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A comparison of natural versus synthetic substrate materials for supporting neuronal cell bioprinting Castanon A1,2*, Glen A1, Haycock JW1
Background
• Peripheral Nerve Injury is a disorder affecting approximately 3-
8% people worldwide.1
• The goal of reconstructive surgery is to design nerve grafts to
span large injury gaps. However, current challenges include
using a material that can successfully mimic the environment of
the natural extracellular matrix to stimulate repair, and thereafter
maintain native cellular morphology and function.2
• Current tissue engineering approaches involve synthetic
polymers such as PCL and PLLA. While these have advantages
in 3D printing for regenerative applications, they have limitations
in stimulating repair.
• Decellularized nerve tissue is an abundant source of natural
matrix. A blend of these materials could be 3D printed & provide
extracellular matrix cues to improve neurite outgrowth.
1Department of Materials Science and Engineering, The University of Sheffield, UK. 2Department of Biomedical Science. *[email protected]
Aim
To investigate whether neuronal cells show preferential
proliferation and maturation/differentiation when grown on
synthetic polymer versus decellularized nerve
Methods and Materials
Results
References • 1Cahill, Lindsay S., Christine L. Laliberté, Xue Jun Liu, Jonathan Bishop, Brian J. Nieman, Jeffrey S. Mogil, Robert E.
Sorge, Catherine D. Jones, Michael W. Salter, and R. Mark Henkelman. "Quantifying blood-spinal cord barrier
permeability after peripheral nerve injury in the living mouse." Molecular pain 10, no. 1 (2014): 60.
• 2Falguni Pati, Jinah Jang, Dong-Heon Ha, Sung Won Kim, Jong-Won Rhie, Jin-Hyuung Shim, Deok-Ho Kim, & Dong-
Woo Cho. "Printing Three-dimensional Tissue Analogues with Decellularized Extracellular Matrix Bioink." Nature
Communications, 2014.
• 3Badylak, Stephen F, Weiss, Daniel J, Caplan, Arthur, & Macchiarini, Paolo. (2012). Engineered whole organs and
complex tissues. The Lancet, 379(9819), 943-952.
• NG108-15 neuronal cell line demonstrated a cell viability of
76% for PLLA and 63% for PCL comparable to that of 65%
for dECM bioink.
• Neurite Outgrowth demonstrated to be greater when NG
108-15 neuronal cells were cultured on dECM bioink (209
µm) in contrast to cultures on matrigel (109 µm ).
• SDS PAGE results demonstrate differences in dECM
bioink proteomic profiling compared to that of matrigel,
suggesting that dECM bioink may contain specific nerve
extracellular matrix proteins.
• This project proposes a new decellularized nerve
biomaterial that has been evaluated as a cell culture
coating specifically for neuronal cells and has
demonstrated to successfully establish neurite outgrowth
on NG 108-15 neuronal cells.
• dECM bioink was successfully bioprinted together with
PEG, demonstrating a novel biomaterial that contains both
natural and synthetic properties which may be supportive
for neurite outgrowth.
Conclusions and Discussion
Figure 7. H&E stain for Pre- and post- Decellularization of porcine sciatic
nerve transverse tissue. Images (A) and (B) display the fascicles of porcine sciatic
nerve prior to decellularization. Image (B) black arrow points to the epineurium,
green arrow points to the perineurium and yellow arrow points to the endoneurium.
Images (C) and (D) display tissue after decellularisation & removal of cell
population.
Figure 9. Neurite Length & Live/Dead Analysis of neuronal cell line
on Matrigel, dECM bio-ink, & tissue culture plastic (TCP). (A) Neurite
length for neuronal cells grown on dECM bioink were over 100 µm longer
than matrigel and 130 µm longer than TCP. (B) However, the viability for
neuronal cells grown on dECM bioink was 20% lower than TCP and
matrigel. This may indicate that dECM bioink may provide longer neuronal
extensions at the expense of cell viability. Cell viability and neurite lengths
were significantly different from each other. **P<0.01, ****P<0.0001.
Figure 8. Time course imaging at 24, 48, & 72-hour proliferation periods of
NG108 neuronal cell line on TCP, Matrigel, and dECM bioink. NG108 neuronal
cells were cultured with serum containing media for time intervals and fixed at the
respective ending time points. All images were taken at the bottom left of each well.
Images were obtained using a Zeiss LSM 510 confocal microscope. TCP and matrigel
supported preferential growth of cells, however cells cultured on dECM bioink (C, F, I)
had the longest neurites in comparison with TCP and matrigel.
Figure 5. NG 108 neuronal cells on Tissue Culture Plastic (TCP), PLLA, and
PCL: 2 days proliferation with serum-containing media and 3 days with
serum-free media. A total of 1.2 x104 neuronal cells were cultured per substrate.
The cells were fixed and stained on day 5. Metabolic Activity results were similar to
those obtained in viability assay, demonstrating that metabolic activity was not due
to cell stress. A One-way ANOVA statistical test was performed to demonstrate cell
viability means to be significantly different from each other. *P<0.05.
Figure 1. Porcine Hind leg dissection. (A) hind leg placed in a horizontal
position and blade is pointed towards the proximal region of the hind leg,
whereas the opposite end represents the distal portion of the hind leg. (B)
displays the nerve and vein bordering one another; yellow arrow points to the
vein & blue arrow points to the sciatic nerve.
Figure 2. Stages of porcine
sciatic nerve: pre- & post-
decellularization. (A) displays
the location of the sciatic nerve
within the porcine hind-leg. (B)
displays nerve after washing the
nerve twice in Phosphate
Buffered Saline (PBS). (C)
displays the sciatic nerve after
decellularization.
Nerve Decellularization. Decellularization is a 3-week process that removes
the cell population from tissue while restricting changes in structure,
composition, or ligand background of the native matrix, including vascular and
lymphatic components.3
Figure 3. Different
stages of dECM
bioink synthesis. (A)
sciatic nerve tissue
after freeze dried for
48 hours (B) tissue
immediately after
pepsin and acetic acid
have been mixed
together with the
freeze-dried sample.
(C) & (D) tissue after
full pepsin digestion
for 24 hours at 37°C.
24 h
ou
rs
48 h
ou
rs
72 h
ou
rs
TCP Matrigel dECM bioink
NG
10
8 n
eu
ron
al c
ell
s
TCP PCL 10% (w/v) PLLA 10% (w/v)
Figure 10. Protein identification of dECM bioink (pre- and post-
digestion) in comparison with matrigel and Fetal Calf Serum (FCS). (A)
shows optimization of sample loading; indicates increasing protein
abundance of post-digested material in correlation to amount of sample
loaded. (B) displays lower molecular weight due to pepsin digestion in
contrast to pre-digested material and different molecular weight bands in
FCS, dECM bioink, and matrigel.
A
B
A B
Cell Culture & Immunostaining. A total of 1.2 x104 NG108-15 neuronal cells
were seeded per well and fixed by adding 5% paraformaldehyde. Cells were
immunostained with mouse monoclonal anti- β3 Tubulin (1:500) and FITC-
conjugated secondary antibodies (1:100). DAPI staining was employed for
nuclear labeling.
MTT & Live/Dead Assay. MTT assay used measures metabolic activity
through reduction of tetrazolium to formazan by the mitochondria. Live/Dead
Cell Trypan Blue assay from Invitrogen was used to measure cell viability.
Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS-PAGE). dECM bioink
(pre and post digestion), matrigel, and Fetal Calf Serum (FCS) samples were
dissolved in SDS sample buffer and subjected to SDS-polyacrylamide gel
(10%) electrophoresis by applying an electric current of 120 V and allowed
protein migration for one and a half hours.
A B
0
10
20
30
40
50
60
70
80
90
100
TCP PLLA PCL
Via
bilit
y P
erc
en
tag
e
Test Substrate
Live/Dead viability assay of neuronal cells on TCP, PLLA, and PCL
TCP
PLLA
PCL
0
10
20
30
40
50
60
70
80
90
100
TCP PLLA PCL
Me
tab
oli
c A
cti
vit
y P
erc
en
tag
e
Test Substrate
MTT assay of neuronal cells on TCP, PLLA, and PCL
TCP
PLLA
PCL
B
A
.00
2 µ
g
.00
5 µ
g
.00
7 µ
g
.01
0 µ
g
198kDa
62kDa
1 2 3 4
1: Pre-digested dECM bioink
2: Post-digested dECM bioink
3: Matrigel
4: Fetal Calf Serum
Acknowledgements
0
10
20
30
40
50
60
70
80
90
100
TCP Matrigel dECM bioink
Via
bilit
y P
erc
en
tag
e
Test Substrate
Live/Dead Viability Assay of neuronal cells on TCP, dECM bioink, and matrigel:
72 hour proliferation with serum-free media
TCP
Matrigel
dECM bioink
0
50
100
150
200
250
TCP Matrigel dECM bioink
Ne
uri
te L
en
gth
(µ
m)
Test Substrate
Average Neurite Length for neuronal cells on TCP, Matrigel, and dECM bioink:
72 hour proliferation with serum-free media
TCP
Matrigel
dECM bioink
This research was funded by Consejo Nacional de Ciencia y Tecnologia (CONACyT). I wish to
acknowledge Martin Frydrych for his help in the freeze-drying process. Special thanks to
Leyla Zilic for her valuable help in dissection & decellularization and all members of the
Haycock laboratory.
B
A 1:1 blend of Poly-ethylene-glycol (PEG)
and dECM bioink was 3D printed using an
open source RepRap machinery. The
biomaterial was successfully extruded into
an octagonal structure. Further work would
involve seeding NG108-15 neuronal cells as
well as primary cell lines to observe cellular
response on the blend of materials.
*
*
*
*
****
****
****
** **
**
