World Journal Of Engineering
NANOCOMPOSITE THIN LAYERS SYNTHESIZED BY ADVANCED
PULSED LASER TECHNOLOGIES FOR BIOMIMETIC IMPLANT
COATINGS
Ion N. Mihailescu
National Institute for Lasers, Plasma and Radiations Physics, Lasers Department, 409
Atomistilor, P.O. Box MG-54, Magurele - Ilfov, RO-077125, Romania;
ion.mihailescu@inflpr.ro
and pressure), the process can continue without
noticeable organic material decomposition [1,3,4].
The collective action of the rapidly evaporating
volatile solvent acts to softly desorb the fragile
solute by soft collisions and deposits the solute as a
uniform thin film which properties, such as
chemical structure and functionality, have been
preserved.
We review herewith recent results on the synthesis
by pulsed laser methods of extracellular matrix
proteins – hydroxyapatite hybrid nanostructures for
biomedical implant applications.
Introduction
Pulsed laser deposition (PLD) proved to be a
competitive method to grow high-quality
nanocomposite thin films. Nevertheless, in case of
very complex delicate biomolecules (such as
organic and biopolymeric materials), PLD fails
because of the irreversible damage of the chemical
bonds and consequent compositional modification
of the deposited film. This disadvantage is
eliminated by matrix assisted pulsed laser
evaporation (MAPLE), capable to transfer
compounds at low temperatures.
Many independent parameters can be changed
under control in MAPLE in order to select the
optimum deposition regimes of some specific
nanostructures and thin films [1,2]. Growing thin
films by the new pulsed laser techniques has
numerous advantages, such as:
i) the laser source is placed outside the deposition
chamber offering increased flexibility in handling
the material, laying out the geometrical setup, and
adjusting deposition parameters;
ii) most of the solid and liquid materials could be
ablated/evaporated;
iii) laser pulses make possible to control the growth
rate of the coatings very accurately (down to a few
fractions of Å);
iv) the stoichiometry of coating materials generally
coincides with that of the target even for complex,
highly unstable compounds;
v) the coatings adhere well onto deposition
substrate due to the high plasma incident energy;
and
vi) species with metastable or nonequilibrium states
and new phases could be synthesized.
MAPLE essentially differs from PLD by target
preparation, laser-material interaction and transfer
mechanisms. It provides a more gentle mechanism
for transferring many different compounds,
including large molecular weight species, such as
polymeric molecules. Specific to MAPLE is the use
of a cryogenic composite target, a dilute mixture of
the organic material to be deposited, and a lightabsorbent, high vapor-pressure solvent matrix. By
optimization of MAPLE conditions (laser
wavelength, repetition rate, fluence, solvent type,
concentration, temperature, background gas nature
Experimental
The general PLD layout used in our experiments of
nanocomposite thin film synthesis can be described
as follows. Laser beam generated by a pulsed UV
excimer laser source enters the reaction chamber
through a quartz window. We used a KrF* excimer
laser source (COMPEXPro 205) generating at 248
nm pulses of 25 ns duration. The beam is focused
onto target surface by means of an AR cylindrical
lens placed outside the deposition chamber. It hits
the target surface at oblique incidence. The target is
rotated and translated during multipulse irradiation
to avoid piercing and improve film uniformity. A
temperature controller is used to monitor substrate
heating and cooling. The reaction chamber is
initially evacuated down to a residual pressure of
10-4 Pa. Dynamic pressure during experiments is
kept constant using a flow controller.
We developed hybrid biomimetic structures
consisting of a thin film of hydroxyapatite (HA)
covered by extracellular matrix proteins in order to
go as close as possible to the actual human bone
structure, composition and functionality. The
nanostructured HA layers were obtained by PLD
onto chemically etched gr. 4 titanium (Ti) disks.
Next, fibronectin (FN) or vitronectin (VN) (1.8
mg/ml in deionized water based saline buffer, 700
mJ/cm2) were transferred on the formed Ti/HA
structures by MAPLE. As negative control, bovine
serum albumin (BSA) onto the Ti/HA structures
were also obtained by MAPLE.
Results and Discussion
Extracellular matrix proteins (ECM) proved to be
efficient in material bio-activation by adsorption
and cell adhesion at the interface.
799
World Journal Of Engineering
The qualitative distribution of proteins during the
MAPLE synthesis was monitored by Ponceau S
Staining Solution on nitrocellulose membranes.
The quantitative reproducibility of FN and VN was
established by colorimetric protein assay using
bicinchoninic acid (BCA) solution. The successful
transfer of either FN or VN was demonstrated by
FTIR studies where the peaks of protein dropcasts
were coinciding with the ones of MAPLE
deposited nanostructures. These results were
supported by antibody staining studies using antihuman FN or VN rabbit polyclonal serum and
FITC-conjugated anti-rabbit IgG when spot-like
fluorescent regions were visualized by fluorescence
microscopy (Figs. 1,2).
structures and small spots in case of VN ones. The
MTT results sustained the higher potential for
spreading and improved viability of protein
covered structures versus Ti/HA and Ti/HA/BSA.
SEM investigations revealed more flattened cell
morphology with long developed filopodia when
cultivated on FN and VN structures (Fig. 3).
Ti/HA
Ti/HA/VN
Fig. 1. Immunofluorescence
fibronectine structures
detection
Ti/HA/BSA
Ti/HA/FN
Fig. 3. SEM images of hOB cells at 7 days from
seeding
of
Conclusion
We concluded that hybrid thin films and structures
(HA- ECM proteins) obtained by PLD and MAPLE
were identical to the starting material, preserving
their chemical structure and very likely their
functionality and biologic activity.
References
Fig. 2. Immunofluorescence
vitronectine structures
detection
[1] Pulsed Laser Deposition of thin films:
applications-lead growth of functional materials,
edited by R. Eason (Wiley & Sons, New York,
USA 2007)
[2] I. N. Mihailescu, C. Ristoscu, A. Bigi, I. Mayer,
Chapter 10 in “Laser-Surface Interactions for New
Materials Production Tailoring Structure and
Properties”, Series: Springer Series in Materials
Science, Vol. 130, Miotello, Antonio; Ossi, Paolo
M. (Eds.), 2010, pp. 235 – 260
[3] D. B. Chrisey, A. Pique‚ R. A. McGill, J. S.
Horwitz, B. R. Ringeisen, D. M. Bubb, and P. K
Wu, Chem. Rev. 103, 553 (2003)
[4] R. Cristescu, I. N. Mihailescu, M. Jelinek, and
D. B. Chrisey, in Functionalized Properties of
Nanostructured Materials, edited by Rainer
Kassing, Plamen Petkov, Wilhelm Kulisch, and
Cyril Popov (NATO Science Series by Springer,
Series II: Mathematics, Physics and Chemistry,
2006) Vol. 223, p. 211
of
Human osteoblast precursor cells were cultured on
all structures for up to 14 days. The cells displayed
a normal morphology, optimal viability and spread.
The synergistic effect of the VN and FN coatings
was evidenced by actin and vinculin staining, MTT
assays and SEM studies. The long elongated actin
filaments proved the excellent induced bioactivity,
also showing a uniform distribution of the cells
over the entire surface. The vinculin staining
evidenced intimate contacts with material surfaces
by focal adhesion patches for FN covered
800