research activities in 2007 - CSEM

Using Microtopography to Study Cell Elasticity
M. Giazzon, N. Matthey, G. Weder, M. Tormen, T. Overstolz, M. Liley
Tissue engineering and regenerative medicine are of increasing importance as global life expectancy increases. The development of surfaces to
control cell adhesion and proliferation can make an important contribution to these fields. In this context, microtopographies are used in studies of
the elasticity of human bone cells and as a tool to influence cell growth and survival.
Micro and nano-topography can be used to promote or inhibit
the proliferation of biological cells. One major field of
application of this effect is that of tissue engineering and
regenerative medicine. The control of cell proliferation is of
increasing importance in medical implants with the potential to
accelerate their integration in the body, to reduce the risk of
infection or to avoid undesired cell growth in specific areas.
For this reason a number of research groups are developing
structured surfaces to improve and control the adhesion and
proliferation of living cells [1] .
CSEM has been developing microtopographic structures in
order to study one specific aspect of cell behaviour that
contributes to cell proliferation or death: cell elasticity. This is
the capability of a cell to bend in response to a physical
stimulus. Indeed cells may bend following a stimulus or when
they encounter a mechanical hurdle, but there is a limit above
which they cannot curve [2] .Varying the microtopography
allows that limit to be found.
Microfabricated quartz surfaces with concentric grooves and
ridges constitute a simple system for investigating cell
elasticity. These substrates, obtained by photolithography,
have ridges and grooves with a width of 2 or 4 µm and a
range of depths from 30 nm to 500 nm. The radius of
curvature of the ridges varies across the quartz substrate
testing cell elasticity under a range of conditions in one
sample.
When bone cells are grown on these surfaces they are found
to assume different morphologies. Where the radius of
curvature is high the cells adopt an elongated morphology
(Figure 1b), following the ridge and groove structure. In
contrast, where the radius of curvature is small the cells
spread over the grooves and ridges (Figure 1a).
Figure 1: Bone cells on concentric grooves with a high curvature (a)
and with a low curvature (b). Cells spread and orient normally on the
flat surface in the top left of b. In the bottom right the cells are
elongated and align to the grooves and ridges.
The cytoskeletal protein, actin, forms fibres that play a crucial
role in cell spreading, movement and adhesion [3] .
Immunofluorescence staining of actin fibres allows the study
of the form and structure of adherent cells on quartz surfaces
(Figure 2). The response of the cells seems to be linked to the
rigidity of the actin fibres. Shallow grooves with a low
curvature result in a partial alignment of the cells, which cross
a few grooves only: the actin fibres are well aligned to the
grooves: close to the centre of the structure the cell is unable
to align to grooves with a high curvature: the actin fibers and
the cell span the ridges with no alignment. However, when the
depth of the grooves is 500 nm or more, a large number of
cells undergo apoptosis, or programmed cell death.
Figure 2: Fluorescence images of actin fibers (green) of bone cells
grown on concentric lines (a) close to and (b) far from the centre of
the grooves. (c) cell nuclei aligned to the circular grooves and
(d) triple fluorescence staining of cells far from the centre with actin in
green, the nuclei in blue and focal points in red.
In conclusion, groove microtopographies influence cell
behavior in vitro. Future work will focus on deep
microtopographies – deeper than 500 nm – and their influence
on cell apoptosis. This may form the basis of a new strategy
for the control of cell proliferation and death.
This work was partly funded by the European Commission in
the context of the Marie Curie research training network
BioPolySurf (www.biopolysurf.net). CSEM thanks them for
their support.
[1] F. Rehfeldt, A. Engler, A. Eckhardt, F. Ahmed, D. Discher,
”Cell responses to the mechanochemical microenvironment-
Implications for regenerative medicine and drug delivery”,
Adv Drug Deliv Rev., 59, (2007)1329
[2] P. Kunzler, C. Huwiler, T. Drobek, J. Vörös, N. Spencer,
“Systematic study of osteoblast response to nanotopography by
means of nanoparticle-density gradients”, Biomaterials, 28,
(2007) 5000
[3] C. Wilkinson, M. Riehle, M. Wood, J. Gallagher, A. Curtis,
“The use of materials patterned on nano-and micro-metric scale
in cellular engineering”, Materials Science and Engineering, 19,
(2002) 263
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