Abstract |
Peripheral nerve injury causes significant morbidity, has an important impact on patients' sensory
activity and significantly deteriorates their quality of life. Albeit the spontaneous regeneration
potential of the peripheral nervous system (PNS), the repair is almost always incomplete and with
limited functional recovery. In order to achieve full recovery of the injured peripheral nerves, the
use of autologous nerve grafts is the golden standard since decades. However, this approach
involves several drawbacks, such as morbidity at the donor site, limited availability of grafts and
more importantly it rarely leads to the full recovery of the patient. As a response to these limitations,
neural tissue engineering (NTE) has emerged as an alternative approach, aiming at the restoration
of lost or damaged neural tissue with the use of cells.
NTE encompasses the development of appropriate scaffolds for the growth of regenerated tissues.
The natural environment of the cells is the extracellular matrix (ECM), a complex environment that
provides the mechanical framework to enable cell growth, intercellular interaction and tissue
formation. During the development of the nervous system, the ECM significantly contributes to the
maturation of neurons and neurites via its surface topography and microenvironment signals.
Consequently, the scaffolds used in NTE play a very important role, as their topography can affect
vital functions of the cells, such as their attachment, proliferation, morphology, migration and
differentiation.
A large variety of fabrication methods and materials have been used for the development of
scaffolds for NTE applications. Here, we fabricated micro- and nano- patterned Si substrates via
direct ultra-fast laser irradiation. The aim of the study was to develop substrates that enable spatial
patterning of the PNS cells in a controllable manner and to explore if and how the underlying
topography can lead to controlled cellular growth and differentiation. The ultimate goal was to
exploit the semiconducting properties of Si in a selective patterning cell culture substrate which
could allow the recording of neuronal signals. In order to achieve this goal, two separate steps were
indispensable: 1. the development of a culture substrate that would support the selective and
controlled cellular adhesion, and 2. the successful differentiation of the neuronal cells on it.
With this aim, Si was irradiated in different environments (reactive gas atmosphere, vacuum and
aqueous environment). Si irradiation with low laser fluence in vacuum and water resulted in the
development of different nano-topographies. By increasing laser fluence, three types of microcones were formed, i.e low, medium and high roughness micro-cones, fabricated both with linear
and circular laser polarization. The resulting micro-cones have been tested with in vitro cell cultures
before selecting the micro-cones fabricated with linearly polarized light to proceed to the
development of the combined nano-rippled and micro-coned (NR-MC) Si substrate. After
considering nano-topographies formed in Si with low laser fluence in both vacuum and water, the
nano-ripples fabricated in water were selected for the NR-MC substrates. The combination of two
different size-scales (micro-cones and nano-ripples) in a single substrate led to the development of
a spatial platform with different pseudoperiodic morphologies. The developed substrates were
characterized regarding surface morphology and wetting properties and were then used as cell
culture substrates in vitro.
The cell models used included mono-cultures of NIH 3T3 fibroblasts cells, glial cells (Schwann –
SW10), and neuronal-like cells (neuro2a – N2a), as well as co-cultures of SW10 and N2a cells.
NIH 3T3 cells were used as a well-studied experimental model for cellular functions, in order to
test some basic parameters of the experiment, such as cell survival and cell density. SW10 and N2a
cells were used as the glia and the neuronal-like cells of the PNS and their co-culture was attempted
because it can better simulate its actual structure. The cell growth was assessed on the different
topographies in comparison to flat Si control surfaces. The neuronal cell adhesion behavior and
differentiation was correlated to the underlying topography and to the presence of glia.
Our results show that by implementing the NR-MC combined topography, the synergistic role of
the glial and neuronal-like cells is impelled and the area where the N2a cells grow can be controlled.
The presence of the glial cells (SW10) influences the adhesion of the neuronal-like cells (N2a).
When they are co-seeded on the Si scaffolds, the N2a cells adhere on top of the oriented Schwann
cells and only on the areas where the Schwann cells adhere. Therefore, the characteristics of the
NR-MC Si surfaces can influence the neural cell growth. The NR-MC topography could then be
designed to comprise predefined areas that can either promote or inhibit neural cell adhesion and
network formation, depending on the therapeutical needs.
Moreover, Si nano- and micro- structures were used as culture platforms to evaluate the effect of
the topography on neuronal differentiation, both in a mono-culture and in a co-culture with glial
cells. The topography of the Si substrates was found to impede the N2a differentiation even in the
presence of a differentiation medium. More specifically, the higher the roughness, the more limited
the differentiation was. The same conclusion is also valid for the differentiation of N2a cells in a
co-culture with SW10 cells, i.e. the N2a differentiation is inhibited as the substrate becomes
rougher. We can therefore conclude that the presence of glia has the potential to change the
adhesion behaviour of the neuronal-like cells, but the ability to differentiate does not alter. This
implies that concerning neuronal differentiation, the topography plays a more important role than
the synergetic role of the glia. Further investigation via protein screening methods can shed light
on cell mechanotransduction, paving the way towards the understanding of how cells translate these
stimuli into biochemical signals that control the differentiation.
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