This document describes a study that aimed to develop a fully decellularized bovine caudal intervertebral disc scaffold. Researchers extracted bovine caudal discs from C4-C5 to C6-C7 and subjected them to a decellularization process involving freezing, ultrasonication, and exposure to a decellularization solution. Tissue samples from the nucleus pulposus and annulus fibrosis were then analyzed to compare glycosaminoglycan and DNA content between decellularized and fresh discs. Histological analysis and DNA gel electrophoresis were also performed to evaluate the decellularization process. The results were meant to determine if a fully decellularized bovine caudal IVD scaffold could be developed.

1. MATERIALS AND METHODS
BACKGROUND
Development of a Fully Decellularized Bovine Caudal IVD Scaffold
A. Hensley, J. Rames, C. Doyle, T. Litzinger, T. Watt, N. Wyman, C. Fernandez, MS, J. Mercuri, PhD
Department of Bioengineering, Clemson University, Clemson, SC
RESULTS
ACKNOWLEDGEMENTS
REFERENCES
Research
support
for
our
lab
has
been
provided
in
part
by
the
Na6onal
Ins6tute
of
General
Medical
Sciences
of
the
Na6onal
Ins6tutes
of
Health
(award
number:
5P20GM103444-­‐07)
and
departmental
start-­‐up
funds.
A
special
thanks
to
Chris
Fernandez
and
Dr.
Jeremy
Mercuri
for
assis6ng
with
this
project.
Much
apprecia6on
to
Publix
for
the
bovine
tails.
This
work
was
supported
in
part
by
the
Clemson
University
Crea6ve
Inquiry
Program.
We
would
also
like
to
thank
the
Office
of
Undergraduate
Studies,
the
Honors
College
and
the
Graduate
School
for
sponsoring
this
event.
A
C
DISCUSSION
C
A
B
C
0.00
50.00
100.00
150.00
200.00
250.00
GAG
Content
(μg/mg)
GAG
Decell
AF
Fresh
AF
Figure
3:
Nucleus
Pulposus
Content.
A)
Comparison
of
average
GAG
content
in
the
NP
of
decelled
IVDs
to
the
NP
of
fresh
IVDs.
(n=6/group)
B)
Comparison
of
average
DNA
content
in
the
NP
of
decelled
IVDs
to
the
NP
of
fresh
IVDs.
(n=6/group)
Figure
4:
Annulus
Fibrosis
Content.
A)
Comparison
of
average
GAG
content
in
the
AF
of
decelled
IVDs
to
the
NP
of
fresh
IVDs.
(n=6/group)
B)
Comparison
of
average
DNA
content
in
the
NP
of
decelled
IVDs
to
the
NP
of
fresh
IVDs.
(n=6/group)
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
GAG
Content
(μg/mg)
GAG
Decell
NP
Fresh
NP
0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
400.00
DNA
Content
(ng/mg)
DNA
Decell
NP
Fresh
NP
0.00
50.00
100.00
150.00
200.00
250.00
DNA
Content
(ng/mg)
DNA
Decell
AF
Fresh
AF
Lower
back
pain
(LBP)
is
a
mul6factorial
problems
associated
with
degenera6ng
intervertebral
discs
(IVDs),
which
provide
flexibility
and
shock
absorbance
for
the
vertebral
column.
IVD
progresses
with
age
with
over
60%
of
those
over
age
70
having
severely
degenerated
IVDs.1
Current
solu6ons
are
pallia6ve.
The
first
goal
of
our
project
is
to
create
a
fully
decellularized
bovine
caudal
IVD
to
be
used
as
a
scaffold
replacement
for
paSents
suffering
from
IVD
degeneraSon
and
lower
back
pain.
Figure
1:
Schema6c
illustra6ng
the
anatomic
loca6on
of
the
IVD
between
the
vertebral
bodies
of
the
spine
from
a
top
view
and
the
two
primary
components
of
the
IVD:
the
nucleus
pulposus
(NP)
and
annulus
fibrosus
(AF).2
Decell
SoluSon
§ Composed
of
50mM
tris,
0.2%
weight
ethylene
diamine
tetraace6c
acid
(EDTA),
0.02%
weight
sodium
azide,
and
1.2%
weight
Triton
X-­‐10
dissolved
into
deionized
water.
DecellurizaSon
Method/TesSng
Protocol
1. Four
of
each
disc
(C4-­‐5,
C5-­‐6,
and
C6-­‐7)
were
extracted.
Two
of
each
were
kept
at
-­‐20°C
for
use
as
fresh,
control
samples.
The
remaining
discs
were
prepared
for
the
decelluriza6on
process.
2. All
of
the
discs
going
through
the
decelluriza6on
process
were
put
in
a
-­‐80°C
freezer
for
2
hours.
3. IVDs
were
subjected
to
ultrasonica6on
for
10
minutes
at
40
kHz.
The
4. IVDs
were
put
in
100
mL
of
decell
solu6on.
These
containers
with
IVD
and
solu6on
were
leh
on
an
orbital
shaker
at
150rpms
at
room
temperature
for
24
hours.
Steps
2
through
4
were
repeated
for
3
cycles.
5. 5mm
6ssue
punches
were
used
to
take
samples
from
the
nucleus
pulposus,
and
the
right,
leh,
top,
and
boiom
por6ons
of
the
annulus
fibrosis.
These
samples
were
frozen
at
-­‐80°C
then
lyophilized
for
use
in
biochemical
analysis
(DMMB
and
PicoGreen
assay).
The
remainder
of
each
IVD
was
placed
in
a
6ssue
casseie
and
placed
in10%
neutral
buffered
formalin
in
prepara6on
for
be
fixed
for
histological
analysis.
6. A
t-­‐test
was
used
to
compare
the
GAG
values
obtained
from
the
DMMB
assay
and
the
DNA
values
obtained
from
the
PicoGreen
assay.
7. Treated
and
Control
disks
were
prepared
with
O.C.T.
and
cryosec6oned.
Standard
ethidium
bromide
live
dead
assays
and
Alcian
blue/
nuclear
red
fast
histology
were
performed.
8. DNA
was
extracted
from
samples
using
a
standard
Qiagen
kit.
Samples
were
run
in
both
using
both
a
1%
and
5%
gel
electrophoresis.
IVD
ExtracSon
Method
1. Betadine
solu6on
was
used
to
coat
the
four
tails
2. Caudal
Discs
(C4-­‐5
to
C6-­‐7)
were
targeted
3. Fascia
and
muscle
were
then
cut
away
with
scalpels,
vertebrae
were
separated
heavy
duty
clippers,
and
scalpels
were
used
to
cut
out
IVDs.
4. IVDs
were
immediately
placed
in
empty
120mL
containers
before
star6ng
decelluriza6on.
Discs
were
labeled
based
on
their
loca6on
in
the
tail.
5. Addi6onally,
a
loop
of
fishing
wire
was
used
to
label
the
right
side
of
the
disc.
q We
evaluated
the
ability
of
a
decellulariza6on
solu6on
to
fully
remove
bovine
cells
from
the
NP
and
AF
regions
of
cow
tail
IVDs,
while
aiemp6ng
to
maintain
as
much
GAG
(an
ECM
component
which
contributes
significantly
to
the
mechanical
func6on
of
the
IVD)
as
possible.
q Our
results
gave:
q Our
qualita6ve
results
are
further
corroborated
qualita6vely.
q Staining:
substan6al
reduc6on
of
nuclear
density
in
AF
and
NP.
q Gel
Electrophoresis:
neither
1%
nor
5%
gels
show
qualita6ve
measurable
DNA.
1Urban,
Jill,
and
Sally
Roberts.
"Degenera6on
of
the
Intervertebral
Disc."
Arthri6s
and
Research
Therapy.
5.3
(2003):
120-­‐130.
Web.
26
Mar.
2014
2hip://www.porcpotlas.hu/en/porckorong.html
3spineuniverse.com
A
A
B
B
Figure
5:
Alcian
Blue
stained
IVD
AF.
An
Alcian
Blue
(GAG)
and
nuclear
fast
red
(DNA)
stain
for
A)
AF
sample
treated
with
decell
protocol
and
B)
fresh
AF
sample.
A
B
A
B
Figure
2:
Macroscopic
images
of
IVDs.
A)
Intact
fresh
sample.
B)
Intact
decelled
sample.
Figure
7:
Alcian
Blue
stained
IVD
NP.
An
Alcian
Blue
(GAG)
and
nuclear
fast
red
(DNA)
stain
for
A)
NP
sample
treated
with
decell
protocol
and
B)
fresh
NP
sample.
A
B
Figure
6:
Ethidium
Bromide
stained
IVD
transiSon
region.
An
ethidium
bromide
live/dead
(DNA)
stain
for
A)
transi6on
region
sample
treated
with
decell
protocol
and
B)
fresh
transi6on
region
sample.
A
B
AF
NP
Treatment
Fresh
Decell
Fresh
Decell
GAG
(µg/mg)
252.70
±
23.17
132.71
±
11.44
677.20
±
95.04
258.21
±
59.32
DNA
(ng/mg)
244.67
±
25.68
65.99
±
4.07
314.28
±
65.40
47.12
±
14.66
GAG
Percent
ReducSon
(%)
47
62
DNA
Percent
ReducSon
(%)
73
85
GAG
T-­‐test
(α=0.05)
0.00421
0.00963
DNA
T-­‐test
(α=0.05)
0.00004
0.00393
Figure
8:
1%
Agarose
gel
electrophoresis.
Lanes
1
and
20
were
loaded
with
300-­‐24,000
kbp
exACTGene
ladder.
Lane
3-­‐5
and
13-­‐17
were
loaded
with
decelled
AF
DNA
Sample.
Lanes
6
and
18
were
loaded
with
decelled
NP
Sample.
Lanes
8-­‐11
were
loaded
with
Fresh
AF
DNA.
Lane
12
was
loaded
with
fresh
NP
sample.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Figure
9:
5%
Agarose
gel
electrophoresis.
Lanes
1
and
20
were
loaded
with
10-­‐300
kbp
exACTGene
ladder.
Lanes
7,
13.
and
19
were
leh
unloaded.
Lane
2-­‐5
and
13-­‐17
were
loaded
with
Decelled
AF
DNA
Sample.
Lanes
6
and
18
were
loaded
with
decelled
NP
Sample.
Lanes
8-­‐11
were
loaded
with
Fresh
AF
DNA.
Lane
12
was
loaded
with
fresh
NP
sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20