Preparation and Characterization of Graphene-

Based 3D Biohybrid Hydrogel Bioink for Peripheral

Neuroengineering
Asli Pinar Zorba Yildiz1,2,4, Hakan Darici3,4,5, Burcak Yavuz2,4,6, Emrah Sefik Abamor2, Ceren Ozdemir2,5, Muge Elif
Yasin4, Melahat Bagirova2,7, Adil Allahverdiyev2,7, Erdal Karaoz3,4,5,8
1 2 3
Vocational School of Health Services, Istinye University Chemical Metallurgy Faculty, Yildiz Technical University Faculty of Medicine, Department of
4 5
Histology & Embryology, Istinye University 3D Bioprinting Design & Prototyping R&D Center, Istinye University Stem Cell, and Tissue Engineering R&D
6 7
Center, Istinye University Vocational School of Health Services, Altinbas University The V. Akhundov National Scientific Research Medical Prophylactic
8
Institute Center for Stem Cells and Regenerative Medicine (LivMedCell), Liv Hospital

Corresponding Author
Abstract
Asli Pinar Zorba Yildiz
aslipinarzorba@gmail.com
Peripheral neuropathies can occur as a result of axonal damage, and occasionally due
to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in
Citation
1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue
Zorba Yildiz, A.P., Darici, H., Yavuz, B., engineering-based approaches, consisting of scaffolds, appropriate cell lines, and
Abamor, E.S., Ozdemir, C., Yasin, M.E.,
Bagirova, M., Allahverdiyev, A., biosignals, have become more applicable with the development of three-dimensional
Karaoz, E. Preparation and
(3D) bioprinting technologies. The combination of various hydrogel biomaterials with
Characterization of Graphene-Based 3D
Biohybrid Hydrogel Bioink for Peripheral stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome
Neuroengineering. J. Vis. Exp. (183),
the existing problems in peripheral nerve regeneration. Accordingly, the production of
e63622, doi:10.3791/63622 (2022).
injectable systems, such as hydrogels, or implantable conduit structures formed by

Date Published various bioprinting methods has gained importance in peripheral neuro-engineering.

May 16, 2022 Under normal conditions, stem cells are the regenerative cells of the body, and
their number and functions do not decrease with time to protect their populations;
DOI
these are not specialized cells but can differentiate upon appropriate stimulation in
10.3791/63622 response to injury. The stem cell system is under the influence of its microenvironment,
called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis,
URL
this microenvironment cannot be fully rescued even after surgically binding severed
jove.com/video/63622
nerve endings together. The composite biomaterials and combined cellular therapies
approach increases the functionality and applicability of materials in terms of various
properties such as biodegradability, biocompatibility, and processability. Accordingly,
this study aims to demonstrate the preparation and use of graphene-based biohybrid
hydrogel patterning and to examine the differentiation efficiency of stem cells into
nerve cells, which can be an effective solution in nerve regeneration.

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 1 of 21
 Introduction
The nervous system, which is the mechanism that bridges
the internal structure of the organism and the environment,
is divided into two parts: the central and peripheral nervous
systems. Peripheral nerve damage is a global problem that
constitutes 1.5%-5% of the patients who present to the
emergency department and develops due to various traumas,
leading to significant job loss1 , 2 , 3 .
Today, cellular approaches to peripheral neuro-engineering
are of great interest. Stem cells come first among the
cells used in these approaches. Under normal conditions,
stem cells are the regenerative cells of the body, and
their number and functions do not decrease with time to
protect their populations; these cells are specialized but
can differentiate upon appropriate stimulation in response to
injury4 , 5 . According to the stem cell hypothesis, the stem
cell system is under the influence of its microenvironment,
called the stem cell niche. The preservation and differentiation
of stem cells are impossible without the presence of their
microenvironment6 , which can be reconstituted via tissue
engineering using cells and scaffolds7 . Tissue engineering
is a multidisciplinary field that includes both engineering
and biology principles. Tissue engineering provides tools
for the creation of artificial tissues that can replace living
tissues and can be used in the regeneration of these
tissues by removing the damaged tissues and providing
functional tissues8 . Tissue scaffolds, one of the three
cornerstones of tissue engineering, are produced using
different methods from natural and synthetic materials9 .
Three-dimensional (3D) printing is an emerging additive
manufacturing technology that is widely used to replace or
restore defective tissues via its simple but versatile production
of complex shapes using various methods. Bioprinting is an
Copyright © 2022 JoVE Journal of Visualized Experiments
additive manufacturing method that enables the coexistence
of cells and biomaterials, called bioinks10 . Considering
the interaction of nerve cells with each other, studies
have shifted to conductive biomaterial candidates such as
graphene. Graphene nanoplates, which have properties such
as flexible electronics, supercapacitors, batteries, optics,
electrochemical sensors, and energy storage, are a preferred
biomaterial in the field of tissue engineering11 . Graphene has
been used in studies where the proliferation and regeneration
of damaged tissues and organs were performed12 , 13 .
Tissue engineering consists of three basic building
blocks: scaffold, cells, and biosignal molecules. There are
deficiencies in the studies on peripheral nerve damage
in terms of providing these three structures completely.
Various problems have been encountered in the biomaterials
produced and used in the studies, such as them containing
only stem cells or biosignal molecules, the lack of a bioactive
molecule that will enable stem cell differentiation, the lack of
biocompatibility of the biomaterial used, and the low effect
on the proliferation of cells in the tissue niche, and, thus,
nerve conduction not being fully realized2 , 13 , 14 , 15 , 16 . This
requires the optimization of nerve regeneration, reducing
muscle atrophy17 , 18 , and creating necessary homing19
with growth factors against such problems.At this point,
the characterization and analysis of the neuro-activity of a
surgical biomaterial prototype, to be transferred to the clinic,
are very important.
Accordingly, this methods study investigates the bioink
hydrogel patterning with graphene nanoplates formed by
a 3D bioprinter and its effectiveness on the neurogenic
differentiation of the stem cells it contains. Also, the effects
jove.com May 2022 • 183 • e63622 • Page 2 of 21
 of graphene on neurosphere formation and differentiation are better visualize the biomaterial-cell interactions to be
investigated. produced21 . The groups to be used in this method can
be created as shown in Table 1.
Protocol

1. Culturing of Wharton's jelly mesenchymal stem

cells

1. Take the Wharton's jelly mesenchymal stem cells (WJ-

MSCs, from ATCC) out of a −80 °C freezer. Culture WJ-
MSCs in DMEM-F12 medium containing 10% fetal calf
serum (FBS), 1% Pen-Strep, and 1% L-glutamine in a
sterile laminar flow at room temperature, as described in

Yurie et al.20 .

2. Cryopreserve some of the cells at 1 x 106 cells/mL with

freezing medium containing 35% FBS, 55% DMEMF-12,
and 10% dimethyl sulfoxide (DMSO). For this, count 1

x 106 cells on a Thoma cell counting slide and add the

freezing solution dropwise. Quickly transfer the slide to a
liquid nitrogen container.

3. When cultured cells are 80% confluent in the flask, pour

out the medium and wash with 5 mL of PBS. Add 5 mL
of 0.25% trypsin and 2.21 mM EDTA-4Na. Incubate in an
incubator at 37 °C for 5 min.

4. Add 10 mL of DMEM-F12 medium with 10% FBS to the

cells removed from the incubator. Suspend it well, collect
the medium, and transfer the medium to a centrifuge
tube.

5. Centrifuge at a rotational speed of 101 x g for 5 min

at room temperature. Discard the supernatant and re-
seed the cells in new flasks with fresh nutrient medium
containing 10% FBS.
NOTE: Commercial WJ-MSCs labeled with the GFP
gene by the transduction method can be used to

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 3 of 21
 Created Groups Reasons to create Number of reps

2D WJ-MSCs (2D-C) 2D Control x5

2D WJ-MSCs & Graphene (2D-G) Graphene toxic dose determination in 2D x 5 reps to each of
different concentrations

WJ-MSCs are included in bioinks (3D-B ) 3D Control x3

WJ-MSCs & 0.1% Graphene 3D Graphene-Bioink Biohybrid Group x3

are included in bioinks (3D-G )

WJ-MSCs are in spheroid 3D Control of Spheroid Form x3

form on the bioinks (3D-BS )

WJ-MSCs & 0.1% Graphene arein spheroid 3D Graphene-Bioink Biohybrid x3

form on the bioinks (3D-GS group) Group of Spheroid Form

3D Bioink drop It is produced for SEM and x5

FTIR characterization analysis.

3D Graphene drop It is produced for SEM and x5

FTIR characterization analysis.

3D Bioink with GFP labeled WJ-MSCs & 0.1% Observation of the movements of x3
WJ-MsCs in the bioink containing
the appropriate dose of graphene.

Table 1. Groups in the method. All 2D and 3D groups in the method are included.

1. Weigh the graphene to create a 1% solution (mg/

2. Graphene toxicity and 2D imaging
mL). Make a stock solution by adding 10 mL of
DMEMF-12 medium with 10% FBS to the weighed
1. Preparation of graphene concentrations and application
100 µg of graphene nanoparticles and label this
to cells
solution as stock solution. Sterilize in an autoclave
NOTE: Raw graphene nanoparticles were purchased
at 121 °C under 1.5 atm pressure for 20 min.
commercially (industrial graphene nanoplates type) and
NOTE: The sterile graphene mixture is stored in the
were also donated. The dimensions of the particles were
refrigerator at 4 °C until use. It may not be suitable for
5-8 nm in thickness, 5 µm in diameter, and 120-150 m2 /
long-term use (maximum 1 month). In these cases,
g in surface area.
the mixture must be reconstituted and sterilized.

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 4 of 21
 2. Prepare a mixture of medium graphene at different
concentrations to determine non-toxic doses. Set
the initial dilutions as 1%, 0.1%, 0.01%, 0.001%, and
0.0001% graphene/media.
3. Starting with the 1% stock solution, take 1 mL
successively from each concentration and transfer
it into a new tube. Add 9 mL of DMEMF-12
medium with 10% FBS to each tube, making five
gradually diluted samples, and shake and vortex the
solutions to get equal distribution. Use only 10 mL of
DMEMF-12 medium with 10% FBS as control.
NOTE: The heavy flakes of graphene precipitate
and, hence, need to be redistributed.
4. Seed the WJ-MSCs in 6-well plates with 2 mL of
fresh medium containing 10% FBS at 5 x 105 cells
per well. Incubate for 1 day at 37 °C. Then, divide
the plates into groups of equal and repeated wells.
Do five repetitions for each concentration.
5. Discard the medium and then replace the medium
with graphene medium concentrations at 2 mL per
well. Use only the medium for the control group.
Incubate the plates at 37 °C for 24 h.
2. Determination of non-toxic concentrations of graphene
with MTT
1. Discard media with graphene after 24 h. Wash each
well with PBS. Add fresh DMEMF-12 medium with
3.
10% FBS at 2mL per well.
NOTE: It is important to wash with PBS following
the application of graphene concentrations on the
cell because graphene nanoparticles, which are not
taken into the cell by endocytosis, are removed from
the environment. This makes the MTT test more
efficient.
Copyright © 2022 JoVE Journal of Visualized Experiments jove.com
2. Use the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide) protocol to determine
the IC50 value indicating 50% cell viability, as
described in Kose et al.22 and in steps 2.2.3.-2.2.6.
3. Firstly, weigh out 5 mg/mL of MTT salt and dissolve
in PBS. Sterilize using a 0.45 µm filter. Wrap the
MTT solution, made in a 15 mL centrifuge tube, with
aluminum foil and store at 4 °C.
4. Add 10 µL of MTT to all the wells and incubate
the plate for 4 h at 37 °C. Observe the formation
of formazan crystals at 10x magnification under the
microscope after incubation.
5. To dissolve the crystals formed in the cells, add 100
µL of DMSO to each well and mix by pipetting. Keep
the plate in the dark at room temperature for 30
min. Insert the plate into the ELISA plate reader.
Set a wavelength of 570 nm from the program
for absorbance measurement and have it read the
plate22 .
NOTE: In addition, while graphene particles alone
are read at 270 nm23 , the read range of 570 nm24
here will be for reading cell viability only.
6. Perform statistical analysis on the obtained results
using one-way ANOVA with Tukey's test in a
statistical analysis program.
Stitch imaging
1. To examine the interaction of different graphene
concentrations with cells, perform a time-lapse of
the cells with the method called stitch imaging.
This method creates a time-lapse image using
image samples taken at regular intervals under the
microscope.
May 2022 • 183 • e63622 • Page 5 of 21
 2. To do this, turn on the computer first. Turn on the
time-lapse imaging incubator and set it to 37 °C.
Place the MTT plate in the time-lapse incubator slot.
3. Open the Stitch program on the computer. Specify
the wells to be read in the system. Bring the reader
to the first well and locate and focus the area at 10x
magnification in white light. Start the program.
NOTE: It is a program for creating high-quality 4
rows x 5 columns multiple photo collages. The 2.
program combines photos taken one after the other
in HD quality. When you press the system start
button, the wells are automatically read, and it takes
and stitches 4 rows x 5 columns multiple photos.
3. Graphene - Bioink biohybrid hydrogel
production and WJ-MSCs differentiation
1. Production of bioinks
NOTE: The lyophilized commercially available Alginate-
Gelatin (3:5) powders are used as the basis of bioinks.
The graphene bioink group (3D-G; 3D-GS) and the
graphene-free control bioink group (3D-B; 3D-BS) are
prepared with the same method (Table 1).
1. Use 50 mL conical tubes for the preparation. First,
weigh 4.5 mg of alginate and 1.5 mg of gelatin
and transfer to a centrifuge tube. Add DMEMF-12
medium containing 10% FBS to the mixture to a
total volume of 50 mL. This is the control (C) group
without graphene.
2. Repeat weighing 4.5 mg of alginate and 1.5 mg of
gelatin and transfer to a centrifuge tube. Then, take
50 µL of the prepared 0.1% graphene from step
2.1.3. and add it to the tube. Add DMEMF-12 with
10% FBS to a total volume of 50 mL.
Copyright © 2022 JoVE Journal of Visualized Experiments jove.com
3. Mix the bioinks first by pipetting and then vortex.
Sterilize in an autoclave at 121 °C under 1.5
atm pressure for 20 min. Alternatively, perform
sterilization by microwaving until boiling point.
4. After the mixtures are autoclaved, centrifuge at 280
x g for 2 min at room temperature to remove formed
bubbles. Keep the bioinks at 37 °C until the cells are
prepared.
Addition of WJ-MSCs and 3D bioprinting
1. For counting, wash the cells when there is 80%
confluence with approximately 5 mL of PBS, and
add 5 mL of 0.25% trypsin and 2.21 mM EDTA 4Na.
Leave for 5 min at 37 °C.
2. Add 10 mL of DMEM-F12 medium with 10% FBS
to the cells after removing from the incubator.
Suspend well, collect the medium, and transfer it
to a centrifuge tube. Centrifuge at 101 x g for 5
min at room temperatures and then discard the
supernatant.
3. Leave approximately 250 µL of medium with the
pellet. Redissolve the pellet in 1 mL of fresh medium.
To 48 µL of medium, add 2 µL of cell suspension and
50 µL of trypan blue (0.4 g trypan blue/100 mL) in a
conical tube (1.5 mL) for counting4 . Pipette it well.
4. Add approximately 10 mL of the prepared stained
cell suspension to a Thoma cell counting chamber.
Calculate the average number of cells falling into the
squares on both sides of the light microscope.
Calculate the vitality percentage (%) = (counted
viable cells/total cells counted) x 100
NOTE: The cell-bioink interaction is studied in two
ways: (1) It can be printed by adding it into the
bioinks (3D-B; 3D-G); (2) The cells are seeded on
May 2022 • 183 • e63622 • Page 6 of 21
 the bioinks after being pressed, and the cells form a
spheroid (3D-BS; 3D-GS).
5. For the cell-bioink interaction, first create the bioink
groups (Table 1). Group 1 includes 3D-B and 3D-G
printed with bioink for bioprinting. Group 2 includes
3D-BS and 3D-GS bioinks on which spheroids were
formed after bioprinting.
6. Count the cells for group 1 so that there are
approximately 1 x 107 cells in 0.5 mL of medium.
Add 4.5 mL of bioink to bring the total volume
to 5 mL. Transfer this to the cartridges in the
sterile cabinet with the help of syringes. Install the
cartridges in the corresponding extruder section of
the bioprinter.
7. For the second group, take 5 mL from each of the
bioink groups and transfer them to sterile cartridges
with the help of an injector.
8. Use the bioprinter with two coaxial printheads and
the pneumatic-driven extrusion technology. Set the
X/Y/Z resolution per microstep to 1.25 µm, the
extrusion width to 400 µm, and the extrusion height
to 200 µm. A 20 mm x 20 mm x 5 mm grid is one of
the most used 3D models for 3D bioprinting work.
9. Create the 3D models using open-source, web-
based CAD programs. Before bioprinting, create
simple 3D models with one of the open-source
platforms (e.g., infill). The model can be linear
(like the model used here), honeycomb, or grid-
shaped.Export and download in .stl format after
creating a 5 mm x 20 mm x 20 mm square.
10. The bioprinter software uses .stl files and converts
them to printable .gcode format using slicer
modules. To get a printable grid shape, disable the
Copyright © 2022 JoVE Journal of Visualized Experiments
outer shell in the slicer module. Wipe the device with
70% ethanol before operation and then sterilize by
UV sterilization.
11. For the bioprinting process, transfer the bioink
groups to the cartridges with the help of an injector.
Install the cartridges in the corresponding extruder
section of the bioprinter.
12. Set the average pressure of the 3D printer to 7.5
psi and the cartridge and bed temperature to 37 °C.
Set the speed to 60% and carry out a standard 3D
printing process.
NOTE: Planning the prepared models with spaces
(like a linear model) allows cell culture to be done in
the spaces after bioprinting.
13. Put the system in the home position during
the writing phase. Position the axes (X, Y, Z)
automatically, select the extruder, and set.Start the
printing process. After the bioprinting process, take
the sample and place it under a laminar flow cabinet.
14. Spray the bioinks with 0.1 N CaCl2 solution after
printing or add 1 mL of the solution with a pipette at
room temperature. Wait for about 10-20 s and wash
the printed patterns 2x with PBS containing Ca2+
and Mg2+ .
15. Add 2 mL of DMEMF-12 with 10% FBS medium
on top of each of the cell-containing bioink groups.
Incubate the plates at 37 °C with 5% CO2. Afterward,
add 2 mL of suspension medium containing 1 x 106
cells to each group for spheroid formation.
16. Incubate the plates at 37 °C with 5% CO2. After 24
h incubation, observe the spheroid formation under
an inverted microscope.
jove.com May 2022 • 183 • e63622 • Page 7 of 21

3. WJ-MSCs differentiation to neuron-like cells
1. Observe and photograph all batches of bioink
after 24 h of incubation. Add 2 mL of neurogenic
differentiation medium per well (except for the
control group) and refresh every 2 days. Follow for
7 days to observe neural differentiation.
4. Graphene-Bioink biohybrid hydrogel
characterization
NOTE: Time-lapse imaging, Fourier transform infrared
spectroscopy (FT/IR), and scanning electron microscopy
(SEM) analyses are performed for the characterization of
graphene-bioink biohybrid hydrogel. The samples are created
from 3D-B and 3D-G bioink groups by the drip method for FT/
IR and SEM analysis.
1. FT/IR analysis
NOTE: FT/IR is a chemical analytical method based on
the mathematical Fourier transform, indispensable for
material characterization. Use an FT/IR device based on
28 °C Michelson interferometer principles, with a halogen
lamp, water-cooled mercury light source, signal aspect
ratio of 4 cm−1 , measured for 1 min, at 2,200 cm−1 . 3.
1. Prepare the FT/IR microscope and make sure the
optics of the instrument are aligned. Calibrate the
device.
2. Since the sample is in a drop, take a piece of 1-2
mm thick hydrogel and place it with forceps on the
sample part. Focus on the sample and raise it until
close contact is made. Collect the sample spectrum
by starting the process from the system.
2. Scanning electron microscopy (SEM) analysis
Copyright © 2022 JoVE Journal of Visualized Experiments jove.com
NOTE: The surface morphology, internal structure, cell
distribution, and bioink-cell interactions can be examined
by SEM analysis in different dimensions.
1. Drop the hydrogel into 6-well plates containing 1
mL of CaCl2 crosslinker using the pipette tip droplet
method (see Figure 1).
2. Wash the plates 2x with approximately 1 mL of
PBS to free the solution of salts. Then, put these
drops into a falcon tube containing 5 mL of 5%
paraformaldehyde with the help of forceps.
3. Make thin sections from the drop bioinks with the
help of a scalpel. Stick the samples on the sticky
side on the metal plate. Insert into the coating device
where the gold palladium, which passes into the
plasma phase by replacing the air with argon gas,
covers the sample.
4. Put it in the electron microscope (SEM). Open the
microscope program that the SEM is connected to.
Perform the scaling and take images of different
parts of the sample. For this protocol, 5 µm, 10 µm,
and 200 µm scales were used. A total of 40 images
were taken for the 3D-B and 3D-G groups.
Time-lapse imaging
NOTE: During time-lapse imaging, not only bioink
samples containing GFP gene transformed cells were
used but also bioinks with spheroids are imaged for 16 h.
Time-lapse imaging is performed to examine the effects
of graphene on stem cells and to monitor cell interactions
within the bioink.
1. Add commercially available 1 x 107 GFP-labeled
MSCs into 5 mL of bioink and bioprint as in step
3.2.11. Add 1 mL of crosslinker, hold for 20-30 s, and
wash 2x with PBS.
May 2022 • 183 • e63622 • Page 8 of 21

2. Turn on the time-lapse monitor and open the
program on the computer. Set approximately 16 h
in Time-Lapse mode and set the temperature to 37
°C. Press the Start button. Videos in the form of
40 s time-lapse video samples are recorded by the
system.
3. Focus the microscope at 10x magnification every 2
h. Also, visualize the spheroids created by following
the same steps.

Figure 1: 3D-B and 3D-G bioink groups produced as drops for use in characterization. (A) Bioink samples (pre-
characterization image) on a plate with a crosslinker. (B) 3D-B drop image of bioink. (C) 3D-G bioink drop image. The
biomaterial to be characterized and the cells it contains can more easily go through processes such as gold plating,
sampling, etc. Please click here to view a larger version of this figure.

3. Collect the spheroids by pipetting, distribute the collected

5. Determination of neurogenic differentiation by
immunostaining method spheroids from the incubator into the wells of the 48-well
plate, and add fresh medium. Then, incubate for 12-24 h.
NOTE: It is quite difficult to distribute the spheroids by
1. Collect the spheroids, without interrupting sphere forms,
counting. Collect the spheroids in a single tube and
and reseed into new plates to restain with an easier
evenly distribute the total volume to the wells. For this
method, instead of embedding in paraffin for tissue
study, each well contained approximately 4-6 spheroids.
sectioning.
NOTE: For 2D samples, pre-stain washing is sufficient,
2. Cover the 48-well plates with about 20 mL of pre-
and no media replacement is required.
autoclaved 0.1% gelatin solution and keep in an
4. Remove the medium and slowly wash the cells with
incubator for at least 1 h. Pipette approximately 500 µL
PBS as the spheroids will be at the top. Fix in 4%
of prepared 0.1% gelatin into 48-well plates. Leave the
paraformaldehyde for 2 h.
gelatinous plates in the incubator for 10 min.
NOTE: This will allow the gelatin to swell slightly, 5. Wash the samples with PBS again and block with about

covering the surface and creating a better environment 10 mL of 2% BSA and 0.1% TritonX for 30 min. Then,

for the collected spheroids to attach. wash with PBS 3x.

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 9 of 21
 6. Dilute selected primer antibodies (N-cadherin-rabbit and
Representative Results
β-III tubulin-mouse) at a 1:100 ratio with antibody diluent
reagent solution. Add 100 µL of antibody solution to each Graphene toxicity and 2D imaging

of the samples and incubate overnight at 4 °C. Statistical analysis of the obtained MTT results was
conducted with a one-way ANOVA with Tukey's test in
7. Wash the samples 3x with PBS. Incubate with anti-
statistical analysis software, and the graph obtained is shown
mouse IgG-FTIC-rabbit for β-III tubulin and anti-mouse
in Figure 2. The graphene percentage compared to control
IgG-SC2781-goat secondary antibody for N-Cad diluted
showed a significant decrease only for the 0.001% graphene
to 1:200 at room temperature for 30 min. Perform all
concentration (**p < 0.01).. There were no significant
operations in the dark.
differences between the other groups and the control (p >
NOTE: Whatever strain is used as the primary antibody
0.05). Therefore, the optimum graphene concentration was
(e.g., mouse) in double staining, a different strain (e.g.,
determined to be 0.1% since the highest viability rates were
goat or rabbit) should be used for the secondary
observed after exposure to this concentration according to the
antibody.
MTT test results and the stitching images.
8. Wash the secondary antibody with PBS 3x and then drip
the DAPI solution (1:1) into each of the samples. Wait for
15-20 min. Perform immunofluorescence imaging.

Figure 2: Investigation of the effect of graphene concentration on cell proliferation. The graphene percentage
compared to control showed a significant decrease only for the 0.001% graphene concentration (**p < 0.01, n = 6). There

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 10 of 21
 were no significant differences between the other groups and the control (p > 0.05; n = 6). Statistical analysis of the obtained
MTT results was conducted with a one-way ANOVA with Tukey's test in statistical analysis software. The error bars
represent the standard deviation. This figure has been modified from28 . Please click here to view a larger version of this
figure.

This part of the presented method demonstrates that the stem tolerated in the 2D system and was uptaken by the cells
cell-graphene interaction can be used in future studies. It was through endocytosis (Figure 3). It has been determined that
observed that, in each tested concentration, graphene was the graphene plates move along the cytoplasm inside the cell.

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 11 of 21
 Figure 3: The cell-graphene interactions are shown in two dimensions in stitched images. (A) Control; (B) 0.0001%;
(C) 0.001%; (D) 0.01%; (E) 0.1%; and (F) 1% concentrations of graphene. It is obtained by combining time-lapse imaging
with high-definition quality settings to obtain a 4 rows x 5 columns collage of multiple photos. This figure has been modified
from 28 . Please click here to view a larger version of this figure.

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 12 of 21
 Furthermore, in this study, it was observed that the graphene-
bioink effect did not create any toxic microenvironment in the
3D system and that the cells were in interaction.
It has been also observed that the use of graphene in
3D systems did not create any toxic microenvironment.
Determining the toxic dose for cells is crucial for the use of
graphene in long-term conduit implants or injectable hydrogel
forms. In addition, since graphene is an effective material in
neuronal communication, its use in nerve tissue engineering
applications has widely increased, as documented in the
literature25 .
Production of composite biomaterials and 3D bioprinting
The formation of gelatin-alginate-based bioink biohybrid
patterning was performed with a non-toxic concentration of
graphene (0.1%). It has been observed that the selected
appropriate dose of graphene interacts with the cells in the
bioink.
Copyright © 2022 JoVE Journal of Visualized Experiments jove.com
Time-lapse imaging
GFP samples were set at 37 °C for approximately 16 h in
time-lapse, and photos and videos were taken (Video 1). GFP
signals were lost when cell death was observed. Here, it was
detected that cells that survived in the 3D graphene medium
maintained their vitalities since GFP brightness was observed
until the end of the incubation.
Video 1. Time-lapse video of GFP-labeled WJ-MSCs. The
interaction of labeled stem cells with each other is observed.
Please click here to download this Video.
Graphene-Bioink biohybrid hydrogel characterization
SEM and FT/IR were used for the characterization of the 3D-
B and 3D-G groups created by the drop bioink method. The
SEM images of the 3D-B and 3D-G bioink groups are given
in Figure 4. Out of the 40 images taken at step 4.2.5., 4
representative images are shown here.
May 2022 • 183 • e63622 • Page 13 of 21
 Figure 4: SEM images of the 3D-B and 3D-G bioink groups. (A) Image of a gold-coated 3D-B bioink section with electron
microscopy. Scale bar: 200 µm. (B) Image of MSC attached to the 3D-B bioink inner surface. Scale bar: 5 µm (C) Image of
3D-G bioink inner and outer surface. Scale bar: 200 µm.(D) Inner surface cell 3D-G bioink adhesion image. Scale bar: 10
µm. Please click here to view a larger version of this figure.

Accordingly, bioink-cell interactions were demonstrated both morphologically round and attached to the material. The FT/
on the surface and internally. There was a cell-biomaterial IR analysis of the 3D-B and 3D-G bioink drop was compared
interaction in both bioinks (3D-B; 3D-G). The cells were with the graph in Figure 5.

Figure 5: FT/IR analysis. (A) 3D-B bioink. (B) 3D-G bioink. Peaks such as 1633.41 cm−1 , 1552.42 cm−1 , and 1033 cm−1
have been found in alginate-gelatin-based hydrogel studies in the literature26 . Also, the 1335.46 cm−1 peak is similar to the
peaks seen in graphene biomaterial studies27 . Please click here to view a larger version of this figure.

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 14 of 21
 Since the control bioink (3D-B) was based on alginate/
gelatin, the most prominent peaks were around 1633.41
cm−1 , 1552.42 cm−1 , and 1033 cm−1 compared with similar
studies in the literature with peaks seen at 1546 cm−1 26 . A
peak of 1399 cm−1 was observed in the graphene group (3D-
G) and a similar peak was found at 1335.46 cm−1 in a study
conducted with graphene27 .
3D neuronal differentiation
The images of spheroids on the bioinks after the 7th-day post
differentiation are represented in Figure 6.

Figure 6: 2D image of control WJ-MSCs and spheroid group samples at day 7 post differentiation. (A) Size of the
sphere from the 3D-BS group bioink with diameters of 160 µm and 200 µm. (B) 2D control cells. Spheroids from (C) the 3D-
BS group and (D) the 3D-GS group. The black materials in (D) are graphene molecules integrated with spheroids. Please
click here to view a larger version of this figure.

It was seen that cells maintained their vitality in both 2D and
3D cultures. It was considered that the borders of the spheroid
cells in both groups (3D-B and 3D-G) were transparent
and lively, and the spheroids in the graphene group were
relatively larger and trapped the graphene inside the cell.
The differentiation was also tested by immunostaining. With
the double staining used here, the activities of cells in 2D
neuronal transformation were compared to 3D culture (Figure
7).

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 15 of 21
 Figure 7: Immunostaining of 2D and 3D cells. (A,B) Immunostaining of cultured spheroids on 3D-BS bioinks. (C,D) 3D-GS
bioink spheroid immunostains. (E) Differentiated 2D positive control WJ-MSCs. (F) Undifferentiated 2D negative control WJ-
MSCs. Please click here to view a larger version of this figure.

Immunofluorescence images of cells cultured for 7 days are
shown in Figure 7. Samples were stained with antibodies
specific to N-cadherin (green) and β-III tubulin (red).
Additionally, DAPI was used for visualization of the nucleus
(blue or purple). Accordingly, the N-cadherins (green) used
in the 2D and 3D samples differed over 7 days as it plays an
important role in signaling mechanisms and the development
of neurons. The green image in Figure 7A-E represents
neuron-like structures (Figure 7). Class III β-tubulin is one
of the seven isotypes known as neuron markers in the
human genome. N-cadherin expression was found to be
higher in the samples that were differentiated for 7 days

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 16 of 21
 compared to β-III tubulin. According to the results of the biomaterial. In the next stages, the effect on migration will be
present experiment, it was established that the 3D systems examined.
created a more suitable microenvironment for the cells to
The biohybrid materials formed by combining two or more
survive and differentiate. The 2D positive control sample
biocompatible materials are becoming more advantageous
(Figure 7E) expressed fewer neuron-like structure markers
in terms of their structural properties compared to
compared to the 3D samples (Figure 7A-D). This shows us
homogeneous materials30 . In a previous study, implantation
that the microenvironment created with the 3D structure is
of a polyglycolic acid-based neuro tube channel into the
more effective in the differentiation of stem cells. In addition,
facial peripheral nerve was performed. Labeled olfactory
instead of cell therapies alone; biomaterial-cell combined
stem cells were inserted into this conduit, resulting in the
treatments appear to be more influential and effective in nerve
successful regeneration of peripheral surgery and injected
damage.
stem cell therapy16 . In another study, the effectiveness of
Discussion the 3D structure obtained from multiple cellular spheroids

The advantages of treatments applied with engineered 3D developed using human normal dermal fibroblasts and the

scaffolds over conventional 2D methods are becoming more silicon nerve canal, which is frequently used in the literature

and more noticeable every day. Stem cells used alone in due to its inert feature, was compared in the regeneration

these therapies or along with scaffolds produced from various of sciatic nerve damage. It was shown that the spheroid-

biomaterials with low biocompatibility and biodegradability based 3D structure increased tissue regeneration significantly

are usually inadequate in peripheral nerve regeneration. and more rapidly in animal models with sciatic damage as

Wharton's jelly mesenchymal stem cells (WJ-MSCs) seem to compared to homogenous materials20 . However, it is known

be a suitable candidate cell line, especially considering the that silicon-based biomaterials, which are frequently used in

optimization of the protocols for acquisition, their proliferation the literature, have some important disadvantages, such as

ability, and their differentiation capacity29 . In this study, we high infection risk and low biocompatibility15 , 30 .

examined the interaction of stem cells with graphene in both

In the present study, the production of biocompatible,
2D and 3D cultures. We also compared the neurogenic
biodegradable hydrogel biohybrid composite tissue with
differentiation in the generated biohybrid hydrogel groups
graphene nanoplatelets, which are known to be effective
with the 2D environment. We demonstrated the neurosphere
in nerve conduction, was carried out by a 3D printing
formation on the bioinks by immunostaining. In further
technique. There are various studies in which graphene
studies, we characterized our candidate prototype, which can
has been used as a biomaterial for nerve conduction25 , 30 .
be injected or implanted as a neural channel, by SEM and
Furthermore, graphene is one of the thinnest and lightest
FT/IR methods. This study characterizes the properties of the
material available, which increases its preference in health
biomaterial produced, the cell-biomaterial interaction, and the
technologies13 . One of the most desirable properties of
neural differentiation of the stem cells in the presence of the
biomaterials is biodegradability. Experimental and molecular
simulation technologies have been applied to investigate the

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 17 of 21
 biodegradation of graphene and its derivatives13 . At this
point, surface modification-functionalization or production in
the form of composite biomaterials can improve or inhibit
biodegradation, largely depending on the properties of the
additives.
Various enzymes, such as manganese peroxidase (MnP),
horseradish peroxidase (HRP), and myeloperoxidase (MPO),
are available in the literature for the biodegradation of
graphene derivatives. The MPO enzyme, which is a
peroxidase released from neutrophils that come to the region
of foreign bodies and perform phagocytosis, is associated
with graphene biodegradation, especially in the human
lung13 . The biodegradability levels of composite biomaterials
containing graphene instead of graphene nanotubes alone
are different from each other. It is easier to achieve
this desired property in composite materials. In addition,
determining the toxic dose of graphene contributes to the use
of clinically applicable biomaterials13 .
An important part of the graphene stage of the protocol
is sterilizing the graphene when it is to be used. The risk
of contamination that may occur during the biohybrid-cell
interaction phase is avoided.
The effect of the obtained biohybrid graphene-bioink hydrogel
patterning on nerve differentiation was investigated and it
was compatible with the literature that the differentiation
was higher in the 3D system. The need for the
production of biomaterials with tissue-engineered cellular-
based therapeutic approaches that can be developed against
various neurodegenerative diseases, such as peripheral
nerve damage, is increasing with the inadequacy of current
treatments day by day, emphasizing the importance of this
study.
Copyright © 2022 JoVE Journal of Visualized Experiments jove.com
In a previous study, the interaction of graphene with cells
in a 2D cell culture medium was investigated28 . With the
experience gained from there, here, graphene was added
to the alginate-gelatin bioink with the known ratio, and a
new and unique biomaterial prototype was produced by a 3D
printing method. This new material was used to study cell
interaction in two different ways. The first is the co-printing of
the cell-composite biomaterial on a 3D printer. The second is
the formation of a cellular spheroid from the biomaterial. In
addition, the interaction of WJ-MSCs containing the tagged
GFP gene with the material was also observed.
In this study, biohybrid bioinks were characterized by
selecting FTIR and SEM methods. These enable the sample
balls formed by the drip method to be examined during the
characterization phase. Especially since we can examine a
hard and dry structure during the SEM gold plating stage,
SEM provides the physical suitability for getting better, thin
sections with a scalpel from the bioink balls we created with
this method.
In this method study, cells were added to the biohybrid
material in two different ways during the experiments: inside
for 3D bioprinting or during spheroid formation. Covering cells
with bioinks and using 3D bioprinters allow researchers to
create any desired 3D shape for nerve regeneration. On the
other hand, it causes more stress on cells due to the printing
pressure and, therefore, loss of cell viability. However, it could
be a more desirable method to increase cell homing when
they are injected or transplanted to a specific area to induce
regeneration.
The creation of spheroids on bioinks creates a more usable
form of artificial tissue in terms of cell interaction and provides
a better niche for cell differentiation. It is also suitable
for mimicking the natural microenvironment and, therefore,
May 2022 • 183 • e63622 • Page 18 of 21
 investigating cellular mechanisms. The low adhesion of the
spheroids to the bioinks also allows easier separation from
the bioinks and versatility of the applications.
The N-cadherins used (shown in green in Figure 7) are part
of cell signaling mechanisms and play an important role in
the development of neurons32 . Class III β-tubulin is one of
the seven isotypes known as neuron markers in the human
genome. It shows that the WJ-MSCs used in this study begin
to form neuron-like structures. In this context, 3D systems
will create a more adequate microenvironment for cells to
maintain their viability.
Finally, due to the expense involved and the clinical
applicability of cell differentiation systems, it is very important
in neuro-engineering to develop systems with controlled
release of scaffolds, cells, and differentiation biosignals33
in the future and adapt these to the clinics as combined
therapies. Therefore, spheroid and bioprinting methods can
also be used in further studies where graphene and other
bio-signals are embedded into hydrogels and released in a
controlled manner. In vitro studies are an important step on
the road to in vivo. When the material, provided here as a
prototype, is produced in accordance with Good Laboratory
Practice standards, the sterilization standards required for
transfer to the clinic can be achieved.
Disclosures
The authors declare that there is no conflict of interest.
The project was performed in collaboration with HD Bioink,
developer of the 3D bioprinting technology.
Acknowledgments
The graphene used in this study was developed at
Kirklareli University, Department of Mechanical Engineering.
Copyright © 2022 JoVE Journal of Visualized Experiments
It was donated by Dr. Karabeyoğlu. The graphene
toxicity test was financed by the project titled "Printing
and Differentiation of Mesenchymal Stem Cells on 3D
Bioprinters with Graphene Doped Bioinks" (Application
No: 1139B411802273) completed within the scope of
TÜBİTAK 2209-B-Industry-Oriented Undergraduate Thesis
Support Program. The other part of the study was
supported by the research fund provided by Yildiz Technical
University Scientific Research Projects (TSA-2021-4713).
Mesenchymal stem cells with GFP used in the time-lapse
imaging stage were donated by Virostem Biotechnology. The
authors thank Darıcı LAB and YTU The Cell Culture and
Tissue Engineering LAB team for productive discussions.
References
1. Kamasak, B. et al. Peripheral Nerve Injuries
and Physiotherapy. Clinical Physiotherapy. 19, Vize
Publishing, Istanbul (2019).
2. Yegiyants, S., Dayicioglu, D., Kardashian, G., Panthaki,
Z. J. Traumatic peripheral nerve injury: A wartime review.
Journal of Craniofacial Surgery. 21 (4), 998-1001 (2010).
3. Mushtaq, S. et al. Frequency of peripheral nerve injury
in trauma in emergency settings. Cureus. 13 (3), e14195
(2021).
4. Allahverdiyev, A. Basic Principles of Somatic and Stem
Cell Culture Systems, 1st Edition. Nobel Medicine
Bookstore, Istanbul (2018).
5. Allahverdiyev, A. M. et al. Adipose tissue-derived
mesenchymal stem cells as a new host cell in
latent leishmaniasis. The American Journal of Tropical
Medicine and Hygiene. 85 (3), 535-539 (2011).
jove.com May 2022 • 183 • e63622 • Page 19 of 21
 nerve defects: Report on a series of 28 lesions. Journal
6. Schofield, R. The relationship between the spleen
of Hand Surgery (European Volume). 37 (4), 342-349
colony-forming cell and the hemopoietic stem cell. Blood
(2011).
Cells. 4 (1-2), 7-25 (1978).
16. Karaaltin, A. B. et al. Human olfactory stem cells for
7. Goodman, S. R. Stem Cells and Regenerative Medicine
injured facial nerve reconstruction in a rat model. Head
(Chapter 13). Goodman's Medical Cell Biology, Fourth
& Neck. 38 (S1), E2011-E2020 (2016).
Edition., 361-380. Academic Press (2021).
17. Bingham, J. R. et al. Stem cell therapy to promote
8. Kaya, T. I. Tissue engineering. International Journal of
limb function recovery in peripheral nerve damage in a
Medical Sciences. 1 (48), 165-9 (2018).
rat model. Annals of Medicine and Surgery. 41, 20-28
9. Sensharma, P., Madhumathi, R. G., Jayant, R. D.,
(2019).
Jaiswal, A. K. Biomaterials and cells for neural tissue
18. Zhuang, H. et al. Gelatin-methacrylamide gel loaded with
engineering: Current choices. Materials Science and
microspheres to deliver GDNF in bilayer collagen conduit
Engineering: C. 7, 1302-1315 (2017).
promoting sciatic nerve growth. International Journal of
10. Hölzl, K. et al. Bioink properties before, during, and after
Nanomedicine. 11, 1383-1394 (2016).
3D bioprinting. Biofabrication. 8 (3), 032002 (2016).
19. Scheib, J., Hoke, A. Advances in peripheral nerve
11. Zheng, Y. et al. 2D nanomaterials for tissue engineering
regeneration. Nature Reviews: Neurology. 9 (12),
and regenerative nanomedicines: Recent advances and
668-676 (2013).
future challenges. Advanced Healthcare Materials. 10
20. Yurie, H. et al. The efficacy of a scaffold-free bio 3D
(7), e2001743 (2021).
conduit developed from human fibroblasts on peripheral
12. Shin, S. R. Graphene-based materials for tissue
nerve regeneration in a rat sciatic nerve model. PLOS
engineering. Advanced Drug Delivery Reviews. 105,
ONE. 12 (2), e0171448 (2017).
255-274 (2016).
21. Guo, Y. et al. Assessment of the green florescence
13. Chen, M., Qin, X., Zeng, G. Biodegradation of carbon
protein labeling method for tracking implanted
nanotubes, graphene, and their derivatives. Trends in
mesenchymal stem cells. Cytotechnology. 64(4),
Biotechnology. 35 (9), 836-846 (2017).
391.-401 (2012).
14. Chen, S. et al. PAM/GO/Gel/SA composite hydrogel
22. Kose, C., Kacar, R., Zorba, A. P., Bagirova, M.,
conduit with bioactivity for repairing peripheral nerve
Allahverdiyev, A. The effect of CO2 laser beam welded
injury, Journal of Biomedical Materials Research Part A.
AISI 316L austenitic stainless steel on the viability
107, 1273-1283 (2019).
of fibroblast cells, in vitro. Materials Science and
15. Chiriac, S., Facca, S., Diaconu, M., Gouzou, S.,
Engineering: C. 60, 211-218 (2016).
Liverneaux, P. Experience of using the bioresorbable
23. Liu, Y. et al. Bio-adenine-bridged molecular design
copolyester poly(DL-lactide-ε-caprolactone) nerve
approach toward non-covalent functionalized graphene
conduit guide Neurolac™ for nerve repair in peripheral

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 20 of 21
 by liquid-phase exfoliation. Journal of Materials Science. guide: Tissue reactions with a focus on collagen III/IV
55., 140-150 (2020). reformation. Journal of Biomedical Materials Research.
69 (2), 334-341 (2016).
24. Rehman, S. Reduced graphene oxide incorporated
GelMA hydrogel promotes angiogenesis for wound 32. Pathre, P. et al. PTP1B regulates neurite extension
healing applications. International Journal of mediated by cell-cell and cell-matrix adhesion molecules.
Nanomedicine. 14., 9603-9617 (2019). Journal of Neuroscience Research. 15, 143-150 (2001).

25. Bei, H. P. et al. Graphene-based nanocomposites for 33. Qing, L., Chen, H., Tang, J., Jia X. Exosomes and
neural tissue engineering. Molecules. 24 (4), 658 (2019). their microRNA cargo: New players in peripheral nerve
regeneration. Neurorehabilitation and Neural Repair. 32
26. Othman, S. A. et al. Alginate-gelatin bioink for bioprinting
(9), 765-776 (2018).
of HeLa spheroids in alginate-gelatin hexagon-shaped
scaffolds. Polymer Bulletin. 78, 6115-6135 (2021).

27. Peng, X. L., Li, Y., Zhang, G., Zhang, F., Fan, X.
Functionalization of graphene with nitrile groups by
cycloaddition of tetracyanoethylene oxide. Journal of
Nanomaterials. 2013, 841789 (2013).

28. Zorba Yildiz, A. P. et al. 3D therapeutic approaches

for peripheral nerve damage. 9th International Molecular

Biology and Biotechnology Congress Abstract Book. E-
ISBN: 978-605-70057-4-8,OP-19, (2020).

29. Liau, L. L., Ruszymah, B. H. I., Ng, M. H., Law,

J. X. Characteristics and clinical applications of
Wharton's jelly-derived mesenchymal stromal cells.
Current Research in Translational Medicine. 68 (1), 5-16
(2020).

30. Yoo, J. et al. Augmented peripheral nerve regeneration

through elastic nerve guidance conduits prepared using
a porous PLCL membrane with a 3D printed collagen
hydrogel. Biomaterials Science. 22, 1-12 (2020).

31. Jansen, K., Meek, M. F., van der Werff, J. F., A.,
van Wachem, P. B., van Luyn, M. J. A. Long-
term regeneration of the rat sciatic nerve through a
biodegradable poly (DL-lactide-Ɛ-caprolactone) nerve

Copyright © 2022 JoVE Journal of Visualized Experiments jove.com May 2022 • 183 • e63622 • Page 21 of 21