona discharge
(LC); the LC source is driven by a nanosecond pulsed
high-voltage generator and can be operated with various
gases (e.g. He, Ar, Air, N2).
In order to increase the treatment area with respect to
the typical pin-to-plate configuration of corona discharges
[12], in the LC plasma source a sharp blade is used as the
high-voltage electrode. The effect of linear corona (LC)
plasma-treatment on electrospun scaffolds was evaluated
through
morphological
and
thermo-mechanical
characterization, as well as by measuring the changes of
the static water contact angle on the scaffold and the
water absorbance percentage after scaffold exposure to
plasma source.
In order to estimate the relative amount of –COOH
functional groups created during the treatment with
atmospheric plasma, a functionalization with fluorescein
isothiocyanate (FITC) was performed. In view of
biomedical applications, the effect of LC plasma
treatment was evaluated using mouse embryonic
fibroblast (MEF) cells cultured on plasma treated and
untreated PLLA scaffolds.
2. Materials and Methods
Scaffold fabrication.
The electrospinning apparatus, made in house, is
composed of a high voltage power supply (Spellman,
SL 50 P 10/CE/230), a syringe pump (KD Scientific 200
series), a glass syringe, a stainless-steel blunt-ended
needle connected with the power supply electrode (inner
diameter: 0.84 mm), and a rotating cylindrical collector
(50 mm diameter; 120 mm length).
PLLA was dissolved in a mixed solvent,
DCM:DMF=65:35 v/v at a concentration of 13% w/v. The
polymer solution was dispensed, through a Teflon tube, to
the needle. PLLA scaffolds were fabricated using the
following conditions: applied voltage = 18 kV, needle to
collector distance = 15 cm, collector rotational speed = 40
rpm, solution flow rate = 0.015 ml/min, at room
temperature (RT) and relative humidity RH = 40-50%.
Electrospun mats (30÷60 μm thick) were kept under
vacuum over P2O5 at RT overnight in order to remove
residual solvents.
Plasma Treatment.
Plasma treatment of PLLA scaffolds was carried out by
means of a LC plasma source mounted on a shaft moved
at a controlled speed by a motorized linear stage. As
shown in Fig. 1, LC plasma source is composed of a
housing made of dielectric material and a sharp stainless
steel blade, 36 mm wide and 0.1 mm thick, as the high
voltage electrode; the housing is provided of a gas inlet
and a compensation chamber and two parallel gas
channels. LC plasma source is driven by a nano-second
pulsed generator having peak voltage (PV) 7-20 kV into a
100-200 Ohm load, pulse repetition frequency (PRF)
83-1050 Hz, pulse width 12 ns and rise time 3 ns. Plasma
treatment was performed at PV 20 kV, PRF 1 kHz; N2
was employed as plasma gas with a 5 slpm flow rate,
plasma treatment time was 20 s. After plasma-treatment
all scaffolds were kept at +4°C in air.
Fig. 1 3D drawings of the LC plasma source: rendering
(left) and section (right)
Scaffold characterization.
Scanning Electron Microscope (SEM) observations were
carried out using a Philips 515 SEM at an accelerating
voltage of 15 kV. The distribution of fiber diameters was
determined through the measurement of about 250 fibers
by means of an acquisition and image analysis software
(EDAX Genesis).
Thermogravimetric analysis (TGA) measurements were
performed with a TA Instruments TGA2950
thermogravimetric analyzer from RT to 600 °C.
Differential
Scanning
Calorimetry
(DSC)
measurements were carried out using a TAInstruments
Q100 DSC equipped with the Liquid Nitrogen Cooling
System (LNCS) accessory.
Stress-strain measurements were carried out with an
Instron 4465 tensile testing machine on rectangular sheets
cut from electrospun mats.
Static water contact angle measurements were
performed under ambient conditions by using an optical
contact angle and surface tension meter KSV’s CAM 100
(KSV, Espoo, Finland). Milli-Q water was used for the
measurements. Drop profile images were collected in a
time range of 0-90 s, every 1 s.
Water absorption was calculated by weighing the
scaffold before and after soaking in distilled water for 24
h at RT.
Surface conjugation and fluorescence characterization.
PLLA
scaffolds
first
was
activated
with
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
and N-Hydroxysuccinimide (sNHS); activated scaffolds
were then incubated with 1,4-diaminobutane, conjugated
with FITC 10-3M in carbonate buffer for 2 hours a RT
and finally shaked and rinsed four times for 5 minutes
each. Untreated (PLLA) and plasma-treated PLLA
(PLLA-LC) FITC conjugate were embedded with
glycerol 0.1% 1,4-phenylendiamine and stored at -20°C
until use. Control samples to evaluate the unspecific
binding of FITC were prepared removing EDC and sNHS
from the conjugation process. FITC conjugated PLLA
and PLLA-LC scaffolds were observed by NIKON
eclipse E600 (Nikon, Italy) equipped with digital CCD
camera Q imaging Retiga 20002V (Q Imaging, Surry, BC,
Canada). The images were analyzed using Imaging NIS
Elements software.
Aging.
The PLLA-LC scaffolds were FITC conjugated at
different times after plasma treatment (0, 6, 24, 48, 72 and
144 hours).
Cell culture on scaffold.
PLLA and PLLA-LC scaffolds were cut and assembled,
by means of CellCrown™ supports (Scaffdex, Tampere,
Finland), in a 24-well and they were sterilized by 15 min
incubation in EtOH 90%, then in EtOH 70% for 30 min.
Mouse embryonic fibroblasts (MEF) were cultured in
Dulbecco’s Modified Eagle Medium (DMEM-F12) in a
humidified atmosphere at 37°C and 5% CO2. MEF were
seeded on different surfaces at a density of 7x 10-4
cells/well.
Immunocytochemistry.
Fibroblasts cultured for 24, 48 and 72 hours were fixed
in 4% PFA in 0.1M phosphate buffer for 20 min at RT,
washed with PBS (2x10 minutes) and incubated with
blocking buffer for 1 hour at RT. Subsequently cells were
incubated with anti Actin primary antibodies (1:200) in
PBS 0.3% triton overnight at 4°C. Cells were rinsed with
PBS and incubated with DyLight488-labeled secondary
antibodies (1:1000) 30 minutes at 37°C followed by
rinses with PBS (2x10 minutes). Finally cells were
incubated with 1 ug/mL Hoechst 33258, 0.2%
Triton-X100 for 20 minutes at RT and were washed two
times with PBS. Finally cells were mounted with glycerol
0.1% 1,4-phenylendiamine. Negative controls were
performed by primary antibody absence. Cell
visualization was performed by NIKON eclipse E600
(Nikon, Italy) equipped with digital CCD camera Q
imaging Retiga 20002V (Q Imaging, Surry, BC, Canada).
3. Results
Morphological structure of PLLA and PLLA-LC
electrospun scaffolds was observed by SEM imaging
technique. Fig. 2 shows both samples at two different
magnifications (1000x, scale bar = 10 µm; 8000x, scale
bar = 1 µm). It was observed a random distribution of
PLLA electrospun scaffolds with uniform sub-micrometer
fibers presenting average fiber diameters of 820 ± 200 nm
and 730 ± 200 nm on bare and on plasma-treated PLLA
scaffolds, respectively. No evidence of morphological
modification, in terms of fiber morphology and fiber
diameter, occurred during or after exposure to the LC
plasma source was observed.
Fig. 2 SEM micrographs of pristine (a) and linear corona
plasma-treated (b) electrospun PLLA scaffolds
TGA analysis demonstrated the absence of residual
solvent after electrospinning fabrication process.
Mechanical properties characterization showed that LC
plasma-treatment reduced around 25-30 % Young’s
modulus and stress at break with respect to pristine PLLA
scaffolds. On the other hand, deformation at break was
proportionally increased after exposure to plasma,
resulting in a loss of rigidity.
WCA measurements resulted unsuitable for electrospun
mats due to the very high void percentage of these
materials. Measurements on pristine samples revealed
high WCA values (>120°); similar values were obtained
when the drop was placed on treated scaffolds, but the
WCA decreased progressively as the drop was absorbed
by the substrate. To carefully measure hydrophilicity of
scaffolds water absorption measurements were adopted.
Measurements to evaluate water adsorption capacity of
PLLA scaffold demonstrated that plasma-treatment
increases of two orders of magnitude the ability to retain
water into the sub-micrometer fibers on PLLA
electrospun scaffolds. In addition, this capacity was kept
for at least 120h, as reported in Fig. 3. The averaged
percentages of water adsorption for the entire period of
study were 7.8 ± 7.4 % and 368.0 ± 33.6 % for bare and
treated PLLA scaffolds, respectively.
At the end of this experiment all scaffolds were kept in
a desiccator at RT overnight, then the samples were
weighted and their thickness was measured again.
Scaffolds were recovered their initial thickness and no
mass loss was evidenced.
Fig. 3 Percentage of water adsorbance measured on bare
and treated PLLA scaffolds during a time-range of 120 h
after plasma-treatment
The introduction of carboxyl groups on PLLA scaffolds
after LC plasma treatment was characterized by
fluorescence analysis, after the chemical conjugation with
FITC molecules. The signal to background ratio of
plasma treated PLLA scaffolds increased of more than
five times compared to untreated scaffolds, while no
relevant value of fluorescence intensity was registered for
control scaffolds (unspecific binding of FITC), as
highlighted in Fig. 4A. In order to determine the aging of
plasma modification, FITC conjugation and fluorescence
analysis were performed at different times after plasma
treatment; Fig. 4B shows that the intensity of fluorescence
remains constant for at least 6 days after plasma
treatment.
A
Cultrex
B
PLLA-LC
Fig. 4 Mean intensity of COOH-FITC binding (A); Mean
intensity of COOH-FITC binding in the time (B)
PLLA
In this study, MEF cells were cultured on untreated
PLLA and treated PLLA-LC surface to investigate the cell
morphology modification induced by plasma treatment
and compared with results for cells cultured on.cultrex
substrates, chosen as control. As shown in Fig. 5,
fibroblasts cell body seeded on cultrex showed the
classical round and spread shape; conversely, fibroblasts
on PLLA were smaller and star-like, while those grown
on PLLA-LC resulted very elongated.
4. Conclusions
Functionalization and biocompatibilization of PLLA
electrospun scaffolds can be achieved using low
temperature atmospheric pressure plasmas. The treatment
with linear corona modified surface properties of
electrospun PLLA scaffolds without alterating their
morphological characteristics. A notable increase of
scaffold wettability after plasma treatment was observed
and measured as an increase of water adsorbance capacity.
Furthermore, a relevant increase of –COOH
concentrations was registered after plasma treatment.
Fig. 5 Fibroblasts on Cultrex, PLLA-LC and PLLA
substrates
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