SUMMARY OF MY PhD
The biocompatibility of materials in implant or biosensor fields
strongly depends on first interactions occurring between a given surface
and a biological environment. It is well-known that a living body brought
into contact with a surface will induce protein adsorption, which creates
the interface, on which proteins or cells will adsorb. The interactions
can be influenced by modifying the surface properties, which are the chemistry,
the surface charge and the topography of the surface. It has been shown
that cells can "sense" the topography, growing along grooves of defined
depth and width at the micrometer scale.
In
the first part of this thesis, the fundamental addressed question is whether
proteins can "sense" the topography, in analogy to what has been observed
for cells on microstructures, because proteins are always present between
the surface and the cells. Topographical modification have to be performed
at the nanometer scale, corresponding to the size of proteins. Using an Atomic
Force Microscope (AFM) and applying Local Anodic Oxidation (LAO) in ambient
air, it is possible to create nanostructures of a height of 1-4nm and a width of 10nm. The characterization of
the surface by X-Ray Photoelectron Spectroscopy (XPS) reveals that this
method assures a modification of the topography of the surface without
change of its chemical composition. Surfaces structured by LAO therefore
represent ideal systems to study the dependence of protein adsorption on
topography. We are able to visualize the created nanostructures by AFM and
successively adsorb proteins in situ, rinse and image the new surface. The
densities of adsorbed proteins on the nanostructured and neat surfaces are
compared and we find that the protein arrangement depends on the underlying
nanostructures. A remarkable specificity of the actin filament (F-actin)
adsorption on the nanostructure height is noticed. On Ti, F-actin is observed
to have a low adsorption on created lines of a height of 4nm and the adsorbed
proteins appear to be randomly oriented. In contrast, high protein adsorption
is observed for structure height between 1 and 2nm, moreover the filaments
adsorb preferentially parallel to the nanostructured pattern. On Si, F-actin
also adsorb preferentially along 1nm high lines, but the density of adsorbed
proteins is higher on neat surface. We have therefore demonstrated that proteins
"sense" the topography of surfaces at the nanometer scale.
|
|
|
Experiments
performed with AFM only permit static measurements giving no information
on the kinetics of protein adsorption. The second part of this thesis is
devoted to the building of a Quartz Crystal Microbalance (QCM), which allows
us firstly to follow the adsorption kinetics, and secondly to get information
about the viscoelastic properties of the adlayer. The QCM is a rather new
but ultrasensitive technique. In liquid the QCM sensitivity is 9ng/cm2
and under vacuum it reaches 0.135 ng/cm2. Nevertheless quantification
of the amount of adsorbed mass is still difficult to determine under liquid
loading, since different phenomena influence the measured parameters. Water
molecules entrapped between adsorbed mass can firstly bring an added measured
mass, and secondly modification of the liquid and mass properties such as
density and viscosity can bring artefacts. In contrary to commercial instruments,
our home-made QCM allows us to measure the maximal oscillation amplitude
of the quartz crystal. This parameter allows us to distinguish the contribution
due to adsorbed mass from the one due to changes of liquid and mass properties.
The interpretation of the QCM measurements is therefore enhanced. During
this thesis work experiments have been performed under liquid with different
systems, such as proteins and cells, and on different surfaces. On Au more
proteins adsorb on the surface, in comparison to Ti. Nevertheless the biological
activity is larger on Ti at high protein concentration. Adsorption of cells
involves complex phenomena, which are not yet fully determined. We demonstrate
that during the first 80min of adsorption, the spreading of the cell influences
mostly the measured parameters. Thereafter the cytoskeleton of the cell
is rearranged, inducing cell stiffening and an increase of the total cell
viscosity. This phenomenon induces variations of the frequency, the amplitude
and the decay time constant, although no real modification of the amount
of adsorbed cell occurs. Using drugs, it has been possible to modify the
polymerisation state of the cytoskeleton, inducing changes of the viscoelastic
properties of the adsorbed cells. Changes of the measured frequency, amplitude
and decay time constant during cell spreading could therefore be reproduced.
In
summary the topography is an important parameter, which has to be taken into
account in the general biomaterial field, because the structure of the
surface at a nanometer scale can influence the response of biological environments.
In order to better analyze the protein adsorption and the cell response
against surfaces, the QCM is a very promising technique, but it is essential
to measure several parameters.
|
|
