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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. *acastanon1@sheffield.ac.uk

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.

*

*

*

*

****

****

****

** **

**