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

2

4

6

8

10

12

14Architect™ Stabilized Collagen MatrixePCM stabilized collagen matrix

0

100

200

300

400

500

600

700

800

Control ePCM

0

100

200

300

400

500

600

700

800

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

10

20

30

40

control ePCM

*

0

10

20

30

40

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.