| Abstract |
Unlike other tissue types, which consist of cells with a much more homogeneous
structure and function, the nervous tissue spans in a complex multilayer environment whose
topographical features display a large spectrum of morphologies and size scales. Because of the
necessity of a multilayer environment, the well established flat tissue culture surfaces are proven
to be insufficient for studying the effect of the topography of the surroundings on nerve cell
morphology and function. In an attempt to approach the complexity of the topographical milieu
of nerve cells, it is necessary to shift to more complex 3D micropatterned surfaces. Micro-and
nanofabrication techniques provide the opportunity to develop new types of cell culture platform,
where the effect of various topographical cues on cellular functions such as proliferation and
differentiation can be studied. Different approaches (regarding the material, fabrication
technique, cell type and assay) have been used in order to fabricate micropatterned surfaces,
where the effect of topography on nerve cell development can be studied.
In this study, the cellular growth on micropatterned Si substrates (comprising arrays of
microcones -MCs) fabricated by ultra-short pulsed laser processing has been investigated. Using
increasing laser fluence, three types of micropatterned Si surfaces, which exhibit different
geometrical characteristics (denoted as low, medium and high roughness substrates,
respectively), have been fabricated and characterized as to surface morphology, wetting
properties and surface chemistry. As roughness increases, among the different geometrical
characteristics of the MCs, intercone distance increases and an anisotropic topography becomes
more pronounced. These three micropatterned Si substrates together with the unpatterned flat Si
have been applied to in vitro cell cultures.
PC12 cells were used as a model of nerve cells in order to study the NGF-induced growth
and differentiation pattern (neuritogenesis) on the micropatterned Si substrates. Upon NGF
treatment, cell differentiation was promoted on low and intermediate roughness substrates,
whereas it was strongly inhibited on the highly rough ones. The obtained results suggested that
the intercone space did effectively influence the NGF-induced PC12 differentiation fate.
Dissociated primary cells of the PNS were used in order to investigate the topographic
guidance of neural outgrowth and network formation of dissociated SCG sympathetic neurons as well as the effect of (surface) topography on Schwann cell morphology. It was shown that the
neuronal network on the low roughness substrates displayed high randomness, whereas the
neurons on intermediate and high roughness substrates exhibited a parallel alignment.
Furthermore, oriented Schwann cell outgrowth was promoted on intermediate and high
roughness substrates. This surface -induced guidance effect was also observed in the whole DRG
explant model, where both Schwann cell migration and axonal outgrowth exhibited a surfacedependent
response. In this model, it was shown that Schwann cells create a cellular “carpet”
onto the substrates. Neurons were, in turn, outgrown on top of them. It is hypothesized that the
plasticity of Schwann cells and their processes allowed a glial “carpet” formation, which served
as a substrate for neurite outgrowth.
Therefore, it can be concluded that the distinct geometrical characteristics of surface
roughness could influence a variety of neuronal and neuroglial cell functions. The laser
micropatterned silicon (Si) substrates presented here could potentially be used as model scaffolds
for the systematic exploration of the role of 3D microtopography on cell differentiation and
neural network outgrowth, where Schwann cell–neuronal interactions could be investigated in
the context of nerve tissue regeneration.
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Material processing via ultra-short pulsed laser : study of the outgrowth and interfacing of neural networks in 3D Si scaffolds
