AN ACELLULAR STABILIZED COLLAGEN MATRIX (ePCM) …AN ACELLULAR STABILIZED COLLAGEN MATRIX (ePCM) DRESSING STRENGTHENS ANTI-MICROBIAL DEFENSES IN HUMAN KERATINOCYTES Abstract Results
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AN ACELLULAR STABILIZED COLLAGEN MATRIX (ePCM) DRESSING STRENGTHENS ANTI-MICROBIAL DEFENSES IN HUMAN KERATINOCYTES
Abstract Results
Shomita S. Steiner1, Piya Ghatak1, Amitava Das1, Sashwati Roy1, and Chandan K. Sen1,2
Characterize the physical and biological characteristics of the
ePCM dressing and its effect on wound healing
Objective
Results
Conclusions and Future Directions
Supported by: Harbor MedTech Inc.
)
1Comprehensive Wound Center, Davis Heart and Lung Research Institute, 2Center for Regenerative Medicine and Cell Based Therapies, Columbus, OH
Methods
A biologically stabilized, acellular, equine pericardial collagen matrix
(ePCM) is clinically used as wound dressing. For the care of chronic
wounds such as diabetic ulcers, ePCM is used as a single application
where the dressing remains embedded in the wound until closure.
Preliminary observations support that in patients, ePCM improves
wound closure. The mechanism of action of ePCM in wound healing
remains unclear. Initial studies from our group characterized human
keratinocyte growth, proliferation and differentiation on ePCM in vitro.
Our studies indicated that human keratinocytes attached to ePCM and
acquired a differentiated phenotype compared to those growing on
glass surfaces. This provided first cues suggesting that ePCM may
serve as a scaffold for cells within the wound microenvironment.
Interestingly, the antimicrobial peptide (AMP) defense system was
significantly upregulated in cells adhered to ePCM compared to those
on glass. AMPs kill a wide variety of microbes including bacteria. Such
upregulation of AMPs in keratinocytes adhered to ePCM could provide
effective defenses against bacterial colonization and wound infection.
Most bacteria are able to attach and grow on biological surfaces leading
to infection. In keeping with this, scanning electron microscopy imaging
identified that ePCM by itself was a suitable substrate for robust
bacterial growth of the primary wound pathogen Pseudomonas
aeruginosa (PA01).This further suggests that effective containment of
wound infection in vivo is likely to be contributed by bolstered epithelial
antimicrobial defense system caused by ePCM. Our observation
demonstrating that an acellular collagen matrix may modify keratinocyte
antimicrobial defenses draws attention to the far-reaching influence of
biological dressings above and beyond their direct physico-chemical
influence on the wound milieu.
In vitro studies:
• Light microscopy (LM) imaging and atomic microscopic
imaging (AFM) of ePCM
• Keratinocyte culture on ePCM: Imaging by scanning electron
microscopy (SEM) and gene expression studies by qPCR
• Bacterial biofilm growth: imaging by SEM
Mouse studies:
• PVA sponge implantation and analysis of immune cell
population recruited at d3 and d7 by flow cytometry. Imaging
of ePCM performed by SEM
Height Map Surface Topography
Microscopy-based characterization of ePCM
A
B
1mm
C
ePCM promotes keratinocyte expression of anti-microbial peptides
Figure 1. Advanced microscopy based characterization
of ePCM. A. Scanning electron microscopic images show
structured organization of collagen fibers in the intact
dressing. B. 3D light microscopic images show uneven
topography of the dressing with randomly oriented collagen
bundles. C. Atomic force microscopy (AFM) images show
surface topography. stiffness measurements were
conducted using wet samples. There was a significant
increase in the sample volume once the sample was
hydrated and it acquired hydrogel-like material properties.
The stiffness measurements are in the range of 1-5 mPa
which is higher, than what it has been reported in the
literature for collagen gels, but closer to skin tissue. The
normal skin modulus distribution peaks at ~322 kPa and
ranges from 25.8 kPa to 1.18 mPa.
Figure 3. AMP expression is significantly
upregulated in human keratinocytes grown on
ePCM. Human keratinocytes were plated on
glass or ePCM for 24h and RNA was extracted
following cell lysis and cDNA was generated.
qPCR analysis indicated that compared to
control (glass), human keratinocytes grown on
ePCM showed upregulation of AMPs – S100A9
and beta-defensin. Data are represented as
mean±SEM. *p<0.05, **p<0.005 n=6
ePCM could act as a possible scaffold/catalyst – serving as a homing base for
host cells such as epithelial, fibroblast, endothelial and immune cells, creating a
gradient of pro-healing cues.
Cells potentially drawing nourishment from dressing itself
Although ePCM serves as a suitable substrate for bacterial attachment and growth
and biofilm formation, it could also act as a decoy, blunting protease mediated
invasion and bolstering host pathogen defenses such as AMPs.
SE
M3D
-LM
AFM
Min. Value 0.0518 kPa
Max. Value 26.9 mPa
modulus (mPa)
co
un
t
Elastic Modulus Distribution
Modulus (MPa)
0 1 2 3 4 5 6
Co
un
t
0
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14Architect™ Stabilized Collagen MatrixePCM stabilized collagen matrix
0
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Control ePCM
0
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Control ePCM
*
**
S100
A9/
-actin r
RN
A(
ct)
-d
efe
nsin
/-a
ctin r
RN
A(
ct)
Macrophages are recruited early in response to ePCM in vivo
Harvest Sponge
and DressingDesign -
sponge
sponge
ePCM
Design of Experiment
d0
sponge
implantation
d3 d7
harvest wound cells
for each time pointSEM imaging
Flow cytometric analysis
A
Figure 4. Enhanced early recruitment of immune cells to ePCM implanted in murine wounds. A. ePCM was sandwiched between
two polyvinyl alcohol sponges and implanted into wounds created on the back of C56Bl6 mice. The sponges were harvested on
days 3 and 7, fixed in glutaraldehyde buffer and processed for SEM imaging. B. Representative SEM images from d3 and d7 are
shown. Increased recruitment of immune cells, particularly macrophages (red arrowheads) are seen at the early time point (d3)
when usually fewer macrophages are known to be present. C. Flow cytometric analysis of F480 (macrophage specific) positive
cells indicated increased macrophage recruitment to the wounds in ePCM treated wounds. Quantitation is shown in the graphs.
*p<0.05, n=4. Additionally, significant remodeling of ePCM was noted at both time points.
B
da
y 3
da
y 7
0
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30
40
control ePCM
*
0
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control ePCM
% F
48
0 p
os
itiv
e c
ells
% F
48
0 p
os
itiv
e c
ells
Cday 3 day 7
ePCM supports growth of human keratinocytesB
Figure 3. Human keratinocyte growth on
ePCM alters the integrity of the dressing. A.
Human keratinocytes were plated on ePCM
or glass for 48h followed by fixation and
processing for SEM imaging. Shown are
representative images of cells on ePCM and
glass substrate. Morphology of the
keratinocytes appear different on the
biologically relevant substrate compared to
glass. B. ePCM was decellularized with 2%
SDS and imaged using SEM. Compared to a
control (top panel), ePCM exposed to
keratinocytes (bottom panel) was found to be
significantly remodeled with loss of integrity.
eP
CM
gla
ss
ePCM control
decellularization post-
keratinocyte growth
A
ePCM supports robust biofilm formationePCM control biofilmwt PA01 biofilmhi PA01
Figure 2. ePCM is a suitable substrate for bacterial growth. Graded
strains of Pseudomonas aeruginosa (PA01) were plated on ePCM
for 48h and imaged using SEM. The biofilmwt and biofilmhi strains
formed robust biofilms. Shown are representative images of a
control and individual PA01 strains grown on ePCM.
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