Efficiency of controlled topical delivery of silver sulfadiazine in infected burn wounds


Shanmugasundaram, N.; Uma, T.S.; Ramyaa Lakshmi, T.S.; Babu, M.

Journal of Biomedical Materials Research. Part A 89(2): 472-482

2008


The present study is designed to assess the potential benefits of controlled delivery of silver sulfadiazine from collagen scaffold (SSDM-CS) in infected deep partial thickness burn wounds in which epidermis is lost completely and the entire papillary dermis and most of the recticular layer of the dermis is lost. Infection induced by inoculating 10(7) colony forming units (cfu) of Pseudomonas aeruginosa caused significant increase in wound size (20%) till day 15, which decreased significantly from day 9 by SSDM-CS treatment, showing complete healing by day 27 (control > or = 37 days). Early subsidence of infection (<10(2) cfu, day 9) by SSDM-CS resulted in faster epidermal resurfacing and fibroplasia, whereas heavy microbial load (>10(7) cfu, day 9) in controls caused severe inflammatory cellular infiltration. Persistent infection triggered early expression of proinflammatory cytokines intereukin-6, intereukin 1-beta, and tumor necrosis factor-alpha, lasting until day 9, whereas cytokine level decreased in SSDM-CS-treated group by day 6. Infection exacerbated expression of active matrix metalloproteinases (MMPs)-2 and -9 in controls (day 15), while SSDM-CS positively modulated MMP-2 and -9 with faster decline in their levels (day 12). Inherent nature of the dressing to maintain drug level at equilibrium therapeutic concentration (51.2 microg/mL) for prolonged time (72 h), below systemic toxic limits (20 microg/dL, serum level), accelerated the magnitude and sequence of reparative events.

Efficiency
of
controlled
topical
delivery
of
silver
sulfadiazine
in
infected
burn
wounds
N.
Shanmugasundaram,
T.
S.
Uma,
T.
S.
Ramyaa
Lakshmi,
Mary
Babu
Biomaterials
Division,
Central
Leather
Research
Institute
-
TICEL
Biopark,
Tharamani
Road,
Tharamani,
Chennai
600
113,
Tamil
Nadu,
India
Received
25
April
2007;
revised
6
December
2007;
accepted
30
January
2008
Published
online
22
April
2008
in
Wiley
InterScience
(www.interscience.wiley.com
).
DOI:
10.1002/jbm.a.31997
Abstract:
The
present
study
is
designed
to
assess
the
potential
benefits
of
controlled
delivery
of
silver
sulfadiaz-
ine
from
collagen
scaffold
(SSDM-CS)
in
infected
deep
partial
thickness
burn
wounds
in
which
epidermis
is
lost
completely
and
the
entire
papillary
dermis
and
most
of
the
recticular
layer
of
the
dermis
is
lost.
Infection
induced
by
inoculating
10
7
colony
forming
units
(cfu)
of
Pseudomo-
nas
aeruginosa
caused
significant
increase
in
wound
size
(20%)
till
day
15,
which
decreased
significantly
from
day
9
by
SSDM-CS
treatment,
showing
complete
healing
by
day,
27
(control
>
37
days).
Early
subsidence
of
infection
(<10'
cfu,
day
9)
by
SSDM-CS
resulted
in
faster
epidermal
resur-
facing
and
fibroplasia,
whereas
heavy
microbial
load
(>10
7
cfu,
day
9)
in
controls
caused
severe
inflammatory
cellular
infiltration.
Persistent
infection
triggered
early
expression
of
proinflammatory
cytokines
intereukin-6,
intereukin
and
tumor
necrosis
factor-a,
lasting
until
day
9,
whereas
cytokine
level
decreased
in
SSDM-CS-treated
group
by
day
6.
Infection
exacerbated
expression
of
active
matrix
metal-
loproteinases
(MMPs)-2
and
-9
in
controls
(day
15),
while
SSDM-CS
positively
modulated
MMP-2
and
-9
with
faster
decline
in
their
levels
(day
12).
Inherent
nature
of
the
dressing
to
maintain
drug
level
at
equilibrium
therapeutic
concentration
(51.2
µg/mL)
for
prolonged
time
(72
h),
below
systemic
toxic
limits
(20
µg/dL,
serum
level),
ac-
celerated
the
magnitude
and
sequence
of
reparative
events.
©
2008
Wiley
Periodicals,
Inc.
J
Biomed
Mater
Res
89A:
472-482,
2009
Key
words:
silver
sulfadiazine;
collagen
scaffold;
Pseudo-
monas
aeruginosa;
proinflammatory
cytokine;
matrix
metal-
loproteinases
INTRODUCTION
Wound
healing
is
a
dynamic,
interactive
process
involving
soluble
mediators,
blood
cells,
extracellular
matrix,
and
parenchymal
cells.
1
These
phases
are
altered
from
their
normal
sequence
in
the
case
of
infection.
Pathogenic
microbes,
especially
Pseudomo-
nas
aeruginosa,
induce
pathogenicity
because
of
extracellular
virulence
factors,
which
exacerbate
the
immunocompromised
state
induced
by
burn
injury.
2
Virulence
factors
trigger
serious
weight
loss,
delay
reepithelialization,
causes
early
dehiscence,
alters
collagen
content,
impede
epidermal
migration,
and
also
spread
systemically
leading
to
sepsis.
3-6
Further,
they
induce
the
production
of
proinflammatory
cyto-
kines,
viz,
intereukin-113
(IL-1(),
tumor
necrosis
fac-
tor-a
(TNF-a),
and
IL-6.
7
These
cytokines
play
a
major
role
in
regulation
of
immune
response,
hema-
topoiesis,
and
inflammatory
reactions.
8
Among
these
Correspondence
to:
M.
Babu;
e-mail:
marybabu@hotmail.
com
or
babumary2000@yahoo.com
©
2008
Wiley
Periodicals,
Inc.
cytokines
IL-6
has
short
peak
time
in
normal
healing
process,
whereas
IL-113
and
TNF-
a
are
shown
to
persist
for
longer
time
9-11
Another
major
detrimental
factor
to
healing
process
is
the
altered
expression
pattern
of
metalloproteinases
(MMPs),
which
impedes
epidermal
migration,
and
degrades
all
components
of
extra
cellular
matrix
(ECM)
with
broad
and
overlapping
specifidties.
12-14
Despite
the
systemic
antibiotic
therapy,
prevention
of
infection
at
the
wound
site
greatly
influences
the
inflammatory
and
remodeling
events.
Topical
treat-
ment
offers
the
advantage
of
immediate
effect
by
lowering
systemic
levels
and
increasing
local
tissue
bioavailability.
Of
all
the
antibacterials
available
for
topical
application,
silver
sulfadiazine
(SSD)
is
the
drug
widely
used.
SSD
is
an
oligodynamic
anti-
microbial,
in
which
Ag
+
component,
even
in
small
amounts,
exerts
an
antimicrobial
effect
in
deep
burn
injury.
15
Later,
it
was
found
that
silver
gets
absorbed
systemically
posing
problems
on
prolonged
use.
Controlled
delivery
system
developed
by
us
main-
tains
therapeutic
level
of
SSD,
which
remains
below
toxic
limits.
In
this
system,
reconstituted
collagen
scaffold
impregnated
with
SSD-loaded
alginate
CONTROLLED
SSD
DELIVERY
IN
BURN
473
microspheres
was
developed
to
deliver
the
drug
in
a
controlled
manner.
The
antibacterial
efficiency
against
P.
aeruginosa
showed
minimal
bactericidal
concentration
level
of
51.2
µg/mL
and
the
system
was
able
to
control
infection
for
extended
time
pe-
riod
with
lesser
dressing
frequencies
and
easier
assessment
of
wound.
16
The
present
in
vivo
experiment
is
aimed
at
investi-
gating
the
efficiency
of
silver
sulfadiazine
from
colla-
gen
scaffold
(SSDM-CS)
on
deep
second
degree
burn
wounds
challenged
with
P.
aeruginosa.
Immunohisto-
chemical
localization
of
proinflammatory
cytokines
and
quantitative
assessment
of
collagen
turn
over,
tissue
level
expression
of
MMP-1, MMP-2,
and
MMP-9
were
performed.
The
influence
of
early
infection
on
various
cascading
phases
during
healing
process
has
been
analyzed
with
special
reference
to
efficient
granulation
tissue
formation
and
effective
remodeling.
MATERIALS
AND
METHODS
Materials
Hydroxyproline,
Glucosamine
HC1,
Para-dimethyl
amino
benzaldehyde,
acetyl
acetone,
chloramine-T,
bicinchoninic
acid,
bovine
serum
albumin,
MMP-2
(EC
3.4.24.24),
and
MMP-9
(EC
3.4.24.35)
from
human
firbroblasts,
alkaline
phosphatase
(AP)
chromogen-Nitro
Blue
Tetrazolium
(NBT),
5-bromo,4-chloro,3-indolylphosphate
(BOP),
and
fast
red
substrate
were
from
SIGMA,
USA.
Primary
anti-
bodies
used
were
mouse
monoclonal
anti-NIMP-1,
goat
polyclonal
anti-MMP-2
and
MMP-9,
polyclonal
rabbit
anti-
IL-10,
IL-6,
and
TNF-a.
Monoclonal
rabbit
anti-mouse
IgG-
AP,
and
polyclonal
donkey
anti-goat
IgG-AP
secondary
antibodies
were
used
to
probe
NIMP-1
and
MMP-2
and
-9,
respectively.
For
immunohistochemical
localization
AP-
tagged
monoclonal
goat
anti-rabbit
IgG
was
used.
All
anti-
bodies
used
were
from
Santa
Cruz
Biotechnology,
CA.
Mueller
Hinton
Broth
and
Mueller
Hinton
Agar
were
obtained
from
HE-MEDIA,
Mumbai,
India
and
P.
aerugi-
nosa
(ATCC
25619)
culture
was
procured
from
IMTECH,
Chandigarh,
India.
All
other
chemicals
used
for
experi-
mental
purpose
were
of
analytical
grade.
Methods
SSD
wound
dressing
A
reconstituted
collagen
scaffold
impregnated
with
SSD-loaded
alginate
microspheres
(SSDM-CS),
capable
of
delivering
the
drug
in
a
controlled
manner
has
been
devel-
oped
as
per
our
previous
protocol.
16
Briefly,
SSD-loaded
alginate
microspheres
were
prepared
by
modified
water-
in-oil
(w/o)
emulsion
technique
through
interfacial
ionic
gelation
of
alginate
using
CaC1
2
.
Microspheres
of
optimum
drug
entrapment
(3.26%)
and
required
size
range
(300-370
gm)
were
prepared.
SSD
release
from
the
scaffold
was
68.8%
(equilibrium
concentration
for
72
h)
with
an
initial
burst
release
of
47.5%
(^-,
-1.5%
of
total
drug
load),
whereby
therapeutic
level
of
SSD
could
be
attained
easily
and
fur-
ther
maintained
for
3
days
(the
percentage
entrapment
of
SSD
obtained
through
our
process
was
optimal
as
it
complies
with
USP
standard,
17
which
recommends
1.5%
of
SSD
for
topical
dosage
forms).
SSD-loaded
microspheres
exhibited
minimal
inhibitory
concentration
and
minimal
bactericidal
concentration
levels
of
44.8
and
51.2
gg/mL
against
P.
aeruginosa
(ATCC
25619).
SSD-loaded
microsphere
impregnated
collagen
scaffold
was
then
developed
by
adding
a
known
amount
(0.5
g)
of
SSD-loaded
microspheres
to
a
known
amount
(300
mg)
of
pepsin-solubilized
collagen
18
and
gently
stirred
to
distrib-
ute
the
spheres
homogeneously
throughout
the
solution.
Following
which
fibril
formation
was
initiated
by
adding
an
appropriate
amount
of
0.2M
phosphate
buffer
(2
mL)
and
adjusting
the
pH
to
6.9-7.2
using
2N
sodium
hydrox-
ide,
until
turbidity
appeared.
After
fibril
formation,
the
viscous
solution
was
uniformly
cast
over
horizontally
placed
polypropylene
platforms
of
10
x
10
x
2
cm
3
dimension.
They
were
allowed
to
dry
at
a
constant
tem-
perature
of
34°C
until
a
thin
scaffold
was
obtained.
The
scaffold
was
then
washed
with
distilled
water
twice
or
thrice
to
remove
excess
of
salt
and
completely
air-dried
and
stored
in
a
light
proof
desiccator
for
further
use.
Burn
wound
model
Female
white
wistar
rats,
weighing
180-220
g
were
pro-
cured
from
Tamil
Nadu
Veterinary
University,
Chennai,
India
(TANUVAS)
and
acclimatized
to
laboratory
condi-
tions
for
1
week.
They
were
fed
with
standard
rat
chow
and
tap
water
ad
libitum.
Institutional
animal
ethical
com-
mittee
approved
all
the
animal
experiment
protocols.
Ani-
mal
maintenance
and
care
were
according
to
the
Commit-
tee
for
the
Purpose
of
Control
and
Supervision
of
Experi-
ments
on
Animals
guidelines,
India.
Deep
second
degree
burn
wound
was
created
by
an
instrument
designed
by
us
using
commercially
available
solder
rod
(500
g)
(Fig.
1).
The
edge
of
the
solder
rod
was
cut
and
replaced
by
a
circular
iron
disc
of
thickness
1.25
cm
and
diameter
of
3
cm.
A
thermal
sensor
was
connected
to
the
non-contacting
surface
and
the
temperature
was
observed
through
a
digital
read-out.
The
desired
tempera-
ture
was
attained
by
electrically
heating
the
solder
rod
and
temperature
was
controlled
using
a
thermostat.
Before
cre-
ation
of
burn
wound,
rats
were anaesthetized
by
intraperi-
tonial
injection
of
thiopental
sodium
(Thiopental),
50
mg/
kg
body
weight.
Hair
in
the
dorsal
side
of
the
rats
were
removed
and
burn
wound
was
inflicted
by
placing
the
circular
iron
disc
(heated
to
82-85°C)
over
the
dorsal
side
for
20
s,
without
exerting
any
external
pressure.
Experimental
design
Rats
were
randomly
divided
and
assigned
equally
(n
=
30)
to
one
of
the
following
groups,
group
1—noninfected
controls
dressed
with
saline
gauze,
group
2—noninfected
rats
treated
with
SSDM-CS,
group
3—infected
controls
Journal
of
Biomedical
Materials
Research
Part
A
SHANMUGASUNDARAM
ET
AL.
474
TEMPERATURE
CONTROLLER
•WOODEN
HANDLE
•SOLDER
ROD
THERMAL
SENSOR
5
HEATING
ELEMENT
8-CIRCULAR
DISC
n
11.5
.am
Figure
1.
Schematic
diagram
of
modified
solder
unit
used
for
infliction
of
burns.
challenged
with
P.
aeruginosa
infection
and
dressed
with
saline
gauze,
and
group
4—infected
rats
treated
with
SSDM-CS.
The
dressing
was
changed
after
every
3
days,
from
day
1.
Bacteriological
methods
Pathogenic
strains
of
P.
aeruginosa
(ATCC
25619)
was
grown
on
Mueller
Hinton
Broth
at
35°C
until
logarithmic
growth
phase
(0.5
McFarland)
19
and
approximately
diluted
to
prepare
the
bacterial
challenge
inoculum
[10
7
colony
forming
units
(cfu)/mL].
For
inducing
infection,
1
mL
of
the
above
inoculum
was
centrifuged
and
resus-
pended
in
100
mL
of
sterile
saline
and
the
suspension
was
injected
carefully
between
the
subcutaneous
skin
and
para-
spinus
muscular
layer.
Rats
with
wounds
colonized
with
>10
6
cfu
(assessed
quantitatively
by
spread
plate
method
from
the
tissue
extract
prepared
from
biopsy
of
1
cm
2
with
1
mL
of
sterile
saline)
and
showing
positive infiltration
of
deep
dermal
neutrophils
after
12
h
of
bacterial
challenge
were
included
in
the
study.
The
severity
of
the
infection
in
groups
3
and
4
was
assessed
at
regular
time
intervals
and
represented
as
cfu/ml
(after
extracting
biopsy
tissue
with
1
ml
of
sterile
saline).
Rate
of
wound
closure
The
rate
of
wound
closure
was
determined
by
tracing
the
margin
of
wound
area
on
to
a
transparent
graph
sheet
and
expressed
as
percentage
surface
area
reduction
at
each
time
interval.
At
these
time
intervals
changes
in
body
weight
was
recorded,
granulation
tissue
was
collected
from
each
group,
and
serum
from
blood
samples
of
SSDM-CS-treated
groups
(2
and
4)
were
collected
and
sub-
jected
to
further
analysis.
Determination
of
collagen
and
hexosamine
content
A
known
amount
of
granulation
tissue
(5
mg)
was
col-
lected
at
regular
time
intervals,
freeze-dried
(Yamato,
Japan)
to
constant
weight,
and
subjected
to
hydrolysis
for
22
h
with
6N
HC1
to
determine
the
collagen
content.
21
Total
hexosamine
content
was
determined
by
the
method
of
Elson
and
Morgan
after
subjecting
the
samples
to
hydrolysis
for
6
h
using
2N
HC1.
Immunohistochemical
analysis
The
early
and
late
course
of
inflammatory
response
during
healing
was
observed
by
the
proinflammatory
cyto-
kines
IL-6,
and
TNF-ce
expression.
Immunohisto-
chemistry
was
performed
according
to
earlier
procedure.
9
The
sections
of
post-burn
days
(until
day
12)
were
incu-
bated
for
1-2
h
at
37°C,
individually
with
polyclonal
rabbit
primary
antibody
for
IL-10,
IL-6,
and
TNF-ce
(1:100
in
2%
bovine
serum
albumin)
followed
by
incubating
the
sections
with
AP-tagged
anti-rabbit
IgG
(1:200
dilutions)
for
45
min
to
1
h
at
37°C.
Then,
sections
were
stained
with
fast
red
substrate
(prepared
with
buffer
provided
in
the
kit
by
manufacturers),
after
washing
overnight
in
water
(in
dark).
Finally,
they
were
counterstained
with
Mayer's
Hematoxy-
lin
for
15
min
and
mounted
using
crystal
mountant.
Determination
of
NIMP
expression
pattern
Expression
of
active
MMPs
in
granulation
tissue
of
burn
wounds
of
all
the
groups
at
various
time
intervals
was
determined
by
gelatin
zymography,
whereas
both
active
and
inactive
forms
combined
were
determined
by
western
blot
analysis.
Gelatin
zymography
In
the
investigation,
the
expressions
of
MMP-2
and
-9
at
various
time
intervals
in
all
the
groups
were
analyzed.
Granulation
tissue
lysate
was
obtained
through
extraction
with
20
mM
HEPES
buffer,
pH
7.2,
at
4°C.
Before
zymo-
graphy,
the
protein
concentration
in
the
samples
were
determined
through
Bicinchoninic
acid
assay.
23
Samples
of
equal
protein
content
(20
lig)
were
mixed
with
nonreduc-
ing
Laemmil's
buffer
(0.125M
Tris,
pH
6.8,
SDS
4%,
glyc-
erol
20%,
and
0.02%
w/v
of
bromophenol
blue)
and
elec-
trophoresed
on
a
10%
polyacrylamide
gel
copolymerized
with
gelatin
(1
mg/mL).
Standard
MMP-2
and
-9
were
used
as
markers.
After
electrophoresis,
the
gel
was
washed
with
2.5%
of
Triton
X-100
for
1
h
and
30
min
and
then
incubated
with
enzyme
buffer
(50
mM
of
Tris-HC1,
150
mM
NaCl,
5
mM
CaC1
2
,
and
0.05%
sodium
azide)
at
37°C
for
20
h
to
allow
reactivation
of
MMP.
Gels
were
then
stained
with
0.5%
of
Coomassie
Brilliant
Blue
R-250
24
and
destained
with
10%v/v
of
acetic
acid
containing
30%v/v
of
methanol.
The
MMPs
were
visualized
as
clarified
bands
corresponding
to
zones
of
digestion
of
substrate
gelatin.
Journal
of
Biomedical
Materials
Research
Part
A
%
CLOSURE
100
SO
25
0
125)
(
75
)
OM)
1125)
1159X)
CONTROLLED
SSD
DELIVERY
IN
BURN
475
Western
blot
analysis
of
IVIMPs
Tissue
lysate
of
all
groups
collected
at
various
time
intervals,
containing
equal
amount
of
protein
(20
lig)
were
mixed
with
Laemmil's
reducing
buffer
and
subjected
to
electrophoresis
on
a
10%
polyacrylamide
resolving
gel.
After
resolving
the
protein
bands,
they
were
transferred
to
nitrocellulose
membrane
(Millipore,
USA)
using
BioRad
minigel
Transfer
apparatus,
for
1
h
and
30
min,
and
were
incubated
with
primary
antibody
for
MMP-1,
-2,
and
-9,
individually
at
4°C
for
16
h.
After
incubation,
blots
were
washed
with
PBS
containing
0.5%
Tween
20
and
then
incubated
with
respective
AP-conjugated
secondary
anti-
bodies
for
1
h
and
visualized
using
NBT/BCIP
(33:66
A
in
10
mL
of
AP
buffer)
as
a
chemiluminescence
substrate.
25
The
immunoblots
were
subjected
to
densitometric
analysis
(BioRad
densitometrix
Geldoc
XR
image
acquisition
equipped
with
quality
one
software
for
1D
electrophoretic
analysis)
to
quantitate
the
total
MMP
expression
(both
active
and
pro
forms
of
MMP).
The
images
were
photodo-
cumented
by
integrating
the
volume
of
each
band
after
background
corrections.
The
volume
intensities
were
ana-
lyzed
through
the
volume
analysis
tool,
where
the
volu-
mograms
were
calculated
in
terms
of
pixels
and
plotted
against
different
time
intervals.
Each
MMP
was
individu-
ally
subjected
to
analysis
twice
by
western
blot
and
the
volumograms
of
two
immunoblots
are
represented
as
mean
number
of
pixels
±
SD
and
statistically
analyzed
(mentioned
below).
Determination
of
silver
content
in
serum
Serum
samples
of
known
volume
(1
mL)
were
digested
with
5
mL
of
0.2N
nitric
acid
and
residue
was
made
up
to
a
standard
volume.
Silver
content
in
the
samples
was
esti-
mated
using
atomic
absorption
spectrophotometer
(Spectra
varian
Atomic
Absorption
Spectrophotometer,
USA)
in
flame
mode
using
an
auto
sampler.
Back
ground
correc-
tions
were
operated
at
lamp
current
of
4
mA,
slit
width
of
0.5
nm,
and
at
a
wavelength
of 328.1
nm.
Statistical
analysis
All
graphical
illustrations
in
this
study
are
represented
as
mean
±
SD
and
analyzed
with
one-way
analysis
of
var-
iance
using
the
Microsoft
Excel
statistical
analysis
tool
pack.
Wherein,
variance
within
each
group
was
compared
individually
and
also
against
two,
three,
and
all
groups
combined
together.
Test
for
significance
was
performed
with
confidence
limit
of
95%,
i.e.,
p
<
0.05
was
considered
statistically
significant.
RESULTS
The
present
investigation
deals
with
several
responses
in
magnitude
and
temporal
pattern
of
wound
repair
in
P.
aeruginosa
(ATCC
25619)
chal-
lenged
deep
second-degree
burn
model
in
rats.
It
was
observed
that
there
exists
a
strong
relationship
between
the
severity
of
early
infection
and
subse-
quent
healing
events.
The
wound
depth
was
assessed
by
staining
the
histological
sections
of
burn
tissue
with
Hematoxylin
and
Eosin
26
(data
not
shown).
Wound
closure
The
efficiency
of
SSDM-CS
against
microbial
chal-
lenge
was
assessed
by
observing
the
wound
healing
pattern
in
treated
rats,
in
comparison
with
their
re-
spective
controls.
There
was
an
observable
increase
in
wound
size
till
day
6
in
all
the
groups
(Fig.
2).
The
noninfected
control
group
(group
1),
showed
an
increase
in
wound
size
till
day
12,
after
which
a
con-
stant
decrease
was
observed
with
complete
healing
occurring
by
day
30
(99%).
On
the
other
hand
nonin-
fected
SSDM-CS-treated
group
(group-2)
exhibited
a
significant
decrease
in
wound
size
as
early
as
day
9
showing
complete
healing
by
day
21.
In
contrast,
infected
rats
(group
3)
showed
a
sig-
nificant
increase
in
wound
size
till
day
15,
followed
by
a
positive
healing,
which
was
slow
when
com-
pared
with
other
groups
and
complete
closure
was
observed
by
37
days.
Infected
rats
(group
4)
treated
with
SSDM-CS
showed
an
increase
in
wound
size
till
day
9,
after
which
it
exhibited
positive
healing
response
achieving
complete
healing
by
day
27.
It
is
observed
that
group
3
(infected
controls)
exhibited
significantly
larger
wound
size
against
other
groups
at
all
time
points
(group
3
vs.
groups
1,
2,
and
4;
p
<
0.05).
The
healing
pattern
in
groups
1
and
4
is
similar
from
day
12
until
day
18.
During
this
time
course
the
wound
size
was
statistically
insignificant
tts
:d...
'•
4
:
7
t
A
—0—
GROUP
1
—s—
GROUP
2
—I—
GROUP
3
—.—
GROUP
4
Figure
2.
Graph
showing
percentage
wound
closure
observed
over
27
days
in
both
infected
and
noninfected
groups
treated
with
SSDM-CS
in
comparison
with
their
respective
controls.
Y-axis
values
in
brackets
indicates
increase
in
percentage
wound
size.*p
<
0.05,
group
3
vs.
group
1,2,
and
4.
NS,
not
significant,
group
4
vs.
group
1.
g
In
1
A
4
A
Journal
of
Biomedical
Materials
Research
Part
A
476
SHANMUGASUNDARAM
ET
AL.
2!O
2LO
12
GI
IN
GAWP
1
Ealli0111
,
9
WIWI'
9
GROUP
Figure
3.
Comparison
of
changes
in
body
weight
observed
in
various
groups:
*p
<
0.05,
group
3
vs.
group
4.
(group
1
vs.
group
4,
p
=
0.237),
after
which
healing
was
observed
to
be
faster
in
group
4
with
significant
difference
until
day
27
(p
=
0.0162).
Similarly,
when
noninfected
groups
were
compared
(group
1
vs.
group
2)
the
healing
rate
matched
on
day
15,
which
was
statistically
insignificant,
after
which
group
2
showed
faster
healing.
Body
weight
assessment
The
average
body
weight
of
the
rats
(n
=
120)
was
184
g.
Comparison
within
noninfected
and
infected
groups
did
not
show
any
significant
(p
<
0.05)
weight
loss
over
a
period
of
21
days
(Fig.
3).
The
weight
of
the
noninfected
controls
remained
same
until
day
9,
after
which
an
increase
of
4%
from
ini-
tial
weight
was
observed.
In
contrast,
the
infected
control
group
(group
3)
showed
a
constant
decrease
(5%)
from
their
initial
weight
till
day
15,
after
which
only
a
marginal
increase
was
seen
but
they
did
not
attain
the
initial
weight
until
complete
healing.
In
SSDM-CS-treated
infected
rats
(group
4)
there
was
marginal
weight
loss
(1%)
initially
but
they
regained
their
initial
weight
and
showed
an
increase
of
3%
from
their
original
body
weight.
When
the
compari-
son
was
made
between
groups
1,
2,
and
4
there
was
variation
in
the
weight,
which
was
not
statistically
significant.
But
the
infected
control
showed
a
signifi-
cant
difference
against
the
noninfected
groups
[day
9
(p
=
0.0247)
and
day
12
(p
=
0.0401)]
(group
3
vs.
groups
1
and
2).
Influence
of
infection
on
healing
Groups
3
and
4
rats
infected
with
P.
aeruginosa
(10
7
cfu)
exhibited
differential
microbial
load
during
the
course
of
healing.
On
day
3,
group
3
showed
a
significant
increase
in
cfu
(from
2
x
10
7
cfu
to
10
9
cfu,
microbial
count
determined
in
the
slough
of
the
wound
since
granulation
was
not
obtained),
whereas
SSDM-CS-treated
rats
did
not
show
a
significant
increase
or
decrease
(from
2
x
10
7
to
4
x
10
7
cfu)
(Fig.
4).
However,
on
day
6,
group
4
showed
a
sig-
nificant
decrease
in
microbial
load
(from
4
x
10
7
to
2
x
10
4
cfu),
which
constantly
reduced
on
post-burn
days.
Group
4
showed
99.9%
decrease
by
day
9
(reduction
of
microbial
load
from
10
7
to
10
2
cfu),
but
group
3
did
not
exhibit
any
significant
decrease
till
day
15.
Almost
75%
of
group
3
showed
mild
to
severe
purulent
discharge,
indicating
the
onset
of
positive
infection.
The
severity
of
infection
was
obvious
in
group
3,
leading
to
mortality
of
3
ani-
mals,
indicating
that
local
infection
has
detrimental
effect
on
healing.
Collagen
and
hexosamine
content
Initial
collagen
expression
levels
are
significantly
different
between
groups
1,
2,
and
4
(p
=
0.00024,
on
day
3).
No
significant
difference
was
observed
when
collagen
turnover
was
compared
within
the
non-
infected
groups
(Fig.
5).
It
is
interesting
to
note
that
SSDM-CS-treated
infected
group
exhibited
a
signifi-
cant
difference
in
collagen
content
until
day
12
when
compared
with
infected
control
(group
4
vs.
group
3,
p
=
0.0116),
showing
peak
concentration
on
day
9.
This
is
much
faster
than
the
noninfected
controls
coinciding
with
the
rate
of
healing
observed
(97.12%).
Throughout
the
study,
intergroup
compari-
sons
exhibited
significant
differences
in
collagen
con-
tent.
The
noninfected
groups
(1
and
2)
were
statisti-
cally
insignificant
when
compared
with
infected
group
4
alone
(groups
1
and
2
vs.
group
4,
p-value
L8.10
1.8+66
1-8+68
LEW/
18+66
11+65
LE1414
111+43
L8+62
1_1141
1_8+66
MOO
2
CROUPS
Figure
4.
Reduction
of
P.
aeruginosa
cfu
observed
in
SSDM-CS
rats
(group
4)
in
comparison
with
infected
con-
trols
(group
3).
*p
<
0.05,
group
3
vs.
group
4.
II
EE
Journal
of
Biomedical
Materials
Research
Part
A
CONTROLLED
SSD
DELIVERY
IN
BURN
477
r
C
O
GROUP
1
GROUP
2.
CROUP
3
GEOU'i
Figure
5.
Collagen
content
determined
in
granulation
tis-
sue
collected
from
infected
and
noninfected
group
rats
treated
with
SSDM-CS
in
comparison
with
their
respective
controls
at
various
time
intervals.
*p
<
0.05.
NS,
not
signif-
icant,
group
4
vs.
groups
1
and
2.
was
within
level
of
confidence,
95%).
The
groups
are
ranked
with
respect
to
their
matrix
deposition,
based
on
collagen
turn
over
as
follows,
group
2
>
group
4
>
group
1
>
group
3.
Proteoglycans
deposition
mainly
hyaluronic
acid,
heparan
sulfate,
chondroitin
sulfate,
and
dermatan
sulfate
in
granulation
tissue
facilitates
an
environment
for
cell
movement
and
collagen
deposition.
Hexosamine
(markers
of
proteo-
glycan)
expression
is
significant
in
all
groups
(Fig.
6)
till
day
12
(p
<
0.05),
after
which
there
was
no
sig-
nificant
difference
between
the
groups
(p
<
0.05
and
<0.01).
Peak
concentrations
were
2-fold
lower
in
infected
groups
(2.83
gg/mg
on
day
12
for
group
3
and
2.206
gg/mg
on
day
9
for
group
4)
in
compari-
son
with
the
noninfected
groups
(4.15
gg/mg
on
day
9
for
group
1
and
4.45
gg/mg
on
day
6
for
group
2)
and
there
was
only
a
marginal
increase
within
the
groups,
indicating
less
deposition
of
matrix
in
infected
groups.
Influx
of
proinflammatory
cytokines
Proinflammatory
cytokines,
IL-6,
IL1-13
and
TNF-a
influence
the
healing
process
by
modulating
the
level
of
IVIMPs.
A
varied
expression
pattern
of
these
cytokines
was
observed
between
treated
and
untreated
groups,
as
well
as
with
respect
to
infection
status.
The
control
groups
exhibited
increased
expression
of
IL1-13
when
compared
with
their
re-
spective
treated
groups
[Fig.
7(A)].
Especially
in
group
3,
persistent
expression
was
seen
until
day
9,
while
group
4
treated
with
SSDM-CS
showed
lesser
expression,
which
subsided
by
day
6.
Another
proin-
flammatory
cytokine
IL-6,
which
generally
shows
a
steep
increase
in
its
level
during
initial
inflammatory
phase
and
quickly
declining
within
short
time,
was
persistent
and
widespread
over
dermal
regions
until
day
6
in
group
3
[Fig.
7(B)].
Whereas
in
group
4
and
group
1
expression
of
IL-6
was
marginal
and
sub-
sided
by
day
3,
indicating
its
transient
expression.
TNF-a
a
key
mediator
of
late
inflammatory
phase
is
generally
expressed
in
cases
of
severe
infection.
Group
3
showed
predominant
expression
of
TNF-a
till
day
9,
whereas
in
other
groups
its
expression
subsided
within
6
days
[Fig.
7(C)].
Especially,
SSDM-CS
treated
groups
(2
and
4)
exhibited
low
level
of
expression
in
comparison
with
their
respec-
tive
controls,
indicating
the
ability
of
SSDM-CS
to
mitigate
infection.
Differential
expression
of
matrix
MMP
Detection
of
active
IVIMPs
by
gelatin
zymography
Expression
of
pro-
and
active
forms
of
IVIMP-2
and
-9
in
the
granulation
tissues
of
post-burn
days
shows
relative
changes
over
time
(Fig.
8).
Group
1
showed
higher
levels
of
both
pro-
and
active
form
of
MMP-2
and
-9
on
day
3,
whereas
on
day
6
the
expression
of
active
form
was
more
than
the
pro-
form.
It
is
also
observed
that
overall
expression
of
MMP-2
was
lesser
than
IVIMP-9.
Noninfected
treated
group
(group
2)
showed
a
proportionate
decrease
in
MMP-2
and
-9
over
time,
but
at
a
faster
rate
than
group
1.
A
distinct
feature
is
the
absence
of
IVIMP-2
expression
from
day
9
and
the
levels
were
compara-
tively
low
(on
day
3)
in
group
2
than
in
group
1.
In
comparison
with
the
noninfected
group,
the
overall
expression
of
both
MMP-2
and
-9
in
infected
group
were
significantly
lower,
thus
hindering
active
remodeling.
The
expression
of
both
IVIMP-9
and
-2,
though
less,
can
be
observed
for
longer
duration
in
case
of
infected
group
(group
3),
until
day
18.
Whereas
group
4
rats
(SSDM-CS
treated)
showed
45
4
AS
:.5
1
0.5
0
2
-.--011011T
1
GROUP
2
-GROUP
S
-
GROUP
4
Figure
6.
Total
hexoseamine
content
determined
in
gran-
ulation
tissue
of
infected
and
noninfected
rats
treated
with
SSDM-CS
in
comparison
with
their
respective
controls
at
various
time
intervals.
*p
<
0.05,
groups
3
and
4
vs.
groups
1
and
2.
(infected
vs.
non-infected).
Journal
of
Biomedical
Materials
Research
Part
A
ti
n6
l
k
\
4
(B)
GROUP-1
GROUP-3
GROUP-4.
D-6
D*;
i
,
d,s
.
"
r
-.
4...
4
jf.
.soi
4
,
!
..
0
1
.7
,i--
1 t
.
i
ir
.
-
.
101
c
.2.1
r
478
SHANMUGASUNDARAM
ET
AL.
(A)
GROUF
1
GRUtlikl
n
3
AZ"
r
GROUF-3
UROVIkt
.•
D-6
D-9
-
-
k
c
•Atk..1.
13-3
D-6
'N.,
t
A
GROUP.I.
GROUP.
(C)
D-3
D.6
:
kr
GROL
P-3
GROUP-4.
D-6
rs
•"••••-
-
•-.,
-••1
Figure
7.
Immunohistochemical
localization
of
proinflammatory
cytolcines
in
granulation
tissue.
(A)
Expression
of
IL-10
(bold
arrow)
detected
in
granulation
tissue
of
various
groups,
at
dermal
(d)
and
sub-dermal—muscular
junction
(sd-m).
Inset
on
day
9
photograph
of
group
3
shows
magnified
image
of
cellular
infiltration
of
IL-113.
(B)
Expression
of
IL-6
(bold
arrow)
seen
on
disrupted
dermal
regions
(d),
Expression
can
be
observed
even
in
muscle
layer
(m)
of
infected
controls
(group
3).
(C)
Expression
of
TNF-a.
(bold
arrows)
observed
in
various
groups
in
dermal
(d)
regions
that
spreads
sparsely
over
muscular
(m)
and
subcutaneous
(sc)
layers.
Inset
on
day
9
photograph
of
group
3
shows
cellular
infiltration
of
INF-a.
gt
,
granulation
tissue.
[Color
figure
can
be
viewed
in
the
online
issue,
which
is
available
at
www.interscience.wiley.com
]
appreciable
levels
of
expression
of
MMPs
until
day
6,
after
which
MMP-2
levels
declined,
while
MMP-9
expression
could
be
still
observed.
After
day
12,
expression
of
both
MMPs
in
group
4
diminished,
which
can
be
attributed
to
the
control
of
micro-
bial
burden
from
day
9.
Expression
of
MMP-9,
in
group
3,
even
on
day
15,
indicates
slower
onset
of
remodeling.
Journal
of
Biomedical
Materials
Research
Part
A
CONTROLLED
SSD
DELIVERY
IN
BURN
479
stn.
GROUP
1-w 4-
GROUP
2
-
Nr
4
.
22237r 4-
GROUP
4
-w
11
a
ME.
1
1
O
a
Figure
8.
Differential
expression
of
active
MMP-2
and
NIMI
3
-9
observed
through
gelatin
zymography
of
granulation
tissue
extract
on
various
post-burn
days.
Analysis
of
matrix
MMP
by
western
blot
MMPs
are
present
along
with
tissue
inhibitors
of
MMP
that
may
not
be
detected
by
zymography.
Young
and
Grinell
27
have
provided
evidence
for
detecting
both
active
and
inactive
forms
MMPs
through
western
blot
analysis
and
shown
it
to
give
a
measure
of
total
MMPs
expressed.
Hence
in
this
study
total
expression
of
MMP-2
and
-9
were
ana-
lyzed
through
western
blot
(Fig.
9).
Volumograms
of
MMP-1, MMP-2,
and
MMP-9
by
densitometry
(Fig.
10)
exhibited
both
active
and
inactive
forms
of
enzymes
at
various post-burn
days.
MMP-llevels
were
found
to
be
high
during
initial
days
of
healing
(3-6)
in
all
the
groups
[Fig.
10(A)].
MMP-1
linearly
decreased
with
time
in
both
the
non-infected
groups,
whereas
level
of
expression
showed
variation
in
both
the
infected
groups.
Group
4
exhibited
significant
increase
in
MMP-1
levels
until
day
9
(group
4
vs.
groups
1
and
2)
and
it
decreased
on
par
with
the
noninfected
group,
which
was
statis-
tically
insignificant
(p
=
0.05293,
p
<
0.05).
Compari-
son
within
groups
does
not
show
significant
level
of
expression
of
MMP-1
on
day
15
(group
1
vs.
group
2,
p
=
0.0881and
group
3
vs.
group
4,
p
=
0.1389).
In
groups
1,
2,
and
4,
expression
of
MMP-2
[Fig.
10(B)]
increased
gradually
with
time
till
day
9,
after
which
it
rapidly
declined,
whereas
infected
control
group
showed
variable
expression
levels
until
day
18.
Rats
in
group
4
(SSDM-CS
treated)
exhibited
higher
levels
of
IVIMP-2
until
day
9
and
the
level
significantly
decreased
from
day
12
onward
in
comparison
with
group
1
(p
=
0.0013).
IVIMP-9
expression
was
high
during
initial
days
in
all
the
groups
[Fig.
10(C)].
Infected
groups
(3
and
4)
exhibited
high
level
of
expression
than
the
noninfected
groups
throughout
the
study.
It
can
be
observed
that
group
3
exhibited
higher
levels
in
comparison
with
other
groups
and
its
expression
was
observed
even
on
day
18.
Croup
2
—6
h
OMR
-14
Group
3
-0.
;
.0
,
-UnorP
4
mu.
IMS-timm
int•
4110
-
A‘•
=t
Figure
9.
Western
blot
analysis
of
both
active
and
inactive
MMP-1,
MMP-2,
and
MMP-9
levels
in
various
post-burn
days.
Journal
of
Biomedical
Materials
Research
Part
A
(E)
900)
2994
1994
1509
1914
DAY
1
DAY
i
DAY
9
DAY
]1.
CLAY
DAY
10
SHANMUGASUNDARAM
ET
AL.
480
(A)
1240
7-1
GROUP
1
ai
GROUP
2
c
GROL7.1
MOLT
(B)
20D
0.0:1111
,
[
O
'UR
GraEt2
D
CR
01:7-5
CiO011R
4
IS
GROUP
-1
0
C190111
,
2
O
011.0111
,
9
CROUP-1
Figure
10.
Densitometric
analysis
of
MMP-1,
MMP-2,
and
MMP-9
levels
observed
in
western
blots.
(A)
Comparison
of
MMP-1
levels
observed
in
infected
and
noninfected
rats
treated
with
SSDM-CS
in
comparison
with
their
respective
controls
at
various
time
intervals.
p
<
0.05,
*group
3
vs.
groups
1
and
2,
**group
4
vs.
groups
1
and
2.
(a)
p
<
0.05.
NS,
not
significant
on
day
6,
groups
3
and
4
vs.
group
1.
(b)
p
<
0.05.
NS,
not
significant
on
day
12,
groups
3
and
4
vs.
group
2.
(B)
Comparison
of
MMP-2
levels
observed
in
infected
and
noninfected
rats
treated
with
SSDM-CS
in
comparison
to
their
respective
controls
at
various
time
intervals.
p
<
0.05,
*groups
1
and
2
vs.
group
3,
**groups
1
and
2
vs.
group
4.
(C)
Comparison
of
MMP-9
levels
observed
in
infected
and
noninfected
rats
treated
with
SSDM-CS
in
comparison
with
their
respective
controls
at
various
time
intervals.
p
<
0.05,
*group
3
vs.
groups
1
and
2,
**group
4
vs.
groups
1
and
2,
NS-not
significant
(group
1
vs.
group
4).
fir
Collectively
in
all
the
groups,
expression
of
IVIMP-
9
was
found
to
be
predominant
in
comparison
with
other
IVIMPs.
It
can
be
observed
from
overall
expres-
sion
of
various
IVIMPs
that
there
exists
a
relationship
between
IVIMP
expression
and
time
of
healing
in
case
of
groups
1,
2,
and
4.
These
groups
exhibit
dif-
ferential
expression
of
various
IVIMPs
till
day
9,
after
which
a
decrease
can
be
observed
reaching
undetect-
able
low
level
by
day
18.
On
the
other
hand,
infected
control
group
(group
3)
showed
variation
in
the
expression
of
all
IVIMPs
at
all
time
periods.
Hence,
it
is
evident
that
the
microbial
load
greatly
influences
remodeling
event.
DISCUSSION
Despite
major
advances
in
burn
wound
manage-
ment
and
other
supportive
care
regimen,
infection
remains
the
leading
cause
of
morbidity
and
mortal-
ity.
The
burn
wound
created
in
this
study
by
the
instrument
designed,
was
deep
second
degree
and
vulnerable
to
infection.
Hence,
the
subcutaneous
injection
of
10
7
cfu/mL
of
P.
aeruginosa
caused
severe
infection
and
its
importance
was
shown
in
previous
studies.
28
Severe
infection
caused
an
enlargement
of
wound
size
during
initial
days
in
both
infected
and
noninfected
groups,
in
particular
the
infected
rats
showed
an
increase
(>50%)
in
size,
implicating
the
effect
of
infection
on
healing
process.
Infection
also
caused
hypermetabolic
response
by
affecting
the
nutritional
balance
and
directly
influ-
encing
the
body
weight.
29
The
infected
rats
(groups
3
and
4)
showed
reduction
in
body
weight,
which
was
significant
in
group
3
when
compared
with
groups
1
and
2
on
day
9,
whereas
group
4
rats
did
not
show
any
significant
weight
loss.
The
later
group
started
to
regain
the
body
weight
once
the
microbial
load
receded
(after
9
days),
because
SSDM-CS
treat-
ment
exerted
antimicrobial
effect
through
controlled
delivery
of
SSD.
A
further
evidence
for
this
effect
was
the
sharp
reduction
from
10
7
to
10
2
cfu
by
day
9
in
group
4
and
increase
in
percentage
wound
closure
on
par
with
noninfected
control,
exhibiting
faster
healing
(15%
more
reduction
in
wound
size
on
day
27).
A
significant
feature
of
thermal
burn
is
slow
re-
epithelialization
in
both
early
and
later
time
points
because
of
loss/damage
of
follicles
residing
in
deep
dermal
regions.
Bacterial
load
aggravates
the
dam-
age
by
gaining
entry
through
the
follicular
path;
re-
side
deep
in
the
follicular-basement
juncture
and
further
infiltrates
into
deep
dermal
and
paraspinal
muscular
layers.
This
is
of
paramount
importance
since
keratinocytes
use
the
same
portal
for
re-epithe-
lialization.
Though
the
normal
wounds
and
Journal
of
Biomedical
Materials
Research
Part
A
CONTROLLED
SSD
DELIVERY
IN
BURN
481
infected
wounds
share
similar
process
of
granulation
characteristics,
existence
of
large
bacterial
load
in
infected
rats
exerted
a
detrimental
effect
on
rate
of
granulation
and
re-epithelialization.
Both
groups
3
and
4
showed
existence
of
infection
during
early
days
of
healing,
sequentially
affecting
epidermal
maturation.
After
day
9
group
4
exhibited
significant
proliferation
and
migration
of
epidermal
cells
over
fibrin
matrix
and
looked
similar
to
groups
1
and
2,
resulting
in
faster
granulation
covering
the
entire
wound
surface.
Whereas,
infected
control
showed
poor
granulation
over
a
long
time
(>18
days)
because
of
persistent
infection.
Another
striking
fea-
ture
is
the
appearance
of
spongious
collagen
layer
as
early
as
day
15
in
group
2
rats,
and
on
day
21
in
case
of
group
4
rats,
while
group
1
rats
exhibited
striated
fibrillar
appearance
on
day
27.
Faster
colla-
gen
deposition
in
SSDM-CS-treated
rats,
in
compari-
son
with
their
respective
controls,
attributed
to
faster
cellular
infiltration
and
fibroblasia.
Glycosaminogly-
can
content
also
reached
the
maximum
at
faster
rate
in
the
treated
groups
indicating
better
remodeling.
Another
vital
event
during
bum
wound
healing
is
inflammatory
response,
which
is
aggravated
due
to
infection.
Early
onset
of
proinflammatory
cytokines,
IL-6,
IL-113,
and
TNF-a
demonstrates
the
wound
sta-
tus
and
especially
persistent
expression
of
IL-113
and
TNF-a
provides
ample
evidence
for
severity
of
infec-
tion
in
triggering
inflammatory
response.
In
normal
healing
process,
IL-6
expression
reaches
the
peak
level
rapidly
and
declines,
whereas
expression
of
IL-113
and
TNF-a
can
be
observed
for
longer
time.
The
immunohistochemical
analysis
shows
prolonged
expression
of
IL-6
(day
6)
along
with
IL-113
and
TNF-a,
in
infected
control,
indicating
the
severity
of
inflammation.
Whereas,
SSDM-CS
treatment
gives
normal
expression
of
proinflammatory
cytokines
similar
to
noninfected
controls.
Prolonged
expression
of
MMPs
is
likely
to
cause
destruction
of
the
early
provisional
matrix,
which
was
further
exacerbated
due
to
heavy
microbial
load
in
group
3,
whereas
SSDM-CS-treated
groups
exhib-
ited
better
remodeling
due
to
positive
modulation
of
MMPs.
Early
expression
of
MMPs
in
groups
1,
2,
and
4
indicates
the
active
clearance
of
dead
tissue
in
these
groups.
Though
the
magnitude
of
expression
varied
within
groups
1
and
4,
the
pattern
of
expres-
sion
was
found
to
be
consistent.
Another
important
observation
is
the
delayed
onset
and
prolonged
expression
of
MMP
1,
2,
and
9
in
infected
controls.
One
of
the
major
factors
that
regulate
MMP
activity
is
the
bacterial
exotoxins
31
along
with
excessive
inflammatory
cell
infiltration
at
the
wound
site.
In
addition
regulation
of
MMP-tissue
inhibitor
of
MMP
association
during
healing
process
plays
a
crucial
role
in
matrix
remodeling.
12
The
SSDM-CS
reduces
the
bacterial
load
and
found
to
lower
the
proinflam-
matory
cytokines
thus
positively
modulating
MMP
activity.
The
results
of
the
current
investigation
affirm
the
influence
of
infection
on
healing
events
and
its
response
to
controlled
delivery
of
SSD.
Perhaps
this
seems
to
be
the
first
report
to
show
the
controlled
delivery
of
SSD
through
collagen
dressing
to
control
early
onset
of
infection,
and
the
subsequent
events.
This
report
correlates
well
with
the
previously
sug-
gested
hypothesis
32
that
controlled
delivery
of
silver
is
effective
in
the
healing
process
by
modulating
the
overall
MMP
activity.
From
silver
incorporated
dressings,
when
Ag
+
ions
are
released
in
bolus
there
is
a
possibility
that
Ag
+
binds
with
serum
proteins
and
chloride
ions
present
in
the
wound
fluids.
This
causes
quick
inac-
tivation
of
a
large
portion
of
Ag
+
as
well
as
causes
accumulation
of
Ag
+
ions
elsewhere
in
the
body
through
systemic
circulation,
whereas
SSDM-CS
treatment
maintains
steady
state
equilibrium
of
drug
at
therapeutic
levels.
These
results
correlate
well
with
our
previous
ex
vivo
release
profiles.
16
Through
serum
analysis,
we
have
proved
that
silver
concen-
trations
were
below
the
toxic
limits
20
gg/dL
(silver
analysis
in
serum
was
below
detectable
limits).
33
Earlier
dressings
developed were
mainly
with
syn-
thetic
polymers
and
do
not
give
clear
evidence
in
controlling
its
release
from
the
base
dressing
mate-
rial.
Recently,
silver
ions
coated
dressing
has
been
developed
and
evaluated
for
its
ability
to
control
infection
silver
concentrations
at
low
levels.
34
In
these
dressings
Ag
+
are
coated
on
the
basis
of
charge
interaction.
Hence,
when
applied
in
vivo
these
silver
ions
are
released
due
to
change
in
electrostatic
flux.
Recent
proposition
to
deliver
silver
ions
in
a
sustained
fashion
through
polyethylene
glycol
based
system
has
presented
no
adverse
effect
on
cell
mi-
gration
or
proliferation.
35
Evidence
has
been
provided
for
their
ability
to
effectively
kill
the
pathogens
that
causes
infection
in
vitro.
Moreover,
incorporation
of
collagen
type
I
into
the
polymer
corrected
the
un-
paired
cell
migration.
35
CONCLUSION
Collectively,
the
present
model
is
designed
taking
into
account
the
situations
where
infection
in
burns
are
severe
and
significantly
affecting
healing
bearing
in
mind
the
narrow
toxicity
limit
(20
gg/dL)
of
SSD
when
used
for
prolonged
period
in
vivo,
providing
a
close
simulation
with
respect
to
clinical
setting.
This
investigation
also
reveals
that
control
of
inflamma-
tion
and
MMP
regulation
depends
on
the
therapeu-
tic
efficacy
of
the
initial
chemoprophylaxis
applied
to
control
infection.
In
addition,
it
would
be
a
con-
structive
proposition
if
the
wound
dressing
product
Journal
of
Biomedical
Materials
Research
Part
A
482
SHANMUGASUNDARAM
ET
AL.
could
assist
in
controlling
the
proteolytic
wound
environment
for
accelerated
remodeling
and
wound
closure.
One
of
the
authors,
N.
S.,
thank
the
Department
of
Biotechnology
(DBT)
for
providing
fellowship.
We
express
our
sincere
thanks
to
Dr.
A.
B.
Mandal,
Director,
CLRI
for
giving
us
the
opportunity
and
encouragement
to
carry
out
the
work.
References
1.
Singer
AJ,
Clark
RAF.
Cutaneous
wound
healing.
N
Engl
J
Med
1999;341:738-746.
2.
Stieritz
DD,
Holder
IA.
Experimental
studies
of
pathogenesis
of
infections
due
to
Pseudomonas
aeruginosa:
Description
of
a
burned
mouse
model.
J
Infect
Dis
1975;131:688-691.
3.
Steinstraesser
L,
Burkhard
0,
Fan
MH,
Jacobsen
F,
Marcus
L,
Su
G,
Daigler
A,
Steinau
HU,
Remick
D,
Wang
SC.
Burn
wounds
infected
with
Pseudomonas
aeruginosa
triggers
wight
loss
in
rats.
BMC
Surg
2005;5:1-7.
4.
Smith
M,
Enquist
IF.
A
quantitative
study
of
impaired
heal-
ing
resulting
from
infection.
Surg
Gyn
Obstet
1967;125:965-
973.
5.
Robson
MC.
A
failure
of
wound
healing
caused
by
an
imbal-
ance
of
bacteria.
Surg
Clin
N
Am
1997;77:637-650.
6.
Singer
AJ,
McLain
SA.
Persistent
wound
infection
delays
epi-
dermal
maturation
and
increased
scarring
in
thermal
bums.
Wound
Repair
Regen
2002;10:372-377.
7.
Freudenberg
MA,
Ness
T,
Kumazawa
Y,
Galanos
C.
The
role
of
cytokines
in
endotoxic
shock
and
in
endotoxin
hypersensi-
tivity.
Inunun
Infekt
1993;21:40-44.
8.
Reddy
RC,
Chen
GH,
Tekchandani
RK,
Standiford
TJ.
Sepsis-
induced
immunosuppression:
From
bad
to
worse.
Inununol
Res
2001;24:273-287.
9.
Moulin
V.
Growth
factors
in
skin
wound
healing.
Eur
J
Cell
Biol
1995;68:1-7.
10.
Neely
AN,
Hoover
DL,
Holder
IA,
Cross
AS.
Circulating
lev-
els
of
Tumour
Necrosis
Factor.
Interleukin
6
and
proteolytic
activity
in
a
murine
model
of
burn
and
infection.
Bums
1996;
22:524-530.
11.
Grellner
W.
Time-dependent
immunohistochemical
detection
of
proinflammatory
cytokines
(IL-
Beta.
IL-6,
TNF-Alpha)
in
human
skin
wounds.
Forensic
Sci
Int
2002;130:90-96.
12.
Armstrong
DG,
Jude
EB.
The
role
of
matrix
metalloproteinases
in
wound
healing.
J
Am
Podiatr
Med
Assoc
2002;92:12-18.
13.
Werb
Z.
ECM
and
cell
surface
proteolysis:
Regulating
cellular
ecology.
Cell
1997;9:1439-1442.
14.
Shapino
SD.
Matrix
metalloproteinases
degradation
of
extrac-
ellular
matrix:
Biological
consequences.
Cell
Biol
1998;10:602-
608.
15.
Monafo
WW,
Freedman
B.
Topical
therapy
for
bums.
Surg
Clin
North
Am
1987;67:133-145.
16.
Shanmugasundaram
N,
Sundaraseelan
J,
Uma
S,
Selvaraj
D,
Mary
Babu.
Design
and
delivery
of
silver
sulfadiazine
from
alginate
microspheres-impregnated
collagen
scaffold.
J
Biomed
Mater
Res
B
Appl
Biomater
2006;77:378-388.
17.
Official
monograph
of
silver
sulphadiazine
cream.
USP
24-NF
19;1999.
p
1567.
18.
Fujii
T,
Kuhn
K.
Isolation
and
characterization
of
pepsin-
treated
type
DI
collagen
from
calfskin.
Hoppe
Seylers
Z
Phys-
iol
Chem
1975;11:1793-1801.
19.
National
Committee
for
Clinical
Lab
Standards
(NCCLS).
Methods
for
dilution
antimicrobial
susceptibility
tests
for
bac-
terial
that
grow
aerobically;
approved
standard,
5th
ed,
2000;
20:M7-A5.
20.
Grzybowski
J,
Janiak
MK,
Oldak
E,
Lasocki
K,
Wrembel-War-
gocka
J,
Cheda
A,
Antos-Bielska
M,
Pojda
Z.
New
cytokine
dressings.
II.
Stimulation
of
oxidative
burst
in
leucocytes
in
vitro
and
reduction
of
viable
bacteria
within
an
infected
wound.
Int
J
Pharm
1999;184:179-187.
21.
Woessner
JF
Jr.
The
determination
of
hydroxyproline
in
tis-
sue
and
protein
samples
containing
small
proportions
of
this
imino
acid.
Arch
Biochem
Biophys
1961;93:440-447.
22.
Elson
LA,
Morgan
WT.
A
colorimetric
method
for
the
deter-
mination
of
glucosamine
and
chondrosamine.
Biochem
J
1931;
27:1824-1828.
23.
Smith
PK,
Krohn
RI,
Hermanson
GT,
Mania
AK,
Gartner
FH,
Provenzano
MD,
Fujimoto
EK,
Goeke
NM,
Olson
BJ,
Klenk
DC.
Measurement
of
protein
using
bicinchoninic
acid.
Anal
Biochem
1985;150:76-85.
24.
Madlener
M,
Parks
WC,
Werner
S.
Matrix
metalloproteinases
(MMPs)
and
their
physiological
inhibitors
(TIMPs)
are
differ-
entially
expressed
during
excisional
skin
wound
repair.
Exp
Cell
Res
1988;242:201-210.
25.
Okada
A,
Tomasetto
C,
Lutz
Y,
Bellocq
JP,
Rio
MC,
Basset
P.
Expression
of
matrix
metalloproteinases
during
rat
skin
wound
healing:
Evidence
that
membrane
type-1
matrix
met-
alloproteinase
is
a
stromal
activator
of
pro-gelatinase
A.
J
Cell
Biol
1997;137:67-77.
26.
Watts
AMI,
Tyler
MPH,
Perry
ME,
Roberts
Al-IN,
McGrouther
DA.
Bum
depth
and
its
histological
measurement.
Bums
2001;
27:154-160.
27.
Young
PK,
Grinell
F.
Metalloproteinases
activation
cascade
after
bum
injury:
A
longitudinal
analysis
of
human
wound
environment.
J
Invst
Dermatol
1994;103:660-664.
28.
Raju
DR,
Jindrak
K,
Weiner
M,
Enquist
IF.
A
study
of
the
critical
bacterial
inoculum
to
cause
a
stimulus
to
wound
heal-
ing.
Surg
Gynecol
Obstet
1977;144:347-350.
29.
Barrow
RE,
Meyer
NA,
Jeschke
MG.
Effect
of
varying
bum
sizes
and
ambient
temperature
on
the
hypermetabolic
rate
in
thermally
injured
rats.
J
Surg
Res
2001;99:253-257.
30.
Schaffer
CJ,
Reinisch
L,
Polis
SL,
Stricklin
GP,
Nanney
LB.
Comparison
of
wound
healing
among
excisional,
laser-cre-
ated
and
standard
thermal
burns
in
porcine
wounds
of
equal
depth.
Wound
Repair
Regen
1997;5:52-61.
31.
Miyajima
S,
Akaike
T,
Matsumoto
K,
Okamoto
T,
Yoshitake
J,
Hayashida
K,
Negi
A,
Maeda
H.
Matrix
metalloproteinases
induction
by
pseudomonal
virulence
factors
and
inflamma-
tory
cytokine
in
vitro.
Microb
Pathogol
2001;31:271-281.
32.
Wright
JB,
Lam
K,
Buret
AG,
Olson
ME,
Burrell
RE.
Early
healing
event
in
a
porcine
model
of
contaminated
wounds:
Effects
of
nanocrystalline
silver
on
matrix
metalloproteinases,
cell
apoptosis,
and
healing.
Wound
Repair
Regen
2002;10:141-
151.
33.
Boosalis
MG,
McCall
JT,
Ahrenholz
DH,
Solem
LD,
McClain
CJ.
Serum
and
urinary
silver
levels
in
thermal
injury
patients.
Surgery
1987;101:40-43.
34.
Gear
AJ,
Hellewell
TB,
Wright
EIR,
Mazzarese
PM,
Arnold
PB,
Rodeheaver
GT,
Edlich
RF.
A
new
silver
sulfadiazine
water
soluble
gel.
Bums
1997;23:387-391.
35.
Babu
R,
Zhang
J,
Beckman
EL
Virji
M,
Pasculle
WA,
Wells
A.
Antimicrobial
activities
of
silver
used
as
a
polymerization
catalyst
for
a
wound-healing
matrix.
Biomaterials
2006;27:
4304-4314.
Journal
of
Biomedical
Materials
Research
Part
A