CSEM Scientific and Technical Report 2008

Measuring Cell Adhesion with Force Spectroscopy
G. Weder, M. Giazzon, N. Matthey, M. Liley
The attachment of human bone cells to medical implant surfaces is a critical step in their integration in the human body. Many bone implants have
roughened surfaces that promote cell, bone and tissue adhesion. However methods to quantify cell adhesion remain limited. CSEM is developing
the use of atomic force microscopy (AFM) to directly measure cell/surface adhesion forces for biological and medical applications.
Cellular adhesion – the binding of a cell to another cell, to a
matrix, or to a surface – is of great clinical importance, and
altered cell adhesion is involved in diseases such as cancer
metastasis and connective tissue disorders. It is also critical in
the osseointegration of bone or tooth implants. The
measurement of cell adhesion in vitro remains a major
challenge due both to difficulties in the manipulation of living
cells in a liquid environment and to the very low adhesion
forces (in the picoNewton range) that require highly sensitive
instrumentation for their measurement.
CSEM is studying cell adhesion on structured surfaces of nonmetallic
bone implants using a combination of optical and
scanning probe microscopy (AFM). In force spectroscopy
mode, AFM can perform force measurements at the single
molecule level as well as stiffness/elasticity measurements on
individual cells.
The micro- and nanostructured surfaces used in these
investigations were made of an acrylate resin. Masters for the
microstructures composed of hemispherical pits with
diameters on the order of tens of microns were fabricated
using photolithography whereas masters for the
nanostructures composed of pillars with heights on the order
of tens of nanometres were obtained by colloidal lithography
(Figure 1).
Figure 1: (a) Hemispherical pits with a diameter of 27 µm and (b)
nanopillars with a height of 45 nm replicated in a non-metallic bone
implant material; (c) Human osteoblasts growing on a microstructured
surface with hemispherical pits.
Living human osteoblasts were captured on a functionalized
AFM cantilever and positioned above the selected structured
surface in a fluidics chamber at 37°C. Each cell was brought
briefly into contact with the microstructured sample and was
then slowly withdrawn from the surface. During the withdrawal,
the deflection of the cantilever was used to measure the
adhesion forces between cell and surface. Both the
mechanical properties of the whole cell and individual
unbinding events corresponding to single proteins or protein
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complexes being released from the surface could be
quantified (Figure 2).
Figure 2: (a) Diagram showing single cell force spectroscopy [ 1]
measurements and (b) a force distance curve acquired during
approach (steps I-II) and retraction (steps III-IV). Step I: in the initial
phase of the approach there is no contact between the cell and the
surface. Step II: the cell is pressed onto the surface until a pre-set
maximal force is attained. During this phase the elastic response of
the cell can be observed. Step III: mechanical parameters related to
the number, position and amplitude of the unbinding events as well
as the maximum force and work of detachment can be quantified.
Step IV: there is no physical contact between the cell and the surface.
(c) A living human osteoblast captured on an AFM cantilever.
Initial work focused on measuring variations in cell adhesion
during the cell cycle. Cell adhesion was found to be similar for
all phases of the cell cycle. It is thus not necessary to
synchronize cells to one phase of the cell cycle to obtain a
single population for future studies of cell/surface interactions.
The method is now well established with different cell lines
and different surfaces under study. The quantitative results
obtained make it a promising approach to surface
characterization and the development of a new generation of
non-metallic bone implants. Future work will focus on methods
to detach adherent cells after long (more than 24 hours)
contact and culture times on structured surfaces.
This work was funded by the Newbone project of the 6th EU
framework program. CSEM thanks them for their support.
[1] J. Helenius, A. C. Heisenberg, H. E. Gaub, D. J. Muller, “Singlecell
force spectroscopy”, Journal of Cell Science, 121 (2008),
1785