CSEM Scientific and Technical Report 2008

Surfaces that Regulate Cell Growth and Death
M. Giazzon, N. Blondiaux, N. Matthey, G. Weder, M. Liley
Microstructured silicon surfaces have been used as substrates for bone cell growth. Cell proliferation and death can be regulated by varying the
height of the microstructures while leaving the other dimensions unchanged. These changes in the surface structure are accompanied by a change
in cell morphology from a 2D shape to a 3D one. This involves modification of the cytoskeleton and a progressive loss of cell-surface adhesion.
Osteoblasts are cells specialized in the production of bone
tissue. They synthesize the two components of bone matrix: a
fibrous matrix that bestows elasticity on the bone and an
amorphous one that provides hardness and resistance to
compression. The correct behaviour of osteoblasts is crucial
to the successful integration of bone and tooth implants. For
this reason there is an increasing effort focused on the search
for new substrates which can mimic the environment that
osteoblasts create around themselves and induce implant
integration.
At CSEM, studies of silicon surfaces with pillar structures,
together with their capacity to influence osteoblast behaviour,
including cell growth, death and morphology are underway [1] .
The surfaces were fabricated by dry etching, using a
structured polymer thin film as mask. By varying the etch time,
different pillar heights could be created while the diameter
(~ 400 nm) and the spacing of the pillars (~ 700 nm) remained
unchanged (Figure 1). This fabrication method has a
significant advantage in that it allows the controlled variation
of the topography while leaving the surface chemistry
unchanged.
Figure 1: SEM images of structured silicon surfaces with pillars of
different heights (a) 200 nm, (b) 500 nm, (c) 1 µm, (d) 2 µm,
(e) 3 µm, and (f) 4 µm
Figure 2: Bone cells (SaOs-2) grown on silicon surfaces with pillars
(a) 200 nm, (b) 500 nm, (c) 1 µm, (d) 2 µm, (e) 3 µm, and (f) 4 µm
The pictures, obtained by confocal microscopy, show the actin fibers
that form the cytoskeleton.
SaOs-2, a human osteosarcoma cell line, has been used as
an osteoblast model. Cells were grown for 5 days on both
structured and flat silicon surfaces. Cells on flat surfaces
showed a good level of proliferation and a low level of
apoptosis (also known as “programmed cell death”). Cells
grown on short pillars had a similar behaviour. However, when
the pillar height increased above 500 nm, the situation
changed, with slow cell proliferation and increased apoptosis
(Figure 2). Thus, it is possible to regulate the delicate
equilibrium between proliferation and death by changing a
single topographical parameter.
In addition, the osteoblast morphology was also influenced by
the change in pillar height [ 2 ] . In vitro studies have
demonstrated that osteoblasts usually assume a flattened
morphology on flat plastic surfaces. This flat form is very
different from their in vivo morphology and represents a
limitation of normal in vitro cell studies. In contrast, a more 3D
morphology, similar to their physiological form was observed
in samples on silicon surfaces with pillars over 500 nm high.
This morphology is the result of internal rearrangements
involving both the cytoskeleton [3] and the cell membrane and
which result in the disruption of focal adhesions culminating in
a reduced cell-substrate adhesion.
In conclusion, these silicon surfaces offer a way to regulate
osteoblast behaviour in an in vitro system. Not only
proliferation and apoptosis but also morphology are strongly
modified. Cell behaviour on these surfaces may be closer to
physiological (in vivo) behaviour, allowing in vitro studies of
osteoblast that are more relevant and useful. Future work will
include the study of bone cell differentiation and the
production of fibrous and amorphous matrices in order to
understand whether osteoblasts on these substrates maintain
their capacity to create bone.
[1] J. M. Rice, J. A. Hunt, J. A. Gallagher, P. Hanarp,
D. S. Sutherland, “Quantitative assessment of the response of
primary derived human osteoblasts and macrophages to a range
of nanotopography surfaces in a single culture model in vitro”,
Biomaterials, 24 (2003), 4799
[2] Y. Wan, Y. Wang, Z. Liu, X. Qu, B. Han, J. Bei, S. Wang,
“Adhesion and proliferation of OCT-1 osteoblast-like cells on
micro- and nano-scale topography structured poly (L-lactide)”,
Biomaterials, 46 (2005) 4453
[3] H. M. Powell, D. A. Kniss, J. L. Lannutti, “Nanotopographic
control of cytoskeletal organization”, Langmuir, 22 (2006), 5087
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