2010 Abstracts-pah[2] - Wound Healing Society

Abstracts
20th Annual Meeting of the Wound Healing Society
SAWC/WHS Joint Meeting
Gaylord Palms Hotel and Convention Center, Orlando,
Florida USA
April 17-20, 2010
STATEMENT OF ASSURANCES:
All authors affirm that any animal studies conform with the “Position of the American Heart
Association on Research Animal Use” (Circulation 1985;71:849A-850A), and that any human
experimentation has been conducted according to a protocol approved by the institutional
committee on ethics of human investigation or, if no such committee exists, that it conforms with
the principles of the “World Medical Association Declaration of Helsinki” (Cardiovascular
Research 1997;35:2-3).
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YOUNG INVESTIGATOR AWARDS
Young Investigator Award Competition – YIA01-09
Sunday, April 18, 2010, 9:15 to 11:30 am
YIA01
BREAKING THE CYCLE OF INJURY: TOPICAL PUMA SILENCING DECREASES
REACTIVE OXYGEN SPECIES GENERATION AND IMPROVES DIABETIC WOUND
HEALING
James L. Crawford, BS, Denis Knobel, MD, Parag Butala, MD, Aleen Kuperman, Shari
Bharrat, Sara B. Immerman, MD, Edward H. Davidson, MA MBBS, Ben R. Roman, MD,
Meredith T. Wetterau, MD, Steven Sultan, BA, Stephen M. Warren, MD, Pierre B. Saadeh, MD.
New York University Langone Medical Center, New York, NY, USA.
INTRODUCTION: Diabetes is characterized by hyperglycemia which yields an elevated
reactive oxygen species (ROS) state causing end organ injury. Furthermore, ROS are increased
in injury while diabetic wounds demonstrate elevated apoptotis and p53 levels. P53 up-regulated
modulator of apoptosis (PUMA) exerts its effects partly by the induction of ROS which, in turn,
is a known modulator of p53 activity. We evaluated this relationship in diabetic mice and
interrupted this pathway in an attempt to improve wound healing. METHODS: Paired 4-mm
stented wounds were created on diabetic db/db mice. Topically-applied PUMA siRNA, evenly
distributed in an agarose-gel matrix, was applied on post-wound days 1 and 7 (nonsense siRNA

served as control). Wounds were evaluated on post-wound day 10 and 20. Wound closure time
was photometrically assessed, and wounds harvested for histology, immunohistochemistry
(IHC), RT-PCR, and ELISA. ANOVA/t-test was used to determine significance (p

demonstrated a 3.5-fold increase in SDF-1 expression in treated wounds. Additionally, RT-PCR
revealed a decrease of the pro-oxidant gene PUMA (33% of control), and to a lesser extent, a
decrease in the anti-oxidant gene GPX1 (64% of control). NAC treatment consistently
accelerated wound closure (17±1 days versus 27±1 days). CONCLUSION: Local reduction of
ROS with NAC reverses apoptotic and vasculogenic derangements associated with diabetic
wounds and improves wound healing.
YIA03
WOUND HEALING IN TGF-β INDUCIBLE EARLY GENE (TIEG) KNOCKOUT MICE
Keijiro Hori, MD, Jie Ding, PhD, Jianfei Wang, PhD, Yvonne Marcoux, BSc, Edward E.
Tredget, MD, MSc, FRCSC.
University of Alberta, Edmonton, AB, Canada.
PURPOSE: TGF-β is a multifunctional growth factor that plays an important role in wound
healing. TGF-β inducible early gene (TIEG) is induced by TGF-β and is a primary response gene
in the TGF-β/Smad pathway. We hypothesize that TIEG plays a role in wound closure and reepithelialization
during cutaneous wound repair.
METHODS: 5mm full thickness punch biopsy wounds were created on the backs of 18 TIEG
knockout (TIEG -/- ) and 18 wild type (TIEG +/+ ) mice. Animals were sacrificed and specimens
collected on postoperative day 3, 5 and 7. Wound closure and re-epithelialization were measured
at each time point. Expression of the myofibroblast marker, alpha smooth muscle actin (αSMA),
was examined by immunohistochemistry. The mRNA level of TGF-β/Smad pathway was
determined by real time RT-PCR. Collagen synthesis was analyzed by LC/MS. Mouse
embryonic fibroblasts and keratinocytes were isolated from the mice and used in an in vitro
contraction assay and scratch migration assay.
RESULTS: Wound closure was slower in TIEG -/- mice at each time point compared to TIEG +/+
mice (4.0±2.1, 16.4±2.9, 36.3±4.8 versus 12.0±1.9, 22.6±2.1, 51.9±3.7% respectively).
Expression of αSMA was decreased in TIEG -/- mice compared to TIEG +/+ mice. Collagen
synthesis was lower in TIEG -/- mice at day 7. In vitro, the fibroblast contraction assay also
showed less contraction with TIEG -/- mice derived fibroblast. Although there were no significant
differences in the mRNA expression of Smad2, Smad7 and TGF-β1, TIEG -/- mice had lower
expression of Smad3 and CTGF compared to TIEG +/+ mice. Re-epithelialization was also slower
in TIEG -/- mice at day3 (18.3±2.6 versus 42.2±3.9%, p

Bianca Chin, MD 1 , Benjamin J. Herdrich, MD 2 , Dustin M. Bermudez, MD 2 , Myron Allukian,
III, MD 1 , Zhe Zhang, PhD 3 , HD Nah, DDS 4 , Kenneth W. Liechty, MD 5 .
1 University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 2 University of
Pennsylvania School of Medicine, Center for Fetal Research at the Children's Hospital of
Philadelphia, Philadelphia, PA, USA, 3 Center for Biomedical Informatics, Children's Hospital of
Philadelphia, Philadelphia, PA, USA, 4 Children's Hospital of Philadelphia, Philadelphia, PA,
USA, 5 University of Mississippi Medical Center, Jackson, MS, USA.
PURPOSE: The fetal response to injury is regenerative and scarless, unlike the reparative, scarforming
response in adults. Scar formation is a fundamental complication of a variety of disease
conditions and surgery that can result in poor functional and aesthetic outcomes. Dermal
regenerative wounds in the fetus can become scar-forming with increased wound size. We
hypothesize that this change is associated with an upregulation of inflammatory genes.
METHOD: To test our hypothesis, we used a fetal sheep dermal model of regenerative and
reparative healing, which removes gestational age as a potential confounding variable. Dermal
wounds are created on the back of 70-75 day fetal sheep (n=7) using a 2mm punch biopsy for
small wounds and 8mm punch biopsy for large wounds. Wounds were harvested 3 days later and
processed for RNA isolation. An ovine-specific microarray gene ship was then used to analyze
differential gene expression. RESULTS: A total of 15,010 genes were analyzed for differential
gene expression. The raw microarray signals were processed by loess normalization and log2transformation.
A gene was considered to be differentially-expressed between small and large
wounds if it had at least a two-fold change and a p-value of t-test less than 0.05. 257 genes were
upregulated in small dermal wounds and 293 genes in large dermal wounds. The gene groups
“wound response” and “inflammatory response” from the gene ontology database were selected
and evaluated to determine if there was differential expression between the two groups. The
principal component analysis (PCA) plots are shown in figure 1. CONCLUSION: Small
regenerative and large reparative dermal wounds can be distinguished by their global gene
expression patterns, as suggested by PCA. Understanding the mechanism of this regulation has
the potential to promote novel gene therapy that could revolutionize wound healing and prevent
complications of fibrosis.
YIA05
KELOID PATIENTS HAVE AN INCREASED PROPENSITY FOR FIBROCYTE
DIFFERENTIATION
Michelle C. Naylor, MD, David A. Lazar, MD, Oren Mushin, BS, Anthony E. Brissett, MD,
Oluyinka O. Olutoye, MD, PhD.
Baylor College of Medicine, Houston, TX, USA.
Keloids are a pathologic form of wound healing characterized by thick collagen bundles in the
dermis that progress beyond the confines of the original wound. The pathogenesis of keloids
remains elusive. Fibrocytes, derived from peripheral blood mononuclear cells (PBMCs), have
been linked to other fibroproliferative diseases, including asthma, interstitial pulmonary fibrosis,
and systemic fibrosis. Serum amyloid P (SAP) has been identified as the active component of
serum that inhibits the transformation of PBMCs to fibrocytes. We hypothesized that PBMCs of

keloid patients would have a higher propensity to differentiate into fibrocytes, and that these
fibrocytes would be more resistant to the effects of serum amyloid protein. To test this
hypothesis, peripheral blood samples were obtained from patients with ear lobe keloids along
with age, sex, and race matched controls. Monocytes were isolated from the peripheral blood and
cultured in serum-free media without SAP, and media with increasing concentrations of SAP.
After 5 days in culture, the plates were rinsed and the adherent cells stained with hematoxylin
and methylene blue. Cells exhibiting known fibrocyte morphology were quantified. Keloid
patients (n=4) had on average ten-times more fibrocytes than controls (n=9) at baseline (1032:93
fibrocytes; Students T-test p

compared to controls. Conclusions: This is the first demonstration of a transgenic HGPS mouse
model of post-operative senescent wound healing, with results suggesting a vasculogenic
dysfunction in wound healing. The Zmpste24-/- mouse can serve as a novel model for the
investigation of underlying theory and validation of new therapies in age-related wound healing
dysfunction.
YIA07
ESSENTIAL ROLE OF SALIVARY VEGF IN PALATAL WOUND HEALING AND
NEOVASCULARIZATION
Louis D. Le, MD, Sundeep G. Keswani, MD, Anna Katz, MD, Foong Y. Lim, MD, Timothy M.
Crombleholme, MD.
Cincinnati Children's Hospital, Cincinnati, OH, USA.
Introduction: Palatal mucosa wound healing proceeds more rapidly than cutaneous wound
healing. High levels of vascular endothelial growth factor(VEGF) are secreted by murine
submandibular salivary glands(SMG). VEGF-induced angiogenesis is critical for timely
cutaneous wound healing however, it has not been studied in the palate. We hypothesize that
SMG resection or selective depletion of salivaryVEGF will impair palatal mucosal
neovascularization and wound healing. To test the role of VEGF in palatal wound healing, SMG
are also cannulated and treated with a novel adenoviral construct that specifically blocks VEGF.
Methods: Saliva was collected from C57/BL6 mice pre and post SMG resection(n=6) or sham
operation(n=5). After recovery, 1.5 mm palatal mucosal wounds were created. Simultaneously,
another group(n=5/group) were treated with 1x10 8 PFU AdVegfTrap, AdLacZ or PBS by SMG
duct cannulation and 1.5 mm wounds were created. Wounds were harvested at 3 days post
wounding and analyzed for epithelial gap closure and capillary density. SalivaryVEGF protein
levels were quantified by ELISA. Data were expressed as mean±SEM; P values were assessed
by ANOVA and correlation by Pearson coefficient. Results: Salivary VEGF levels are
significantly reduced after SMG resection(136pg/mg±15.9 vs. Sham472pg/mg±40;p

NOVEL MOLECULAR TECHNIQUES TO CHARACTERIZE THE MICROBIAL
FLORA IN CHRONIC WOUNDS
Anne Han, M.D. 1 , Jonathan M. Zenilman, M.D. 1 , Johan H. Melendez, Ph.D. 1 , Mark E. Shirtliff,
Ph.D. 2 , Alessandra Agostinho, D.D.S., M.S., Ph.D. 3 , Garth James, Ph.D. 3 , Emmanuel F.
Mongodin, Ph.D. 4 , Alexander H. Rickard, Ph.D. 5 , Gerald S. Lazarus, M.D. 1 .
1 Johns Hopkins Medical Institutions, Baltimore, MD, USA, 2 University of Maryland, Baltimore,
MD, USA, 3 Montana State University, Bozeman, MT, USA, 4 Institute for Genome Sciences,
University of Maryland, Baltimore, MD, USA, 5 State University of New York, Binghamton,
NY, USA.
Background: Chronic wounds contain persistent microbial populations which may function as
benign colonizers or pathogenetic impediments to wound healing. Until recently, the complexity
of wound microflora was underappreciated because standard culture techniques did not identify
the full inventory of bacteria or define relationships between various species. Purpose: To
characterize the microbial flora in chronic wounds
Methods: We obtained tissue samples of chronic wounds from 8 patients presenting to the Johns
Hopkins Wound Center. We utilized quantitative culture and molecular techniques such as realtime
polymerase chain reaction (RT-PCR) and 16s rRNA gene pyrosequencing to identify the
bacterial species within the wound. We employed confocal microscopy and fluorescent in-situ
hybridization (FISH) to visualize the spatial relationships of organisms in the biofilm. We
collected data on quorum sensing molecules to understand signaling patterns among bacterial
colonies. Institutional review board approval was obtained. Results: Standard quantitative culture
and RT-PCR techniques demonstrated the presence of an average of 3 common bacterial species
in our samples. High-throughput pyrosequencing of the 16S rRNA gene revealed unsuspected
bacterial diversity, including previously unrecognized anaerobes. In addition, these molecular
techniques resulted in precise bacterial identification, as demonstrated by consonance among the
data sets. Confocal microscopy localized bacteria within highly organized biofilms, and FISH
visualized the spatial relationship between S. aureus, P. aeruginosa, and C. albicans in the
wound. We discovered elevated levels of autoinducer-2 (AI-2), a quorum sensing molecule
involved in density-dependent inter- and intra-species signaling. Quorum sensing molecules, like
AI-2 and acyl-homoserine lactone (AHL), are linked to shifts in types and concentrations of
microbial populations, resulting in a transition from health to disease. Conclusion: These data
provide new insights into the identity, organization, as well as synergistic and inhibitory
interactions of microorganisms within the chronic wound. Such information may provide
important clues to effective strategies in wound healing.
YIA09
The first place winner of the 2009 ETRS Young Investigator competition (Limoges, France,
August 2009):
THE RAC2 GTP-ASE CONTROLS BETA 2-INTEGRIN DEPENDENT MACROPHAGE
FUNCTIONS DURING WOUND HEALING
Sindrilaru A 1 , Moepps B 2 , Peters T 1 , Treiber N 1 , Gierschik P 2 , Scharffetter-Kochanek K 1

1 Department of Dermatology and Allergology, University of Ulm, Ulm, Germany
2 Institute of Pharmacology and Toxicology, University of Ulm, Germany
Proper migration and function of leukocytes to wound sites during the inflammatory phase is a
prerequisite for physiologic wound healing and depends on adhesion molecules-mediated cellcell
and cell-matrix interactions. β2-integrins are adhesion and signalling molecules which
essentially control leukocyte functions by triggering cytoskeletal rearrangements via
RhoGTPases. We have previously shown that β2-integrins and their downstream target
molecule, the GTPase ativator Vav3, mediate the adhesion-dependent phagocytosis of apoptotic
neutrophils by macrophages, and their subsequent release of TGF-β1, the major growth factor
promoting wound contraction. As a result, both β2-integrins- and Vav3-deficient mice presented
with severely delayed wound healing when compared with wildtype mice. However, it remains
unclear which GTPase(s) control the CD18-Vav3-dependent activation of macrophages to
release TGF-β1 during inflammation. As Rac2 is the most abundant GTPase in macrophages, we
here investigated the role of Rac2 in leukocytes functions required for wound healing.
In “full-thickness” wound healing experiments, neutrophil recruitment to wound sites was
reduced to 50% in Rac2-/- mice compared to wildtype mice, while recruitment of macrophages
was not affected. Rac2-/- macrophages revealed a 30% to 40% reduction of their capacity to
adhere to and phagocytose apoptotic neutrophils in in vitro co-culture experiments, suggesting a
role of Rac2 in β2-integrin-dependent phagocytosis of apoptotic neutrophils by macrophages.
Rac2 pull-down assays revealed a β2-integrin-specific increase in active GTP-Rac2 in
macrophages after adhesion onto the specific ligand ICAM-1. Rac2-/- mice presented with
significantly delayed wound closure compared to wildtype mice, similarly with β2-integrins- and
Vav3-deficient mice. Taken together, these data are suggestive for a role of Rac2 in b2-integrindependent
signalling in macrophages during wound healing. Targeting of molecules signalling
downstream of β2-integrin receptors may provide valuable tools to selectively modulate wound
healing and inflammation.
PODIUM PRESENTATIONS
Mini Symposia 1 - Burns and Ischemic Wounds - MS1.01-MS1.08
Sunday, April 18, 2010, 4:45 to 7:00 pm
MS1.01
EXPLORATION OF THE SOURCE OF HYPOXIA IN THE BURN WOUND
Dongmei Xing, MD, Ph.D, Xianjie Zhang, MD, Ph.D, Lixin Liu, Ph.D, Guy Marti, MD, Gregg
L. Semenza, MD, Ph.D, John W. Harmon, MD, FACS.
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
The role and importance of hypoxia in wound healing remains elusive and controversial. Using
Pimonidazole we have identified hypoxia in the healing margin of burn wounds in a murine
model beginning at 48 hours after injury. Using immunohistochemistry we have co-localized
increased levels of Hypoxia Inducible Factor 1 alpha (HIF-1 alpha) and Stromal Derived Factor-

1 in the same regions over the same time course. In this model wound blood flow peaks at 7 days
after wounding suggesting a physiologic relevance for HIF-1 alpha in angiogenesis in the
wounds. We report now immunohistochemical assessment of the cells in the healing edge to
explore the cause of hypoxia in that region. Searching for inflammatory cells that might be
hypermetabolic in the area, myeloperoxidase and F4/80 staining showed that there were rare
neutrophils and macrophages in the region of hypoxia on days 2 and 3 after burn. Searching for
cellular proliferation in that region, Ki 67 staining showed that the proliferative zone was
proximal, not co-localized with the hypoxic zone. Pancytokeratin staining was positive for the
entire regenerating layer including the proximal normoxic and the distal hypoxic regions.
Finally, Keratin 17 staining was positive just in the hypoxic region. K 17 is an intermediate
filament protein which is required for the stimulation of mTOR activity for cell growth and
tissue remodeling. In conclusion, we propose that the rapidly growing, differentiating, and
migrating cells in the leading edge of the healing zone of the burn wound utilize aerobic
metabolism and become hypoxic. This hypoxia stabilizes HIF-1 alpha and leads to angiogenesis.
In contrast the adjacent proliferating zone, as noted in other tissues, may utilize anaerobic
metabolism, explaining why it does not become hypoxic.
MS1.02
POSSIBLE ROLE OF STROMAL CELL-DERIVED FACTOR 1 (SDF-1)/CXCR4
SIGNALING IN THE DEVELOPMENT OF HYPERTROPHIC SCAR (HTS) AFTER
BURN INJURY
Jie Ding, MD., PhD, Keijiro Hori, MD., PhD, Rainny Zhang, MD., PhD, Jianfei Wang, MD.,
PhD, Edward E Tredget, MD., MSc., FRCSC.
University of Alberta, Edmonton, AB, Canada.
PURPOSE: Recent data revealed that SDF-1 might be responsible for the recruitment of bone
marrow-derived stem cells to wound sites during skeletal, myocardial and lung repair via its
receptor CXCR4, and that SDF-1/CXCR4 contributed to lung fibrosis. We explore the role of
SDF-1/CXCR4 in wound healing and the formation of HTS following burn injury. METHODS:
The expression of SDF-1 and CXCR4 were examined by immunohistochemistry in skin biopsies
from burn patients. SDF-1 mRNA levels were determined by real time RT-PCR in dermal
fibroblasts in vitro. SDF-1 protein levels in patient serum and in fibroblast-conditioned medium
were measured by ELISA. As well, we determined the effect of SDF-1 and fibroblastconditioned
medium on the mobility of PBMCs. RESULTS: In proportion to scattered
expression of SDF-1, CXCR4 is diffusedly expressed in HTS and normal skin and was elevated
in HTS compared to normal skin. In vitro, deep dermal fibroblasts constitutively expressed more
SDF-1 than superficial fibroblasts (Relative expression, 2.0±0.2 vs 1.0±0.1, p=0.015). Although
no significant difference, LPS increased the SDF-1 expression (1.7±0.2 vs 1.0±0.2), and
following LPS stimulation, IFNα2b down-regulated SDF-1 expression (1.1±0.1 vs 1.7±0.2 at
24hr, p=0.059). Burn injury caused an acute increase in the serum SDF-1 level of burn patients,
and the appearance of the SDF-1 peak seemed to be related to total body surface area (TBSA)
and age. SDF-1 and LPS-stimulated fibroblast-conditioned medium significantly increased the
migration of PBMCs, and IFNα2b decreased the migration following LPS stimulation (50.3±2.9
at 24hr, 39.3±3.7 at 48hr, 40.0±2.5 at 72hr, vs 66.0±2.0, p=0.001). CXCR4 expression was

decreased during IFNα2b treatment in HTS from burn patients. CONCLUSIONS: SDF-1 was
up-regulated by proinflammatory factors in fibroblasts from the deep dermis and it promoted the
migration of CXCR4-expressing cells from the blood to the wounds, where they may be
involved in wound healing and HTS formation.
MS1.03
HEME OXYGENASE-2 MODULATES DERMAL WOUND CONTRACTION AND
RESOLUTION OF INFLAMMATION
Alwin Scharstuhl, PhD 1 , Bas Pennings, BSc 1 , Jeroen te Paske, BSc 1 , Koen Scheerders, BSc 1 ,
Raymond Regan, PhD 2 , René van Rheden, BSc 1 , Frans Russel, PhD 1 , Frank Wagener, PhD 1 .
1 Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, 2 Thomas Jefferson
University, Philadelphia, PA, USA.
Following dermal wounding, the severity of scar formation seems to be inversely related to the
speed of wound contraction. Therefore, we set out to identify novel factors that are involved in
dermal wound contraction. We previously demonstrated that heme oxygenase (HO)-activity, via
generation of carbon monoxide and bilirubin, can modulate in vitro wound contraction. Here, we
studied the role of HO in more detail using in vivo models of wound contraction. Four excisional
wounds (4 mm) were made on the dorsal skin of HO-2 knock-out (KO) mice and wild type (WT)
controls, and additionally, wounds were made in hyperbilirubinemic Gunn-UDPTGTJ and WT
Gunn-UDPTGT+ rats. Wound size was documented daily up to day 7 by digital photography.
Our results show that in HO-2 KO mice wound contraction was significantly slower on day 1, 2,
5 and 7 compared to WT controls. In contrast, in hyperbilirubinemic rats wound contraction was
significantly faster than in WT rats in the early phases of wound healing, suggesting that HOeffector
molecules can modulate wound contraction also in vivo. Leukocyte infiltration and HO-
1 expression in the wound area were significantly higher in HO-2 KO animals compared to WT
controls at day 7 following wounding. Surprisingly, serum bilirubin levels in HO-2 KO mice
were significantly higher at baseline than in WT controls and also HO-1 mRNA levels on day 2
were significantly increased when compared to WT controls. Since abrogation of HO-2 results in
a delay of dermal wound closure and hampers resolution of inflammation despite increased HO-
1 expression, our data suggest that not only HO-1, but also HO-2 proteins are crucial in the
resolution of inflammation. In conclusion, we show that both HO isoforms and its effector
molecules are involved in dermal wound contraction and therefore likely in the process of scar
formation.
MS1.04
INFLUENCE OF HEAVY METAL PROTOPORPHYRINS ON BURN WOUND
CONVERSION
Dorne Yager, PhD, Kimberly Oh, MS, Katherine Braun, MS, Michael Feldman, MD, Andrea
Pozez, MD.
Virginia Commonwealth University, Richmond, VA, USA.

Heavy metal protoporphyrins are well characterized modulators of the stress response protein,
heme oxygenase. This study tested the hypothesis that cobalt protoporphyrin, an inducer of heme
oxygenase-1 expression, and stannous protoporphyrin, an inhibitor of heme oxygenase-1
activity, can influence the level of burn wound conversion and that the level of conversion would
correlate with expression of markers of inflammation.
Methodology: Animals were placed into three groups of eighteen. One group received burn
injury only. The animals in a second group received a single bolus of cobalt protoporphyrin and
were then burned on the following day. Animals in the third group received a bolus of stannous
protoporphyrin the day before injury. Burn injury was created using a comb template that
generated three regions of full-thickness burns with two intervening spaces representing zones of
stasis. Wound morphology (necrosis), heme oxygenase-1 expression, and markers of
inflammation were examined on days 1, 3, and 7 post-injury. In a follow up study, a fourth group
of animals cobalt protoporphyrin was administered thirty minutes post-wounding. Results:
Necrosis within the intervening space was increased in animals pretreated with stannous
protoporphyrin. Pretreatment with cobalt protoporphyrin exhibited significantly less necrosis
than animals of the other two groups. Levels of myeloperoxidase activity and matrix
metalloproteinase-9 correlated with levels of necrosis. Examination of VCAM-1 revealed
significantly lower levels in animals pretreated with cobalt protoporphyrin. In the post-wounding
group, the mean areas of necrosis were smaller in the animals receiving cobalt protoporphyrin
although this difference (p=0.055) was not statistically significant. Conclusion: This study
demonstrated the ability of heavy metal protoporphyrins, and by inference, heme oxygenase-1, to
modulate burn wound conversion. Heme oxygenase-1 generates products with potent
antioxidant, anti-inflammatory and anti-apoptotic actions. Modulation of heme oxygenase-1 or
use of its products may provide a means for controlling burn conversion.
MS1.05
USING MATHEMATICAL MODELING TO ASSESS THE EFFICACY OF OXYGEN
FOR PROBLEM WOUNDS: USE OF HYPERBARIC OR TOPICAL OXYGEN
THERAPIES
Richard C. Schugart, Ph. D. 1 , Jennifer Flegg, Ph. D. 2 , D.L.S. McElwain, Ph. D. 2 .
1 Western Kentucky University, Bowling Green, KY, USA, 2 Queensland University of
Technology, Brisbane, Australia.
We extend a previously developed mathematical model (Schugart, R.C., Friedman, a., Zhao, R.,
Sen, C.K., Wound angiogenesis as a function of tissue oxygen tension: a mathematical model,
PNAS USA 105: 2628 - 33, 2008) for acute wound healing to investigate the application of
hyperbaric and topical oxygen therapies to treat acute, delayed, and chronic wounds. The
mathematical model is a nonlinear system of partial differential equations, which describes the
complex interactions in both space and time of inflammatory cells, endothelial cell tips,
endothelial cell sprouts, fibroblasts, extracellular matrix, oxygen, and growth factors. The
equations were solved in Matlab using pdepe. A sensitivity analysis was conducted on the model
to indentify the parameters that most affect the healing response. From the analysis, we identified
oxygen uptake by inflammatory cells as a parameter that can create a delayed healing response,
and the chemotactic response of inflammatory cells as a parameter that can create a chronic

healing response, as defined by a persistence of inflammatory cells at the wound site. For the
acute wound, our model suggests that hyperbaric oxygen heals the wound at a faster rate than
topical oxygen therapy. For the delayed healing response, the wound healed better with topical
oxygen than with hyperbaric oxygen therapy. For the chronic wound, both therapies decrease the
inflammatory response and improve the healing response, with a slightly faster healing response
for hyperbaric oxygen than topical oxygen therapy. The model suggests that the different
therapies change the oxygen gradients in the wound, which affects how the wound heals. We
conclude that mathematical models can not only provide potentially useful insights into the
mechanisms that contribute to the healing response, but also suggest which therapeutic strategy
might best heal the wound.
MS1.06
CD109, A NOVEL TGF-β ANTAGONIST, DECREASES EXTRACELLULAR MATRIX
PROTEIN PRODUCTION IN THE SKIN UNDER ISCHEMIC CONDITIONS
Sebastian Winocour, M.D., Joshua Vorstenbosch, B.Sc., Alissa Trzeciak, B.Sc., Lucie Lessard,
M.D., Anie Philip, Ph.D..
Division of Plastic Surgery, Department of Surgery, McGill University, Montreal, QC, Canada.
Background:Our group has identified a novel TGF-β antagonist CD109 which inhibits
extracellular matrix (ECM) protein production in vitro. Since some fibrotic skin disorders such
as hypertrophic scarring and scleroderma are associated with hypoxia, we examined whether
CD109 is able to regulate ECM deposition under low oxygen tension in vivo. Specific Aim:To
determine whether CD109 regulates ECM production during ischemic wound healing using
transgenic mice overexpressing CD109 in the skin. Methods:Transgenic mice and wild-type
littermates were used to generate an ischemic wound model by creating dorsal bipedicle skin
flaps with centrally located excisional wounds. Excisional wounds were made in each
experimental group: (i) without skin flaps (n=6), (ii) with skin flaps but no underlying silicone
sheet (n=6), and (iii) with skin flaps having an underlying silicone sheet (n=6). Mice were
sacrificed at 7 or 14 days after surgery, and tissues were harvested for analysis. The degree of
ischemia was evaluated by HIF-1α levels and histological changes were analyzed by H&E and
Masson's Trichrome staining. Finally, alterations in ECM protein levels and TGF-β signaling
pathway components were determined by Western blot. Results:Both transgenic and wild-type
mice on days 7 and 14 post-wounding showed increased levels of HIF-1α in ischemic excisional
wounds, validating this animal model in mice. Transgenic mice on day 7 post-wounding
demonstrated decreased collagen I and fibronectin deposition in ischemic wounds as compared
to their wild-type counterparts, and to non-ischemic internal control wounds. However, no
difference in angiogenesis was observed in wounds of transgenic mice as compared to those in
their wild-type littermates. Conclusions:Ischemic wounds in transgenic mice display decreased
collagen I and fibronectin content, suggesting that CD109 inhibits ECM production under low
oxygen tension. As a result, manipulating this molecule may have potential therapeutic value for
the treatment of fibrotic skin disorders which are associated with poor oxygen delivery.
MS1.07

CARDIAC PROGENITOR CELL RECRUITMENT IS ASSOCIATED WITH
RESTORATION OF CARDIAC FUNCTION IN FETAL REGENERATIVE HEALING
Myron Allukian, III, MD 1 , Benjamin J. Herdrich, MD 2 , David H. Stitelman, MD 2 , Dustin M.
Bermudez, MD 2 , Marcus G. Davey, PhD 3 , Robert C. Gorman, MD 1 , Joseph H. Gorman, MD 1 ,
Kenneth W. Liechty, MD 4 .
1 University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 2 University of
Pennsylvania School of Medicine, Center for Fetal Research at the Children's Hospital of
Philadelphia, Philadelphia, PA, USA, 3 Center for Fetal Research at the Children's Hospital of
Philadelphia, Philadelphia, PA, USA, 4 University of Mississippi Medical Center, Jackson, MS,
USA.
Purpose: We have previously shown that following fetal myocardial infarction (MI), the heart
has the ability to heal by way of regeneration thus restoring left ventricular function and
promoting normal morphology of the myocardial tissue. The mechanism of fetal regenerative
cardiac healing is unclear. However, it is believed that proliferating, cardiac progenitor cells
surround the infarct. We hypothesized that following fetal MI; there would be migration of nkx
2.5+ cardiac progenitor cells to the area of infarction. Methods: Fetal anteroapical MI
encompassing approximately 20% of the LV mass was created in early gestation fetal sheep. The
fetal heart was harvested 3 days following MI. Immunohistochemistry utilizing confocal
microscopy was used to identify the presence of nkx 2.5+ cells in the infarct, the border zone,
and the uninfarcted myocardium. Age matched uninfarcted fetal sheep heart was the control.
Results: Three days following fetal MI, the presence of nkx 2.5+ cells was preferentially
increased at the endocardial, epicardial, and perivascular borders within the anteroapical infarct.
Nkx 2.5+ cells were also seen in the border zone and the uninfarcted myocardium; however, the
numbers decreased significantly away from the infarct. Nkx 2.5+ cells were identified in control
hearts; however, there was no evidence of increased numbers in the apex or clustering around
blood vessels within the fetal myocardium. Conclusions: Following fetal MI, increased numbers
of nkx 2.5+ cells were found at the area of infarction. This suggests that cardiac progenitor cells
participate in the regenerative capacity of the fetal heart following MI. In addition, the increased
numbers of nkx 2.5+ cells surrounding blood vessels suggests that some of these cardiac
progenitor cells are recruited from the peripheral circulation. We hope to better understand the
mechanisms that underlie the fetal regenerative response to healing so that we can alter the postnatal
response to MI.
MS1.08
HYPOXIA ENHANCES SELF-RENEWAL OF MULTIPOTENTIAL STROMAL CELLS
THROUGH HIF-DEPENDENT AND HIF-INDEPENDENT MECHANISMS
Kenichi Tamama, MD, PhD, Haruhisa Kawasaki, Ph.D., Ramesh K. Ganju, Ph.D., Chandan K.
Sen, Ph.D..
The Ohio State University Medical Center, Columbus, OH, USA.
Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising
approach to promote wound healing and tissue regeneration. MSCs could be expanded in vitro;

however, both differentiation and therapeutic potentials of MSCs are gradually lost during in
vitro expansion. Ambient O2 (20% as opposed to 2-9% in vivo) has been recognized as an
excessive O2 condition that limits the life span of cultured primary cells. Hypoxia promotes MSC
self-renewal through preserving early progenitors and maintaining undifferentiated phenotypes;
however, key signaling pathways remain unknown. Hypoxia inducible factor (HIF) pathway is a
crucial signaling pathway activated in hypoxic condition. We hypothesize that HIF plays a
pivotal role to enhance MSC self-renewal in hypoxic condition. Immunoblot analysis suggested
that HIF-2α was stabilized whereas that of HIF-1α was unaltered under hypoxic condition;
however, immunofluorescent images demonstrated that both HIF-1α and HIF-2α were
translocated to the nucleus only under hypoxic condition. Furthermore, our studies revealed that
hypoxic condition enhanced MSC colony formation. It was neither reversed by HIF-1β shRNA
nor reproduced by introduction of stable HIF-1α and HIF-2α, suggesting that hypoxia increases
MSC colony formation in a HIF-independent manner. Both stable HIF-1α and HIF-2α increased
cell cycle arrest. Osteogenic differentiation, default differentiation pathway for MSCs, was
reversibly inhibited by hypoxic exposure. This inhibition was further reversed by HIF-1β shRNA
and reproduced by stable HIF-2α, indicating that hypoxia inhibits osteogenic differentiation in a
HIF-dependent manner. Overall, these data demonstrate that hypoxic condition enhances MSC
self-renewal through HIF-independent as well as HIF-dependent mechanisms.
Mini Symposia 2 - Wound Therapies: Mechanisms and Approaches - MS2.01-MS2.08
Sunday, April 18, 2010, 4:45 to 7:00 pm
MS2.01
INTRACELLULAR DELIVERY OF ATP-VESICLES ON INCISIONAL SKIN WOUNDS
IN RABBITS
Jianpu Wang, PhD, Alexander Bajorek, MD, Sufan Chien, MD.
University of Louisville, Louisville, KY, USA.
Every year, there are over 50,000,000 elective surgical incisions made in the US. Even after
strict precautions, wound dehiscence and incisional hernia occur. Hundreds of substances have
been tried to increase tensile strength, yet no agent has shown clearly effective for augmentation
of the wound healing in humans. We have used highly fusogenic lipid vesicles for intracellular
ATP delivery and tested the skin tensile strength in incisional wounds. Using 12 New Zealand
White rabbits, two full thickness 10 cm incisions were made on both sides of the spina, and
closed with 4-0 sutures. The wound on one side received ATP-vesicles mixed with a neutral
cream and other side received the neutral cream only as controls. The wounds were covered with
a piece of gauze and Tegaderm TM . Dressing changes were made daily, and the animals were
sacrificed at days 4, 7, and 14. The sutures were removed and skin strips (5 mm) with the
incisional wound at the center were taken for tissue tensile measurements and histological
analyses using the MTS Insight Material Testing System with 1000N load cell. Tissue samples
were placed between two tensile clampers and stretched up to rupture, and the tensile was
captured by computer software. At 7 days post-operation, the wound breaking force was much
higher in the wounds treated by ATP-vesicles than those treated by vehicles (p

vehicles but the different was not statistical significant. The result indicated that incisional
wound breaking strength was increased with the application of ATP-vesicles.
MS2.02
INCREASED RATE OF FULL THICKNESS DERMAL WOUND CLOUSURE AFTER
TREATMENT BY PULSED RADIO FREQUENCY ENERGY (PRFE) FIELDS IN THE
PORCINE MODEL.
John Moffett, Ph.D. 1 , Naomi M. Gades, D.V.M., M.S. 2 .
1 Regenesis Biomedical, Inc, Scottsdale, AZ, USA, 2 Mayo Clinic Arizona, Scottsdale, AZ, USA.
Abstract: The objective of the research is to establish an animal model that will allow for the
identification of optimal treatment parameters of PRFE fields to accelerate wound healing.
Understanding of the biological mechanism(s) associated with PRFE field treatment will lead to
further application of this modality to the broad range of wounds. In our pilot study we used a
full-thickness porcine model. We show that wounds treated with the PRFE fields exhibit an
increased rate of closure over that of sham treated controls. Materials and Methods: Animals
(Sus Scrofa Domestica) were acclimatization and trained for at least 7 days. All studies were
performed in accordance with standard animal procedures. Wounding was performed under
general anesthesia with sterile conditions. Three quadrates were used one which was used as a
control. One quadrant per animal was used as an untreated control. The animals were
photographed and biopsied during the study. The wounds were traced and rates of wound closure
were determined (mm 2 /day of wound closure). Histological analysis was performed using H & E
staining to determine the anatomical and gross cell infiltration of the wound site. Results: The
data shows that the animal treated with Provant shows faster rates of wound healing as judged by
wound closure measured in mm 2 /day (p

insulin-induced improved healing are not well known. We have recently shown that insulin acts
early in wound healing by stimulating keratinocytes through the insulin receptor, PI3K, Akt, src,
SREBP, LN5 and a3integrin, accelerates their migration in vivo. Furthermore, insulin stimulates
angiogenesis in vivo by increasing tube formation and also the number of periendothelial cells
suggesting a role not only in stimulating blood vessel formation but also their stabilization.
Stromal cell-derived factor-1α (SDF1α) is a chemokine that play roles in bone marrow cell
mobilization that are thought to be important in wound healing. Thus, we hypothesized that a
combination of both insulin and SDF1α may greatly improve the overall wound healing process.
To test this possibility, mice were wounded and treated with single or combined therapy for 15
days. The wound areas were recorded daily and statistical analysis was then performed. We show
that early in the healing process insulin alone accelerates wound healing, but insulin treatment
followed by SDF1a further accelerates wound closure. Histological observations show that the
combined sequential treatment also increases the quality of healing. We are currently testing the
effects of SDF1a on wound cells to elucidate its effects on these cells. In summary, our data
suggest that using insulin followed by SDF1α leads to a beneficial treatment combination for
wound healing and has the potential to treat a variety of wounds, including burns and chronic
wounds.
MS2.04
OPEN CAVITY USE OF ACELLULAR XENOGRAFT WOUND MATRIX
Aamir Siddiqui, MD, Cheryln Bayer, RN, Judith Flanz, RN, Shirley Peebles, RN.
Henry Ford Hospital, Detroit, MI, USA.
Introduction; Pressure ulcers that do not respond to evidence-based treatment guideline
interventions present a dilemma for caregivers. One particularly challenging problem is a large
cavity defect with a small skin opening. For high output chronic wounds, healing may be
impeded by the inability to control the volume of drainage. For patients not interested in surgical
intervention, very few options remain. We used acellular wound matrix material to treat these
wounds. Methods; Patient wounds that failed to progress for a minimum of 6 weeks were offered
this intervention. Eight wounds were ischial and 4 were trochanter pressure ulcers. The Oasis®
was cut into 1 centimeter squares and placed into the cavity. This is done 4 weeks in a row and
then stopped for 4 weeks. If there was no response noted, a second course of Oasis® treatment
was performed. Results; At the start of treatment the average cavity size was 45± 37 cm2. Nine
patients received a total of 8 weeks of acellular wound matrix therapy. At one year after
treatment 6 wounds were healed. Five of the six wounds that healed received a total of 8 weeks
of Oasis® treatment. Average wound size for unhealed wounds was 18 ±32 cm2. None of the
trochanter wounds healed. Conclusion; Deep cavity placement of Oasis® stimulated healing for
5 ischial ulcers that failed to respond to evidence-based interventions. We hypothesize that
acelluar wound matrices can decrease drainage and increase tissue plane adherence. This in turn
may shrink the cavity a sufficient amount to jumpstart the healing process. Further research is
needed to determine specific indication for use of in deep cavity high output wounds.
MS2.05

SUCCESSFUL CLOSURE OF LARGE, RECALCITRANT VENOUS LEG ULCERS
USING COMBINED SPLIT-THICKNESS SKIN GRAFTING AND POST-OPERATIVE
COMPRESSION THERAPY
George Perdrizet, MD, PhD 1 , Brian M. Allen, MD 2 , Alan Babigian, MD 2 .
1 Morristown Memorial Hospital, Morristown, NJ, USA, 2 Hartford Hospital, Hartford, CT, USA.
Historically, surgeons have been reluctant to offer split-thickness skin grafting (STSG) for the
closure of chronic venous leg ulcers (VLU) because of the high rate of skin graft break-down and
ulcer recurrence. Most VLU’s (80%) can be closed by the diligent application of the standard
principles of comprehensive wound care including, moist wound care, topical antimicrobial
therapy to control bioburden, frequent debridement to control biofilm, and multilayer
compression therapy to control lower extremity edema. Purpose- To describe the role of STSG in
the management algorithm for patients with large (> 30cm 2 ) , recalcitrant (> 6 months of
comprehensive wound care)VLU’s. For patients with large, recalcitrant VLU’s we utilized the
application of STSG’s to effect their closure and employed early (day 5) post-operative
compression therapy to achieve success in these patients. Results- Retrospective review of the
clinical outcomes in 138 patients referred to our Comprehensive Wound Care center with the
diagnosis of VLU over a 3 year period (2005-2008). A total of 360 wounds were treated during
this time period. Of these, 287 (80%) wounds were treated by standard comprehensive
techniques with an 89% rate of closure and a mean time to closure of 118 days. An additional 73
(20%) wounds (large, recalcitrant group) underwent closure using STSG with an 84% rate of
closure and a mean time to closure of 345 days. Post-operative care was provided by the Wound
Care Center to ensure continued, effective compression therapy was in place and a plan for
ongoing monitoring and follow-up established. Conclusion- Surgeons can improve outcomes for
patients with large, recalcitrant VLU’s by wound coverage using standard STSG techniques.
Wound Care Centers can ensure success by incorporating aggressive post-operative compression
therapy and long-term follow-up.
MS2.06
A ROLE FOR ADAMTS-5 IN GRANULATION TISSUE REMODELING AND
FIBROSIS
Jennifer Velasco, B.S., Jun Li, M.D., Anne-Marie Malfait, Ph.D., John Sandy, Ph.D., Anna
Plaas, Ph.D..
Rush University Medical Center, Chicago, IL, USA.
ADAMTS-5 is a metalloproteinase which has high specificity for the cleavage of large matrix
proteoglycans aggrecan, brevican and versican, resulting in the loss of chondroitin sulfate from
tissues such as cartilage. We have developed a murine model of osteoarthritis, which involves
intra-articular injections of TGFβ-1 and daily treadmill running. After 2 weeks, the joints of
wild-type (WT) mice exhibit a robust synovitis along with cartilage erosion. In contrast,
ADAMTS-5 knockout mice (C57Bl6, adamts5Δexon2) are protected from these changes,
suggesting that ADAMTS-5 activity is required for synovial fibrosis in response to injury. The
synovial fibrosis seen in WT mice is preceded by the formation of granulation tissue, in a

process which has features similar to those seen in dermal wound-repair. In order to examine the
role of ADAMTS-5 in the fibrotic response to joint injury, we followed the standard excisional
punch-wound model. Macroscopic photography and comparative histology showed that WT
mice exhibited the normal repair steps of inflammation, matrix remodeling and dermal closure.
However, the ADAMTS-5 (TS-5) KO mice achieved re-epithelialization and failed to restore an
organized functional dermis. As a result, granulation tissue was observed at Day 15, at which
rupture of the epithelial layer also occurred. Confocal microscopy showed marked differences
between the WT and TS-5 KO mice, in the spatial and temporal distribution of ADAMTS-5, its
substrates (aggrecan and versican) and the matrix-retained products of cleavage (aggrecan G1-
NITEGE and versican G1-DPEAAE). In addition, Western blot analysis confirmed the temporal
differences between WT and KO mice in the abundance of aggrecan, versican and its cleavage
products in the wound site. It appears that ADAMTS-5-mediated proteolysis of large chondroitin
sulfate-substituted proteoglycans plays a central role in the inflammation and granulation tissue
remodeling which are required for functionally-effective dermal reconstruction in wound
healing.
MS2.07
ANTIBODY MICROARRAY PROFILING UNCOVERS MOLECULAR ANOMALIES
IN CHRONIC VENOUS LEG ULCERS
Hannah Trøstrup, MD 1 , Rasmus Lundquist, MSc 1 , Lars Jorgensen, MD, DrMedSci 1 , Tonny
Karlmark, MD, DrMedSci 1 , Brian Haab, PhD 2 , Magnus S. Ågren, DrMedSci 1 .
1 Bispebjerg Hospital, Copenhagen, Denmark, 2 Van Andel Research Institute, Grand Rapids, MI,
USA.
Increased levels of pro-inflammatory cytokines, chemokines and tissue destructive proteinases
but deficiencies of angiogenic and other growth factors, antimicrobial peptides, proteinase
inhibitors and extracellular matrix components have been proposed to explain delayed healing of
chronic wounds. We still lack information on the relative levels of these various regulatory
proteins. The aim of this study was to determine unique protein alterations in chronic venous leg
ulcers compared to acute wounds using high-throughput antibody-based microarray proteomics.
The study was approved by the local Ethics Committee. Wound fluids were collected for 24
hours under occlusion from 8 patients (4 females, 82 ± 7 years [mean ± SEM]) with 9 chronic
(154 ± 91 months) venous leg ulcers (35 ± 11 cm2) at 1-week intervals for one month and from
22 patients (4 females, 27 ± 2 years) with 7-day-old open granulating acute wounds (9.8 ± 1.1
cm2) after excision of pilonidal disease. A validated antibody microarray method using twocolor
rolling-circle amplification was used to profile the relative levels of 50 proteins
representing the above-mentioned categories of wound-healing modulators. The chronic ulcers
showed no healing tendency over the 1-month sampling period (5 ± 11 %). Unexpectedly the
relative levels of several of the examined proteins were significantly (p

(250 ± 20 µg/ml versus 450 ± 20 µg/ml; p

Human lactoferrin (hLF), a member of the transferrin family, has been shown to stimulate wound
repair through its antimicrobial activities. To study the direct effects of hLF on wound healing,
we used a rice-derived recombinant human LF (holo-rhLF) to examine the function of hLF in
keratinocyte proliferation and migration, which are two essential processes in reepithelialization.
Our studies revealed that holo-rhLF significantly stimulates keratinocyte
proliferation which can be blocked by MAP kinase pathway inhibitor (PD98059, MEK/ERK
inhibitor). Compared with control cells without adding holo-rhLF, the total cell number was
increased by 28.57% and 26.08% with the supplementation of 200 µg/mL holo-rhLF on day 3
and 5, respectively (both p

(p

MS3.04
BACTERIAL ANTIGENS ARE PRESENT IN CLOSED FRACTURE GAPS WITH
DELAYED BONE UNION
Waldemar L. Olszewski, Professor 1 , Grzegorz Szczesny, Dr 1 , Bozenna Interewicz, Dr 1 , Ewa
Swoboda-Kopec, Dr 2 , Andrzej Górecki, Professor 3 .
1 Dept of Surgical Research &Transplantology, Medical Research Center, Polish Academy of
Sciences, Warsaw, Poland, 2 Department of Microbiology, Medical University, Warsaw, Poland,
3 Departament of Orthopedics & Traumatology, Medical University, Warsaw, Poland.
Introduction. More than 1% of closed fractures of lower limbs and 6% of implanted materials are
complicated by inflammation despite all efforts to avoid infection. Aim.The question arises
whether this clinical complication is not caused by bacteria dwelling in limb tissues. Methods.
Skin, subcutaneous fat, muscle and fracture gap callus were obtained from 155 adult patients
operated on because of closed comminuted fractures of tibia or femur, 75 because of nonalignment
of bone axis and 80 due to delayed fracture healing. Results. Aerobic bacteria were
isolated from gap callus of 12% healing and 31% non-healing fractures. In the subcutaneous
tissue and muscles isolates were found only sporadically. No anaerobic bacteria were detected.
PCR amplifications of 16s rRNA were found positive in 40% of callus specimens proving
presence of bacterial DNA even when no isolates were detected. The 95% similarity of the
genetic pattern of some strains from foot skin and callus, estimated with RAPD technique,
suggested their foot skin origin. Conclusions. The colonizing bacterial cells and their DNA were
detected in fracture callus but not other deep tissues. Contamination was precluded by lack of
isolates in disinfected skin, subcutis, muscles and materials used for sampling and cultured after
surgery. We suggest that certain strains of bacteria dwell in normal tissues of lower limbs and
may cause inflammation upon stimulation by trauma. Their source may be tissue fluid,
superficial and deep lymphatics, and lymph serving the physiological transport to the regional
lymph nodes of microorganisms penetrating foot skin during microinjuries.
MS3.05
MARKERS OF MUCOSAL HEALING IN AIRWAY SECRETIONS EVALUATED IN A
RABBIT MODEL OF SUBGLOTTIC STENOSIS FOLLOWING ANTI-
INFLAMMATORY THERAPY
Selma Cetin-Ferra, MD, Allison B. Tobey, MD, Mark Barsic, BS, Joseph E. Dohar, MD,
Patricia A. Hebda, Ph.D..
University of Pittsburgh, Pittsburgh, PA, USA.
Mucosal injury is associated with up-regulation of inflammatory genes and parallel increases in
secretion levels of IL-1β and prostaglandin E2 (PGE-2), a key product of the COX-2 pathway.
Inflammation follows its classical wound healing cascade in airway mucosa after injury,
resulting in stenosis. We examined the effects of inhibition of COX-2 on wound healing in
airway mucosa. New Zealand white rabbits received cricothyroidotomy and CO2 laser injury
causing subglottic stenosis (SGS). Experimental groups received either topical or systemic COX-

2 inhibitors versus vehicle treatments. Animals were euthanized at 3 or 8 weeks post-wounding,
and airway wounds were macroscopically and microscopically examined. Secretions were
collected with gelatin foam sponge swabs before injury (day0), on post-wounding days 1, 3, 7,
and at the time of euthanasia, then IL-1β and PGE-2, inflammatory markers, and matrix
metalloproteinase (MMP)-8, a fibrotic healing marker, were measured using ELISA kits and
were standardized with secretion wet weight, protein level or urea level. COX-2 inhibition
reduced the severity of stenosis, and correlated with reduced levels of IL-1β and PGE-2 at
intermediate timepoints, while MMP-8 levels were down-regulated in the treatment group at
later timepoints. Quantification with urea concentration in airway secretions showed close
agreement with protein and wet weight in all treatments for each mediator. Inflammatory
mediator levels in secretions for each animal correlated well with histological findings of
stenosis severity. In conclusion, anti-inflammatory therapy resulted in promising reduction of
fibrosis and stenosis and correlated with reduction of markers for inflammation and fibrotic
healing in secretions. Standardization to urea concentration in airway secretions is consistent
with protein levels and wet weight, and is more robust. Further experimentation will focus on
temporal correlation of airway secretion markers with degree of stenosis and potential clinical
use in predicting long term prognosis. This work was supported by NIH grant DC007437.
MS3.06
MECHANICAL FORCE ACTIVATES FOCAL ADHESION KINASE/MCP-1
SIGNALING IN DERMAL FIBROBLASTS
Kristine C. Rustad 1 , Victor W. Wong 1 , Jason P. Glotzbach 1 , Michael Januszyk 1 , Melanie R.
Major 1 , Anna A. Kuang 2 , Michael T. Longaker 1 , Geoffrey C. Gurtner 1 .
1 Stanford University, Stanford, CA, USA, 2 Oregon Health & Science University, Portland, OR,
USA.
Mechanoresponsive focal adhesion kinase (FAK) pathways associated with monocyte
chemoattractant 1 (MCP-1) production were implicated in our murine model of hypertrophic scar
(HTS) formation. Thus we sought to elucidate the role of FAK-mediated MCP-1 pathways in
dermal fibroblasts using novel in vitro and in vivo biomechanical systems. We utilized an in
vitro equibiaxial strain system to examine FAK phosphorylation and MCP-1 secretion in human
fibroblasts. Small molecule inhibition of fibroblast FAK signaling was employed to confirm a
role in mechanical activation of MCP-1 signaling. For in vivo studies, we generated dermal
layer-specific FAK KO mice using the Cre/loxP system. Our validated mechanical load model
for HTS was applied to both wildtype and dermal FAK KO mice, and MCP-1 gene expression
and protein production were assayed, as well as wound apoptosis and macrophage quantity.
Mechanical strain activated FAK phosphorylation and induced significantly higher MCP-1
secretion in dermal fibroblasts. Dermal FAK KO scars demonstrated significantly decreased scar
area, weaker MCP-1 staining, and increased keratinocyte apoptosis. Dermal KO scars exhibited a
marked decrease in MCP-1 gene expression and a trend toward decreased MCP-1 production as
compared to wildtype scars. Mechanical forces may modulate HTS formation through FAKmediated
release of MCP-1 from dermal fibroblasts, which appears to both recruit macrophages
and promote keratinocyte proliferation. These effects are abrogated by deletion of FAK signaling

in dermal fibroblasts. Strategies that target FAK mechanotransduction and/or MCP-1 signaling
may lead to novel anti-fibrotic therapies.
MS3.07
COMBINATORIAL ANTI-INFLAMMATORY THERAPY FOR MORE
REGENERATIVE HEALING IN EXPERIMENTAL SKIN WOUNDS
Tianbing Yang, PhD, Joseph E. Dohar, MD MS, Patricia A. Hebda, PhD.
University of Pittsburgh, Pediatric Otolaryngology, Pittsburgh, PA, USA.
Early inflammation helps determine long-term healing outcome, and in general, more
inflammation is thought to produce more scar. However, our lab has previously shown that the
inflammatory mediator prostaglandin (PG)E2 inhibits fibroblast migration and collagen
production and hence may help reduce scar. We hypothesized that PGE2 may be a beneficial
inflammatory signal, and that, if used with an anti-inflammatory agent that blocks potentially
detrimental mediators, regenerative, less fibrotic healing will ensue. Therefore, we investigated
short duration, early anti-inflammatory therapy with Nimesulide, a COX-2 inhibitor, plus PGE2
(NP). We evaluated tensile strength and collagen content and organization in rat wounds. At
PWD(post-wound day)14, the tensile strength reached 77±37 (g/sq-mm) in the NP group, while
only 50±12 (g/sq-mm) in the vehicle group (p=0.003). Interestingly, each agent alone did not
produce this beneficial effect. To explore the mechanism, wound were biopsed at PWD 4,7,14,
and 21. Total collagen was evaluated as hydroxyproline, and collagens I and III by Western
blotting. NP treatment increased total collagen, but decreased collagen I. Other biomarkers were
tested by Western Blotting and among them TGFβ1 was increased only at PWD21. Polarized
light microscopy of picrosirius-stained wounds was used to evaluate collagen organization. NPtreated
wounds showed greater organization of collagen (9.8% at PWD14 and 21% at PWD21
(normalized to uninjured dermis), which was higher than vehicle treated wounds (3.3% at
PWD14 and 12% at PWD21). Infiltration of inflammatory cells and fibroblasts was evaluated
using morphometric analysis. There was decreased inflammatory cell infiltration for both
Nimesulide and NP groups, and a slight increase with PGE2 alone, compared with the vehicle. In
conclusion, combinatorial anti-inflammatory therapy with a COX-2 inhibitor and PGE2
produced beneficial effects in the quality of healing, through changes in cell infiltration, cytokine
expression and collagen production and organization.
Supported by the Armed Forces Institute for Regenerative Medicine
MS3.08
GLUTATHIONE METABOLISM IN YOUNG AND AGED ISCHEMIC WOUNDS
Andrea N. Moor, PhD 1 , Lisa J. Gould, MD, PhD 2 .
1 University of South Florida, Tampa, FL, USA, 2 James A. Haley Veteran's Hospital, Tampa, FL,
USA.
Hyperbaric oxygen (HBO) is often used to treat chronic wounds, however the mechanism of
action remains unclear. We examined the impact of HBO on the glutathione antioxidant defense

system in ischemic wounds of young and aged Fisher rats. Rats underwent surgery to create an
ischemic flap with 6mm circular wounds made within (ischemic) and adjacent to the flap (nonischemic).
Daily HBO (90 minutes, 2.4 atm) was compared to normoxia. Non-ischemic wounds
were healed by day 14, while ischemic wounds remained open through day 21 despite HBO
treatment. Wounds were harvested at 7, 14 and 21 days. Wound lysates were analyzed for the
regulatory subunit of γ-glutamylcysteine synthetase (GCLM), glutathione reductase, glutathione
peroxidase and total glutathione. In young rats, non-ischemic wound glutathione decreased over
time and GCLM did not change significantly. In aged rats, non-ischemic wound glutathione and
GCLM peaked at day 14 and declined as wounds healed. Conversely, in ischemic wounds,
glutathione was nearly undetectable at day 7, rose rapidly by day 14 and remained elevated.
Aged ischemic wounds treated with HBO showed a marked but transient induction of GCLM at
day 14 with a 10-fold increase in glutathione by day 21. Glutathione peroxidase and reductase
levels were not different between groups. GCLM was expressed in epidermis, hair follicles,
muscle, fibroblasts and endothelial smooth muscle but not in endothelial cells of normoxic
tissue, while in HBO-treated ischemic tissue, GCLM was expressed in endothelial cells of neovessels
in granulation tissue. These results suggest that wound healing requires induction of
GCLM in aged rats. In contrast, there is minimal de-novo synthesis of glutathione in wounds of
young rats, indicating sufficient pre-existing antioxidant defense. HBO treatment of ischemic
wounds in aged rats results in a late but substantial increase of de-novo glutathione synthesis,
suggesting a marked, albeit delayed adaptive response to oxidative stress.
Mini Symposia 4 -Fibrosis and Scarring - MS4.01-MS4.08
Tuesday, April 20, 2010, 8:00 to 10:00 am
MS4.01
OVEREXPRESSION OF CD109 IN THE EPIDERMIS ALTERS EPIDERMAL-
DERMAL INTERACTIONS TO PROMOTE WOUND REPAIR
Joshua Vorstenbosch, Albane Bizet, Anie Philip.
McGill University Health Centre, Montreal, QC, Canada.
TGF-β plays a critical role during wound healing and its elevated expression at the wound site
has been linked to wound healing pathologies such as hypertrophic scarring. Our group has
recently identified CD109 as a novel TGF-β antagonist in skin cells in vitro. To determine the
role of CD109 in regulating TGF-β action in the skin in vivo during wound healing, we
developed transgenic mice overexpressing CD109 in the epidermis, and performed wound
healing studies. Our results demonstrate that elevated epidermal expression of CD109 promotes
wound healing and reduces scar formation, with enhanced reepithelialization, faster granulation
tissue resolution and decreased inflammation. To examine the mechanism underlying this
phenotype, we determined extracellular matrix production in whole skin extracts from CD109
transgenic mice and wild-type littermates. We show that whole skin extracts of CD109
transgenic mice have increased levels of fibronectin and collagen I expression with decreased
levels of ALK5 and phosphoSmad2/3, and increased levels of ALK1 and phosphoSmad1/5/8.
We then analyzed the role of keratinocytes and fibroblasts in this model using in vitro monolayer

culture. Transgenic keratinocytes in culture display decreased fibronectin expression and
phosphoSmad2/3 levels, as compared to wild type keratinocytes. To analyze epidermal-dermal
interactions to regulate ECM synthesis in this model, we treated wild-type and transgenic
fibroblasts in monolayer culture with conditioned media from wild-type keratinocytes (WT-
KCM) or transgenic keratinocytes (TG-KCM). Our results show that TG-KCM induces more
extracellular matrix production in fibroblasts than WT-KCM. Together, these results demonstrate
that CD109 promotes wound healing and reduces scarring in vivo and that it exerts these effects
by altering epidermal-dermal interactions. These findings suggest that CD109 is a potential
therapeutic target to improve healing and reduce scarring in vivo in the skin.
MS4.02
KERATINOCYTE TIMP-1 SECRETION DURING HYPERTROPHIC SCAR
FORMATION
Veronique J. Moulin, PhD 1 , Franck Simon, Msc 1 , Danielle Bergeron, Msc 1 , Carlos A. Lopez-
Valle, MD 2 , Hervé Genest, MD 3 , Alexis Armour, MD 4 .
1 LOEX-Université Laval, Quebec, QC, Canada, 2 Hopital de Chicoutimi, Chicoutimi, QC,
Canada, 3 CHA, Quebec, QC, Canada, 4 CHUM, Montreal, QC, Canada.
Hypertrophic scars (Hsc) are disfiguring pathological scars whose pathogenesis is still not well
understood. Myofibroblasts, cells appearing during healing process, allow contraction of the
wound and produce new extracellular matrix (ECM). The most accepted hypothesis of the Hsc
formation is that these myofibroblasts do not disappear at the end of the healing process and
continue to produce ECM and contract scars. We hypothesize that other cells besides
myofibroblasts can play a role in the hypertrophic scar development. The use of the tissue
engineering approach allows placing cells in a very similar context to that found in vivo with the
presence of major elements as fibroblasts or myofibroblasts, keratinocytes and ECM. Our model
used the property of mesenchymal cells to produce ECM similarly to that found in vivo. The
thickness of the reconstructed dermis is thus a reflection of fibrotic capacity of the cells. We
have demonstrated that this thickness was increased when keratinocytes isolated from Hsc were
used in comparison with keratinocytes from normal biopsies. Collagen, MMP and cell growth
variation can explain the fibrotic response of cells depending on keratinocyte origin. Using
proteomic methods, we have determined that hypertrophic scar keratinocytes secreted a
significantly higher level of TIMP-1, a protein inhibitor of metalloproteinases, than normal skin
keratinocytes. Validation of the action of TIMP-1 increase on fibrotic dermal formation,
independently of the origin of the mesenchymal cells, has then been obtained. Pathological
keratinocyte secretion of a higher quantity of TIMP-1 that inhibits the degradation of several
types of collagen could thus be partly responsible for the accumulation of ECM in hypertrophic
scarring.
MS4.03
SPHINGOSINE-1-PHOSPHATE ACTIVATION OF NON MUSCLE MYOSIN II IN
DUPUYTREN’S FIBROBLAST CONTRACTILITY

Issei Komatsu 1 , L Scott Levin, MD 2 , Angelica Selim, MD 3 , Howard Levinson, MD 3 .
1 Duke University, Durham, NC, USA, 2 University of Pennsylvania School of Medicine,
Philadelphia, PA, USA, 3 Duke University Medical Center, Durham, NC, USA.
Purpose: Dupuytren’s disease is putatively caused by fibroblast and myofibroblast contractility
within Dupuytren’s nodules; however, what stimulates cell contractility is unknown.
Sphingosine-1-phosphate (S1P) is a serum derived lysophospholipid mediator that enhances cell
contractility in scar. It is hypothesized that S1P stimulates Dupuytren’s fibroblast contractility
through the activation of calcium dependent, Rho-associated kinase (ROCK), and calcium
independent, myosin light chain kinase (MLCK), to activate non muscle myosin II (NMMII).
This investigation examines the role of S1P signaling and NMMII activation in Dupuytren’s
disease progression and highlights potential targets for disease prevention. Methods:
Dupuytren’s fibroblasts were enmeshed into fibroblast populated collagen lattices (FPCL) and
S1P stimulated FPCL contraction assayed in the presence of the S1P2 receptor inhibitor, JTE-
013, the ROCK inhibitor, Y-27632, the MLCK inhibitor, ML-7, and the NMMII inhibitor,
blebbistatin. Tissues from Dupuytren’s fascia (n=10) and normal palmar fascia (n=10) were
immunostained for NMMIIA and NMMIIB. Results: S1P stimulated FPCL contraction in a dose
dependent manner. Inhibition of S1P2, ROCK, MLCK, and NMMII prevented S1P stimulated
FPCL contraction. Dupuytren’s nodule fibroblasts robustly expressed NMMIIA and NMMIIB, in
comparison to quiescent appearing cords and normal palmar fascia. Conclusions: S1P promotes
Dupuytren’s fibroblast contractility through S1P2, which stimulates ROCK and MLCK
activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytren’s
nodules suggesting that nodule fibroblasts are primed to respond to S1P stimulation to cause
contracture formation. S1P promoted activation of NMMII may be a target for preventing
Dupuytren’s disease progression.
MS4.04
REDUCING CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE SUBUNIT
ETA (CCT-ETA) DECREASES ALPHA-SMA EXPRESSION IN ADULT SKIN
DERIVED FIBROBLASTS
Latha Satish, Ph D, Sandra Johnson, B.S., J C. Post, MD PhD, Garth D. Ehrlich, PhD, Sandeep
Kathju, MD PhD.
Center for Genomic Sciences, Pittsburgh, PA, USA.
Adult mammalian tissues heal skin injury with formation of scar; this process though quickly
seals an injured area, however, excessive scar formation can become a source of persistent
pathology, interfering with numerous vital functions. In contrast, certain mammalian fetal tissue
can heal without scar formation. Our previous studies demonstrated that the eta subunit of the
chaperonin containing T-complex polypeptide (CCT-eta) is specifically reduced in healing fetal
wounds in a rabbit fetal model. We also found that CCT-eta is underexpressed in fetal fibroblasts
compared to adult (postnatal) fibroblasts, whereas the CCT-beta subunit showed no such
differential expression. Futhermore, downregulation of CCT-eta using siRNA in adult fibroblasts
reduced both basal and growth factor-induced cell motility and inhibited growth factor-induced
cellular contractility. In contrast, downregulation of CCT subunit beta had no such effects. In the

present study, we examined the effect of CCT-eta modulation on alpha-smooth muscle actin (α-
SMA) expression, a gene product well known to play a critical role in adult wound healing and
to mediate fibroblast contractility. Fetal fibroblasts were found to constitutively express less α-
SMA than adult cells. Reduction of CCT-eta with siRNA in adult fibroblasts had minimal effect
on cellular beta-actin but markedly decreased α-SMA; in contrast, reduction of CCT-beta had
minimal effect on either actin isoform. Direct inhibition of α-SMA with siRNA in adult
fibroblasts reduced both basal and growth factor-induced fibroblast motility. These results
indicate that CCT-eta is a specific modulator of fibroblast motility and contractility and may be a
key determinant of the scarless wound healing phenotype by means of its specific regulation of
α-SMA expression.
MS4.05
INDUCTION OF NONMUSCLE MYOSIN II EXPRESSION AND MIGRATION IN
KELOID-DERIVED FIBROBLASTS BY ANGIOTENSIN II: IMPLICATIONS FOR
THE TREATMENT OF KELOIDS
Peter C. Thurlow, BS 1 , Maria Angelica Selim, MD 1 , Jennifer Bond, PhD 1 , Anna A. Kuang,
MD 2 , Salvatore V. Pizzo, MD, PhD 1 , L Scott Levin, MD 3 , Howard Levinson, MD 1 .
1 Duke University School of Medicine, Durham, NC, USA, 2 Oregon Health and Science
University, Portland, OR, USA, 3 University of Pennsylvania School of Medicine, Philadelphia,
PA, USA.
Aberrant fibroblast migration in response to fibrogenic cytokines is believed to play a primary
role in the pathophysiology of keloids. Recent evidence has implicated angiotensin II (AngII) as
a mediator of cutaneous wound healing and pathologic scarring; however, AngII signaling in
keloid fibroblasts remains poorly understood. We investigated the effect of AngII signaling on
migration and nonmuscle myosin II (NMMII) expression in keloid fibroblasts as a potential
mechanism of disease formation. Keloid fibroblasts were stimulated with AngII in the presence
of type 1 angiotensin receptor (AT1) antagonist EMD66684, type 2 angiotensin receptor (AT2)
antagonist PD123319, or myosin II inhibitor blebbistatin. Protein expression was quantified by
Western blot. Migration was quantified using a modified Boyden chamber assay. Keloid tissue
sections were then immunostained for NMM IIA, NMM IIB, AT1, and AT2. Ang II increased
keloid fibroblast migration in a dose-dependent manner, an effect inhibited by EMD66684 and
blebbistatin, but unaffected by PD123319. AngII induced dose- and time-dependent increases in
NMM IIA and IIB expression. This effect was completely inhibited by EMD66684 and
unaffected by PD123319. Immunostaining of keloid tissue demonstrated markedly increased
NMM IIA, NMM IIB, and AT1 expression. No change in the expression of AT2 was observed in
dermal fibroblasts of keloid tissue. These data demonstrate that Ang II stimulates myosin IIdependent
migration and NMMII expression in keloid fibroblasts through AT1, but not AT2, -
dependent signaling pathways. Taken together with our observation of increased NMMII and
AT1, but not AT2, expression in vivo, these findings suggest a putative mechanism for Ang II in
keloid pathogenesis in which Ang II drives both activation and upregulation of NMM II, thereby
functioning in a synergistic manner to drive fibroblast migration and keloid progression.
Selective inhibition of components of this pathway may offer a novel approach to the treatment
of keloid disease.

MS4.06
TRANSPLANTED FIBROBLASTS CORRECT DSYFUNCTIONAL REPAIR OF
WOUNDS IN AGED MICE LACKING CXCR3 SIGNALING AXIS
Cecelia C. Yates, PhD, Diana Whaley, M.S., Alan Wells, M.D., DMs.
University of Pittsburgh, Pittsburgh, PA, USA.
Failure to heal wounds is a major problem in persons of advanced age; compounded by common
pathologies that cause limited mobility or metabolic imbalances. In skin, the regeneration of the
ontogenically distinct mesenchymal-epithelial compartments must proceed coordinately
orchestrated by extracellular signaling networks. We have recently found that repair is
coordinated by ELR-negative CXC chemokines that bind the common CXCR3 receptor. These
ligands affect not only the cells of the innate and acquired immune system but also the
fibroblasts, endothelial cells, and keratinocytes critical to tissue regeneration. If this signaling is
disrupted wounds proceed to a chronic hypercellular and hypertrophic state characterized by
ongoing inflammation--a wound in which the regenerative processes are not curbed. To
investigate the effects of CXCR3-signaling in wound healing of the elderly, full-thickness
excisional wounds were created on 12month old CXCR3-/- or wild-type mice and examined for
30days. Wounds were analyzed for re-epithelialization, epidermal-dermal maturation, collagen
remodeling and organization. The CXCR3-/-mice exhibited a significant delay in healing in all
areas compared to wild-type mice. Interestingly, the combined lack of blood vessels coupled
with fibroblast death in dermis during the stage in which one normally sees fibroplasia would
cause the observed diminution of granulation tissue. To test a cell-therapy, these full-thickness
wounds were transplanted with fibroblasts derived from newborn CXCR3-/- or wild-type mice.
The transplanted fibroblasts were labeled with a fluorescent dye(CM-DiI) and suspended in a
hyaluronic-acid gel. Wild-type fibroblasts transplanted into CXCR3-/-mice wounds reversed the
delay and dysfunction previously seen in CXCR3-/-wounds; this correction was not noted with
transplanted CXCR3-/-fibroblasts. The transplanted fibroblasts exhibited strong survival and
migration patterns and led to an increase in tensile-strength. Expression of matrix proteins and
collagen in transplanted CXCR3-/-wounds resembles normal wild-type healing. These finding
have intriguing implications for rational cellular interventions aimed at promoting wound healing
via cell-therapy.
MS4.07
HMG-COA REDUCTASE INHIBITORS (STATINS) REDUCE HYPERTROPHIC SCAR
FORMATION IN A RABBIT EAR WOUNDING MODEL
Jason Hyunsuk Ko, MD, Yanan Zhao, MD, Seok Jong Hong, PhD, Xian-zhong Ding,
MD/PhD, Peter Seihwan Kim, MD, Sonya Paisley Agnew, MD, Mauricio De la Garza, MD,
Thomas A. Mustoe, MD.
Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Background: HMG-CoA reductase inhibitors, also known as “statins,” are the most commonly
prescribed cholesterol-lowering medications in the world. Simvastatin has been shown to inhibit

connective tissue growth factor (CTGF) gene and protein expression, but this has never been
proven in an in vivo model. Previous studies in our laboratory have demonstrated that CTGF
plays a significant role in wound healing and scarring. Given the potent inhibition of CTGF seen
with simvastatin in vitro, we explore whether administration of various statins to healing wounds
has any effect on hypertrophic scar formation using an established rabbit ear wounding model.
Methods: Seven-millimeter punch wounds were made on the ears of 30 rabbits (n=360 total
wounds). Treatment wounds were injected with either simvastatin, lovastatin, or pravastatin at
low, medium, or high doses on post-wounding days 15, 20, and 25, whereas control wounds
were injected with vehicle at corresponding doses and time points. Wounds were harvested after
35 days for histomorphometric analysis using the scar elevation index (SEI). Results: Low-dose
simvastatin, lovastatin, and pravastatin each demonstrated significant reductions in SEI when
compared to control--21.9% (p=0.03), 25.8% (p=0.02), and 22.8% (p=0.01), respectively. There
were no significant differences in SEI in the medium- and high-dose statin groups compared to
controls. Analysis of mRNA in a subset of rabbits treated with low simvastatin demonstrated a
significant reduction in CTGF expression (p=0.009), consistent with the in vitro work of other
laboratories while providing a potential mechanism for the reduction in hypertrophic scarring
seen with simvastatin. Conclusions: HMG-CoA reductase inhibitors, or statins, reduce
hypertrophic scar formation via CTGF inhibition when administered at low doses, and the novel
application of these commonly prescribed medications may lead to innovative and effective antiscarring
therapies.
MS4.08
FOCAL ADHESION KINASE IS CRITICAL IN MAINTAINING MECHANICAL
HOMEOSTASIS DURING HYPERTROPHIC SCAR FORMATION
Victor W. Wong, MD, Kristine C. Rustad, BA, Ivan N. Vial, BA, Jason P. Glotzbach, MD,
Michael Januszyk, MD, Melanie R. Major, Josemaria Paterno, MD, Michael T. Longaker, MD,
MBA, Geoffrey C. Gurtner, MD.
Stanford University, Stanford, CA, USA.
We have recently shown that the application of mechanical loading to incisional murine wounds
results in hypertrophic scar (HTS) formation, which was associated with focal adhesion kinase
(FAK) mechanotransduction processes. Given that global FAK deletion results in embryonic
lethality, we generated full thickness skin FAK knockout (KO) mice using the Cre/loxP system
to evaluate the role of in vivo FAK signaling during HTS formation. After the application of our
HTS model to FAK KO and wildtype mice, wounds were evaluated for scar formation, gene
expression profiles, scar matrix phenotype, and biomechanical strength and stiffness. Full
thickness FAK KO scars were significantly smaller than wildtype scars, which could not be
attributed to wound cellularity or apoptosis. FAK-mediated alterations in matrix profile were
associated with increased connective tissue growth factor (CTGF), and decreased collagen III
and matrix metalloproteinase 9 (MMP9) gene expression, which was confirmed with
immunohistochemistry. Based on polarizing light, confocal, and scanning electron microscopy
analyses, we determined that FAK KO scars were comprised of thinner collagen fibers that were
less oriented against the axis of mechanical load. Finally, loss of FAK signaling was associated
with a weaker and less stiff scar in response to mechanical loading, as compared to wildtype

scars. These data support a defined role for FAK in maintaining wound mechanical homeostasis
during HTS mechanofibrosis. Thus, targeting of FAK signaling pathways may result in novel
therapies to treat fibrotic contractures, adhesions, and encapsulation.
Mini Symposia 5 - Biologicals (Gene Therapy / Growth Factors / Stem Cells) and
Angiogenesis - MS5.01-MS5.08
Tuesday, April 20, 2010, 8:00 to 10:00 am
MS5.01
ANGIOGENIC GENE EXPRESSION IN RAT ISCHEMIC SKIN FLAPS AFTER AAV2-
VEGF GENE THERAPY
Xiao Tian Wang, MD, Heather Durfee, Jin Bo Tang, MD, Paul Y. Liu, MD.
Roger Williams Medical Center, Providence, RI, USA.
Purpose: Necrosis of surgical flaps is a difficult clinical problem. Angiogenesis is an essential
process in flap survival. Previously, we reported that delivery of VEGF gene by adenoassociated
viral type 2 (AAV2) vectors improved flap survival. Here we explored expression of a
series of genes using a novel technique_PCR array to assess genetic profile in AAV2-VEGF
treated skin flaps. Methods: Twenty rats were divided into an experimental and a control group
(n=10 each). In the experimental group, 10 10 -10 11 AAV2-VEGF particles were injected
intradermally into a 7 cm x 3 cm area on the rat back. The control group received saline
injection. Two weeks later, a flap of 10cm x 3 cm including the injection area was raised and
sutured back. One week post-surgery, the flap viability was evaluated. The injected and
surviving flap tissues were harvested, and samples of three randomly selected rats from each
group were used. We performed qPCR arrays by using Rat Angiogenesis RT 2 Profiler PCR
Array (SABiosciences). Expression of a total of 84 genes was analyzed. The data were analyzed
by using a web-based software (SABiosciences). Results: Multiple genes showed up-regulation
in the AAV2-VEGF treated flap tissue. Notably, these up-regulated genes included VEGFb (12fold),
PDGFα (3-fold), EGF(3-fold), chemokine (C-X-C motif) ligand 2 (CXCL2)(15-fold) and
matrix metallopeptidase 9 (MMP9) (13.5-fold). In contrast, FGF2 gene expression was downregulated
substantially. Conclusion: We found up-regulation of a series of endogenous growth
factor and cytokine genes in AAV2-VEGF treated flaps. The findings indicated that endogenous
genes relating to angiogenesis and wound healing are activated by AAV2-VEGF therapy and can
play a role in enhancement of flap survival. This novel technique, qPCR array analysis, provides
an efficient tool for screening changes in gene expression relating to wound healing. The study
on the gene expression of AAV2-GFP treated rat flaps is ongoing.
MS5.02
MICRO-RNAS ATTENUATE GROWTH FACTOR SIGNALING IN CHRONIC
VENOUS ULCERS
Irena Pastar, PhD 1 , Olivera Stojadinovic, MD 1 , Elizabeth Leburn, BS 1 , Aly Azeem Khan,
PhD 2 , Christina Leslie, PhD 2 , Harold Brem, MD 3 , Marjana Tomic-Canic, PhD 1 .

1 University of Miami Miller School of Medicine, Miami, FL, USA, 2 Memorial Sloan Kettering
Cancer Institute, New York, NY, USA, 3 NYU School of Medicine, New York, NY, USA.
MicroRNAs (miRNAs) are short (~22 nt) noncoding RNAs that can suppress the expression of
protein-coding genes and are found disregulated in various diseases. To determine the role of
miRNAs in inhibition of wound healing we utilized biopsies obtained from non-healing venous
ulcers (VUs) and molecular biology and bioinformatics approaches. miRNA fraction was
isolated from full thickness skin biopsies obtained after surgical debridement of ten VUs and
healthy skin. All VU biopsies were verified for established histological criteria for non-healing
edges and nuclear presence of biomarker, β-catenin. Quantification of specific miRNAs was
performed using TaqManMicroRNA Assays, and miRNA expression was normalized between
different samples based on the values of U48 RNA expression. PCR results revealed induction of
miR-21 and miR-20a expression in VUs. To validate these findings we used ex-vivo human
wound model in which we applied topically specific miRNAs and followed the rate of healing.
Using dye-conjugated mimic we documented efficient penetration of miRNAs through epidermis
and dermis within first 24 hrs. Mimic-mir-21, but not mimic-control or a vehicle, significantly
inhibited wound healing. Next, we used bioinformatics approach to identify genes that may be
targeted by both, miR-21 and miR-20a. Interestingly, we found highest prediction for growth
factor signaling molecules, including TGFβ and EGF. To further determine if these targets are
indeed affected in VUs we used qPCR and immunohistochemistry. Indeed, we found that all
three TGFβ and EGF receptors were down-regulated in non-healing edges of VUs. Interestingly,
TGFβRI and III were also suppressed at mRNA level whereas TGFβRII mRNA levels remained
unchanged. We conclude that induction of specific miRNAs in VUs contribute to loss of growth
factor signaling leading to overall non-healing phenotype. Thus, targeting specific miRNA
molecules may be intriguing novel approach to accelerate wound healing in patients.
MS5.03
IMPROVED WOUND HEALING IN ANGIOGENIC PROVISIONAL MATRIX
OCCURS IN THE ABSENCE OF INCREASED TGF-BETA EXPRESSION
Swathi Balaji 1 , Bradley A. King 1 , abdul Q. sheikh 1 , Hongkwan Cho 1 , Michelle Bloomer 1 ,
Foong Y. Lim 2 , Timothy M. Crombleholme 2 , Daria A. Narmoneva 1 .
1 University of Cincinnati, Cincinnati, OH, USA, 2 Cincinnati Childrens Hospital Medical Center,
Cincinnati, OH, USA.
Objective: Delayed diabetic wound healing is characterized by increased proteolysis, scarce
provisional matrix and impaired neovascularization. TGF-β and myofibroblasts (myoFBs) play
regulatory roles in wound healing. We previously showed that wound treatment with
proteolytically-stable angiogenic nanoscaffold in diabetic db/db mice resulted in formation of
robust in situ tissue engineered provisional matrix (ISTEPM). The ISTEPM resulted in
significantly reduced inflammation, enhanced early neovascularization and improved wound
morphology (days-7&14). The objective of this study was to determine if improved healing
associated with ISTEPM resulted from enhanced neovascularization, increased TGF-β and
myoFB signaling, or both. Methods: Paired 8mm excisional wounds created on diabetic db/db
mice were treated with nanoscaffold or control solutions PBS and hyaluronic acid (HA) (n=7).

Wounds were harvested at post-wound days-7, 14, & 28 to quantify granulation tissue area,
epithelial gap, capillary lumen density (CD31 and tail-vein lectin injection), myoFBs (α-SMA),
expression and distribution of TGF-β1, -β2, -β3, and wound strength. Results: The increased
early neovascularization of ISTEPM in nanoscaffold-treated wounds persisted through day-28
with a significant increase in capillary lumen density (p

endothelial cells and fibroblasts are vital during the development of normal and pathological
scars. We thus hypothesize that myofibroblasts from the wounds could play a more important
role that originally thought on neovascularization.
MS5.05
AUTOLOGOUS ADIPOSE DERIVED STEM CELLS DIFFERENTIATE INTO
EPITHELIAL AND VASCULAR CELLS
Shanmugasundaram Natesan, PhD 1 , Ge Zhang, MD, PhD 2 , David G. Baer, PhD 1 , Thomas J.
Walters, PhD 1 , Laura G. Suggs, PhD 3 , Robert J. Christy, PhD 1 .
1 US Army Institute of Surgical Research, Fort Sam Houston, TX, USA, 2 University of Akron,
Akron, OH, USA, 3 University of Texas, Austin, TX, USA.
Thermal injury accounts for approximately 5% of combat casualties and continue to be a
significant source of morbidity. Combat burn injuries are often full-thickness burns, involving
large total body surface areas (TBSA) of skin and are often compounded by multiple injuries.
While a wide variety of dermal matrices have been developed for the treatment of burns injuries,
the lack of a host cell source and the time involved for cell expansion have limited their clinical
application. We hypothesize that autologous adipose derived stem cells (ASC) can be used to
produce a clinically relevant tissue engineered skin equivalent. ASC possess a heterogeneous cell
population with the potential to differentiate into endothelial and stromal cell lineages. In this
study we describe the differentiation of adipose derived stem cells into epithelial cells and
vascular cells which can be used to develop tissue engineered wound healing treatments.
Adipose derived stem cells were differentiated into epithelial cells using a combination of
growth factors and a collagen matrix. The differentiated ASC show a squamous cell-like
structure morphology by day 5 and become stratified when exposed to the air-medium interface.
Using immunocytochemistry and RT-PCR the stratified epithelium were shown to express
keratins 10, 14 and involucrin. Additionally, we have developed a biodegradable vasculature
matrix mimic by modifying fibrinogen with a PEG derivative. We found by 7 days, ASC seeded
in PEGylated fibrin have formed vascular tube-like networks in the biomatrix in the absence of
additional soluble cytokines. Similar to microvessels in vivo, the ASC in the PEG-fibrin matrix
express both endothelial (e.g. vWF, CD31) and pericyte (e.g. NG2, SMA) specific markers. The
ability of ASC to differentiate into epithelial and vascular cells provides a system for
development of epidermal and vascularized tissue, using appropriate conditions and biomaterials,
for healing burn wounds.
MS5.06
RADIATION-INDUCED FIBROSIS IS RESCUED BY SIRNA BLOCKADE OF SMAD3
Benjamin R. Roman, MD, Judy W. Lee, MD, Richard A. Zoumalan, MD, John P. Tutella, MD,
Gina K. Paek, BS, S Immerman, MD, Denis Knobel, MD, Meri Wetterau, MD, James Crawford,
BS, Stephen M. Warren, MD, Pierre B. Saadeh, MD.
NYU Medical Center, New York, NY, USA.

Purpose: Cutaneous radiation injury occurs during the treatment of cancer, or in rare
environmental exposure. As the acute wound heals, fibrosis is induced and extracellular matrix
(ECM) is deposited. The fibrotic pathway is mediated by the transforming growth factor-β
(TGF-β) cascade, and is dependent on Smad3, a transcription factor for ECM. We characterized
gene expression of this cascade after radiation injury and performed in vitro and in vivo gene
silencing of Smad3 in an attempt to reverse the fibrotic pathway. Methods: Wild-type murine
dermal fibroblasts were irradiated with 20Gy and harvested at serial time-points. RT-PCR was
performed for known regulators and mediators of fibrosis. Smad3 was silenced by transfection
with siRNA. For the in vivo experiment, dorsal skin of wild-type mice was irradiated with 45Gy.
Five weeks later, siRNA was applied to the fibrotic areas for one week. Skin was harvested and
tissue analyzed by RT-PCR and Western blotting, as well as tissue tensiometry, which
quantitatively measures rigidity. Results: Following irradiation, there was a steady increase in
mRNA expression of Smad3, TGFβ, and ECM genes collagen 1A1, metalloprotease2, and tissue
inhibitor of metalloprotease-1, with peak expression at 12-24 hours. Inhibition of Smad3 with
siRNA significantly decreased expression of Smad3, TGFβ, and ECM genes. In the mouse
model, topical treatment with siRNA again significantly decreased expression of these genes.
Tensiometry demonstrated decreased stiffness in Smad3 siRNA treated skin, with a Young’s
modulus nearer to normal compared to untreated and nonsense siRNA treated skin. Conclusion:
Following initiation of the fibrotic pathway by radiation, Smad3 siRNA treatment both in vitro
and in vivo effectively reversed gene expression. Furthermore, cutaneous Smad3 inhibition
mitigated radiation-induced fibrotic stiffening. These findings suggest a therapeutic role for
Smad3 silencing for cancer patients treated with radiation as well as those accidentally exposed
to radiation.
MS5.07
INHIBITION OF CONNECTIVE TISSUE GROWTH FACTOR/CCN2 EXPRESSION IN
HUMAN DERMAL FIBROBLASTS BY INTERLEUKIN-1 α AND β
Elizabeth Kiwanuka, M.D. 1 , Anita Koskela 2 , Elof Eriksson, M.D. PhD. 1 , Bengt Gerdin, M.D.
PhD. 3 , Mikael Ivarsson, PhD. 2 , Daniel Nowinski, M.D. PhD. 3 .
1 Division of Plastic Surgery, Boston, MA, USA, 2 Clinical Research Center, University Hospital,
Orebro, Sweden, Örebro, Sweden, 3 Department of Surgical Sciences, Plastic Surgery, Uppsala
University, Uppsala, Sweden.
Introduction: Connective tissue growth factor (CTGF/CCN2) is a matricellular protein intimately
involved with wound healing and tissue repair. CTGF is induced by transforming growth factor
(TGF)-β and is believed to mediate several of the downstream actions of TGF-β.
We previously showed that keratinocytes in vitro down regulate TGF-β-induced expression of
CTGF in fibroblasts by an interleukin (IL)-1 α-dependent mechanism. Here, we investigated
further the mechanisms of this down regulation by both IL-1 α and β. Methods: Human dermal
fibroblasts or NIH 3T3 mouse fibroblasts were treated with IL-1 α or β in presence or absence of
TGF-β1. mRNA levels were measured with real-time PCR and protein expression was measured
with Western blotting. NIH 3T3 cells were transfected with cDNA constructs to study CTGF
promoter activity, and the activity of a TGF-β1-inducible minimal promoter construct (CAGA).
Results: A suppression of basal and TGF-β-induced CTGF mRNA expression of about 30 to

50% was seen at IL-1 concentrations in the range of 10-200 pg/ml. There was no detectable
CTGF protein when cells were cultured without TGF-β. Incubation with TGF-β resulted in a
strong induction of the CTGF protein and IL-1 inhibited this up regulation at concentrations of
50-600 pg/ml. IL-1 α and β inhibited TGF-β-induced CTGF promoter activity, and the activity of
the TGF-β1-inducible minimal promoter construct. Conclusions: IL-1 α and β suppress CTGF
mRNA and protein, and down regulate CTGF gene transcription, in fibroblasts. These results add
to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated,
which in turn may have implications for the pathogenesis of scar.
MS5.08
MICRORNA-27B RESCUES IMPAIRED ENTOTHELIAL PROGENITAL CELL-
INDUCED ANGIOGENESIS AND IMPROVE WOUND HEALING IN TYPE 2
DIABETIC MICE
Jie-Mei Wang, MD, Alex F. Chen, MD, PhD.
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Background: Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is
dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that
regulate gene expression at the posttranscriptional level. However, whether miRNAs regulate
EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b
rescues impaired EPC angiogenesis in vitro and in vivo via suppressing anti-angiogenic protein
thrombospondin-1 (TSP-1) in diabetes. Methods and Results: Bone marrow-derived EPCs from
adult male type 2 diabetic db/db and their normal littermates db/+ mice (both C57BLKS/J, 9
weeks) (glucose 437.6±36.1 vs. 162.9 ±19.4 mg/dL, n=38, p

MS6.01
INSULIN AND ITS EFFECTS ON MULTIPLE PROCESSES IN WOUND HEALING
Darcie L. McClelland, Manuela Martins-Green, PhD.
University of California, Riverside, Riverside, CA, USA.
Insulin is essential for the growth and differentiation of many cell types. We and others have
shown that wound treatment with insulin accelerates healing. Our studies have further shown that
insulin increases both keratinocyte migration and cell adhesion molecule expression and
expression of ECM molecules, facilitating migration. It also stimulates angiogenesis, improves
dermal regeneration, and reduces scarring (Liu et al., 2008; Liu et al., 2009). These observations
led us to hypothesize that insulin affects monocytes/macrophages and fibroblasts in the wound
tissue, altering inflammatory responses and promoting dermal repair. Using ELISA and western
blot analysis, we show that insulin levels decline initially after wounding for 48 hours and then
increase, peaking at 5 days before returning to basal levels. In contrast, the insulin receptor levels
increase through five days, decline through day 9, and then increase again upon wound closure
around day 14. We also show that insulin stimulates monocyte and fibroblast proliferation,
chemotaxis and migration. The effect on monocytes is prevented with a insulin receptor (IR)
neutralizing antibody, showing that increased monocyte chemotaxis results from insulin
signaling through the IR. In fibroblasts, the effect of insulin is reversed by the IR neutralizing
antibody when a low dose of insulin (10-7) is used, but requires both IR neutralizing antibody
and IGF1 receptor inhibitor when a high dose of insulin (10-6) is applied, indicating that insulin
signals through both receptors in fibroblasts. These results suggest that insulin affects monocyte
behavior of during the inflammatory phase of healing. Insulin may also recruit fibroblasts to the
wound site. Our results suggest that insulin may stimulate wound healing early on by promoting
necessary inflammatory responses and dermal restoration. We are currently deciphering the
insulin-induced signaling mechanisms in monocyte/macrophages and fibroblasts to improve
healing and reduce scar formation.
MS6.02
HYPERLEPTINEMIA CONTRIBUTES TO OBESITY-IMPAIRED WOUND HEALING
IN OBESE, NON-DIABETIC MICE
Janelle Wagner, MD, Phuong D. Nguyen, MD, Robert J. Allen, Jr., MD, Edward H. Davidson,
MA MBBS, Orlando Canizares, MD, Pierre B. Saadeh, MD, Stephen M. Warren, MD.
NYU Institute of Reconstructive Plastic Surgery, New York, NY, USA.
Background: Leptin functions via reactive oxygen species (ROS) intermediates. At physiologic
levels, leptin improves wound healing. Obese individuals have leptin levels 5 times greater than
non-obese, yet wound healing is impaired. We hypothesize that excessive leptin levels may
paradoxically impair wound healing in obese, non-diabetic mice. Methods: 3 groups of mice had
stented wounds placed on their dorsa. Group 1 (moderate hyperleptin) were obese, non-diabetic
TallyHo/JngJ mice (n=5). Group 2 (super-hyperleptin) were obese, non-diabetic TallyHo/JngJ
mice that received 200 ug/g of leptin i.p. daily (n=5). Group 3 (normoleptin) were SWR/J

controls (n=5). Group 1 have circulating leptin levels 2.8 times greater than wild-type mice. In
order to mimic the 5x leptin elevation present in obese humans, Group 2 mice received
supplemental leptin. Wounds were photographed on day 0, 7, 10 and 14. Time to closure was
measured. Results: Wounds of control mice closed 71% at day 7, 90% at day 10 and 100% at day
14. Time to closure was impaired in both moderately hyperleptin and super-hyperleptin TallyHo
mice. Wounds of Group 1 were 5% closed at day 7, 23% at day 10 and 58% at day 14. Group 2
had poor granulation tissue formation and their wounds had not even begun to heal at day 7, with
13% closure at day 10, and only 20% closure at day 14. Conclusions: Elevated levels (both 2.8
and 5-fold) of an adipose-derived hormone can impair wound healing. We hypothesize that
excess leptin signaling increases the ROS response and impairs wound healing.
MS6.03
TOPICAL ANGIOGENIC GENE THERAPY: PHD2 SIRNA IMPROVES DIABETIC
WOUND HEALING
Meredith T. Wetterau, MD, Finny George, MD, Denis Knobel, MD, Phuong D. Nyugen, MD,
Stephen M. Warren, MD, Pierre B. Saadeh, MD.
New York University Langone Medical Center, New York, NY, USA.
Background: Prolyl hydroxylase domain 2 (PHD2) is a cytoplasmic protein that regulates the
expression of hypoxia inducible factor (HIF)-1alpha, a key transcription factor in the cellular
response to hypoxia. Normoxia results in hydroxylation of HIF-1alpha on specific proline
residues by PHD2, rendering it inactive. However in the absence of oxygen, HIF-1alpha
activates factors such as VEGF and FGF-2 to promote angiogenesis. Since a central impairment
in diabetic wound healing is impaired vascularity, we hypothesized that siRNA to PHD2 will
increase angiogenic mediators and improve diabetic wound healing. Methods: 4mm paired
stented wounds were created on the dorsum of diabetic db/db mice. Mice were treated with
PHD2 siRNA evenly distributed in an agarose-gel matrix (nonsense siRNA served as control).
PHD-2 siRNA or nonsense siRNA was reapplied on days 7, 14, and 21. Wounds were measured
photometrically on days 0, 3, 7, 10, 14, 21 and 28. Additionally, wounds were harvested for
histology, immunohistochemistry, and for protein and RNA analysis of angiogenic mediators
(VEGF/FGF2).
Results: Diabetic wounds treated with siRNA closed within 21 +/-2 days while those not treated
closed in 28+/- 3 days. PHD protein expression was nearly completely suppressed and HIF-
1alpha was elevated two fold on Western blot by day 7. Furthermore, RT-PCR demonstrated the
angiogenic mediators VEGF and FGF-2 were elevated by a fold induction of 14.76 ± 0.96 and
15.68 ± 1.96, respectively. Compared to 0.11 ± 0.42 for controls, this is significant. This
corresponds to increased CD31 staining in the treated versus control groups. No off-target effects
were identified, and silencing was transient (28 days). Conclusions: siRNA mediated silencing of
PHD2 increases HIF-1alpha and other mediators of angiogenesis, corresponding to improved
time to closure in diabetic wounds compared to controls. These findings suggest that impaired
wound healing in a diabetic model can by ameliorated by therapeutic angiogenesis.
MS6.04

THE EFFECTS OF COMBINED EPIDERMAL GROWTH FACTOR AND
ERYTROPOIETIN TREATEMENT AGAINST RAT DIABETIC FULL THICKNESS
WOUNDS.
Sung Woo Park, MS, Joon Pio Hong, MD, PhD, MBA.
Asan Medical Center University of Ulsan, Seoul, Korea, Republic of.
The purpose of this study is to evaluate the combined effect of erythropoietin (EPO) and
epidermal growth factor (EGF) against full thickness diabetic wounds and whether synergistic
effect exist over single growth factor treatment. A total of 50 Sprague-Dawley rats were divided
into 5 groups which were respectively treated with rh-EPO vehicle, rh-EPO, rh-EGF vehicle, rh-
EGF or combined rh-EPO and rh-EGF. Drug application and dressing were performed twice a
day with a 12 hour interval until wound was fully epithelialized. Analysis of wound size, 50%
Healing time, hematoxylin and eosin stain, immunohistochemical staining for Ki-67 and EGF
receptors were performed. The rh-EPO and rh-EGF combined treatment showed significantly
faster 50% Healing time and higher expression for Ki-67 compared to all 4 groups (p

WT ECs, angiogenic peptide nanoscaffold was used as an in vitro 3D-system to quantify EC
angiogenic response, and as an in vivo treatment for excisional wounds in db/db mice to quantify
neovascularization (CD31), proliferation (ki67) and VEGF expression (ELISA) in the wounds.
Results: In vitro: Diabetic ECs demonstrated significant impairment in chemotactic migration
(p

MS6.07
SUSTAINED EXPRESSION OF ANGIOGENIC FACTORS STIMULATES
VASCULARIZED GRANULATION TISSUE DEPOSITION IN DIABETIC MICE
Allen Comer, Ph.D. 1 , Joely Straseski, Ph.D. 2 , Sara Pirnstill, B.S. 1 , Kristi Schaeve, B.S. 1 , Maggie
Bach 1 , Jennifer Murphy, M.D. 2 , Lynn Allen-Hoffmann, Ph.D. 1 .
1 Stratatech Corporation, Madison, WI, USA, 2 University of Wisconsin-Madison, Madison, WI,
USA.
Introduction: Insufficient vascularization is a limiting factor in the healing of chronic wounds.
Strategies to promote the formation of vascularized granulation tissue are promising approaches
to promote wound healing. Topical application of individual angiogenic factors is limited by
rapid degradation or clearance from the wound environment. This study describes the
development of human skin substitutes designed to express sustained, elevated levels of
angiogenic factors to promote deposition of highly-vascularized granulation tissue. ethods: NIKS
keratinocytes were stably transfected with non-viral plasmid vectors encoding either a single
angiogenic factor (VEGF) or a transcriptional regulator that induces multiple angiogenic factors
(HIF-1α). Genetically-modified skin substitutes were evaluated for secretion of angiogenic
factors and stimulation of endothelial cell proliferation, as well as the ability to promote
deposition of vascularized granulation tissue after engraftment on nude or diabetic mice. Results:
Stably-transfected cells are genetically stable and non-tumorigenic. Skin substitutes modified to
express VEGF secreted 100 fold more VEGF than unmodified tissue. Tissue modified to express
HIF-1α secreted elevated levels of several pro-angiogenic chemokines. Conditioned medium
from tissue expressing VEGF stimulated the proliferation of human microvascular endothelial
cells compared to medium from unmodified skin substitutes. Tissue expressing either VEGF or
HIF-1α promoted enhanced vascularization compared to unmodified skin substitutes after
grafting on nude mice. Skin substitutes expressing VEGF also enhanced the deposition of
vascularized granulation tissue in diabetic mice. Cell banks were produced to support GMP
production. Conclusions: Skin substitutes expressing elevated levels of angiogenic factors
enhance vascularization in animal models of impaired wound healing. Uniform, non-viral
genetic modification of NIKS keratinocytes offers safety and consistency advantages compared
to heterogeneous modification of primary keratinocytes with viral vectors. The ability of skin
substitutes expressing elevated levels of angiogenic factors to promote vascularization in vivo
suggests that these substitutes may accelerate the vascularization and healing of chronic wounds.
MS6.08
GRAFTING OF DIABETIC WOUNDS WITH MINCED SKIN
Florian Hackl, MD, Juri Bergmann, MD, Taro Koyama, MD PhD, Pejman Aflaki, MD,
Elizabeth Kiwanuka, MD, Emily Waisbren, BS, Scott Granter, MD, Elof Eriksson, MD PhD.
Brigham and Women's Hospital, Boston, MA, USA.
Introduction: Transplantation of autologous skin grafts is the dominant technique in the treatment
of non-healing wounds. Previous experiments in our lab showed successful expansion of skin in

a 1:100 ratio using minced skin. To translate theses results to an impaired wound healing model
experiments were performed in streptozotocin induced diabetic pigs. Methods: 2.5cm x 2.5cm
full-thickness wounds have been created on the dorsum of streptozotocin induced pigs and
transplanted with very small autologous skin pieces (0.8mm x 0.8mm) in a 1:100 ratio. Wounds
were covered with polyurethane wound chambers followed by injection of saline to create a wet
wound environment. Untransplanted full-thickness wounds served as controls. Biopsies were
taken on days 10, 14 and 18. Reepithelialization of the wounded area was evaluated in H&E
stained slides. Sections were also stained for Ki-67, Cytokeratin and von Willebrand factor
(vWF). Results: Biopsies of day 10 showed that 75% of the wound surface area of the wounds in
the transplanted group was epithelialized and only 27.5% in the untransplanted control group. By
day 14 transplanted wounds were fully healed versus only 50% of the surface area of the
untransplanted controls was epithelialized (Fig1). The Ki-67 antibody staining demonstrates that
keratinocytes proliferate from the basal layer and the skin appendages of the skin pieces.
Histologies illustrate fibroblast proliferation from the wound bed with proliferating skin particles
riding on top of the granulation tissue (Fig2). The vWF staining showed well developed blood
vessels in the subepidermal plexus (Fig3). Conclusion: Results obtained in healthy pigs were
reproduced in the diabetic pig model. Skin grafts can be expanded 100-times in the operating
room and heal experimental wounds in a diabetic pig model within 14 days. The ability to
accelerate wound healing using this technique may offer new options in the treatment of nonhealing
diabetic ulcers.
ORAL POSTERS
Poster Talk 1 - Biomaterials and Bioengineering - PT1.01 to PT1.10
Monday, April 19, 2010, 3:30 to 5:30 pm
PT1.01
A PRIMING IN VITRO STEP GREATLY ENHANCES THE REPERTOIRE OF KEY
GENES INVOLVED IN EPIDERMAL PROLIFERATION AND MIGRATION IN A
BIOENGINEERED SKIN
Xiaofeng Lin, MD, PhD, Polly Carson, CWS, Vincent Falanga, MD.
NIH Center of Biomedical Research Excellence (COBRE), Roger Williams Medical Center,
Providence, RI, USA.
We have continued to intensively study a living bilayered skin construct (BSC), which consists
of neonatal foreskin fibroblasts and keratinocytes. BSC has been extensively used for the
treatment of human difficult-to-heal chronic wounds. We investigated several in vitro conditions
to induce“BSC priming”, in which key genes involved in proliferation and migration are
induced. Four experimental BSC groups were analyzed in vitro: A) control; B) meshed; C)
incubated in DMEM; and D) incubated in DMEM, then meshed. Total RNA was isolated from
each group. Gene expression analysis was performed using Affymetrix HG-U133 Plus 2.0 Array
and the Ingenuity Pathway Analysis program. Microarray results were filtered according to Log2
ratio (> 1.5) and false discovery rate adjusted p-value (

quantitative real-time PCR. Key genes activated by the in vitro manipulation were selected, and
the BSC epiboly assay (migration of the epidermal over its dermal component) was tested in the
presence of neutralizing antibodies against selected genes. In all, and upon further analysis, 1119
and 1175 genes were differentially expressed when the BSC was incubated in DMEM with or
without meshing, respectively, compared to the control. Using ingenuity functional analyses, we
found that the genes whose expression was the most statistically significant were those involved
in epidermal proliferation and migration. BSC epiboly was significantly inhibited when certain
concentrations of neutralizing antibodies against the selected key genes were present during
incubation with DMEM. Therefore, our study demonstrates that numerous genes related to
wound healing are significantly induced by this novel in vitro priming of the BSC. The findings
indicate that the treatment efficacy of the BSC could be increased by simple in vitro
manipulation prior to its application to human wounds.
PT1.02
BIOMATERIALS MODULATE IL-8 AND OTHER INFLAMMATORY PROTEINS
DURING RE-EPITHELIALIZATION IN CUTANEOUS PARTIAL-THICKNESS
WOUNDS IN PIGS
Kyle R. Kleinbeck, B.S., Lee D. Faucher, M.D., Weiyuan John Kao, Ph.D..
University of Wisconsin-Madison, Madison, WI, USA.
Acute and chronic cutaneous wounds remain a clinical challenge that requires a mechanistic
understanding to advance treatment options. For example, the role of inflammatory mediators
during wound healing is not completely understood. Biomimetic materials, such as an in situ
photopolymerizable semi-interpenetrating network (sIPN) derived from extracellular matrix
components, show great potential in improving healing through the delivery of therapeutic
agents and the function as a temporary tissue scaffold. In this study we characterized the
temporal profile of porcine cutaneous partial-thickness wound healing in response to
Xeroform TM and sIPN treatment via histological and inflammatory protein analyses in epidermal,
remodeling dermal, and dermal regions. Generally, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70,
IFN-γ, and TNF-α, but not IL-8, were expressed in the epidermis and remodeling dermis in a
time course that followed the progression of epidermal maturation in response to both treatments.
Differences in cellularity and protein expression were observed between treatments in a time-
and region-dependent manner. In particular, the healing response to sIPN exemplified a key
relationship between IL-8 expression and re-epithelialization. These results provide insights into
the expression of inflammatory mediators and the time course of cutaneous healing and the
capacity for biomaterials to further modulate this relationship.
PT1.03
COMPARISON OF FIBRINOLYTIC PROTEASE ACTIVITY AND COLLAGEN
PRODUCTION OF KERATINOCYTES AND FIBROBLASTS CULTURED IN 3-D
FIBRIN MATRICES

Erik Reinertsen, B.S., Haison Duong, M.S., Ben Wu, D.D.S., Ph.D, Bill Tawil, MBA, Ph.D.
UCLA, Los angeles, CA, USA.
Introduction: Dermal wound healing involves a coordinate effort between keratinocytes,
fibroblasts, and other resident cell types- all having unique roles in the regeneration of the
injured site. Subsequent to injury, keratinocytes migrate to the wound edge and proliferate within
a fibrin matrix to re-epithelialize the dermis; simultaneously, fibroblasts maintain the structural
integrity of the dermis by synthesizing new extracellular matrix proteins (ECM). Ultimately, the
fibrin matrix is degraded and replaced by new tissue matrix, collagen. The dynamic process
between the degradation of the fibrin matrix and production of new collagen is orchestrated by
the fibrinolytic capacity of keratinocytes and ECM production of fibroblasts respectively. To
date, it is still unclear how much do either keratinocytes or fibroblasts are involved in the
clearance of fibrin and production of new collagen. In this study, we’ve constructed 3-D fibrin
matrices to compare the fibrinolytic capacity and ECM production of keratinocytes and
fibroblasts. Methods: Fibroblasts and keratinocytes were seeded (200,000 cells/ml) in individual
fibrin matrices of varying formulations and cultured in respective growth medium. At days 1,3,7,
and 14, the condition medium were obtain and assayed for expression of plasminogen activators,
uPA or tPA. Collagen production was assessed by hydroxyproline assay. Results: Plasminogen
activator activities were observed in both keratinocytes and fibroblasts 3-D fibrin matrices;
however, the activity was higher in keratinocyte cultures; in addition, the activity level varied
with varying fibrin matrix composition. Collagen production was observed only in fibroblast
cultures, and this was also corresponded to fibrin matrix composition. Conclusion: Both
keratinocytes and fibroblast participated in the fibrinolytic process; whereas, the production of
new collagen is coordinated solely by fibroblasts. Understanding the role of keratinocytes and
fibroblasts in the rate of fibrin breakdown versus rate of collagen production may enable us to
engineer products that could enhance or expedite wound regeneration.
PT1.04
COMPOSITE SCAFFOLDS FOR IMPROVED ENGINEERED SKIN STRENGTH AND
VIABILITY
Heather M. Powell, PhD, Jason W. Drexler, BS.
The Ohio State University, Columbus, OH, USA.
It is essential that the mechanical properties of engineered skin (ES) approximate normal human
skin to ease surgical application and improve range of motion after engraftment. To fulfill this
goal, major advances in ES design are needed. Ideally, the use of co-axial electrospinning
generates composite scaffolds with a core of high strength polymers and a shell of bioactive
polymers combining advantageous properties of both. To study the ability to precisely control
scaffold strength while utilizing minimal amounts of synthetic polymer, scaffolds were
fabricated using co-axial electrospinning with variable polycaprolactone (PCL)-core flow rates
(0.5-4ml/hr) and core solution concentrations (4-8 wt./vol.%). A 12wt./vol.% gelatin shell was
used for all scaffolds. Monofiber gelatin and PCL scaffolds served as controls. Scaffolds were
mechanically tested, subsequently inoculated with human fibroblasts and keratinocytes and
cultured at the air-liquid interface for 18 days. ES organization via histology, surface electrical

capacitance and cellular metabolism (MTT assay) were evaluated at days 6, 12 and 18 with
mechanical properties quantified at day 18. Core flow rate positively correlated with scaffold
mechanical strength with core rates of 1ml/hr and 2ml/hr producing scaffolds with average
tensile strengths of 210+43 kPa and 314+52 kPa, respectively. Core diameter also positively
correlated with core flow rate (1ml/hr and 2ml/hr produced 886+141 nm and 974+62 nm,
respectively)(Fig. 1). ES fabricated with core-shell scaffolds were significantly stronger than
pure gelatin or PCL alone with ES strength scaling proportionally with composite scaffold
strength. Cell metabolism was significantly higher in core-shell scaffolds compared to PCL
alone; however were on average lower than pure gelatin. The ability to control the mechanical
properties of engineered skin by modifying scaffold properties allows highly viable/functional
skin to be engineered with increased biomimetic mechanics. These improvements to engineered
skin may facilitate surgical application, and provide a platform for further advances in ES
mechanics.
PT1.05
BRIDGING THE GAP BETWEEN RESEARCH AND MANUFACTURING: THE ROLE
OF PRODUCT AND PROCESS DEVELOPMENT
Katie Faria, Vincent Ronfard, PhD.
Organogenesis Inc., Canton, MA, USA.
The field of cellular therapy has emerged as an important approach to regenerative medicine.
However, bench to bedside translation is long, fastidious and costly requiring both
comprehensive in vitro and in vivo studies to get the approval by regulatory bodies. One of the
greatest difficulties is to bridge the gap between research and manufacturing is to develop good
manufacturing processes (GMP) and scalable designs and to apply these principles into the
development of a safe and effective product. Using the Organogenesis experience we will
discuss the process flow from research and development, process optimization, validation, FDA
registration and through to implementation into manufacturing. Apligraf ® was the 1 st tissue
engineered living product to gain FDA approval and be manufactured at a commercial scale. The
manufacturing process is unique and highly complex involving the production of key raw
materials (i.e. fibroblast and keratinocyte cell banks and collagen type I) as well as the core
process manufacturing the construct, Apligraf ® . The core process requires 7 different culture
medias, 12 separate feeds, 2 different cell types and 20 days in culture. The organization has
been very successful developing process modification, gaining FDA approval for and
implementing manufacturing efficiencies. Over the last 11 years we have successfully
implemented more than 51 process efficiencies. Specific process changes that will be detailed
are; 1. Scaling up from monolayer expansion system for fibroblasts to a suspension system based
on microcarriers, both spinner flask and Wave Bioreactor cultures. 2. Scaling up media
production and implementing disposable technologies to improve efficiency. 3. Development
and implementation of automated processing into the Apligraf production process. In the end, the
key challenge is to produce a safe and effective product which is economically viable for the
industry and the society that uses it.
PT1.06

ADAPTATION OF BENCH-TOP ASSAYS FOR USE AS RAPID BED-SIDE TESTS FOR
HUMAN NEUTROPHIL ELASTASE ACTIVITY AND NITRIC OXIDE METABOLITE
LEVELS IN CHRONIC WOUND FLUIDS
Daniel J. Gibson, M.S., Gregory S. Schultz, Ph.D..
University of Florida, Gainesville, FL, USA.
Introduction: We have previously presented work on rapidly detecting matrix metalloproteinase
(MMP) activity in wound fluids. Recently, we have developed two additional assays that detect
human neutrophil elastase (hNE) activity and nitric oxide metabolite levels (NOx) within 10
minutes and with the same type of read out as the MMP detector. Purpose: Using the previously
developed system for measuring MMP activity, to test the feasibility of developing assays for
hNE activity and NOx levels that can be performed in the same manner and read by the same
device. Methods: For the hNE assay, a commercially available green fluorescence resonance
energy transfer (FRET) peptide was used to generate a standard curve. The fluorescence
generated was continuously measured over an 11 minute period. For the NOx levels, a
commercially available green fluorophore precursor was used to generate a nitrite standard
curve. The standards were incubated with the substrate under oxidizing conditions for 0, 2.5, 5.0,
7.5, or 10.0 minutes, the reaction was then stopped by neutralization with sodium hydroxide. The
standard curve was then measured with a fluorescent plate reader. Finally, a standard curve of
MMP-9 was generated with our previously published system to serve as a basis for comparison.
Results: Both the hNE and NOx techniques yielded a measurement within 10 minutes that fell
within the dynamic range of the MMP detector. Additionally, the number of steps and a
processes for all three systems are similar enough at present to be unified under a common
protocol that would vastly simplify the implementation and integration of these techniques into
the clinical environment. We now have the foundation for a well integrated system of assays that
measure different molecular aspects of a wound including the wound’s response to therapy and
its healing trajectory.
PT1.07
A NEW STRATEGY FOR WOUND HEALING PEPTIDES
Timothy Falla, Ph.D, Lijuan Zhang, Ph.D.
Helix BioMedix, Bothell, WA, USA.
The innate immunity peptide HB107 has been demonstrated to accelerate wound healing
(Wound Repair. Regen. 2004; 12: 351-8). Our goal was to determine if smaller, more stable and
more cost effective peptide fragments within the sequence were capable of exhibiting
bioactivities beneficial to the healing wound. Such peptides would provide specific directed
activity that could be delivered temporally during wound healing avoiding unwanted, deleterious
or untimely activities. Thirty overlapping peptide fragments of the eighteen residue parent
molecule were synthesized and assayed for effects upon cell proliferation, cell migration,
collagen synthesis, angiogenesis and cytokine (IL6 and IL8) levels. A seven amino acid
sequence, HB1061, was found to stimulate the proliferation of keratinocytes in a dose (0.2ug/ml
to 50ug/ml) dependant manner to 200% of control while exhibiting no such effect on

melanocytes or fibroblasts. In the scratch test HB1061 also induced a significant acceleration in
keratinocyte migration at 6 hours and 10 hours compared to controls. A number of fragments,
including HB1061, were positive in the endothelial cell tube formation assay and produced an
increase in collagen synthesis in fibroblasts. Such activities were observed in the low ug/ml
range for tetra-, penta- and hexapeptides. IL6 and IL8 down-regulation was observed for two
peptides HB802 and HB1410. The phenomenon was pronounced in keratinocytes in an inflamed
state and characterized using UV irradiated cell cultures. This work demonstrated that specific
bioactivities can be isolated from innate immunity peptides and such a technique could provide a
strategy for delivering wound healing activities in a more controlled and specific way. By the use
of time release delivery such peptides could be sequentially administered providing time relevant
activity to the wound.
PT1.08
BIOLOGICAL ACTIVITIES OF CYTOKINE-NEUTRALIZING HYALURONIC ACID-
ANTIBODY CONJUGATES
Liang Tso Sun 1 , Sidi A. Bencherif, Ph.D. 1 , Thomas Gilbert, Ph.D. 2 , Adam A. Farkas, Ph.D. 2 ,
Michael T. Lotze, M.D. 2 , Newell R. Washburn, Ph.D. 1 .
1 Carnegie Mellon University, Pittsburgh, PA, USA, 2 University of Pittsburgh, Pittsburgh, PA,
USA.
Wound healing represents a highly regulated, orchestrated response of cells recruited to sites of
injury. High molecular weight hyaluronic acid was conjugated with monoclonal antibodies to the
cytokines interleukin-1β to create a matrix-forming polymer capable of modifying healing. Using
gel electrophoresis and fluorescence immunosorbent assays, we measured a degree of antibody
functionalization per hyaluronic acid chain of 13.6 ± 1.6%. The biological activity of the
conjugate in vitro, measured using a nuclear factor-κB translocation assay in activated THP-1
monocytes, was comparable in inhibiting cytokine signaling to a similar level as the
unconjugated antibody. Subcutaneous implantation studies in Sprague-Dawley rats indicates that
viscous hyaluronic acid solutions were immunologically active, but covalent functionalization
with antibodies against tumor necrosis factor-α and interleukin-1β resulted in significant
reductions in the inflammatory response. Covalent attachment of cytokine-neutralizing
antibodies to matrix-forming polymers could lead to the development of materials capable of
locally regulating wound healing and inflammatory responses in the setting of tissue
regeneration.
PT1.09
NOVEL PROGENITOR CELL THERAPY AUGMENTS ISCHEMIC OSSEOUS
WOUND HEALING
Edward H. Davidson, MA MBBS, Xiaoxia Wang, MD, Parag Butala, MD, Steven M. Sultan,
BA, Robert J. Allen, Jr., MD, John Paul Tutela, MD, Orlando Canizares, MD, Denis Knobel,
MD, Lukasz Witek, BA, Pierre B. Saadeh, MD, Stephen M. Warren, MD.
NYU Institute of Reconstructive Plastic Surgery, New York, NY, USA.

Purpose Bone fractures and distraction osteogenesis are characterized by an ischemic
microenvironment that limits healing. We hypothesize that mobilization of bone marrow-derived
endothelial progenitor cells (EPCs), involved in new blood vessel formation, can be utilized to
enhance neovascularization, alleviate ischemia, and augment healing. Methods Fractures: 3-mm
defects were created on murine parietal bone. AMD3100 (a partial CXCR4 agonist and
mobilizing agent of bone marrow-derived EPCs) or sterile saline was injected intraperitoneally
daily post-operatively, and bony regeneration was assessed using micro-CT. CD31 and
osteocalcin staining were performed to assess for vascular density and osteoblast density.
Distraction osteogenesis: Rats were subjected to mandibular distraction. From the beginning of
the consolidation phase, animals received daily injections of AMD3100 or sterile saline.
Mandibles were harvested and bony regeneration was assessed using micro-CT,
immunohistochemistry (CD31 for vascular density and osteocalcin for bone formation), BMP-2
ELISA and mechanical testing. Results Flow cytometry confirmed an increase in circulating
EPCs in response to AMD3100 Fractures: AMD3100-treatment improved bony healing at postop
week 8 (34.8±11.5% vs. 50.3±11.5%, p=0.017) and week 12 (36.0±5.7% vs. 61.8±11.4%,
p

Only 32 patients completed the study, 18 in the experimental treatment and 14 in the control
group. The mean age was 60 years and the mean evolution time 38 months. The mean surface
reduction was 6.29 cm2 (IC95 % 3.28-9.29) (p = 0. 0001) in the MTC-2G group and 5.85 cm2
(95 % CI 3.58-8.12) (p = 0. 001) in the hydrogel group. There was no statistical significance
between both groups (p = 0. 815). There was no statistical significance when patients were
grouped depending on the size of the ulcer in ≤10cm2 and ≥10cm2 (p = 0. 42). There was no
change in the laboratory parameters at the end of the study. In the pathology evaluation only
neutrophilic infiltrates improved in the MTC-2G group compared to the hydrogel group
(p=0.05). Discussion: MTC-2G was not superior to hydrogel in venous leg ulcers treatment.
Poster Talk 2- Biologicals (Cell and Molecular Mechanisms) – PT2.01 to PT2.10
Monday, April 19, 2010, 3:30 to 5:30 pm
PT2.01
ANALYSIS OF CARP FUNCTION IN VIVO AND IN VITRO BY GENE DELETION
Susan Samaras, Ph.D. 1 , Stephen Koch, MS 1 , Nanjun Wu, Ph.D. 1 , Jeffrey M. Davidson, Ph.D. 2 .
1 Vanderbilt University School of Medicine, Nashville, TN, USA, 2 Vanderbilt University School
of Medicine, VA Tennessee Valley Healthcare System, Nashville, TN, USA.
Cutaneous wounding rapidly induced expression of ankrd1 and its translation product, cardiac
ankyrin repeat protein (CARP), in multiple cell types in the skin. Adenovirus transduced CARP
overexpression in vascular endothelial cells in vitro increased cell survival during serum
starvation and during exposure to two apoptosis inducing agents, TGF-β and adriamycin. In vivo,
overexpression of CARP in experimental wounds significantly increased neovascularization. To
study how CARP affects endothelial survival in vitro and to deduce its role in tissue regeneration
and wound healing in vivo, we engineered a conditional CARP knockout mouse. Global
knockout was achieved by breeding ankrd1 flox/+ or ankrd1 flox/flox mice with mice expressing a
Sox2-driven Cre-recombinase. Mice with partial or complete CARP depletion were viable and
fertile and exhibited a normal phenotype. The effect of CARP depletion was revealed following
injury. Ischemic dorsal skin flaps (1.5 cm by 2.0 cm) in mice with normal CARP expression
healed completely with little necrosis, while about 30-50% of the skin flap on mice heterozygous
for ankrd1 deletion became necrotic. Complete depletion of CARP resulted in 70-75% flap
necrosis. To study the mechanism by which CARP affects cell survival we have isolated and
transformed (SV40 T antigen) dermal fibroblasts from CARP expressing and CARP deplete
mice. The rate and magnitude of adriamycin induced caspase activity was significantly different
between these cell lines. These results indicate that cells derived from the CARP knockout mice
will be useful for identifying the mechanism by which CARP affects cell function and survival.
This work was supported by the Department of Veterans Affairs and NIH grant DK65656.
PT2.02
EARLY GENE EXPRESSION OF FIBRONECTIN SPLICED VARIANTS,
INTERLEUKIN-1β, AND COLLAGEN PROTEINS IN VOCAL FOLD MUCOSA
DURING LASER-INDUCED SUBGLOTTIC INJURY IN RABBITS

Ha-Sheng Li-Korotky, MD PhD, Vlad C. Sandulache, MD PhD, Nancy Lo, BS, Brynn Saeler,
BS, Chia-Yee Lo, MS, Mark Barsic, BS, Joseph E. Dohar, MD MS, Patricia A. Hebda, PhD.
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Background: Fibronectin (FN), one of the main components of connective tissue matrix is
comprised of a family of about 20 isoforms generated by alternative gene splicing. Recently,
there has been significant progress made to identify the functions of alternatively spliced FN
isoforms. Our group previously reported that the age-dependent activation of FN-EDA splice
variant may play a fundamental role in differentiating fetal wound regeneration from postnatal
wound scar formation during the early events of airway mucosal wound healing. Purpose: To
delineate molecular activities of the variant-inclusion FN transcripts, inflammatory and scarringassociated
molecules in vocal fold mucosa during early events of laser-induced airway injury.
Methods: A cricothyroidotomy was performed in the subglottic region of adult rabbits. CO2 laser
at a wavelength of 10,600 nm at a level of either 2 or 5 watts was used to induce subglottic
injury, which also extends into the adjacent vocal folds. Vocal fold mucosal tissues were
harvested at 12, 24, 48, and 72hrs post-injury. Expression levels of mRNA transcripts were
assessed with gene-specific primers coding for total FN and alternatively spliced variants EDA
and V, collagen components (Col1a1, Col1a2, Col3a1) and interleukin-1 beta (IL-1β) using realtime
qPCR. Results: Dose-dependent induction of EDA was observed in vocal fold mucosa at 48
and 72hrs after 5-watts injury and at 72hrs after 2-watts injury. At 72 hrs, Col1a1 and Col1a2
were up-regulated whereas the Col3a1c gene was selectively suppressed. Inflammatory cytokine
IL-1β was significantly induced at 24hrs in 5-watts injured wounds by comparison with 2-watts
injured wounds, and remained elevated in 5-watts injured wounds at 72hrs. Conclusion: Dosedependent
inclusion of the FN-EDA domain correlates with induction of IL-1β and increased
ratio of collagen I/III transcripts, suggesting that the FN spliced variant EDA may be a key
component leading to vocal fold scarring. Acknowledgement: Supported by NIH
Grant#DC007437
PT2.03
CD109, A NOVEL TGF-β CO-RECEPTOR, ENHANCES COLLAGEN
ORGANIZATION AND DECREASES DERMAL THICKNESS IN A MOUSE MODEL
OF SKIN FIBROSIS
Hasan Alajmi, MD, joshua vorstenbosch, BSc, Sebastian Winocour, MD, Alissa Trzeciak, BSc,
Lucie Lessard, MD, FRCSC, FACS, Anie Philip, PhD.
McGill University, Montreal, QC, Canada.
Background: Transforming Growth Factor-β (TGF-β) is a multifunctional growth factor
important in wound healing. However, when in excess, it can cause skin scarring and fibrosis.
Our group has previously identified CD109, a novel TGF-β co-receptor as an inhibitor of TGFβ1
signaling and extracellular matrix (ECM) protein production in vitro. In the current study, we
examined whether endogenous TGF-β action in vivo in the skin can be manipulated by CD109 in
a bleomycin-induced model of skin fibrosis. Objective: To determine whether CD109 regulates
TGF-β signaling and ECM deposition during bleomycin-induced skin fibrosis in vivo. Methods:
Transgenic mice overexpressing CD109 in the epidermis and wild-type littermates were injected

intradermally with bleomycin or PBS control, every second day for 28 days to induce skin
fibrosis. Mice from each experimental group were sacrificed at 21 and 28 days after the initial
injection and tissues were harvested for analysis. Histological changes were analyzed by H&E
and Masson's Trichrome staining, and alterations in TGF-β signaling pathway components were
determined by Western blot. Results: Our results show that intradermal injection of bleomycin
increases collagen deposition in the skin in both transgenic and wild-type littermates.
Importantly, transgenic mice injected with bleomycin for 28 days demonstrate decreased dermal
thickness and display more compact and organized collagen deposition compared to wild-type
littermates. Furthermore, bleomycin treated transgenic mice exhibited decreased fibronectin
deposition and a reduction in TGF-β signaling (phosphoSmad2) as compared to bleomycin
treated wild type littermates. Conclusion: CD109 transgenic mice display decreased dermal
thickness and enhanced collagen organization during bleomycin-induced skin fibrosis as
compared to their wild-type counterparts. Collectively, our data illustrate the potent regulatory
effect of CD109 on skin fibrosis, and suggest that this molecule may have a therapeutic value for
the treatment of skin disorders such as hypertrophic scarring and scleroderma.
PT2.04
WHOLE GENOME ANALYSIS OF CHRONIC WOUNDS TREATED WITH TOPICAL
NEGATIVE PRESSURE USING RETICULATED OPEN CELL FOAM
Kathleen L. Derrick, M.S. 1 , Raymund E. Horch, M.D. 2 , Amy McNulty, Ph.D. 1 , Mareike
Leffler, M.D. 2 .
1 KCI, San Antonio, TX, USA, 2 University of Erlangen-Nürnberg, Erlangen, Germany.
Chronic wounds impair quality of life and are a considerable worldwide health care expense,
costing billions of dollars per year. In order for chronic wounds to heal, these wounds must be
altered to a more acute wound state to begin the healing process. Topical Negative Pressure
(TNP) with reticulated open cell foam (ROCF), also known as negative pressure wound therapy
(NPWT), has been shown to promote healing in chronic wounds and to help aid in primary
closure. To better elucidate TNP/ROCF healing in these wounds, 11 chronic wound samples
from patients were obtained during routine debridements prior to and 7 - 12 days after
continuous TNP with -125 mmHg. Whole genome microarray analyses were performed on these
samples in order to better comprehend how TNP/ROCF facilitates wound healing in chronic
wounds. There were 606 genes expressed on day 0 and 851 genes expressed for day 7 following
TNP/ROCF. Of these, only 65 genes were commonly expressed between time points (p

PT2.05
AN INVESTIGATION OF EFFICIENCY OF AAV2-VEGF GENE DELIVERY TO
ENHENCE STRENGTH OF INJURED TENDONS: AN IN VIVO STUDY
Ya Fang Wu, MD 1 , Chuan Hao Chen, MD 1 , Xiao Tian Wang, MD 2 , Paul Y. Liu, MD 2 , Jin Bo
Tang, MD 2 .
1 Department of Hand Surgery, Nantong Unversity, Nantong, China, 2 Roger Williams Medical
Center, Providence, RI, USA.
Introduction Delivery of growth factor genes into tissues may enhance the healing strength. We
have found that bFGF gene transfer can increase the strength of the healing tendon. In this study,
we investigated efficiency of human recombinant VEGF gene delivery to the injured tendons in
promoting the tendon strength. Methods Sixteen flexor digitorum profundus tendons of 16
chickens were divided into two groups: AAV2-VEGF injection group and non-injection control
group. In the experimental group, AAV2-VEGF was injected into 4 sites of the tendon stump
immediately after tendon transection. The tendon was subsequently repaired with modified
Kessler method. In the control group, the tendon was cut and repaired similarly, without
injection of vectors. Four weeks later, the tendons were harvested and were subjected to load-tofailure
test in an Instron tensile testing machine, and the tendon samples were analyzed for
expression of transgene and extracellular matrix genes by real-time PCR. Results Compared with
the controlled tendons, the tendons injected with AAV2-VEGF had a significantly greater tensile
strength; the increase in the strength was drastic, to 220% of that of the controls. Since the
transgene is of the human origin, we could detect the presence in all tendon samples. Real-time
PCR analysis showed increased expression of tissue inhibitor of metalloproteinase (TIMP) gene.
Conclusions AAV2-VEGF can effectively improve the healing strength of the injured digital
flexor tendon in an in vivo animal model. However, morphologically adhesions still formed
around the tendon. TIMP may serve to decrease the activities of metalloproteinase and ensure
accumulation of tendon collagen. Whether this gene therapy strategy significantly increases
peritendinous adhesions requires further investigations.
PT2.06
NOVEL MOUSE MODEL TO STUDY RESOLUTION OF INFLAMMATION DURING
WOUND HEALING
Melissa L. Petreaca, PhD, Avo Serafino, Milton Ferreyro, Neil Schiller, PhD, Manuela
Martins-Green, PhD.
University of California, Riverside, Riverside, CA, USA.
Defective regulation of inflammation, granulation tissue formation, and/or remodeling phases of
wound healing can lead to impaired healing or the development of chronic wounds. We have
found that wounds in Tumor Necrosis Factor Superfamily Member 14 (TNFSF14/LIGHT)deficient
mice exhibit a prolonged inflammatory phase accompanied by defective healing.
Histological examination of the LIGHT -/- wounds 14 days after wounding revealed that, while
these wounds had re-epithelialized, the wound tissue was immature. The nascent epithelium

lacked a continuous basement membrane, as shown by collagen IV immunostaining, and failed
to form strong interactions with the dermis. Furthermore, we observed excessive inflammation in
the underlying tissue. These findings are consistent with our previous work showing that LIGHT
promotes macrophage apoptosis in vitro. Taken together, our results suggest important roles for
LIGHT in the resolution of inflammation and wound healing in vivo. Indeed, some LIGHT -/-
wounds become chronically inflamed, non-healing wounds. These chronic wounds contain many
macrophages and T cells in the underlying tissue. Furthermore, they exhibit a more severe
epithelial defect than in acute wounds; the chronic wound epithelia have discontinuous basement
membranes, and the wounds re-opened multiple times following re-epithelialization. The
inflammation appears to be largely responsible for the healing defects, as prolonged treatment
with anti-inflammatory and anti-bacterial compounds ultimately promoted wound healing. These
results also suggested the possibility that the chronic wounds were contaminated with bacteria.
We found that, while the bacterial content of acute wounds was minimal, LIGHT -/- chronic
wounds were heavily contaminated with Staphylococcus aureus and epidermidis, species
frequently found in chronic human wounds. Our data suggests a predisposition of LIGHT -/-
wounds to become highly inflamed and bacterially contaminated, thereby increasing the
likelihood of chronic wound development. We are currently investigating the possibility that the
absence of LIGHT-mediated resolution of inflammation increases the risk of systemic
inflammation and intravascular coagulation.
PT2.07
MAST CELLS CONTRIBUTE TO SCAR FORMATION IN FETAL WOUNDS
Traci Wilgus, PhD, Allison Parent, BS, Melissa Meleski, Brian Wulff, PhD.
The Ohio State University, Columbus, OH, USA.
Mast cells are resident innate immune cells important for initiating acute inflammation. While
mature skin responds to injury by mounting a vigorous inflammatory response and producing
scar tissue, immature fetal skin heals without inflammation and without forming a scar. Using a
mouse model of fetal wound healing, we showed previously that fetal skin development is
associated with an increase in mast cell numbers and mast cell maturity. We also showed that
scar-forming or fibrotic wounds generated at embryonic day 18 (E18) contain more total mast
cells and more degranulated (activated) mast cells than scarless E15 wounds. To determine
whether the presence of mast cells is important for the fibrotic healing phenotype found in more
developed fetal skin, scar formation was assessed in E18 fetuses from mast cell-deficient (kit W/Wv
) and wild-type mice. Repair of E18 wounds in mast cell-deficient fetuses resulted in scars that
were over two times smaller in size compared to those in wild-type litter mates. While additional
studies are needed to identify the precise mechanism(s) by which mast cells stimulate scar tissue
production, our results underscore the importance of mast cells in fibrosis and suggest that the
release of mast cell-derived mediators upon degranulation contributes to the transition from
scarless to fibrotic healing in fetal skin.
PT2.08
TESTING OF FISH OIL-DERIVATIVES IN WOUND HEALING - A PILOT STUDY

Jodi McDaniel, PhD.
The Ohio State University, College of Nursing, Columbus, OH, USA.
Recent studies report that circulating n-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA)
polyunsaturated fatty acids rapidly appear in inflammatory exudates and are converted to lipid
mediators that resolve inflammation by diminishing polymorphonuclear leukocyte (PMN) levels.
The purpose of this pilot study was to evaluate effects of oral supplements containing EPA +
DHA on lipid-derived mediators of inflammation (eicosanoids) and PMN in acute wound fluid
and wound re-epithelialization. The study was a prospective, randomized, experimental design
conducted on 18 eligible individuals. Subjects were randomized to 2 groups: 28 days of daily
oral supplementation with EPA+DHA+aspirin (1.6 g/d of EPA + 1.1 g/d of DHA + 81 mg/d of
aspirin) or daily supplementation with vehicle + aspirin (mineral oil + 81 mg/d of aspirin). The
specific aims were to: 1) compare eicosanoid levels in blister wound fluid at 12 and 24 hours
post blister formation across the 2 groups with LC/ESI-MS/MS; 2) compare PMN levels in
blister fluid by quantifying myeloperoxidase, a PMN marker; and 3) assess wound reepithelialization
using Single Digital Photogrammetry. After 4 weeks the EPA +DHA Group had
significantly higher plasma concentrations of EPA (t=9.23, df=16, p=.000) and DHA (t=9.71,
df=16, p=.000) when compared with the Placebo Group. There was a direct effect on 15lipoxygenase
(LOX) products of n-6 fatty acids [eicosanoids: 9- hydroxyoctadecadienoic acid
(HODE), 13-HODE, 12-hydroxyeicosatetraenoic acid (HETE), 15-HETE and 15-
hydroxyeicosatrienoic acid (HETrE)]. The EPA + DHA Group had significantly lower 13-HODE
(t= -3.38, df=12, p=.005) and 15-HETE (t= -4.23, df=16, p=.001) in blister fluid at 24 hours post
blistering than the Placebo Group. On average, the EPA+DHA Group had lower PMN levels at
12 hours post blistering when compared to the Placebo Group and healed one day faster,
although not statistically significant. EPA/DHA supplementation could potentially target local
inflammatory mediators of inflammation and facilitate wound healing.
PT2.09
DHEA STIMULATES CONTRACTION AND MIGRATION OF CULTURED HUMAN
DERMAL FIBROBLASTS: EVIDENCE FOR NON-GENOMIC SIGNALLING
INDEPENDENT OF CONVERSION TO ESTRADIOL
Nanda Kandamany, MD, David Sharpe, MD, Desmond Tobin, PhD, M Julie Thornton, PhD.
Centre for Skin Sciences, University of Bradford, United Kingdom.
The adrenal androgen dehydroepiandrosterone (DHEA), an estrogen precursor improves wound
healing. Adrenarche, distinctive in humans, results in high plasma DHEA levels, but with age is
significantly reduced in both sexes. Since dermal fibroblasts are critical wound healing cells we
evaluated whether DHEA directly modulates human fibroblast contraction and migration, or
whether its effects are mediated by aromatisation to estradiol. Primary cultures from female
breast skin (mean age 27; n=8 patients) were mechanically wounded and incubated with vehicle
control, DHEA (10nM-10uM), DHEA plus an aromatase inhibitor (Arimidex), or DHEA with
pertussis toxin (PTX). Cell migration was determined using the scratch wound assay between 4
and 48hours. Cell contraction was assessed by fibroblast populated collagen lattice (FPCL) gels
at intervals between 2 and 10 days. DHEA significantly increased contraction, while Arimidex

alone had no effect. However, Arimidex inhibited the DHEA-stimulated contraction at days 2, 4,
6, 8 and 10. DHEA significantly increased migration of mechanically wounded fibroblasts as
early as 4hrs. Although the stimulatory effect of DHEA was significantly reduced in the presence
of Arimidex, migration was still higher than vehicle control, suggesting only partial inhibition.
At 48h Arimidex did not inhibit DHEA stimulated migration. Incubation with PTX, which
blocks G-protein coupled receptor signalling, reversed the stimulatory effect of DHEA on cell
migration; PTX alone had no effect on migration. These results demonstrate that DHEA has
direct effects on human fibroblast wound healing responses in vitro. Although inhibition of
DHEA by Arimidex indicates metabolism of DHEA to estradiol, the early migratory response at
4hrs, the partial inhibition by Arimidex, and the complete inhibition with PTX suggest that
DHEA may also be activating non-genomic signalling pathways. A greater understanding of
DHEA signalling may help develop wound-healing therapies, without the risks of estrogen
therapy, benefiting the elderly and patients with impaired wound healing.
PT2.10
BIOFILM-FORMING STAPHYLOCOCCUS AUREUS SELECTIVELY IMPAIRS
WOUND HEALING IN THE RABBIT DERMAL ULCER MODEL
Anandev N. Gurjala, MD 1 , Matthew Geringer, BA 1 , Seok Jong Hong, PhD 1 , Robert D.
Galiano, MD 1 , Kai P. Leung, PhD 2 , Thomas A. Mustoe, MD 1 .
1 Northwestern Feinberg School of Medicine, Chicago, IL, USA, 2 Microbiology Branch, Walter
Reed Army Research Institute, Great Lakes, IL, USA.
Bacterial biofilms are purported to be a key component in the etiology of chronic wounds,
including pressure ulcers, diabetic foot ulcers, and venous stasis ulcers. Although the presence of
biofilm has been demonstrated across all chronic wound types, there is a dearth of evidence
definitively establishing a causal relationship between biofilm-infected wound beds and their
delayed healing. The purpose of this study was to investigate the hypothesis that the biofilm
phenotype is critical to the ability of bacterial infection to cause an aggressive, persistent wound
healing impairment and to confer resistance to antibiotic treatment, both hallmarks of recalcitrant
human chronic wounds. Partial-thickness wounds were created in the ears of New Zealand
rabbits and either kept as controls or inoculated with biofilm-forming wild-type S. aureus
bacteria, or its biofilm-impaired mutant counterpart. After 10 days of wound healing, animals
were sacrificed and wounds were assessed for presence of biofilm, bacterial load, and measures
of wound healing. A second cohort was treated with topical mupirocin antibiotic. Scanning
electron microscopy confirmed the formation of mature biofilm architecture in wild-type
inoculated wounds, while predominantly planktonic phase bacteria were visualized in the mutant
inoculated wounds. Quantitative PCR counts of bacterial load revealed that the host immune
response and mupirocin treatment were able to clear mutant S. aureus in an additive fashion, but
were ineffective against wild-type bacteria. Histological analysis demonstrated near-complete
wound healing in the mutant + antibiotic group, a mild wound healing impairment in the mutant
group, and severely impaired wound healing in both the wild-type and wild-type + antibiotic
groups. This work demonstrates that the biofilm phenotype specifically is responsible for the
ability of bacteria to establish persistent infection resulting in severe delays in wound healing,
and provides further evidence for a causative role of biofilm in impaired chronic wound healing.

Poster Talk 3 - Fibrosis and Scarring – PT3.01 to PT3.10
Monday, April 19, 2010, 3:30 to 5:30 pm
PT3.01
MATRIX COMPLIANCE REGULATES EXPRESSION OF SMOOTH MUSCLE
ALPHA-ACTIN IN MYOFIBROBLASTS VIA ACTIN DYNAMICS AND MYOCARDIN
RELATED TRANSCRIPTION FACTOR-A
James J. Tomasek, PhD, George M. Risinger, PhD, Carol J. Haaksma, BS.
University of Oklahoma - Health Sciences Center, Oklahoma City, OK, USA.
Myofibroblasts are specialized fibroblasts that facilitate wound closure through expression of
smooth muscle alpha-actin (SMAA) and generation of contractile force on the surrounding
matrix. We have previously demonstrated that expression of SMAA in myofibroblasts is
mechanoregulated; however, the underlying mechanism of this regulation is unclear. We
hypothesize mechanoregulation of SMAA expression in myofibroblasts is mediated by actin
dynamics regulating localization of myocardin related transcription factor-A (MRTF-A). MRTF-
A is a serum response factor binding partner that is essential to promote SMAA transcription. In
addition, G-actin can bind MRTF-A, thus sequestering MRTF-A and blocking SMAA
expression. In this study we have used three-dimensional collagen lattices of varying compliance
to test this hypothesis. Rat fibroblasts (RF) cultured in compliant lattices in the presence of
transforming growth factor-beta1 (TGF-β1), conditions that maintain a fibroblast phenotype and
low SMAA expression, have decreased F-actin assembly and MRTF-A localized to the
cytoplasm. In contrast, RFs cultured in stiff collagen lattices in the presence of TGF-β1,
conditions that promote the myofibroblast phenotype and SMAA expression, have stress fibers,
with increased F-actin assembly, and MRTF-A localized to the nucleus. Jasplakinolide and
cytochalasin D, drugs that reduce G-actin levels, promote SMAA expression and nuclear
localization of MRTF-A even on a compliant substratum. In contrast, latrunculin B, which
increases G-actin, reduces SMAA expression and promotes cytoplasmic localization of MRTF-
A. Expression of a constitutively nuclear mutant MRTF-A promotes SMAA expression on a
compliant substratum. This work demonstrates that actin dynamics, in response to the
mechanical environment, regulates nuclear localization of MRTF-A and thereby expression of
SMAA and myofibroblast formation and function. Funded by NIH grant R01 GM60651.
PT3.02
NON-ER OUTSIDE-IN FUNCTIONS OF THE ER CHAPERONE CALRETICULIN IN
DIABETIC WOUND REPAIR
Fares Samra, BA 1 , Sara-Megumi Naylor, BA 1 , Daniel Gorovets, BA 1 , savvas Pavlides, PhD 1 ,
Joanne E. Murphy-Ullrich, PhD 2 , Jamie P. Levine, MD 1 , Stephen M. Warren, MD 1 , Leslie I.
Gold, PhD 1 .
1 New York University School of Medicine, New York, NY, USA, 2 University of Alabama,
Birmingham, AL, USA.

We previously reported that topically applied calreticulin (CRT), a calcium-binding ER
chaperone protein comprising N, P, and C domains, markedly enhances diabetic murine (db/db)
and porcine cutaneous wound healing. Consistent with the potent wound healing effects, we
further showed, in vitro, that exogenous CRT stimulated proliferation of keratinocytes and
fibroblasts, induced concentration-dependent migration of these cells and monocytes and
macrophages, and upregulated protein expression of collagen, fibronectin, and TGF-β-3 in
fibroblasts. Notably, all these broad-ranging effects purport novel non-ER functions for CRT that
act from outside the cell inward. The current studies address: 1) whether the ER chaperone
function of CRT is required for its extracellular functions, 2) the molecular structure(s) of CRT
that function in its biological activities and 3) the in vitro effects of CRT on diabetic compared to
normal mouse and human fibroblasts. Using CRT null mouse embryo fibroblasts (K42)
compared to wild type (K41) in proliferation and migration assays (scratch plate and chamber),
we show that exogenous CRT stimulates proliferation of null K42 cells to a similar extent as K41
cells (2-fold at 10 pg/ml). However, K42 cells require 100 times more CRT for a peak migratory
response (1 vs 100 ng/ml), with a 20% decreased response. We also show that the C domain
stimulates fibroblast proliferation to the same extent and peak response as the entire molecule.
Finally, we show that fibroblasts isolated from db/db mouse skin and human fibroblasts cultured
in high glucose, to simulate type II diabetes, respond to CRT by migration and proliferation
albeit with 1/3 less robust response requiring 10-fold more CRT for peak responses compared to
controls. The breath of novel non-ER functions of CRT, structure-function relationships, and
effects on diabetic cells in vitro underscore this molecule as a potential potent agent for the
topical treatment for healing diabetic wounds.
PT3.03
EFFECT OF STIMVASTATIN ON TRANSFORMING GROWTH FACTOR-BETA
INDUCED CONNECTIVE TISSUE GROWTH FACTOR PROTEIN EXPRESSION
LEVELS IN HUMAN CORNEAL FIBROBLASTS
Paulette M. Kuznia, Ph.D, Alfred Lewin, Ph.D, Gregory Schultz, Ph.D.
University of Florida, Gainesville, FL, USA.
The transforming growth factor-β (TGF-β) system plays a key role in the scar formation in the
eye. Furthermore, it is now known that connective tissue growth factor (CTGF) is a secreted
fibrogenic cytokine that is a downstream mediator of many of the fibrotic actions of TGF-β,
including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into
myofibroblasts. Watts et al. (2005) showed that the addition of Simvastatin, a hypolipidemic
drug, to idiopathic pulmonary fibrosis (IPF) lung fibroblasts reduced both RNA and protein
levels of CTGF. In this study, we examined the effects of Simvastatin on TGF-β1 induced CTGF
protein expression levels in human corneal fibroblasts (HCF). HCF were serum starved for 48hrs
with or without the addition of various concentrations of Simvastatin (5, 10, and 20uM). After
48hrs, the HCF were stimulated with 5ng/mL of TGFβ-1, and all cultures received various
concentrations of Simvastatin (5, 10, and 20uM). The addition of Simvastatin, both continuously
and at the time of stimulation, significantly inhibited (P

that Simvastatin may be effective at reducing the stimulation of CTGF after trauma to the cornea,
which leads to scar formation and ultimately haze.
PT3.04
THE ROLE OF SMALL-LEUCINE RICH PROTEOGLYCANS, DECORIN,
BIGLYCAN, FIBROMODULIN, AND LUMICAN IN THE DEVELOPMENT OF
HYPERTROPHIC SCAR AFTER SURGERY AND BURN WOUND
Dariush Honardoust, PhD, Heather Shankowsky, Jie Ding, PhD, Edward E. Tredget, PhD.
University of Alberta, Edmonton, AB, Canada.
PURPOSE: Small leucine-rich proteoglycans (SLRPs) are structurally related extracellular
matrix (ECM) molecules that regulate collagen fibrillogenesis and inhibit transforming growth
factor-β (TGF-β) fibrogenic activity and as a result, SLRPs may play a critical role in wound
healing and scar formation. Hypertrophic scar (HSc) occurs in over 70% of burn patients and can
have devastating consequences that range from disfigurement to organ dysfunction. Studying the
role of SLRPs may provide information that could improve wound healing and clinical
therapeutic approaches that minimize fibrosis and scar formation following injury. METHODS:
We used immunohistochemistry as well as single and double immunostaining to study the ECM
and cell-associated localization of SLRPs in normal skin and HSc. We determined the protein
and mRNA expression levels of SLRPs in normal skin and HSc from the same patient and
examined the medium and cultured fibroblasts treated with TGF-β using immunoblotting and
reverse transcriptase-polymerase chain reaction. RESULTS: In normal, uninjured skin, there was
more decorin and fibromodulin accumulation in the superficial layers than in the deeper areas.
Compared to normal skin, the levels of decorin and fibromodulin were lower in HSc, while
biglycan was slightly higher. There was an increased expression of biglycan, fibromodulin and
lumican in the basement membrane and around basal epithelial cells, whereas, they were absent
or weekly expressed in HSc. CONCLUSION: Down-regulation of SLRP expression after
surgical or burn wound healing may contribute to TGF-β1 fibrogenic activity and the
development of fibrosis and HSc. Acknowledgments: The research is funded through grants
from the Canadian Institutes of Health Research (Tredget) and the Firefighters' Burn Trust Fund
of the University of Alberta.
PT3.05
CONNEXIN 43 LEVELS IN CHRONIC WOUNDS:A TARGET FOR ACCELERATED
WOUND HEALING?
Thomas E. Serena, MD FACS.
Serena Group, Warren, PA, USA.
Intercellular communication plays a pivotal role in the healing of acute and chronic wounds.
Cellular interaction is controlled in part by a family of connexin molecules: proteins that
assemble to form gap junctions between cells permitting the exchange of small molecules (

1000 Daltons). Connexin 43 is down-regulated in the epithelial cells at the leading edge of a
healing wound. In fact, this down-regulation appears to be essential to normal healing. In chronic
wounds it is postulated that elevated levels of connexin 43 impair wound healing. In one study
down-regulation of connexin 43 accelerated healing. 1 We investigated the altered expression of
connexins in a variety of chronic wounds. Sixty total patients with diabetic, venous, arterial or
pressure ulcers underwent biopsies of their wound margins along with a matched forearm full
thickness biopsy. Histological and quantitative analysis revealed abnormal elevations in
connexin 43 expression in all of the chronic wounds studied. There was also variation between
individuals and wound types. This study suggests that connexin 43 may be a good target for a
medication designed to down-regulate this protein. Wang C.M., Lincoln, J., Cook, J.E., and
Becker, D.L., “Abnormal connexin expression underlies delayed wound healing in diabetic
skin,” Diabetes 56:2809-2817 (2007).
PT3.06
TEMPORAL SPATIAL EXPRESSION AND FUNCTION OF NON MUSCLE MYOSIN
II ISOFORMS NMMIIA AND NMMIIB IN SCAR REMODELING
Trung Q. Ho, BA, Angelica Selim, MD, Jennifer Bond, PhD, Edith Bowers, BS, Howard
Levinson, MD.
Duke University Medical Center, Durham, NC, USA.
Introduction: Scar contracture is believed to be caused by cell contractility during the remodeling
phase of repair. Cell contractility is medicated by activation of non muscle myosin II, isoforms
NMMIIA (NMMIIA) and NMMIIB (NMMIIB), but the temporal-spatial expression profile of
NMMII during the remodeling phases of repair and the role of NMMII in enhancing scar
fibroblast tissue remodeling are unknown. This investigation charaterizes the temperal-spatial
expression of NMMIIA and NMMIIB in scar fibroblasts throughout the remodeling phase of
repair and investigates the role of NMMII in scar fibroblast contractility. Methods and Materials:
Human scar tissues of various ages, with normal surrounding skin, were immunostained for
NMMIIA and NMMIIB to determine the temporal-spatial expression pattern of protein
expression during the remodeling phase of repair. Patient matched scar and normal fibroblasts
were analyzed for NMMII expression levels with flow cytometry, and subsequently enmeshed in
collagen lattices to analyze the role of NMMII in collagen matrix remodeling. Blebbistatin, a
specific NMMII inhibitor, was used to inhibit NMMII function in collagen matrix contraction.
Results: NMMIIA and NMMIIB was highly expressed in scar tissue during the remodeling
phase of wound healing and expression levels returned to normal after the remodeling phase of
repair. NMMIIA and NMMIIB was widely distributed throughout the cytoplam of scar fibroblast
but not uninjured dermal fibroblasts. Increased expression of NMMIIA and NMMIIB in scar
fibroblasts correlated with increased collagen lattice contraction rates. Blockade of NMMII
prevented scar and normal fibroblast collagen lattice contraction. Conclusion: NMMII
expression changes coordinately with the remodeling phase of repair. NMMII overexpression in
scar fibroblasts correlates with increasing cell contractility. NMMIIA and NMMIIB is
upregulated during the remodeling phase of repair to promote cellular contractility, but as the
remodeling phase resolves so do NMMIIA and NMMIIB.

PT3.07
PERSISTENT REGENERATIVE SIGNALING IN TUBULE EPITHELIUM WITH
FAILED DIFFERENTIATION LEADS TO VICARIOUS CYTOKINE PRODUCTION,
FIBROBLAST PROLIFERATION AND INTERSTITIAL FIBROSIS AFTER
ISCHEMIC KIDNEY INJURY
Rongpei Lan, PhD, Hui Geng, PhD, Pothana Saikumar, PhD, Manjeri A. Venkatachalam,
MBBS.
University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Regenerating tubules fail to re-differentiate during late recovery after ischemic kidney injury;
this is associated with interstitial fibrosis. Both are corrected by TGF-beta signaling inhibition
(Am J Pathol 174:1291, 2009). We investigated connections between regenerative signaling and
fibrosis. Rats were studied serially after 45 minutes renal ischemia. Cultured proximal tubule
epithelium was studied after wounding with a device that we reported (Wound Rep Reg 17:A10,
2009). Both models were studied by morphology, immunohistochemistry and immunoblotting
for signaling proteins. Expression of PDGF-B, a chemotactic and fibrogenic peptide, remained
persistently high in a sub-population of regenerating tubules 14 days after reperfusion, when
azotemia was corrected. The same population exhibited flat epithelium, little expression of
differentiation markers, decreased expression of PTEN, little labeling for proliferation marker
Ki67 and high labeling for cdk-inhibitor p21. Interstitium surrounding these tubules showed
numerous (myo)fibroblasts expressing PDGFR-beta, the receptor for PDGF-B and increased
Collagen I. Compared to these abnormal tubules in areas of fibrosis, the remainder of recovering
tubules were phenotypically normal and differentiated, without expression of PDGF-B;
associated interstitium was also normal. Parallel studies on wounded regenerating epithelium
showed increased expression of PDGF-B and secretion of phlogistic cytokines. Signaling studies
showed that at least two major signaling pathways - lysophosphatidic acid mediated GPCR and
TGF-beta - had separate and independent, as well as interdependent and cooperative effects on
the control of epithelial differentiation and cytokine expression. Our studies suggest that
following ischemia-reperfusion, a sub-population of regenerating tubules fail to re-differentiate.
These tubules with abnormal phenotype exhibit amplified signaling initiated physiologically as
early reparative responses that have persisted pathologically during recovery and prevent
differentiation. Persistent signaling by de-differentiated epithelium results in increased
expression of phlogistic and fibrogenic cytokines such as PDGF-B, causing fibrosis through
paracrine stimulation of receptors such as PDGFR-beta on interstitial cells.
PT3.08
SYSTEMICALLY ADMINISTRATED CURCUMIN CAN SIGNIFICANTLY PROMOTE
WOUND HEALING AND IMPROVE HYPERTROPHIC SCARRING
Shengxian Jia, MD, PHD 1 , Yanan Zhao, MD 1 , Adam Singer, MD 2 , Richard A. F. Clark, MD 2 ,
Thomas A. Mustoe, MD 1 .
1 Northwestern University, Chicago, IL, USA, 2 Stony Brook University, Stony Brook, NY, USA.

Curcumin, the major chemical in the curry spice tumeric, is widely used in alternative medicine
for its purported anti-inflammatory and antioxidant activities. The goal of this study was to test
the efficacy of curcumin on wound healing and scar reduction in a rabbit ear scar model. Fifteen
young New Zealand White rabbits were used. 20µg or 100µg of curcumin were given
intravenously with vehicle used as control. Four 7-mm full-thickness dermal punches were then
made on the inner surface of each ear down to perichondrium. Animals were sacrificed and the
wounds were collected and processed for histological analysis at 7 and 28 days, respectively.
Results: Treatments with 20µg or 100µg curcumin promoted wound healing significantly versus
control in terms of epithelial gap (3643±326 and 1954±292µm vs 4990±230µm, p

vivo compared with controls. The results suggest that ESS prepared using keloid-derived cells
represents a suitable organotypic model for studies of keloid scarring. A major advantage of this
model is the ability to achieve stable engraftment in mice, providing an animal model of human
keloid scarring that can be utilized for further studies. By advancing our understanding of keloid
formation, novel therapeutic interventions can be developed.
PT3.10
REPOSITIONING FOR THE PREVENTION OF PRESSURE ULCERS AN ECONOMIC
ANALYSIS
Zena E. Moore, PhD 1 , Seamus Cowman, PhD 2 .
1 Royal College of Surgeons in Ireland, Dublin, Ireland, 2 Royal Colleg of Surgeons in Ireland,
Dublin, Ireland.
Background: An economic analysis was conducted to explore the cost implications of
repositioning for the prevention of pressure ulcers. Aim: To compare the cost implications of
repositioning individuals using 2 different repositioning regimes - the experimental group (n=99)
were repositioned 3 hourly at night, using the 30 degree tilt; the control group (n=114) received
standard care (6 hourly turning using the 90 degree lateral rotation).Methods: Ethical approval
was received. Cost analysis focussed on the number of nurses per turn, time per turn, cost of a
nurse per minute and cost of dressing treatments and nurse time for dressing changes for pressure
ulcers that developed during the study period. Data were collected for a 4 week period. Results:
The mean daily nurse time was 18.5 minutes (experimental) and 24.5 minutes (control)
(p

INCIDENCE OF METHEMOGLOBINEMIA IN PATIENTS RECEIVING CERIUM
NITRATE AND SILVER SULFADIAZINE FOR THE TREATMENT OF BURN
WOUNDS: A BURN CENTER’S EXPERIENCE
Melissa A. Kath, MD, Jeffrey W. Shupp, MD, Sarah E. Matt, MD, James C. Jeng, MD, Marion
H. Jordan, MD.
The Burn Center, Washington Hospital Center, Washington, DC, USA.
In 1976, the combination of cerium (cerous) nitrate and silver sulfadiazine (SSD) was introduced
as a topical therapy for burn wounds. Experience with a locally-prepared combination agent has
shown physical change (dessication) of the eschar and delayed sub-eschar bacterial colonization.
A potential systemic complication of cerium-SSD application is methemoglobinemia (Met-Hb)
due to due to the oxidizing nature of Ce(NO3)3. Met-Hb has a spectrum of clinical
consequences, from headache and cyanosis to cardiac ischemia, hypotension, and even death.
There are only a few isolated case reports in the literature that describe met-Hb complications
subsequent to using cerium-SSD. Given the frequent use of this agent at this burn center a
retrospective review was conducted. A query of the pharmacy records revealed 167 patients from
January 2005 to October 2009 that had been treated with cerium-SSD. Charts for these patients
were reviewed for age, burn size, and the development and subsequent treatment of met-Hb.
Sixteen patients in this group developed methemoglobinemia as noted on arterial blood gas (met-
Hb concentration greater than 3%, range 3.3% to 23.5%). In the majority of cases, there was no
clinical indicator of met-Hb development. The patients ranged in age from 18 to 93 years;
percent total body surface area burned was 20% to 95%. Most patients’ relative hypoxia resolved
with the cessation of cerium-SSD and only one person required multiple treatments with
methylene blue. With appropriate monitoring of methemoglobin concentrations during treatment
with cerium-SSD, the development of met-Hb can be realized early and morbidity and mortality
related to hypoxia can be avoided. Cerium-SSD remains a valuable treatment option for deep
partial and full-thickness burn wounds.
PT4.02
ANALYTICAL APPROACH TO DECODING INFORMATION ON INTERSTITIAL
FLUID DYNAMICS FROM MAGNETIC RESONANCE IMAGES (MRI) OF BURNED
DERMIS
Maria P. McGee, MD, Michael Morykwas, PhD, Louis Argenta, MD.
Wake-Forest University, Winston-Salem, NC, USA.
The interstitial edema that follows heat-injury reflects fluid-transfer anomalies putatively of local
origin, for which the exact pathogenesis remains speculative. We investigate structure-function
correlations of pathological fluid-transfer in dermal interstitium by comparing MRI intensity and
water’s influx-times distributions in both undamaged controls and ex-vivo heat-damaged (2
°C/min to a 60 °C target temperature then maintained for 30 min;

gravidometrically. Mean intensities of T2 maps’ histograms were 78 and 50 (grey value) with
variation-coefficients of 16 and 39 % for burned and control dermis, respectively; the
corresponding images’ fractal dimensions, obtained by the box-counting method were 1.52 ±
0.02 and 1.72 ± 0.05, suggesting changes toward lesser structural complexity in damaged
compared to controls. Like the image-intensity histograms, the distribution of water influx-times
constructed from dermal-swelling kinetics were narrower in burned skin with times to half
maximal volume of 41 ± 17 and 113 ± 37 minutes, again, consistent with decreased complexity
of flow-paths. Qualitatively, these changes in fluid-transit kinetics are as predicted from a
theoretical model relating transit-times to imposed randomness and consequent fractal
dimensions of flow-paths (Karshafian et al, Phys. Med. Biol.2003). Together, these ex-vivo
results indicate abnormalities in the distribution of water-potentials in burned tissue occurring
independently of vascular, neurological and hormonal influences, and are therefore, due to local
physicochemical changes in the interstitial matrix; increased intensity in T2 images does not
imply increased interstitial water-volume. These data further suggest that changes in fluidtransfer
dynamics across interstitial spaces can be surmised from fractal dimensions differences
in T2 maps.
PT4.03
IMPROVED HEALING OBSERVED IN A PARTIAL THICKNESS BURN INJURY
TREATED BY INFLAMMATION-MODULATING POLYMERS
Liang Tso Sun 1 , Shan Natesan, Ph.D. 2 , Robert J. Christy, Ph.D. 2 , Newell R. Washburn, Ph.D. 1 .
1 Carnegie Mellon University, Pittsburgh, PA, USA, 2 United States Army Institution for Surgical
Research, San Antonio, TX, USA.
Burn injuries are characterized by intensive inflammatory responses, that further tissue injury
and delay healing. Hyaluronic acid (HA) based material conjugated with interleukin-1beta and
tumor necrosis factor alpha neutralizing antibodies was fabricated, and rat partial thickness burn
model was utilized to examine the effect of this material on healing. Partial thickness burn was
induced on the dorsal of rat. Eschar was removed the next day, followed by treatments with
saline, HA, and HA-mAb conjugates. The treatments were re-applied every two days until the
third treatment was applied, and the rats were sacrificed 1 day, 4 days, 7 days, and 21 days after
first treatment to follow healing advance of the burn wounds. The progression of healing was
determined by wound contraction and histological analysis. Preliminary results suggest that the
wounds treated with HA-mAb conjugates contract in a faster rate than that treated with HA and
saline. Masson’s trichrome images of the injury sites show less inflammatory progression, tissue
damage, and earlier granulation in wounds treated HA-mAb conjugates than that treated with
controls. With these preliminary observations, we concluded that injuries treated with
inflammation-modulating polymers can lead to faster and more efficient healing in a burn model.
More experiments will be performed to establish statistical significance.
PT4.04
IMPACT OF CHRONIC VENOUS DISEASE AND PERIPHERAL ARTERIAL
DISEASE ON PHYSICAL ACTIVITY FOR ADULTS IN DRUG TREATMENT

Barbara A. Pieper, PhD, RN 1 , Thomas N. Templin, PhD 1 , Thomas J. Birk, PhD, MPT 1 , Robert
S. Kirsner, PhD, MD 2 .
1 Wayne State University, Detroit, MI, USA, 2 University of Miami, Miami, FL, USA.
Purpose: We examined how chronic venous disease (CVD), peripheral arterial disease (PAD)
and other risk variables (i.e., health conditions, body mass index, age, years of opioid addiction,
site of injection drug use, and motivation for physical activity) impacted physical activity in
adults in methadone-maintained therapy. Methods: The study used a three-drug group, crosssectional,
comparative design with stratification (age, gender, and ethnicity). Participants
completed physical activity questionnaires and had their legs assessed for CVD and PAD.
Results: Participants (N=569) were female (53%), African-American (61%), and had a mean age
of 46 years. Ninety-two percent of them had CVD and 18.5% had PAD (ABI < .90 in either leg).
Advanced CVD was highly related to type of drug use [χ 2 (2, N=569)=105.62, p

was 0.71 mm/week for the entire cohort. Two of the patients achieved complete wound closure.
None of the patients developed additional ulcers or other adverse events while using the latexfree
tubular bandages. Conclusion: This study indicates that compression with a latex free
tubular elastic bandage can be safely used in patients with VLU requiring frequent dressing
changes.The healing rate and decrease in ulcer area obtained with this type of compression
suggest that the ultimate healing of the ulcer may not be compromised, and that this compression
regimen can be used in experimental protocols.
PT4.06
INFERRING MECHANISM FROM MORPHOLOGY: UNDERSTANDING PRESSURE
ULCER FORMATION IN SPINAL CORD INJURY PATIENTS USING AGENT-BASED
MECHANISTIC SIMULATIONS
Cordelia Ziraldo 1 , Alexey Solovyev 1 , M. Kristi Henzel 1 , Gwendolyn A. Sowa 1 , David Brienza 1 ,
Gary An 2 , Qi Mi 1 , Yoram Vodovotz 1 .
1 University of Pittsburgh, Pittsburgh, PA, USA, 2 Northwestern University, Chicago, IL, USA.
Purpose: Pressure ulcers are complications of spinal cord injury (SCI), and are hypothesized to
form either due to ischemia/reperfusion or shear force. We hypothesized that agent-based
mechanistic simulations can elucidate the relative impact of these two inducing stimuli, as well
as the roles of inflammation and healing, in pressure ulcer formation. Methods: We constructed
our model using a novel agent-based modeling framework (SPARK) developed in our group to
facilitate translational modeling applications, The model simulates epithelial cells, inflammatory
cells, blood vessels, inflammatory cytokines, and danger signals, and incorporates mechanistic
rules for pressure-induced ischemia/reperfusion and shear stress-mediated injury. Results: We
simulated two types of initial injury: 1) a circumscribed area of pressure whose intensity
decreases radially, simulating the effect of a bony protrusion against soft tissue, followed by
ischemia/reperfusion injury, activation of an inflammatory cascade, and production of oxygen
free radicals; or 2) shear force injury, in which epithelial cells are damaged by tissue stretching.
In both cases, tissue damage stimulates inflammatory cells to produce cytokines, and further
damage caused by cytokines leads to ulceration. The shapes of the simulated ulcers qualitatively
match patterns seen clinically, including position and size relative to the protrusion and the
development of secondary satellite ulcers that eventually coalesce with the primary ulcer.
Simulated ulcers induced by ischemia/reperfusion injury are predicted to be circular, whereas
ulcers induced by shear stress are predicted to be elliptical. Photos of ulcers taken at early
timepoints often show an elliptical shape. Conclusions: Our studies suggest that agent-based
modeling may be a powerful tool for elucidating mechanisms of post-SCI ulcer formation.
Modeling provides a means of linking cellular and molecular mechanistic knowledge to
clinically evident phenomena, namely the shape and evolution of pressure ulcers. Models of this
type may serve as a platform for the rational development of therapies.
PT4.08
CHRONIC VENOUS DISEASE, LEG PAIN, AND WALKING MOBILITY IN
METHADONE TREATED ADULTS

Barbara A. Pieper, PhD, RN 1 , Thomas N. Templin, PhD 1 , Thomas J. Birk, PhD, MPT 1 , Robert
S. Kirsner, PhD, MD 2 .
1 Wayne State University, Detroit, MI, USA, 2 University of Miami Miller School of Medicine,
Miami, FL, USA.
Purpose: We examined the bidirectional relationship between walking mobility and chronic
venous disease (CVD) for persons in drug treatment. Leg pain, chronic venous disease, and
walking mobility were expected to form a negative feedback loop in which each variable acted
as a mediator for the other two. Methods: We used a three-drug group, cross-sectional,
comparative design with stratification (age, gender, and ethnicity). Participants completed
questionnaires, rated their leg pain, had their legs assessed for CVD, and performed Tinetti
Balance and Gait and walk speed testing. Results: Participants (N=713) included men (46.9%)
and African-Americans (61.7%) (Mean age=46.26 years). Those who injected drugs (n=518) did
so for a mean of 13.08 years. Participants (92.3%) had clinical changes associated with CVD.
The overall mean leg pain score was 3.69 (possible range = 0 to 10). Balance and gait scores
were high indicating generally good stand balance and gait. Walk speed was very slow and
similar to a household ambulator. The structural equation model pathway from injection site to
CVD was .40 (p

Results: IPT and VPT produced similar wound bed characteristics. IPT and VPT caused a
gradual increase in wound bed contraction, while this effect was immediate for continuous
NPWT. IPT and VPT resulted in more granulation tissue formation than continuous NPWT.
Furthermore, leukocyte infiltration, tissue disorganization, i.e. the disruption of the contacts
between the cells and differences in cell size, was more prominent under IPT and VPT than
under continuous NPWT. These findings suggest faster conversion of the cells in the wound bed
to become fibroblasts and lay down collagen in the process of forming granulation tissue under
IPT and VPT. Conclusions: The effects of IPT and VPT on the wound bed are simialr. IPT and
VPT produce more granulation tissue than continuous NPWT. The mode of negative pressure
application may be selected depending on desirable effects on the wound bed. VPT may have the
clinical advantages of maintaining the negative pressure atmosphere throughout the therapy.
PT4.10
OFFLOADING SURFACE TENSION EFFECTS LOCAL CHANGES IN THE DERMAL
ARCHITECTURE OF SEQUESTERED MURINE SKIN
Michael Januszyk, MD, Ivan Nick Vial, BA, Kristine C. Rustad, BS, Victor W. Wong, MD,
Melanie R. Major, Jason P. Glotzbach, MD, Mackenzie R. Wehner, BS, Sarah A. Long, BA,
Michael T. Longaker, MD, MBA, Geoffrey C. Gurtner, MD.
Stanford University, Stanford, CA, USA.
Redundant skin margins, or “dog ears,” are a frequent byproduct of surgical closures, and their
appearance often provides a source of undue post-operative patient anxiety. Although
considerable anecdotal evidence suggests that these imperfections will settle over time, the
mechanisms underlying this phenomena remain poorly understood. Recently, the rise of bariatric
surgery has highlighted the natural atrophy, over time, of excessive skin folds following rapid
weight loss, further supportive of a role for local mechanical forces. Our laboratory has
developed a murine model of excessive skin sequestration, which allows us to examine the
nature and time course of this process. Healthy CD1 female mice were tattooed dorsally using a
plastic template device to achieve two concentric circles (areas: 8 cm2 and 4 cm2). A doublelayered
purse string suture was run in a circular fashion along the outer tattoo perimeter of each
mouse, circumscribing an area of 8 cm2. One hour later, this suture was either tightened to 2 cm2
or left at its original configuration (control). In the former group, the affected skin assumed an
elevated, “goblet-like” topography. Sutures remained in each mouse for 1 day, 2 weeks, or 8
weeks, with gross photographs taken at regular intervals. Mice were then sacrificed at 1 day, 2
weeks, or 8 weeks after suture removal, at which point final gross measurements were recorded.
Each circular skin region was then processed for histological analysis. Resting skin area in the
“compressed” group returned to levels intermediate to those of pre-wounded skin. Dermal
thickness was considerably increased in this group, although total dermal volume remained
relatively constant over time. Further, cellular density and composition did not change
significantly in either group, suggesting that thickening was not attributable to inflammation.
These data demonstrate a direct link between mechanical offloading and sustainable changes to
dermal architecture.
Poster Talk 5 - Diabetic Wounds: Novel Mechanisms and Development of Potential

Therapies – PT5.01 to PT5.10
Monday, April 19, 2010, 3:30 to 5:30 pm
PT5.01
HYPERGLYCEMIA TIME IS A MAJOR FACTOR IN SKIN WOUND HEALING
Yiqun Mo, MD, PhD, Rong Wan, MD, PhD, Jiangpu Wang, MD, PhD, Sufan Chien, MD,
Qunwei Zhang, MD, MPH, PhD.
University of Louisville, Louisville, KY, USA.
Many wound studies have been performed in rabbits with diabetic times shorter than 2 months,
but diabetic wounds in humans are the result of decades of diabetic times. This study was
designed to test the short and long term effects of hyperglycemia on the wound healing in the
rabbits. In the first study, a total of 16 young adult New Zealand white rabbits were divided two
groups in which 8 of them were induced hyperglycemia by alloxan (100mg/kg, IV); another 8
were used as control (normoglycemia) group. Hyperglycemia (great than 350 mg/dl) developed
within 48 hours in all animals and stabilized thereafter. Insulin (1-4U/kg, SC) was given daily to
control hyperglycemia. After two weeks, four circular full-thickness wounds were created on the
ventral side of each ear with a 6-mm stainless steel punch. Normal saline was used and the
wounds were covered with Tegaderm. Dressing changes were made and digital photos were
taken daily until all wounds were healed. The closure times were 12 to 22 days (mean 16.4±3.4)
in the diabetic wounds and 13 to 18 days (mean 15.7±1.2) in the control group. There were no
significant differences in closure times between the two groups (p= 0.3865). In the second study,
12 rabbits were used in which 6 of them were induced hyperglycemia by alloxan and 6 were
used as controls. After 9 months of hyperglycemia, wounds were created and managed the same
way as above. Wound closure times were 19.3±3.2 days in diabetic group and 15.1±1.0 days in
control group. There was significant difference between the normal and long diabetic rabbits. In
summary, long term, but not short term hyperglycemia significantly affects the wound healing in
the rabbit ear model.
PT5.02
INFLUENCE OF WOUND CARE CENTER VISIT FREQUENCY ON WOUND
HEALING OUTCOME OF DIABETIC FOOT AND VENOUS LEG ULCERS
Robert A. Warriner, III, M.D. 1 , James R. Wilcox, R.N. 1 , Deborah Stewart, M.D. 1 , Marissa J.
Carter, Ph.D. 2 .
1 Diversified Clinical Services, Jacksonville, FL, USA, 2 Strategic Solutions, Inc., Cody, WY,
USA.
Methods: Data were collected prospectively on the healing of 56 Wagner grade I/II diabetic foot
ulcers (DFU, 1 wound per patient) that were examined on a weekly basis (could be seen more
frequently than once per week) and 69 similar wounds examined on a biweekly basis defined as
more than 10 days between any visits in the first 4 weeks. No amputations or surgically closed

wounds were included. Data were also collected on 39 venous leg ulcers (VLUs) examined
weekly and 38 VLUs examined biweekly. Ulcers had to have a length/width < 5 cm and a depth
≤ 0.2 cm. For each type of DFU, the following variables were compared between the weekly and
biweekly groups using independent t tests with statistical significance adjusted using a full
Bonferroni correction: wound healing (%) at 4 weeks, time to heal (weeks), and number of
excisional debridements per wound. VLUs were compared similarly except for wound healing
(%) at 4 weeks. Results: For the DFUs, the means of the 3 parameters were 91% and 47%, 24
and 101 days, and 0.95 and 2.56 excisional debridements per wound, respectively. After
Bonferroni adjustment, the significance (P) of each of these results was < .001, < .001, and .006.
For the VLUs, the means for time to heal and number of excisional debridements perwound were
21 and 90 days, and 0.47 and 1.54 respectively for the weekly and biweekly examination groups.
After full Bonferroni correction, the former parameter between the groups was significant (P <
.001) but the latter parameter was not significant.
Conclusion: In this simple analysis examining the wound at least twice as frequently improved
healing rates dramatically. It is likely that several factors were involved in producing the marked
difference in outcome reported.
PT5.03
GRANULOCTYE-MACROPHAGE COLONY STIMULATING FACTOR ENHANCES
WOUND HEALING IN DIABETES VIA UPREGULATION OF PROINFLAMMATORY
CYTOKINES
Yong Fang, MD, PhD 1 , Jie Shen, MD, PhD 2 , Min Yao, MD, PhD 1 , Kenneth W. Beagley 3 , Brett
D. Hambly, MBBS, PhD 2 , Shisan Bao, MD, PhD 2 .
1 Institute of Traumatic Medicine, Shanghai Jiao Tong University, Shanghai, China, 2 Discipline
of Pathology, The Bosch Institute, The School of Medical Sciences, Faculty of Medicine, The
University of Sydney,, Sydney, Australia, 3 Institute of Health and Biomedical Innovation,
Queensland University of Technology, Kelvin Grove, Australia.
Chronic ulceration, especially in diabetes remains a substantial clinical problem. Exogenous
GM-CSF is efficacious in the treatment of chronic wound healing in both animal models and
patients, but its role in diabetic wounds remains to be explored. Using a diabetic mouse model,
the role of GM-CSF in wound healing has been investigated, with clinical observation,
histopathology, immunohistochemistry and cytokine assays. There is a significant reduction
(50%) GM-CSF production in the wounds of the diabetics compared to non-diabetics.
Exogenous GM-CSF substantially enhanced the wound healing in diabetes, accompanied by
increased IL-6 and MCP-1 production. The elevated cytokines correlated with increased
neovascularization, infiltration of macrophages and neutrophils. GM-CSF showed no beneficial
effects in non-diabetic wound healing. Our results provide useful guidelines for the clinical
management of chronic ulceration in diabetes.
PT5.04
TIME COURSE STUDY OF BIOFILM INFECTED WOUNDS IN DIABETIC MICE

Ge Zhao, M.D., Ph.D. 1 , Marcia Usui, B.S. 1 , Robert Underwood, B.S. 1 , Pradeep Singh, M.D. 1 ,
Garth James, Ph.D. 2 , Philip Stewart, Ph.D. 2 , John Olerud, M.D. 1 , Philip Fleckman, M.D. 1 .
1 University of Washington, Seattle, WA, USA, 2 Montana State University, Bozeman, MT, USA.
A reproducible and consistent animal model is critical in studying the mechanism and
management of chronic wound healing. Our previous work demonstrated that Pseudomonas
aeruginosa (PAO-1) biofilm infection on wounds in diabetic (db/db) mice significantly delayed
wound healing: control 6 mm wounds on db/db mice healed by 4 weeks, while scabs from
biofilm infected wounds at 4 weeks showed abundant P. aeruginosa colonies. In the present
study, we compared time-to-closure of 6 mm wounds between control db/db mice and mice with
biofilm infection. PAO-1 bacteria were cultured on polycarbonate membrane filters for 72h to
form bacterial biofilms. The biofilms were transferred onto 2 day wounds (6 mm) created on the
dorsal surface of the experimental groups in db/db mice. All the wounds were covered with an
occlusive dressing for 12 days. Wounds were harvested at 4, 6 and 8 weeks post-wounding. Five
of 6 (83%) control wounds healed by 4 weeks; none of 5 (0%) biofilm-infected wounds healed
by 4 weeks; 4 of 5 (80%) of the biofilm-infected wounds healed by 6 weeks; and 4 of 4 (100%)
of the biofilm-infected wounds healed by 8 weeks. Four mice died during the course of the study.
Colony forming units (CFU) on biofilm infected wounds only contained 10 3 -10 4 P. aeruginosa
after 4 weeks. In contrast, scabs from the same wounds had bacterial load of 10 7 CFU. The
bacteria in the scabs were more resistant to ofloxacin treatment than planktonic PAO-1. In
conclusion, our research further demonstrated a delayed wound healing process in diabetic mice
in response to applied pseudomonas biofilm. The presence of antibiotic resistant biofilms was
observed in the scabs. Bacterial biofilm may be a factor in chronic wounds, and the db/db mouse
biofilm wound model will be a useful test bed for exploring new strategies to control biofilm in
vivo.
PT5.05
CHARACTERIZATION OF HOST RESPONSES TO STAPHYLOCOCCUS AUREUS
DERIVED BIOFILM IN A NOVEL POLYGENIC MURINE MODEL OF DIABETES
Da P. Jin, BS, Seok Jong Hong, PhD, Thomas A. Mustoe, MD, Robert D. Galiano, MD.
Northwestern University, Chicago, IL, USA.
Background: It is clinically appreciated that biofilm plays an important role in the delay of
healing in chronic wounds in diabetic patients. We investigate the host responses of two different
murine models of diabetes mellitus (db/db and polygenic NONcNZO10) to Staphylococcus
aureus biofilm, in hopes of elucidating key mechanisms that impair wound healing and defenses
against biofilm formation in diabetes.
Methods: Bilateral splinted excisional wounds were created in the dorsum of db/db,
NONcNZO10, and wild type mice. Wounds were inoculated with Staphylococcus aureus biofilm
at the time of wounding. On post-wounding day 10, mice were euthanized and the wound beds
were harvested with 1 mm surrounding skin for routine histology, immunofluorescence, and
gene expression analysis.
Results: Routine histology reveals impaired wound healing in the diabetic mice when compared
with the wild type. Immunofluorescence and gene expression analyses reveal key differences in

the innate immune response between the db/db and wild type, for example, in the TLR2 and
TLR4 pattern recognition receptors. In this ongoing study, we are also investigating whether
these differences also hold true for the polygenic NONcNZO10 murine model.
Conclusions: In this study of biofilm established wounds in diabetes, we reveal key differences
in the diabetic host response when compared to non-diabetic mice. These differences are seen in
the pattern recognition receptors of the innate immune system, and reveal mechanistic failures in
the healing of chronic wounds.
PT5.06
BLADDER OUTLET OBSTRUCTION: PROGRESSION FROM INFLAMMATION TO
FIBROSIS
Peter Metcalfe, MD, Jian Fei Wang, Ph. D., Keijiro Hori, MD, Haiyan Jiao, B.Sc., Yue Huang,
B.Sc., Ron Moore, MD, PhD, Edward Tredget, MD.
University of Alberta, Edmonton, AB, Canada.
Introduction: Partial bladder outlet obstruction (pBOO) results in a progression of
pathophysiologic changes, which results in a risk for renal damage. We hypothesize that
inflammation and upregulation of inflammatory cytokines occurs in response to the physical
stress of pBOO and this results in smooth muscle hypertrophy, and decompensation to fibrosis.
Methods: Healthy, adult, female Fischer rats underwent surgical creation of a pBOO for either
2,4,8, or 13 weeks. Urodynamic measurements were used to compare bladder volumes and
pressure. Tissue was grossly analyzed with light microscopy and bladder weights and
thicknesses were compared. RT-PCR for collagen, TGF-β, CTGF, HIF-1α, and PDGF-A was
performed on all samples, as well as immunohistochemistry for α-SMA. Mass spectrometry was
used to quantify the collagen content of the bladders. Results: Initially, urodynamics
demonstrated an increase in capacity while maintaining normal pressures, but then deteriorated
into small capacity, high-pressure bladders. H+E demonstrated an initial inflammatory response,
and this was confirmed with significantly increased mRNA levels of TGF-β, CTGF, HIF-1α, and
PDGF. The progression to smooth muscle hypertrophy was evident on H+E and confirmed with
increased bladder mass and thickness. IHC for α-sma demonstrate the increase in myofibroblasts
associated with the increased bladder pressures. Masson’s Trichrome and mass spectrometry
showed a progressive increase in collagen to 13 weeks. Conclusion: With this model, we have
effectively replicated the distinct clinical phases of bladder hypertrophy and decompensation
after pBOO. The time course of the inflammatory markers implicates their role in this process
and implies the need for early intervention to prevent this cascade. Novel strategies targeting
these observed physiologic responses could lead to preventative therapeutic strategies warranting
further exploration. In addition, the resulting end-stage bladder model allows for the potential
investigation of tissue-engineered bladder substitutes
PT5.07
CLINICAL EFFICACY: KNOWING WHAT WORKS

Laura Bolton, PhD, Adjunct Assoc. Professor.
UMDNJ Dept. of Surgery (Bioengineering), Metuchen, NJ, USA.
Rationale: Clinicians use evidence of product efficacy to supplement clinical judgment as they
strive to deliver consistent quality patient and wound outcomes. The scientific method that
proves efficacy is clear, but frequent misleading use of the word “efficacy” in conclusions and
interpretations can confuse clinical decision makers. More accurate efficacy communication can
improve professional capacity to support and implement accurate care decisions. Objective:
Review wound care efficacy literature to define efficacy and clarify its use in informing sound
patient-oriented practice decisions. Methods: MEDLINE, Joanna Briggs, AHRQ and Google
websites were reviewed respectively for scientific studies, best practice guidelines, evidencebased
practice and lay literature definitions and interpretations of wound care efficacy. The best
available evidence of efficacy was summarized for an example wound to illustrate a simple path
for using evidence of efficacy to inform decisions about what may work best for a given patient.
Results: Evidence of wound care modality efficacy was summarized in the context of recognized
AHRQ “strength of evidence” definitions. Conclusions supportable by each type of study design
were illustrated with examples of best available evidence from published literature for common
modalities. Some wound care modalities had clear evidence of efficacy. Several incorrectly used
case studies or series to support “efficacy” claims. Few modalities had clear evidence of efficacy
compared to current best practice. Conclusion: Improved uniformity and accuracy of efficacy
interpretations could reduce confusion and improve consistency of wound care practice and
outcomes, elevating the credibility of wound care as a specialty.
PT5.08
TAILORED PATIENT EDUCATION FOR ADULTS WITH CHRONIC WOUNDS
Ranjita Misra, PhD 1 , Lynn Lambert, MS 2 , David Vera, MS 3 , Ashley Mangaraj, BS 3 , Suchin R.
Khanna, BS 3 , Chandan K. Sen, PhD 3 .
1 Texas A&M University, College Station, TX, USA, 2 National Healing Corporation, Boca
Raton, FL, USA, 3 2The Ohio State University Comprehensive Wound Center, Columbus, OH,
USA.
Background: Rising healthcare costs have generated significant interest and growth in educating
patients with chronic wounds. Effective patient education is supported by the Joint Commission
on the Accreditation of Healthcare Organizations and considered a critical factor in the
prevention of illness by managed care organizations. Identifying population-specific barriers and
deficits, especially for individuals with diabetes, can help improve current practice with respect
to designing tailored education for preventable complications and amputations. Purpose/Method:
This retrospective study examined differences in patient’s knowledge and health behaviors by
gender, diabetic status, and race/ethnicity. The sample comprised of 1003 patients (72 % whites,
53% females) treated for acute, acute traumatic or chronic wounds at a hospital-affiliated
Comprehensive Wound Center in the Midwest. Results: The mean age and number of wounds
was 55.2±17 years and 1.9± 1.4 wounds respectively. The majority was high school graduates
(48%), on Medicare/Medicaid insurance (57%), and overweight/obese (69%). Thirty seven
percent of the patients had diabetes and 30% had infected wounds. Health behaviors indicated

20% were current smokers, 73% did not exercise, and 34% stated they had poor or fair appetite.
Furthermore, 51% self-reported their health to be fair/poor and 60% reported low/medium
knowledge of their health problems. Significant differences were noted by gender, diabetic
status, and racial/ethnic groups with females, minorities, and diabetics indicating a lower
knowledge of health problems, sedentary lifestyle, current or prior history of tobacco use, and
higher rate of infection. Expectedly, co-morbidities (hypertension, obesity) were higher among
individuals with diabetes and African American patients. Discussion/Conclusions: Effective
wound prevention programs should be grounded in health behavior change theory and tailored
content by patient’s gender/ethnicity/ diabetes status to promote behavior changes (physical
activity, nutritional habits, and smoking cessation). A strong knowledge base regarding how and
why these programs are effective can improve services and healing outcomes of patients.
PT5.09
A STATISTICAL APPROACH FOR AVOIDING PSEUDOREPLICATION AND
INCREASING POWER IN WOUND HEALING STUDIES
William G. Haag, Ph.D 1 , Obsidiana Abril-Horpel, PhD 1 , Sophie D. Becquerelle, PharmD 1 ,
Patricia M. Mertz, BA 2 , Stephen C. Davis, BS 2 .
1 Exoxemis, Inc., Little Rock, AR, USA, 2 University of Miami, Miami, FL, USA.
In analyzing categorical data, the use of contingency tables is appropriate when all data are
independent. In many animal models, results from multiple samples within multiple animals are
used to obtain information regarding animal variability or to satisfy statistical power
requirements. Contingency table analyses that do not take into account the animal-to-animal
variability result in pseudoreplication. Kaiser, et al., overcome pseudoreplication by adjusting the
inter-animal variation estimate to correct for the inter-animal covariance yet this solution is
limited by a loss of statistical power. An alternative method is described using historical data
from wound-healing studies conducted by Mertz, et al. This statistical approach initially
determines, for each treatment, the time for half of the wounds within each animal to be
completely healed (TCH50). These TCH50 values are subsequently analyzed using a model with
fixed treatments and random animals. Power calculations are made based on the estimated
variance components. This method generates unbiased estimates of treatment means and
differences between means, and provides accurate p-values for these differences and for the
overall model. It resolves the issue of pseudoreplication, increases statistical power, overcomes
inflation of false positive risk, and provides an accurate treatment error estimate. Comparisons of
differences between TCH50 values for different treatments is a reliable method of evaluating
potential products on wound healing with concomitant efficient animal use. This method is
suited for analysis of healing outcomes over time from any animal model having fixed treatments
on multiple sites crossed with animals. It has sufficient power to detect treatment differences
with fewer animals resulting in more accurate estimate of therapeutic outcomes and reduces
costs associated with study execution.
PT5.10
OBESITY IMPAIRS WOUND HEALING VIA A VASCULOGENIC MECHANISM

Janelle Wagner, MD 1 , Robert J. Allen, Jr., MD 2 , Edward H. Davidson, MA MBBS 2 , Orlando
Canizares, MD 2 , Pierre B. Saadeh, MD 2 , Stephen M. Warren, MD 2 .
1 New York University Institute of Reconstructive Plastic Surgery, New York, NY, USA, 2 New
York University Institute of Reconstructive Plastic Surgery, NY, NY, USA.
Background: Obesity is an epidemic. Since bone marrow-derived endothelial progenitor cells
(EPCs) are responsible for 35% of new blood vessel formation in healing wounds, we
hypothesize that impaired wound healing in non-diabetic obesity is due to EPC dysfunction.
Methods: Peripheral blood was obtained from non-diabetic obese patients (BMI>30, n=10), and
non-obese controls (BMI

cell-cell and cell-matrix interactions, TGFb I&III receptor (CD105), PDGFb receptor (CD140b)
for cellular response to these fibroblast proliferative cytokines, EpCAM (CD326) and Heat
Stable Antigen (CD24), both present on epithelial cells, Aminopeptidase (CD13) as it’s
expression facilitates matrix digestion and neutral endopeptidase (CD10). Cell samples harvested
at passage 4 for keratinocytes and passage 6 fibroblasts, were collected and analyzed by flow
cytometry. Staining of these markers have shown a clear population of fibroblasts expressing
CD105, CD61, CD10, CD140b on fibroblasts only; CD24, CD326, CD104 on keratinocytes only
whereas CD13 and the alpha integrins are expressed at varying levels on both cell types. CD29,
the alpha-integrins beta subunit is expressed equally high on both populations. These markers are
capable of defining one population from the other in culture and can indicate cell function.
CD61, fibrinogen receptor is only expressed on the fibroblasts, the cells that secrete matrix,
where as CD104, beta integrin responsible for cells anchoring to the basement membrane and
each other are only expressed on keratinocytes. CD49b, d and e, all receptors for collagen,
laminin and fibrinogen are present on both cell types at varying levels and indicate their
interactions with tissue formation and basement membranes. The expression of CD13 is
consistently greater on fibroblasts as these cells make and remodel matrix. Overall these markers
can define one population from the other and their levels expression can assist in identifying
cellular activity as it contributes to the generation of a bilayered living cell-based treatment.
BRC03
EVALUATION OF PREVENA TM INCISION DRESSING ON THE MITIGATION OF
HEMATOMA/SEROMA
Deepak V. Kilpadi, PhD, MBA.
Kinetic Concepts, Inc, San Antonio, TX, USA.
Background and Objective Hematomas and seromas can be undesirable consequences of surgery
and can have a negative impact on healing. The objective of this study was to evaluate the effect
of negative pressure therapy (NPT) using Prevena TM Incision Dressing on the quantity of
hematoma/seroma collected from subcutaneous voids beneath sutured incisions in a porcine
model. Methods In each of 8 female domestic swine, 4 subcutaneous voids (8 cm x 8 cm) with
overlying sutured incisions (5 cm) were created using liposuction tools on the abdomen/ventral
side abutting mammary tissue, with 2 on the right and 2 on the left of the mid-line. The incisions
in each set of contralateral incisions were assigned randomly to Prevena TM Incision Dressing
(NPT; KCI, San Antonio, TX) with continuous -125 mmHg and standard-of-care (SOC;
3M TM Tegaderm TM Dressing, 3M, St. Paul, MN), respectively. After 4 days of therapy, the
hematoma/seroma in each defect was weighed; differences between the SOC sites and
contralateral NPT sites were averaged for each animal. A paired-difference t-test was used to
determine if the means differed significantly from zero. Fluid levels in the negative pressure
canister were also monitored. Results and Discussion The mean difference in mass of
hematoma/seroma between the Prevena TM Incision Dressing (NPT) and contralateral SOC
groups was -25 (mean) ± 8 (SE) g (p=0.012). The weight of the hematoma/seroma was 63% less
with Prevena compared to SOC (Prevena: 15 ± 3 g, SOC: 41 ± 10 g). Fluid did not accumulate in
the canister, suggesting the underlying mechanism is something other than removal of fluid.
Studies are currently underway to understand alternative mechanisms. Conclusion This decrease

in mass of hematoma/seroma suggests that the Prevena TM Incision Dressing could potentially
help incisions with underlying voids heal in a more normal and timely manner.
BRC04
DEVELOPMENT OF A NOVEL MULTIDIMENSIONAL PRODUCT FOR POTENTIAL
WOUND HEALING APPLICATIONS
Necrisha Roach, BS 1 , Dorne Yager, PhD 1 , Dave Vachon, PhD 2 .
1 Virginia Commonwealth University, Richmond, VA, USA, 2 IASIS Molecular Sciences, LLC,
Spokane, WA, USA.
A tailorable family of macromolecular compounds possessing several properties that make them
potentially useful for the treatment of complex wounds have been identified. Methodology: The
parent macromolecule was tested for its ability to inhibit a number of proteases that are
associated with chronic wounds. The parent compound was found to efficiently inhibit the
activities of neutrophil-derived elastase, cathepsin G, MMP-8 and MMP-9. This compound is
water soluble and retained its effectiveness against several relevant proteases even at relatively
low concentrations (80% inhibition of elastase at14 nM). The MTT assay was used to examine
the effects of the parent compound on the viability of human dermal fibroblasts. Concentrations
as high as 31 micromolar did not have an affect on viability as reflected by the levels of
formazan generated. Various salts of this macromolecular compound were generated and tested
for their abilities to inhibit the growth of clinical isolates of Staphylococcus aureus,
Pseudomonas aeruginosa, and Acinetobacter baumanii. A silver salt exhibited MIC50s ranging
from 357 nM to 929 nM. with MIC90s ranging from 886 nM to 1.7 microM. A chlorhexidine salt
exhibited MIC50s ranging from 129 nM to 1.5 microM. A salt containing doxycycline exhibited
MIC50s ranging from 2.86 nM to 857 nM. We have demonstrated an ability to formulate a variety
of formulations that can release a number of clinically relevant agents including antibacterial,
anti-inflammatory, antifungal, antioxidant, and/or nutritive compounds directly into a wound bed
in a controlled fashion. Furthermore, it is straightforward to engineer formulations that contain
multiple substitutions in order to address any variety of “targets” as well as to tailor quantitative
strength. In light of the phenotypic heterogeneity of chronic wounds, this feature of this family of
compounds make them particularly attractive.
BRC05
PHYSIOLOGICAL PARAMETERS FOR EFFECTIVE COMPRESSION THERAPY OF
SWOLLEN LOWER LIMBS- SKIN TONOMETRY, TISSUE FLUID PRESSURE AND
FLOW
Waldemar L. Olszewski, Professor, Marzanna Zaleska, MSc, Marta Cakala, Dr.
Dept of Surgical Research &Transplantology, Medical Research Center, Polish Academy of
Sciences, Warsaw, Poland.
Introduction. Removal of excess of tissue fluid (TF) from injured tissue is indispensable for
healing, irrespective whether it is an open or closed wound. Aim. Mechanical compression is at

present the most effective method enabling tissue fluid to overcome tissue resistance and flow to
non-swollen regions. Methods. We studied hydraulics of tissue fluid in swollen lower limb
(lymphedema, venous ulcers, posttraumatic hematoma) using sequential pump with no deflation
of distal segments. Skin tonometry, TF pressure and flow were measured in calf and thigh.
Results. TF pressure generated by massage depends on skin rigidity. In advanced cases of
lymphedema with fibrotic skin, pressures in the sleeve had to be raised to as high as 150mmHg
to obtain the transmural pressure of 40 mmHg. This was the minimum pressures necessary for
TF flow. Newly proposed tonometry helped to choose proper sleeve inflation pressures.
Tonometer piston was pressed against swollen tissue to a depth of 10mm and applied force read
off on the scale. Simultaneously, TF pressure was measured under tonometer. Force plotted
against pressure gave hints how high massage pressure is required to move TF. Tonometer force
of 1000g/sq.cm generated average TF pressures of 30mmHg, of 2000g/sq.cm 50mmHg, above
2000g/sq.cm 70mmHg. Massage pressures had to be set accordingly. Strain gauge applied
around calf showed increase in circumferences depending on applied sleeve pressures. TF flow
calculated from girth increase ranged from 1 to 20 ml per inflation cycle. Conclusions.
Pneumatic compression to be effective should be based on prior skin tonometry and TF
pressure/flow measurements.
BRC06
ASSESSMENT OF KERATINOCYTE MIGRATION ON FIBRIN AND COLLAGEN
MATRICES OF DIFFERENT COMPOSITIONS IN A MODIFIED SKIN EQUIVALENT
WOUND MODEL
Haison S. Duong, M.S., Nasim Zarkesh, B.S, Ben Wu, D.D.S., Ph.D., Bill Tawil, MBA, Ph.D..
University of California, Los Angeles, CA, USA.
INTRODUCTION: The re-epithelialization of the dermis is an essential event in the dermal
wound repair process. Keratinocytes play an important role in this process [1]. Understanding the
relationship between matrix compositions and the speed of which keratinocytes can re-establish
the dermis may reduce our knowledge gap in the biology of wound repair. To explore ways to
accelerate keratinocyte re-epithelialization of wounds, we’ve established a skin equivalent
wound model (SEWM) to study keratinocyte migration on wound fillers of different matrix
compositions, namely: collagen and fibrin. In addition, we’ve looked at how incorporating
growth factors such as: keratinocyte growth factor (KGF) [2], basic fibroblast growth factor
(FGF), and the chemokine CTACK/CCL27 [3] into fibrin and collagen-based wound fillers can
affect the rate of keratinocyte migration and re-epithelialization of the SEWMs. METHOD:
SEWMs were prepared, in a Transwell® insert, by incorporating fluorescent labeled human
dermal fibroblasts into a collagen matrix. Fluorescent labeled human keratinocytes were
dispensed atop the fibroblast-collagen matrix, and the entire construct was cultured for 48 hours
in a defined skin equivalent medium. On day 3, simulated wound defects in the SEWM were
created by a 2 mm biopsy punch. The defects were filled with various formulations of
fibrinogen-thrombin or type I collagen. Photomicrographs of all SEWMs were taken at a focal
point of the filler areas at time points ranging from day 1 to day 14. Cell migration was
quantified by image analysis software (BioQuant®). RESULTS: Fibrin-based wound fillers of
different fibrinogen compositions exhibited different rates of keratinocyte migration. Collagen-

ased wound fillers also showed some degree of enhanced keratinocyte migration. Cell
migration was also enhanced with KGF and CTACKK/CCL27 while bFGF showed insignificant
effect on cell migration. CONCLUSION: Fibrin and collagen-based wound fillers with KGF and
CTACK/CCL27 enhanced keratinocytes migration and the re-epithelialization of the SEWM
defects
BRC07
ULTRASONIC TRANSPORT OF DISSOLVED OXYGEN IN TISSUE USING SAW
TECHNOLOGY
Michael Morykwas, Phd 1 , Terry Williams 2 , Paul Schubert 2 .
1 Plastic and Reconstructive Surgery Wake Forest University School of Medicine, Winston-
Salem, NC, USA, 2 Trinity Wound Institute, LLC, Raleigh, NC, USA.
A follow-up study using hyper-oxygenated saline and SAW (surface acoustic waveform) low
frequency ultrasound technology substituted for a cymbal array flexural ultrasonic transducer
utilized in a previous experiment was performed to measure the transport of oxygen in tissue.
METHODS: 5 cm by 5 cm wounds were created in the epidermis on the dorsum of anesthetized
swine (n=3) lateral to the spine. Probes measuring dissolved oxygen by fluro-optical fibers were
inserted approximately 2-3 mm below the surface of the wound in the center and in the
periwound area. Dressings fitted with drip solutions were affixed to the wounds. Hyperoxygenated
saline (35-40 ppm) solution was dripped as a control and interstitial dissolved
oxygen levels were documented. A thin and flexible SAW transducer was then affixed to wound
sites atop the dressings and sonication was delivered at 90 kHz at 60mW/cm2 with a continuous
drip of hyperoxygenated saline. RESULTS: Baseline dissolved oxygen levels did not
significantly change with control drip of hyper-oxygenated saline. When sonication was
activated, PaO2 levels increased from 40 mm Hg to 100+ mm Hg in wound tissue directly under
the transducer and increased from 20 mm Hg to 60 mm Hg in the periwound area which was 3
cm lateral to the transducer. CONCLUSION: Use of SAW ultrasound technology in conjunction
with oxygenated solutions can predictably increase interstitial oxygen in and around a wound
bed. It should be noted that prior experiments with cymbal array transducers only registered
increase in oxygen levels directly under the transducer array. This technique may be a refined
method of treatment for hypoxic wounds such as diabetic foot ulcers.
BRC08
EVALUATION OF ANTI-INFLAMMATORY EFFECT OF CLOSTRIDIUM
COLLAGENASE AND ITS COLLAGEN DEGRADATION PRODUCTS
Lei Shi, PhD, Brett Kiedaisch, MS, Ryan Ermis, BS, Duncan Aust, PhD.
Healthpoint Ltd., Fort Worth, TX, USA.
Clostridium collagenase has been widely used in biomedical research to dissociate tissues and
isolate cells. Since 1965, the enzyme has been used as a therapeutic drug* for the removal of
wound necrotic tissues. Although evidence exists for a chemoattractant effect on human

neutrophils, C. collagenase is not known for its inflammation related activities. Our previous
studies showed that the enzyme could effectively degrade various types of collagens found in
wounds. Therefore, the inflammation activity of C. collagenase used in wound will extend to its
collagen degradation products. The purpose of this in vitro study was to investigate the level of
inflammation induced by bacterial lipopolysacchride (LPS) with pre-treatment of C. collagenase
and its degradation products of type I and III collagens. In this study, human monocytic leukemia
cells (THP-1) were chosen as the cellular target. THP-1 is a highly differentiated monocytic cell
line that will secrete proinflammatory cytokines IL-1, IL-6, and TNF-α in response to LPS
stimulation. In culture THP-1 cells were pre-treated for one hour with various test articles,
including C. collagenase, digests of human type I and III collagen, and controls. After pretreatment,
LPS was added to stimulate inflammation. Following four hours of exposure a TNF-α
ELISA assay was used to determine the anti-inflammatory effects of the various test articles.
Collagenase did not demonstrate any increase in TNF-α compared to LPS. Instead, it performed
similar to epigallocatechin gallate (EGCG), a bioactive component of green tea, which is wellknown
for anti-inflammatory effects, with significantly lowered TNF-α level. The digests from
collagen I and III demonstrated enhanced anti-inflammatory property, significantly better than
EGCG. The data suggest that collagenase treatment may provide anti-inflammatory activity by
the enzyme itself and by the collagen digests produced by C. collagenase. * Santyl ® Collagenase
Ointment (Healthpoint Ltd.)
BRC09
A VERSATILE IN VITRO BIOFILM MODEL USING TWO WOUND PATHOGENS TO
SCREEN FORMULATIONS
Catherine Van Der Kar, B.A., Eric Roche, Ph.D., Aleksa Jovanovic, Ph.D., Dennis Carson,
Ph.D..
Healthpoint Ltd., Fort Worth, TX, USA.
New therapies are needed to effectively treat biofilm infections in chronic wounds and thereby
enable wound healing. A simple in vitro colony model was used to evaluate the biofilm efficacy
of topical formulations against two prominent wound pathogens, Staphylococcus aureus and
Pseudomonas aeruginosa. Bacteria were spotted onto a collagen matrix resting on a filter on a
blood agar plate and incubated to allow biofilm formation. The model mimics in vivo wound
biofilms in that nutrients are provided from below the biofilm while topical treatments are
applied at the air interface above. The two wound pathogens were inoculated separately to form
monospecies biofilms and together in an attempt to generate polymicrobial biofilms. Stable
polymicrobial biofilms were not formed as P. aeruginosa predominated, maintaining steady
viable counts after biofilm establishment while the level of S. aureus decreased over time.
Efficacy studies were therefore focused on monospecies biofilms. Commonly used topical
antimicrobials were effective in preventing biofilm formation by both species if applied when the
filter was inoculated. However, when the biofilm was allowed 24 hours to form, the effect of
these topical agents was substantially reduced. Several differences in efficacy were observed for
topical agents between the two species; for example, triple antibiotic ointment was more
effective against S. aureus than P. aeruginosa whether treatment was initiated early or late. The
rank order of efficacy for topical antimicrobials against S. aureus biofilms corresponded to the

anking observed in an in vivo model of MRSA wound biofilm. The model enabled
identification of novel formulations with superior biofilm efficacy. These results demonstrate the
utility and validity of a simple in vitro wound biofilm model for screening biofilm efficacy prior
to in vivo studies.
BRC10
DEVELOPMENT OF TWO RAPID SCREENING MODELS OF MONOSPECIES
BIOFILM INFECTION IN SUPERFICIAL MURINE WOUNDS
Eric Roche, Ph.D. 1 , Paul Renick, M.S. 1 , David Valtierra, B.A. 2 , Shannon Tetens, B.A. 1 , Dennis
Carson, Ph.D. 1 .
1 Healthpoint Ltd., Fort Worth, TX, USA, 2 UNT Health Science Center, Fort Worth, TX, USA.
With the growing evidence of bacterial biofilms disrupting wound healing, the search for new
agents to combat these types of infections has intensified. We have developed low cost, rapid
models to evaluate the efficacy of agents in treating monospecies biofilms produced by MRSA
or Pseudomonas aeruginosa in partial-thickness mouse wounds. Infected wounds were generated
by applying a Dremel tool with a course sanding attachment to the backs of anesthetized mice
followed by inoculation of the wounds with either MRSA or P. aeruginosa. Mice were treated
for two days with treatment initiated either the same day wounds were inoculated or after 24
hours when the infection became established. At the end of the treatment period, the mice were
humanely euthanized and samples were obtained by tissue biopsy. Samples were either
homogenized and used to enumerate bacterial counts on selective agars or fixed and examined
microscopically. The presence of a biofilm was demonstrated by observation of dense bacterial
communities and the substantial numbers of bacteria present per gram of homogenized tissue.
Small molecule therapies used for model validation had substantial efficacy when treatment was
initiated early but reduced efficacy against the established biofilm. Silver based therapies showed
little to no efficacy against biofilms. A consistent rank ordering of antimicrobials was seen
between the murine model and an in vitro biofilm model used for antimicrobial screening. These
models should facilitate the rapid and efficient development of new antimicrobial agents to treat
gram positive and gram negative biofilm infections.
BRC11
ANTIMICROBIAL EFFECTIVENESS AND EXUDATE MANAGEMENT FROM A
NOVEL SILVER-CONTAINING TRANSFORMING POWDER DRESSING
John V. StJohn, PhD.
ULURU, Inc, Addison, TX, USA.
Silver-containing antimicrobial dressings are typically characterized in terms of silver
concentration or perceived concentration in a wound. We present the data comparing a novel Agcontaining
Transforming Powder Dressing to other silver products evaluating fluid management,
strength, adhesion to wounds and preclinical porcine wound healing studies in addition to
antimicrobial effectiveness measured in in vitro models.

The data demonstrates that silver concentration in vitro is invariably the same for every dressing
studied in physiological fluid. In addition, the relative antimicrobial effectiveness as measured in
extended Kirby-Bauer testing is the same for all dressings as tested. The variables for dressing
performance are therefore limited to the physical properties of the dressings themselves. In this
study, the antimicrobial effectiveness was studied with dressings on a simulated wound. the
results show dramatic differences that appear to stem from the degree of contact between the
wound dressing and simulated wound bed. A hypothesis is developed that differences in the
volume of fluid between a dressing and the underlying wound bed can affect the degree of
antimicrobial effectiveness. Results are shown that test this hypothesis and derive conclusions
about dressing performance.
BRC12
ULINASTATIN IMPROVES PULMONARY FUNCTION IN SEVERE BURN-INDUCED
ACUTE LUNG INJURY BY ATTENUATING INFLAMMATORY RESPONSE AND
MICROVASCULAR PERMEABILITY
Yong Fang, MD, PhD, Peng Xu, MD, Chuan Gu, MD, Wei-Rong Yu, MD, PhD, Min Yao, MD,
PhD.
Institute of Traumatic Medicine, Shanghai Jiao Tong University, Shanghai, China.
Objective: Ulinastatin is potentially an effective intervention that attenuates systemic
inflammatory response induced by endotoxin and improves myocardial and pulmonary functions
during ischemic shock and reperfusion. Our hypothesis is that ulinastatin improves pulmonary
function in burn-induced acute lung injury by inhibiting inflammatory mediators and reducing
pulmonary microvascular damage. Interventions: Animals were subjected to full-thickness burn
wound (30% total body surface area) using boiling water, followed by resuscitation with
Ringer’s solution. The treatment group received 50,000 U/kg of ulinastatin and the control group
was given the same amount of saline solution immediately following burn injury. Additionally, a
sham group was subjected to an identical procedure as the burn group using water at room
temperature. Blood samples and lungs were collected at 8, 24, and 48 h after injury for further
laboratory investigations. Measurements and main Results: Compared with the burn group,
administration of ulinastatin significantly decreased both local and systemic levels of
inflammatory mediators, such as TNF-α, IL-1β, IL-6, and IL-8. Ulinastatin also markedly
reduced the secretion of neutrophil elastase and myeloperoxidase in the lung, demonstrating that
ulinastatin inhibited activation of neutrophils. Additionally, immunohistochemical analysis
revealed diminished expression of intercellular adhesion molecule-1 on the surface of lung
epithelium in the ulinastatin-treated animals. Furthermore, the pulmonary oxygenation was
investigated by blood-gas analysis. Administration of ulinastatin dramatically ameliorated the
PaO2 and DPa-vO2 as compared with that in the burn animals. Likewise, ulinastatin attenuated
pulmonary edema, as demonstrated by decreased pulmonary microvascular permeability in vivo
and the ratio of wet/dry lung weight in the ulinastatin-treated animals. Conclusions:
Administration of ulinastatin protects against pulmonary dysfunction in severe burn-induced
acute lung injury by limiting local and systemic inflammation mediators and neutrophil
activation, as well as attenuating microvascular permeability.

BRC13
INCREASED PRESENCE OF BIOFILM AND A GREATER DELAY IN HEALING
OBSERVED IN AN IMPROVED PORCINE MODEL OF MRSA-INFECTED FULL-
THICKNESS WOUNDS
Eric Roche, Ph.D. 1 , Paul Renick, M.S. 1 , Shannon Tetens, M.S. 1 , Sarah Ramsay, M.S. 1 , Egeenee
Daniels, D.V.M. 2 , Dennis Carson, Ph.D. 1 , Duncan Aust, Ph.D. 1 .
1 Healthpoint Ltd., Fort Worth, TX, USA, 2 UNT Health Science Center, Fort Worth, TX, USA.
The idea that microbial biofilms are a major cause of non-healing ulcers has growing acceptance
in the wound care community, but data in support of this concept remain limited. A model was
previously established in which full-thickness wounds on the backs of pigs were inoculated with
MRSA, resulting in wound infection and delayed healing. At the end of one study, a wound
remaining open was sampled and an MRSA strain was isolated that had the same antibiotic
resistance profile as the parent inoculation strain. This pig-passaged strain was used as the
inoculating strain in several subsequent studies. The resulting MRSA wound infections exhibited
a greater, more stable tissue bioburden than observed in studies with the parent strain.
Furthermore, a greater delay in healing relative to control wounds was observed in terms of
wound area and wound closure for the passaged strain. To understand whether these changes
corresponded to an increased biofilm character of the wound infection, gram-stained wound
biopsy samples from studies using the parent or passaged MRSA strains (20 of each) were
examined microscopically. Evidence of biofilm was observed for both strains, as most samples at
a minimum had multiple isolated, dense microcolonies of bacteria. Colonies were observed
chiefly at or near the surface of the wound tissue. However, the passaged MRSA resulted in
bacterial colonies that were of greater frequency and size, and that more often occurred in
concatenated fashion to generate extended sections of biofilm. These results provide a model
case where increasing biofilm character of a wound infection corresponded with a greater delay
in wound healing.
BRC14
EFFECT OF GROWTH ARRESTING CELLS BY GAMMA IRRADIATION ON
GROWTH FACTOR SECRETION AND LONGEVITY WITHIN A FIBRIN MATRIX
Brett Kiedaisch, MS 1 , Kathi Mujynya-Ludunge, MS 2 , Dennis Carson, PhD 1 , Eric Rolland,
PhD 2 .
1 HealthPoint Ltd, Fort Worth, TX, USA, 2 DFB Bioscience, Lausanne, Switzerland.
HP-802 is a product containing growth arrested allogenic fibroblast, and keratinocytes that are
delivered to the wound site within a fibrin matrix. This allows for the local delivery of growth
factors and matrix components, secreted from the cells, to improve wound healing. The cells are
growth arrested to maintain a controlled dose of secreted growth factors by preventing cell
proliferation. Growth arresting the cells is accomplished by exposing them to low doses (80 Gy)
of gamma irradiating with Cesium-137. This low dose of irradiation disrupts cell proliferation
without interrupting cellular functions. Initial testing compared cell proliferation of non-

irradiated and irradiated cells using a BrdU cell proliferation assay. Results showed that up to 12
days after irradiation both fibroblast and keratinocytes remained stable without proliferating.
To further investigate the effects of irradiation, growth factor secretion and cell longevity within
the fibrin matrix was studied. VEGF, GM-CSF, and b-FGF secretion was compared in nonirradiated
and irradiated samples of HP-802. A single dose of HP-802 was added to each well of
a 24-well plate and media was added after the matrix had formed. After a 24 hour period the
matrix and media was collected and analyzed with ELISA assays for growth factor secretion. For
longevity testing, HP-802 samples were added to 24-well plates as above and the media was
collected over a two week period and VEGF secretion was analyzed. In conclusion, the dose of
gamma irradiation these cells are exposed to is sufficient to prevent cell proliferation without
inhibiting cellular function. In addition growth factor secretion of VEGF, GM-CSF, and b-FGF
was similar in non-irradiated and irradiated samples, with VEGF secretion continuing for up to
two weeks in culture. This confirms our initial testing demonstrating that relevant cellular
functions are not affected by irradiation.
BRC15
Please see abstract MS2.02
BRC16
INSPIRE RECOMBINANT HUMAN TM TYPE-I COLLAGEN WOUND DRESSING, A
NOVEL SCAFFOLD FOR CUTANEOUS WOUND HEALING.
Shani Shilo, D.M.D Ph.D 1 , Sigal Roth, B.S.C 1 , Or Dgani, Ph.D 1 , Olga Mizrahi, B.Med.Sc 2 ,
Dima Sheyn, MSc 2 , Gadi Pelled, D.M.D Ph.D 2 , Dan Gazit, D.M.D Ph.D 2 , Hagit Amitai, Ph.D
MBA 1 , Oded Shoseyov, Ph.D 3 .
1 CollPlant Ltd, Ness-Ziona, Israel, 2 Skeletal Biotech Laboratory, Hebrew University of
Jerusalem – Hadassah Medical Campus, Jerusalem, 91120, Israel, Jerusalem, Israel, 3 The Robert
H. Smith Institute of Plant Science and Genetics and the Minerva Otto Warburg Center for
Agricultural Biotechnology. The Robert H. Smith Faculty of Agricultural, Food and
Environmental Quality Sciences, the Hebrew University of Jerusalem P.O.B., Rehovot, Israel.
Type-I Collagen wound dressings are thought to be the best scaffolds for the medical device
market, providing an optimal mimic environment for cell proliferation and tissue repair. To date,
the collagen used is of animal origin, entailing several risks including pathogens and allergic
reactions. The purpose of this study was to produce a safe and efficient scaffold based on novel
human recombinant type-I collagen. Using genetic engineering we have successfully expressed
five human genes in tobacco plants to produce fully functional triple helical recombinant human
type-I collagen, Collage-rh TM , identical to human collagen. A 100% human recombinant
collagen type-I wound dressing sheet, "Inspire-rh TM " wound dressing sheet was fabricated by
fibrillogenesis and freeze drying. Thermal dehydration treatment was applied to physically crosslink
the collagen monomers in order to increase chemical and mechanical stability. A highly
porous cohesive structure was obtained, capable of absorbing large quantities of fluids, up to 40
times its own weight, in a few seconds. This characteristic is particularly important and highly
desirable for wound dressing products, due to the need to absorb exudates. In tissue culture the

io-functionality of "Inspire-rh TM " wound dressing sheet was studied using key cell models that
participate in the wound healing process, human dermal fibroblasts, human endothelial cells and
human bone marrow-derived mesenchymal stem cells. Proliferation of cells on Inspire-rh TM
scaffolds was compared to commercially available collagen containing products for cutaneous
wound healing. Inspire-rh TM sheets showed superior biological performance in supporting
human dermal fibroblasts, human endothelial cells and human mesenchymal stem cell
proliferation, therefore expected to jump start tissue repair and accelerate wound healing.
Collage-rh TM can be used to produce additional types of safe and efficient scaffolds for other
applications such as orthopedic bone devices, leading to a new era in regenerative medicine.
BRC17
HUMAN KERATINOCYTES ARE STIMULATED BY OWN TISSUE FLUID/LYMPH
AND PROLIFERATE
Waldemar L. Olszewski, Professor, Anna Domaszewska-Szostek, Dr, Maria Moscicka-
Wesolowska, Dr, Marzanna Zaleska, MSc.
Dept of Surgical Research &Transplantology, Medical Research Center, Polish Academy of
Sciences, Warsaw, Poland.
Introduction. Keratinocytes (KC) are needed for covering large denuded skin areas. They can be
cultured in artificial media, however, the yield is always low and viability is low. However, in
vivo they can proliferate actively as it is observed in lymphedematous skin characterized by
hyperkeratosis. In this condition tissue fluid and lymph (TF/L) contain high levels of growth
factors and cytokines. The question arises what effect do lymph cytokines have on proliferation
of KC and whether they can be used for obtaining large numbers of cells. Aim. To study the
effect of human TF/L obtained from patients with obstructive lymphedema on expression of
proliferation markers of lower limb keratinocytes . Methods. KC were cultured in various
concentrations of TF/L. Lymph contained IL1, IL6, TNFalpha, KGF, EGF, VEGF, TIMP1,2 at
higher levels than serum. To block the effect of cytokines on KC proliferation antibodies against
IL1, IL6,TNFalpha, KGF, FGF and TGFβ and their receptors on KC were used. After 7 days
phenotypes were defined using moAbs against p63 (stem cells), CD29 (transient daughter cells),
markers of proliferation: Ki67 and PCNA and differentiation: keratins 1,6,10,16 and 17. Results:
seven day culture in lymph showed increased percentage of p63 and CD29 positive cells.
Moreover, there was increased number of dividing cells in lymph. The total yield after 7 days
was 2- 3x higher than initial number of KC. Blocking of all but TGF cytokines decreased KC
proliferation by 30%. Similar results were obtained after blocking of receptors. Conclusion. KC
cultured in skin tissue fluid/lymph reveal high proliferation tendency mediated by a combination
of locally produced cytokines.
BRC18
A SKIN SUBSTITUTE TISSUE BIOENGINEERED USING A NON-VIRAL VECTOR
TO EXPRESS TIMP-1 INHIBITS PROTEASE ACTIVITY

Christina Thomas-Virnig, Ph.D. 1 , Cathy Rasmussen, Ph.D. 1 , Nick Simon, B.S. 1 , Sarah Sisson,
B.S. 1 , Melissa Taylor 2 , Sandy Schlosser, B.S. 2 , Lynn Allen-Hoffmann, Ph.D. 2 .
1 Stratatech Corporation, Madison, WI, USA, 2 University of Wisconsin, Madison, WI, USA.
Purpose: Successful wound closure requires balanced matrix metalloproteinase (MMP) and
tissue inhibitor of metalloproteinase (TIMP) activity for proper granulation tissue formation and
keratinocyte growth and migration. Chronic cutaneous wounds exhibit a highly proteolytic
environment due to an increase in MMP activity and a corresponding decrease in TIMP activity
inhibiting the healing process. The goal of this study is to develop a genetically modified
therapeutic skin substitute with a non-viral vector that reduces the proteolytic imbalance of
chronic wounds by expressing elevated levels of TIMP-1. Results NIKS ® cells stably transfected
with TIMP-1 (NIKS TIMP-1 ) were previously described to form a fully-stratified skin substitute
tissue (ExpressGraftShield) that secreted 18-fold higher TIMP-1 levels than unmodified tissue
(StrataGraft ® ). Conditioned medium from ExpressGraftShield inhibited MMP-2 activity by 66%
compared to StrataGraft ® in an in vitro gelatin cleavage assay. Additional assessment of
cutaneous barrier function and tensile strength has demonstrated that the physical properties of
ExpressGraftShield are comparable to clinically-tested StrataGraft ® . Furthermore, NIKS TIMP-1 cells
possess a stable karyotype with transgene insertion on chromosome 3q. Preliminary studies have
also evaluated the effectiveness of ExpressGraftShield-secreted inhibitor activity against clinical
chronic wound exudate samples. Testing of chronic wound fluid from a venous ulcer patient
using the gelatin cleavage assay has revealed a 30% inhibition of protease activity by
ExpressGraftShield conditioned medium compared to unmodified StrataGraft ® . Conclusions:
NIKS TIMP-1 cells produce fully-stratified skin tissue possessing cutaneous barrier function and
tensile strength comparable to clinically-tested StrataGraft ® , that inhibits both MMP-2 and
chronic wound fluid proteases in vitro. Confirmation of a stable karyotype and identification of
the transgene insertion site are important for ultimate regulatory approval by the FDA. We will
next test this therapeutic in an in vivo chronic wound model to obtain preclinical data supporting
translation of this technology into the clinic to combat the highly proteolytic environment of
chronic cutaneous wounds.
BRC19
IMPROVED HEALING IN TISSUE CULTURE MODEL OF VESICANT INJURY
SHOWN BY HIGH FEATURE ANTIMICROBIAL DRESSING
Bernd Liesenfeld, Ph.D. 1 , Albina Mikhaylova, Ph.D. 1 , William Toreki, Ph.D. 1 , David Moore,
B.Sc. 1 , Gregory Schultz, Ph.D. 2 .
1 Quick-Med Technologies, Gainesville, FL, USA, 2 University of Florida, Gainesville, FL, USA.
Quick-Med Technologies (QMT) has developed a dressing for the treatment of vesicant injuries
following debridement under an army SBIR. This dressing is designed to enable optimal wound
healing by providing a moist wound healing environment with antimicrobial protection, protease
inhibiting properties through sustained delivery of an antibiotic (doxycycline) that also acts as a
protease inhibitor, as well as antioxidants and a growth factor (EGF). Sustained antimicrobial
activity and protease inhibition have been previously documented through in vitro experiments.

Tissue culture models were used to assess healing of both physical and chemical injury models.
Epiderm FT (Mattek) full thickness dermal tissue cultures were physically injured by excision of
epidermal layer, and chemically injured by vapor phase exposure to ‘half-mustard’ (CEES) gas.
Injured tissue cultures treated with test dressing improved healing in a statistically significant
manner as evaluated by cell proliferation assay. Pathology showed upregulated cell growth and
decreased detachment in the treated groups. Treatment with the dressings appeared to prevent the
characteristic dermal - epidermal separation induced by vesicant injury. These results have
prompted progress of the research project to animal experiments.
BRC20
EPIGENETIC REPRESSION OF MMP GENES IN TISSUE FIBROSIS
Yuan-Ping Han, PhD.
University of Southern California, Los Angeles, CA, USA.
Wound healing and tissue repair are vital for survival and well being, while abnormal healing
may lead to chronic wounds or excessive scars in the opposite directions. In response to injury
signals such as inflammatory cytokines, a group of extracellular proteases called matrix
metalloproteinase (MMPs) are induced to cope with cell activation and ECM degradation.
Excessive amount of MMPs is associated with chronic wounds and even lethal in systemic
diseases. Conversely, in fibrotic tissue the MMP genes are profoundly repressed in favor of ECM
accumulation to form fibroplasias. We are intrigued by these two extreme opposite directions to
know how MMP genes are turned on in acute injury, and how the same MMP genes are
repressed in fibrosis. In the field of MMPs, most attentions in past decades have been given to
address how the MMP genes are turned on in diseases such as injury, cancer, and arthritis.
Surprisingly unknown is that how MMP genes are repressed or silenced in fibrogenesis. Using
skin and liver models, we found that epigenetic regulations through class II HDACs may set the
mesenchymal cells in transcriptionally permissive state, capable to express MMPs under injury
signals. Although many cell types can produce some MMPs under stress, mesenchymal cells
including dermal fibroblasts and hepatic stellate cells are unique for their restrained manner to
express MMPs, since both ECM and injury signals are required to turn on many MMP genes in
injured tissues. We found that ECM does not directly contribute to the key signal transduction
pathways for the MMP genes; rather, the matrix down-regulates class II HDACs, conferring the
genes in permissive state, ready for injury signals. On the other hand, in myofibroblasts, many
class II HDACs are stabilized to repress the MMP genes among others, rendering the cells into
fibrotic state.
Blue Ribbon General Posters 1 – BRG01-BRG10
Sunday, April 18, 2010, 7:30 to 9:00 am
BRG01
PRODUCTION OF FULL-THICKNESS HUMAN SKIN SUBSTITUTE TISSUES
WITHOUT ANIMAL-DERIVED COLLAGEN

Kenneth R. Gratz, Ph.D., Barry M. Steiglitz, Ph.D., John C. Pirnstill, B.S., Nick J. Simon, B.S.,
Sara C. Pirnstill, B.S., Sara L. Sisson, B.S., Allen R. Comer, Ph.D., B Lynn Allen-Hoffmann,
Ph.D..
Stratatech Corporation, Madison, WI, USA.
Introduction: Currently-available full-thickness skin substitutes contain a stratified epidermal
layer grown on a dermal equivalent (DE) composed of human dermal fibroblasts (NHDFs) in an
animal-derived (often bovine) collagen gel. These autopolymerized collagen matrices do not
provide the normal composition, structure, or function of the human dermis and despite rigorous
screening and quality control, introduce process variability and the potential for adventitious
agent transmission (e.g. bovine spongiform encephalopathy). Alternatively, long-term cultures of
NHDFs direct the formation of tissues de novo, from the cells and their secreted extracellular
matrix (ECM). The objective of this study was to generate NHDF-derived DEs and use them for
production of skin substitute tissues. Methods: NHDFs were seeded into porous tissue culture
inserts and allowed to secrete and assemble ECM components into a cohesive DE. NIKS
keratinocytes were seeded onto DEs and cultured at the media/air interface to promote formation
of fully-stratified epidermal layers. DEs and mature skin substitutes were analyzed for thickness,
histological appearance, collagen content, cell density, tissue viability, barrier function, and
tensile properties and compared to skin substitutes generated using animal-derived collagen gels.
Results: NHDF cultures deposited significant quantities of extracellular matrix (collagen density
>100 µg/cm 2 ), forming DEs >50 µm in thickness, with substantial mechanical strength (>1
MPa). In contrast, collagen-gel DEs remained composed primarily of the input collagen and had
negligible mechanical properties. NHDF-derived DEs supported formation of mature skin
substitutes containing fully-stratified epidermal layers with viability, mechanical properties, and
barrier function comparable to tissues produced with gelled collagen DEs. Conclusions: NHDFderived
DEs have properties closer to those of normal human skin and support the production of
full-thickness skin substitutes. Elimination of animal-derived collagen from tissue-engineered
skin substitutes, simplifies the manufacturing process, improves product safety, and may also
reduce immunological responses to the tissue.
BRG02
OPTIMINIZATION OF A HUMAN KERATINOCYTE ADULT STEM CELL ASSAY
TO PREDICT PROPER EPIDERMAL DEVELOPMENT IN BILAYERED LIVING
CELL BASED TREATMENT
Zongyou Guo, Ph.D, Dolores Baksh, Ph.D, Steven Zabroski, Brian Gardell, Kshama Doshi,
Vincent Ronfard, Ph.D.
Organogenesis Inc, Canton, MA, USA.
Selection of the optimal cell populations in the generation of a bilayered living cell-based
treatment (BLCT) comprising both a dermal and epidermal compartment is essential in
producing a consistent product. BLCTs are composed of human keratinocytes and fibroblasts
derived from neonatal foreskin that promotes wound healing through matrix proteins and growth
factors produced by the selection of optimal living cells.
We hypothesize that the presence of the keratinocytes stem cells in the seed pool plays an

important role in the compatibility with human dermal fibroblasts (HDF) and in the proliferation
and differentiation of keratinocyte cells during BLCT production and, thus the quality of the
finished product. We have developed a colony-forming efficiency (CFE) assay to estimate the
percentage of adult stem cells in candidate keratinocyte cell populations to be used in the
generation of quality of BLCTs. This assay allowed to assess proliferation and migration
potential of keratincocyte cell population by measuring the size of each individual colony. We
found that different media and media components have different sensitivity to discriminate the
potency of different keratinocyte lines to produce a stratified epithelium and that the colony sizes
were more powerful to distinguish cell quality then CFE alone. Additionally, we observed that
the CFE assay is very sensitive to different EGF lots. Taken together, we have developed a CFE
procedure to assay keratinocyte adult stem cells, which not only estimates the potency of
keratinocyte to produce BLCTs, but also reflects the quality of key media components (e.g,
rhEGF) used in the media. On-going studies are designed to determine the baseline CFE required
for keratinocyte cells to produce a well-developed epithelium in BLCTs. Furthermore, the
mechanisms through which EGF affects keratinocyte CFE remains to be elucidated in our
system.
BRG03
IN VITRO FIBROBLAST GENE ARRAY ANALYSIS OF AN ACTIVE WOUND
DRESSING.
Patrick Parks, MD PhD 1 , Michele Anderson, PhD 2 , Marnie Peterson, Pharm D, PhD 2 .
1 3M Company and University of Minnesota, St Paul, MN, USA, 2 University of Minnesota,
Minneapolis, MN, USA.
Selective gene array analysis was performed in vitro on fibroblasts exposed to the components of
a wound dressing designed to normalize the wound environment. The absence of generally
accepted preclinical models of wound healing result in a need to examine potential therapies for
the treatment of non-healing wounds using alternative experimental systems and fibroblast
cultures represent one such model. The development of wound therapies with molecular effects
also dictates a need for analysis at the subcellular level. Tegaderm Matrix is a wound dressing
that is being used for wounds that have ‘stalled’ in their progression to healing. The Tegaderm
Matrix effect is the result of selective cation interaction with the healing environment and
superarray technology was used to examine the effect of the cations on genes pertinent to the
healing process. WS 1 skin derived fibroblasts were grown in essential medium with 10% fetal
calf serum with pen/strep and gentamicin. At ~80% confluency, cells were switched to serum
free, antibiotic free media for 18 hours. Prior to measurement, media was replaced with fresh
media. Either platelet derived growth factor-BB (PDGF-BB) at 40ng/ml or cations at
concentration ranges expected with Tegaderm Matrix were added. RNA was collected using
RNeasy Plus and cDNA was measured using real time PCR with two different superarrays
measuring genes associated with angiogenesis and extracellular matrix. The results at 7 hours
indicate that the cations down-regulate the genes associated with Interleukin 8 and with matrix
metalloproteinases in a selective manner, without concomitant reduction in tissue inhibitors of
matrix metalloproteinases. The time course of reaction also indicates a blunting in the increase of
matrix metalloproteinases compared to PDGF-BB at 24hrs and 48hrs after addition of either

material to the cultures. The results indicate that selective cation interaction reduces
inflammmatory components that interfere with healing of chronic wounds.
BRG04
NOVEL NANOSPHERE COMPOUNDS WITH CUTANEOUS WOUND HEALING
PROPERTIES
Zhiguo Zhou, PhD 1 , Steve Joslin, PHD 1 , Anthony Dellinger, BS 1 , Marion Ehrich, PhD 2 , Brad
Brooks, BS 1 , Qing Ren, MD 3 , Ulrich Rodeck, MD 3 , Christopher L. Kepley, PHD MBA 1 .
1 Luna Innovations Inc, Danville, VA, USA, 2 Virginia-Maryland Regional College of Veterinary
Medicine, Virginia Tech, Blacksburg, VA, USA, 3 Thomas Jefferson University, Philadelphia,
PA, USA.
Impaired wound healing is a major complication underlying several disease processes (such as
diabetes). Efficient wound healing is hampered by a wide variety of processes including hypoxia
(oxygen deprivation), inflammation, infection, and oxidative stress through the generation of
harmful reactive oxygen species (ROS). The inherent complexity of the healing wound has led to
limited efficacy of most therapies that target single parameters involved in the slow healing
wound. Fullerenes are carbon nanospheres previously shown to exhibit a wide range of
biological activities. Given that these molecules have been shown to be potent antiinflammatories
and antioxidants we hypothesized that fullerenes could aid in wound healing
based on these properties. We designed and synthesized a panel of fullerene derivatives and
investigated their ability to accelerate wound healing using a modified scratch assay, an ex vivo
human skin model, and a mouse model of skin irritation. Several fullerene derivatives supported
cell migration, induced wound closure in human skin explants, and greatly accelerated the rate at
which wound healing occurred in vivo. Therefore, fullerene derivatives may represent a new
class of wound healing therapies that may aid in wound healing treatment.
BRG05
ALLOGENEIC ADULT BONE MARROW-DERIVED SOMATIC CELLS ENHANCE
WOUND HEALING AND REDUCE SCARRING IN YUCATAN MINIATURE SWINE.
Vanessa Ragaglia, PhD 1 , Baron Heimbach, BS 1 , Randall Mack, BSc 2 , Gene Kopen, PhD 1 .
1 Garnet BioTherapeutics, Inc., Malvern, PA, USA, 2 Malvern Consulting Group, Malvern, PA,
USA.
Adult bone marrow-derived somatic cells (ABM-SC) represent a homogenous population of
non-hematopoietic trophic support cells derived from human and animal sources. Previous work
has shown that administration of ABM-SC to sites of tissue injury results in host tissue
preservation and repair without long term cell engraftment. Here we explored the potential of
allogeneic ABM-SC to augment the healing process of thermal burns and full thickness wounds
created in Yucatan miniature swine. On Day 0, 12 full thickness excision and 12 thermal burn
wounds were created per miniature swine. The wounds were treated once on Day 0 with ABM-
SC or vehicle or twice with ABM-SC on Days 0 and 7. On Day 7, ABM-SC and vehicle

treatments were repeated for four excision and thermal burn wounds per animal. Animals were
maintained on study for 56 days. All wound sites were evaluated during the study and included
modified Draize scoring, photographic documentation, and histopathology. The photographs
were used to conduct quantitative image analysis of wound density, size and texture. There were
no statistically significant differences in Draize scoring, demonstrating no host-mediated
rejection of the grafted allogeneic cells or anaphylactic response to the excipients (vehicle). The
density and area of the thermal wounds treated 2X with ABM-SC was reduced compared to
controls. The largest differences of 62% (p=0.075) and 67% (p=0.080) respectively, were seen at
day 56. Compared to controls, there was also a mild decrease in dermal fibrosis after 2x
treatment with ABM-SC; 88% of controls compared with 50% 2X treated ABM-SC had mild to
moderate fibrosis. Inflammation in the dermis was also slightly decreased in incidence at sites
treated with ABM-SC. This study demonstrated that treatment of thermal or excisional wounds
with allogeneic ABM-SC, especially after 2 doses, appears to enhance wound healing and reduce
scaring and inflammation in the dermis.
BRG06
REDUCTION OF SCAR FORMATION THROUGH LAYERING OF ACELLULAR
BOVINE DERMAL CONNECTIVE TISSUE IN WOUNDS
John S. Starinski, DPM.
Northwood Health Services, Easton, PA, USA.
Existing data and clinical belief states that layering of acellular zenograft-like tissue substitutes
has limited clinical benefit. This study was designed to test if layering of Tissue Mend a bovine
dermal graft replaces lost dermal and subdermal tissue and limits scar development in stage 3-4
wounds. Patients with diabetic, stasis and traumatic wounds were treated by application of a
various number of layers of Tissue Mend after preparation of the graft site. A cap layer was then
applied and stapled or sutured in place. The cap layer was allowed to dry and form an artificial
eschar. The wounds were then observed on weekly or biweekly intervals until the eschar began
to detach. After detachment of the eschar the wounds with the remaining layers of graft, which
were now integrated with the host tissue, were treated with conventional wound management. In
some cases reapplication of the layered graft was required. This study demonstrated that a
gradual vascularization of the graft occurs followed by granulation and epithelization. The
wounds after healing demonstrated a limited amount of scar tissue with corresponding
replacement of innervation and microvascularity. These data challenge the conventional clinical
belief the layering of biological replacement grafts in not beneficial. Also, this process appears to
limit the scar formation stage of normal wound healing by creating a stable platform for cell
migration and subsequent matrix production and tissue remodeling.
BRG07
ANTIMICROBIAL BARRIER WOUND DRESSING OPTIMIZED FOR
PROPHYLACTIC USE.

Bernd Liesenfeld, Ph.D. 1 , Albina Mikhaylova, Ph.D. 1 , David Moore, B.Sc. 1 , William Toreki,
Ph.D. 1 , Jillian Vella, B.Sc. 1 , Gregory Schultz, Ph.D. 2 .
1 Quick-Med Technologies, Gainesville, FL, USA, 2 University of Florida, Gainesville, FL, USA.
Antimicrobial dressings are becoming increasingly attractive to aid in the prevention of
nosocomial infections, partly based on increasing pressures on healthcare systems to drive down
costs. Currently, the most prevalent antimicrobial dressings in the marketplace are silver based.
These function well in controlling microbes and are a very good option for some types of
wounds. We have previously reported on the antimicrobial capacity of the NIMBUS bound
polymeric quaternary microbicide, including testing of bacterial resistance generation - where it
was shown that the bound polymeric cationic technology did not induce bacterial resistance. We
have further developed this technology to commercialization, and present new finding
correlating zones of inhibition testing and cytotoxicity assessments. The primary intended use of
this dressing is to provide an antimicrobial barrier dressing to prevent pathogen transfer. In order
to allow broadest application of this technology, the dressing was designed to be economical,
effective, and have the highest safety and biocompatibility possible. While currently marketed
antimicrobial dressings typically function by leaching microbicides from the dressing, the bound
polymeric microbicide utilized in the NIMBUS dressing is permanently attached to the substrate.
This design permits strong and durable efficacy because the antimicrobial is concentrated only at
the dressing surface - the dressing stays effective even in the presence of highly proteinaceous
bodily fluids. Cytotoxicity testing using L929 fibroblast cell lines showed that the NIMBUS
dressings were non-cytotoxic. A silver dressing was compared in the same model, showing that
leached silver was significantly more aggressive towards the cells. The NIMBUS dressing is
targeted to wounds that require protection from pathogens in the healthcare environment, in
wounds that are not infected. Mammalian cell testing demonstrates the highest levels of
biocompatibility demonstrating that the dressing can be applied prophylactically in situations
where other antimicrobial dressings might be unsuitable
BRG08
IN VITRO AND IN VIVO INTERACTION OF MATRIX METALLOPROTEINASES
WITH SMALL INTESTINE SUBMUCOSA (SIS) WOUND MATRIX
Lei Shi, PhD, Ryan Ermis, BS, Sarah Ramsay, MS, Dennis Carson, PhD.
Healthpoint Ltd., Fort Worth, TX, USA.
Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix
components and are considered to play an important role in wound healing. Various studies have
confirmed the presence of high MMP levels in chronic wound fluid. 1 These MMPs include
interstitial collagenase (MMP-1), gelatinase A (MMP-2), and gelatinase B (MMP-9). The Small
Intestine Submucosa (SIS) Wound Matrix* is a bioactive extracellular matrix for wound healing
that contains critical components of the extracellular matrix such as collagens, proteoglycans,
and glycosaminoglycans. Previous studies have indicated that MMPs in cell culture medium
inhibit keratinocyte migration and that the inhibition was significantly reduced if the MMP was
pre-incubated with SIS. The purpose of the current study is to investigate the interaction between
SIS and human MMPs-1, 2 and 9 in vitro and in vivo. SIS materials were incubated with each

MMP and the MMP activity in the solution phase was determined using a series of fluorogenic
substrates. The binding curves of the MMPs to SIS material were determined. MMPs-1, 2 and 9
displayed different binding affinities with 46.2%, 14.1% and 63.1% reduction in activity during
24 hours, respectively. A diabetic mouse wound healing study was conducted using the SIS
wound matrix. Biopsy samples were collected on different days for analysis of MMP levels by
gelatin zymography. The MMP activity was found to be attenuated by SIS testament on day 3.
On day 7, the attenuation became less significant, indicating that the MMP binding capability of
SIS was saturated. The results of this study suggest that SIS wound matrix may minimize MMP
activity during the early days immediately after treatment, and could reduce the MMP’s
inhibitory effect on keratinocyte migration.
* Oasis ® Wound Matrix (Cook Biotech)
1. Trengove NJ, Stacey MC, Macauley S, Bennett N, Gibson J, Schultz G, Wound Rep Reg
1999;7:442-52
BRG09
COMPARISON OF BACTERIAL AEROSOLIZATION DURING WOUND CLEANSING
VIA TWO METHODS: PULSED LAVAGE AND NORMAL SALINE INSTILLATION
IN CONJUNCTION WITH NEGATIVE PRESSURE WOUND THERAPY
Diwi Allen, Masters, Ioana L. Bondre, Amy K. McNulty, PhD.
KCI, San Antonio, TX, USA.
Wound cleansing is a part of proper wound bed preparation. A particularly important step for the
healing of wounds is regular debridement, which consists of removing necrotic tissue, wound
exudates and bacterial biofilm. One of the most commonly used debridement methods is pulsed
lavage, a technique consisting of the delivery of an irrigant under an optimal pressure range. Its
foremost disadvantage, though, is concern regarding bacterial aerosolization. Given that the
pressure delivered to the wound must be high enough to remove the microbes at least from the
surface of the wound bed, it is reasonable to express concern regarding the possibility of
bacterial cross contamination. The level of bacterial aerosolization during wound cleansing was
compared between pulsed lavage and instillation in conjunction with negative pressure wound
therapy (NPWT). Sacral pressure ulcers on wound care models were inoculated with 6 x 10 7
particles of heat killed, fluorescently-conjugated Escherichia coli and Staphylococcus aureus.
Fluorescence spectrometry was used to quantitate the aerosolized particles collected at 3 in. and
6 in. from the edge of the wound. Several cleansing solutions with differing antimicrobial
properties were used with the pulsed lavage trials. Different types of foam dressings were used
with the instillation plus NPWT trials. Approximately one third of the inoculated particles
aerosolized 3 in. and 6 in. from the wound during pulsed lavage cleansing. There was no
evidence of bacterial aerosolization during wound cleansing via instillation plus NPWT. This
study shows that wound cleansing via instillation in conjunction with NPWT allows for wound
bed preparation while decreasing the risk of bacterial aerosolization associated with other
common wound lavage techniques.
BRG10

PRECLINICAL DATA IN SUPPORT OF HP-802, A CELL-BASED PRODUCT FOR
THE TREATMENT OF VENOUS LEG ULCERS
Brett Kiedaisch, MS 1 , Kathi Mujynya-Ludunge, MS 2 , Dennis Carson, PhD 1 , Eric Rolland,
PhD 2 .
1 HealthPoint Ltd, Fort Worth, TX, USA, 2 DFB BioScience, Lausanne, Switzerland.
The use of advanced therapies is becoming more common in the treatment of hard to heal
wounds attributed to venous ulcers. Conventional therapies either do not work or reoccurrence of
the ulcer is extremely high. HP-802 is a combination of living growth arrested allogenic
fibroblast and keratinocytes contained within a fibrin based biological matrix. These skin cells
are known to produce a large variety of factors that induce wound healing and by using both cell
types, the level of growth factor production and matrix component expression is enhanced
compared to using just a single cell type. The fibrin matrix maintains the cells close to the wound
surface ensuring the local delivery of these growth factors into the wound. This influx of growth
factors into the wound should stimulate angiogenesis, reepitheliazation, and granulation tissue
formation leading to wound closure. Preclinical testing has focused on the secretion of various
growth factors from both cell types in the fibrin matrix. VEGF, b-FGF, GM-CSF, and KGF were
analyzed in HP-802 samples at various cell concentrations and ratios of fibroblasts to
keratinocytes. In brief, a single dose of HP-802 was added per well to a 24-well plate and media
was added after the matrix had formed. After a 24 hour period the matrix and media was
collected and analyzed with ELISA assays for growth factor secretion. The data revealed that
growth factor secretion is highly dependent on cell concentration and cell ratio. The results also
demonstrated that synergistic effects are observed on growth factor release when both
keratinocytes and fibroblasts are present. In conclusion, HP-802 is a cell loaded biological matrix
providing controlled, local delivery of necessary growth factors and matrix components to restart
the wound healing process in venous leg ulcers.
Blue Ribbon General Posters 2 – BRG11-BRG15
Sunday, April 18, 2010, 7:30 to 9:00 am
BRG11
APLIGRAF® STIMULATES EPITHELIAL MIGRATION IN AN TRIDIMENSIONAL
IN VITRO WOUND HEALING ASSAY
Katie Faria, Batchelors of Science 1 , Christophe Egles, PhD 2 , Brian Gardell 1 , Steve Zabroski 1 ,
Kiel Peck 1 , Jon Leman 1 , Jonathan Garlick, DDS, PhD 2 , Vincent Ronfard, PhD 1 .
1 Organogenesis Inc., Canton, MA, USA, 2 Tufts University, Boston, MA, USA.
The goal of this research was to evaluate the ability of a bilayered living cell-based treatment
(Apligraf ® ) to stimulate epithelial migration in a 3D in vitro human wound healing assay and
begin to dissect the role played by Apligraf components (i.e. cells, soluble factors and matrix).
Contrary to 2D culture wound assays a 3D tissue-engineered wound healing model more closely
mimics the form and function of human skin undergoing wound healing response. A

ioengineered tissue, comprised of a dermis (fibroblasts + collagen matrix) and a stratified
epithelium that serves as a model platform which allows the study of the temporal and spatial
response of wounded epithelium to Apligraf. This more complex system better approximates the
complexities of the wound healing microenvironment allowing for a more meaningful
correlation between in-vitro and in-vivo response. To better elucidate the interaction between
Apligraf and the wound site in vitro. We applied Apligraf in 3 different ways: (1) Intact, (2) With
barrier; (3) devitalized. Conditions were evaluated at 48/96 hours. Tissue morphology and
migration of the epidermal tongue from wound edge was evaluated using hematoxylin and eosin
staining. The tissue phenotype was analyzed by immunohistochemical: detection of BrdU to
establish the impact on cell proliferation, extracellular matrix proteins and specific proteins from
the epidermis to assess the differentiation of restored epithelium, detection of the presence of
laminin 5 linked to cell migration and keratin 1/10. Our experiments reveal that the Apligraf,
when placed on the wounds, aids the healing by decreasing the time of wound closure. All
constructs containing cells stimulated epithelial migration and were better than the control group
(no Apligraf) or the devitalized Apligraf, indicating the importance of cells in this model. Thus,
our results demonstrate that Apligraf are well-suited for the treatment of cutaneous wounds by
stimulating the activation of wound repair.
BRG12
OVINE FORESTOMACH MATRIX (ENDOFORM) STIMULATES CELL
DIFFERIENTATION AND VASCULARIZATION
Barnaby Charles Hough May 1 , Sharleen Irvine 2 , Evan Floden 1 , Stan Lun 1 , Brian Roderick
Ward 1 .
1 Mesynthes Limited, Lower Hutt, New Zealand, 2 Otago University, Dunedin, New Zealand.
We have generated a new biologically-derived extracellular matrix to meet existing needs within
wound care clinics and emerging needs for tissue regeneration scaffolds. The ovine forestomach
matrix (OFM or Endoform) retains the complex collagenous micro architecture of native ECM,
as well as, biologically relevant macromolecules including fibronectin, GAGs, heparin sulphate,
laminin and FGF2. We have shown that extracts of OFM stimulate differentiation of rat
pheochromocytoma cells (PC12), and that the stimulatory effect could be inhibited, in part, by an
anti-FGF2 antibody. OFM produced a dramatic increase in microvasculature in a rat aortic ring
assay. Further in vivo studies using a porcine full thickness wound model demonstrated
increased vascularity in OFM treated wounds, relative to OaSIS® and untreated wounds. These
findings demonstrate that OFM is biologically functional and has promising characteristics for
clinical applications. Further studies are underway to identify the stimulatory factors responsible
for the observed effects.
BRG13
THE RELATIONSHIP BETWEEN MVTR AND TISSUE ENGINEERED SUBSTRATES
John V. St. John, PhD.
ULURU, Inc, Addison, TX, USA.

Numerous tissue-engineered substrates have been developed as advanced therapies in the
treatment of chronic wounds. These substrates typically contain extracellular matrix, viable or
nonviable cells and signaling molecules or growth factors. The substrates are typically placed
within a prepped wound and either have a laminated secondary membrane in place over the
surface or a secondary dressing is applied over the material. Failures of these products can occur
if the total amount of moisture beneath the substrate or trapped beneath the secondary dressing
causes maceration or allows favorable microbial growth. Data is presented that shows the
relationship between moisture vapor transpiration rates and fluid management above and below
tissue engineered substrates. Dressing MVTR's were calculated and a simulated wound was
developed. Pooling of simulated wound fluid was studied above and below the substrates and the
relationship between the pooling and MVTR is presented.
BRG14
BANKING AND SCREENING OF CELLS FROM MULTIPLE PIG BREEDS FOR THE
PRODUCTION OF A BIOENGINEERED LIVING TISSUE AND ITS USE IN A
PRECLINICAL WOUND HEALING MODEL
Thomas Bollenbach, Ph.D., Vincent Ronfard, Ph.D..
Organogenesis Inc., Canton, MA, USA.
Rodent species have been used as models to study wound healing, but major differences in skin
morphology and physiology make results obtained with these models difficult to compare with
human. Morphologic and functional similarities between porcine and human skin have long been
recognized. Our goal is to develop a pig model for research of cell-based therapies for wound
repair and tissue regeneration. Challenges associated with achieving this goal include the
possibilities of: rejection and transplant cell death during xenotransplantation of human cellbased
therapies onto pigs and significant differences in the ability of cells from various pig
breeds to be serially passaged and cast into a transplantable living cellular construct. The
immediate goal of our work was to screen fibroblasts and keratinocytes from multiple pig breeds
starting with Yucatan, Sinclair and Hanford for a) their performance during isolation and serial
passage, b) the ability of fibroblasts to produce a contracted collagen gel or to secrete a selfassembling
matrix in vitro, and c) the ability of keratinocytes to replicate the stratified and
differentiated epidermis of the host breed. Histological staining of back skin revealed significant
differences in skin morphology between the three breeds. Morphology of isolated fibroblasts also
differed between breeds, whereas there were no obvious differences in keratinocyte morphology.
Fibroblasts and keratinocytes from all three breeds performed well during isolation and serial
passage. Fibroblasts were screened for their ability to produce a contracted collagen gel, and
keratinocytes are being tested for their ability to differentiate, stratify and produce a stratum
corneum.
Our results demonstrate that pig cells can be isolated, serially cultured, and used to produce a
living cell-based treatment similar to the one obtained with human cells. The next step is to apply
these constructs to porcine soft tissue defects and validate this model for use in wound healing
studies.
BRG15

DEVELOPMENT OF AN INJECTABLE TRANSFORMING GEL DRESSING
SUITABLE FOR SINUS TRACT FILLING WITH HIGH TENSILE STRENGTH AFTER
AGGREGATION AND THE ABILITY TO PROVIDE CONTROLLED RELEASE OF
THERAPEUTICS
John V. StJohn, PhD.
ULURU, Inc, Addison, TX, USA.
A critical area of wound care is the resolution of sinus tracts or tunneling wounds. For these
complicated wounds, care can include surgical excision and opening, injectable gel filler
materials, and the packing of ropes or gauze materials into the tunnel. A novel technology has
been developed that uses a shape conforming viscous gel. The gel contains a formulation of a
technology based on the aggregation of tiny polymer particles that aggregate irreversibly when
contact is made with physiological fluid. The resulting shape retentive aggregate can be tailored
through formulation to provide a material with maximum tensile strength and provide intimate
contact with the tract walls. In addition, the formulations were developed to contain pore sizes
that prevent tissue infiltration while in place within the wound tunnel. Data is presented that
demonstrates the mechanism and physical properties of aggregation, and the effects of
formulation on strength and moisture uptake. Of critical importance is the development of a
material that has sufficient tensile strength and rapid enough aggregation that removal using
forceps results in the entire filler releasing from the wound as a single piece of material. The
underlying technology is an advanced drug delivery system. Data is presented showing the
release of antibiotics as demonstration of the drug delivery capability of the future tunnel filling
system. This is an important development as this type of wound often has underlying
contamination or infection associated with the tunnel or tract.
POSTER GENERAL SESSION
General Posters – GP01 to GP41
Sunday, April 18, 2010, 7:30 to 9:00 am
GP01
WOUND HEALING MEDIATES PROTECTION OF CHEMOTERAPY-INDUCED
ALOPECIA
Olivera Stojadinovic, MD, Marjana Tomic-Canic, PhD, Joaquin Jimenez, MD.
University of Miami Miller School of Medicine, Miami, FL, USA.
Loss of hair is one of the most dreaded side effect of cancer therapy. Despite many attempts in
the research community, currently, there is no effective method for preventing chemotherapyinduced
alopecia. Wound healing is a process that mediates secretion of plethora of growth
factors, triggers cell cycling and recruitment of various types of progenitor cells, all of which
may stimulate hair cycle. Therefore, we hypothesize that the induction of a wound healing
phenotype of skin cells may lead to protection of chemotherapy-induced alopecia. To study this

phenomenon we used a newborn rat model of chemotherapy-induced alopecia. We introduced
wound phenotype using 5mm incisional wounds on the one side of dorsal back skin of
anesthetized 3 day old pups. The contra-lateral dorsal site served as an internal control. At
postnatal day 11, pups were intra-peritonealy injected with a frequently used anti-neoplastic
agent, Etoposide (1.5 mg/kg) for three days. Hair growth was assessed qualitatively, by visual
observation, and quantitatively using hematoxylin and eosin staining at 11 and 18 days postchemotherapy.
Interestingly, we noted hair re-growth after chemotherapy-induced alopecia at the
wound site in 25 days old pups, whereas hair was absent from the rest of the body. This was
confirmed by H&E. Most of the hair follicles at the wound site where in anagen stage. We
conclude that induction of wound healing phenotype may be an efficient method of induction of
the hair re-growth after the chemotherapy-induced alopecia. We are in the process of
determining which component(s) of wound healing pehontype and specific molecular events
guide this process. Taken together, the new concept that wounding prevents chemotherapyinduced
alopecia will lead to development of more effective cancer therapeutics without such
devastating side effects.
GP02
AMNION-DERIVED CELLULAR CYTOKINE SOLUTION (ACCS) ACCELERATES
HEALING OF EXPERIMENTAL PARTIAL-THICKNESS BURNS
Yvonne N. Pierpont, M.D. 1 , Wyatt G. Payne, M.D. 2 , Thomas L. Wachtel, M.D. 3 , Charlotte A.
Smith, M.S. 3 , M. Georgina Uberti, M.D. 2 , Francis Ko, B.S. 2 , Martin C. Robson, M.D. 4 .
1 Institute for Tissue Regeneration, Repair, & Rehabilitation,Bay Pines VA Health Care System,
Bay Pines, FL, USA, 2 Institute for Tissue Regeneration, Repair, and Rehabilitation, Bay Pines
Veterans Healthcare System, Bay Pines, FL, USA, 3 Stemnion, Inc., Pittsburgh, PA, USA,
4 Division of Plastic Surgery, University of South Florida, Tampa, FL, USA.
Introduction: Amnion-derived multipotent progenitor cells (AMP cells), unlike most stem cells,
have been demonstrated to be non-tumorigenic and non-immunogenic. Amnion-derived cellular
cytokine solution (ACCS), a secreted product of AMP cells, is a cocktail of cytokines existing at
physiological levels. This study evaluates the influence of ACCS to accelerate epithelialization
of experimental partial-thickness burns.
Materials & Methods: Using modifications of Zawacki’s guinea pig partial-thickness scald burn
model, a total of 65 animals were treated with ACCS, ACCS + AMP cells, unconditioned
medium (UCM) + AMP cells, or UCM alone or saline as controls. Dosage times ranged from
every other day to once a week. Percent epithelialization was serially determined from acetate
wound tracings. Histology was performed on wound biopsies. Results: ACCS, UCM+AMP cells,
and ACCS+AMP cells improved epithelialization compared with the two control groups
(P

GP03
A NOVEL PDMS DEVICE TO PREVENT CONTRACTION IN RODENT EXCISIONAL
WOUNDS
Fang Yu, MD. Ph.D, Susan R. Opalenik, Ph.D, Phillip C. Samson, David K. Schaffer, MS, John
P. Wikswo, PhD, Jeffrey M. Davidson, Ph.D.
Vanderbilt University, Nashville, TN, USA.
Aim: The goal of this work was to design, fabricate and implement a novel, implantable device
to prevent contraction in rodent excisional wounds. Materials and Methods: The device consists
of a flexible, circular disc/flange with a circular array of perpendicular posts to retain the initial
wound dimensions. Brass molds were machined into desired geometries by CNC, and
polydimethylsiloxane (PDMS) was used to form the stents, with flanges structurally supported
by incorporated Nytex mesh. The flange was designed for either superficial/external or
subcutaneous/internal placement. Center holes and when required suture holes were introduced
with a standard biopsy punch. Excisional wounds were created on the dorsum of male Sprague
Daley rats with a 10 mm biopsy punch. Wounds were stented with manufactured standard
stainless steel rings, external PDMS rings, internal PDMS rings, or left unstented. Wounds were
harvested 7, 14, and 21 days post injury, fixed and processed for routine histologic methods.
Contraction, reepithelization and granulation tissue area was quantified by morphometric
analyses. Results: Superficial placement of the device permitted removal at any time after
surgery, and as compared to metal stents or no treatment, the PDMS posts significantly reduced
wound contraction. The spacing of the posts had a strong influence on the degree and rate of
reepithelization. Subcutaneous insertion of the device flange beneath the panniculus carnosus,
with the posts projecting upwards, produced a dramatic reduction in the rate of wound repair.
This was likely due to mechanical pressure of the flange on the overlying skin and creation of an
ischemic wound margin. Use of a thinner flange supports this concept and suggests that
subcutaneous flanges can provide a chronic wound model. Conclusions: We have developed a
convenient, biocompatible device for significantly reducing rodent wound contraction. Surgical
placement affects the rate of epithelization and granulation tissue formation.
GP04
SILVER-IMPREGNATED NANOMETER-THICK POLYMER FILMS INTEGRATED
IN WOUND-BEDS DO NOT IMPAIR HEALING
Ankit Agarwal, PhD, Harpreet Singh, BS, Kathleen M. Guthrie, DVM, Charles J. Czuprynski,
DVM, PhD, Michael J. Schurr, MD, PhD, Christopher J. Murphy, DVM, PhD, Jonathan F.
McAnulty, DVM, PhD, Nicholas L. Abbott, PhD.
University of Wisconsin, Madison, WI, USA.
Introduction: We report a new approach that broadly enables engineering the surface chemistry
and nanostructured topography of the wound bed. The approach uses nanometer-thick polymer
films to achieve nanoscopic localization of bioactive molecules onto the wound bed. Silver
nanoparticles have been used as an example of an antimicrobial agent and impacts on wound

healing from the mechanical transfer of silver-impregnated polymer films to the wound surface
were investigated in this initial study. Method: Nanometer-thick polymer multilayer films were
prepared on elastomeric substrates and impregnated with silver nanoparticles. Polymer films
were then mechanically transferred onto the full-thickness 8 mm diameter wounds created using
biopsy punch on the flanks of wild or diabetic mice. Diabetic mice exhibited delayed wound
healing similar to chronic wounds. Impairment of wound healing was monitored by imaging the
wounds at intervals. Result: Successful integration of the silver-loaded nanometer-thick polymer
films on the wounds beds was demonstrated. The size of wounds in mice stamped with silverloaded
polymer films was compared to that of untreated wounds in control mice. No significant
difference (n>10, p

Hyaluronic acid inclusion in the scaffolds improved adhesion between the material and wound
bed by absorbing excess moisture, and its presence may have augmented the local healing
response. In addition to providing a template for tissue regeneration, growth factors may be
incorporated into the scaffolds to enhance healing by local delivery. Prefabricated scaffolds
containing rhPDGF-BB showed accelerated cell infiltration, material degradation, and tissue
remodeling in excisional wounds. These biodegradable, tunable PUR scaffolds demonstrate
potential to support regeneration of both form and function of wounds in a range of tissues,
including bone and skin.
GP07
ASSESSMENT ON THE HEALING QUALITY OF DEEP PARTIAL THICKNESS SKIN
BURNS TREATED WITH DIFFERENT SKIN GRAFT TRANSPLANTATION AT
DIFFERENT POST-WOUNDING TIME
Chiyu Jia, M.D, Ph.D.
Burn and Plastic Surgery Center, 309 Hospital of PLA, Beijing, China.
Object: Investigate the correlation between quality of healing and different method of skin
transplantation in different point of time after deep partial thickness burns. Establish an optimal
time frame and method of skin transplantation for such wounds. Materials and methods: 108 SD
rats were divided into five groups by different method of skin transplantation such as razor graft,
intermediate split thickness skin graft, full-thickness skin graft, xenogenous dermis graft and
combined skin graft (xenogenous dermis plus autologous epidermis). Each group was subdivided
into five subgroups by time span such as 0, 1, 3, 7, 14 days after burns. The other eight rats were
divided into two groups, named simply burns and normal skin (control) group. Firstly dorsal
deep partial thickness skin burns model in rats were established. Then skin transplantations were
performed with different free skin graft in different time after burns to cover wound surfaces. 28
days after burns mechanical conformability and tension breaking strength of wound surface
tissue were evaluated and tested by using Vancouver Scar Scale and Instron 5848 micro tension
tester, respectively. Results and Conclusions: As for quality of wound healing, among methods
of treatment for deep partial thickness skin burns in rats, skin transplantation is better than
spontaneous closure and early treatment results in better outcome. As to skin graft used in skin
transplantation, best healing quality is achieved with autologous full thickness skin graft whereas
autologous intermediate split thickness skin graft result in moderate healing quality. When
autologous skin is not enough for skin transplantation, combined skin graft and xenogenous
dermis should be optional.
GP08
MANAGEMENT OF BURNS, OF SECOND AND THIRD DEGREE WTH
BIOTECHNOLOGY.
Fernando F. Uribe, Sr., M.D. , Ph.D., Professor in Surgery.
Asociaciòn Mexicana de Heridas A.C., Guadalajara, Jalisco, Mexico.

Under a prospective study in the Angeles del Carmen Hospital and San Javier Hospital, we are
received patients with burns of special areas and non majors of 20% and non smaller areas of 8%
of second degree burns and third degree, by ignition, deflagration and direct bonding. In a period
of 24 months with ten patients, an average of age of 26 years, seven patients of males and three
of females. Material and Methods: All patient who is received to urgencies, make dermotomies
and fasciotomies, to avoid the compartamental syndrome,for the extremities; the solutions
support, to receive the support of intensive therapy, diminishing the antibioticotherapy, with the
application of equipment of negative pressure, application of sponge with silver and handy foams
for the injuries in hand, with periods of change of three days during the first and second weeks,
and later the combination of polivinil sponges and initiating with the application of factors of
growth in fasciotomies, and the denude site in them of sequential form,and clean with
superoxidant solutions, with jetox machine each change, the equipment of negative pressure is
applied to sheets of queratinocites cultivated over sheets of matrix and with changes each 3 days
during two or three weeks and end with the application of skin graftings of average thickness and
to finalize one week with the equipment of negative pressure with sponges of polivinil. Results:
The handling of the patients with the combination of biotechnology and negative pressure to
modulating the 75 pressure from 50 mmHg, give to faster results in the reorganization of the
extremities, limiting the times of cutaneous covers and early rehabilitation in the first 6 to 7
weeks, of handling, obtaining total closing and handling with hyperbaric oxygenation and
rehabilitation for the minimum sequels.
GP09
EFFECT OF WOUND LOCATION IN A DIABETIC MOUSE MODEL
Stephanie F. Bernatchez, PhD, Patrick J. Parks, MD, PhD, Bruce P. Ekholm, MS.
3M, St. Paul, MN, USA.
Impaired healing animal models are frequently used to study wound healing. We have used the
homozygous db/db mouse to study wound healing over the last several years and analyzed our
results to compare different wound locations on the body and the consistency of the healing
process in this model across experiments. We found that contraction on the back was less than
what was observed on the head. A small difference in the healing is visible from Day 1-4
between the upper back and the lower back of the animal. Head wounds healed more quickly
than back wounds, although there seemed to be more variability between experiments over time.
Histology observations were performed and were more difficult to quantitate than originally
designed. As the wound closes, it becomes difficult to identify the area where the original wound
boundaries were located and to section every wound exactly in the center when processing the
tissue. This misalignment can result in an overestimation of healing, especially when the
remaining wound area at 7 days is minimal. Gross examination of the wound healing site at
various points in time may be a more reliable indicator of overall wound size reduction than a
single microscopic image corresponding to the end point of the experiment. Histology remains
the only way to observe the tissues at the cellular level and to assess the overall quality of the
healing response using parameters such as tissue density, cellular infiltration, matrix deposition,
and fluid accumulation. There is a general pattern of healing that includes ‘layers’ of
proliferative elements varying in apparent staining intensity, reflecting the difference in

cytoplasm (darker with more protein and lighter with more fluid). In conclusion, a mouse model
using back wounds is a good model to study the tissue reaction to various wound care materials.
GP10
LEG ULCERS IN INTRAVENOUS DRUG ADDICTS: CLINICAL
CHARACTERISTICS, COMPLICATIONS AND CHALLENGES.
Karsten Fogh, MD, DMSci.
Aarhus University Hospital, Aarhus, Denmark.
Leg ulcers are frequent in patients with previous intravenous drug abuse due to repeated injection
of drugs into leg veins. As a result, both superficial and deep venous insufficiency develop
leading to severe skin changes and leg ulcerations. Patients often have a life style that is not
compatible with a sustained treatment effort and recurrences and complications are frequent. The
purpose of the present study was to describe patients with leg ulcers related to intravenous drug
abuse. In a retrospective study nine patients were treated during the period 1999-2009 at our leg
ulcer clinic. Patients were evaluated for local skin changes, ulcerations, venous and arterial
system, associated diseases, medication, compliance and life style. Results: All patients had
clinical signs of venous insufficiency (leg oedema, stasis eczema and leg ulcers as well as
superficial scarring). All patients had an ABPI larger than 0.8. All patients were treated with
moist wound healing and compression. All patients had recurring wound infections and reported
severe wound pain, all patients were smokers and all patients were offered free methadone. Five
patients were hepatitis C-positive and all patients were HIV-negative. All patients are on social
welfare and all patients had a prolonged healing process due to several complicating infections
and due to lack of compliance. Currently 4 patients have stopped treatment at the leg ulcer clinic
due to healing (or minor ulceration), one patient died of severe pulmonary insufficiency and
endocarditis. Four patients continue treatment due to not yet healed ulcers. Conclusions: The
present study shows that this group of patients demands special attention and treatment effort
from physicians, wound specialists and social workers. Both the health care system and the
social welfare system face huge challenges dealing with these patients as their life style is not
compatible with adherence to relevant treatment and follow up.
GP11
A PROSPECTIVE STUDY OF THE PUSH TOOL IN DIABETIC FOOT ULCERS
Sue E. Gardner, PhD, RN 1 , Rita A. Frantz, PhD, RN 1 , Stephen L. Hillis, PhD 2 .
1 The University of Iowa College of Nursing, Iowa City, IA, USA, 2 Center for Research in the
Implementation of Innovative Strategies in Practice (CRIISP), Iowa City VA Medical Center,
Iowa City, IA, USA.
The Pressure Ulcer Scale for Healing (PUSH) has been validated as a useful for monitoring
healing in venous ulcers and pressure ulcers. The purpose of this study was to examine the
validity of PUSH (v. 3.0) to monitor healing in a sample of DFUs. The study employed a
prospective research design. The DFUs of study subjects were assessed every two weeks with

the PUSH (v. 3.0) by a member of the research team for a maximum of thirteen weeks. Followup
ended after the ulcer healed. An ulcer was assessed as healed when the wound surface was
judged re-epithelialized by visual inspection. 18 subjects comprised the sample. The median
follow-up times was 4.0 weeks. A plot of the PUSH means at each time point was examined to
obtain some idea of the PUSH trajectory. The graphical display suggested that the trajectory was
approximately piecewise linear, with a constant negative slope until two weeks before healing,
and then a steeper slope in the last two weeks. We evaluated trajectory using a random slopesand-intercepts
model. PUSH decreases at a rate of .6539 per week up until two weeks before
healing, and then it decreases at an estimated rate of 2.2585 per week for the last two weeks.
Slope1 and slope2 are significant (p < .0001), and t the two slopes differ significantly from each
other (slope differential: p < .0001). The findings suggest that PUSH total scores decrease in a
predictable trajectory over time when used to monitor diabetic foot ulcer healing. Because this
tool is relatively easy to use, it provides a useful means for clinicians to track DFU healing or
lack of healing in a manner that can inform management and treatment decisions.
GP12
HUMAN DIABETIC SKIN HAS IMPAIRED BIOMECHANICAL PROPERTIES AT
BASELINE
Dustin M. Bermudez, MD 1 , David P. Beason, MS 2 , Louis P. Soslowsky, PhD 2 , Kenneth W.
Liechty, MD 3 .
1 University of Pennsylvania School of Medicine, Center for Fetal Research at the Children's
Hospital of Philadelphia, Philadelphia, PA, USA, 2 University of Pennsylvania School of
Medicine, McKay Orthopaedic Research Laboratory, Philadelphia, PA, USA, 3 University of
Mississippi Medical Center, Jackson, MS, USA.
Purpose: After injury the healing of diabetic skin is impaired. We have previously shown that
murine diabetic skin has impaired biomechanical properties at baseline compared to non-diabetic
skin. This impairment may play a role in the susceptibility of diabetics to develop chronic
wounds. We hypothesize that human diabetic skin at baseline has a similar biomechanical
impairment, which may predispose this tissue to injury.
Methods: With institutional review board approval, we collected 11 samples of human skin postmortem.
All patients were between 55 and 75 years of age. Four were diabetics and 7 were nondiabetics.
All specimens were collected within 8 hours of death. 5 x 5 cm tissue specimens from
the anterior portion of the lower extremity were flash-frozen in liquid nitrogen and later analyzed
for cross-sectional area and tensile tested to failure and to provide maximum stress and elastic
modulus. Results: The mean cross sectional area between the two groups was not significantly
different. The maximum stress for diabetic skin was significantly lower than that for the nondiabetic
skin (6.5±1.7 v. 4.1±1.9 MPa, p = 0.03). In addition, the modulus for diabetic skin was
also significantly lower than that for non-diabetic skin (25±12 v. 13±5.3 MPa, p = 0.04).
Conclusions: The biomechanical properties of human diabetic skin are dramatically impaired at
baseline, similar to our findings in our animal model. This new finding has implications in the
susceptibility of human diabetic skin to injury and in the pathogenesis of the diabetic wound
healing impairment. Further investigation is needed to identify the factors responsible for these
impaired biomechanical properties.

GP13
PORCINE MODELS OF DELAYED WOUND HEALING: REVIEW, CHALLENGES
AND OPPORTUNITIES
M. Christian Lessing, Ph.D., Kenneth C. Norbury, Ph.D..
Kinetic Concepts, Inc., San Antonio, TX, USA.
Chronic recalcitrant wounds are a socioeconomic problem that demands improved options for
clinical therapeutic intervention. Human clinical factors implicated in impaired wound healing
include reduced perfusion, metabolism, infection, neuropathy, stalled inflammatory response,
delayed keratinocyte migration, elevated levels of MMPs, and altered extracellular matrix and
growth factor involvement. However, a limited amount of this knowledge has been translated
back to pertinent experimental models for preclinical testing of medical devices. A recent
PubMed search for delayed wound healing revealed 693 references pertaining to humans, 218 for
mice, 114 for rats and only 35 for swine. Because of their low cost, limited intraspecies
variability, amenability to genetic manipulation, and availability of biochemical reagents, rodent
models are commonly used in wound healing research. Nevertheless, rodents are limited in their
similarities to human physiology, including skin architecture and healing/disease progression,
and can be of inadequate size for testing of medical devices. Pigs, on the other hand, have a
human-like dermal anatomy and physiological systems that respond more similarly to humans;
the larger size of these animals allows testing of dressings and devices unmodified from their
clinical design. Delayed porcine wound healing, observed with slower wound granulation and reepithelialization
and increased susceptibility to infection, has been achieved to varying degrees
in swine by local irradiation, administration of corticosteroids, and induction of ischemia and
diabetes. However, these delayed wound healing models are currently not adequately
characterized in terms of pertinent cellular and molecular characteristics, such as growth factor
and MMP expression/receptor profiles, extracellular matrix deposition, and participating cell
types. The purpose of this presentation is three-fold: to review the current state of research in
porcine models of delayed wound healing; to highlight the biochemical and cellular
characteristics of porcine wound healing; and to outline various ways in which effective porcine
delayed wound healing models may be achieved.
GP14
WOUND HEALING BY RACIAL/ETHNIC CATEGORIES
Ranjita Misra, PhD 1 , Lynn Lambert, MS 2 , David Vera, MS 3 , Ashley Mangaraj, BS 3 , Suchin R.
Khanna, BS 3 , Chandan K. Sen, PhD 3 .
1 Texas A&M University, College Station, TX, USA, 2 National Healing Corporation (NHC),
Boca Raton, FL, USA, 3 The Ohio State University Comprehensive Wound Center, Columbus,
OH, USA.
Purpose: Wound healing rates are not homogenous in the population and there is a paucity of
information on healing outcomes by racial/ethnic subgroups. Hence, this retrospective study
examined differences in healing outcomes among 1003 patients (53% females, 37% diabetics)

from three major racial/ethnic groups i.e., Non Hispanic White (NHW; 72%), African American
(14%) and other (14%; Asian/Hispanic/Am Indian, Biracial). Patients were treated for acute,
acute traumatic or chronic wounds at a hospital-affiliated Comprehensive Wound Center that
provide advanced outpatient care for 16 weeks. All wound patients were evaluated for healing
with expected rate set at 25%, 50%, 75%, and 100% at the end of 4,8, 12, and 16 week treatment
period. Every four weeks, the wound care physician and case manager reviews the progress of
the patient and assess the cause of why they have not reached the targeted progress rate. Results:
The mean age and number of wounds was 55.2±17 years and 1.9± 1.4 wounds respectively.
Thirty seven percent of the patients had diabetes, 30% had infected wounds, and 69%
overweight/obese. Individuals with diabetes were more likely to be overweight/obese, used
tobacco, and had ulcer related amputations as compared to their non-diabetic peers; 53% had
HbA1c ≥ 7.0 (poor metabolic control). Significant differences were noted in healing rates; 61%
of patients’ wounds healed during the treatment period (56% Males, 66% females, 59% White,
61% African Americans, 55% other race, 55% diabetics, 60% non-diabetics). Ulcer-related
amputation also varied (10% diabetics, 3% non-diabetics, 7% males, 4% females, 5.6% White,
5.7% African American, 5.3% Other Race category). Differential healing pattern was noted by
racial/ethnic category, gender, and diabetes status. Diabetics, African Americans, and males had
delayed healing as compared to others. Conclusion: Results provide important information for
evidence based wound care interventions and developing culturally appropriate education
strategies among high risk groups.
GP15
WILL ICD-10-CM IMPROVE CLASSIFICATIONS OF CHRONIC LOWER LIMB
WOUNDS IN PATIENTS WITH DIABETES?
Jeanne R. Lowe, PhC, RN, CWCN, Gregory J. Raugi, MD, PhD, Gayle E. Reiber, MPH, PhD,
JoAnne D. Whitney, PhD, RN, CWCN, FAAN.
University of Washington, Seattle, WA, USA.
OBJECTIVE: To determine the sensitivity and specificity of ICD-9-CM and ICD-10-CM codes
for people with diabetes and foot ulcers.
RESEARCH DESIGN AND METHODS: Health history and wound variables were
prospectively determined using gold standard data from prospective research and medical
records in a cohort of 49 patients with 65 foot ulcer episodes and a total of 81 incident foot
ulcers. Code sets were mapped to compare the ability of ICD-9-CM and ICD-10-CM to correctly
classify individuals with diabetes and foot ulcers. RESULTS: Compared to the gold standard
data, frequencies for health history variables were similar in both systems. For ulcer
characteristic variables, ICD-9 was unable to capture any data on laterality (left or right) or
depth/severity of ulcer. ICD-9 captured only 69 of 81 incident ulcers (85%), whereas ICD-10
captured 78 of 81 incident ulcers (96%). For ulcers located on the heel or midfoot, ICD-9
captured 94% of incident ulcers and ICD-10 captured 100%. Ulcers on other parts of foot (i.e.
toe or dorsal foot) were captured 82% of the time in ICD-9 and 95% in ICD-10. Sensitivity and
specificity for ulcer characteristics were consistently slightly higher in ICD-10 vs. ICD-9,
ranging from 75.0 to 100 percent. CONCLUSIONS: With “ideal” coding, ICD-9 and ICD-10 are

similar for data capture on health history variables, but wound variables are captured more
frequently in ICD-10. Increased specificity of ICD-10 for ulcer location improves identification
of multiple ulcers during a single episode of care, and also provides ulcer history and severity. In
the future ICD 10 can be used to stratify foot ulcer risk based on a history of foot ulcers.
GP16
EFFECTS OF CLAUDIN-2 KNOCKDOWN IN CULTURED KERATINOCYTES
Dale Telgenhoff, PhD, Amber Anderson, Lynn Lemmons, Tahmina Zaman, Matt Cvitanovich.
Tarleton State University, Fort Worth, TX, USA.
The purpose of this study was to examine the effects of changes in claudin-2 protein in cell
monolayers following knockdown with claudin-2 siRNA. Previous studies have shown that
claudin-2 is increased in wounded epidermis, both in vitro and in vivo. In order to investigate the
role of claudin-2 in wound healing we developed a model which was deficient in claudin-2.
siRNA, transfection medium, and control siRNA (nonsense siRNA) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Human keratinocytes (Cascade Biologics, Oregon) were
cultured to confluence in 6 well plates. Multiple replicates were treated with either claudin-2
siRNA, nonsense siRNA, or remained untreated at 37° C. Cultures were rinsed after 6 hours,
fresh medium was added, and plates were returned to the incubator. After 48 hours the cells were
lysed and the lysate was assayed for protein levels using the BCA assay (Thermo Scientific,
Rockland IL). Equal amounts of protein were loaded on an 8% acrylamide gel, electrophoresed,
and transferred to nitrocellulose for Western blot analysis. Claudin-2 antibody (abcam,
Cambridge MA) was used for detection; bands were quantified using the Kodak Imaging system.
The bands in the knockdown cells were greatly decreased compared to the two controls.
Utilizing the same knockdown procedures we then analyzed the effects of knockdown on cell
migration using a scratch test assay. Monolayer keratinocyte cultures showed a decrease in cell
migration into scratch areas following claudin-2 knockdown. We have developed a successful
knockdown procedure for claudin-2 in keratinocytes, and have used this method to show a
relation between claudin-2 levels and wound healing in vitro.
GP18
EFFECTS OF EPIDERMAL GROWTH FACTOR ON THE EXPRESSION OF EGF
RECEPTORS IN DIABETIC HUMAN FIBROBLASTS
Guang Li, MD, Sung Woo Park, MS, Joon Pio Hong, MD, PhD, MBA.
Asan Medical Center University of Ulsan, Seoul, Korea, Republic of.
EGF plays an important role in wound healing and its clinical use in diabetic foot has been
reported. However the effect of EGF against EGF receptors of diabetic fibroblasts has not been
studied in detail. Also we researched the relationship of EGF to EGF receptors in regards to
levels of hyperglycemia. We hypothesized that the number of EGF receptor would decrease in
higher level of blood sugar and thus the response to EGF would decrease leading to suppressed
healing as seen in clinical situations. The fibroblast EGF receptors from both normal control

group and diabetic group showed positive feedback increasing the expression of EGFR in
response to EGF treatment. In both groups, there was no change of expression regardless of
various concentration of glucose. We first hypothesized that the glucose concentration might
influence expression of EGFR, but the effect was minimal. Therefore, higher level of glucose
leading to suppressed healing may be influence by other mechanism than decreased receptors for
EGF. Further study is warranted to seek out the mechanism behind this finding.
GP19
LOCALIZED INJURY TO THE RABBIT KNEE LEADS TO SYSTEMIC EFFECTS:
IMPACT ON GENE EXPRESSION IN THE CORNEA
David A. Hart, PhD, Kyla Huebner, Yamini Achari.
University of Calgary, Calgary, AB, Canada.
Purpose: Determine the minimal injury following a localized injury to the knee and during
wound healing that evokes a systemic response using the cornea of the eye as the target.
Methods: Injuries to the female rabbit knee were induced by opening the capsule and then
subjecting the femoral bone to a coring procedure with release of bone marrow into the joint
space (experimental) , or just opening the capsule (shams). Total RNA was isolated from the
central 6 mm of the cornea and the remaining periphery, and then mRNA levels for a subset of
relevant molecules were assessed at 3, 6 and 9 weeks post-injury using RT-PCR. Results:
Induction of the coring injury led to signficant depressions in mRNA levels for a subset of the
molecules assessed in the central cornea (matrix molecules, growth factors, MMPs, and hormone
receptors) during the healing response. The response of the peripheral cornea differed from that
of the central cornea, with primarily significant elevations in mRNA levels for an over-lapping
set of the molecules assessed. There was no detectable response of the cornea to the sham
operation, indicating a "minimal" knee injury is require to elicit a corneal response. The changes
were noted by 3 weeks post-injury, with partial resolution by 9 weeks. Conclusions: A localized
knee injury led to prolonged alterations in corneal cell metabolism. Comparison of the present
results to previous knee injury studies (Cornea, 2008; Cornea, in press) indicated that the pattern
of changes in the cornea (location, molecules affected, elevations/depressions) was injuryspecific.
The findings are consistent with involvement of inflammatory mediators induced at the
injury site with subsequent cornea effects. The findings raise the possiblity that distal injuries
and subsequent wound healing responses could affect the integrity of vision, and confirms the
cornea is a susceptible responsive target tissue.
GP20
WOUND HEALING - LOCAL LYMPHATIC SYSTEM DOWN-REGULATES
REACTION TO MICROBES AND OWN ANTIGENS?
Waldemar L. Olszewski, Professor 1 , Marta Cakala, Dr 1 , Zdzislaw A. Machowski, Professor 2 ,
Grzegorz Szczesny, Dr 1 .
1 Dept of Surgical Research &Transplantology, Medical Research Center, Polish Academy of

Sciences, Warsaw, Poland, 2 Dept. of Surgery, Limpopo University, Polokwane Campus,
Polokwane, South Africa.
Introduction. Open and closed wounds cause response of regional lymphoid tissue. What kind of
reaction proceeds in lymph nodes and whether it reflects cellular and molecular events in healing
wound. Aim. To study phenotypes of cells in wound and draining lymph nodes after primary and
secondary injury. Material. Group 1a. Incisional wound of dorsum of paw and stiching. Group
2a. Subdermal injection into paw of 10 8 Staph.epidermidis for 6 days. Group 3a. Closed fracture
of tibia. Follow-up period lasted in all groups for 7 days. Group 1b. Incisional wound followed
after 7 days by another incision. Group 2b. Re-injection of 10 8 Staph.epidermidis. Group 3b. Refracture
of tibia. Results. Groups 1a, 2a,3a. Paw skin.In all groups, 7 days after primary injury,
multiple (++) MHCII+, ED1+ and CD54+ cells were found in subepidermal region or fracture
gap. Lymph nodes. In all groups an increase in lymph node weight and cell concentration was
observed. The W3/25+, MHC II+, ED1+, HIS48+, OX7+ and OX62+ and CD54+ cells
accumulated in the follicles, paracortex and medulla. The percentage of W3/25+CD25+ (T
regulatory) was not increased. The medulla extracellular matrix was stained OX12+. Endothelial
cells stained strongly for OX6 and OX7. There was not more HIS48 granulocytes compared with
controls. Groups 1b,2b,3b. In all groups evidently less of infiltrates in the paw and positively
stained cells in lymph nodes were seen. Conclusions. There were no qualitative changes in
infiltrates and lymph node cell subtypes depending on the type of primary injury. Surprisingly,
secondary injury caused less inflammatory reaction.
GP21
A NEW SIMPLE AND ACCURATE TEST FOR ZINC DEFICIENCY
Mitchell V. kaminski, MD 1 , James Drinane 2 , Deena Horn 2 .
1 Paradigm Shift Wound Care, Niles, IL, USA, 2 Rosalind Franklin University of Medicine and
Science, North Chicago, IL, USA.
Aim: Zinc is the most abundant trace mineral in the human body. It is critical to ~300 enzyme
reactions and its requirement for wound healing has been established. The "metalo" in matrix
metalloproteinase is zinc. Scientists in the European Union have formed a group called the
Zincage Study. Their findings make testing for a zinc deficiency a wise clinical decision. Zinc
is predominantly intracellular, expensive and difficult to assay. A valid functional diagnostic test
is the Zinc Tally Test which is cumbersome. We report a simple and accurate test for zinc
deficiency using two puffs of a zinc solution from an over the counter cold remedy. (Zicam,
Phoenix, AZ.) Methodology: 24 nursing home patients were studied using a cross over design.
A zinc deficiency is diagnosed if the subject does not develop a wry facial expression within
seconds of oral administration. The two methods proved equivalent. Then, a zinc taste test was
added as part of a wound care consult. A serum zinc level was drawn to confirm the diagnosis
and repeated serially to track efficacy of a 220 mg zinc sulfate/day supplement and monitor for
overload. (n=60 to 130 mcg/dL) 79 patients have been studied. The mean initial serum value
was 48.8 mcg/dL. The mid-study and post-study mean serum zinc levels were 54.5 and 61
mcg/dL respectively. The average serum zinc level was found to increase in patients over time
with few reaching normal. Results and Conclusions: All patients with glossitis are zinc deficient.

The two without glossitis were also zinc taste test positive. None recovered from hypogusia
despite some achieving low normal serum values. The simplicity of the oral spray zinc taste test
should make it an essential part of a patients work up.
GP22
ORAL AND CUTANEOUS SIGNS OF VITAMIN, ZINC AND FATTY ACID
DEFICIENCY. A BED SIDE ASSESSMENT
Mitchell V. kaminski, MD 1 , James Drinane 2 , Deena Horn 2 .
1 Paradigm Shift Wound Care, Niles, IL, USA, 2 Rosalind Franklin University of Medicine and
Science, North Chicago, IL, USA.
The clinical efficacy of therapeutic modalities used to heal chronic wounds is related to the
nutritional status of the patient. In 1989 a nutritional assessment of the residents in two nursing
homes was done including photographs of the oral and cutaneous signs of vitamin deficiencies
and serum levels. Marasmus and Kwashiorkor was diagnosed by anthropometric measurement
as well as serum albumin and total lymphocyte count. The incidence of malnutrition was
substantial and further analysis of the data revealed that patients with pressure ulcers were all
severely malnourished. The conclusion was that "a pressure ulcer is a grave sign of
malnutrition". It is therefore imperative that a chronic wound consult include diagnosis of both
macro and micro nutrient deficiencies to guide supplementation. To neglect supplementation
will retard healing. Using recent digital images, the presentation of glossitis (B deficiencies),
magenta tongue and angular stomatitis (B2), hypertrophic lingular papilla (B12), cellophane
skin, purpura and skin tears (C, Scurvy), para-follicular hyperkeratosis (A) and superficial "thin
ice" epidermal cracking (B3, Pellagra) will be illustrated. Also, "snow flake" dandruff (essential
fatty acid), and the Zinc Taste Test (Dx for zinc deficiency) will be presented. This paper is an
update of the chapter on malnutrition published in the Agency for Health Care Policy and
Research: "Treating Pressure Ulcers" Volume 1, Guideline Technical Report, Number 15.
GP23
FROM THE TOP DOWN: DECREASING UNIT-ACQUIRED PRESSURE ULCER
RATES - A CLEVELAND CLINIC EXPERIENCE
Kathleen M. Hill, RN, MSN, CCNS-CSC, Jennifer L. Brinkman, RN, BSN, WOCN, Susan
Wilson, RN, CCRN, Meghan Pishnery, RN, BSN, CCRN.
Cleveland Clinic, Cleveland, OH, USA.
This presentation will elaborate on the process and outcomes associated with a deliberate,
structured approach to pressure ulcer reduction that resulted in a 70% decrease in prevalence.
Recent regulatory changes in the way that hospital acquired pressure ulcers are reimbursed puts
nursing practice front-and-center in the skin care arena. The rules may have tightened, but
nursing interventions remain the key in preventing and treating pressure ulcers. The agencies that
judge fitness for reimbursement and accreditation focus on prevalence, and compliance with
assessment, treatment, and documentation. The 42-bed Surgical Intensive Care Unit of this

international tertiary referral medical center formed a project team to address escalating pressure
ulcer prevalence. The goal was to identify issues that impact the incidence of unit-acquired
pressure ulcers in a setting where every patient is at high-risk for their development. From the
five hour collaborative session thirteen most wanted improvements (MWI) were identified by the
team. The MWIs served as a bundle of interventions - each having individual merit, but
collectively created a synergy that fueled the project. Each MWI was assigned an owner selected
from the team members. Time, resources and cooperation across service lines were critical to the
team member’s progress. The dramatic reductions in unit-acquired pressure ulcer rates were
attributed to the thirteen initiatives that formed the core of the pressure ulcer bundle. The project,
which could be translated to any practice setting, emphasized and achieved collaborative
relationships that increased the strength of the change and served as the source of energy to
sustain the project now at its conclusion.
GP24
POTENTIAL Novel treatment for reversing injury to the liver
Hongwei Yuan, Manuela Martins-Green, Ph. D.
University of California, Riverside, CA, USA.
Western sedentary life style and habits have associated with them serious health problems,
among them the increase of non-alcoholic fatty liver disease (NAFLD), which is the hepatic
hallmark of the metabolic syndrome. Fatty liver or hepatic steatosis has been classified as the
first stage of NAFLD. The detailed mechanisms for the accumulation of lipids in hepatocyte and
the subsequent injury to the liver, remain unclear. Our previous data showed that mice exposed
to second-hand cigarette smoke (SSW), a mixture of more than 4000 chemicals and 80% of
environmental smoke, have lipid accumulation in the liver tissue. Using ApoB100 transgenic
mice fed high fat diet, we proved that SSW can modulate the activity of 5'-AMP-activated
protein kinase (AMPK) and sterol response element binding protein-1 (SREBP-1), two critical
molecules involved in lipid synthesis. Therefore, we hypothesized that drugs that stimulate
AMPK reverse lipid accumulation and the corresponding liver damage caused by second hand
smoke. In order to test this possibility, we fed the ApoB100 transgenic mice with high fat diet
and exposed them to sidestream whole (SSW) smoke. We found that the activity of AMP kinase
in the liver was recovered, with subsequent inactivation of SREBPs, leading to decline in
accumulation of fat in the liver, in response to stimulation of SSW for only 5 days. We are
currently investigating whether this drug affects fatty acid influx, another possible way of
accumulation of fat in the liver, as well as the increased lipolysis in insulin-resistant adipose
tissue. These processes reflect predisposition of individuals to the development of insulin
resistance when exposed to smoke. Our findings may lead to new treatments for injury in the
liver that result in NAFLD and development of inflammation and cirrhosis, a major cause of
liver failure.
GP25
A SIMPLE METHOD TO QUANTIFY INTERSTITIAL SOLUBLE PROTEIN
DIFFERIENTIATES "LYMPHEDEMA" AS HYDROSTATIC EDEMA

Mitchell V. Kaminski, MD 1 , James Drinane 2 , Deena Horn 2 .
1 Paradigm Shift Wound Care, Niles, IL, USA, 2 Rosalind Franklin University of Medicine and
Science, North Chicago, IL, USA.
Reported here is a simple method to sample interstitial fluid from dependent edematous
tissues of an extremity for routine soluble protein analysis. The soluble proteins, both in
circulation and in the interstitial space, consist of albumin, globulin and fibrinogen the sum of
which is reported as total protein. The concentration of these proteins, particularly albumin
because of its smaller size, determines colloid osmotic pressures and the fluid volume in that
space. Over 100 years ago Starling published a formula that describes these relationships and is
known as Starling’s Equation. Sampling method: Place the edematous extremity in a dependent
position, use maximum pitting to identify the site to be accessed with a 21ga. needle inserted at a
45 degree angle after alcohol prep. As the interstitial fluid fills the hub it is collected by
aspiration into a syringe; milking helps. One half cc of fluid is required for analysis.
Observations: Edema secondary to profound hypoprotenemia, as seen in severe Kwashiorkor or
massive blood loss replaced with only RBCs and crystalloid; as well as high Pc as seen in CHF
or locally high venous pressures as in chronic venous insufficiency contains a dilute Πif as low
as albumin 0.4 gm% and total protein 0.6 gm% (n=2 gm% albumin) but, lymphedema
secondary to a post mastectomy, lymph node dissection and tissue necrosis from excess radiation
therapy revealed a Πif of 1.4 gm% albumin and 2.4 gm% total protein consistent with the
literature. Conclusion: Doppler flow studies for reflux in the deep venous system need to be
done. What is currently called "lymphedema" may rather be a compromise of the unidirectional
valves in the deep venous system. If reflux and a dilute interstitial protein is demonstrated, the
edema is secondary to locally elevated Pc and not obstruction to lymph flow.
GP26
ASSESSING ADULT LERANING PREFERENCE FOR SUCCESSFUL WOUND CARE
IN A COMPREHENSIVE WOUND CENTER
Ranjita Misra, PhD 1 , Lynn Lambert, MS 2 , David Vera, MS 3 , Ashley Mangaraj, BS 3 , Suchin R.
Khanna, BS 3 , Chandan K. Sen, PhD 3 .
1 Texas A&M University, College Station, TX, USA, 2 National Healing Corporation (NHC),
Boca Raton, FL, USA, 3 The Ohio State University Comprehensive Wound Center, Columbus,
OH, USA.
Background: Understanding the adult learning principles as well as assessing learning
preferences can aid clinicians and health providers for successful plan of care designed to initiate
a change in behavior, knowledge, and skills. Patient motivation is also an important pedagogical
and andragogical strategy for adult learns. Purpose/Method: The overall goal of this retrospective
study was to examine adult patient’s learning preference and motivation by gender, age, and
race/ethnicity. The sample comprised of 1003 patients (72 % whites, 53% females) treated for
acute, acute traumatic or chronic wounds at a hospital-affiliated Comprehensive Wound Center
in the Midwest. Results: Demographic characteristics indicated 99% adults (mean age =
55.2±17; 29% ≥ 65 years), with low levels of education (48% HS education) and socioeconomic
status (57% on Medicare/Medicaid). The majority of patients indicated their learning preference

as explanation (59%) or demonstration (56%) by their health care providers. Printed materials
was preferred by only one-third (34%) of the patients. Use of video and group/class room
environment was not favored by 99%. Assessment by a nurse manager showed half (51%) were
eager to learn and asked questions (69%). However, 11% were anxious, and 6% were
uninterested/confused/uncooperative. Knowledge of their health problems was low to medium
by 60% of the patients. Learning preference and motivation varied with females having lower
knowledge of health problems, did not prefer to ask questions, were anxious, and preferred the
demonstration/printed materials than their male peers (p

USE OF GRANULOCYTE COLONY STIMULATING FACTOR (GCSF) FOR THE
HEALING OF CHRONIC WOUNDS IN HUMANS
Scott B. Hammerman, M.D., Satori Iwamoto, M.D., Polly Carson, CWS, Xiaofeng Lin, M.D.,
David Fiore, B.A., Vincent Falanga, M.D..
NIH Center of Biomedical Research Excellence (COBRE), Roger Williams Medical Center,
Providence, RI, USA.
Purpose: Even with recent advances up to 50% of chronic wounds that have been present for
more than a year remain resistant to treatment, creating an urgent need for better treatments to be
developed. An attractive idea is to treat these wounds with a younger population of cells that
would be more responsive to growth factors. Therefore, our objective was to determine whether
bringing a patient’s own (autologous) stem cells to their chronic wound lead to healing.
Methods: Stem cell mobilization was accomplished via systemic administration (SQ injections)
of GCSF, for 4 consecutive days, with a dose of 10ug/kg. Bone marrow cell recruitment was
stimulated by the concomitant application of fibrin spray to the wound itself. In addition, the
subject received local wound care and compression therapy. Blood was drawn, at various time
points, to check for the presence of stem cell markers and blood count. In addition, biopsies of
the wound and thigh were performed for histological examination, tissue culture, and frozen
section. The subject was followed on a weekly basis for 28 weeks. At each visit, the wound was
examined, traced for planimetry, and photographed. Results: One 63 y/o Caucasian male with a
chronic venous leg ulcer was enrolled and completed the treatment phase. The subject’s white
blood cell count rose from 8.2 10n 3 ul (screening) to 42.6 10n 3 ul (after 3 injections). Stem cell
markers CD34 and CD117 increased between the 1 st and 3 rd GCSF injection. The wound size
decreased from 7.9 cm 2 (screening) to 3.5 cm 2 by 28 weeks after treatment. Clinically, the wound
became more superficial, granulation tissue increased, and less crusted at the wound edges. No
adverse events occurred. Conclusions: Early data shows GCSF could improve the clinical
appearance of chronic wounds. Stem cell markers indicate GCSF facilitates the recruitment of
stem cells.
GP29
ADIPOSE-DERIVED STEM CELLS ENHANCE WOUND HEALING AND FAT
REGENERATION
Sadanori Akita, MD, PhD.
Nagasaki University, Nagasaki, Japan.
Intractable and prolonged wounds after irradiation, there are clinical evidences that the local
somatic stem cells are expressed in the adjacent to of the injured wounds. Among such somatic
stem cells, adipose-derived regenerative cells (ADRCs) are highly expected cell source, because
the donor-site morbidity is minimal and quickness and easiness of cell processing. As our series
of the radiation-induced wound treatment with ADRCs redeemed the regenerative wound
healing and local tissue regeneration including tendon, bone, fat and muscle as well as skin resurfacing.
In vitro cell culture derived from the patients’ own ADRCs demonstrated proliferative
and can be differentiated to the adipocytes by induction with IBMX, insulin, indomethacin and

dexamethasone and confirmed by oil red O staining. With these clinical and laboratory proofs,
systemically impaired fat distribution associated to HIV-lipodystrophy, was sought to be
investigated for fat regeneration with the patient’s own ADRCs. Thousands of Hemophilia
patients and their wives are victimized by HIV-viral contaminated factors VIII and IXuntil
middle of 80s are targeted for this therapy. Lipodystrophy comprise both fat accumulation and
fat atrophy, which causes remarkable adverse impact social life of the patients. Thus, first, three
dimensional CT (3DCT) analysis focused on the patients’ subcutaneous fat tissue for patients
analyzed and quantified with fat volume analyzing software. The fat atrophy was observed in
cheeks, temporal fossae, below elbows and knees and sacral areas. Female patients, who were
secondarily infected HIV from their partners demonstrated fat accumulation in their torsos and
abdomens. Regenerative surgery with male hemophilia-induced HIV-lipoatrophy in his cheek
with his own ADRCs demonstrates remarkable improvement of the deficit fat tissue.
GP30
GCSF STEM CELL MOBILIZATION CAN ACCELERATE MOUSE AND HUMAN
WOUND HEALING
Satori Iwamoto, MD, PhD, Xiaofeng Lin, MD, PhD, Kendra Kobrin, Scott Hammerman, MD,
Tatyana Yufit, MD, Polly Carson, CWS, John Morgan, PhD, Nicola Kouttab, PhD, Faina Zak,
David Fiore, Deborah Greer, Alex Iwamoto, Vincent Falanga, MD.
NIH Center of Biomedical Research Excellence (COBRE), Roger Williams Medical Center,
Providence, RI, USA.
Innovative approaches such as stem cell therapy may be required to stimulate the healing of
difficult-to-heal chronic wounds. Granulocyte colony stimulating factor (GCSF) is a cytokine
used to mobilize bone marrow-derived stem cells to the peripheral blood. We studied the effects
of systemic GCSF on wounds in mice and in a human subject. Mice were injected for five days
with two formulations of GCSF and compared to controls. (1). To monitor stem cell
mobilization, flow cytometric measurements of Sca-1 and c-Kit, and colony forming cell (CFC)
assays were performed. (2). Full thickness tail wounds were created and monitored for clinical
evidence of healing. (3). To measure connective tissue formation, polyvinyl alcohol sponges
were implanted to monitor collagen content as a function of time. (4). To monitor bone marrow
stem cell homing to wound sites, chimeric mice transplanted with GFP bone marrow cells were
scanned by live imaging. (5). In addition, one human subject with a chronic wound refractory to
standard care was treated with systemic GCSF. Results were as follows: (1). Peripheral blood
stem cells appeared to rise between three to five days following the initiation of GCSF
administration, as confirmed preliminarily by CFC assays. (2). GCSF treatment resulted in
cleaner, less crusted wound beds in mouse-tail wounds. (3). There was a small increase of
connective tissue formation in GCSF treated mice. (4). Live imaging revealed an increasing
accumulation of bone marrow-derived cells at the tail wound for at least eight days after
wounding. (5). The wound of our single human subject treated with systemic GCSF showed an
increase in granulation tissue, followed by nearly a 50% decrease in the ulcer area. GCSF stem
cell mobilization shows promise for stimulating wound healing and treating wounds.
GP31

TOPICALLY APPLIED RABBIT ADIPOSE-DERIVED STEM CELLS CAN
SIGNIFICANTLY PROMOTE WOUND HEALING IN A RABBIT EAR MODEL
Yanan Zhao, M.D., Shengxian Jia, MD, PHD, Jennifer Nunez, MD, PHD, Seok Jong Hong,
PHD, Marina Vracar-Grabar, MS, Thomas A. Mustoe, MD.
Wound Healing Lab,Department of Surgery,Northwestern University, Chicago, IL, USA.
Stem cell application has shown very promising therapeutic effects for many diseases. The goal
of this study is to test the effects of rabbit adipose-derived stem cells on wound healing with a
rabbit ear wound model. Three young New Zealand White rabbits were used in this study. Four
7-mm full-thickness dermal punches were then made on the inner surface of each ear down to
perichondrium. Each wound in one ear was topically applied with 6×10 5 rabbit adipose-derived
stem cells in 15µl saline with vehicle or same amount of rabbit dermal fibroblasts served as
controls and applied in the other ear. At 7 days post wounding, the animals were sacrificed and
the wounds were collected and processed for histological analysis. Results: rabbit stem cell
treatment significantly promote granulation tissue synthesis in terms of granulation tissue
distance ( 1723±146 vs 846±71 or 1292±139µm, p

elongs into a class of compounds that disrupt the bacterial cell membrane structure and kill the
bacterial and hence bactericidal. The results will be discussed in detail.
GP33
PREDILECTION TO SEPSIS, ACUTE TISSUE INFECTIONS AND DELAYED
INFECTED WOUND HEALING MAY DEPEND ON THE SAME GENETIC
POLYMORPHISMS AT TNFΑ G308A, TNFΒ G252A, CCR2 G190A, CD14 C159T, TLR2
G2259A AND C2029T, TLR4 A1036G AND T1336C, AND TGFΒ G25C
Waldemar L. Olszewski, Professor 1 , Marek Durlik, Dr 1 , Joanna Rutkowska, Dr 1 , Bozenna
Interewicz, Dr 1 , Krystyna Stepien, Dr 2 , Zanetta Czapnik, MSc 1 , Malgorzata Zagozda, Dr 1 .
1 Dept of Surgical Research &Transplantology, Medical Research Center, Polish Academy of
Sciences, Warsaw, Poland, 2 Central Clinical Hospital, Ministry of Internal Affairs, Warsaw,
Poland.
Introduction. Most published studies on infections and patients’ genetic polymorphisms are
dealing with sepsis. Only few analyze the genetic predilection to less fulminant inflammatory
processes as acute circumscribed organ or tissue infections and infections causing delayed
healing of large wounds. Aim. We studied polymorphisms of allele of cytokines and TLRs at 9
polymorphic sites in groups of patients with sepsis, acute tissue infections and prolonged wound
suppuration. Results: 1) in entire group presenting systemic and local infections, we found higher
frequency of TNFαG308A GG, TNFβ G525A mutated homozygote AA, and CCR2 G190A
mutated homozygote AA than in controls (all p

Introduction: The genetics of microbial pathogens have been extensively studied, but there is
little work on genetic susceptibility to surgical site infection (SSI). Chronic Granulomatous
Disease, which results in recurrent, severe infections with wound pathogens such as Staph
aureus, is caused by a mutation in one of four sub-units of the phagosomal oxidase (phox). The
possibility of less severe mutations that increase susceptibility to SSI through a milder reduction
in oxidative bacterial killing has not been investigated. We studied familial contribution to SSI
using a genealogical database that has links to healthcare records for 2 million individuals.
Methods: With IRB approval (pending), we identified individuals with ICD-9 codes indicating a
diagnosis of SSI. These patients were analyzed for evidence of excess relatedness. To properly
account for characteristics that could affect the quality and quantity of genealogy data, all cases
were assigned to sex-, birthyear- and birthplace cohorts. Cohort-matched hospital controls were
randomly selected. The relative risk for SSI in first-, second- and third-degree relatives was
estimated by comparing the number of affected relatives of cases to the number of affected
relatives of controls. To test for excess relatedness, the average pairwise relatedness of cases was
compared to the same measure on 1,000 sets of matched controls. Results: Adequate subjects and
controls with linked genealogical data can be identified. The cohort is ready for analysis. Similar
analysis using this resource has shown a genetic contribution to phenotypes including rotator
cuff disease and benign pituitary tumors using hospital data. Conclusion: Familiality of SSI
suggests a genetic component, although environmental factors may also contribute. The
population database has an approved mechanism for contacting identified families to obtain
samples for genetic analysis of the identified phenotype, which has successfully identified a
number of genetic mutations linked to cancer.
GP36
INNOVATIVE WOUND CARE PROGRAM TO PREVENT INCONTINENCE
DERMATITIS
Selina Hune, MSc. FNP 1 , Ana Stanesic, RN 2 , Sonia Lounds, BSc. N 3 .
1 Sunnybrook Health Sciences Centre, Willowdale, ON, Canada, 2 Sunnybrook Health Science
Centre, Toronto,, ON, Canada, 3 Sunnybrook Health Science Centre, Toronto, ON, Canada.
Purpose: To develop an innovative, evidenced base skin care protocol and system to prevent
pressure ulcers and incontinence dermatitis in our residents. Background: Incontinence is a
common and costly clinical problem in long-term care. Many nursing homes use only soap and
water, or continent product inconsistently for perineal care. Clinically soap and water interfere
with skin PH resulting in dryness of skin, which can precipitate skin break down. A 530 longterm
care facility, committed to providing high quality resident focused care, has recently
completed an evaluation of an incontinence care system and protocol to prevent incontinence
dermatitis. Methods: A purposeful sampling of 20 residents, suffer from slightly red to highly
irritated skin conditions, was selected as study group. The control group consisted of five
residents from a different complex continuing care units. Initial assessment was done on all 20
residents as a baseline for post trial comparison. A tracking form was developed to track changes
in skin condition. Results: Skin condition had improved moderately in 19 residents after two
weeks; one resident declined any skin product use. In the control group, three residents
experienced worsening skin condition, one has improved with medicated cream use, and one

emained the same. No new cases of dermatitis were noted since the implementation of new
products use. Conclusion: Application of a three-option system including skin cleanser, durable
barrier cream, and liquid barrier film helped to maintain skin integrity in the elderly population.
A custom designed plastic caddy system were effective organizer to store products at designated
residents’ bedside. It helps to reduce risk of cross infection, control wastage/cost, prevent extra
bottle/tube left opened at bedside, and to eliminate excess product use.
GP37
THE PALLIATIVE SURGERY OF ADVANCED FUNGATING SKIN CANCERS
Masaki Fujioka, Dr..
National Hospital Organization Nagasaki Medical Center., Ohmura, Japan.
Introduction: Approximately 5-10% of patients with usual advanced skin cancer will develop a
fungating wound. When the cancer had already advanced, curative treatment is often not chosen,
and the goal of treatment is to optimize the quality of life (QOL). However, fungating wound
sometimes prevents the patients from the life at home. Methods: We present two cases of large
fungating ulcers resulting from breast cancer (case 1) and malignant melanoma (case 2), which
were treated with palliative amputation with a free skin grafting. Results: Wound in case 1 was a
23.0 x 21.0-cm, hard tumor in the right breast which developed a fungating wound with
infection, odors, discharge and bleeding. CT showed that the mass had invaded to the pectoralis
major muscle and ribs. 4 weeks after palliative amputation with a free skin grafting, the patient
was discharged, remained law surface was treated with ointment at home. The patient had visited
our hospital once or twice a month aiming to be received palliative care for 7 months. The
wound in case 2 was a 13.0 ×12.0-cm, hard tumor which developed a fungating wound with
infection, unpleasant odors, discharge and bleeding. It was resurfaced by 3 weeks after skin
grafting and the patient was discharged 4 weeks after the surgery without wounds. The patient
had visited our hospital once or twice a month aiming to be received palliative care for 4 months.
Both patients resulted in successful outcome in the meaning of improvement of patient’s QOL.
Conclusions: Simple palliative surgery can improve QOL of terminal patients such as the
decrease of secretion, odors, the risk of infection, consequently, improve the nutrition and
general condition. Palliative abrasion is sometimes a reasonable option, especially for the
patients with malignant fungating complex ulcers.
GP38
ZINC-OXIDE BOOT CUMARINE+ MULTILAYER SHORT-STRETCH BANDAGE
(SOM)
Eugenio O. Brizzio, Sr., MD.
GIC- International Compression Group, Buenos Aires, Argentina.
Introduction: UNNA boot and Fisher’s modification are the precursors of the complete treatment
in venous ulcers since legs and ulcers are treated. Zinc-oxide Cumarine boot and a multilayer
short-stretch bandage (SOM) make a system which acts in tissues and hemodynamic

compensation. Objectives: To show the beneficial action of the system - To evaluate the healing
rate of ulcers treated with this system - To detect the responsible risk factors of recalcitrant
ulcers. Materials : 672 patients between January 2000 and January 2009 and 202 leg ulcers. The
different types of ulcers were: recurrent venous ulcer (78); primary venous ulcer (51); mixed
ulcer (27); diabetic ulcer (7); arteriosclerotic ulcer (6); post-traumatic ulcer (6); Martorell Ulcers
(6); vasculitic ulcer (5); ulcerated white atrophy (4); post-thrombotic venous ulcer (3); UPP (2);
Pyoderma gangrenosum (2); lymphatic ulcer (3); spider poison ulcer (1); lipidical Necrobiosis.
Methods: Arterial and venous control assessed with Color Duplex Scan - Photographic
documentation - Evolution control of ulcers areas and depth (Visitrak® - Smith and Nephew) All
patients were treated with zinc-oxide Cumarine boot and multilayer bandage. Results: Prevalence
of female sex - Most ulcer incidence in left legs - Risk factors for recalcitrant venous ulcers are:
ulcers initial size, ulcers age, and CVI age. Conclusions: The beneficial actions of the system are
showed by the high healing rate and healing percentage before 90 days. Also it indicates the risk
factors that facilitate recalcitrant ulcers.
GP39
MANAGEMENT OF ACUTE SEVERE MOIST DESQUAMATION RADIATION
DERMATITIS WITH BIAFINE (TROLAMINE), AN OIL-IN-WATER EMULSION,
AND MEPILEX LITE, A THIN SOFT SILICONE DRESSING
MARGARITA SIMON, MS, FNP-BC, CWCN 1 , SONYA SIMMONS, RN 2 .
1 Simon Wound Consulting, PLLC, Virginia Beach, VA, USA, 2 GENTIVA HOME HEALTH
SERVICES, Virginia beach, VA, USA.
Purpose: There are no specific practice guidelines for the treatment of radiation dermatitis, only
recommendations for topical skin agents with varying degrees of evidence of success. The
objective of this case study is to report an effective therapy for acute moist desquamation
radiation dermatitis. Clinical Problem: A 66-year-old female, former smoker, underwent
radiation and chemotherapy for left piriform sinus squamous cell carcinoma. Two months later,
she had developed severe moist desquamation of the entire neck circumference. She also
developed mucositis, had a 36 lb. weight loss and dehydration, requiring PEG tube placement.
Home health care was ordered for wound care after discharge from the hospital. The silvadene
and xeroform gauze applied in the hospital prior to discharge were adhered, causing significant
pain and restricting neck movement. Clinical Approach: Based on some of the literature and case
studies reviewed, the wound consultant developed a plan of care. It took 2 visits for the nurse to
remove the adhered dressings with Skintegrity spray wound cleanser to saturate the dressings
and remove them without causing the patient further trauma, bleeding, and pain. Then Biafine
(which also has non-steroidal anti inflammatory properties) was applied and covered with
Mepilex Lite daily. Case consultation via phone and e-mailed photography between the home
health nurse and wound consultant was done at least weekly to monitor progress. Patient
Outcomes: Patient reported instant pain relief once Biafine and Mepilex Lite were applied. After
two weeks, there was no need to cover the area with Mepilex Lite and only Biafine was applied
daily after showering. She experienced rapid healing. All the radiation dermatitis was completely
healed in 4 weeks, with no adverse events and improved quality of life. Conclusion: This case

study shows the effectiveness of a treatment for acute severe moist desquamation radiation
dermatitis with Biafine and Mepilex Lite.
GP40
RADIOFREQUENCY ABLATION OF A COMPETENT LESSER SAPHENOUS VEIN
FOR THE MANAGEMENT OF A NON-HEALING VENOUS STASIS ULCER ON THE
POSTERIOR CALF RELATED TO INCOMPETENT PERFORATOR VEIN
Erik A. Maus, MD.
University of Texas Health Science Center at Houston, Houston, TX, USA.
Venous insufficiency ulcers account for more than 50% of the ulcers seen at a general Wound
Center. We present the case of a 60 year old woman with a venous stasis ulcer on the posteromedial
aspect of the right calf that had been present for over two years and who had failed to
respond to standard wound care including compression bandaging and use of biological skin
substitutes.The patient had disabling pain and was on large doses of narcotic medication. Patient
was found to have a dilated incompetent perforator vein two cm above the ulcer. The perforator
communicated the lesser saphenous vein with the posterior tibial vein. The perforator could not
be directly targeted due to the proximity with an artery. While the lesser saphenous vein was
competent and would in other circumstances not been consider amenable for treatment we
decided to ablate it in order to block venous flow through the incompetent perforator. This led to
immediate resolution of pain and to complete wound closure within the following weeks. We
present here an alternative approach to the management of venous stasis ulcers related to
incompetent perforators. A review of the literature and current options for the diagnosis and
treatment of incompetent perforating veins is presented.
GP41
USE OF 10% POVIDONE-IODINE MOISTENED GAUZE PRIMARY DRESSINGS IN
THE TREATMENT OF VENOUS STASIS ULCERS THAT HAVE FAILED TO HEAL
WITH STANDARD WOUND CARE
Mark Lipman, MD, Inna Udall, ARNP.
Sarasota Memorial Hospital, Sarasota, FL, USA.
Conventional wound care recommended for the treatment of venous stasis ulcers typically
includes compression therapy using a multi-layer wrap or graduated compression stockings to
control edema, over a foam or gauze dressings to control exudate. Use of dressings containing
povidone-iodine is controversial because of the cytotoxic properties associated with povidoneiodine
and concern for potential absorption of free iodine after prolonged topical exposure.
Recent data suggest that primary dressings consisting of cadexomer iodine were well tolerated
and may improve healing of venous stasis ulcers. We present a series of several patients with
venous stasis ulcers that had failed to heal using standard therapy, yet healed completely once the
primary dressing was changed to a 10% povidone-iodine, (Betadine Solution), moistened gauze
primary dressing used beneath a multilayer compression wrap. Use of a 10% povidone-iodine

moistened gauze primary dressing beneath a multilayer compression wrap is a cost effective
alternative to other primary dressings and should be considered as an alternative dressing in
patient’s with venous stasis ulcers that have failed to heal with more conventional primary
dressings and compression wraps or graduated compression stockings. Further investigation of
the safety and efficacy of the use of 10% povidone-iodine moistened gauze dressings for the
treatment of venous stasis ulcers is indicated.
GP42
COMPARISON OF LOW-STRENGTH COMPRESSION STOCKINGS WITH
BANDAGES FOR THE TREATMENT OF RECALCITRANT VENOUS ULCERS - A
RANDOMIZED TRIAL
Eugenio O. Brizzio, Sr., MD.
GIC- International Compression Group, Buenos Aires, Argentina.
Objectives: To evaluate the effects of compressive therapy in 2 groups of patients with leg
venous ulcer. One group used low compression medical stockings (MCS) and the other group
used multilayer short-stretch bandages. Healing rate and time to healing, pain progress and
quality of life were assessed. Methods: A randomised, single-centre, open-label study was
performed on 60 legs of 56 consecutive patients with no prior compression therapy. Sigvaris
prototype MCS providing 15-20 mmHg at the ankle were compared with multi-layer shortstretch
bandages. Eccentric padding was used in all patients. Wound treatment was individually
tailored. Endpoints were healing within 90d and 180d, time to healing, and quality of life
measured monthly with the chronic venous insufficiency questionnaire (CIVIQ). Results: 60
patients were enrolled. 55 patients completed the study protocol: 28 in the bandage group and 27
in the bandage group. Healing within 90 days occurred in 36% using MCS and in 48% using
bandages. Healing within 180 days occurred in 50% using stockings and in 67% using bandages.
Healing rate was identical in both groups. Pain initially scored 44 and 46 (in a scale where 100
means maximum pain and 0 absence of pain). Pain decreased to 20 and 28 within the first week,
in MCS and bandage groups, respectively. Quality of life showed no differences between both
treatment groups. In both groups, pain decreased semi independent with regard to healing, at 90
days. Conclusion: Our study illustrated the difficulty to bring such large and long-standing
venous ulcers to healing. Compression effects were similar with either stockings or bandages.
Pain was alleviated rapidly in both treatment groups, even when low compression was applied.
With the information provided by this study, it is suggested that compressive therapy with two
superposed stockings is probably the most reasonable choice (19).