Introduction
The biological, neurochemical and anatomical complexity of the human
nervous system challenges attempts to achieve neuronal repair or
regeneration after disease or traumatic injury. The over-arching goal is
to restore the functional properties of the nervous system, compensate
or substitute for tissue defects, and restore neural transmission
[1]. In relation to these goals, it is of considerable interest that
bioengineered scaffolds can induce topographical, chemical and
biological cues that effectively stimulate nerve regeneration [2].
However, there is consensus that natural microenvironments and theirin vivo physiological equivalent conditions cannot be fully
represented using two-dimensional (2D) cell cultures. Three-dimensional
(3D) cell models are a more reliable representation of the physiological
environment of living tissue which suitably replicate the in vivonative matrix and mimic the biophysical properties of remnant tissue
[3, 4].
The 3D cell culture products in tissue engineering development include
porous scaffolds, scaffold-free constructs, (self-assembling) hydrogels,
and microchips. Among these, pre-fabricated porous scaffolds are
recognized as the most promising platform to advance cell therapy and
drug discovery [5]. These scaffolds are used as a supportive matrix,
replicating the extracellular matrix of the central nervous system
(CNS). In vitro proof-of-principle work shows that 3D interstices
are conducive to cell attachment, migration, and infiltration [6]. A
tissue-engineered scaffold with suitable biocompatibility,
biodegradability and interconnected porosity is favourable in neural
tissue engineering (NTE) applications [7].
The literature presents multiple examples of polymer-based materials of
relevance to the fabrication of 3D scaffolds. Of these, the non-toxic,
biodegradable and biocompatible properties of alginate, a natural
biopolymer, have been extensively investigated for neural applications
[8]. The 3D alginate-based scaffolds of Ansari et al. (2017)
demonstrated efficacy to sustainably release neurotrophic factors and
enhance the proliferation and neurogenic differentiation of encapsulated
mesenchymal stem cells (MSCs) in vitro [9]. Another in
vitro study by Wang et al. (2017) demonstrated the aptitude of hybrid
scaffolds composed of chitosan and alginate to promote olfactory
ensheathing and neural stem cell (NSC) viability [10]. Similarly, anin vivo study by Prang et al. demonstrated the compatibility of
alginate-based scaffolds loaded with NSCs to dampen inflammatory
response in experimental spinal cord injury necessary to encourage
axonal regrowth [11]. Despite promising results, in vitro andin vivo , alginate-based constructs suffer from weak mechanical
strength, high degradation rate and electrical insulation at biological
frequencies, which might be attuned through combination with other
biomaterials [12].
Recently, graphene-based scaffolds have attracted intense interest for
their use in NTE due to their unique properties including large surface
area, excellent electrical conductivity, suitable biocompatibility,
chemical stability, and mechanical properties [13]. Importantly, the
high electrical conductivity of graphene provides a great electrical
coupling between regenerating nerve cells which is conducive to the
regeneration of excitable tissues [14]. Two graphene derivatives,
namely graphene oxide (GO) and reduced graphene oxide (RGO) are endowed
with unique physicochemical properties of interest to functional NTE
[15, 16]. Serrano et al. [17] showed that GO scaffolds improve
the differentiation of NSCs into mature neurons replete with axons,
dendrites and synapses and supportive glial cells. In addition,
researchers observed that biological properties and cytotoxicity of
graphene-based composites could be enhanced with respect to cell type,
the interaction between graphene and the matrix, graphene concentration
and composite production method [18]. These data suggest that
graphene-based composites may warrant closer investigation.
Details on the
physicochemical characterization of the GO powder used as well as
of the obtained rGO scaffolds were published elsewhere.
[ 35 ]
Numerous studies showed that coating reagents such as poly-l-lysine
(PLL) and laminin (LAM) on the surface of the scaffolds induce signals
regulating cell responses, adhesion and growth [19]. However, it is
interesting to point out that various coating reagents have different
impacts on cellular behaviour according to scaffold biomaterial and cell
type [20]. Culture medium is another consideration, with more
information required about the interaction between serum, protein
corona, and scaffold properties [21-23]. These data suggest a
requirement for well-designed laboratory protocols, taking into account
these considerations.
We have previously reported the mechanical, electrical and physical
properties of engineered 3D composite scaffolds consisting of GO and
sodium alginate (SA) (GOSA) [24]. Our study revealed that GOSA
composite porous scaffolds combine the known advantages of alginate
(including non-toxicity, biocompatibility, biodegradability) and
graphene (including hydrophilicity, excellent mechanical strength,
suitable biocompatibility, good electrical conductivity). The next step
was to realize that the incorporation of GO into SA to produce a
composite scaffold introduces excellent chemical properties, mechanical
strength and electrical conductivity, which perhaps can be harnessed to
exploit the CNS physiology. In our previous work, it was shown that GOSA
and RGOSA scaffolds with 0.5 and 1 wt. (%) concentrations and mean pore
sizes of 147.4 µm (GOSA0.5), 142.5 µm (GOSA1), 116.0 µm (RGOSA0.5), and
114.7 µm (RGOSA1) can accommodate stem cells, as an effective and
promising cell source in regenerative therapies, in culture. Therefore,
it will be important to establish cellular viability and cytotoxicity
data based upon potential mechanisms of graphene-based material
incorporation into the scaffold.
Various studies have
demonstrated the significant neurotrophic expression and
secretion of DPSC encompassing nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-
3), glial cell-line derived neurotrophic factor (GDNF), vascular
endothelial growth factor (VEGF) and platelet-derived neuro-
trophic factor (PDGF) [12, 25–27].
Neural crest-derived human dental pulp stem cells (hDPSCs), are a rich
source of mesenchymal stem cells (MSCs), with the ability for high
proliferation and multi-lineage differentiation capacity [25]. These
cells can be easily harvested from human exfoliated deciduous teeth,
permanent and primary teeth, and supernumerary teeth [26]. It has
also been shown that proliferation and cell number of DPSCs are greater
than bone marrow‐derived MSC [27]. Studies showed that hDPSCs can
differentiate into neuron-like cells and form functionally active
neurons, under the direction of appropriate environmental cues [28,
29]. DPSCs also appear to induce axonal guidance via stromal-derived
factor-1 (SDF-1) secretion, encouraging further exploration. Another
study by Nosrat et al. [30] showed that DPSCs express repertoire of
neurotrophic factor and stimulate neurogenesis and angiogenesis [31,
32]. Similarly, implantation of hDPSCs has been shown to significantly
improve forelimb sensorimotor function in cerebral ischemia rodent model
[33]. For all these reasons, hDPSCs represent a candidate stem cell
population for in vitro investigation of neural tissue repair.
Following the fabrication of composite graphene-based scaffolds, in this
paper, we present our investigations into the influence of graphene
incorporation, coating conditions, and DPSC donor type on the viability
and functions of DPSCs. The viability and cytotoxicity of DPSC-loaded
GOSA and RGOSA scaffolds have been assessed using the Alamar blue (AB)
and lactate dehydrogenase (LDH) activity assays. Furthermore, defined
serum-free media has been developed for the culture of DPSCs on the
fabricated scaffolds to overcome the problematic issues of using fetal
bovine serum (FBS) and make efficient clinical translations of stem
cell-based approaches.
In previous work, we engineered vascular networks on biocom-
patible and biodegradable poly(-lactic acid)(PLLA)/polylactic-
glycolic acid (PLGA) scaolds, using a coculture of endothelial
cells and support cells