# 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.
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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) scaolds, using a coculture of endothelial
cells and support cells