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\title{Biodegradable and Biocompatible Graphene-based Scaffolds for Functional
Neural Tissue Engineering: A Strategy Approach Using Dental Pulp Stem
Cells and Biomaterials}



\author[1]{Negar Mansouri}%
\author[2]{Said Al-Sarawi}%
\author[1]{Dusan Losic}%
\author[2]{Jagan Mazumdar}%
\author[2]{Jillian Clark}%
\author[2]{S. Gronthos}%
\author[3]{Ryan Doig}%
\affil[1]{University of Adelaide}%
\affil[2]{The University of Adelaide}%
\affil[3]{South Australian Health and Medical Research Institute}%


\vspace{-1em}



  
  \date{April 25, 2026}


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\selectlanguage{english}
\begin{abstract}
Neural tissue engineering aims to restore function of nervous system
tissues using biocompatible cell-seeded scaffolds. Graphene-based
scaffolds combined with stem cells deserve special attention to enhance
tissue regeneration in a controlled manner. However, it is believed that
minor changes in scaffold biomaterial composition, internal porous
structure, and physicochemical properties can impact cellular growth and
adhesion. The current work aims to investigate in vitro biological
effects of 3D graphene oxide (GO)/sodium alginate (GOSA) and reduced
GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of
cell viability and cytotoxicity. Herein, the effects of the 3D
scaffolds, coating conditions, and serum supplementation on DPSCs
functions are explored extensively. Biodegradation analysis revealed
that addition of GO enhanced the degradation rate of composite
scaffolds. Compared to the 2D surface, the cell viability of 3D
scaffolds was higher (p \textless{}0.0001), highlighting the optimal
initial cell adhesion to the scaffold surface and cell migration through
pores. Moreover, the cytotoxicity study indicated that the incorporation
of graphene supported higher DPSCs viability. It is also shown that when
the mean pore size of scaffold increases, DPSCs activity decreases. In
terms of coating conditions, poly-l-lysine (PLL) was the most robust
coating reagent that improved cell-scaffold adherence and DPSCs
metabolism activity. The cytotoxicity of GO-based scaffolds showed that
DPSCs can be seeded in serum-free media without cytotoxic effects. This
is critical for human translation as cellular transplants are typically
serum-free. These findings suggest that proposed 3D GO-based scaffolds
have favourable effects on the biological responses of DPSCs.%
\end{abstract}%



\sloppy


\textbf{Biodegradable and Biocompatible Graphene-based Scaffolds for
Functional Neural Tissue Engineering: A Strategy Approach Using Dental
Pulp Stem Cells and Biomaterials}

Negar Mansouri \textsuperscript{a, b}, Said
Al-Sarawi\textsuperscript{a}, Dusan Losic \textsuperscript{b, c}, Jagan
Mazumdar\textsuperscript{a}, Jillian Clark \textsuperscript{d, e}, Stan
Gronthos\textsuperscript{f, g}, Ryan O'Hare Doig \textsuperscript{h}

\textsuperscript{a} School of Electrical and Electronic Engineering, The
University of Adelaide, Adelaide, Australia.

\textsuperscript{b} ARC Research Hub for Graphene-Enabled Industry
Transformation, The University of Adelaide, Adelaide, Australia.

\textsuperscript{c} School of Chemical Engineering and Advanced
Materials, The University of Adelaide, Adelaide, Australia.

\textsuperscript{d} Centre for Orthopaedics and Trauma Research,
Adelaide Medical School, Faculty of Health and Medical Sciences, The
University of Adelaide, Adelaide, Australia

\textsuperscript{e} South Australian Spinal Cord Injury Research Centre,
Hampstead Rehabilitation Centre, Lightsview, Adelaide, Australia.

\textsuperscript{f} Mesenchymal Stem Cell Laboratory, Adelaide Medical
School, Faculty of Health and Medical Sciences, The University of
Adelaide, Adelaide, Australia.

\textsuperscript{g} Precision Medicine Theme, South Australian Health
and Medical Research Institute (SAHMRI), Adelaide, Australia.

\textsuperscript{h} Neil Sachse Centre for Spinal Cord Research,
Lifelong Health, South Australian Health and Medical Research Institute
(SAHMRI), Adelaide, Australia.

\section*{Abstract}

{\label{abstract}}

Neural tissue engineering aims to restore function of nervous system
tissues using biocompatible cell-seeded scaffolds. Graphene-based
scaffolds combined with stem cells~deserve special attention to enhance
tissue regeneration~in a controlled manner. However, it is believed that
minor changes in scaffold biomaterial composition, internal porous
structure, and physicochemical properties can impact cellular growth and
adhesion. The current work aims to investigate \emph{in vitro}
biological effects of 3D graphene oxide (GO)/sodium alginate (GOSA) and
reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in
terms of cell viability and cytotoxicity. Herein, the effects of the 3D
scaffolds, coating conditions, and serum supplementation on DPSCs
functions are explored extensively. Biodegradation analysis revealed
that addition of GO enhanced the degradation rate of composite
scaffolds. Compared to the 2D surface, the cell viability of 3D
scaffolds was higher (p \textless{}0.0001), highlighting the optimal
initial cell adhesion to the scaffold surface and cell migration through
pores. Moreover, the cytotoxicity study indicated that the incorporation
of graphene supported higher DPSCs viability. It is also shown that when
the mean pore size of scaffold increases, DPSCs activity decreases. In
terms of coating conditions, poly-l-lysine (PLL) was the most robust
coating reagent that improved cell-scaffold adherence and DPSCs
metabolism activity. The cytotoxicity of GO-based scaffolds showed that
DPSCs can be seeded in serum-free media without cytotoxic effects. This
is critical for human translation as cellular transplants are typically
serum-free. These findings suggest that proposed 3D GO-based scaffolds
have favourable effects on the biological responses of DPSCs.

\textbf{Keywords:} Neural tissue engineering, 3D scaffolds, Graphene,
Stem cell, Biocompatibility

\section*{Introduction}

{\label{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
their\emph{in 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 \emph{in
vivo}native 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). \emph{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) \emph{in vitro} {[}9{]}. Another \emph{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,
an\emph{in 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, \emph{in vitro}
and\emph{in 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 \selectlanguage{greek}µ\selectlanguage{english}m (GOSA0.5), 142.5 \selectlanguage{greek}µ\selectlanguage{english}m (GOSA1), 116.0 \selectlanguage{greek}µ\selectlanguage{english}m (RGOSA0.5), and
114.7 \selectlanguage{greek}µ\selectlanguage{english}m (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 \emph{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

\section*{Experimental Section}

{\label{experimental-section}}

\textbf{2.1. Materials}

Sodium alginate and Calcium chloride dried were purchased from
Chem-Supply. Alpha-MEM (Gibco, Life Technologies, Australia, Cat. No.
12561056) was supplemented with fetal bovine serum (FBS, USA origin,
Life Technologies, Australia), penicillin /streptomycin (Gibco),
L-ascorbic acid-2-phosphate (Sigma-Aldrich), and 2mM L-Glutamine (Life
Technologies, Australia). Natural mouse Laminin (Gibco-Life
Technologies) and 0.01\% Poly-l-lysine (PLL) solution (Sigma) were used
as coating reagents. Trypsin-EDTA was supplied by Gibco Life
Technologies, Australia. Clear-bottom 24-well and 96-well
plates~(Costar\selectlanguage{ngerman}®,~Corning, NY, USA)~were used throughout the study. The
cytotoxicity assay was performed following the manufacturer's protocol
using LDH kit from Promega Corporation (WI, USA). The
AlamarBlue\textsuperscript{TM} cell viability reagent was supplied by
Invitrogen--ThermoFisher Scientific, USA.

\textbf{2.2. GOSA/RGOSA Scaffold Fabrication and Preparation}

The graphene-based composite scaffolds were fabricated by a technique
involving solution mixing, freeze-drying, crosslinking, and
bio-reduction as described previously {[}24{]}. Briefly, 2 wt. \% sodium
alginate powder was dissolved in deionized water until a transparent
solution is obtained. Then, GO (synthesized by modified hummers' method
as described earlier {[}34{]}) was added to the solution to get a final
concentration of 0.5 and 1 wt. \%. The mixture was frozen overnight at
-18 °C and fabricated into a porous structure by the freeze-drying
technique. The obtained aerogels were crosslinked in 1 M calcium
chloride (CaCl\textsubscript{2})~solution for 1 hour and freeze-dried
again to form GOSA scaffolds. The obtained scaffolds are named as
GOSA0.5 and GOSA1 according to the concentration of GO in the composite
sample. SA scaffolds were fabricated without the addition of GO. In
order to make conductive RGOSA scaffolds, the obtained GOSA0.5 and GOSA1
scaffolds were treated with gelatin solution (1 mg
mL\textsuperscript{-1}) at 95°C to produce conductive RGOSA0.5 and
RGOSA1 scaffolds, respectively. The prepared scaffolds were cut in 2 mm
thickness (diameter \selectlanguage{english}[?]14 mm) until further use. Details on the
physicochemical characterization of the GOSA and RGOSA scaffolds can be
found in {[}24{]}.

\textbf{2.3. Scanning Electron Microscopy (SEM)}

The cross-sectional area of scaffolds was captured by a scanning
electron microscope (SEM, Hitachi, SU1510 high technologies) at an
acceleration voltage of 30 kV to observe the morphology and porosity of
samples.

\textbf{2.4. \emph{In vitro} biodegradation study}

The \emph{in vitro} ~biodegradation study aims to investigate the
biodegradation rate of prepared scaffolds {[}35{]}. Briefly, each
scaffold (\(n=3\)) with known weight (\(W_{0}\)) was
incubated at 37degC in alpha-MEM culture media containing 1\% (v/v)
penicillin/streptomycin to prevent bacterial growth. At specific time
points, scaffolds were rinsed with ddH\textsubscript{2}O. Then, samples
were dried under vacuum and weighed (\(W_{1}\)). The extent of
biodegradation was calculated using the following formula.

\emph{In vitro} Biodegradation (\%)\(=\frac{W_{0}-W_{1}}{W_{0}}\times 100\)

\textbf{2.5. Sterilization and coating of scaffolds}

Before cell seeding, the scaffolds were sterilized with 80\% ethanol for
24 hours, then allowed to dry overnight and washed twice with DPBS. For
coating, scaffolds were incubated at room temperature in poly-l-lysine
(PLL) (10 \selectlanguage{greek}µ\selectlanguage{english}g/mL) overnight followed by incubation at 37 \selectlanguage{ngerman}°C in laminin
(LAM) solution (10\selectlanguage{english} \selectlanguage{greek}µ\selectlanguage{english}g/mL) overnight. Non-coated (NC) scaffolds, which
were wetted but not incubated with any coating material, served as
control.

\textbf{2.6. Cell culture}

Donor DPSCs (passage 6-7) were cultured in Alpha modification of Eagle's
medium (\selectlanguage{greek}α-\selectlanguage{english}MEM) supplemented with 10\% FBS, 1\% penicillin (100
U/mL)/streptomycin (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL), 100 \selectlanguage{greek}μ\selectlanguage{english}M L-ascorbic acid 2-phosphate, and
2 mM L-glutamine at 37 \selectlanguage{ngerman}°C in 5\% CO\textsubscript{2} humidified
atmosphere. Cells reaching 80--90\% confluency were harvested using
0.05\% (w/v) trypsin/ethylenediaminetetraacetic acid (EDTA) solution and
seeded onto scaffolds in 24-well culture plates (100 \selectlanguage{greek}µ\selectlanguage{english}L/well) at 1, 2,
4, 8, or 16\selectlanguage{ngerman}×10\textsuperscript{4} cells/sample. The scaffolds were
incubated for 3 hours at 37 °C with 5\% CO\textsubscript{2} to allow
diffusion of cells through the network of pores before adding culture
media (550 \selectlanguage{greek}µ\selectlanguage{english}L/well). DPSCs cultured on the surface of the well plate
(i.e. No scaffolds) were used as 2D control.

\textbf{2.7. Cell viability using Alamar Blue assay}

Alamar Blue (AB) assay was used to quantitively measure the metabolic
activity of living cells on the scaffolds by detecting the
oxidation-reduction rate of AB reagent {[}36{]}. The effects of cell
seeding density and coating condition of the various 3D porous scaffolds
on the viability of DPSCs were evaluated. Briefly, after 24 and 48 hours
of cell seeding on scaffolds, 1 mL of 10\% AB solution was added to each
well (250 \selectlanguage{greek}µ\selectlanguage{english}L/well). The plates were shaken gently (200 rpm) for 5 min
and incubated for 4h at 37 \selectlanguage{ngerman}°C with 5\% CO\textsubscript{2}. After
incubation, 100 \selectlanguage{greek}μ\selectlanguage{english}L of each sample was transferred to a 96-well plate and
the fluorescence intensity was recorded at an excitation wavelength of
540 nm and an emission wavelength of 600 nm using a spectrophotometer
(BioTek Synergy H1 multi-mode reader). Non-seeded scaffolds supplemented
with 10\% AB dye were used as a negative control to confirm that the
fluorescence intensity of scaffolds alone did not interfere with the
assay. Each experimental condition was conducted in triplicates, and
experiments were replicated twice.

\textbf{2.8. Cytotoxicity study using Lactate Dehydrogenase (LDH) assay}

The cytotoxic effects and level of toxicity of fabricated 3D scaffolds
seeded with DPSCs, considering various coating conditions and
inclusion/exclusion of serum in media, were measured by lactate
dehydrogenase (LDH) assay for up to two days under proliferation
conditions. The LDH kit (CytoTox 96\selectlanguage{ngerman}® non-radioactive cytotoxicity assay,
Promega) was used and LDH assay was performed according to
manufacturer's instructions in which the number of cytotoxic cells is
quantified by measuring cytosolic LDH enzyme leakage into the culture
medium as a result of cell membrane damage {[}37{]}. Density 3
(4×10\textsuperscript{4} cells/sample) was chosen to compare the~DPSCs
toxicity of scaffolds. Briefly, 50 \selectlanguage{greek}µ\selectlanguage{english}L of cell culture media was
collected from 24-well plates after 24 and 48h following DPSC seeding
and transferred into a fresh 96-well plate. 50 \selectlanguage{greek}µ\selectlanguage{english}L of LDH assay mixture
was then added to each well containing the supernatant and incubated at
room temperature in dark for 30 minutes. After 30 min, the reaction was
stopped with HCl (1 N, 10 vol \%) and absorbance values were obtained at
490 nm using a 96-well plate reader (GloMax Discovery microplate
reader). DPSCs grown without scaffolds (2D controls) were incubated with
lysis solution for 45 min and used as positive controls (100\% dead,
maximum LDH release control). The cytocompatibility performance of the
scaffolds was analysed by using the absorbance of the experimental
groups and the negative control group (scaffold only, with no cells).
Cytotoxicity data are presented as the average of three replicates. Two
different donors were selected to determine the effects of donor type on
cytotoxic effects of DPSCs with and without FBS in culture media.

\textbf{2.9. Statistical analysis}

Data are graphically reported as mean~\selectlanguage{ngerman}±~SEM (standard error of mean) of
at least three independent samples. Statistical analysis was performed
by two-way analysis of variance (ANOVA) with a significance level
of\(p<0.05\) followed by post hoc Dunnett test. The analysis was
carried out on GraphPad Prism software.

The correlation between cell viability and mean pore size was determined
by the Spearman Rank Order Correlation test.

\section*{Results}

{\label{results}}

\textbf{3.1. Structural analysis}

The microstructure of fabricated scaffolds was assessed using SEM images
(Fig. 1a-e). The data revealed that all scaffolds exhibited homogeneous
porous structures with interconnected pores. As reported in our previous
study {[}24{]}, the average pore sizes were measured as 162.5, 147.4,
142.5, 116, and 114.7 \selectlanguage{greek}µ\selectlanguage{english}m for SA, GOSA0.5, GOSA1, RGOSA0.5 and RGOSA1,
respectively. These observations show the dependency of composite
scaffold pore size on GO concentration.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 1. SEM images of (a) SA, (b) GOSA0.5, (c) GOSA1, (d) RGOSA0.5 and
(e) RGOSA1 scaffolds showing connected porous structure of fabricated
scaffolds to facilitate 3D DPSCs culture.

\textbf{3.2. Biodegradation study}

To evaluate the effect of GO addition on the degradation rate of
composite scaffolds, weight loss was expressed as the percentage (\%) of
biodegradation within three weeks (Fig. 2). The biodegradation of GOSA
and RGOSA scaffolds containing different percentages of GO were analysed
over a three-week period. At day 3, there was gradual biodegradation of
all GOSA and RGOSA scaffolds by approximately 20\% of initial weight. In
contrast, SA scaffolds showed degradation percentages of approximately
31\%. The difference between all GO-based scaffolds and the SA scaffold
(no-graphene control) was statistically significant (\(p<0.0001\))
from day 2 to day 21. Fig. 2 presents \emph{in vitro} biodegradation
data for all five scaffolds. The mean (SEM) weight loss between day-0
and day-21 was approximately 24.86\%\selectlanguage{ngerman}±1.34, 32.42\%±0.68, 23.19\%±1.60,
and 31.42\%±1.20 for GOSA0.5, GOSA1, RGOSA0.5, and RGOSA1 scaffolds,
respectively. The SA scaffold (containing no graphene) showed the
highest weight loss of approximately 44.02\% at day 21, while hybrid
scaffold weight loss stabilised after 7 days.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 2. The biodegradation rate expressed as a percentage (SEM) of SA,
GOSA0.5, GOSA1, RGOSA0.5 and RGOSA1 scaffolds from 0 to 3 weeks.

\textbf{3.3. Enhancement of cell viability using 3D culture system}

The viability of DPSCs grown in 2D or 3D scaffolds was assessed using
the AB assay. After 24 h of cell culture (Fig. 3), there was a
significant increase in the cellular activity of SA and GOSA scaffolds
seeded with DPSCs compared to DPSCs grown under 2D conditions at all
cell densities (\(p<0.0001\)). Cells seeded directly on the
surface had an average (SEM) AB reduction of 53.7\%±1.00 across all five
seeding densities. Furthermore, cells on 3D scaffolds were found to be
more viable when compared to the no scaffold (2D) condition (Fig. 3).

\textbf{3.4. Comparison based on cell densities}

Both scaffolds (SA and GOSA) supported cell viability across various
cell densities with no negative effect on seeding efficiency, after 24h
of cell culture (Fig. 3). The data showed that AB reduction increased
significantly, as an indication of metabolic activity, in SA and GOSA1
scaffolds at all five cell densities.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 3. 24-hour Alamar Blue reduction (\%) of 2D surface (No Scaffold),
SA and GOSA scaffolds (means, SEM) at five cell seeding densities; *
indicates statistical significance (***p \textless{} 0.001, ****p
\textless{} 0.0001).

\textbf{3.5. Increased DPSCs viability on scaffolds coated with PLL}

As DPSCs viability was not affected by seeding density conditions,
densities 1, 3 and 5 were selected to conduct AB analysis over 24- and
48-hours. In order to determine the effects of coating conditions on
seeded DPSCs, three different coating conditions (NC, PLL, and PLL+LAM)
were used. The effect of cell seeding densities was impacted by various
coating conditions following 24 hours of culture. Significantly higher
AB reduction percentage was observed for SA and GOSA scaffolds at all
three densities compared to the 2D control condition (Fig. 4a-c). After
48 h of DPSCs culture, statistically significant differences were
detected in the proliferation profiles of cells growing in 2D and 3D
under various coating conditions (Fig. 4d-f). In addition, PLL coating
significantly increased the cellular activity of GOSA scaffolds at all
three cell densities within 48 hours of DPSCs culture (Fig. 4).

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 4. (a-c) 24- and (d-f) 48-hour change (means, SEM) in Alamar Blue
reduction percentage of No scaffold, SA and GOSA1 scaffolds across (a,
d) Density 1 (1×10\textsuperscript{4}cells/scaffold), (b, e) Density 2
(4×10\textsuperscript{4}cells/scaffold), and (c, f) Density 5
(16×10\textsuperscript{4}cells/scaffold); * indicates statistical
significance (**p \textless{} 0.01, ***p \textless{}0.001, ****p
\textless{} 0.0001).

\textbf{3.6. Scaffold biomaterial composition plays a key role in
cellular functions}

To explore the effects of scaffold biomaterials on cellular functions,
the metabolic activity of cultured DPSCs on various scaffolds was
measured using the AB assay. In SA scaffolds, AB reduction (\%) was
maintained at the 2D cell culture (Fig. 5). In contrast, the AB
reduction was significantly increased for GOSA1, RGOSA0.5 and RGOSA1
scaffolds compared to SA only scaffolds across all coating conditions. A
significantly higher degree of reduction of the AB dye was observed in
DPSCs grown on PLL-coated graphene-based scaffolds compared to those
cultured on a 2D surface. This highlights the importance of PLL coating
(as previously proved in coating conditions), where the highest AB
reduction percentage was recorded for PLL-coated RGOSA1 as 93.66\%±5.88.

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 5. Alamar Blue reduction (\%, means, SEM) of DPSCs seeded on 2D
surface (No scaffold), SA, GOSA0.5, GOSA1, RGOSA0.5 and RGOSA1 scaffolds
after 48h; * indicates statistical significance (*p \textless{} 0.05,
**p \textless{} 0.01, ****p \textless{} 0.0001).

\textbf{3.7. Lower cytotoxic effects of graphene-based scaffolds}

The cytotoxicity of fabricated scaffolds was examined using analysis of
lactate dehydrogenase (LDH) release in culture media. In this study, LDH
release was taken as a marker of DPSCs membrane damage. At 24 hours of
DPSCs culture, all biomaterials showed a relatively low level of
released LDH (Fig. 6a). However, significant increase in the LDH levels
of DPSCs were observed for uncoated SA (41.65\%±7.35) and PLL-coated
GOSA0.5 (34.31\%±5.31) scaffolds after 24 hours of cell culture. It is
interesting to note that, after 48 hours of culture, DPSCs toxicity
obtained by LDH assay on graphene-based scaffolds were not significantly
higher than the 2D surface (Fig. 6b). However, DPSCs cultivated onto
pure SA scaffolds displayed the highest levels of cytotoxicity as
compared to 2D control (no scaffold) (\(p<0.0001\)).

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 6. Cell cytotoxicity (mean, SEM) of each scaffold measured by LDH
assay in no coating, PLL coating and PLL+LAM coating conditions at (a)
24 and (b) 48 hours of DPSCs culture; * indicates statistical
significance (*p \textless{}0.05, ****p \textless{}0.0001).

\textbf{3.8. Better cellular behaviour in scaffolds with smaller mean
pore size}

The AB and LDH assay results were utilized to determine the effects of
scaffold pore size on cellular behaviour. The data showed that cellular
activity decreases when the mean pore size increases on PLL-coated
scaffolds (Fig. 7a). The lowest AB reduction percentages were measured
for PLL+LAM coated SA and GOSA0.5 scaffolds with the largest mean pore
sizes as 36.12\% and 38.33\%, respectively. Accordingly, the relatively
lowest cytotoxicity was observed for RGOSA0.5 (116.0 \selectlanguage{greek}µ\selectlanguage{english}m) and RGOSA1
(114.7 \selectlanguage{greek}µ\selectlanguage{english}m) scaffolds with smaller mean pore sizes compared to SA and
other composite scaffolds (Fig. 7b). Overall, a negative correlation
(Spearman R\(=-0.83\)) was observed between mean pore size of
scaffolds and cellular activity (obtained by AB assay). Conversely, a
weak positive correlation (Spearman R\(=0.06\)) was seen
between the mean pore size of scaffolds and cytotoxicity (obtained by
LDH assay). These findings indicate that increasing mean pore size
decreases DPSCs metabolic activity with no corresponding relationship to
cytotoxicity.

\textbf{Hosted file}

\verb`image7.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 7. Effect of mean pore sizes on (a) cell activity and (b) cell
toxicity (mean, SEM) within various scaffolds (n=3) 48h after incubation
(mean pore sizes: SA=162.5 \selectlanguage{greek}µ\selectlanguage{english}m, GOSA0.5=147.4 \selectlanguage{greek}µ\selectlanguage{english}m, GOSA1=142.5 \selectlanguage{greek}µ\selectlanguage{english}m,
RGOSA0.5=116.0 \selectlanguage{greek}µ\selectlanguage{english}m and RGOSA1=114.7 \selectlanguage{greek}µ\selectlanguage{english}m). There is a strong negative
correlation between cellular activity and mean pore size (Spearman R =
-0.83) and only a weak positive correlation between cell toxicity and
mean pore size (Spearman R = 0.06), that is, the enlargement of mean
pore size led to a decrease in cellular activity and did not correlate
with cellular toxicity.

\textbf{3.9. Serum supplemented scaffolds greatly increased
cytotoxicity}

DPSCs cultured for 24h in serum-free media on all graphene-based
scaffolds caused no significant increases in cytotoxicity levels
compared to cell only (2D) control, irrespective of coating conditions
(Fig. 8a). The data also indicated significantly lower cell toxicity of
PLL+LAM-coated RGOSA1 scaffolds (1.81\%\selectlanguage{ngerman}±3.57), compared to no scaffold
condition. Furthermore, quantitative LDH activity measurements (Fig. 8b)
showed no significant differences after 24 hours between all 3D
graphene-based scaffolds seeded with DPSCs using serum-containing media,
when compared to 2D control. These cytotoxic effects were not influenced
by coating conditions. However, the percentage of cytotoxic effects of
PLL+LAM-coated SA scaffolds (24.83\%±4.77) was the highest amongst other
scaffolds assessed across all coating conditions.

There are no significant increases in the percentage of cell toxicity of
DPSCs exposed to coated and uncoated GOSA and RGOSA scaffolds in
comparison to the 2D control (Fig. 8c). However, after 48 h of cell
culture, a significant elevation of LDH release was detected when DPSCs
were cultured on SA scaffolds with serum deprivation, with the highest
cell toxicity percentage of 36.22\%±1.26 for PLL+LAM coating. The cell
toxicity percentage of almost all samples tested with no serum was found
to have increased in comparison to the corresponding conditions at the
24 h time point. In addition, when DPSCs in serum-rich media were seeded
onto fabricated 3D scaffolds, cell toxicity of scaffolds increased
significantly in comparison with no scaffold culture, regardless of
coating conditions (Fig. 8d). After 48h of DPSCs culture with serum,
cell cytotoxicity was the highest in SA (20.78\%±2.95), GOSA1
(16.95\%±3.38), and RGOSA1 (16.43\%±3.58) matrices coated with PLL.
Furthermore, at 48 h of cell culture with serum, the cell toxicity
percentage of all evaluated samples was reduced when compared with the
corresponding 24 h time point.

\textbf{Hosted file}

\verb`image8.emf`  available at \url{https://authorea.com/users/389095/articles/503728-biodegradable-and-biocompatible-graphene-based-scaffolds-for-functional-neural-tissue-engineering-a-strategy-approach-using-dental-pulp-stem-cells-and-biomaterials}

Fig. 8. Comparative evaluation of LDH assay (mean of two different
donors, SEM) by DPSCs grown on SA, GOSA1 and RGOSA1 scaffolds at (a, c)
24- and (b, d) 48-hours post-seeding in (a, b) serum-free and (c, d)
serum-containing media; * indicates statistical significance (*p
\textless{}0.05, **p \textless{} 0.01, ***p \textless{} 0.001, ****p
\textless{} 0.0001).

\section*{Discussion}

{\label{discussion}}

Significant complexity has been uncovered in the interaction between
stem cells and engineered scaffolds {[}38{]}. In this study, we report
the profiles of DPSCs cultured on fabricated 3D GOSA and RGOSA scaffolds
with 2D, coating and media controls.

Our \emph{in vitro} biodegradation data revealed an inverse relationship
between GO concentrations and weight loss. This relationship is
explained by accessibility of water molecules to GO composites as a
function of GO concentration. As discussed in the water contact angle
measurements in our previous paper {[}24{]}, the incorporation of GO
increases the interaction of composite scaffolds with water and this
effect is GO concentration-dependent. Therefore, as hydrophilicity
accelerates with higher GO concentration, water-mediated scaffold
degradation increases {[}39{]}. Notably, a reduced degradation rate is
assumed to be beneficial for tissue regeneration {[}40{]}. Collectively,
our results confirm that the degradability of composite 3D R/GOSA
scaffolds is adjustable and controlled by graphene content.

It is noteworthy that when cultured onto 3D SA and GOSA scaffolds, DPSCs
viability was enhanced in relation to 2D culture plates. This
relationship clearly signifies the optimal condition of initial cell
adhesion to the scaffold surface to promote subsequent cell
proliferation and infiltration. Our observation of an increase in the
total metabolic activity of cell-seeded scaffolds provides
proof-of-principle support for cell growth and proliferation in a 3D
matrix which can act as a delivery system for seeded cells. Thus, it is
reasonable to conclude that an artificial 3D scaffold is an acceptable
approach to mimic the natural architecture of the native tissue and
crease a microenvironment conducive to DPSCs engraftment.

The cell-cell interaction within the matrix of 3D cell culture systems
has a profound influence on cellular functions including viability,
migration and proliferation in contrast to 2D culture {[}41{]}. For
example, one report showed that 3D polymer-based scaffolds seeded with
hepatic cells had less cytotoxic effects than those cultured in 2D
{[}42{]}. In another study, the 3D culture of dental stem cells was
found to support their neuronal characteristics and maintain cell
phenotypes {[}43{]}. Extending this, we have confirmed that a superior
proliferative ability of DPSCs, as measured by metabolic activity, can
be obtained when cells are cultured on 3D porous scaffolds.

Regarding cell seeding density, we have shown that when cells are seeded
on 3D scaffolds at all densities, the cell proliferation rate is
significantly increased in comparison to 2D. Moreover, we found that
increased seeding density in 3D scaffolds could be achieved without
inducing cytotoxic effects, as determined by LDH assay. In this study,
we also showed that the addition of graphene to 3D composite scaffolds
improved cellular behaviours and this was seen across all DPSCs seeding
densities examined. Notably, the degree of cellular metabolic activity
did not differ significantly between the different cell densities
tested. This result is important because the implication is that seeding
efficiency can be achieved even at high cell densities, at least within
a 48-hour period. More work would be required to determine if high
seeding densities might have different effects on longer-term culture.

Assessment of coating reagents revealed that PLL+LAM coating did not
affect cell viability as indicated by AB reduction percentage. After 48
hours of DPSCs culture, both laminin coating and no coating conditions
decreased cell viability on both SA and GOSA scaffolds. Possible
explanations involve interactions between coating properties and DPSCs
adherence or cell aggregation which can decrease proliferation {[}44{]}.
Interestingly, PLL was identified as the coating reagent that enhanced
cell-matrix adherence. This enhancement might be due to the larger
number of cationic sites offered by PLL coating on the 3D surface. In
agreement with another study {[}44{]}, our results show that PLL is
superior to laminin coating. In addition, the effect of all three
coating conditions on DPSCs was irrespective of cell seeding density.

The AB assessment of metabolic activity shows that biomaterial
composition can modulate DPSCs responses to fabricated scaffolds. Our no
scaffold controlled LDH results also indicate that SA, GOSA and RGOSA
scaffolds materials are nontoxic to DPSCs in short-term culture. Based
on previous work, it might be inferred that the composition of a
scaffold material can have a direct effect on the biodistribution of
secreted factors~that~in turn influence the stem cell fate. The results
appear to suggest that different scaffolds with varying material
properties (such as blend ratio, swelling index, or microstructure)
elicit diverse DPSCs behaviours. The increase in cell viability
observed\emph{in vitro} upon the incorporation of graphene in composite
scaffolds is consistent with other studies {[}36, 45{]}. Published data
suggest that the outstanding surface properties and adsorption capacity
of graphene-based nanomaterials are the main contributors to the
observed DPSCs responses.

Our results showed a strong influence of pore size, material composition
and substrate dimensionality on cell viability, in accordance with the
findings of Domingos et al. {[}46{]}. Comparisons of AB reduction in SA
(97.2\%) and graphene-based scaffolds including GOSA0.5 (97.5\%), GOSA1
(98.0\%), RGOSA0.5 (99.05\%), and RGOSA1 (99.18\%) revealed the
differences in DPSCs proliferation markers across various graphene-based
scaffolds. These differences appear to be explained by variations in
scaffold porosity (\%) such that scaffolds with higher porosity (RGOSA \selectlanguage{english}[?]
99\%) are able to accommodate higher numbers of viable cells.
Furthermore, it was shown that scaffolds with smaller mean pore sizes
induce relatively less toxicity. This can be explained by the available
surface area of scaffolds for cultured cells or applying the principle
that the mean pore size and specific surface area are inversely
proportional. In consideration of the specific surface area and mean
pore size of a scaffold, biophysical properties can affect cell adhesion
{[}47{]}. It follows that the low levels of cell adhesion are observed
on scaffolds with larger pore size and less specific surface area {[}48,
49{]}. As a result, the available specific surface area per unit volume
for cell adhesion of each fabricated scaffolds can be calculated using
mean pore sizes {[}50{]}. Accordingly, the normalized specific surface
area of GOSA and RGOSA scaffolds, as shown in Table 1, can be obtained
by dividing the mean pore size of each scaffold by mean pore size of SA
scaffold (3D control). Thus,~the higher AB reduction observed in our
RGOSA1 scaffold can be explained by the higher specific surface area in
comparison with GOSA1 and SA scaffolds. These data indicate that higher
pore size facilitates increased DPSCs migration and proliferation, in
agreement with a previous report on culturing DPSCs into 3D
poly-L-lactic acid-based scaffolds {[}51{]}. In addition, the mechanical
properties of scaffolds with overly large pores are compromised, whereas
higher cellular proliferation within large pore sizes can have
implications for differentiation {[}52{]}.

Table 1. Estimates of the specific surface area of graphene-based
scaffolds relative to SA scaffold\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Sample & Mean pore size (\selectlanguage{greek}µ\selectlanguage{english}m) & Normalized Specific Surface
Area\tabularnewline
\midrule
\endhead
SA & 162.5 & 1\tabularnewline
GOSA0.5 & 147.4 & 0.907\tabularnewline
GOSA1 & 142.5 & 0.876\tabularnewline
RGOSA0.5 & 116.0 & 0.713\tabularnewline
RGOSA1 & 114.7 & 0.705\tabularnewline
\bottomrule
\end{longtable}

Our cytotoxicity findings are consistent with several other studies
using GO layer cultured with mesenchymal stromal cells and GO/chitosan
scaffolds seeded with human adipose-derived stem cells {[}53, 54{]}. In
our study, LDH measurements showed significantly higher levels of DPSCs
toxicity for uncoated SA scaffolds which can be attributed to poor
ability and lack of efficient sites to support cell adhesion and
proliferation. This result is supported by another study utilizing other
materials mixed with alginate to create biocomposites {[}55{]}. However,
the incorporation of GO into GOSA scaffolds did not elicit significant
differences in cytotoxic effects after 2 days of DPSCs culture. Overall,
the GO-enriched scaffolds exhibited cytotoxicity of 15--27\% after 48h
of culture, suggesting that these materials are biocompatible with
DPSCs. Therefore, GO has no apparent cytotoxic effect but exhibits
positive effects on cell function in long-term DPSCs culture.

The use of serum-free or serum-rich culture media for biological assays
is a contentious issue {[}56{]}. The associated clinical uncertainties
with the use of FBS include immune rejection, batch-to-batch variations,
ethical concerns, interfering effects of unidentified growth factors and
proteins, and viral contamination {[}57{]}. Regarding DPSCs stability,
there is clear evidence about the positive effects of serum-free culture
media on retaining stemness {[}58, 59{]}. Furthermore, serum-free
cultured DPSCs within chitosan scaffolds expressed stem cell markers
(Nestin and Sox2) and survived successfully after transplantation into
the rodent spinal cord {[}60{]}. The present study investigated whether
serum depletion facilitates the attachment of DPSCs onto hybrid
scaffolds. Thus, the effects of serum on the cytotoxicity of DPSCs were
tested using 2D and 3D culture systems across three different coating
conditions at a single cell density of 4 \selectlanguage{ngerman}×
10\textsuperscript{4}cells/sample. We have indicated that the presence
of serum caused significantly higher cytotoxic behaviour of coated 3D
scaffolds which could be due to suboptimal surface-cell attachment
{[}20{]}. This was also shown for the combination of PLL and LAM
coatings in the presence of serum on 3D structures. It is surmised that
coatings lower the available surface area of 3D scaffold biomaterials
for the attachment of cell receptors, which subsequently causes poor
adhesion. In contrast, coated 2D surface was found to result in better
attachment of DPSCs which is due to the unfavourable bare tissue culture
surface. Moreover, our study found that the cytotoxicity of DPSCs
cultured in serum-rich media on 3D scaffolds increased significantly in
comparison with the 2D control. The increased level of cytotoxicity in
serum-containing medium could be attributed to the surface oxygen
content of GO and RGO, which favours FBS adsorption. This could be due
to the formation of protein corona on the GO surface which in turn
influence the toxicity of GO-based materials {[}61, 62{]}. Furthermore,
studies have indicated that serum proteins can have an interfering
effect on interactions between nanoparticles, cells and biological
molecules {[}63-65{]}. Accordingly, Lesniak et al. {[}64{]} demonstrated
that various protein corona formed on silica nanoparticles modified cell
adhesion, cellular uptake, and toxicity, which is determined by the
serum protein concentration. It was also shown that serum-containing
media resulted in lower cell adhesion and internalization efficiency of
silica nanoparticles. The present study found DPSCs can be seeded in
serum-free media onto GOSA and RGOSA scaffolds with no cytotoxic
effects, showing promising potential for clinical translation as
cellular transplants are typically serum-free.

\section*{Conclusion}

{\label{conclusion}}

Alginate-based scaffolds have been extensively investigated for NTE,
however, they faced some limitations that hampered their further
developments. Importantly, alginate-based scaffolds have poor mechanical
strength and degradation rate, which are not matched with native tissue
microenvironment. It was found that incorporation of graphene within the
alginate matrix can address these drawbacks. However, it is important to
investigate cellular viability and cytotoxicity of 3D graphene-based
composite scaffold. In addition, it is revealed that coating of
scaffolds induces cell functions, adhesion and growth. Therefore, this
study examined the biodegradation, biocompatibility, bioactivity and
cytotoxicity of neural crest-derived DPSCs loaded graphene-based 3D
composite scaffolds, using three different coating conditions.

It was shown that the composite GOSA and RGOSA scaffolds have controlled
biodegradability which is effective in therapeutic tissue engineering
applications. DPSCs viability cultured onto SA and GOSA scaffolds was
higher than that of on 2D controls thus signifying surface cell adhesion
followed by cell infiltration through the porous matrices. Therefore,
superior proliferative ability of DPSCs can be obtained when cells are
cultured on 3D porous scaffolds. The LDH assay showed comparable DPSCs
toxicity on the GOSA and RGOSA scaffolds to that obtained on a 2D
surface in the absence of the biomaterial, highlighting no significant
cytotoxic effects of graphene incorporation after 2 days of DPSCs
culture. Furthermore, smaller mean pore size of scaffolds resulted in
higher cellular activity and relatively less cytotoxicity, which is due
to more available specific surface area on scaffolds with smaller mean
pore sizes. In terms of coating conditions, PLL was the most robust
reagent that improved cell-matrix adherence and affected metabolism
activity of DPSCs, being superior to combined PLL+LAM coating.
Furthermore, the cytotoxicity of GOSA and RGOSA scaffolds in the
presence of serum is increased compared to serum-free condition,
indicating that DPSCs can be cultured in serum deprivation onto the
fabricated scaffolds for clinical translation. The findings from the
current study suggest that the proposed 3D graphene-based composite
scaffolds had a favourable effect on the biological responses of DPSCs
which could be exploited in further DPSCs differentiation and electrical
stimulation for functional NTE.

\section*{Declaration of Competing
Interest}

{\label{declaration-of-competing-interest}}

The authors declare no conflict of interest.

\section*{Acknowledgement}

{\label{acknowledgement}}

This study was supported by AOSpine Asia Pacific Research Grant 2019
(AOSAUNZ(R) 2019-05). The first author wishes to acknowledge the
University of Adelaide for awarding Adelaide Scholarship International
(ASI) for her PhD study. The authors also appreciate the support from
the Australian Research Council Research Hub for Graphene Enabled
Industry Transformation (project no. IH 150100003). Also, special thanks
go to Mr Arash Mazinani for his support with SEM imaging.

\section*{References}

{\label{references}}

{[}1{]} C.E. Schmidt, J.B. Leach, Neural tissue engineering: strategies
for repair and regeneration, Annual review of biomedical engineering
5(1) (2003) 293-347.

{[}2{]} L. Ghasemi-Mobarakeh, M.P. Prabhakaran, L. Tian, E.
Shamirzaei-Jeshvaghani, L. Dehghani, S. Ramakrishna, Structural
properties of scaffolds: crucial parameters towards stem cells
differentiation, World journal of stem cells 7(4) (2015) 728-744.

{[}3{]} J.S. Joseph, S.T. Malindisa, M. Ntwasa, Two-dimensional (2D) and
three-dimensional (3D) cell culturing in drug discovery, Cell Culture 2
(2018) 1-22.

{[}4{]} J. Wu, L. Xie, W.Z.Y. Lin, Q. Chen, Biomimetic nanofibrous
scaffolds for neural tissue engineering and drug development, Drug
discovery today 22(9) (2017) 1375-1384.

{[}5{]} P. Jaiswal, Allied Market Research, 3D Cell culture Market,
Report code LI171474, 2017.

{[}6{]} B. Larson, 3D cell culture: A review of current techniques,
BioTek 6 (2015) 1-10.

{[}7{]} A. Subramanian, U.M. Krishnan, S. Sethuraman, Development of
biomaterial scaffold for nerve tissue engineering: Biomaterial mediated
neural regeneration, Journal of Biomedical Science 16(1) (2009) 108.

{[}8{]} E. Ansorena, P. De Berdt, B. Ucakar, T. Simón-Yarza, D. Jacobs,
O. Schakman, A. Jankovski, R. Deumens, M.J. Blanco-Prieto, V. Préat,
Injectable alginate hydrogel loaded with GDNF promotes functional
recovery in a hemisection model of spinal cord injury, International
journal of pharmaceutics 455(1-2) (2013) 148-158.

{[}9{]} S. Ansari, I.M. Diniz, C. Chen, P. Sarrion, A. Tamayol, B.M. Wu,
A. Moshaverinia, Human periodontal ligament\selectlanguage{english}-and gingiva\selectlanguage{english}-derived
mesenchymal stem cells promote nerve regeneration when encapsulated in
alginate/hyaluronic acid 3D scaffold, Advanced healthcare materials
6(24) (2017) 1700670.

{[}10{]} G. Wang, X. Wang, L. Huang, Feasibility of chitosan-alginate
(Chi-Alg) hydrogel used as scaffold for neural tissue engineering: a
pilot study in vitro, Biotechnology \& Biotechnological Equipment 31(4)
(2017) 766-773.

{[}11{]} P. Prang, R. Muller, A. Eljaouhari, K. Heckmann, W. Kunz, T.
Weber, C. Faber, M. Vroemen, U. Bogdahn, N. Weidner, The promotion of
oriented axonal regrowth in the injured spinal cord by alginate-based
anisotropic capillary hydrogels, Biomaterials 27(19) (2006) 3560-3569.

{[}12{]} J. Sun, H. Tan, Alginate-based biomaterials for regenerative
medicine applications, Materials 6(4) (2013) 1285-1309.

{[}13{]} O. Akhavan, Graphene scaffolds in progressive
nanotechnology/stem cell-based tissue engineering of the nervous system,
Journal of Materials Chemistry B 4(19) (2016) 3169-3190.

{[}14{]} N. Li, Q. Zhang, S. Gao, Q. Song, R. Huang, L. Wang, L. Liu, J.
Dai, M. Tang, G. Cheng, Three-dimensional graphene foam as a
biocompatible and conductive scaffold for neural stem cells, Scientific
reports 3 (2013) 1604.

{[}15{]} W.C. Lee, K.P. Loh, C.T. Lim, When stem cells meet graphene:
Opportunities and challenges in regenerative medicine, Biomaterials 155
(2018) 236-250.

{[}16{]} W. Lee, C.Y.X. Lim, H. Shi, L.A.L. Tang, Y. Wang, C. Lim, K.
Loh, Origin of enhanced stem cell growth and differentiation on graphene
and graphene oxide, ACS nano 5(9) (2011) 7334-7341.

{[}17{]} M.C. Serrano, J. Patino, C. Garcia-Rama, M.L. Ferrer, J.L.G.
Fierro, A. Tamayo, J.E. Collazos-Castro, F. del Monte, M.C. Gutierrez,
3D free-standing porous scaffolds made of graphene oxide as substrates
for neural cell growth, Journal of Materials Chemistry B 2(34) (2014)
5698-5706.

{[}18{]} A.M. Pinto, I.C. Goncalves, F.D. Magalhaes, Graphene-based
materials biocompatibility: a review, Colloids and Surfaces B:
Biointerfaces 111 (2013) 188-202.

{[}19{]} X. Zhang, T. Viitala, R. Harjumaki, A. Kartal-Hodzic, J.J.
Valle-Delgado, M. Osterberg, Effect of laminin, polylysine and cell
medium components on the attachment of human hepatocellular carcinoma
cells to cellulose nanofibrils analyzed by surface plasmon resonance,
Journal of Colloid and Interface Science 584 (2020) 310-319.

{[}20{]} D. Liu, N. Pavathuparambil Abdul Manaph, M. Al-Hawwas, L.
Bobrovskaya, L.-L. Xiong, X.-F. Zhou, Coating Materials for Neural
Stem/Progenitor Cell Culture and Differentiation, Stem Cells and
Development 29(8) (2020) 463-474.

{[}21{]} V. Serpooshan, M. Mahmoudi, M. Zhao, K. Wei, S. Sivanesan, K.
Motamedchaboki, A.V. Malkovskiy, A.B. Goldstone, J.E. Cohen, P.C. Yang,
Protein corona influences cell--biomaterial interactions in
nanostructured tissue engineering scaffolds, Advanced functional
materials 25(28) (2015) 4379-4389.

{[}22{]} I. Lynch, A. Salvati, K.A. Dawson, Protein-nanoparticle
interactions: What does the cell see?, Nature nanotechnology 4(9) (2009)
546-547.

{[}23{]} B. Andree, H. Ichanti, S. Kalies, A. Heisterkamp, S. Strauss,
P.-M. Vogt, A. Haverich, A. Hilfiker, Formation of three-dimensional
tubular endothelial cell networks under defined serum-free cell culture
conditions in human collagen hydrogels, Scientific reports 9(1) (2019)
1-11.

{[}24{]} N. Mansouri, S.F. Al-Sarawi, J. Mazumdar, D. Losic, Advancing
fabrication and properties of three-dimensional graphene--alginate
scaffolds for application in neural tissue engineering, RSC Advances
9(63) (2019) 36838-36848.

{[}25{]} X. Lan, Z. Sun, C. Chu, J. Boltze, S. Li, Dental pulp stem
cells: an attractive alternative for cell therapy in ischemic stroke,
Frontiers in neurology 10 (2019) 824.

{[}26{]} S. Gronthos, M. Mankani, J. Brahim, P.G. Robey, S. Shi,
Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo,
Proceedings of the National Academy of Sciences 97(25) (2000)
13625-13630.

{[}27{]} D.L. Alge, D. Zhou, L.L. Adams, B.K. Wyss, M.D. Shadday, E.J.
Woods, T. Gabriel Chu, W.S. Goebel, Donor\selectlanguage{english}-matched comparison of dental
pulp stem cells and bone marrow\selectlanguage{english}-derived mesenchymal stem cells in a rat
model, Journal of tissue engineering and regenerative medicine 4(1)
(2010) 73-81.

{[}28{]} H. Li, A.Q. Ye, M. Su, Application of stem cells and advanced
materials in nerve tissue regeneration, Stem cells international 2018
(2018).

{[}29{]} A. Arthur, G. Rychkov, S. Shi, S.A. Koblar, S. Gronthos, Adult
human dental pulp stem cells differentiate toward functionally active
neurons under appropriate environmental cues, Stem cells 26(7) (2008)
1787-1795.

{[}30{]} I.V. Nosrat, C.A. Smith, P. Mullally, L. Olson, C.A. Nosrat,
Dental pulp cells provide neurotrophic support for dopaminergic neurons
and differentiate into neurons in vitro; implications for tissue
engineering and repair in the nervous system, European Journal of
Neuroscience 19(9) (2004) 2388-2398.

{[}31{]} I.V. Nosrat, J. Widenfalk, L. Olson, C.A. Nosrat, Dental pulp
cells produce neurotrophic factors, interact with trigeminal neurons in
vitro, and rescue motoneurons after spinal cord injury, Developmental
biology 238(1) (2001) 120-132.

{[}32{]} K. Sakai, A. Yamamoto, K. Matsubara, S. Nakamura, M. Naruse, M.
Yamagata, K. Sakamoto, R. Tauchi, N. Wakao, S. Imagama, Human dental
pulp-derived stem cells promote locomotor recovery after complete
transection of the rat spinal cord by multiple neuro-regenerative
mechanisms, The Journal of clinical investigation 122(1) (2012) 80-90.

{[}33{]} W.K. Leong, T.L. Henshall, A. Arthur, K.L. Kremer, M.D. Lewis,
S.C. Helps, J. Field, M.A. Hamilton-Bruce, S. Warming, J. Manavis, Human
adult dental pulp stem cells enhance poststroke functional recovery
through non\selectlanguage{english}-neural replacement mechanisms, Stem cells translational
medicine 1(3) (2012) 177-187.

{[}34{]} D.C. Marcano, D.V. Kosynkin, J.M. Berlin, A. Sinitskii, Z. Sun,
A. Slesarev, L.B. Alemany, W. Lu, J.M. Tour, Improved Synthesis of
Graphene Oxide, ACS Nano 4(8) (2010) 4806-4814.

{[}35{]} X. Zhou, Y. Pan, R. Liu, X. Luo, X. Zeng, D. Zhi, J. Li, Q.
Cheng, Z. Huang, H. Zhang, Biocompatibility and biodegradation
properties of polycaprolactone/polydioxanone composite scaffolds
prepared by blend or co-electrospinning, Journal of Bioactive and
Compatible Polymers 34(2) (2019) 115-130.

{[}36{]} F. Rostami, E. Tamjid, M. Behmanesh, Drug-eluting PCL/graphene
oxide nanocomposite scaffolds for enhanced osteogenic differentiation of
mesenchymal stem cells, Materials Science and Engineering: C (2020)
111102.

{[}37{]} G. Lalwani, M. D'agati, A. Gopalan, M. Rao, J. Schneller, B.
Sitharaman, Three\selectlanguage{english}-dimensional macroporous graphene scaffolds for tissue
engineering, Journal of Biomedical Materials Research Part A 105(1)
(2017) 73-83.

{[}38{]} S.M. Willerth, S.E. Sakiyama-Elbert, Combining stem cells and
biomaterial scaffolds for constructing tissues and cell delivery,
StemJournal 1(1) (2019) 1-25.

{[}39{]} H. Samadian, S. Farzamfar, A. Vaez, A. Ehterami, A. Bit, M.
Alam, A. Goodarzi, G. Darya, M. Salehi, A tailored polylactic
acid/polycaprolactone biodegradable and bioactive 3D porous scaffold
containing gelatin nanofibers and Taurine for bone regeneration,
Scientific reports 10(1) (2020) 1-12.

{[}40{]} S.D. Purohit, R. Bhaskar, H. Singh, I. Yadav, M.K. Gupta, N.C.
Mishra, Development of a nanocomposite scaffold of
gelatin--alginate--graphene oxide for bone tissue engineering,
International journal of biological macromolecules 133 (2019) 592-602.

{[}41{]} B.M. Baker, C.S. Chen, Deconstructing the third dimension--how
3D culture microenvironments alter cellular cues, Journal of cell
science 125(13) (2012) 3015-3024.

{[}42{]} C. Jensen, Y. Teng, Is It Time to Start Transitioning From 2D
to 3D Cell Culture?, Frontiers in Molecular Biosciences 7 (2020) 33.

{[}43{]} A. Pisciotta, L. Bertoni, M. Riccio, J. Mapelli, A. Bigiani, M.
La Noce, M. Orciani, A. de Pol, G. Carnevale, Use of a 3D floating
sphere culture system to maintain the neural crest-related properties of
human dental pulp stem cells, Frontiers in physiology 9 (2018) 547.

{[}44{]} M.S. Liberio, M.C. Sadowski, C. Soekmadji, R.A. Davis, C.C.
Nelson, Differential effects of tissue culture coating substrates on
prostate cancer cell adherence, morphology and behavior, PLoS One 9(11)
(2014) e112122.

{[}45{]} Y. Qian, J. Song, X. Zhao, W. Chen, Y. Ouyang, W. Yuan, C. Fan,
3D fabrication with integration molding of a graphene
oxide/polycaprolactone nanoscaffold for neurite regeneration and
angiogenesis, Advanced Science 5(4) (2018) 1700499.

{[}46{]} M. Domingos, F. Intranuovo, T. Russo, R. De Santis, A. Gloria,
L. Ambrosio, J. Ciurana, P. Bartolo, The first systematic analysis of 3D
rapid prototyped poly (\selectlanguage{greek}ε-\selectlanguage{english}caprolactone) scaffolds manufactured through
BioCell printing: the effect of pore size and geometry on compressive
mechanical behaviour and in vitro hMSC viability, Biofabrication 5(4)
(2013) 045004.

{[}47{]} V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial
scaffolds and osteogenesis, Biomaterials 26(27) (2005) 5474-5491.

{[}48{]} F.J. O'Brien, B.A. Harley, I.V. Yannas, L.J. Gibson, The effect
of pore size on cell adhesion in collagen-GAG scaffolds, Biomaterials
26(4) (2005) 433-441.

{[}49{]} F.J. O'Brien, B.A. Harley, M.A. Waller, I.V. Yannas, L.J.
Gibson, P.J. Prendergast, The effect of pore size on permeability and
cell attachment in collagen scaffolds for tissue engineering, Technology
and Health Care 15(1) (2007) 3-17.

{[}50{]} C.M. Murphy, F.J. O'Brien, Understanding the effect of mean
pore size on cell activity in collagen-glycosaminoglycan scaffolds, Cell
adhesion \& migration 4(3) (2010) 377-381.

{[}51{]} C.M. Conde, F.F. Demarco, L. Casagrande, J.C. Alcazar, J.E.
N\selectlanguage{ngerman}ör, S.B.C. Tarquinio, Influence of poly-L-lactic acid scaffold's pore
size on the proliferation and differentiation of dental pulp stem cells,
Brazilian dental journal 26(2) (2015) 93-98.

{[}52{]} R.A. Morsy, H. Beherei, M. Ellithy, H.E. Tarek, M. Mabrouk, The
odontogenic performance of human dental pulp stem cell in 3-dimensional
chitosan and nano-bioactive glass-based scaffold material with different
pores size, Journal of The Arab Society for Medical Research 14(2)
(2019) 82.

{[}53{]} S. Dinescu, M. Ionita, S.-R. Ignat, M. Costache, A. Hermenean,
Graphene Oxide Enhances Chitosan-Based 3D Scaffold Properties for Bone
Tissue Engineering, International journal of molecular sciences 20(20)
(2019) 5077.

{[}54{]} M. Kalbacova, A. Broz, J. Kong, M. Kalbac, Graphene substrates
promote adherence of human osteoblasts and mesenchymal stromal cells,
Carbon 48(15) (2010) 4323-4329.

{[}55{]} Y. Luo, A. Lode, A.R. Akkineni, M. Gelinsky, Concentrated
gelatin/alginate composites for fabrication of predesigned scaffolds
with a favorable cell response by 3D plotting, RSC Advances 5(54) (2015)
43480-43488.

{[}56{]} C.F. Jones, D.W. Grainger, In vitro assessments of nanomaterial
toxicity, Advanced drug delivery reviews 61(6) (2009) 438-456.

{[}57{]} J. Van der Valk, D. Mellor, R. Brands, R. Fischer, F. Gruber,
G. Gstraunthaler, L. Hellebrekers, J. Hyllner, F. Jonker, P. Prieto, The
humane collection of fetal bovine serum and possibilities for serum-free
cell and tissue culture, Toxicology in vitro 18(1) (2004) 1-12.

{[}58{]} V. Bonnamain, R. Thinard, S. Sergent-Tanguy, P. Huet, G.
Bienvenu, P. Naveilhan, J.-C. Farges, B. Alliot-Licht, Human dental pulp
stem cells cultured in serum-free supplemented medium, Frontiers in
physiology 4 (2013) 357.

{[}59{]} L. Xiao, T. Tsutsui, Characterization of human dental pulp
cells\selectlanguage{english}-derived spheroids in serum\selectlanguage{english}-free medium: Stem cells in the core,
Journal of cellular biochemistry 114(11) (2013) 2624-2636.

{[}60{]} J. Jung, J.-W. Kim, H.-J. Moon, J.Y. Hong, J.K. Hyun,
Characterization of neurogenic potential of dental pulp stem cells
cultured in xeno/serum-free condition: in vitro and in vivo assessment,
Stem cells international 2016 (2016).

{[}61{]} W. Hu, C. Peng, M. Lv, X. Li, Y. Zhang, N. Chen, C. Fan, Q.
Huang, Protein corona-mediated mitigation of cytotoxicity of graphene
oxide, ACS nano 5(5) (2011) 3693-3700.

{[}62{]} X.-Q. Wei, L.-Y. Hao, X.-R. Shao, Q. Zhang, X.-Q. Jia, Z.-R.
Zhang, Y.-F. Lin, Q. Peng, Insight into the interaction of graphene
oxide with serum proteins and the impact of the degree of reduction and
concentration, ACS applied materials \& interfaces 7(24) (2015)
13367-13374.

{[}63{]} A. Lesniak, A. Campbell, M.P. Monopoli, I. Lynch, A. Salvati,
K.A. Dawson, Serum heat inactivation affects protein corona composition
and nanoparticle uptake, Biomaterials 31(36) (2010) 9511-9518.

{[}64{]} A. Lesniak, F. Fenaroli, M.P. Monopoli, C. Aberg, K.A. Dawson,
A. Salvati, Effects of the presence or absence of a protein corona on
silica nanoparticle uptake and impact on cells, ACS nano 6(7) (2012)
5845-5857.

{[}65{]} O. Karaman, Z.B. Yaral\selectlanguage{polish}ı, \selectlanguage{english}Determination of minimum serum
concentration to develop scaffold free micro-tissue, The European
Research Journal 4(3) (2018) 145-151.\selectlanguage{english}
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