The biologic and mechanical testing of carbon scaffolds as a graft substitute for the repair of critical sized tendon defects and their potential use as a biologics carrier for tendon repair and regeneration

1. Carbon
Engineered
Scaffolds
May
Provide
An
Op9mum
Balance
Of
Perspec(ve
Biologic
And
Mechanical
Proper9es
For
Use
In
Tendon
Repair
Surgery
Advantage
Solu(ons,
LLC
RM.
Joseph
DPM
PhD1,3,
JS.
Czarnecki
MS2,
K.
Lafdi
PhD
DSc2,3,
PA.
Tsonis
PhD3
1
Perspec@ve
Advantage
Solu@ons,
LLC,
2
University
of
Dayton
Research
Ins@tute,
3
Center
for
Tissue
Regenera@on
&
Engineering
at
Dayton
Purpose:
The
purpose
of
this
study
is
to
inves9gate
the
mechanical
and
biologic
proper9es
of
carbon
fiber
Figure
1:
Reconstructed
Cri9cal
Size
Defect
of
Achilles
Tendon
Figure
5:
Load
to
Failure
of
Carbon
Scaffolds
Figure
9:
Confocal
Microscopy
of
Fibroblast
Adhesion
on
Carbon
Scaffolds
Results:
scaffolds
as
poten9al
graSs
for
repairing
cri9cal-­‐size
Achilles
tendon
defects.
Light
Microscopy
Immunoflourescence
1) CF
and
CV
demonstrate
less
variability
in
physical
alributes
(Fig.
2)
and
load
failure
(Fig.
5)
than
the
Microscopy
GJ
controls.
2) CF
and
CV
were
more
porous
than
GJ
and
demonstrated
greater
tensile
modulus
than
GJ
control
(Figs.
* 2,
4).
Regression
analysis
showed
porosity
and
thickness
to
be
a
predic9ve
factors
in
load
failure
of
GJ
Introduc9on:
Surgical
repair
of
cri9cal
size
tendon
defects
associated
with
Achilles
tendon
ruptures
are
Denotes
Sta9s9cal
Significance
Between
Groups
Sample
1
control
but
not
CF
of
CV
(Fig
.
8).
Stress
and
modulus
were
predic9ve
factors
in
carbon
scaffold
load
formidable
challenges
in
reconstruc9ve
surgery.
Cri9cal
defects
occur
when
a
tendon
can
not
be
re-­‐approximated
or
failure.
1c
sufficiently
lengthened
to
reconstruct
the
tendon
following
injury.
When
this
occurs,
there
is
a
tendon
gap
that
must
3) Micro-­‐CT
imaging
showed
a
more
random
organiza9on
of
CV
fibers
than
CF.
The
fibrous
nature
of
GJ
be
bridged
to
restore
tendon
structure
and
func9on
(Fig
1a).
Tendon
transfers
(Fig.
1b
Flexor
Hallucis
transfer)
and
CV
extracellular
matrix
could
not
be
appreciated
on
Micro-­‐CT
(Fig.
3).
tendon
allograSs
(Fig.
1c
Semitendonosis
allograS)
have
been
u9lized
to
bridge
deficits
however
both
techniques
4) Load
to
failure
of
CF
most
closely
resembled
that
of
GJ
controls.
CV
load
to
failure
was
less
than
CF
have
limita9ons
(1,
2).
AllograSs
pose
a
risk
of
infec9on
and
have
no
capacity
for
repair
given
their
acellular
nature
1a
1b
1d
1e
and
GJ
(Fig.
5).
Carbon
modulus
was
greater
the
GJ
control
and
more
closely
resembles
modulus
of
(3,
4).
Tendon
transfers
provide
a
func9onal
muscle
tendon
complex
however
the
strength
and
power
of
commonly
Sample
2
Achilles
tendon
reported
in
the
literature
(Fig.6).
transferred
tendons
are
inferior
to
the
Achilles
tendon.
There
are
no
biologically
ac9ve
graSs
with
FDA
approved
5) Max.
load
to
failure
of
carbon
was
greater
than
GJ
control
and
reported
values
of
load
failure
of
indica9ons
for
use
in
cri9cal
defect
repair
however
several
commercial
scaffolds
such
as
GraS
Jacket
(GJ)
(Fig.
1d)
are
Figure
6:
Tensile
Behavior
of
Carbon
Scaffolds
rou9nely
used
to
re-­‐enforce
Achilles
tendon
repair
surgery
(5).
In
the
most
severe
cases,
all
of
the
above
techniques
Figure
2:
Physical
Proper9es
of
Carbon
Scaffolds
and
GraS
Jacket
Controls
Achilles
tendon
(Figs.
5
&6
).
6) Morphology
of
fibroblast
adhesion
on
CF
consistently
demonstrated
cell
clustering
and
diffuse
ac9n
may
be
applied
to
reconstruct
the
Achilles
tendon
(Fig.
1e).
There
are
currently
no
graS
implants
available
that
organiza9on
similarly
observed
with
adhesion
to
the
vascular
surface
of
GJ
controls
while
CV
provide
the
mechanical
strength
,
durability
and
cellular
ac9vity
needed
for
tendon
repair
and
func9on.
Limited
Sample
1
demonstrated
less
clustering
and
focal
filamentous
ac9n
organiza9on
more
similar
to
cell
adhesion
to
studies
have
explored
carbon
as
a
biocompa9ble
structural
graS
for
Achilles
repair
yet
none
have
explored
its
the
epidermal
surface
of
GJ
control.
(Fig.
10).
poten9al
dual
func9ons
as
a
structural
graS
and
vehicle
for
delivering
cells
and
biofactors
(6).
7) Growth
rate
of
fibroblasts
on
carbon
was
less
than
GJ
control
GJ>CF>CV.
Growth
rate
on
CF
Carbon
scaffolds
seeded
with
fibroblasts
or
mesenchymal
stem
cells
may
have
the
poten9al
to
enhance
tendon
healing
through
synthesis
of
anabolic
growth
factors
and
extracellular
matrix
(10,
11,
12).
Although
carbon
is
not
a
CF
8)
approached
that
of
GJ
control
(Fig.
10).
Fibroblast
adhesion
on
carbon
scaffolds
was
significantly
lower
than
GJ
controls
cultures
(Fig.
7).
new
component
of
medical
implants,
technologies
that
enable
nanoscale
engineering
of
carbon
and
its
physical
proper9es
are.
Carbon
scaffolds
can
be
engineered
to
specific
dimensions,
fiber
orienta9on,
porosity
as
well
as
Sample
2
mechanical
strength
and
texture
at
a
nanoscale.
Extracellular
matrix
interac9ons
with
cells
also
occur
on
this
same
scale
to
regulate
cell
morphology.
Fibroblast
morphology
influences
cell
anabolism
and
prolifera9on,
two
key
Conclusions
&
Discussion:
1) Carbon
engineering
has
poten9al
to
yield
tendon
subs9tutes
with
mechanical
proper9es
similar
to
the
process
that
regulate
9ssue
repair
and
healing.
The
organiza9on
of
Poly(D,L-­‐lac9c-­‐co-­‐glycolic
acid)
and
collagen
Achilles
tendon
and
biologic
ac9vity
that
promotes
tendon
healing.
Factors
that
influence
variance
in
meshes
have
been
shown
to
influence
fibroblast
morphology
however
it
is
unknown
whether
carbon
similarly
load
failure
in
carbon
are
different
from
factors
affec9ng
load
failure
in
GJ.
These
differences
may
be
influences
morphology
or
if
carbon
can
be
specifically
manipulated
to
geometrically
stabilize
or
induce
cell
Figure
3:
Micro-­‐CT
Imaging
of
Scaffold
Organiza9on
*Achilles
modulus
data
extracted
from:
Abrahams,
M.
Mechanical
Behavior
of
Tendon
IN
VITRO:
A
Preliminary
Report.
Med.
&
Biol.
Eng
1967;5(5):433-­‐443
Sample
1
related
to
unique
proper9es
of
carbon
in
comparison
with
9ssue
derived
scaffolds.
morphologies
that
promote
9ssue
repair
and
regenera9on
(7,
8,
9).
In
na9ve
9ssues,
disrup9on
or
loss
of
Dermal
Surface
2) Scaffold
porosity
was
shown
to
have
an
insignificant
affect
on
load
failure
variance
in
carbon
but
did
extracellular
matrix
results
in
9ssue
degenera9on
and
failure
of
9ssue
repair.
The
poten9al
ability
of
carbon
to
account
for
significant
variance
in
GJ
load
failure.
This
suggests
that
carbon
may
have
advantages
support
9ssue
repair
processes
in
an
environment
deficient
of
extracellular
matrix
may
provide
an
unique
opportunity
to
create
a
graS
with
the
strength
of
a
mature
9ssue
without
the
matrix
structure
and
organiza9on
of
Figure
7:
Fibroblasts
Adhesion
and
Survival
to
Scaffolds
In
Vitro
GJ
over
some
natural
9ssue
scaffolds
that
allow
maximiza9on
of
scaffold
porosity
to
promote
graS
vasculariza9on
without
compromising
the
mechanical
strength
necessary
to
restore
Achilles
func9on
mature
9ssue.
Sample
2
* when
repairing
cri9cal
size
tendon
defects.
This
study
compared
the
mechanical
strength,
physical
proper9es
and
biologic
capacity
of
two
carbon
scaffolds
CV
CV
Epidermal
Surface
Denotes
Sta9s9cal
3) Carbon
scaffolds
demonstrate
a
capacity
to
support
fibroblast
growth
and
prolifera9on.
This
makes
against
GraS
Jacket
test
controls.
The
ability
of
carbon
to
support
cell
growth
and
act
as
a
substrate
for
9ssue
repair
(Surface)
(Deep)
Significance
Between
Groups
carbon
a
poten9al
carrier
of
living
cells
for
sustained
delivery
of
growth
factors
and
extracellular
was
assessed
by
examining
fibroblast
adhesion,
survival
and
prolifera9on
on
carbon
scaffolds.
The
ability
of
carbon
matrix
synthesis
to
a
site
of
tendon
repair.
Some
refinement
in
CF
and
CV
proper9es
will
likely
be
to
stabilize
fibroblast
morphology
was
examined
by
confocal
microscopy.
The
physical
and
mechanical
proper9es
of
(Red
=
Ac9n
Filaments,
Blue
=
Nuclei)
necessary
to
op9mize
and
prolong
fibroblast
growth
and
adhesion
proper9es
to
carbon.
carbon
were
examined
by
Micro-­‐CT
and
MTS
tes9ng.
GJ
CV
CF
GJ
CF
CF
Future
Studies:
Materials&Methods:
Carbon
Scaffolds:
Two
polyacrylonitrile
(PAN)
carbon
fiber
materials
were
engineered
(Surface)
(Deep)
Figure
10:
Rates
of
Fibroblast
Prolifera9on
on
Scaffolds
In
Vitro
(A 450nm-­‐A690nm)
1) Characteriza9on
of
growth
factor
and
matrix
molecule
synthesis
of
fibroblasts
cultured
on
carbon
to
make
a
Carbon
Veil
(
CV)
and
Carbon
Fabric
(CF)
scaffold
by
conven9onal
processing
techniques
of
spinning,
scaffolds
stabiliza9on
and
carboniza9on.
Carbon
scaffolds
were
fabricated
by
combining
PAN
fibers
with
10%
w/w
2) Further
modifica9on
and
design
a
carbon
scaffolds
proper9es
to
op9mize
mechanical
strength
and
polycaprolactone
(14kDa,
SIGMA)
in
acetone
.
CV
is
a
heterogeneous
organiza9on
of
fibers
while
CF
fibers
are
more
ability
to
support
cell
adhesion
and
prolifera9on
homogenously
aligned.
The
sequen9al
processes
of
spinning
and
carboniza9on
were
used
to
make
carbon
scaffolds.
Figure
4:
Rela9onship
of
Carbon
Scaffold
Porosity
to
Tensile
Stress
Mechanical
Tes9ng:
Load
failure,
stress
and
modulus
were
compared
across
hydrated
scaffolds
with
an
MTS
using
a
Disclosure
loading
rate
of
2.54
mm
/min,
(N=10).
Micro-­‐CT
Tes9ng:
A
micro-­‐ct
x-­‐ray
system
(Scanco
Medical,
Switzerland)
was
*Test
Control
Samples
of
GraSJacket
were
donated
by
Wright
Medical
Technology*
used
to
characterize
scaffold
thickness
and
porosity
with
7
um
sec9ons.
Confocal
Microscopy:
Fibroblasts
(CRL
2703
Stepwise
Mul9variate
Regression
of
Carbon
Scaffolds’
Load
Failure
as
cell
line)
were
cultured
on
scaffolds
in
vitro
with
in
DMEM
with
10%
FBS.
Confocal
microscopy
was
used
to
evaluate
Figure
8:
a
Func9on
of
Tensile
Modulus,
Porosity,
Stress,
Scaffold
Thickness
References
cell
adhesion,
morphology
and
viability
of
fibroblast
cultures
at
several
9me
points
over
a
period
of
96
hrs
using
Carbon
Scaffolds
(Combined
Scaffolds
N=40
)
GraS
Jacket
Control
(N=20)
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P.
and
P.
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S226-­‐31.
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was
also
confirmed
by
fibroblast
absorbance
measured
at
450nm
and
690
nm
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Z.J.,
B.G.
Loder,
and
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tendon
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u9lizing
an
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144-­‐8;
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2564-­‐71.
using
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viability
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Applied
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per
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each
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et
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p.
1073-­‐87.
scaffold.
Step-­‐wise
mul9variate
regression
was
used
to
examine
the
rela9onship
between
scaffold
thickness
and
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J.L.,
A.G.
Gona,
and
H.
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vitro
tendon
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on
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J.L.,
et
al.,
Efficacy
of
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in
kniQed
silk-­‐collagen
scaffold
for
tendon
9ssue
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and
their
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p.
9438-­‐51.
significance
with
alpha
of
0.05.
GraS
Jacket
(GJ)
control
samples
were
donated
by
Wright
Medical
Technologies.
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X.,
et
al.,
Stepwise
differen9a9on
of
human
embryonic
stem
cells
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tendon
regenera9on
by
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