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Three-Dimensional Macroporous Graphene Scaffolds for Tissue Engineering

Article  in  Journal of Biomedical Materials Research Part A · August 2016

DOI: 10.1002/jbm.a.35867

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Three-dimensional macroporous graphene scaffolds
for tissue engineering

Gaurav Lalwani,1 Michael D’agati,1 Anu Gopalan,1 Manisha Rao,1

Jessica Schneller,2 Balaji Sitharaman1
1
Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York 11794-5281
2
Department of Bioengineering, National Institutes of Health, Bethesda, Maryland 20892

Received 27 February 2016; revised 28 July 2016; accepted 10 August 2016

Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35867

Abstract: The assembly of carbon nanomaterials into three-
dimensional (3D) porous scaffolds is critical to harness their
unique physiochemical properties for tissue engineering and
regenerative medicine applications. In this study, we report
the fabrication, characterization, and in vitro cytocompatibil-
ity of true 3D (>1 mm in all three dimensions), macroscopic
(3–8 mm in height and 4–6 mm in diameter), chemically
cross-linked graphene scaffolds prepared via radical initiated
thermal cross-linking of single- and multiwalled graphene
oxide nanoribbons (SWGONRs and MWGONRs). SWGONR
and MWGONR scaffolds possess tunable porosity (65-80%)
comparable to 3D poly(lactic-co-glycolic) acid (PLGA) scaf-
folds. Confocal live-cell imaging showed that cells were met-
abolically active and could spread on SWGONR and
MWGONR scaffolds. Immunofluorescence imaging showed
the presence of focal adhesion protein vinculin and expres-
sion of cell proliferation marker Ki-67 suggesting that cells
could attach and proliferate on SWGONR and MWGONR
scaffolds. These results indicate that cross-linked SWGONR
and MWGONR scaffolds are cytocompatible and opens-
avenues toward the development of 3D multifunctional gra-
phene scaffolds for tissue engineering applications. V C 2016

and interconnected macro-, micro-, and nanoscale pores. Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2016.
Human adipose derived stem cells (ADSCs) and murine
MC3T3 preosteoblast cells show good cell viability on Key Words: three-dimensional, graphene, scaffolds, cytotox-
SWGONR and MWGONR scaffolds after 1, 3, and 5 days icity, tissue engineering

How to cite this article: Lalwani G, D’agati M, Gopalan A, Rao M, Schneller J, Sitharaman B. 2016. Three-dimensional
macroporous graphene scaffolds for tissue engineering. J Biomed Mater Res Part A 2015:00A:000–000.

INTRODUCTION bottom–up approaches such as vacuum filtration, substrate

The two-dimensional (2D) carbon nanomaterial graphene pos-
sess unique nanoscopic physiochemical properties such as
high electrical conductivity (electron mobility >15,000 cm2
V21 s21,  106 3 copper),1 mechanical strength (Young’s mod-
ulus > 1 TPa),2 thermal stability (1500–2500 W m21 K21),3
and optoelectronic response.4 The multifunctional characteris-
tics of graphene nanoparticles of various shapes and sizes such
as graphene oxide (a.k.a. graphene nanoplatelets), graphene
oxide nanoribbons, and nanosheets have been exploited for
several biomedical applications such as bioimaging,5–7 drug
and gene delivery,8–10 stem cell tracking,11,12 photodynamic
therapy,13,14 and cancer therapeutics.15 They have also been
used as reinforcing agents to improve the mechanical proper-
ties of tissue engineering scaffolds 16 or as contrast agents to
enable noninvasive structural and functional monitoring of
polymeric scaffolds under physiological conditions.17,18 2D
thin-films of graphene fabricated using various top–down and
patterning,19 spray coating,20 and chemical vapor deposition
(CVD) 21 have been reported as cytocompatible substrates to
direct stem cell proliferation and differentiation,22 accelerate
osteogenic differentiation of mesenchymal stem cells,23
enhance neuronal impulse conduction,24 and neural-network
development.25 However, the assembly of graphene nanopar-
ticles into 3D porous architectures with tunable porosity and
mechanical properties is desired to harness their unique physi-
ochemical properties for tissue engineering across various
length scales. 3D graphene assemblies would permit the devel-
opment of next-generation multifunctional scaffolds with abil-
ity to induce/guide cellular processes such as protein
expression or cell differentiation,23 stimulus-based drug deliv-
ery (electroceuticals),26 and noninvasive longitudinal in vivo
monitoring of tissue regeneration.27
Current methods to assemble graphene nanoparticles
into 3D macroscopic architectures have not been

This article was published online on 27 August 2016. An error was subsequently identified. This notice is included in the online and print ver-
sions to indicate that both have been corrected 06 September 2016.
Additional Supporting Information may be found in the online version of this article.
Correspondence to: B. Sitharaman, PhD; e-mail: balaji.sitharaman@stonybrook.edu
Contract grant sponsor: National Institutes of Health; contract grant number: 1DP2OD007394-01
Contract grant sponsor: U.S. Department of Energy, Office of Basic Energy Sciences; contract grant number: DE-AC02–98CH10886

C 2016 WILEY PERIODICALS, INC.

V 1
demonstrated to be suitable scalable processes for fabricat-
ing macrosized scaffolds (>1 mm in all three dimensions).
Graphene has been assembled into foams using CVD and
sacrificial template transfer methods and their cytotoxicity
has been investigated for tissue engineering applica-
tions.28,29 However, these methods have several limitations.
CVD requires specific substrates capable of withstanding
high temperatures and pressure. Sacrificial template transfer
method requires complex procedures of etching using harsh
acids. Other methods such as vacuum filtration and spray
coating are time-consuming and require significant effort to
build 3D architectures layer-on-layer. Furthermore, these
aforementioned methods do not allow tunable control over
porosity and pore sizes of the 3D architecture. Thus, most
of the cytocompatibility studies using these 3D scaffolds are
limited to their surface. Additionally, the propensity of gra-
phene structures fabricated by these above methods to
allow cellular infiltration, an important characteristic of
scaffolds for tissue engineering, still needs to be demon-
strated. These approaches also present challenges toward
fabrication of macroscopic 3D scaffolds due to scalability or
operational costs. Additionally, due to absence of strong
chemical bonds between individual nanomaterials, 3D archi-
tectures fabricated using these methods mainly rely on
weak Van der Waal forces or physical entanglement of
nanomaterials for structural integrity and are prone to dis-
sociation under physiological shear forces. Conventional
methods such as solvent casting/particulate leaching,30 gas
foaming,31 phase separation,32 electrospinning,33 and stereo-
lithography 34 used for the fabrication of polymeric scaffolds
present limitations for the fabrication of 3D all-carbon
porous architectures. These methods typically require a
polymeric binder phase that would invariably shield/embed
the nanomaterial in the polymeric matrix,35–37 thereby limit-
ing the interactions between cells and the underlying nanoto-
pography. Therefore, the assembly of graphene nanoparticles
into 3D macroscopic (>1 mm in all three dimensions) chemi-
cally cross-linked, macroporous scaffolds without a polymeric
matrix would constitute a significant advancement.38
Herein, towards the development of 3D graphene scaf-
folds for tissue engineering, we report fabrication, characteri-
zation, and comprehensive cytotoxicity evaluation (cell
viability, adhesion, and proliferation) of 3D macroscopic
chemically cross-linked graphene scaffolds fabricated using
single- and multiwalled graphene oxide nanoribbons
(SWGONRs and MWGONRs).
MATERIALS AND METHODS
Fabrication of 3-D cross-linked graphene scaffolds
SWGONRs and MWGONRs were synthesized by the longitudi-
nal unzipping of single- and multiwalled carbon nanotubes
(SWCNTs and MWCNTs), respectively.8 The detailed synthesis
protocol and characterization of SWGONR and MWGONR using
transmission electron microscopy (TEM), atomic force micros-
copy (AFM), and Raman spectroscopy have been reported pre-
viously.16,39,40 The 3D SWGONR and MWGONR scaffolds were
fabricated by mixing SWGONRs or MWGONRs with benzoyl
peroxide (BP) at a mass ratio of nanoribbon: BP 5 1:0.5. One
2 LALWANI ET AL.
milliliter of CHCl3 was added to the mixture to dissolve, and
ensure uniform dispersion of BP. The BP–carbon nanomate-
rial mixture was subjected to bath sonication (30 min,
Ultrasonicator FS30H, Fischer Scientific, Pittsburgh, PA),
poured in custom machined Teflon molds (length 5 1.2 mm,
diameter 5 0.5 mm), and incubated at 608C for 24 hr. Postin-
cubation, the molds were disassembled to obtain the cross-
linked 3D carbon scaffolds. Five scaffolds were prepared for
each experimental group. As a purification step after cross-
linking, scaffolds were gently washed with CHCl3 and placed
in an oven at 1008C for 20 min to remove the excess BP.
Scanning electron microscopy (SEM)
To characterize the morphology of scaffolds, SEM imaging
was performed using a JOEL 7600F Analytical high-resolution
SEM at the Center for Functional Nanomaterials, Brookhaven
National Laboratory, New York. SWGONR and MWGONRs
scaffolds were placed on double-sided conductive carbon
tape and sputter coated with 3 nm of silver (Ag). SEM was
operated at 5 kV accelerating voltage and images were cap-
tured using a secondary electron imaging (SEI) detector.
Microcomputed tomography (microCT)
MicroCT was used to quantify the porosity of SWGONR and
MWGONR scaffolds. High-resolution microCT scanning was
performed using a microCT 40 system (Scanco Medical AG,
Bassersdorf, Switzerland) at an energy and intensity level
corresponding to 55 kV voltage and 145 lA current with
300 ms integration time for 1000 projections. A Gaussian
filter was used to suppress noise. The scaffold was isolated
from the background, using a thresholding procedure that
was specific to each material. The values to segment scaf-
fold from background were optimized individually by com-
paring the 2D gray scale image of a single slice of a
material with the thresholded image. Three different regions
covering a circular area of 1 mm2 and a depth of 0.5 mm
were chosen in the center with an effort to minimize the
inclusion of edge artifacts, and total volume (TV), scaffold
volume (SV), and scaffold volume fraction (SV/TV) were
determined for each scaffold. The mean and standard devia-
tions for these three different regions were used for statisti-
cal comparisons. Porosity values were determined as
  
SV
Porosity ð%Þ5 12 3100
TV
SEM image processing for porosity analysis
To quantify nanoporosities in MWGONR and SWGONR scaf-
folds, SEM images were cropped to remove scale bars and
subjected to a series of image processing steps (thresholding,
edge detection, median filtration, erosion, and dilation and
quantification of region properties) using the image process-
R
ing toolbox in MatLab (MathWorksV, MA, USA). Porosity was
calculated (n 5 5 images) using the following formula:
X . 
Porosity ð%Þ5 Area of voids ðarea of imageÞ 3100
THREE-DIMENSIONAL GRAPHENE SCAFFOLDS FOR TISSUE ENGINEERING

Cell culture
Human ADSCs were cultured in ADSC basal media (ADSC
Growth Media BulletKitTM, Lonza, USA) and MC3T3 preos-
teoblasts were cultured in minimum essential media alpha
(MEM-a, Gibco, USA) supplemented with 10 vol % heat-
inactivated fetal bovine serum (FBS, Gibco, USA) and 1%
antibiotics (penicillin–streptomycin, Gibco, USA). Media was
changed twice a week and cells were maintained at 378C in
a humidified atmosphere of 5% CO2 and 95% air. For cyto-
compatibility studies, purified SWGONR and MWGONR scaf-
folds were washed with CHCl3 and placed in an oven at
1008C to remove excess BP. The scaffolds were washed
(33) with CHCl3 and gradients of ethanol (100–70%) and
air-dried overnight inside a fume hood. PLGA scaffolds were
only subjected to ethanol washes. The scaffolds (PLGA,
SWGONR, and MWGONR) were sterilized by UV radiation
for 24 h followed by washes (33) with phosphate-buffered
saline (PBS) and cell culture media (ADSC basal media for
ADSCs and MEM-a for MC3T3 cells). For prewetting, the
scaffolds were then incubated with cell culture media for
24 h. ADSCs and MC3T3 cells were trypsinized using tryp-
sin–EDTA (13, Gibco, USA), resuspended in fresh cell cul-
ture media and seeded onto scaffolds at a density of
250,000 cells per scaffolds in 60 lL media (added in 4
intervals of 15 lL increments). Cells were allowed to adhere
to the scaffolds in a 24-well plate for 2 h before the addi-
tion of cell culture media (1 mL in each well). ADSCs and
MC3T3 cells were cultured for 1, 3, and 5 days to character-
ize cell viability, attachment, and proliferation. ADSCs were
cultured for additional two time points (days 15 and 30) to
characterize cell spreading on the scaffolds.
Lactate dehydrogenase (LDH) assay
LDH is an intracellular cytosolic enzyme that is released into
cell culture media upon rupture of cell membrane during cell
death by apoptosis or necrosis. Therefore, LDH released by
cells with compromised membrane integrity is quantified as a
measure of cell death. LDH assay was performed using a com-
mercially available LDH kit (Tox-7, Sigma Aldrich, NY, USA)
according to manufacturer’s instructions. Briefly, 50 lL of cell
culture media was collected from 24-well plates after 1, 3,
and 5 days and transferred to a fresh 96-well plate. To this,
100 lL of LDH assay mixture was added to each well and
incubated in dark for 45 min. HCl (1 N, 10 vol %) was added
to stop the reaction and absorbance values were recorded at
490 nm using a 96-well plate reader (Molecular Devices, CA,
USA). ADSC or MC3T3 cells grown on TCPS were treated with
lysis solution for 45 min and used as positive controls (100%
dead) and cells cultured on PLGA scaffolds were used as base-
line controls. Total LDH release (% of positive control) was
reported as
Total LDH released ð% of positive controlÞ
! RESULTS
ðODtest 2ODblank Þ
5   3 100
ODpositive 2ODblank
where ODtest is the absorbance of cells cultured on PLGA,
SWGONR, or MWGONR scaffolds, ODpositive is the absorbance
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2016 VOL 00A, ISSUE 00
ORIGINAL ARTICLE
of positive control (100% dead cells) and ODblank is the
absorbance of blank 96-well plate. Absorbance of blank cell
culture media was recorded for baseline correction.
Calcein-AM fluorescence imaging
Calcein acetoxymethyl ester (calcein-AM) is a cell-permeant
dye, which is converted to calcein (green fluorescence) due
to hydrolytic cleavage of acetoxymethyl ester group by
intracellular esterase. Therefore, to selectively stain live cells
for fluorescence imaging, PLGA, SWGONR, and MWGONR
scaffolds were stained with calcein-AM. After each time
point, the scaffolds were washed with PBS (33) and 1 mL
of calcein-AM working solution (4 lM) was added to each
well. Post 45 min of incubation in dark, the scaffolds were
removed and placed on 35 mm glass-bottom petridishes
(Mattek Corporation, Ashland, MA, USA). The samples were
imaged using a laser-scanning confocal microscope (Zeiss
LSM 510 Meta NLO) using Zeiss LSM Image Browser
software.
Immunofluorescence staining for cell attachment
and proliferation
Immunofluorescence staining was performed as described
previously.41 PLGA, SWGONR, and MWGONR scaffolds con-
taining glutaraldehyde-fixed cells were washed with PBS
(33) and incubated with glycine (2% for blocking) for 5
min. The scaffolds were then transferred to permeabilizing
buffer (0.5 mL Triton-X-100, 10.3 g sucrose, 0.29 g NaCl,
0.06 g MgCl2, and 0.4 g Hepes buffer in 100 mL of DI
water) for 30 min. After permeabilization, scaffolds were
washed (33) with immunofluorescence buffer (IFB, 0.1%
BSA and 0.1% triton-X-100 in PBS). Scaffolds were then
incubated with primary monoclonal antibodies (2 lg/mL in
IFB, see Table I) for 1 h followed by washes with IFB (33)
and incubation with secondary antibodies (2 lg/mL in IFB,
see Table I) for 1 h. Subsequently, scaffolds were rinsed
with IFB (33) and cytoplasm was stained to visualize actin
using FITC-conjugated phalloidin (2 lg/mL in PBS). Scaf-
folds were then imaged using a laser-scanning confocal
microscope (Zeiss LSM 510 Meta NLO) using Zeiss LSM
Image Browser software.
Statistical analysis
Data is reported as mean 6 standard deviation. Normal dis-
tribution of data was checked by Kolmogorov–Smirnov (K–
S) test in SPSS (LDH assay, N 5 3, n 5 5). Statistical analysis
was performed using Students t-test using a 95% confi-
dence interval (alpha level 0.05). To analyze difference
between groups, one-way ANOVA followed by Tukey Kramer
post hoc analysis was performed. p values <0.05 were con-
sidered as statistically significant.
Fabrication of PLGA, SWGONR, and MWGONR scaffolds
A previously described thermal cross-linking particulate
leaching procedure was used to fabricate PLGA scaffolds
with 85% porosity.42 SWGONR and MWGONR scaffolds
were fabricated using a radical initiated thermal cross-
3
TABLE I. List of Antibodies Used for Immunofluorescence Studies
Cell Type Antigen Primary Antibodies
ADSC Ki-67 Monoclonal antiproliferating cell nuclear
antigen antibody produced in mouse
(P8825, Sigma Aldrich, USA)
Vinculin Antivinculin antibody produced in rabbit
(V4139, Sigma Aldrich, USA)
MC3T3 cells Ki-67 Monoclonal Ki-67 antibody produced in
rabbit (MA5-14520, Thermo Fisher,
USA)
Vinculin Antivinculin antibody produced in rabbit
(V4139, Sigma Aldrich, USA)
linking procedure using BP as the radical initiator.6 The 3-D
scaffolds were macroscopic cylinders with a diameter of 4–
5 mm and height of 5–9 mm (Fig. 1). The scaffold dimen-
sions were smaller than the molds because the molds were
not completely filled to facilitate easy disassembly and
retrieval of the scaffolds post-cross-linking. To ensure uni-
formity between groups, scaffolds were cut into smaller 3D
cylinders (3–4 mm height) prior to cytocompatibility
studies.
Characterization of PLGA, SWGONR,
and MWGONR scaffolds
Scanning electron microscopy (SEM). SEM was performed
for morphological characterization of scaffolds. The SEM
images of PLGA scaffolds (85% porosity, 50–350 lm pore
sizes) have been reported previously.41 Figure 2 shows the
representative SEM images of 3D MWGONR [Fig. 2(A,B)]
and SWGONR [Fig. 2(C,D)] scaffolds. The cross-sections
clearly show interconnected MWGONR and SWGONR net-
works that form the macroscopic 3-D architecture. The SEM
images also depict the formation of nanoscale cross-links or
junctions between MWGONRs or SWGONRs [red arrows,
Fig. 1(B,D)]. Furthermore, nanoscale porosity can also be
observed [yellow circles, Fig. 1(B,D)]. The pores are irregu-
larly shaped and appear interconnected.
FIGURE 1. Optical images of representative three-dimensional porous
poly(lactic-co-glycolic) acid, single-walled graphene oxide nanorib-
bons, and multiwalled graphene oxide nanoribbons scaffolds pre-
pared as cylinders (4–5 mm diameter, 5–9 mm length).
4 LALWANI ET AL.
Secondary Antibodies
Antimouse Rhodamine antibody
produced in rabbit (SAB3701218,
Sigma Aldrich, USA)
Antirabbit IgG – TRITC antibody
produced in goat (T6778, Sigma
Aldrich, USA)
Antirabbit IgG – TRITC antibody
produced in goat (T6778, Sigma
Aldrich, USA)
Antirabbit IgG – TRITC antibody
produced in goat (T6778, Sigma
Aldrich, USA)
Microcomputed tomography (microCT). MicroCT is a well-
established method to determine the porosity of 3D poly-
meric and carbon nanotube scaffolds.6,42 The microCT
images of PLGA scaffolds have been reported previously.41
Figure 3A,B shows the reconstructed microCT images of
cylindrical sections (1 mm2 area, 0.5 mm height) of
MWGONR and SWGONR scaffolds. The pore sizes deter-
mined by analysis of microCT images were between 150–
400 lm for MWGONR and 50–350 lm SWGONR scaffolds.
The porosity values of SWGONR and MWGONR scaffolds
were 80.02 6 5.43% and 63.10 6 1.49%, respectively. It
should be noted that the white and grey solid interconnect
structures in Figure 3A,B possess nanoscale pores that can-
not be visualized by microCT due to a resolution of 6 lm.
These pores are clearly visualized by SEM imaging [Fig.
2(A,B)].
SEM image processing using MatLab. A widely accepted
SEM image processing technique was used to characterize
the nanoscale surface porosities of MWGONR and SWGONR
scaffolds.6,41,43,44 Nanoscale porosities are vital for efficient
nutrient transport and waste exchange inside 3D tissue
engineering scaffolds. The porosity values calculated using
this method were 43.95 6 4.85% for MWGONR scaffolds
and 35.38 6 5.57% for SWGONR scaffolds (Table II). The
pore sizes for MWGONR and SWGONR scaffolds were
between 35–900 nm and 20–650 nm, respectively. PLGA
scaffolds have macroscopic pores (100–500 lm pore diame-
ter) and their porosities are accurately determined using
microCT as porosities are greater than the resolution limit
of microCT system (6 lm). Furthermore, PLGA scaffolds lack
nanoporosity, and therefore, SEM image processing was only
used for MWGONR and SWGONR scaffolds.
Cytocompatibility analysis of MWGONR
and SWGONR scaffolds
Lactate dehydrogenase (LDH) assay. LDH assay quantifies
the cell death as a measure of the amount of cytosolic
enzyme LDH released in the media by apoptotic or necrotic
cells with compromised cell membranes. The released LDH
catalyzes the conversion of lactate to pyruvate that in turn
converts iodonitrotetrazolium (INT, present in the assay
THREE-DIMENSIONAL GRAPHENE SCAFFOLDS FOR TISSUE ENGINEERING
 ORIGINAL ARTICLE

FIGURE 2. Representative scanning electron microscopy images of (A, B) multiwalled graphene oxide nanoribbon and (C, D) single-walled gra-
phene oxide nanoribbon scaffolds. Red arrows in images B and D correspond to the formation of nanoscale junctions (cross-links) between gra-
phene nanoribbons. Yellow circles in images B and D correspond to irregularly shaped pores.

reagent) to formazan, a red-colored product with an absorb-
ance at 490 nm that can be quantified using a plate reader.
Figure 4A,B shows the total LDH released from ADSCs and
MC3T3 cells after 1, 3, and 5 days of culture on PLGA,
SWGONR, and MWGONR scaffolds, respectively. The data are
normalized to positive controls (100% dead cells). No sig-
nificant differences in the amount of LDH released were
observed between all the experimental groups (PLGA,
SWGONR, and MWGONR scaffolds) compared to tissue cul-
ture polystyrene (TCPS) live controls at all time points
(p > 0.05). LDH released was between 25 and 45% for
ADSCs and 30 and 50% for all groups.
Calcein-AM live staining. Calcein-AM staining is widely
used in cytocompatibility studies to selectively stain healthy
eukaryotic cells.7,41 Calcein-AM is a nonfluorescent dye that
upon internalization by living cells is converted to green flu-
orescent calcein due to cleavage of the acetoxymethyl ester
group by intracellular esterases. Figure 5 shows the repre-
sentative calcein-AM-stained ADSCs cultured on PLGA [Fig.

FIGURE 3. Representative 3D microcomputed tomography reconstructions of (A) multiwalled graphene oxide nanoribbon and (B) single-walled
graphene oxide nanoribbon scaffolds. The blue color in the images represents void spaces. Scale bars are 200 lm.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2016 VOL 00A, ISSUE 00 5

TABLE II. Porosity and Pore Sizes of Scaffolds
Porosity
Scaffold MicroCT (%) SEM Image Processing (%)
PLGA 85.75 6 3.37 – 100–500
SWGONR 63.10 6 1.49 35.38 6 5.57
MWGONR 80.02 6 5.43 43.95 6 4.85
5(A–E)], MWGONR [Fig. 5(F–J)], and SWGONR [Fig. 5K–O)]
scaffolds after 1, 3, 5, 15, and 30 days. Live ADSCs were
observed on all scaffold groups after 24 h of culture [Fig.
5(A,F,K)]. For each scaffold group, an increase in the cell
density can be observed at later time points (days 3 and 5)
with a characteristic spindle-shaped elongated cell morphol-
ogy. At days 15 and 30, ADSCs appear confluent and com-
pletely spread out on all scaffold groups. Significant
increases in the number of green fluorescent cells can be
observed between days 1 and 30 for all scaffold groups sug-
gesting that ADSCs can proliferate on PLGA, MWGONR, and
SWGONR scaffolds. Supporting Information, Figure S1 shows
the calcein-AM-stained MC3T3 cells on PLGA, MWGONR,
and SWGONR scaffolds after 1, 3, and 5 days of culture.
Similar to ADSCs, an increase in MC3T3 cell number was
observed between days 1 and 5 on all scaffold groups.
FIGURE 4. Total LDH release after 1, 3, and 5 days of (A) ADSC and (B)
MC3T3 cell culture on poly(lactic-coglycolic) acid, single-walled gra-
phene oxide nanoribbon, and multiwalled graphene oxide nanoribbon
scaffolds. Total LDH release (%) is normalized to positive controls
(100% dead cells). Data are represented as means 6 standard deviation.
6 LALWANI ET AL.
Pore sizes
MicroCT (lm) SEM Image Processing (nm)
–
50–350 20–650
150–400 35–900
Immunofluorescence analysis for cell attachment (focal
adhesion vinculin). Vinculin is a membrane-cytoskeletal pro-
tein associated with cell–cell and cell–matrix junctions.45,46 It
is present in focal adhesion complexes responsible for the link-
age of integrin proteins to the underlying actin cytoskeleton
and has been widely used as a marker to characterize cell
attachment.41,45,46 Figure 6 shows the representative confocal
immunofluorescence images of ADSCs stained for vinculin pro-
tein after 5 days of culture on PLGA [Fig. 6(A–C)], MWGONR
[Fig. 6(D–F)], and SWGONR [Fig. 6(G–I)] scaffolds. ADSCs were
stained with FITC-phalloidin [green fluorescence, Fig. 6(A,D,G)]
for actin cytoskeleton and fluorescently labeled antibodies for
vinculin protein [red fluorescence, Fig. 6(B,E,H)]. Merged
images are also presented [Fig. 6(C,F,I)] to confirm the colocali-
zation of actin filaments and focal adhesion complexes (vincu-
lin protein expression). Figure 6(B,E,H) confirm the expression
of vinculin and the presence of focal adhesion complexes by
ADSCs cultured on PLGA, MWGONR, and SWGONR scaffolds.
Supporting Information, Figure S2 shows the expression of vin-
culin proteins by MC3T3 cells after 5 days of culture on PLGA
(Supporting Information, Figure S2A–C), MWGONR (Support-
ing Information, Figure S2D–F), and SWGONR (Figure S2G–I)
scaffolds. For both ADSCs and MC3T3 cells, the vinculin protein
is colocalized with actin filaments and uniformly distributed
throughout the cytoplasm.
Immunofluorescence analysis for cell proliferation (ki-67
expression). Ki-67 is a nuclear antigen associated with ribo-
somal RNA transcription and a marker of cell prolifera-
tion.41,47,48 It is absent during the resting phase of cell cycle
(G0) and expressed only during all active phases of cell cycle
(G1, S, G2, and M).49 Figure 7 shows the representative con-
focal immunofluorescence images of ADSCs stained for Ki-
67 antigen after 5 days of culture on PLGA [Fig. 7(A–C)],
MWGONR [Fig. 7(D–F)], and SWGONR [Fig. 7(G–I)] scaffolds.
ADSCs were stained with FITC-phalloidin [green fluores-
cence, Fig. 7(A,D,G)] for actin cytoskeleton and fluorescently
labeled antibodies for Ki-67 antigen [red fluorescence, Fig.
7(B,E,H)]. Figure 7(C,F,I) shows the merged images of actin
cytoskeleton and Ki-67 expression. Figure 7(B,E,H) confirms
the expression of Ki-67 antigen by ADSCs cultured on PLGA,
MWGONR, and SWGONR scaffolds. Supporting Information,
Figure S3 shows the expression of Ki-67 antigen by MC3T3
cells after 5 days of culture on PLGA (Supporting Informa-
tion, Fig. S3A–C), MWGONR (Supporting Information, Fig.
S3D–F), and SWGONR (Supporting Information, Fig. S3G–I)
scaffolds. For both cell types—ADSCs and MC3T3 cells—Ki-
THREE-DIMENSIONAL GRAPHENE SCAFFOLDS FOR TISSUE ENGINEERING
 ORIGINAL ARTICLE

FIGURE 5. Representative calcein-AM-stained green fluorescence images of ADSCs on (A–E) poly(lactic-coglycolic) acid, (F–J) multiwalled gra-
phene oxide nanoribbon, and (K–O) single-walled graphene oxide nanoribbon scaffolds after 1, 3, 5, 15, and 30 days of culture. Live cells (green
fluorescence) can be observed on all scaffold groups. Scale bars are 200 lm.

67 antigen can be observed throughout the cytoplasm and
nucleus suggesting that cells on PLGA, MWGONR, and
SWGONR scaffolds were proliferating.
DISCUSSIONS
The objective of this study was to fabricate, characterize,
and investigate the in vitro cytocompatibility of 3D macro-
sized macroporous chemically cross-linked graphene scaf-
folds fabricated using SWGONRs and MWGONRs as
nanoscale building blocks. Figure 1 shows the optical
images of 3D SWGONR and MWGONR scaffolds fabricated
using radical (benzoyl peroxide, BP) initiated thermal-cross-
linking method as reported previously.41 The scaffolds are
macroscopic (>1 mm in all three-dimensions) free-standing
cylindrical architectures. BP has been used in free-radical
polymerization reactions to fabricate polymeric scaffolds for
tissue engineering applications. BP yields benzoyl and ben-
zoyloxyl free radicals that attack the C@C bonds present on

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2016 VOL 00A, ISSUE 00 7

FIGURE 6. Representative immunofluorescence images of ADSCs cultured on (A–C) poly(lactic-co-glycolic) acid, (D–F) multiwalled graphene
oxide nanoribbon and, (G–I) single-walled graphene oxide nanoribbon scaffolds after 5 days. Cells are stained green for actin cytoskeleton and
red for focal adhesions complexes (vinculin protein). Images C, F, and I show colocalization of actin filaments and vinculin protein signals. Scale
bars are 20 lm.

FIGURE 7. Representative immunofluorescence images of ADSCs cultured on (A–C) poly(lactic-co-glycolic) acid, (D–F) multiwalled graphene
oxide nanoribbon, and (G–I) single-walled graphene oxide nanoribbon scaffolds after 5 days. Cells are stained green for actin cytoskeleton and
red for cell proliferation marker (Ki-67 protein). Images C, F, and I show colocalization of actin filaments and Ki-67 fluorescence signals. Scale
bars are 20 lm.

8 LALWANI ET AL. THREE-DIMENSIONAL GRAPHENE SCAFFOLDS FOR TISSUE ENGINEERING


carbon nanomaterials and creates reactive sites that serve
as internanomaterial cross-linking centers forming 3D archi-
tectures of cross-linked carbon nanomaterials. For efficient
nutrient transport, waste exchange, and ECM deposition,
macroporous tissue engineering scaffolds with intercon-
nected porosity are required. In this study, a nanomater-
ial:BP ratio of 1:0.5 was used to fabricate macroporous
graphene scaffolds. PLGA scaffolds were fabricated using a
thermal cross-linking particulate leaching method with NaCl
as the porogen. Chemical characterization of 3D all-carbon
scaffolds fabricated using BP as the radical initiator has
been reported in our previous study.50 Elemental analysis
using X-ray photoelectron spectroscopy (XPS) showed that
3D all-carbon scaffolds comprised carbon (94.1%) and
oxygen (5.5%) as the primary elements. The disruption of
C@C bonds by BP during the fabrication process leads to
the formation of several oxygen-containing functional
groups such as carbonyl, oxides, epoxides, phenyl, and ben-
zoyloxyl along with termination by-products. During thermal
annealing of the scaffolds, these by-products along with
other volatile components are removed which results in the
partial restoration of the sp2 hybridization of C@C double
bonds.
The formation of nanoscale cross-links between individ-
ual and bundled carbon nanomaterials due to radical initi-
ated thermal cross-linking has been characterized in our
previous study using high-resolution TEM and SEM.50 In
this study, SEM imaging (Fig. 2) was performed to confirm
the presence of nanoscale cross-linking [red arrows, Fig.
2(B,D)] and characterize the morphology and pore architec-
ture [yellow circles, Fig. 2(B,D)] of MWGONR and SWGONR
scaffolds. Irregularly shaped macro-, micro-, and nanoporos-
ities of MWGONR and SWGONR scaffolds are clearly visible.
We additionally used microCT to quantify the porosity of
MWGONR and SWGONR scaffolds. MicroCT is a well-
established method to characterize the porosity of tissue
engineering scaffolds and 3D all-carbon architectures.41,42
Figure 3 shows the microCT reconstruction of cylindrical
sections (circular area of 1 mm2 and a depth of 0.5 mm) of
MWGONR and SWGONR scaffolds. These images confirm the
presence of interconnected irregularly shaped pores distrib-
uted randomly throughout the scaffolds. Due to a resolution
limit of 6 lm, the white solid interconnected structures in
the microCT reconstructions can have nanoporosities, which
may not be visualized. To characterize the nano- and micro-
scale porosities, a widely accepted method of SEM image
processing was used.41,43,44 SEM image processing con-
firmed the presence of interconnected nano- and micro-
porosities, which is vital for efficient nutrient exchange,
waste removal, ECM deposition, and neovascularization in
tissue engineering scaffolds upon in vivo implantation.
Comprehensive in vitro cytotoxicity evaluation is the
first step before significantly elaborate and expensive in
vivo studies to assess biocompatibility of tissue engineering
scaffolds.51,52 As our long-term focus is to employ the 3D
macroporous SWGONR and MWGONR scaffolds for bone tis-
sue engineering applications, they will primarily interact
with mesenchymal stem cells (MSCs) and osteoblasts cells
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2016 VOL 00A, ISSUE 00
ORIGINAL ARTICLE
in vivo. Therefore, in this study, human MSCs derived from
adipose tissue along with MC3T3 pre osteoblast cells were
used as model cell lines for cytocompatibility studies.
LDH assay is a widely accepted method to quantitatively
analyze the cytotoxicity of carbon nanomaterials.7,8,41 Other
cytotoxicity assays such as MTT and XTT produce erroneous
results due to strong binding of formazan crystals with car-
bon nanomaterials.53 LDH assay measures the cell death by
quantifying the amount of intracellular enzyme LDH
released in the cell culture media by apoptotic or necrotic
cells with compromised cell membrane. Therefore, no inter-
ference was observed. We have previously established the
suitability of LDH assay to evaluate the cytotoxicity of car-
bon nanomaterials.7,8,41,54 Figure 4 shows the total LDH
released by ADSCs and MC3T3 cells after 1, 3, and 5 days of
culture on SWGONR and MWGONR scaffolds. Porous 3D
PLGA scaffolds were used as baseline controls. Results show
no significant differences in the amount of LDH released by
cells on PLGA, SWGONR, and MWGONR scaffolds suggesting
that SWGONR and MWGONR scaffolds are cytocompatible,
comparable to FDA-approved polymer PLGA. To corroborate
LDH results and confirm the presence of live cells on
MWGONR and SWGONR scaffolds, we performed confocal
fluorescence imaging of calcein-AM-stained ADSCs cultured
for 1, 3, 5, 15, and 30 days (Fig. 5) and MC3T3 cells for 1,
3, and 5 days (Supporting Information, Fig. S1). Calcein-AM
dye (nonfluorescent) is converted to green-fluorescent cal-
cein due to the cleavage of acetoxymethyl ester group by
intracellular esterases and is retained in the cytoplasm
imparting green fluorescence to healthy cells. Therefore,
calcein-AM staining has been extensively used to selectively
stain live cells.7,20,41 Figure 5(A–O) and Supporting Informa-
tion, Figure S1(A–I) show the green-fluorescent ADSCs and
MCT3T cells at all time points confirming that SWGONR and
MWGONR scaffolds are cytocompatible. Furthermore, an
increase in cell number was observed at every successive
time point suggesting that ADSCs and MC3T3 cells can pro-
liferate on SWGONR and MWGONR scaffolds.
Immunofluorescence studies were performed to charac-
terize the formation of focal adhesion complexes (vinculin)
and the expression of cell proliferation marker (Ki-67). Vin-
culin is a membrane-cytoskeletal protein found in focal
adhesion complexes that play a key role in the linkage of
integrin proteins to the actin cytoskeleton of cells and is
responsible for the development of cell–cell and cell–matrix
junctions. The absence of vinculin impacts several cellular
functions such as cell adhesion and spreading, reduced
stress fiber development, and inhibition of lamellopodia for-
mation thereby governing actin polymerization, cell signal-
ing, adhesion, maturation, motility, and cytoskeletal
mechanics.45,46,55 ADSCs and MC3T3 cells cultured on
SWGONR and MWGONRs scaffolds express vinculin (red flu-
orescence, Fig. 6 for ADSCs and Supporting Information, Fig.
S2 for MC3T3 cells) suggesting the formation of focal adhe-
sion complexes and confirming the attachment of ADSCs
and MC3T3 cells with the underlying SWGONR and
MWGONR networks. Ki-67 is a nuclear antigen expressed
during all active phases of cell cycle (G1, S, G2, and M) and
9
absent during the resting phase (G0).49 Therefore, Ki-67 has
been extensively used as a marker of cell proliferation.47–49
ADSCs and MC3T3 cells cultured on SWGONR and
MWGONRs scaffolds express Ki-67 (red fluorescence, Fig. 7
for ADSCs and Supporting Information, Fig. S3 for MC3T3
cells) suggesting that cells are metabolically active and pro-
liferating on the scaffolds.
To the best of our knowledge, this is the first study to
comprehensively assess the in vitro cytocompatibility of
macroporous 3D graphene scaffolds fabricated via radical-
initiated thermal cross-linking of graphene nanoribbons. Cel-
lot et al. have reported enhanced neuronal impulse conduc-
tion on CNT-coated glass cover slips because cells form tight
contacts with the underlying nanotube matrix that favors
electrical shortcuts between proximal and distal ends of
neurons.56 Pryzhkova et al. have demonstrated control of
human pluripotent stem cell fate on CVD-deposited CNT
substrates by altering surface roughness, topography, and
stiffness.57 Nayak et al. have reported enhanced osteogenic
differentiation of human mesenchymal stem cells on rough
graphene coated glass coverslips compared to smooth non-
graphene-coated counterparts.23 Li et al. have reported
increased neuronal stem cell differentiation toward astro-
cytes on CVD-deposited graphene substrates.29
The fabrication and in vitro results of 3D graphene scaf-
folds reported in this work opens avenues for in vivo stud-
ies to assess their safety and efficacy. The 3D graphene
scaffolds may offer several benefits for tissue engineering
applications. The formation of covalent bonds between indi-
vidual nanomaterials could ensure mechanical stability
under shear stress conditions post-in vivo implantation. Fur-
thermore, graphene scaffolds with nanotopography and sur-
face roughness can ensure good host-implant integration.
Additionally, as discussed in the introduction, 3D graphene
scaffolds could exhibit multifunctional capabilities. There-
fore, the results of this study lay the foundation toward the
development of novel 3D multifunctional graphene scaffolds
for the next generation of tissue engineering and regenera-
tive medicine applications.
CONCLUSIONS
3D macroporous graphene scaffolds were fabricated via rad-
ical initiated thermal cross-linking method using SWGONRs
and MWGONRs as building blocks. SWGONR and MWGONR
scaffolds are macroscopic architectures (>1 mm in all three
dimensions) and possess interconnected macro-, micro-, and
nanoporosities. Human ADSCs and murine MC3T3 preosteo-
blast cells show good cell viability (comparable to PLGA
controls) on SWGONR and MWGONR scaffolds. The cells
express vinculin (focal adhesion proteins) and Ki-67 (cell
proliferation marker) suggesting that SWGONR and
MWGONR scaffolds permit cell attachment and proliferation.
Taken together, these results indicate that 3D SWGONR and
MWGONR scaffolds are cytocompatible matrices that show
good cell viability, adhesion, and proliferation. The results
open avenues for the development of multifunctional 3D
10 LALWANI ET AL.
graphene scaffolds for tissue engineering and regenerative
medicine applications.
ACKNOWLEDGMENTS
Research was carried out in part at the Center for Functional
Nanomaterials, Brookhaven National Laboratory, New York,
which is supported by the U.S. Department of Energy, Office of
Basic Energy Sciences, under Contract No. DE-AC02–98CH10886.
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