Biomimetic 3D bacterial cellulose-graphene foam hybrid scaffold regulates neural stem cell proliferation and differentiation

Abstract

Neural stem cell (NSC)-based therapy is a promising candidate for treating neurodegenerative diseases and the preclinical researches call an urgent need for regulating the growth and differentiation of such cells. The recognition that three-dimensional culture has the potential to be a biologically significant system has stimulated an extraordinary impetus for scientific researches in tissue engineering and regenerative medicine. Here, A novel scaffold for culturing NSCs, three-dimensional bacterial cellulose-graphene foam (3D-BC/G), which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam has been reported. 3D-BC/G not only supports NSC growth and adhesion, but also maintains NSC stemness and enhances their proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G induces NSCs to selectively differentiate into neurons, forming a neural network in a short amount of time. The scaffold has good biocompatibility with primary cortical neurons enhancing the neuronal network activities. To explore the underlying mechanisms, RNA-Seq analysis to identify genes and signaling pathways was performed and it suggests that 3D-BC/G offers a more promising three-dimensional conductive substrate for NSC research and neural tissue engineering, and the repertoire of gene expression serves as a basis for further studies to better understand NSC biology.

Full text

Colloids and Surfaces B: Biointerfaces 200 (2021) 111590
Available online 27 January 2021
0927-7765/© 2021 Elsevier B.V. All rights reserved.
Biomimetic 3D bacterial cellulose-graphene foam hybrid scaffold regulates
neural stem cell proliferation and differentiation
Rongrong Guo
a
,
b
,
d
,
e
,
1
, Jian Li
d
,
1
, Chuntao Chen
g
,
1
, Miao Xiao
f
,
h
,
1
, Menghui Liao
d
,
e
,
Yangnan Hu
d
,
e
, Yun Liu
d
,
e
, Dan Li
c
,
d
, Jun Zou
i
, Dongping Sun
g
, Vincent Torre
h
, Qi Zhang
a
,
**,
Renjie Chai
d
,
e
,
j
,
k
,
*, Mingliang Tang
d
,
e
,
f
,
*
a
School of Radiation Medicine and Protection and School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation
Medicine of Jiangsu Higher Education Institutions, Medical College of Soochow University, Suzhou, Jiangsu, 215123, China
b
Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming,
Yunnan, 650500, China
c
State Key Laboratory of Bioelectronics, School of Life Sciences and Technology, Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast
University, Nanjing, 210096, China
d
Key Laboratory for Developmental Genes and Human Disease, Ministry of Education, School of Life Sciences and Technology, Southeast University, Nanjing, 210096,
China
e
Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226001, China
f
Institute for Cardiovascular Science & Department of Cardiovascular Surgery of the First Afliated Hospital, Medical College, Soochow University, Suzhou, 215000,
China
g
Institute of Chemicobiology and Functional Materials, Key Laboratory for Soft Chemistry and Functional Materials of Ministry Education, School of Chemical
Engineering, Nanjing University of Science and Technology, 200 Xiao Ling Wei Street, Nanjing, 210094, Jiangsu Province, China
h
International School for Advanced Studies (SISSA), via Bonomea 265, Trieste, 34136, Italy
i
Department of Orthopaedic Surgery, The First Afliated Hospital of Soochow University, 188 Shizi Street, Suzhou, Jiangsu, 215006, China
j
Institute for Stem Cell and Regeneration, Chinese Academy of Science, Beijing, China
k
Beijing Key Laboratory of Neural Regeneration and Repair, Capital Medical University, Beijing, 100069, China
ARTICLE INFO
Keywords:
Graphene
Bacterial cellulose
Three-dimensional culture
Neural stem cell
Differentiation
ABSTRACT
Neural stem cell (NSC)-based therapy is a promising candidate for treating neurodegenerative diseases and the
preclinical researches call an urgent need for regulating the growth and differentiation of such cells. The
recognition that three-dimensional culture has the potential to be a biologically signicant system has stimulated
an extraordinary impetus for scientic researches in tissue engineering and regenerative medicine. Here, A novel
scaffold for culturing NSCs, three-dimensional bacterial cellulose-graphene foam (3D-BC/G), which was prepared
via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam has been
reported. 3D-BC/G not only supports NSC growth and adhesion, but also maintains NSC stemness and enhances
their proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G induces NSCs to selectively
differentiate into neurons, forming a neural network in a short amount of time. The scaffold has good
biocompatibility with primary cortical neurons enhancing the neuronal network activities. To explore the un-
derlying mechanisms, RNA-Seq analysis to identify genes and signaling pathways was performed and it suggests
that 3D-BC/G offers a more promising three-dimensional conductive substrate for NSC research and neural tissue
engineering, and the repertoire of gene expression serves as a basis for further studies to better understand NSC
biology.
* Corresponding authors at: Key Laboratory for Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University,
Nanjing, 210096, China.
** Corresponding author.
E-mail addresses: qzhang2012@suda.edu.cn (Q. Zhang), renjiec@seu.edu.cn (R. Chai), mltang@suda.edu.cn (M. Tang).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
https://doi.org/10.1016/j.colsurfb.2021.111590
Received 13 October 2020; Received in revised form 29 December 2020; Accepted 22 January 2021

Colloids and Surfaces B: Biointerfaces 200 (2021) 111590
2
1. Introduction
An increasing number of studies have shown that neural stem cells
(NSCs) have the potential for wide use in cell-based therapy to both
protect and restore damaged neurons in neurodegenerative diseases
such cervical spinal cord injury [1], Parkinson’s disease (PD) [2] and
Alzheimer’s disease [3]. However, NSC transplantation faces many ob-
stacles in its transition from the lab to the clinic, especially issues related
to the uncontrolled differentiation and functional engraftment of
implanted tissues [4]. By further developing our knowledge of the mo-
lecular, cellular, and developmental biology of NSCs, we can better
understand the organization and function of the complex brain. Under
normal physiological conditions, NSCs reside in neurogenic niches, and
neurogenesis occurs in a spatially and temporally regulated fashion
through many physiological stimuli, including culture media, co-culture
with other cells, physicochemical conditions, surface biochemistry,
surface topography, mechanical signals, and 3D culture scaffolds [5].
Consequently, signicant efforts have been made to develop tunable
three-dimensional NSC microenvironments that can regulate and con-
trol NSC fate in the desired direction, and such porous scaffolds show
great promise for manipulating cell behavior in the areas of tissue en-
gineering and regenerative medicine.
Electrically conductive scaffolds have found applications in neural
regeneration through their ability to guide NSCs to differentiate into
neural lineages, and graphene lm has been shown to induce stem cells
to preferentially differentiate into specic lineages [6–8]. In addition,
other stimulation could also been used to increase the number of neu-
rons as compared to glia, such as electrical [9,10], chemical [7,11],
morphological [12,13], pulsed laser [14], NIR laser [15], visible light
[16] and photoelectrical stimulation [17]. This issue has been well
demonstrated in a review [18]. Of clinical interest, three-dimensional
graphene foam (3D-G) has been widely accepted as a good scaffold
material in the eld of tissue engineering by virtue of its excellent
biocompatibility and electrical properties along with its porous structure
and precisely tunable properties [19–23]. Previous research by our
group has shown that 3D-G is a biocompatible and conductive scaffold
for use with NSCs [9] and that the material improves skin wound healing
by promoting the growth and proliferation of mesenchymal stem cells
[24].
Taken together, many of the properties of three-dimensional gra-
phene make it a highly favorable microenvironment for neural cells to
reside in and respond to. However, these materials have pore sizes of
100–300
μ
m such that cells seeded into them typically attach, prolifer-
ate, and differentiate along the walls of the pores rather than lling the
entire space [9]. This observation has motivated us to develop a method
to functionalize these unoccupied cavities in order to simulate a more
realistic and suitable three-dimensional microenvironment.
Three-dimensional bacterial cellulose (3D-BC), which is an un-
branched polymer of β-1, 4-linked glucopyranose residues [25,26], has
long been used as an effective biomaterial for use in full-thickness skin
tissue repair, blood vessel grafts [27], bone tissue engineering [28,29],
and meniscus replacement [30] with the advantage of easy purication
and manipulation, good biocompatibility, high tensile strength and
elastic modulus, high hydrophilicity, efcient biodegradation, and
similar morphological characteristics to collagenous bers [26,31–34].
It has thus been extensively researched as a suitable substrate material
for tissue engineering and biomedical applications [35]. Several studies
have shown that 3D-BC alone or with specic surface modications can
maintain the biological activity of cultured cells and can direct the
morphology and differentiation of various cells, including
adipose-derived stem cells, glial cells, neurons, and skeletal muscle cells
[36].
These unique properties of bacterial cellulose have opened up new
perspectives for developing nanober scaffolds for tissue engineering.
However, bacterial cellulose fermented directly by Acetobacter xylinum
forms a dense network of nanobers with pore sizes of 0.02–10
μ
m [37],
which are smaller than the dimensions of mammalian cells such that
these cells cannot penetrate into the pore and form three-dimensional
structures [38–40]. The preparation methods of scaffold structures
with bacterial cellulose or natural nanober materials have been re-
ported to form nanobrous structures [41–43]. To address this issue, we
fermented A. xylinum on 3D-G to create a novel microporous,
nanober-based, and electrically conductive scaffold (3D-BC/G) that
integrates the advantages of graphene foam and bacterial cellulose, and
this method enhances the topographic properties and decreases the pore
diameter of 3D-G in order to increase its usefulness in tissue engineering
[44].
2. Results and discussion
2.1. Fabrication and characterization of 3D-BC/G for NSC culture
Multilayer 3D-G foams were fabricated by chemical vapor deposition
using 3D-Ni scaffolds as both catalysts and templates. Nickel was sub-
sequently removed by FeCl
3
etching. The 3D-BC/G was obtained by
culturing A. xylinumon the 3D-G surface. As shown in Fig. 1A, the 3D-BC
nanobers were bunched together on the surface of the graphene skel-
eton. The scanning electron microscopy (SEM) images (Fig. 1B, a–d)
showed a robust, porous, and nanober-embedded 3D-BC/G foam. The
inclusion of BC did not compromise the structural integrity of the gra-
phene foams, and this is important for future applications because it is
critical that the three-dimensional microstructures provide sufcient
physical support in order for NSCs to homogenously distribute and
expand. Moreover, the BC nanobers on the surface of the graphene
skeleton signicantly improve the nano-sized topological structures,
leading to enhanced cell-scaffold interaction [45].
Fig. 1C shows the Raman spectra of the 3D-BC, 3D-G, and 3D-BC/G
materials. The prominent 2D band located at ~2700 cm
−1
and the G
band centered at ~1580 cm
-1
were observed in the spectrum of 3D-G,
indicating the overall high quality of the graphene. These two peaks
were also observed in the spectrum of 3D-BC/G but were weakened
because of the presence of BC on the surface of the graphene skeleton.
Similar to BC, a Raman band located at ~1090 cm
−1
for 3D-BC/G was
assigned to C
–
O stretching ring modes. The bands at 1337 cm
-1
and
1377 cm
-1
were assigned to C
–
H deformation and OH
–
deformation,
respectively, which were also seen in the spectrum of 3D-BC/G. X-ray
powder diffraction (XRD) and Raman spectroscopy were used to verify
the construction of the 3D-BC/G scaffold. The intrinsic peaks of 3D-G (2θ
=27.5◦and 55.2◦), corresponding to its diffraction planes (002) and
(004), were seen in the XRD patterns (Fig. 1D). Three major peaks of BC
located at 14.5◦, 16.8◦, and 22.7◦, corresponding to the (110), (110),
and (200) diffraction planes of cellulose I, respectively, were also
observed. The characteristic peaks of both BC (2θ =14.5◦) and G (2θ =
27.5◦) were inherited in the 3D-BC/G composite material, but with
weakened intensities. Furthermore, the incorporation of nanobrous BC
on the surface of the graphene skeleton increased its surface area,
reduced its pore size, and provided a broad array of oxygen groups. All
of these features suggest that this novel 3D-BC/G material has the ability
to enhance cell-cell and cell-matrix interactions.
2.2. Biocompatibility of 3D-BC/G
For the successful clinical application of scaffold biomaterials,
biocompatibility is a critical issue. We rst evaluated the biocompati-
bility of 3D-BC/G using a live/dead assay. We found that NSCs cultured
on 3D-BC/G for 3 days had a viability of more than 99 %, which was
comparable to that of NSCs grown on 3D-G (Fig. 2A). SEM images
showed that NSCs on these two scaffolds had normal morphology and
normal extensions and protrusions (Fig. 2B). SEM analysis of the cul-
tures also allowed investigation of the interactions of such protrusions
with the three-dimensional materials. As shown in the high-
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magnication images, the bers appeared tightly anchored to the sur-
face of scaffolds with evident development of membrane-substrate
junctions and growth cones. These ndings suggest the biocompati-
bility of 3D-BC/G in vitro.
2.3. Maintaining the stemness of NSCs on 3D-BC/G
Neurogenesis consists of several developmental stages, including
proliferation, differentiation, maturation, migration, and integration.
The foundation of all of these processes is the maintenance of NSC
stemness on the scaffold biomaterials because NSCs themselves are key
determinates of neurogenesis. To test whether 3D-BC/G maintains NSC
stemness, the expression of nestin, a protein that is essential for main-
taining the stemness of NSCs, was evaluated by immunouorescence
staining (Supplementary Fig. S5). Almost all of the cells cultured on 3D-
BC/G were nestin-positive (green), with no obvious differences
compared to those cultured on 3D-G and 3D-BC, indicating that NSCs
cultured on 3D-BC/G maintained physiological levels of stemness.
2.4. Focal adhesion of NSCs on 3D-BC/G
It has been demonstrated that spatiotemporal and topographical cues
can regulate multiple cellular behaviors, including survival, prolifera-
tion, differentiation, and migration, by modulating integrin-based focal
adhesion and subsequent changes in cytoskeletal organization and
mechanosensitive signaling cascades [46–48]. The interactions between
cells and scaffold biomaterials are very complex multi-step processes,
but these can be studied in vitro by studying the adsorption of extra-
cellular matrix (ECM) proteins, such as laminin, collagen, and bro-
nectin, onto the surfaces of the materials. The recognition of cell-binding
domains of the ECM by cell surface receptors focuses primarily on the
integrin superfamily, and the collection of focal adhesion proteins,
including vinculin, FAK, and paxillin, is followed by cytoskeletal rear-
rangements that lead to changes in cell behavior [49]. In this study,
qPCR showed that NSCs cultured on 3D-G had greater expression levels
of the focal adhesion proteins vinculin, FAK, and paxillin than NSCs
cultured on 3D-BC/G (Fig. 3B and C), which indicated that 3D-G was
more effective than 3D-BC/Gin facilitating focal adhesion development
of NSCs, likely by providing more focal adhesion points.
2.5. NSC proliferation on 3D-BC/G
To evaluate the structural and topographical effects of 3D-BC/G on
the proliferation of NSCs, NSC proliferation on 3D-BC/G was determined
by measuring the percentage of EdU-positive cells, which indicate early
S-phase cells. We found that NSCs cultured on 3D-BC/G had greater
proportions of EdU-positive cells compared to 3D-G (Fig. 4A and B)
suggesting that NSCs cultured on 3D-BC/G have greater proliferative
ability compared to cells cultured on 3D-G. To further verify this
phenotype, an alamarBlue assay was carried out to measure NSC pro-
liferation (Fig. 4C). The NSCs cultured on 3D-BC/G had a greater
reduction in alamarBlue than those cultured on 3D-G, which was similar
to the EdU assay. qPCR experiments also supported this result by
showing that the gene expression levels of Ki67, MCM2, and PCNA,
which are all markers of proliferation, were higher in NSCs cultured on
3D-BC/G compared to those cultured on 3D-G (Fig. 4D).Together these
observations indicate that the addition of bacterial cellulose onto a
graphene skeleton can signicantly increase the proliferative ability of
NSCs.
The proliferative potential of NSCs is tightly regulated by extrinsic
signals – such as growth factors, ECM components, neurotransmitters,
and cellular focal adhesion molecules – in order to maintain the ho-
meostasis of the stem cell pool. The deletion or decrease in cellular focal
adhesion proteins enhances NSC proliferation by integrin-β1-mediated
signaling [50], and this is consistent with our results (Fig. 3A). We
further speculate that this improvement is because the bacterial cellu-
lose simulates the self-assembly process of protein brillogenesis in vivo,
which leads to the formation of ECM.
2.6. NSC differentiation on 3D-BC/G
To further investigate the phenotypic differences of differentiated
Fig. 1. (A) The schematic diagram for the preparation of 3D-BC/G. (B) The SEM images of 3D-BC (a),3D-G (b), and 3D-BC/G (c, d). (C) Raman images of 3D-BC, 3D-
G, and 3D-BC/G. (D) XRD spectra of 3D-BC, 3D-G, and 3D-BC/G.
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NSCs, we induced NSCs to differentiate for 7 days and visualized their
offspring by immunouorescence staining for Tuj-1 (Fig. 5). The images
indicated that the differentiated neurons cultured on 3D-BC/G formed
an obvious three-dimensional neural network, while the differentiated
neurons on 3D-G had shorter neurite outgrowths. These images of cells
grown on 3D-BC/G and 3D-G were all obtained by confocal microscopy,
Fig. 2. Biocompatibility of 3D-BC/G and the morphology of NSCs cultured on 3D-BC/G. (A) Cell viability assay of NSCs cultured on 3D-G and 3D-BC/G after 5 days
of culture. Cell-permeating calcein-AM produced an intense green uorescence within live cells. EthD-1, producing red uorescence, entered into dead cells with
damaged membranes. (B) Low-, medium-, and high-magnication SEM images of NSCs cultured on 3D-G and 3D-BC/G under proliferation conditions. The insets
illustrate the interaction between the cell growth cone and the three-dimensional scaffold.
Fig. 3. NSC adhesion on 3D-BC/G. (A) NSCs were immuno-stained with anti-integrin β1 (red), a transmembrane receptor that mediates the connection between cells
and their microenvironment. DAPI (blue) was usedto stain nuclei. (B,C) The relative mRNA expression of the four adhesion-related molecules integrin β1, vinculin,
FAK, and paxillin. The data are presented as the mean ±SEM, * p <0.05, ** p <0.01, *** p <0.001.
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Colloids and Surfaces B: Biointerfaces 200 (2021) 111590
5
in which every picture is an optical slice. The resulting projections of
several optical slices show that 3D-BC/G can quickly organize a three-
dimensional neural network.
Mounting evidence suggests the importance of mechanosensitivity,
in which biophysical signals are transduced into biochemical signals
[51,52], for modulating the physiological functions of stem cells or
neurons, including their adhesion, motility, differentiation, and neurite
outgrowth. The addition of BC to 3D-G compensates for many of the
limitations of 3D-G’s mechanical properties. For example, the Young’s
modulus of 3D-BC is about 80 GPa, while that of 3D-G is about
1000–2000 MPa, and thus 3D-BC/G shows a Young’s modulus that is
similar to soft-tissue membranes. At the same time, the micro- and
nano-scale topography of biomaterials has been shown to be very
important for determining neural differentiation and for inuencing
neurite outgrowth by mimicking the topography of native collagen lms
[53]. The 3D-BC/G scaffold showed more complicated nano-topography
than 3D-G (Fig. 1B), and therefore it is tempting to speculate that the
neurites of newborn neurons adapt to the distinctive morphological and
mechanical features of scaffold materials and that this might explain
why 3D-BC/G induced longer neurites to form a neural network.
Fig. 4. NSC proliferation on 3D-BC/G. (A) Representative immunouorescence images of NSCs labeled with EdU and DAPI. (B) The percentage of EdU-labeled NSCs.
(C) The percent reduction of alamarBlueas measured by microplate reader. (D) Relative mRNA expression of the proliferation markers Ki67, MCM2, and PCNA.
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2.7. Construction of functional 3D neural networks on 3D-BC/G and 3D-
G
The novel 3D-BC/G scaffold was also found to be compatible with
neuronal culture. Primary cortical neurons isolated from Wistar rats
(postnatal day 1–3) were used to compare 3D neuronal network for-
mation on both 3D-BC/G and 3D-G. After 8 days of culture, the cells
were stained with antibodies against Tuj-1 to identify neurons and with
antibodies against glial brillary acidic protein (GFAP) to identify glial
cells. Confocal images of cortical neuronal networks grown on 3D-BC/G
and 3D-G (Fig. 6A) showed that the neurons grew primarily along the
walls of the 3D-G scaffold and developed a 3D neural network that
followed the scaffold’s topology. In contrast, neurons grown on 3D-BC/
G formed a denser network with the support of BC, which was conrmed
by the cell density analysis (Fig. 6B). After 8 days in culture, nucleus
counting showed an almost 3-fold increase in cell density on 3D-BC/G
compared to 3D-G. The decreased porosity of 3D-BC/G scaffolds
compared to the porosity of the 3D-G scaffolds enabled the retention of a
larger number of neurons and glia cells inside the 3D-BC/G scaffolds,
thus better mimicking the in vivo situation.
Spontaneous calcium transients occur extensively throughout the
development of the nervous system, and these transients operate over a
wide temporal range to inuence proliferation, migration, and differ-
entiation [54]. To determine whether neurons grown on the 3D-BC/G
scaffolds are alive and functionally active, we performed calcium im-
aging experiments using the calcium indicator Fluo-4 AM. The uores-
cence images of Fluo-4-loaded cortical cells cultured on 3D-BC/G and
3D-G are shown in Fig. 6C and D, respectively. Spontaneous calcium
transients (DF/F) associated with the electrical ring of neurons were
obtained by acquiring images at 3–5 Hz for 10–20 min (Fig. 6E and F).
After 8 days in culture, calcium transients with an amplitude of up to 1
DF/F were observed. The neuronal activity on 3D-BC/G scaffolds was
~50 % more frequent than that on 3D-G scaffolds (Fig. 6G), and the
synchronization of neuronal activity could be represented by the mean
correlation coefcient of the calcium transients. However, the syn-
chrony of neuronal activity did not show any obvious difference be-
tween the 3D-BC/G and 3D-G substrates. Considering the complex
features of the brain, such highly-packed neurons and glial cell culture
system with complex functional neuronal networks constructed on
3D-BC/G present a better model for studying the physiological and
pathological processes in the brain.
2.8. Differential gene expression on the three-dimensional biomaterials
RNA-Seq was performed to identify differences in the gene expres-
sion proles of NSCs cultured on 3D-G and 3D-BC/G substrates, and we
explored the most abundantly expressed genes in NSCs cultured on these
substrates. Fig. S1 shows the expression levels of the top 200 most
abundant genes in NSCs cultured on 3D-BC/G (red bar). For comparison,
the expression levels for the same transcripts in NSCs cultured on 3D-G
(green bar) and the abundance rankings for these transcripts are also
illustrated. As shown in the gures, the majority of the transcripts that
were richly expressed in one group were also abundantly expressed in
the other. We compared the expression levels of all of the transcripts in
these two groups and selected the top 40 differentially expressed genes
(Fig. S2A and B). The differentially expressed genes were categorized as
those whose expression levels were above background and at least 2-fold
different between the two groups (p <0.01). Among these differentially
expressed genes, Nhlh1, Epcam, Cidea, Pdzph1, 2310069G16Rik, Ser-
pinf1, Emb, Cacng6, Vwc2, Flywch2, Ptgs1, Pgm5, Kcnip2, Fut4, Gjd2, Fhl2,
Fam213a, Dbpht2, Krt1, Gal3st1, Ppp1r1b, Mroh3, and Insc were prefer-
ably expressed in the NSCs cultured on 3D-BC/G.Pkn3, BC030867, Tes,
1700001L05Rik, Frmd3, Slc3a1, Arhgef3, and 1700012D01Rik were
preferably expressed in NSCs cultured on 3D-G.
2.9. Cell cycle analysis
Our results showed that NSCs cultured on 3D-BC/G had signicantly
greater proliferative ability than NSCs cultured on 3D-G. However, the
detailed mechanism behind this difference remains unknown. In order
to identify the possible cell-cycling genes regulated by 3D-BC/G, we
compared our differentially expressed genes with the KEGG pathway
database and identied 31 differentially expressed genes related to the
cell cycle (Fig. S3A). The genes that were highly expressed in NSCs
cultured on 3D-G included Stag1, Rad21, Mad1l1, Gsk3b, Smad3,
Gadd45a, Mad2l1, Prkdc, Ep300, Cdk6, Atm, Smad2, Ccnd1, Crebbp, E2f1,
Cdc20, Bub1b, and Plk1 (Fig. S3A). Silencing of Rad21 has been shown to
induce cell cycle arrest and apoptosis in breast tumor cells [55], and
Fig. 5. NSC differentiation on 3D-BC/G. Representative immunouorescence images of differentiated NSCs stained with Tuj-1 (red) and DAPI (blue).
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Smad2 and Smad3 can create an appropriate chromatin landscape to
allow enhancer transcription. Smad2 and Smad3 are also the effectors of
TGFβ signaling, which is important for NSC proliferation and develop-
ment [56,57]. The histone acetyltransferase gene EP300 is critical for
sustaining the proliferation of human leukemia and lymphoma cell lines
[58]. Mad2l1 is an essential component of the spindle assembly check-
point, and deletion of a single Mad2l1 allele results in defective mitotic
checkpoint activation in mouse embryonic broblasts and in human
HCT116 cancer cells [59]. Gadd45a is involved in DNA repair and helps
to maintain genomic stability.
The highly expressed genes in NSCs cultured on 3D-BC/G included
Cdkn1c, Ccne1, Cdc25b, Mcm2, Gadd45g, Anapc13, Mcm4, Ccnh, Rbx1,
Cdkn2b, and Cdkn1a (Fig. S3A). Interestingly, we identied three cyclin-
dependent kinase inhibitors –Cdkn1c, Cdkn2b, and Cdkn1a. Cdkn1c is a
major regulator of cell cycle progression and inhibits cyclin/cyclin-
dependent kinase complexes in the mid-G1 phase [60,61]. Cdkn1a can
inhibit the progression from G1 to S phase by interacting with the
N-terminal domain or by interfering with the phosphorylation of CDK1
and CDK2 [62], and similarly Cdkn2b induces G1 arrest. Mcm2 and
Mcm4 are essential protein components of pre-replicative complexes
and catalyze the unwinding of DNA duplexes, and their activation re-
quires the actions of cyclin-dependent kinases [63,64]. Most of the
remaining differentially expressed cell cycle-regulating genes we iden-
tied have not been characterized in-depth in NSCs and need to be
further studied.
2.10. Transcription factor analysis
Transcription factors (TFs) are associated with neurogenesis, prolif-
eration, differentiation, and epigenetic control in NSCs. We identied
seven TF genes (Bmp4, Id3, Id2, Id1, Map2k2, Id4, and Fzd2) that were
signicantly highly expressed in NSCs cultured on 3D-BC/G and 25 TF
genes (Map2k1, Smad2, Jarid2, Wnt5a, Apc, Fzd10, Skil, Fzd1, Jak3,
Zfhx3, etc.) that were signicantly highly expressed in NSCs cultured on
3D-G (Fig. S3B).What is striking is that Jak3 was richly expressed in
NSCs cultured on 3D-BC/G,but not in NSCs cultured on 3D-G. Jak3 in-
hibition has been shown to induce neuronal differentiation, [65] and the
highly expressed TF genes Rest and Skil have the ability to promote the
self-renewal of NSCs and other stem cells [66,67]. Thus we speculate
that 3D-G has reduced ability to maintain the stemness of NSCs and thus
results in low proliferation ability.
Fig. 6. Three-dimensional functional neuronal networks were constructed on 3D-BC/G and 3D-G. (A) Representative immunouorescence images of cortical cells
after 8 days of culture on 3D-BC/G (left) and 3D-G (right) stained for neurons (with Tuj-1, red), astrocytes (with GFAP, green), and nuclei (with DAPI, blue). (B) Box
plot of cell density for cortical cultures grown on 3D-BC/G and 3D-G (n =8, **p <0.01 from the Mann–Whitney U-test). (C, D) Representative uorescence images of
cortical cultures after 8 days loaded with Fluo-4 AM and grown on 3D-BC/G (C) and 3D-G (D). (E, F) Spontaneous calcium transients on 3D-BC/G and 3D-G from
three selected neurons after 8 days of culture. (G) Frequency of neuronal calcium spikes in NSCs cultured on 3D-BC/G and 3D-G (n =121 neurons from ve in-
dependent cultures for 3D-BC/G and n =76 neurons from three independent cultures for 3D-G; *p <0.05 from Mann-Whitney test). (H) Mean correlation coefcient
of neuronal calcium spikes in NSCs cultured on 3D-BC/G and 3D-G (data are from ve independent cultures for 3D-BC/G and three independent cultures for 3D-G; ns:
no signicant difference).
R. Guo et al.

Colloids and Surfaces B: Biointerfaces 200 (2021) 111590
8
2.11. Signaling pathway analysis
Signaling pathways such as Notch, Wnt, and growth factor-mediated
pathways have been shown to play important roles in stem cell biology.
In the current study, we examined each of these pathways in greater
depth. Fig. S4 summarizes all of the different signaling pathways and the
differential gene expression among these pathways. As shown in
Fig. s4A, we identied many signicant signaling pathways that play
important roles in regulating NSC homeostasis and neurogenesis, such as
FoxO [68], insulin [69], HIF-1 [70], estrogen, cAMP [71], ErbB, Ras,
Wnt [72], Notch [73], and TGF-β [74]. Remarkably, the FoxO signaling
pathway, which dominates the stem cell fate decision by regulating
critical cellular events such as apoptosis, cell-cycle progression, glucose
metabolism, oxidative stress resistance, and longevity, was the leader in
top rankings (Fig. S1A) [75]. The FoxO family has four isoforms in
mammals – FoxO1, Fox3a, Fox4, and the more distantly related FoxO6 –
and we found that FoxO4 was highly expressed in NSCs cultured on
3D-BC/G but FoxO3 and FoxO6 were highly expressed in NSCs cultured
on 3D-G.Most importantly, the FoxO signaling pathway has been shown
to cross talk with other signaling pathways such as the insulin, IGF, EGF,
Akt, and Wnt signaling pathways [76,77]. Therefore, we speculate that
the top position of the FoxO signaling pathway is the result of the cu-
mulative effects of various signaling pathways.
3. Conclusion
The addition of bacterial cellulose to graphene foam signicantly
enhanced the biocompatibility, proliferation, differentiation, and for-
mation of neural networks differentiated from NSCs. Moreover, primary
cortical neurons cultured on 3D-BC/G formed an intense neuronal
network with greater network activity than that which was formed on
3D-G. We investigated the transcriptome differences between NSCs
cultured on the 3D-BC/G and 3D-G scaffolds and found signicantly
differentially expressed genes that might regulate NSC differentiation
and proliferation. This provides a better understanding of the regulatory
patterns of NSCs. However, the clinical application of these materials
needs further investigation. Our 3D-BC/G culture system is not
restricted to NSCs and primary cortical neurons, and it has the potential
to be optimized for use with different cells or in different areas of
regenerative medicine.
Funding sources
This work was supported by grants from the Major State Basic
Research Development Program of China (2015CB965000), the Na-
tional Natural Science Foundation of China (No. 81970883, 31571530,
81622013, 81470692, 31500852, 31871322), National Natural Science
Foundation of China (91839101, Nos. 82030029, 81970882,
51803092), the Strategic Priority Research Program of the Chinese
Academy of Science (XDA16010302, XDA16010303), the Jiangsu
Province Natural Science Foundation (BK20200862, BK20181435,
BE2019711), Boehringer Ingelheim Pharma GmbH, the Yingdong Huo
Education Foundation, the Fundamental Research Funds for the Central
Universities (2242017K41042, 2242017K3DN23, 2242017K41041), the
Scientic Research Foundation of the Graduate School of Southeast
University (YBJJ1739), Shenzhen Fundamental Research Program
(JCYJ20190814093401920), and “the Fundamental Research Funds for
the Central Universities” and “Postgraduate Research & Practice Inno-
vation Program of Jiangsu Province” (KYCX17_0050).
CRediT authorship contribution statement
Rongrong Guo: Data curation. Jian Li: Data curation. Chuntao
Chen: Data curation. Miao Xiao: Data curation. Menghui Liao: Data
curation. Yangnan Hu: Data curation. Yun Liu: Data curation. Dan Li:
Data curation. Jun Zou: Data curation. Dongping Sun: Data curation.
Vincent Torre: Data curation. Qi Zhang: Data curation. Renjie Chai:
Data curation. Mingliang Tang: Data curation.
Declaration of Competing Interest
The authors report no declarations of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2021.111590.
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