22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Plasma processing of scaffolds for tissue engineering and regenerative medicine
F. Intranuovo1, R. Gristina2, L. Lacitignola3, A. Crovace3 and P. Favia1,2
1
Department of Chemistry, University of Bari “Aldo Moro”, Bari, Italy
2
Institute of Inorganic Methods and Plasmas, CNR, Bari, Italy
3
Veterinary Surgery, Department Emergency and Organ Transplantation, University of Bari, Italy
Abstract: Plasma processes are largely employed in the biomedical field for different kind
of materials. The challenge is to modify 3D scaffolds in order to be used for in vivo tissue
regeneration in Regenerative Medicine. The correct 3D biointegration inside the living
tissue is the crucial objective, towards which many aspects are directed, from the material
engineering to its surface modification and affinity with the biological environment.
Keywords: plasma deposition, plasma treatment, scaffold, wettability, porosity, cell
affinity, in vivo
1. Introduction
In the last thirty years, Tissue Engineering (TE) has
been proposed and developed as a potential therapeutic
approach in addressing the repair, replacement, and/or
regeneration of vital tissues and organs, becoming an
interesting alternative both to artificial prostheses and
transplants of living tissues in the human body [1]. In
particular, TE of bone tissues is a very attractive strategy
suggesting novel reconstructive approaches for repairing
large bone defects resulting from traumas, pathological
degenerations, or congenital malformations. This is
replacing different strategies in reconstructive surgery,
that often are limited by several critical constraints (e.g.,
risk of infection at the body/tissue interface and chronic
prosthesis rejection) that could be minimized, in principle,
by TE [2]. The challenge is very high, since two hundred
thousand surgical cases of bone-grafting procedures are
performed annually.
This strongly developing field is based on the use of
cells (stem cells particularly) seeded in a threedimensional (3D) scaffold, that provides the initial
structural integrity and organizational backbone for cells
to assemble and ignite several other processes, up to the
development of a functional tissue [3]. For successful
bone TE, a scaffold needs to be osteoconductive, porous,
biocompatible and biodegradable (functional properties
similar to those of the native tissue for minimizing
premature failure), thus able to support cell attachment
and proliferation and to guide the regeneration of the
bone. Scaffolds characterized by high porosity, proper
pore size, shape, interconnectivity, surface area, stiffness
and mechanical integrity, are generally required.
Nowadays, several fabrication techniques have been
employed
to
realize
scaffolds
with suitable
biocompatibility [4]. The Solvent Casting/Particulate
Leaching (SCPL) technique, for instance, used in this
research, is the easiest, time-saving and cheap
conventional scaffolding technique. This is based on the
leaching of water-soluble particulates (e.g., NaCl) from a
polymer blend creating the pore network in the scaffold.
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It allows the control of micro-structural characteristics
such as total porosity, pore interconnection and pore size
[5].
Often, a good micro-/nanostructure is not enough to
guarantee proper cell growth and valuable tissue/material
interfacial adhesion. Indeed, the surface properties of the
scaffold material are known to influence cell-material
interactions. Another significant weakness can be the
inhomogeneity of cell colonization inside the scaffolds.
When cells are seeded in vitro into 3D scaffolds, in fact,
cell adhesion is favoured at the periphery of the
constructs, more accessible than the inner surfaces,
resulting poorly populated inside, because the external
surfaces of the scaffolds are more accessible than the
inner ones.
In one of the most promising strategies for scaffold
engineering, the adsorption of proteins from the cell
culture medium has to be granted homogeneously through
the entire inner structure of the scaffold in order to
consequently improve cell motility and ensure
homogeneous colonization. This result can be achieved
by properly modifying and controlling the internal and
external surface composition and properties of the
scaffolds. Gradient chemical surface compositions are
utilized sometimes, e.g., more hydrophilic inside the
scaffold with respect to the external part, for this purpose
[6, 7].
Cold plasma processes both at low and atmospheric
pressure have been shown to be very useful to
functionalize material surfaces, with no change of the
bulk, to tailor the surface composition of scaffolds and
improve their cytocompatibility, as well as to synthesize
functional surfaces for direct cell growth and
biomolecules immobilization, for depositing non-fouling
coatings, nanocomposite bacterial resistant coatings or
micro-/nanostructured surfaces [8-11].
2. Results & Discussion
In this contribution, several plasma modification
approaches on scaffolds will be presented, showing how
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the creation of suitable chemistries through different
porous scaffolds could improve cell biocompatibility in
vitro and help the regeneration of tissues in vivo.
Plasma technology will be showed as the drawing
strength to produce cutting-edge materials by means of a
straightforward combination of innovative and
environmental-friendly techniques and strategies in the
field of Regenerative Medicine.
Firstly, in order to create the right healing environment
and a good mechanical and micro-morphological stability,
particular attention was paid to the production of
scaffolds, tuning the technique (conventional or CAD),
the material (polymers, composites), the structural
properties (porosity, pore size and connectivity).
Many plasma-based approaches are used for the surface
modification of polymer properties; however, it is still
quite challenging to properly and homogeneously address
such processes with efficiency when applied to complex
3D porous structures. Since synthetic polymers utilized
for scaffolds are quite hydrophobic, plasma processes at
low and atmospheric pressure are utilized to tailor the
hydrophilic character of their surfaces, as well as their
chemical composition, with the aim of improving their
biocompatibility, in vitro with cells and in vivo with the
living tissues they have to be integrated with. Plasma
discharges fed with different gas/vapours mixtures (N 2 ,
O 2 , carboxylic acid and derivatives, alkyl amines, etc.)
have been performed in controlled conditions, in order to
decorate the inner and external surfaces of scaffolds with
nitrogen- or oxygen-based chemical moieties. Such
chemical groups are known to be good binding sites for
cell adhesion and growth. Plasma parameters were
properly tuned in order to minimize damages to the
scaffold and to improve the penetration of the plasma
species inside the scaffolds pores.
A step-by-step material characterization is essential to
control each stage of the scaffold production, before and
after plasma modification, from the chemical (with X-ray
Photoelectron Spectroscopy, XPS), wettability with
dynamic Water Contact Angle (WCA) analyses and
titrations, to the morphological characterization with
Scanning Electron Microscopy (SEM) and MicroComputed Tomography (µ-CT).
In vitro viability and morphological tests were also
performed to check the cytocompatibility of the plasma
modified
scaffolds
when
cultured
with
fibroblast/osteoblast cell lines or mesenchymal stem cells
(MSC). In particular, MSCs are attractive candidate
progenitor cell types for bone TE, since they are
characterized by extensive ability to proliferate and
differentiate along various lineages of mesenchymal
origin, such as cartilage, bone, muscle, ligament, and
tendon. In vitro experiments allowed to understand if the
chosen materials were cell-adhesive and if the produced
scaffolds were able to load cells homogeneously inside
their 3D structure. These results were verified by
observing the cell morphology with confocal fluorescence
microscopy and by studying the cell viability with MTT
2
assays.
To verify the feasibility of the produced materials as
bone and cartilage substitutes, in vivo implants in ovine
models were carried out.
Integration and biomineralization process of the implanted scaffolds were
then investigated in vitro by means of µ-CT, in order to
follow the formation of new bone and cartilage tissues
around the implanted scaffolds and their gradual
absorption and degradation. Results will be shown in
terms of differences between untreated and plasma
processed scaffolds.
In Fig. 1a summary of the 3D analysis of an ovine
condile embedding a plasma treated polycaprolactone
(PCL) scaffold of proper morphology and chemistry, is
shown. This condile was scanned six months after the
surgical implant by means of µ-CT. Then, scanned
projection images were reconstructed with a step by step
procedure with different softwares.
On the top right part of the figure, there is the shadow
projection image of the condole, with a cylindrical
scaffold embedded, visible because it is a lower density
material, thus it absorbs less X-ray radiation respect to the
surrounding bone tissue. The section on the red line is
then represented on the three axes on the three images at
the top left side. Here it is possible to distinguish the
native bone, from the newer formed bone tissue just
around the scaffold that is the less dense porous material
in the middle of the image. At the bottom of the figure
there is a 3D image of the entire reconstructed condile.
Here it is possible to notice the point where the scaffold
was implanted (red circle).
3. Summary
In this talk plasma processes of 3D porous scaffolds
will
be
described,
along
with
surface/bulk
characterization and evaluation of in vivo experiments
performed on ovine models. In most examples cold
plasma processes at low/atmospheric pressure have shown
the ability of functionalizing the substrates in several
ways, tuning chemical composition, wettability and other
surface properties improving the cytocompatibility as well
as the osteointegration of the scaffolds.
4. Acknowledgements
The following projects are acknowledged for funding
and supporting this research: RIGENERA (Aiuti a
Sostegno dei Partenariati Regionali per l’Innovazione, n°
P9Y0834); RINOVATIS (Regenerating Nervous and
Osteocartilagineous Tissues by Means of Innovative
Tissue
Engineering
Approaches,
PON
02_00563_3448479); LIPP (Rete di Laboratorio 51,
Regione Puglia); SISTEMA (PONa3_00369 MIUR,
Laboratorio per lo Sviluppo Integrato delle Scienze e
delle TEcnologie dei Materiali Avanzati e per dispositivi
innovativi). Ms Edda Giuseppina Francioso (University
of Bari), Mr Savino Cosmai (IMIP-CNR) and Mr Danilo
Benedetti (University of Bari) are acknowledged for
technical support.
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K.M. Shakesheff and M.R. Alexander.
Acta
Biomater., 3336, 7 (2011)
[8] P. Favia, E. Sardella, L.C. Lopez, S. Laera,
A. Milella, B. Pistillo, F. Intranuovo, M. Nardulli,
R. Gristina, R. d’Agostino. S. Guceri and
B. Smirnov (Eds.). NATO-ASI series (2008)
[9] F. Intranuovo, P. Favia, E. Sardella, C. Ingrosso,
M. Nardulli, R. d’Agostino and R. Gristina.
Biomacromolecules, 380, 12 (2011)
[10] M. Domingos, F. Intranuovo, A. Gloria, R. Gristina,
L. Ambrosio, P.J. Bartolo and P. Favia. Acta
Biomater., 5997, 9 (2013)
[11] F. Intranuovo, R. Gristina, F. Brun, S. Mohammadi,
G. Ceccone, E. Sardella, F. Rossi, G. Tromba and
P. Favia. Plasma Proc. Polymers, 184, 11 (2014)
Fig. 1. Top: µ-CT images (left) of a reconstructed 2D
section (red line on the top-right shadow projection
image) along the 3 axes of an ovine condile with a
plasma-processed PCL scaffold implanted (Data Viewer
software). The condile was explanted after 6 months.
Bottom: 3D reconstruction image of the same condile
with the PCL scaffold inside (CTVox software).
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