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Response of macrophages and neural cells in

Cite this: DOI: 10.1039/c8bm00902c
contact with reduced graphene oxide microfibers
M. C. Serrano, *a M. J. Feito,b A. González-Mayorga,c R. Diez-Orejas,d
M. C. Matesanzb and M. T. Portolés*b

Received 31st July 2018,
Accepted 14th September 2018
DOI: 10.1039/c8bm00902c
rsc.li/biomaterials-science
Graphene-based materials are revealing a great promise for biomedical applications and demonstrating
attractiveness for neural repair. In the context of neural tissue damage, the dialogue between neural and
immune cells appears critical for driving regeneration, thus making the understanding of their relations
pivotal. Herein, the acute response of RAW-264.7 macrophages on nanostructured reduced graphene
oxide (rGO) microfibers has been evaluated through the analysis of cell parameters including proliferation,
viability, intracellular content of reactive oxygen species, cell cycle, apoptosis, and cell size and complex-
ity. The influence of the direct contact of rGO microfibers on their polarization towards M1 and M2 phe-
notypes has been studied by analyses of both M1 (CD80) and M2 (CD163) markers and the secretion of
the inflammatory cytokines TNF-α and IL-6. Finally, the capability of these rGO microfibers to regulate
neural stem cell differentiation has been also evaluated. Findings reveal that rGO microfibers inhibit the
proliferation of RAW-264.7 macrophages without affecting their viability and cell cycle profiles. The pres-
ence of M1 and M2 macrophages on these microfibers was confirmed after 24 and 48 h, respectively,
accompanied by a decrease in TNF-α and an increase in IL-6 cytokine secretion. These rGO microfibers
were also able to support the formation of a highly interconnected neural culture composed of both
neurons (map2+ cells) and glial cells (vimentin+ cells). These findings encourage further investigation of
these microfibers as attractive biomaterials to interact with immune and neural cells, attempting to
support wound healing and tissue repair after implantation.

1. Introduction trauma exerted on the neural tissue, leading to the loss of

Tissue engineering involves the regeneration of damaged
tissues and organs1,2 and requires the development of func-
tional 3D substitutes in vitro with appropriate biological,
physicochemical and mechanical characteristics that allow to
mimic natural tissues in vivo.3,4 These substitutes must
possess the capability for guiding cell growth, delivering bio-
molecules and stimulating physicochemical signal mecha-
nisms of native tissues.5 At the central nervous system (CNS),
damage and degeneration occur as both direct mechanical
injury and secondary pathophysiological mechanisms of a
a
Group of Materials for Health, Instituto de Ciencia de Materiales de Madrid
(ICMM), Consejo Superior de Investigaciones Científicas (CSIC), 28049-Madrid,
Spain. E-mail: mc.terradas@csic.es
b
Department of Biochemistry and Molecular Biology, Faculty of Chemistry,
Universidad Complutense de Madrid, Instituto de Investigación Sanitaria del
Hospital Clínico San Carlos (IdISSC), 28040-Madrid, Spain.
E-mail: portoles@quim.ucm.es
c
Laboratory of Interfaces for Neural Repair, Hospital Nacional de Parapléjicos,
Servicio de Salud de Castilla-La Mancha, 45071-Toledo, Spain
d
Department of Microbiology and Parasitology, Faculty of Pharmacy,
Universidad Complutense de Madrid, 28040-Madrid, Spain
numerous neural elements such as neurons, supportive glial
cells and neural connections.6 Regeneration at the peripheral
nervous system is enhanced by the presence of Schwann cells,
which mediate reparative responses through their conversion
to repair-promoting phenotypes induced by nerve injury.7
Conversely, as these cells are not present at the CNS, regener-
ation is hampered in both the brain and the spinal cord.
Current strategies to promote repair and overcome limitations
in the injured spinal cord include pharmacological
approaches,8 cell therapy,9 immunotherapies,10 and biomater-
ials,11 to cite a few.
Diverse biomaterials have been investigated to date for
mediating regeneration at the injured spinal cord including
polysaccharides (e.g., hyaluronic acid, chitosan, agarose), pro-
teins (e.g., collagen, gelatin) and organic polymers (e.g., poly
(lactic-co-glycolic) acid, polycaprolactone).12 In the last decade,
graphene-based materials (GBMs) are revealing as a great
promise for biomedical applications including tissue engineering
due to their superior physicochemical properties mainly derived
from their chemical structure and features at the nanoscale.13
Although the toxicity of GBMs is still an open debate,14,15 most
studies published to date support the existence of a safe range

This journal is © The Royal Society of Chemistry 2018 Biomater. Sci.


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of sizes, shapes and concentrations for their use in biological
scenarios.16 Biomedical applications of these materials
include drug/gene delivery, imaging, biosensors, stem cell
differentiation, and photothermal therapy.17,18 Regarding con-
figuration, GBMs can be prepared and assembled in diverse
configurations such as nanosheets, nanoparticles, 2D films,
and 3D aerogels and scaffolds, among others.19 Their confor-
mation in the shape of fibers and microfibers of diverse
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dimensions is attracting increasing attention due to their prac-
tical relevance in terms of applicability.20 Although the use of
both GBM-coated and GBM-based fibers for neural growth and
regeneration is still in its infancy, recent evidences are
encouraging the utility of these platforms for this type of
applications.21–23
In the recent years, outstanding progress in the field has
started unraveling the impact of GBMs on immune cells.24 For
instance, amine groups-functionalized graphene oxide (GO)
polarizes T-cell and monocyte activation toward a T-helper-1/
M1 immune response.25 GO also modulates B cell surface phe-
notype by influencing CD80, CD86 and CD40 receptors and
antigen presenting molecules.26 Moreover, it reduces the
secretion of immunoglobulins in terminal differentiated
plasma cells. When interfaced with neural components, GBMs
are demonstrating an enormous attractiveness in vitro27–30 and
in vivo,31 thus becoming interesting candidates for spinal cord
repair.32 Both in vitro and in vivo models are currently used for
the understanding of neural tissue responses to this kind of
materials.33,34 For instance, porous 3D reduced graphene
oxide (rGO) scaffolds, implanted in the injured rat spinal cord,
induce immunomodulatory and angiogenic responses without
systemic toxicity.35,36 On the other hand, nanostructured rGO
microfibers regulate the neural stem cell differentiation into
neurons, evidencing their potential as an artificial neural
tissue engineering scaffold for neural regeneration.37
Concerning the healing process, a recent investigation with
normal and diabetic rats shows that rGO scaffolds enhance
angiogenesis and collagen synthesis, shortening the inflam-
mation phase and recruiting macrophages to enhance the
early phase of wound healing.38
Macrophages, as pivotal cells in the immune response, play
a key role in tissue regeneration by preparing the surrounding
parenchyma.39 During nerve injury, it has been recently
observed an enhanced macrophage recruitment by Toll-like
receptor 4 activation.40 However, macrophages can also
produce tissue damage if their response is excessive.39
Specifically, macrophages display a spectrum of activation phe-
notypes ranging from two opposite extremes: the classically
activated M1 macrophages ( pro-inflammatory phenotype) and
the alternatively activated M2 macrophages (reparative pheno-
type).41 Both M1 and M2 macrophages are characterized by the
expression of distinct cell surface markers and by the secretion
of different cytokines that allow them to respond to changes in
their microenvironment.42 In this sense, although the pres-
ence of macrophages plays essential roles during regeneration
in many tissues,43,44 the modulation of macrophage phenotype
at the site of injury can be crucial, influencing the regenerative
Biomater. Sci.
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outcomes.39,45 In the present study, the acute response of
RAW-264.7 macrophages cultured for short time points (24
and 48 h) on rGO microfibers has been evaluated through
different cell parameters including: proliferation, viability,
intracellular content of reactive oxygen species (ROS), cell
cycle, apoptosis, and cell size and complexity. Additionally, the
influence of the direct contact of rGO microfibers on their
polarization towards M1 and M2 phenotypes has been studied
through both the observation of specific M1 (CD80) and M2
(CD163) markers and the analysis of the secretion of the
inflammatory cytokines TNF-α and IL-6. Finally, the capability
of these rGO microfibers to support neural progenitor cell
growth and differentiation has been evaluated.
2. Materials and methods
2.1. rGO microfiber preparation and characterization
GO was purchased as a slurry from Graphenea, S.A. rGO micro-
fibers were prepared as described elsewhere.23,46 Briefly, a GO
nanosheets suspension (5 mg mL−1 in distilled water) was col-
lected into standard Hirschmann capillary tubes (1.35 mm of
inner diameter and 120 mm in length), which were thereafter
sealed up at their ends and thermally treated at 220 °C for 2 h.
The resulting rGO microfibers were then extracted from the
capillary tubes and allowed to dry overnight under light weight
to avoid curving. Microfibers morphology was evaluated by
scanning electron microscopy (SEM) by using a Hitachi
S-3000N microscope. Surface roughness of the microfibers was
studied by atomic force microscopy (AFM, Bruker multimode
Nanoscope III A). XPS studies were performed using a K-Alpha
(Thermo Scientific) electron spectrometer equipped with an Al
Kα (hν = 1486.68 eV) Watts X-ray source (12 kV and 6 mA) (n =
40 scans for complete spectra, n = 75 for C 1s and O 1s and n =
300 for N 1s). The flood gun option was active during the ana-
lyses for charge compensation, and the pressure in the analysis
chamber was maintained at 2.4 × 10−7 mbar. The pass energy
of the analyzer was set at 200 eV for complete spectra and 40
eV for zone spectra. The binding energies were referenced to
the binding energy of the C 1s core-level spectrum at 285 eV.
Data processing was performed with the XPS peak-fit program
in Advantage 4.87 software. Spectra were decomposed with the
least-squares fitting routine provided by the software with the
Gaussian/Lorentzian (90/10) product function and after sub-
tracting a Shirley background. Atomic fractions were calculated
using peak areas normalized on the basis of sensitivity factors
provided by the manufacturer.
2.2. Culture of RAW-264.7 macrophages
RAW-264.7 cells (2.5 × 104 cells) in 20 μL of Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 10%
fetal bovine serum (FBS, Gibco, BRL), 1 mM L-glutamine
(BioWhittaker Europe, Belgium), penicillin (200 μg mL−1,
BioWhittaker Europe, Belgium), and streptomycin (200
μg mL−1, BioWhittaker Europe, Belgium) were seeded on bare
rGO microfibers glued to standard glass coverslips placed into
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Biomaterials Science
12 well culture plates (CULTEK S.L.U., Madrid, Spain) and
maintained at room temperature for 5 min after seeding.
Then, a volume of 1.5 mL of culture medium was added in
each well and cells were maintained at 37 °C under a CO2 (5%)
atmosphere. After 24 h and 48 h in culture, the medium was
aspirated and frozen at −20 °C for inflammatory cytokine
detection. After washing each well containing rGO microfibers
and RAW-264.7 macrophages with phosphate buffered saline
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(PBS), cells attached on rGO microfibers were observed by con-
focal fluorescence microscopy in order to characterize macro-
phage phenotypes (as indicated below) and cells around rGO
microfibers were harvested using cell scrapers. Cell suspen-
sions were centrifuged at 310g for 10 min and resuspended in
fresh medium for the analysis of cell viability, intracellular
ROS content, cell cycle and apoptosis by flow cytometry as
described below. In order to determine the effects of rGO
microfibers on cell proliferation, cells were counted with a
Neubauer hemocytometer after harvesting.
2.3. Neural cells isolation and culture
Embryonic neural progenitor cells (ENPCs) were obtained
from cerebral cortices of E18 Wistar rat embryos as pre-
viously described.47 Pregnant female Wistar rats were pro-
vided by the animal facilities of the Hospital Nacional de
Parapléjicos. All the experimental protocols used for rat
embryos extraction adhered to the regulations of the
European Commission (directives 2010/63/EU and 86/609/
EEC) and the Spanish government (RD53/2013 and ECC/
566/2015) for the protection of animals used for scientific
purposes and followed institutional guidelines at the
Hospital Nacional de Parapléjicos. These experimental proto-
cols were approved by the Animal Research and Well-Being
Committee at the Hospital Nacional de Parapléjicos
(144CEEA/2016), the corresponding Enabled Organ at the
Hospital Nacional de Parapléjicos (11-OH/2016) and the
Department of Agriculture, Environment and Rural
Development of the region of Castilla-La Mancha (Spain,
21-2016). ENPCs were only used when viability of the iso-
lated cells reached values superior to 85%.
Prior to ENPCs culture, rGO microfibers were coated with
poly(L-lysine) (PLL) by immersion in an aqueous solution con-
taining the polymer (45 μg mL−1) for 1 h at room temperature.
After a gentle wash in sterile distilled water, PLL-coated micro-
fibers were allowed to dry in a biological cabinet for 2 h and
sterilized under UV radiation for 30 min. A total of 2.5 × 104
cells contained in a small fraction of media (typically 10 μL)
was seeded on the top part of each microfiber and allowed to
attach for 10 min. Immediately after, samples were completely
covered with 500 μL of complete Neurobasal™ media contain-
ing: B-27 supplement (2%), streptomycin (100 UI mL−1), peni-
cillin (100 UI mL−1), and L-glutamine (1 mM). After 2 h of
adhesion in a sterile incubator at 37 °C under a CO2 atmo-
sphere (5%), culture media was replaced and cultures main-
tained for up to 21 days. Culture media was half replaced every
4 days and completely replaced every 7 days.
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2.4. Morphological studies of M1 and M2 macrophages by
confocal laser scanning microscopy
Macrophages were cultured for 24 and 48 h on rGO microfi-
bers in order to evaluate their polarization towards M1 and M2
phenotypes in direct contact with these microfibers. Cell were
then fixed with 3.7% paraformaldehyde (Sigma-Aldrich
Corporation, St Louis, MO, USA) in PBS for 10 min, washed
with PBS and permeabilized with 0.1% Triton X-100 (Sigma-
Aldrich Corporation, St Louis, MO, USA) for 5 min. The
samples were then washed with PBS and pre-incubated with
PBS containing 1% bovine serum albumin (BSA, Sigma-
Aldrich Corporation, St Louis, MO, USA) for 30 min to prevent
non-specific binding. Samples were incubated in 1 mL of
staining buffer with either phycoerythrin (PE) conjugated anti-
mouse CD80 antibody (2.5 µg mL−1, BioLegend, San Diego,
CA, USA), as an M1 marker, or anti-mouse CD163 conjugated
to Alexa Fluor® 488 (2.5 µg mL−1, BioLegend, San Diego,
CA, USA), as an M2 marker, for 30 min at 4 °C in the dark.
Then, cells were incubated during 20 min with either FITC–
phalloidin or rhodamine–phalloidin (dilution 1 : 40, Molecular
Probes), respectively, to stain F-actin filaments. Samples were
then washed with PBS and the cell nuclei were stained with
DAPI (4′-6-diamidino-2′-phenylindole, 3 μM in PBS, Molecular
Probes) for 5 min. Samples were examined by a LEICA SP2 con-
focal laser scanning microscope (CLSM). The fluorescence of
both FITC and Alexa Fluor® 488 was excited at 488 nm and the
emitted fluorescence was measured at 491–586 nm. PE fluo-
rescence was excited at 488 nm and measured at 575–675 nm.
DAPI fluorescence was excited at 405 nm and measured at
420–480 nm.
2.5. Inflammatory cytokine detection
The amounts of TNF-α and IL-6 present in the culture medium
were quantified by ELISA (Gen-Probe, Diaclone) with pre-
coated strip plates, biotinylated secondary antibodies and
streptavidin–avidin conjugated to horseradish peroxidase for
colorimetric reaction, according to the manufacturer’s instruc-
tions. Recombinant cytokines were used as standards. The
absorbance at 450 nm of the samples and standards was
measured using an ELISA Plate Reader. The sensitivity of these
assays was 25 pg mL−1 and 10 pg mL−1, respectively, and their
inter-assay variation coefficients were <10%.
2.6. Flow cytometry studies on macrophages in contact with
rGO microfibers
The effect of the direct contact of RAW-264.7 macrophages
with rGO microfibers was evaluated by flow cytometry.
Specifically, cell parameters such as viability, intracellular ROS
content, cell cycle, apoptosis, and cell size and complexity
were investigated. After incubation with the different probes,
the conditions for data acquisition and analysis were estab-
lished using negative and positive controls with the CellQuest
Program of Becton Dickinson. For statistical significance, at
least 10 000 cells were analyzed in each sample.
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2.6.1. Cell viability. Cell viability was evaluated by exclusion
of propidium iodide (PI; 0.005% wt/vol in PBS, Sigma-Aldrich,
St Louis, MO, USA). PI was added to the cell suspensions in
order to stain the DNA of dead cells. The fluorescence of PI
was excited by a 15 mW laser tunning to 488 nm and the
emitted fluorescence was measured with a 530/30 band pass
filter in a FACScalibur Becton Dickinson flow cytometer.
2.6.2. Intracellular ROS content. Cells were incubated at
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cells including glial cells. The secondary antibodies selected
were: Alexa Fluor® 488 goat α-mouse IgG (H + L) and Alexa
Fluor® 594 goat α-rabbit IgG (H + L) (Life technologies). Both
primary and secondary antibodies were dissolved in PBS con-
taining saponin (0.25%) and fetal goat serum (2%) to guaran-
tee cell permeability and block non-specific binding, respect-
ively. Each antibody was incubated for 1 h at room temperature
in darkness. Cell nuclei were labeled with DAPI (3 μM, 5 min).

37 °C for 30 min with 100 μM of 2′,7′-dichlorofluorescein di- The fluorescence in the confocal SP5 microscope were col-
acetate (DCFH/DA, Serva, Heidelberg, Germany) for directly lected as follows: Alexa Fluor® 488 excitation at 488 nm with
measuring the intracellular content of ROS. DCFH/DA diffuses an argon laser and detection in the range 507–576 nm, Alexa
into cells and is deacetylated by cellular esterases to non-fluo- Fluor® 594 at 594 nm with a helium–neon laser and detection
rescent DCFH, which is rapidly oxidized to highly fluorescent in the range 625–689 nm and DAPI excitation at 405 nm with a
DCF by ROS. To measure the intracellular concentration of diode UV laser and detection in the range 423–476 nm.
ROS, the DCF fluorescence was excited by a 15 mW laser Reflection images were simultaneously captured to localize the
tuning to 488 nm and the emitted fluorescence was measured microfibers.
with a 530/30 band pass filter in a FACScalibur Becton
Dickinson flow cytometer. 2.9. Statistics
2.6.3. Cell-cycle analysis and apoptosis detection. Cells
Data are expressed as the mean ± standard deviation of one
were centrifuged at 310g for 10 min, resuspended in PBS
representative experiment out of three carried out in triplicate.
(0.5 mL) and incubated with 4.5 mL of ethanol 70% during 4 h
Statistical analysis was performed using the Statistical Package
at 4 °C. Then, cells were centrifuged at 310g for 10 min,
for the Social Sciences (SPSS) version 19 software. Statistical
washed with PBS and resuspended in 0.5 mL of PBS with
comparisons were made by one-way analysis of variance
Triton X-100 0.1%, PI 20 mg mL−1 and RNAsa 0.2 mg mL−1
(ANOVA). Scheffé test was used for post hoc evaluations of
(Sigma-Aldrich, St Louis, MO, USA). After incubation at 37 °C
differences between groups. P < 0.05 was considered as statisti-
for 30 min, the fluorescence of PI was excited by a 15 mW
cally significant.
laser tunning to 488 nm and the emitted fluorescence was
measured with a 585/42 band pass filter in a FACScalibur
Becton Dickinson flow cytometer. The cell percentage in each
cycle phase (i.e., G0/G1, S and G2/M) was calculated with the 3. Results and discussion
CellQuest Program of Becton Dickinson. The SubG1 fraction 3.1. rGO microfiber characterization
was used as indicative of apoptosis.
rGO microfibers were prepared by a one-step dimensionally
2.6.4. Cell size and complexity analysis. Forward angle
confined hydrothermal method previously described.47 Fig. 1
(FSC) and side angle (SSC) scatters were evaluated as indicative
illustrates the morphology of the resulting microfibers at
of cell size and complexity, respectively, using a FACScalibur
different magnifications as observed by SEM. Typically, micro-
Becton Dickinson flow cytometer.
fibers diameter was 106.0 ± 2.5 μm, with rGO sheets packed
2.7. ENPCs morphological studies on rGO microfibers forming compact fibers. Conversely to previously reported rGO
microfibers,23,37 their surface was highly rough due to the
ENPCs cultured on rGO microfibers were rinsed in PBS twice
packaging of large-sized rGO sheets, eventually standing out
and fixed with glutaraldehyde (2.5% in PBS) for 45 min as a
from the surface. When analyzed by AFM, the surface rough-
conventional fixation method for examination by SEM. After
ness was characterized by a root-mean-square (rms) value of
washing in distilled water, dehydration was performed by
399.3 ± 69.6 nm (Fig. 2A). We anticipate that this microfiber
using series of ethanol solutions for 15 min (2 washes) and a
roughness will necessarily influence cell responses as cells are
final dehydration in absolute ethanol for 30 min. Samples
able to detect and respond to surface topography and rough-
were then dried at room temperature for at least 24 h. After
ness features.48,49 Typically, increased cell responses are found
mounting in stubs and gold coating in vacuum, the mor-
phology of the samples was characterized by a Hitachi S-3000N
microscope working at 15 kV with secondary electrons.

2.8. ENPCs differentiation studies on rGO microfibers

The differentiation of ENPCs in culture on rGO microfibers
was investigated by specific immune-labeling and CLSM. After
fixation with paraformaldehyde (4% in PBS) at room tempera-
ture for 10 min, fixed samples were incubated with primary
antibodies against the following cell proteins: map-2 for Fig. 1 Morphological characterization of rGO microfibers by SEM.
somas and dendrites in neurons and vimentin for non-neuron Scale bars: 100, 20 and 10 μm.

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Fig. 2 Physic-chemical characterization of the rGO microfibers. (A)
Surface roughness characterization by AFM. Representative surface plot
for an area of 100 μm2. (B) C 1s spectrum by XPS.
in rougher substrates as a function of their phenotype.49 For
instance, titanium alloy with topography roughness in the
order of 40 nm induced significant osteogenic gene expression
and morphological changes in human skeletal stem cells.50
Importantly, there is a significant synergy between substrate
roughness and surface chemistry that remarkably influences
cell responses, as demonstrated with NIH/3T3 fibroblasts cul-
tured on micro- and nanostructured Si substrates.51
Specifically, although adhesion was favored on hydrophilic
substrates, cell spreading was maximum on low-rough surfaces
with independence of their wettability. In a different work, tita-
nium hierarchical substrates with microholes of 10–30 μm in
diameter and nanopits of tens of nm obtained by NH4OH and
H2O2 treatment boosted cell attachment, proliferation and
osteogenic differentiation.52 Given this close relation between
topography and surface chemistry, the degree of GO reduction
and chemical composition at the microfiber surface was then
characterized by XPS (Fig. 2B and Table 1). When compared
with the GO slurry, the thermal treatment at 220 °C for 2 h
induced a significant reduction of GO, characterized by a
remarkable decrease in oxygen-containing groups (e.g.,
Table 1 XPS characterization of GO slurry and rGO microfibers
Sample %C %O %N C sp2
GO slurry 67.7 29.8 0.3 41
rGO microfiber 86.0 13.4 0.6 59
Fig. 3 Effects of rGO microfibers on proliferation, viability and intracellular ROS content in RAW-264.7 macrophages after 24 h in culture. Controls
in the absence of microfibers were carried out in parallel (white). Statistical significance: *** p < 0.005.
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hydroxyl groups) accompanied by the subsequent increase in
carbon content (i.e., carbon atoms representing 67.7% in GO
slurry and 86.0% in rGO microfibers). The final carbon to
oxygen ratio augmented from 2.3 to 6.4 as a consequence of
the thermal reduction. These values are in agreement with pre-
vious reports on rGO microfibers fabricated by a similar
methodology.23
3.2. Effects of rGO microfibers on RAW-264.7 macrophage
proliferation, viability and intracellular ROS content
Fig. 3 shows the effects of rGO microfibers on the prolifer-
ation, viability and intracellular ROS content of
RAW-264.7 macrophages after 24 h in culture. As can be
observed, the presence of these microfibers produced a signifi-
cant delay in macrophage proliferation ( p < 0.005), without
alterations in cell viability but inducing a pronounced increase
in the intracellular ROS content ( p < 0.005). In agreement with
these findings, previous studies evidenced a significant
decrease in proliferation, cell cycle alterations, apoptosis, and
oxidative stress in RAW-264.7 macrophages, but also Saos-2
osteoblasts and MC3T3-E1 preosteoblasts, after internalization
of GO nanosheets.53 Importantly, this nanomaterial was pre-
ferentially localized on F-actin filaments, thus altering cell pro-
cesses involving cytoskeleton proteins. As the proliferation
delay and the enhanced ROS production induced by rGO
microfibers on RAW-264.7 cells in these studies could be also
related to alterations in macrophage cell cycle phases, apopto-
sis, size, complexity and morphology, these cell parameters
were further investigated as described below.
3.3. Effects of rGO microfibers on RAW-264.7 macrophage
cycle phases, apoptosis, size, complexity and morphology
To study the effects of rGO microfibers on macrophage divi-
sion and growth in more detail, we analyzed the cell cycle
C sp3 C—OH OvC–O Trans. π C/O (at.) C/N (at.)
— 48 10 1 2.3 247
11 11 10 8 6.4 137
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Fig. 4 Effects of rGO microfibers on cell cycle profiles, cell size (FSC)
and cell complexity (SSC) of RAW-264.7 macrophages after 24 h in
culture. Cell cycle profiles: M1 = SubG1 fraction (apoptotic cells with
fragmented DNA), M2 = G0/G1 phase (Quiescence/Gap1), M3 = S phase
(Synthesis), and M4 = G2/M phase (Gap2 and Mitosis).
phases by flow cytometry after 24 h of culture in the presence
of this biomaterial. This analysis allowed us to detect the per-
centage of cells in the progressive stages of the cycle: G0/G1
phase (Quiescence/Gap1), S phase (Synthesis) and finally G2/M
phase (Gap2 and Mitosis). The SubG1 fraction was attributed
to the percentage of apoptotic cells with fragmented DNA. As
it can be observed in the left panels of Fig. 4, no alterations
were observed in the cell cycle profile of RAW-264.7 cells cul-
tured in the presence of rGO microfibers in comparison with
control cells. The percentage of cells in each stage of the cell
cycle has been depictured in Fig. 5. Importantly, the presence
of this biomaterial did not induced alterations in G0/G1, S and
G2/M phases of the cell cycle in comparison with control cells.
More importantly, macrophage apoptosis (SubG1 fraction) did
Fig. 5 Effects of rGO microfibers on the percentage of
RAW-264.7 macrophages at the different stages of the cell cycle after
24 h in culture. Controls in the absence of microfibers were carried out
in parallel (white). SubG1 fraction = apoptotic cells with fragmented
DNA, G0/G1 phase = Quiescence/Gap1, S phase = Synthesis, G2/M phase
= Gap2 and Mitosis.
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Fig. 6 Morphology of RAW-264.7 macrophages on rGO microfibers
after 24 h in culture. Representative CLSM images showing the cyto-
skeletal actin filaments labelled with FITC-phalloidin (green) and cell
nuclei stained with DAPI (blue).
not increase in the presence of rGO microfibers. On the other
hand, no changes in either cell size (FSC) or cell complexity
(SSC) of RAW-264.7 macrophages were detected after 24 h in
culture on these microfibers (Fig. 4, right panels).
The morphology of RAW-264.7 macrophages was then
observed by CLSM after 24 h of culture in direct contact with
rGO microfibers. Fig. 6 shows the actin filaments after staining
with FITC-phalloidin (green) and the nuclei stained with DAPI
(blue). These images evidence that macrophages adhered and
proliferated on rGO microfibers without suffering morphologi-
cal alterations.
3.4. Effects of rGO microfibers on RAW-264.7 macrophage
polarization towards M1 and M2 phenotypes
In order to evaluate the effect of the direct contact of rGO
microfibers on the polarization of RAW-264.7 macrophages
towards M1 and M2 phenotypes, the expression of CD80 and
CD163 as specific M1 and M2 markers, respectively, was
observed by CLSM after staining with specific fluorescent anti-
bodies. In Fig. 7, pro-inflammatory M1 macrophages (CD80+)
on an rGO microfiber after 24 h of culture are shown. These
merged images show the combination of the CD80 (red)
expression of M1 macrophages detected with a PE-conjugated
anti-mouse CD80 antibody, the actin cytoskeleton after stain-
ing with FITC-phalloidin (green), the nuclei stained with DAPI
(blue) and the rGO fiber (reflection mode, black and white).
The colocalization of CD80 (red) and actin (green) results in a
yellow coloration. Fig. 8 shows reparative M2
RAW-264.7 macrophages (CD163+) on rGO microfibers after
48 h of culture observed by CLSM. These merged images sim-
ultaneously display the CD163 (green) expression of M2 macro-
phages labelled with a Alexa Fluor®-conjugated anti-mouse
CD163 antibody, the actin cytoskeleton after staining with
rhodamine-phalloidin (red), the nuclei stained with DAPI
This journal is © The Royal Society of Chemistry 2018

Biomaterials Science
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Fig. 7 Pro-inflammatory M1 RAW-264.7 macrophages on rGO
microfibers after 24 h in culture. Representative CLSM images showing
the combination of the CD80 (red) expression of M1 macrophages
detected with a PE-conjugated anti-mouse CD80 antibody, the actin
cytoskeleton after staining with FITC-phalloidin (green), the nuclei
stained with DAPI (blue) and the rGO microfiber (reflection mode, black
and white).
Fig. 8 Reparative M2 RAW-264.7 macrophages on rGO microfibers
after 48 h in culture. Representative CLSM showing the combination of
the CD163 (green) expression of M2 macrophages labelled with a Alexa
Fluor®-conjugated anti-mouse CD163 antibody, the actin cytoskeleton
after staining with rhodamine-phalloidin (red), the nuclei stained with
DAPI (blue), and the rGO fiber (reflection mode, black and white).
(blue), and the rGO fiber (reflection mode, black and white). It
is important to highlight that the macrophage phenotype
polarization has been related to changes in cell shape46 that
were also observed in the present study. Thus, CD80+ macro-
phages, polarized towards the M1 phenotype, showed more
spherical shapes (Fig. 7) than CD163+ cells, polarized towards
the M2 phenotype, which show more elongated shapes
(Fig. 8), in agreement with other authors54 and previous
studies.55 Taken together, these results evidence that, although
mainly M1 macrophages were observed on rGO microfibers
after 24 h, a polarization towards an M2 phenotype was
induced by the microfibers after 48 h.
Potential positive and negative effects of macrophages in
disease and tissue remodeling have been described depending
on their appropriate polarization towards either an M1 or an
M2 phenotype and their ability to shift polarized responses.56
For instance, concerning the essential role of macrophages in
the prevention of bacterial and fungal infections, previous
studies have evidenced that GO nanosheets induced a signifi-
This journal is © The Royal Society of Chemistry 2018
View Article Online
Paper
cant increase of Candida albicans phagocytosis and killing by
both pro-inflammatory (M1/stimulated with LPS/IFN-γ) and
reparative (M2/stimulated with IL-4) macrophages, suggesting
a beneficial role of this nanomaterial during infection.57,58 In
the context of biomaterials implantation, the ability of the
host innate immune system to resolve a polarized macrophage
response after implantation may be of critical importance for
the replacement and/or repair of biologic structures.56 In
normal tissue repair, macrophages exhibit an M1 pro-inflam-
matory phenotype secreting pro-inflammatory cytokines at
early stages, while a preferential M2 pro-healing phenotype is
found at later stages to promote extracellular matrix synthesis
and cell proliferation.59 The fact that M1 macrophages appear
at early stages of normal wound healing followed by
M2 macrophages has suggested that the control of this M2/M1
balance could be a key step in the design of immuno-informed
biomaterials to enhance positive tissue remodeling, inte-
gration and regeneration.60 The results obtained in the present
study confirm that rGO microfibers first promote the M1 phe-
notype of RAW-264.7 macrophages followed by the M2 pheno-
type, thus suggesting the potential of these microfibers for
inducing wound healing and tissue repair after implantation.
3.5. Effects of rGO microfibers on TNF-α and IL-6 secretion
in RAW-264.7 macrophages
The analysis of TNF-α and IL-6 as inflammatory cytokines was
carried out in the culture medium after 24 and 48 h in culture
of RAW-264.7 macrophages in the presence of rGO microfi-
bers. As it can be observed in Fig. 9, a time-dependent
decrease of both cytokines was observed in the absence of the
biomaterial (control cells). The presence of rGO microfibers
induced a decrease of both TNF-α ( p < 0.005) and IL-6 (not sig-
nificant) secretion in RAW-264.7 macrophages after 24 h of
culture with respect to control cells. However, a significant
increase of IL-6 ( p < 0.05) was observed after 48 h in culture in
the presence of these rGO microfibers.
Contrarily to these results, previous studies demonstrated
that the internalization of GO nanosheets induced a signifi-
cant decrease of IL-6 levels in Saos-2 osteoblasts53 and primary
splenocytes.61 However, GO treatment significantly increased
the secretion of TNF-α by RAW-264.7 macrophages without
altering IL-6 and IL-1β levels.61 As oxidative stress-sensitive
generated molecules, cytokines play an important role in the
Fig. 9 Effects of rGO microfibers on TNF-α and IL-6 secretion in
RAW-264.7 macrophages. Controls in the absence of microfibers were
carried out in parallel (white). Statistical significance: *p < 0.05, ***p <
0.005 (comparisons with respect to the control) and #p < 0.05, ###p <
0.005 (comparisons between time points).
Biomater. Sci.

Paper
maintenance of cellular homeostasis.62 Particularly, IL-6 has
also been shown to protect against different compound-
induced cytotoxicity.63 The significant decrease of TNF-α and
increase of IL-6 observed in the medium of
RAW-264.7 macrophages cultured in the presence of rGO
microfibers (Fig. 9) could be related to the significant cell pro-
liferation decrease and ROS increase induced by these micro-
fibers, respectively (Fig. 3). In line with these findings, electro-
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spun microfiber scaffolds coated with self-assembled colloidal
graphene suppressed microglia and astrocyte activation levels
when implanted in the striatum and the subventricular zone
of adult rats.64 Although the concrete mechanisms behind
most of these modulatory effects of GBMs on immune cells are
still unknown, studies on RAW-264.7 macrophages exposed to
GO nanosheets indicate Toll-like receptor signaling cascades
(e.g., TLR4 and TLR9) as target pathways within inflammatory
responses.65
3.6. Effects of rGO microfibers on neural progenitor cell
growth and differentiation
We finally tested if these rGO microfibers could support
neural progenitor cell growth, as previously described for
similar rGO microfibers.23,37 Initial studies with bare rGO
microfibers revealed a poor adhesion of ENPCs, which were
unable to properly grow and differentiate on their surface
(data not shown). Conversely, confluent ENPCs cultures were
formed on top of rGO microfibers when coated with PLL, as
depicted in Fig. 10. Morphological studies by SEM demon-
strated the formation of a highly interconnected neural culture
on top of the microfibers (left images). Further differentiation
studies by immunofluorescence and CLSM revealed the pres-
ence of both neurons (map2+ cells) and glial cells (vimentin+
cells) on the microfibers (right images). Regarding microfibers
degradation, it is worth to mention that no signs of substrate
degradation or disassembly were observed during these long-
term cultures (i.e., 21 days). When handled for SEM and con-
focal studies, microfibers were confirmed to retain their initial
Fig. 10 Highly interconnected ENPCs cultures on rGO microfibers as
illustrated by representative SEM (left) and CLSM (right) images. On the
right micrographs, neurons appear in green (map-2 labeling), non-
neural cells including glial cells in red (vimentin labeling) and cell nuclei
in blue (DAPI).
Biomater. Sci.
View Article Online
Biomaterials Science
mechanical stability. Micron-sized aggregates of rGO
nanosheets released from the microfibers were not either
appreciated in the culture media.
4. Conclusions
In this work, we have demonstrated the capacity of rGO microfi-
bers to inhibit the proliferation of RAW-264.7 macrophages,
without affecting their viability and cell cycle profiles. Moreover,
this biomaterial mediate the polarization of these cells towards
M2 pro-healing phenotype after 48 h in culture, accompanied
by a decrease in TNF-α and an increase in IL-6 cytokine
secretion. Regarding their interaction with neural progenitor
cells, these rGO microfibers support the formation of highly
interconnected neural cultures composed of both neurons and
glial cells. These findings encourage further investigation of
these biomaterials for wound healing and tissue repair in
injured neural tissues where the combined actions of immune
and neural cells are essential for regeneration.
Conflicts of interest
Authors declare no conflicts of interests.
Acknowledgements
This work was supported by the Ministerio de Economía y
Competitividad and the Fondo Europeo de Desarrollo Regional
(MAT2016-75611-R and MAT2016-78857-R, MINECO/FEDER,
UE). M.C.M. is greatly indebted to the Ministerio de Economía y
Competitividad for a predoctoral fellowship. Dr Jose Ángel
Rodríguez and Dr Javier Mazarío from the Service of Microscopy
and Image Analysis at the Hospital Nacional de Parapléjicos are
acknowledged for assistance with CLSM studies. Authors are
thankful to Dr Enrique Rodríguez from the Servicio
Interdepartamental de Investigación at the Universidad Autónoma
de Madrid for assistance with SEM studies and Dr Daniel
Gamarra from the SAIUEx for XPS studies. Thanks also to the
staff of the Centro de Citometría y Microscopía de Fluorescencia
of the Universidad Complutense de Madrid (Spain) for the
assistance in flow cytometry and CLSM studies.
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