Controlled Tuning of Cell Ingrowth Inside Polycaprolactone Scaffolds by
C2H4/N2 Plasma Deposition
F. Intranuovo1, E. Sardella2, R. Gristina2, M. Nardulli1, R. d’Agostino1,2,3, P. Favia1,2,3
1
2
Department of Chemistry, University of Bari, Italy
Institute of Inorganic Methodologies and Plasma (IMIP) CNR, Italy
3
Plasma Solution Srl, Spin off of the University of Bari
[email protected]
Abstract: Poly ε-caprolactone (PCL) scaffolds with controlled pore dimensions
(100-200 µm) were processed in low pressure RF (13.56 MHz) Glow Discharges.
C2H4/N2 mixtures were used to plasma deposit cell-adhesive coatings with
controlled conditions to increase the affinity of the Saos2 osteoblast cell line.
Very promising results were obtained in terms of N-based species penetration, by
plasma processes, inside the core regions of scaffolds, that increased the
metabolic activity of cells grown on modified scaffolds, respect to untreated
ones.
Keywords: plasma deposition, tissue engineering, cell adhesion, scaffold.
1. Introduction
Tissue engineering strategies are based on the use of
three-dimensional (3D) porous polymer scaffolds,
homogeneously seeded with proper cells, according
to the living tissue or organ to replace or repair. To
accomplish this task, both scaffolding and seeding
techniques are decisive. A number of fabrication
technologies have been applied to process
biodegradable materials into 3D polymeric scaffolds
of high porosity, proper pore size, shape, surface
area, stiffness and mechanical integrity [1]. Several
methods have been used to improve cell seeding,
bioreactor cultivation and conditioning, in order to
guide the ingrowth of cells into the scaffolds [2].
However, 3D matrices provide physical and
chemical cues that could be strengthened and
exploited to create cell-binding sites to support and
guide cell attachment and spreading. The main
drawback with 3D scaffolds is that cell adhesion is
often favored at the scaffold peripheries, because
more accessible respect to the core, resulting poor
cell populated. By controlling the chemical stimuli
and surface properties of PD,LLA scaffolds, with
proper allyl amine plasma depositions, improvement
of cell colonization was achieved [3]. Although
plasma deposition processes in radiofrequency (RF,
13.56 MHz) glow discharges have been widely
exploited for the modification of 2D surfaces [4],
when applying to 3D scaffolds, the challenge is the
improvement of plasma depositing species ability to
penetrate into the scaffold pores, in order to
uniformly coat the 3D structures. This could support
the cell ingress and homogeneous colonization, from
the top to the bottom of the scaffolds.
With this aim, this study was focused on the control
of the chemical composition of poly(ε-caprolactone)
(PCL) scaffolds, fabricated with the Solvent
Casting/Particulate Leaching technique, by means of
low pressure plasma deposition processes. In order
to improve the cell colonization of the internal
scaffold surfaces, the hydrophobic PCL polymer
composition was enriched with nitrogen containing
coatings, with plasma deposition from a C2H4/N2
mixture feed. Post treatments with H2 plasmas were
aimed to the reduction of nitrogen containing groups
to NH2 functionalities.
2. Materials and Methods
2.1 Solvent Casting/Particulate Leaching and
scCO2 processes
PCL (Mw 65kDa) scaffolds were produced by
Solvent Casting/Particulate Leaching, dissolving
PCL in CHCl3 (20/80 wt/wt), with NaCl crystals
(150-300 µm size) as porogen (5/95 wt/wt
PCL/NaCl). Teflon moulds were used to cast the
polymer solution. The scaffolds were then dipped in
ethanol to extract the solvent. The leaching process
was performed in distilled H2O.
2.2 Surface Modification by Plasma Deposition
PCL scaffolds were modified in a stainless steel
parallel-plate RF (13.56 MHz) plasma reactor. A
first 30 min deposition was performed in a C2H4/N2
mixture (1:3), at 40 Pa and 50 W in continuous
mode, obtaining a nitrogen-doped plasma
polymerized ethylene (PPE:N) film, whose thickness
on flat PCL substrates was 500 nm. Then the
scaffolds were plasma treated in H2 (53 Pa, 10 sccm,
10 W, 30 s).
reference). Statistical analyses were performed by a
paired Student’s t-test (p<0.01).
3. Results and Discussion
In this study, PCL scaffolds (10 mm diameter, 4 mm
depth) were modified by means of a PPE:N/H2
plasma process. This strategy allowed to create a
nitrogen-rich coating inside the scaffolds, while the
periphery was characterized by a different surface
chemical composition, e.g. rich of –NH2 groups
(short H2 plasma treatment time) or rich of
hydrocarbon moieties (long H2 plasma treatment
time) [6]. For this work, the exposure time of PCL
scaffolds to the two plasma processes was not
varied. For PPE:N depositions on scaffolds, different
plasma parameters (pressure, power, time, feed ratio,
etc.) were varied (data not shown) and only one
condition has been applied in this work. XPS
attested that before being plasma modified, PCL
scaffolds had a chemical composition typical of PCL
in literature (Table I and Fig. 1A), that was kept
constant in all the scaffolds thickness (data not
shown).
2.3 Chemical analysis (X-Ray Photoelectron
Spectroscopy)
X-ray Photoelectron Spectroscopy (XPS) analysis
was performed on PCL scaffolds by a Theta Probe
Thermo VG instrument (monochromatic Al Kα Xray source; 1486.6eV; take-off angle 53°). Binding
energies were charge corrected by setting the C1s
aliphatic carbon signal at 285.0eV [5]. The C1s peak
fitting for PCL samples was performed using the
four components at 285.0 (1), 285.6 (2), 286.5 (3)
and 289.1eV (4), considering the chemical formula
of PCL. After plasma modification, two further
components were added, at 286.0 (C-N/C-O) and
288.0eV (N-C=O). The nitrogen content was used to
identify the presence of the plasma coatings.
2.4 In vitro cell culture on PCL scaffolds
PCL scaffolds were seeded with human Saos2
osteoblasts (3x104 cells per scaffold) at three cell
culture times (18, 48 and 96 h); the cell
mitochondrial activity was determined with the MTT
colorimetric assay; data were presented as mean
optical densities (n=3) at 570 nm (690 nm
PCL
C%
76±1
O%
24±1
N%
/
PPE:N
85±2
5±2
10±0.5
PPE:N/H2
81±0.5
9±0.5
10±0.5
Table I. Chemical percentage composition of untreated PCL
(A); PPE:N coated PCL (B); PPE:N/H2 treated PCL (C) scaffold
surfaces.
The scaffold chemistry changed after the deposition
of PPE:N coatings. N-C=O and C-N bonds appeared
in the C1s spectra (Fig. 1B), with a corresponding
decrease of carboxylic moieties. This was confirmed
by the presence of N% with a reduction of O% for
the scaffold top surfaces (Table I). After H2
treatment on PPE:N coated PCL scaffold surfaces,
no significant change in the C1s was noted (Fig.
1C), while an increase of O% with constant N%
were measured (Table I). The presence of O-groups
was due to oxygen uptake from atmosphere. XPS
was also performed on the scaffolds section along
the axis perpendicular to the top surface, previously
cut with a scalpel blade, in order to evaluate the
plasma polymer penetration inside. After the PPE:N
deposition, C% and N% decreased from the top to
the bottom of the PCL scaffolds, while O%
increased (Fig. 1D), confirming the formation of a
nitrogen-rich film onto the scaffold surfaces, that
decreased towards the bottom, but increased again
on the outer bottom surface. After the H2 treatment,
C% remained almost constant in depth, while O%
and N% inverted their trends. This confirmed that a
different chemistry (NH2 groups, hydrocarbon
moieties, oxygen uptake) was achieved between the
inside and the outside of the scaffolds. Except for
few variations, an almost constant N% from the top
to the bottom of PCL scaffolds, with the formation
of an almost uniform nitrogen-rich coating inside,
was obtained before and after H2 treatment.
of cell proliferation within the first 48 h, followed by
a decrease from 48 h on, probably due to cell
differentiation. However, at all three culture times,
the cells on plasma coated scaffolds showed a higher
metabolic activity respect to untreated scaffolds
(p<0.01).
Figure 2. MTT test of Saos-2 cells cultured on untreated and
PPE:N/H2 coated PCL scaffolds, at 18, 48 and 96 h culture
times.
4. Conclusion
Figure 1. C1s high resolution spectra of untreated PCL (A),
PPE:N coated PCL (B); PPE:N/H2 treated PCL (C). Chemical
composition of plasma modified PCL scaffold sections along the
z-direction (D).
On PCL scaffolds, untreated and plasma modified,
the behavior of the Saos2 osteoblast cell line was
studied.
In Fig. 2, the metabolic activity of Saos-2 cells
cultured on the two scaffold typologies, at three
culture times (18, 48 and 96 h), is represented.
Actually, the PPE:N/H2 coated scaffolds allowed the
best cell colonization of scaffolds. The general trend
of MTT data in both two scaffolds, is an increasing
This study provides a simple and effective method to
make porous scaffolds more cell-adhesive,
independently from the material, suitable for tissue
engineering
applications.
Indeed,
different
successfully plasma processes are proposed to
chemically modifying PCL scaffolds. By properly
control the plasma parameters, an almost uniform
plasma treatment inside the scaffolds has been
accomplished, with a nitrogen-rich coating present
from the top to the bottom of the scaffolds, even if
its presence (N%) decreased with the scaffold depth,
due to morphological constraints of scaffolds.
Furthermore, the chemical composition after plasma
modification, evidently stimulated Saos2 osteoblast
proliferation into PCL scaffolds. Further analyses
are in progress to visualize cells inside the scaffolds
by means of Micro-Computed Tomography and
fluorescence visualization, in order to get more
information on the distribution of cells inside the
scaffolds and understand if the plasma modifications
significantly improve the cell colonization in the
core regions respect to the scaffold peripheries.
Acknowledgements
Mr. Savino Cosmai is gratefully acknowledged for
his technical assistance. The research was supported
by PRIN 2008 prot. 9CWS4C_002 project.
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