Table Of ContentHyaluronic acid biomaterials for perspective
peripheral nerve regeneration
A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy
in the Faculty of Medical and Human Sciences
2011
Sihem Ouasti
School of Biomedicine
Contents
List of Contents
1.
Introduction......................................................................................................................21
1.1.
Hyaluronan....................................................................................................................22
1.2.
HA
physical
chemistry................................................................................................23
1.3.
Derivatization
chemistry
of
HA..............................................................................29
1.3.1.
Dihydrazide-HA
conjugate......................................................................................29
1.3.2.
Methacrylated
HA.......................................................................................................31
1.3.3.
Haloacetate-modified
HA.........................................................................................32
1.3.4.
Aldehyde-modified
HA..............................................................................................33
1.3.5.
Tyramine
modified-
hyaluronic
acid...................................................................34
1.3.6.
HA-sulfhydryl
derivatives........................................................................................34
1.4.
Clinical
applications...................................................................................................35
1.4.1.
Visco-separation.........................................................................................................36
1.4.2.
Visco-supplementation.............................................................................................37
1.4.3.
Visco-protection..........................................................................................................38
1.4.4.
Visco-surgery...............................................................................................................38
1.5.
HA
hydrogels.................................................................................................................39
1.5.1.
Relation
between
cell
matrix
stiffness
and
cellular
phenotype/proliferation.......................................................................................................40
1.5.2.
HA-based
hydrogels
for
tissue
regeneration....................................................42
1.5.3.
Creation
of
niches
or
micro-environment
to
modulate
stem
cell
fate......50
1.6.
HA
receptors..................................................................................................................52
1.6.1.
CD44................................................................................................................................52
1.6.2.
RHAMM...........................................................................................................................55
1.6.3.
LYVE-1.............................................................................................................................63
1.6.4.
HARE...............................................................................................................................66
1.6.5.
Soluble
receptors........................................................................................................67
1.7.
Aims
and
objectives....................................................................................................69
1.8.
References......................................................................................................................75
2.
Hyaluronic
acid
/
PEG
hydrogels.
Network
connectivity,
mechanical
properties
and
cell
adhesion...........................................................................................82
2.1.
Introduction..................................................................................................................84
2.2.
Experimental
section..................................................................................................89
2.2.1.
Materials.......................................................................................................................89
2.2.2.
Preparative
procedures...........................................................................................90
2
Contents
2.2.3.
Physico-chemical
characterization......................................................................93
2.2.4.
Biological
characterization....................................................................................95
2.3.
Results
and
discussion...............................................................................................98
2.3.1.
Preparation
and
cytotoxicity
of
thiolated
HA
(HA-SH)..................................98
2.3.2.
Properties
of
the
photopolymerized
networks...............................................100
2.3.3.
Further
investigations
on
type
II
gels...............................................................107
2.4.
Conclusions..................................................................................................................118
2.5.
Acknowledgements...................................................................................................119
2.6.
References....................................................................................................................120
2.7.
Supplementary
Information..................................................................................124
3.
Receptor
binding
and
re-presentation
in
the
uptake
of
a
targeted
carrier:
RGD-functionalized
hyaluronic
acid
and
its
interactions
with
CD44
/
integrins
143
3.1.
Introduction................................................................................................................145
3.2.
Materials
and
methods............................................................................................151
3.2.1.
Materials.....................................................................................................................151
3.2.2.
Physico-chemical
characterization....................................................................152
3.2.3.
Preparative
procedures.........................................................................................152
3.2.4.
Biological
investigations.......................................................................................154
3.3.
Results
and
discussions...........................................................................................159
3.3.1.
Preparation
of
HA
derivatives.............................................................................159
3.3.2.
Uptake
kinetics
and
intracellular
localization
of
HA
and
HA-RGD.........161
3.3.3.
Mechanism
of
uptake..............................................................................................168
3.4.
Conclusions..................................................................................................................176
3.5.
Acknowledgements...................................................................................................177
3.6.
References....................................................................................................................178
3.7.
Supporting
Information..........................................................................................185
4.
Hyaluronic
acid
(HA)
receptors
and
their
role
in
the
motility
of
adipose-
derived
stem
cells
differentiated
into
a
Schwann
cell
phenotype........................188
4.1.
Introduction................................................................................................................190
4.2.
Experimental
section................................................................................................193
4.2.1.
Materials.....................................................................................................................193
4.2.2.
Methods........................................................................................................................195
4.3.
Results
and
discussions...........................................................................................203
4.3.1.
Presence
of
HA
and
HA
receptors
in
peripheral
nerves..............................203
4.3.2.
HA
receptors
as
possible
markers
for
Schwann
cell
differentiation?....205
3
Contents
4.3.3.
Influence
of
HA
receptors
on
Schwann
cell
motility
and
morphology...209
4.4.
Conclusions..................................................................................................................214
4.5.
Acknowledgements...................................................................................................214
4.6.
References....................................................................................................................215
4.7.
Supplementary
Information..................................................................................221
4.8.
Appendix.......................................................................................................................224
4.8.1.
Appendix
references................................................................................................235
5.
Conclusions.....................................................................................................................224
4
Contents
List of Figures
Figure
1.1-1.Structure
of
hyaluronan.
The
mere
unit
of
the
polysaccharide
is
composed
by
glucuronic
acid
(with
a
charged
carboxylic
acid
at
physiological
pH)
and
N-acetyl
glucosamine.
As
highlighted
in
red
in
this
figure,
a
cluster
of
hydrogen
atoms
is
on
one
face
of
the
disaccharide,
which
may
confer
a
more
hydrophobic
tendency
to
one
face
of
the
polymer
as
it
is
bearing
on
the
other
face
hydrophilic
groups
highlighted
in
blue. ______________________23
Figure
1.2-1:
Probability
of
each
of
the
intramolecular
hydrogen
bond
being
form
in
water,
β
1-4
linkage
(top),
and
β
1-3
linkage
(bottom)
[10].______________________________________________26
Figure
1.2-2.“Dependence
of
the
specific
viscosity
on
interactions
between
macromolecules,
as
expressed
by
the
four-term
equation
(Eq.
1).
Each
term
depends
on
the
coil
overlap
factor
to
a
different
power
The
term
k′c[η]
is
a
measure
of
the
degree
to
which
the
domains
of
the
polymer
coils
overlap
in
dilute
or
semi-dilute
solution”
[14].____________________________________28
Figure
1.3.6-1:
Composite
structure
of
HA
derivatives
illustrating
selected
primary
modifications
discussed
above:
thiopropionyl
hydrazide
from
DTPH
modification;
bromoacetate;
adipic
dihydrazide
for
use
in
further
crosslinking
via
acrylamide
or
hydrazone
linkages;
methacrylate
on
primary
6-hydroxyl
group;
benzyl
ester;
glycidyl
methacrylate;
tyramide
for
peroxidase
crosslinking;
2-
thiolethyl-ether;
dialdehyde
obtained
by
dihydrazide
chemistry
and
periodate
oxidation;
unmodified
disaccharide
mere
unit[30].__________________35
Figure
2.3.1-1.
1H-NMR
(left)
and
IR
(right)
spectra
of
HA
(64
kDa)
before
and
after
its
functionalization
with
cysteamine.
The
structure
of
the
thiol-bearing
copolymer
is
shown
above
with
the
assignment
of
the
proton
resonances.
The
general
structure
of
the
NMR
spectrum
appears
considerably
altered
by
the
functionalization:
the
resonances
of
the
two
anomeric
protons
H1
and
H7
have
merged
in
one
broad
signal
at
4.3-4.4
ppm
and
the
fine
structure
of
the
cluster
between
3
and
4
ppm
is
significantly
altered.
The
IR
spectrum
shows
a
significant
decrease
of
the
peak
at
1610-1620
cm-1,
which
can
be
associated
to
the
HA
carboxy
groups,
and
an
increase
in
the
amide
I
band._____________________________________________________99
Figure
2.3.1-2.
Cytotoxicity
of
HA
and
of
HA-SH
on
L929
murine
fibroblasts.
The
viability
of
the
cells
was
measured
with
the
MTS
metabolic
assay
after
an
exposure
of
24
hours
to
different
concentrations
of
the
polymers
in
full
medium,
normalized
for
the
protein
content
(measured
through
the
BCA
assay)
and
expressed
in
reference
to
the
value
of
cells
grown
on
TCPS
under
identical
conditions
without
the
presence
of
HA
or
HA-SH.________________________99
Figure
2.3.2-1.Relative
hardening
rates
as
a
function
of
the
type
of
gels
and
of
HA
molecular
weight
and
content.
The
rates
were
calculated
as
the
ratios
between
values
of
the
HA
formulations
and
that
of
the
control
(PEGDA
5%wt.
with
no
HA).
G’
was
differentiated
numerically
utilizing
a
G’
vs.
time
curve
obtained
averaging
three
experimental
curves.
We
estimate
an
error
of
±20%.______________________________________________________________________101
Figure
2.3.2-2.Left:
storage
modulus
(G’)
as
a
function
of
HA
concentration
for
mixtures
containing
5
%wt.
of
PEGDA.
HA
was
used
in
its
native
form
(Type
I)
and
as
thiolated
derivative
polymerized
without
incubation
(Type
II),
or
with
incubation
at
slightly
basic
pH
(Type
III);
as
a
control,
the
polymerization
of
Type
II
was
also
performed
in
the
presence
of
TCEP
as
a
reducing
agent
(Type
IIRed).
Right:
tan
δ
(=G”/G’)
for
the
same
systems
shown
in
the
left
panel.
Tan
δ
values
of
TypeIIRed
overlap
with
those
of
Type
II
and
are
not
shown
here.
Average
and
SD
calculated
on
four
repeats.____________________________________________________102
Figure
2.3.2-3.Left:
swelling
behavior
of
type
I-III
gels
at
4°C
upon
24
hours
incubation
in
PBS
buffer
containing
5%NaN ;
type
II
gels
were
also
incubated
in
the
presence
of
TCEP
in
order
to
3
highlight
the
presence
of
elastically
active
disulfide
bonds.
Right:
magnified
view
of
the
effect
of
the
presence
of
a
reducing
agent
(TCEP)
on
the
swelling
of
type
II
gels,
which
demonstrates
the
presence
of
elastically
active
disulfides. ____________________________________________________102
Figure
2.3.2-4.Plots
of
G’
vs.
swelling
ratio
for
all
the
gels
investigated
in
this
study.
Type
I
gels
showed
a
clearly
distinct
behavior
from
the
others,
suggesting
a
more
rigid
character
of
the
network.
Type
II
and
type
III
gels
showed
a
certain
overlap,
which
can
be
ascribed
to
the
higher
compatibility
between
HA
and
PEG
achieved
in
these
systems;
however,
a
difference
between
them
is
visible
too.
The
lines
represent
linear
fittings
of
the
data
points
of
each
class
of
gels. ____________________________________________________________________________________________103
Figure
2.3.2-5.
Equilibrium
swelling
ratios
of
the
three
different
hydrogel
types
based
on
2%
wt.
HA
234
kDa
during
a
period
of
seven
days
of
incubation
at
37°C
buffer
containing
5
Contents
200U/mL
ovine
testis
hyaluronidase
and
0.5%NaN ;
the
medium
was
changed
every
day
in
3
order
to
allow
for
the
continuous
presence
of
active
hyaluronidase.
The
lines
are
just
guides
for
eyes.___________________________________________________________________________________________107
Figure
2.3.2-6.
Equilibrium
swelling
ratios
of
all
gel
types
at
37°C
in
PBS
buffer
containing
200U/mL
ovine
testis
hyaluronidase
and
0.5%NaN ;
the
medium
was
changed
every
day
in
3
order
to
allow
for
the
continuous
presence
of
active
hyaluronidase.
Left:
1
day
of
incubation.
Right:
7
days
of
incubation.______________________________________________________________________107
Figure
2.3.3-1.
Examples
of
nanoindentation
curves.
The
experiments
were
performed
using
a
cantilever
of
elastic
constant
0.14
N/m
on
“as
prepared”
type
II
gels
in
air
with
100%
humidity
to
avoid
during.________________________________________________________________________110
Figure
2.3.3-2.
Nanoindentation
measurements
showed
that
the
surface
Young’s
modulus
qualitatively
followed
a
trend
similar
to
the
storage
modulus
obtained
from
oscillatory
rheology.
Left:
comparison
of
Young’s
modulus
(E)
and
storage
modulus
(G’).
Right:
E/G’
ratio
plotted
as
a
function
of
content
and
molecular
weight
of
HA.
Since
G’~G,
this
ratio
should
be
in
ideal,
isotropic
hydrogels.________________________________________________________________________110
Figure
2.3.3-3.
Examples
of
force
maps.
Type
II
gels
are
likely
characterized
by
a
partially
phase
separated
structure,
but
on
a
length
scale
relevant
for
cell
experiments
the
Young’s
modulus
was
reasonably
homogeneous:
the
modulus
of
PEGDA
control
varied
within
24-30
KPa,
and
type
II
gels
showed
even
lower
relative
variations.__________________________________111
Figure
2.3.3-4.
The
ratio
of
the
formazan
absorption
(MTS
assay)
at
different
days
was
used
an
indication
of
the
=
metabolic
activity
and
proliferation
of
L929
cultured
on
top
of
type
II
gels.
Prior
to
any
photopolymerization
experiment,
PEGDA
was
conjugated
to
GCGRGDS
via
Michael-type
addition,
with
1:20
thiol:acrylate
ratio;
all
the
samples
featured
the
same
density
of
RGD
peptides.
Left:
comparison
between
day
6
and
day
3,
showing
that
in
this
period
the
activity
of
cells
on
64
kDa
samples
and
on
PEGDA
was
much
increased,
while
cells
on
234
kDa
samples
did
not
show
significant
increases.
Right:
the
opposite
was
observed
in
the
day
9
/
day
6
comparison. ____________________________________________________________________________________113
Figure
2.3.3-5.
Phase
contrast
pictures
of
L929
fibroblasts
cultured
under
standard
conditions
at
day
3
(top)
and
day
6
(bottom)
on
different
type
II
hydrogels
and
on
5%
wt.
photopolymerized
PEGDA
and
on
TCPS
as
controls.
The
density
of
RGD
peptide
was
kept
constant
for
all
hydrogels
(1:20
thiol/acrylate
molar
ratio).__________________________________114
Figure
2.3.3-6.
Quantitative
descriptors
of
cell
shape
and
spreading
at
day
3.
Prior
to
any
photopolymerization
experiment,
PEGDA
was
conjugated
to
GCGRGDS
via
Michael-type
addition,
with
1:20
thiol:acrylate
ratio;
all
the
samples
featured
the
same
density
of
RGD
peptides.
At
day
3
fibroblasts
reached
already
confluency
on
TCPS
and
this
prevented
a
reasonable
image
analysis
of
the
TCPS
samples.
A:
percentage
of
gel
surface
covered
by
cells
ad
day
3.
B:
Relative
number
of
cells
on
type
II
gels
at
day
3
in
comparison
to
those
on
PEGDA.
C,
D:
mean
area
and
mean
circularity
of
the
fibroblasts
present
on
the
gel
surfaces.
The
parameters
for
A,
C
and
D
were
calculated
through
the
particle
analysis
routines
of
ImageJ;
cell
numbers
were
obtained
from
manual
count.
Error
bars
were
omitted
from
all
graphs
for
clarity
of
presentation;
for
mean
cell
area
and
mean
circularity,
the
error
bars
are
presented
in
Figure
2.3.3-7._________________________________________________________________________________115
Figure
2.3.3-7.
Dependency
of
the
mean
cell
area
(left)
and
the
mean
circularity
(right)
on
the
surface
Young’s
modulus
for
type
II
gels
and
control
PEGDA
for
L929
fibroblasts
at
day
3.
The
two
parameters
provide
a
similar
picture
of
increasing
spreading
on
harder
materials.
Please
realize
that
the
Young’s
modulus
values
are
measured
through
nanoindentation
on
the
gels
as
prepared
in
a
humid
air
environment.
The
actual
modulus
of
the
gels
in
culture
media
will
be
lower,
with
a
larger
decrease
for
234
kDa
gels
(larger
swelling,
see
Figure
2.3.2-4). 116
Figure
2.3.3-8.
The
metabolic
activity
per
cell
was
evaluated
by
dividing
the
results
of
the
MTS
assays
(mitochondrial
reductase
activity)
at
day
3
by
the
mean
cell
density
on
the
gels
at
the
same
time;
both
quantities
were
normalized
in
reference
to
TCPS,
which
therefore
showed
an
activity
per
cell
equal
to
100%.
Left:
relative
metabolic
activity
per
cell
as
a
function
of
the
concentration
of
HA
in
the
type
II
gels.
Right:
relative
metabolic
activity
per
cell
as
a
function
of
the
Yong’s
modulus
of
type
II
gels.____________________________________________________________117
Figure
2.7-1:
Cysteamine
calibration
curve
for
Ellman’s
assay.______________________________124
Figure
2.7-2.
Evolution
of
storage
(G’)
and
loss
(G”)
moduli
during
and
after
irradiation
for
two
of
the
formulations
investigated
in
this
study _____________________________________________125
Figure
2.7-3.
Time
dependency
of
the
swelling
ratio
for
all
gels
used
in
this
study._________126
6
Contents
Figure
2.7-4.Swelling
behavior
of
type
I-III
gels
at
4°C
upon
24
hours
incubation
in
deionized
water
containing
5%NaN .______________________________________________________________________127
3
Figure
2.7-5.
Swelling
degree
of
hydrogels
containing
5%wt.
PEGDA,
and
0,
0.5,
1,
or
2%
wt.
HA
(Type
I
gels),
incubated
for
seven
days
at
37°C,
in
PBS
buffer
containing
0.05%NaN3.__128
Figure
2.7-6.
Swelling
degree
of
hydrogels
containing
5%wt.
PEGDA,
and
0,
0.5,
1,
or
2%
wt.
HA-SH
(Type
II
gels),
incubated
for
seven
days
at
37°C,
in
PBS
buffer
containing
0.05%NaN3.
____________________________________________________________________________________________________128
Figure
2.7-7.
Swelling
degree
of
hydrogels
containing
5%wt.
PEGDA,
and
(pre-incubated)
0,
0.5,
1,
or
2
wt.%
HA-SH
(Type
III
gels),
incubated
for
seven
days
at
37°C,
in
PBS
buffer
containing
0.05%NaN .__________________________________________________________________________129
3
Figure
2.7-8.
The
metabolic
activity
per
cell
showed
a
clear
inverse
relationship
with
cell
spreading,
which
can
be
expressed
through
the
mean
cell
circularity
(inversely
related
to
spreading,
left)
or
the
mean
cell
area
(directly
related
to
spreading,
right)._________________129
Figure
3.1-1.
Surface
distribution
of
integrin
αv
subunit
(red)
and
CD44
(blue)
on
J744.2
murine
macrophages
imaged
through
the
use
of
polyclonal
rabbit
antibodies.
CD44
is
rather
uniformly
present
on
the
membrane
with
no
apparent
clustering.
On
the
contrary,
integrin
αv
subunits
appear
more
aggregated
and
less
evenly
distributed. _______________________________149
Figure
3.3.1-1.Above:
sketch
of
the
chemical
reactions
with
proton
numbering
for
the
assignment
of
1H-NMR
peaks.
A:
amidation
with
2-aminopropyl
methacrylamide;
B:
Michael-
type
addition
of
the
cysteine-containing
peptide
and
MeOGCGRGDSNH
onto
the
2
methacrylamide
double
bond.
Below:
1H-NMR
(left)
and
IR
(right)
spectra
for
64
kDa
HA.
The
presence
of
the
double
bond
after
reaction
A
is
confirmed
by
the
presence
of
the
typical
vinyl
resonances
above
5
ppm;
the
shift
to
lower
delta
values
of
the
protons
of
the
methacrylamide
groups
(H13-H15)
confirms
the
occurrence
of
the
Michael-type
addition.
The
peptide
signals
overlap
with
those
of
HA
with
the
exception
of
the
cluster
of
signals
immediately
below
3
ppm,
which
can
be
attributed
to
the
resonances
of
the
side
chain
methylenes
of
cysteine
and
aspartic
acid.
In
the
IR
spectrum
the
larger
changes
appear
in
the
carbonyl
region
and
affect
the
relative
ratio
between
amides
and
caboxylates,
which
increases
after
both
reaction
A
and
B:
this
determes
the
relative
decrease
of
the
carboxylate-associated
peaks
(asymmetric
stretching
band
at
1604
cm-1
and
symmetric
stretching
band
at
1404
cm-1),
and
the
relative
increase
of
the
amide
ones
(e.g.
the
amide
I
at
1636
cm-1);
peaks
above
1700
cm-1
are
recorded
after
the
addition
of
the
peptide
and
are
associated
to
the
guanidine
group
and/or
the
presence
of
protonated
carboxylic
acids._______________________________________________________160
Figure
3.3.2-1.
Uptake
kinetics
and
intracellular
localization
of
HA-FA
(MW:
64,
234,
1100
kDa,
0.2
mg/mL
in
full
DMEM
medium)
in
J.744.2
macrophages
followed
through
widefield
fluorescence
microscopy.
After
the
completion
of
incubation,
the
cells
were
extensively
washed
with
PBS
and
fixed.
The
samples
incubated
for
24
hours
were
also
co-stained
with
LysoTracker
Red.
In
the
pictures,
a
white
line
highlights
the
cell
perimeter.________________________________162
Figure
3.3.2-2.
Quantification
of
the
uptake
of
HA
derivatives
in
J774.2
macrophages
using
the
fluorescence
of
cell
lysates.
HA-FA
(Panel
A)
and
HA-RGD-FA
(Panel
B)
were
incubated
at
a
concentration
of
0.2
mg/mL
for
10
min,
1,
4
and
24hours
in
full
medium.
At
the
completion
of
the
incubation,
the
cells
were
carefully
washed
with
PBS
and
lysed
with
0.5%TritonX-100
in
PBS
before
reading.
The
readings
were
normalized
in
respect
to
the
amount
of
total
protein,
assessed
with
the
BCA
assay.
Panel
C
presents
the
ratio
of
the
uptake
data
shown
in
Panel
B
and
A:
with
the
exception
of
two
data
points
with
larger
errors,
the
ratio
is
rather
constantly
in
the
range
1.5-2.
(N=3) ________________________________________________________________________163
Figure
3.3.2-3.
Top.
Western
blotting
analysis
of
CD44
and
integrin
αv
in
comparison
to
β-
tubulin
for
J744.2
murine
macrophages
whole
cell
lysates.
The
cells
were
incubated
with
HA
and
HA-RGD,
respectively
for
the
analysis
of
CD44
and
integrin,
for
10
minutes,
1
hour
or
4
hours
in
full
medium.
The
4+20
hours
band
corresponds
to
lysates
of
cells,
which
were
incubated
with
HA
derivatives
for
4hours,
washed
and
incubated
further
for
20
hours
in
standard
conditions
for
cell
culture.
Bottom.
The
semi-quantitative
analysis
of
the
density
of
the
Western
blotting
bands
presented
in
the
top
part
was
performed
using
The
density
of
the
bands
was
assessed
using
the
Image
j
gels
analyzer
tool,
normalizing
the
integrals
of
the
peaks
in
respect
to
the
β-tubulin
band
density
and
expressed
as
a
percentage
of
the
control
sample.
____________________________________________________________________________________________________167
Figure
3.3.3-1.
Relative
inhibition
of
the
uptake
of
HA-FA
(left)
and
HA-RGD-FA
(right)
as
a
function
of
the
selective
inhibition
of
the
two
receptors.
J.744.2
murine
macrophages
were
incubated
with
anti-CD44
and/or
cyclic
RGD
blocking
agents
for
30
minutes
and
then
7
Contents
incubated
for
1
hour
incubation
in
full
medium
containing
HA-FA
or
HA-RGD-FA.
The
readings
refer
to
the
fluorescence
of
cell
lysates
normalized
with
regard
to
the
amount
of
total
protein
and
compared
with
the
values
of
the
control.
(N=4)___________________________________________168
Figure 3.3.3-2. Relative uptake of HA-FA (left) and HA-RGD-FA (right) as a function of
the pharmacological inhibition of endocytic mechanisms. J744.2 murine macrophages
were pre-treated with Cytochalasin D, Nocodazole, Ethylisopropylamiloride (EIPA),
Chlorpromazine, Bafilomycin and Filipin (see experimental section for pre-treatment
conditions). ______________________________________________________________________________________170
Figure
3.3.3-3.
Immunostaining
of
CD44
(blue)
as
a
function
of
incubation
time
and
of
the
presence
of
Bafilomycin
for
J744.2
murine
macrophages
incubated
in
standard
conditions
for
cell
culture
in
full
medium
containing
234
kDa
HA
(green).
In
the
control
samples
the
CD44-
associated
fluorescence
promptly
disappears,
indicating
the
quick
internalization
of
the
CD44/HA
complex,
and
re-appears
only
after
additional
20
hours
incubation
without
HA;
the
rather
weak
surface
HA
fluorescence
shows
no
significant
co-localization
with
that
of
CD4
after
2hours.
In
Bafilomycin-treated
samples,
CD44
appears
to
remain
on
the
surface
with
substantial
co-localization
with
HA.____________________________________________________________171
Figure
3.3.3-4.
The
co-localization
of
CD44
immunostaining
and
HA/HA-RGD
(234
kDa)
fluorescence
was
colour
coded
in
a
scale
from
black
(fluorescence
of
CD44
stain
alone)
to
red
(max
co-localization
of
the
CD44
stain
and
HA/HA-RGD
fluorescence).______________________173
Figure
3.3.3-5.
Immunostaining
of
macrophages
after
treatment
with
Bafilomycin
and
3
hours
incubation
with
HA-RGD.
Green:
HA-RGD;
Blue:
CD44;
Red:
integrin.
Green
and
red
dots
appear
co-localized
only
in
a
very
limited
number
of
points.__________________________________174
Figure
3.3.3-6.
Co-localization
for
the
stain
of
CD44
and
HA-RGD
fluorescence
(top
left,
colour
coded
black
(only
CD44)
to
red),
for
the
stains
of
CD44
and
integrins
(top
right,
colour
coded
black
(only
CD44)
to
green),
for
the
stain
of
integrins
and
HA-RGD
fluorescence
(bottom
left,
colour
coded
black
(only
integrin)
to
purple)
and
for
three
signals
together.
The
latter
panel
is
colour
coded
to
show
the
predominance
of
CD44/HA-RGD
co-localization
in
a
yellow
to
red
scale,
and
that
of
integrin/HA-RGD
co-localization
in
a
blue
scale.
When
similar
levels
of
co-
localization
are
obtained
for
the
two
couples,
the
resulting
colour
is
green._________________175
Figure
3.7-1.
Mechanism
of
the
Ugi
reaction,
which
was
employed
for
the
functionalization
of
HA
with
fluoresceinamine._______________________________________________________________________185
Figure
3.7-2.
Evaluation
of
the
immunostaining
fluorescence
associated
to
CD44
as
a
function
of
time
for
the
pictures
shown
in
Figure
3.7-3._________________________________________________186
Figure
3.7-3.Immunostaining
of
CD44
(blue)
as
a
function
of
incubation
time
and
of
the
presence
of
Bafilomycin
for
J744.2
murine
macrophages
incubated
in
standard
conditions
for
cell
culture
in
full
medium
containing
HA-RGD
(green)._______________________________________187
Figure
3.7-4.Staining
of
HA
and
HA-RGD
(green)
(after
3
hours
incubation)
and
integrin
αv
of
murine
macrophages
J744.2.____________________________________________________________________187
Figure
4.3.1-1.
HA
and
CD44
in
cross-sections
of
sciatic
nerves
of
adult
male
SD
rats
by
fluorescence
histocytochemistry.
Top
(merge):
overlaid
fluorescence
pictures
providing
localization
of
cell
nuclei
(DAPI,
blue),
of
HA
(HABP,
green)
and
of
CD44
(mouse
monoclonal
anti-CD44,
red).
The
two
insets
are
magnified
in
pictures
A
and
B.
Bottom:
same
pictures
as
above,
with
visually
enhanced
co-localization
of
CD44
and
HA.
The
pictures
were
obtained
overlaying
CD44
and
HABP
fluorescence,
colour-coding
them
to
show
in
blue
=
prevalence
of
CD44
fluorescence,
in
light
purple
=
prevalence
of
HABP
fluorescence,
dark
purple
=
similar
intensities
for
both._______________________________________________________________________________204
Figure
4.3.1-2.
Distribution
of
HA
and
RHAMM
in
cross-sections
of
sciatic
nerve
from
adult
male
SD
rats
by
fluorescence
histochemistry.
Top
(merge):
overlaid
fluorescence
pictures
providing
localization
of
cell
nuclei
(DAPI,
blue),
of
HA
(HABP,
green)
and
of
RHAMM
(mouse
monoclonal
anti-CD44,
red).
The
two
insets
are
magnified
in
the
pictures
A
and
B.
Bottom:
same
pictures
as
above,
with
visually
enhanced
co-localization
of
RHAMM
and
HA.
The
pictures
were
obtained
overlaying
RHAMM
and
HABP
fluorescence,
colour-coding
them
to
show
in
blue
=
prevalence
of
RHAMM
fluorescence,
in
light
purple
=
prevalence
of
HABP
fluorescence,
dark
purple
=
similar
intensities
for
both._______________________________________205
Figure
4.3.2-1.
Immunostaining
of
CD44
and
RHAMM
in
non-permeabilised
nSC
(primary
neonatal
Schwann
cells),
aSC
(primary
adult
Schwann
cells),
uADSC
(undifferentiated
adipose
stem
cells),
dADSC
(differentiated
adipose
stem
cell
into
a
Schwann
cell
phenotype),(x
60).Blue:
CD44,
red:
RHAMM. ___________________________________________________________________206
8
Contents
Figure
4.3.2-2.
Left:
RT
(reverse
transcriptase)-PCR
of
CD44,
RHAMM
and
actin
in
dADSC
(differentiated
adipose
stem
cell
into
a
Schwann
cell
phenotype),
uADSC
(undifferentiated
adipose
stem
cells),
aSC
(primary
adult
Schwann
cells),
nSC
(primary
neonatal
Schwann
cells).
Right:
Western
blotting
of
CD44
and
RHAMM
for
the
same
cells______________________________207
Figure
4.3.2-3.
Western
blotting
of
RHAMM
on
subcellular
fractions
of
aSC.
CL:
cell
lysate,
CYTO:
cytoplasmic
fraction
MEM:
membrane
fractions
(plasmic
membrane,
reticulum
endoplasmic
and
golgi),
CK:
cytoskeleton
fraction,
SNE:
soluble
nuclear
extract,
CBE:
chromatin-bound
extract.
See
Figure
4.7-4
for
controls._______________________________________208
Figure
4.3.3-1.
Relative
wound
closure
rates
for
aSCs,
nSCs,
uADSCs
and
dADSCs;
the
actual
would
closure
rates
(see
those
for
neonatal
and
adult
SCs
as
examples
in
Supplementary
Information,
Figure
4.7-5)
were
obtained
as
the
%
decrease
of
the
wound
area
per
time
unit
through
the
use
of
the
Tscratch
software,
developed
in
the
Laboratory
of
Computational
Science
and
Engineering,
ETH
Zurich,
and
available
through
http://chaton.inf.ethz.ch/software/,
using
images
recorded
every
10
minutes
through
24
hours.
The
relative
wound
closing
rates
were
obtained
my
normalizing
the
actual
rates
by
dividing
them
by
the
closing
rate
of
the
control.(N=3)_________________________________________211
Figure
4.3.3-2.
Immunostaining
data
of
SCs,
uADSCs
and
dADSCs
located
at
the
edge
of
the
scratch,
1
hour
and
24
hrs
after
the
scratch
(x60).
The
cells
were
pre-treated
with
anti-
RHAMM
(2nd
column,
red)
or
anti-CD44
(3rd
column,
red),
co-staining
tubulin
(blue). ______213
Figure
4.7-1.
Distribution
of
HA,
CD44
and
RHAMM
in
cross-sections
of
sciatic
nerve
from
adult
male
SD
rats
by
fluorescence
histocytochemistry.
Left:
DAPI
(blue):
localization
of
cell
nuclei.
HABP
(green):
localization
of
HA
(fluorescein-labeled
streptavidin
bound
to
biotinlylated
hyaluronic
acid
binding
protein,
HABP).
CD44
(red):
localization
of
CD44
(Alexa
Fluor
568-labelleld
goat
secondary
anti-mouse
antibody
bound
to
mouse
monoclonal
anti-
CD44).
Right:
as
for
the
left
column,
replacing
CD44
with
RHAMM. __________________________221
Figure
4.7-2.
Distribution
of
HA
in
cross-sections
of
sciatic
nerve
from
adult
male
SD
rats
by
fluorescence
histocytochemistry.
DAPI
(blue):
localization
of
cell
nuclei.
HABP
(green):
localization
of
HA
(fluorescein-labeled
streptavidin
bound
to
biotinlylated
hyaluronic
acid
binding
protein,
HABP).__________________________________________________________________________222
Figure
4.7-3.
Glial
markers
expression.
After
2
weeks
of
culture
in
a
differentiation
medium
containing
glial
growth
factors,
dADSC
were
found
to
express
GFAP,
p75
and
S100.
Green:
GFAP,
p75NTR,
and
S100.
Blue:
DAPI.
Courtesy
of
Alessandro
Faroni._____________________222
Figure
4.7-4.
Controls
for
the
intracellular
fractionation
experiments.
MEM:
membrane
fractions
(plasmic
membrane,
reticulum
endoplasmic
and
golgi),
CK:
cytoskeleton
fraction,
SNE:
soluble
nuclear
extract,
CBE:
chromatin-bound
extract._________________________________223
Figure
4.7-5.
Wound
closure
rate
for
neonatal
(left)
and
adult
Schwann
cells
as
a
function
of
the
presence
of
HA
or
of
antibodies
for
RHAMM
or
CD44.
(N=3)______________________________223
Figure
4.8-1.
(Left)
uADSC,
(center)
dADSC,
right
(aSC),
cultured
on
TCPS
(pre-treated
with
PDL
for
aSC
only)
in
standard
conditions
for
cell
culture._____________________________________225
Figure
4.8-2.
MTS
assay
data
of
SC,
when
cultured
on
top
of
HA-based
hydrogels
containing
5%
wt.%
PEG-DA
and
containing
or
not
0.5%,
1%,
2%
wt.%
SH-HA
[MW:
64,
234
kDa],
after
3,
6,
9
days
after
cell
seeding
(5000
cells/well
of
96well/plate)
of
incubation
in
standard
conditions
for
cell
culture._______________________________________________________________________226
Figure
4.8-3.
Brightfield
images
of
uADSC
when
cultured
on
top
of
HA-based
hydrogels
containing
5%
wt.%
PEG-DA
and
containing
or
not
0.5%,
1%,
2%
wt.%
SH-HA
[MW:
64,
234
kDa],
after
3,
6,
9
days
after
cell
seeding
(5000
cells/well
of
96well/plate)
of
incubation
in
standard
conditions
for
cell
culture.
The
contrast
of
the
pictures
was
equalized
using
a
Coreldraw
plugin
in
order
to
enhance
visibility
of
cell
culture.________________________________228
Figure
4.8-4.
(a)
MTS
data
of
uADSC
when
cultured
on
top
of
HA-based
hydrogels
containing
5%
wt.%
PEG-DA
and
containing
or
not
0.5%,
1%,
2%
wt.%
SH-HA
[MW:
64,
234
kDa],
after
3,
6,
9
days
after
cell
seeding
(5000
cells/well
of
96well/plate)
of
incubation
in
standard
conditions
for
cell
culture.
(b)
The
graph
shows
the
proliferation
rate
(between
day
3
and
day
6)
of
dADSC
cultured
on
top
of
hydrogels
in
function
of
the
elastic
modulus
of
substrate.
(c)
This
graph
shows
the
proliferation
rate
of
dADSC
(between
day
3
and
day
6)
based
on
MTS
data
(normalized
with
MTS
data
on
cells
seeded
on
TCPS),
(d)
this
graph
shows
the
proliferation
rate
of
dADSC
(between
day
6
and
day
9)
based
on
MTS
data _________________229
Figure
4.8-5.
Brightfield
images
of
dADSC
when
cultured
on
top
of
HA-based
hydrogels
containing
5%
wt.%
PEG-DA
and
containing
or
not
0.5%,
1%,
2%
wt.%
SH-HA
[MW:
64,
234
kDa],
after
3,
6,
9
days
after
cell
seeding
(5000
cells/well
of
96well/plate)
of
incubation
in
9
Contents
standard
conditions
for
cell
culture.
The
contrast
of
the
pictures
was
equalized
using
a
Coreldraw
plugin
in
order
to
enhance
visibility
of
cell
culture.________________________________231
Figure
4.8-6.
(a)
MTS
data
of
dADSC
when
cultured
on
top
of
HA-based
hydrogels
containing
5%
wt.%
PEG-DA
and
containing
or
not
0.5%,
1%,
2%
wt.%
SH-HA
[MW:
64,
234
kDa],
after
3,
6,
9
days
after
cell
seeding
(5000
cells/well
of
96
well-plate)
of
incubation
in
standard
conditions
for
cell
culture.
(b)
The
graph
shows
the
proliferation
rate
(between
day
3
and
day
6)
of
dADSC
cultured
on
top
of
hydrogels
in
function
of
the
elastic
modulus
of
substrate.
(c)
This
graph
shows
the
proliferation
rate
of
dADSC
(between
day
3
and
day
6)
based
on
MTS
data
(normalized
with
MTS
data
on
cells
seeded
on
TCPS),
(d)
this
graph
shows
the
proliferation
rate
of
dADSC
(between
day
6
and
day
9)
based
on
MTS
data _________________232
10
Description:cell numbers were obtained from manual count. Error bars were .. equalized using a. Coreldraw plugin in order to enhance visibility of cell culture.
