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Abstracts
7th Joint Meeting of the European Tissue Repair Society (ETRS)
with the Wound Healing Society
25th Annual Meeting of ETRS
Scandic Copenhagen
Copenhagen, Denmark
October 21–23, 2015
From Bed to Bench
Abstracts were competitively selected by six blinded peer reviewers. Abstracts are in order of the First Author’s last
name. Presenting Authors are underlined.
MONITORING EPIDERMAL WOUND HEALING IN HUMANS BY
OPTICAL COHERENCE TOMOGRAPHY
M. Ahlstr€om1,2, H. F. Larsen1, L. M. R. Gjerdrum3, A. L. Sørensen4,
J. L. Forman4, L. N. Jorgensen5, M. Mogensen1, M. S. Ågren1,5
1
Department of Dermatology and Copenhagen Wound Healing Center,
University of Copenhagen, Copenhagen NV, Denmark; 2The National Allergy
Research Centre, Gentofte Hospital, University of Copenhagen, Hellerup,
Denmark; 3Department of Clinical Medicine, Region Sjælland, Roskilde,
Denmark; 4Section of Biostatistics, Department of Public Health, University of
Copenhagen, Copenhagen K, Denmark; 5Digestive Disease Center, Bispebjerg
Hospital, University of Copenhagen, Copenhagen NV, Denmark
Introduction: Non-invasive monitoring of wound healing is warranted. Optical
coherence tomography (OCT) enables instant visualization of the epidermis and
upper dermis. We have studied the healing of uniform epidermal wounds in
humans using OCT.
Methods: Thirty-two, 16 females and 16 males, non-smoking healthy volunteers aged
18–49 years were enrolled (NCT02116725). Suction blisters were raised on each buttock by applying chambers with a 10 mm opening at 2380 mmHg and surface temperature of 408C. The blister roofs comprising the entire epidermis were excised. The
60 wounds were scanned by OCT on day 0 (baseline), and post-wounding days 2 and
4. Full-thickness skin biopsies (12 mm 3 3 mm) of the 60 wounds were excised and
processed for routine histology. In two volunteers, wound healing was followed over
14 days by OCT and transepidermal water loss. Data were analyzed in linear mixed
models for repeated measurements and by Bland-Altman.
Results: The complete loss of epithelium after wounding was verified by OCT.
Inflammation, measured by OCT as blood vessel dilation in underlying dermis, rose
14-fold (95% confidence interval: 8-26, p < 0.001) from baseline to day 2 and then
tended to decline (p 5 0.090). The thickness of neoepithelium increased from days 2
to 4 by 19 mm (6–31 mm, p 5 0.01) but was still thinner than normal epidermis. Quantitative histology showed epithelial coverage of 39% (36–43%) on day 4. Percentage
re-epithelialization measured by OCT was biased by 10% (4–16%, p < 0.001) compared to histology with wide limits of agreement (235–54%). On day 7, the clinically
healed wounds were accompanied by steep drop in TEWL and by wound neoepithelium that was thicker than adjacent intact epidermis.
Conclusions: OCT is a promising noninvasive technique in the assessment of
inflammation and epithelial thickness during human epidermal wound healing.
CAN LOSARTAN AND/OR ATORVASTATIN IMPROVE THE
HEALING OF FULL THICKNESS BURN WOUNDS?
J. Akershoek1,2, W. Talhout1,2, M. Vlig2, K. Brouwer1,2, E. Middelkoop1,2,
M. Ulrich1,2
1
Department of Plastic, Reconstructive and Hand Surgery, VU University
Medical Center, Amsterdam, The Netherlands; 2Association of Dutch Burn
Centres, Beverwijk, The Netherlands
Introduction: Scars negatively affect the quality of life of burn wound patients
and often require multiple surgical reconstructions. The renin angiotensin sys-
A1
tem (RAS) is one of the pathways which could play a role in scar formation.
RAS is locally activated upon tissue damage in various organs. Inhibition of
RAS was shown to reduce fibrosis of these organs after injury. Angiotensin
receptor blockers (e.g., Losartan) and statins (e.g., Atorvastatin) and the combination of these drugs are able to reduce RAS activation. The aim was to investigate the effects of Losartan, Atorvastatin, or the combination of both drugs on
wound healing of full-thickness burn wounds (FTBW).
Methods: Six FTBW were induced on the flanks of pigs. Systemic treatment
with Losartan, Atorvastatin, and combination was started one day after infliction
of the burn wounds and continued for 28 days. FTBW were excised and transplanted with autologous meshed split skin grafts (STSG) 14 days later. Biopsies
were taken for histology. Contraction of the wounds was measured by planimetry and scar quality was scored at day 56.
Results: Treatment with Losartan resulted in a statistically significant reduced
STSG take 8 days after transplantation. In addition, induced redness, increased
influx of neutrophils (day 14) and contraction were observed for Losartan compared to control or Atorvastatin. The deteriorating effects of Losartan were partially reduced in the combination therapy. Atorvastatin reduced redness in FTBW
compared to the control (day 28/day 56). Atorvastatin-treated wounds showed
increased STSG take compared to control, although not statistically significant.
Conclusions: Oral administration of Losartan deteriorated healing of FTBW
through reduced STSG take which probably resulted in increased wound
contraction and poor scar outcome. Treatment with Atorvastatin reduced redness
of FTBW and showed indications of improved STSG take. Further research is
needed to explore the possible positive effects of Atorvastatin.
POSITIVE IMPACT OF BOTH PLASMA-BASED FIBRIN MATRIX
AND ITS RELEASED FACTORS ON EPIDERMAL SUBSTITUTE
PROLIFERATION, CLONOGENICITY AND ENGRAFTMENT IN VIVO
M. Alexaline1,2, M. Nivet2, A. Zuleta1,2, T. Leclerc3, E. Bey4, P. Duhamel4,
C. Bernard5, L. Jean-Jacques6, M. Trouillas2
1
HRA Pharma, Paris, France; 2Institut de Recherche Biom"edicale des Arm"ees,
Clamart, France; 3CTB (Burn Treatment Unit), Percy Hospital, Clamart, France;
4
Plastic Surgery Department, Percy Hospital, Clamart, France; 5Paris Sud
University, UMRS 1197 INSERM, IRBA/CTSA - Lab Recherche & Therapie
Cellulaire, Clamart, France; 6Unit"e de Th"erapie Cellulaire Et R"eparation
Tissulaire, H^
opital Percy, Clamart, France
Introduction: Cultured epithelial autografts (CEAs) represent a lifesaving surgical technique in case of full-thickness skin burn covering more than 50% total
body surface area. CEAs present numerous drawbacks, among them the high
fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal
junction leading to heavy cosmetic and functional sequelae. To overcome these
weaknesses, we developed a new human epidermal substitute (ES) cultured
over a human-plasma-based fibrin matrix (hPBM).
Methods: Phenotypical, functional (clonal analysis, long-term culture, or in
vivo grafting), and released factors analyses were used to compare ES cultured
on hPBM, fibrin from purified fibrinogen or no matrix.
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Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
Results: The use of hPBM for ES production induced more proliferation,
improved cell organization and clonogenicity, enhanced dermal-epidermal junction protein deposition, and prevented their degradation while FPF specifically
improved keratinocyte migration potential. Keratinocyte differentiation was
decreased using both fibrin matrices. The growth factors released from hPBM
in culture medium were different from FPF. We showed that released insulinlike growth factor-1 from hPBM enhanced proliferation in ES in vitro. Finally,
the use of hPBM as a culture support for ES allowed better engraftment directly
on a NOD-SCID model of acute wound with the formation of a functional
dermal–epidermal junction.
Conclusions: Together, these results show the positive impact of fibrin matrices,
modulated by released growth factor, on ES phenotype and grafting efficiency.
We hope that this new strategy could improve the current medical treatment of
full-thickness burn patients in the future.
A NOVEL NUDE MOUSE MODEL OF HYPERTROPHIC SCARRING
USING SCRATCHED FULL-THICKNESS HUMAN SKIN GRAFTS
S. Alrobaie1, J. Ding1, Z. Ma1, E. Tredget1
University of Alberta, Edmonton, Canada
1
Introduction: Hypertrophic scar (HTS) is a dermal form of fibroproliferative
disorder that develops following deep skin injury that cause deformities, functional disabilities, and aesthetic disfigurements (1). The pathophysiology of
HTS is not well understood due to the lack of an ideal animal model (2). We
hypothesize that human skin graft with deep dermal wounds grafted onto the
back of an athymic nude mouse will develop a more promising scar. Our aim is
to develop an ideal animal model that represents human HTS.
Methods: Thirty-six nude mice were xenografted with full-thickness human skin
with scratch wound before or 2 weeks after grafting or without wound. The scratch
on the human grafts was made using a specially designed jigsaw that helps to
make an approximately 0.56 mm deep wound. Grafts were morphologically analyzed by digital photography. Mice were euthanized at one, two, and three months
postoperatively for histology and immunohistochemistry analysis.
Results: The mice developed red, raised, and firm grafts. Compared to the nonscratch wounds, the scratch wounds before and after grafting resulted in more graft
contraction at 1 month (wound area: 1.84 6 0.235 cm2 versus 2.5 6 0.16 cm2 and
2.6 6 0.012 cm2), showed increase in a-SMA expressing myofibroblasts at two
months (22.8 6 2.33 versus 35.0 6 1.51 and 33.80 6 2.35) and recruited more macrophages at three months (10.2 6 0.663 and 10.2 6 1.39 versus 6.0 6 0.547). Collagen bundles orientation and morphology were constant with HTS in all xenografts.
Conclusions: Scratched human full-thickness skin onto the back of nude mice
developed mature scars and the grafts were morphologically and histologically
similar to human HTS.
References
1. Armour A, Scott PG, Tredget EE. Cellular and molecular pathology of
HTS: basis for treatment. Wound Repair Regen 2007; 15 Suppl 1: S6-17.
2. Ramos ML, Gragnani A, Ferreira LM. Is there an ideal animal model to
study hypertrophic scarring? J Burn Care Res 2008; 29: 363-8.
THE BIOCHEMICAL POTENTIALS IN MAGGOTS—WILL THEY
EVER FLY?
A. S. Andersen1
1
Business Creation R&D, Novozymes A/S, Bagsvaerd, Denmark
The use of maggot debridement therapy in chronically infected wounds has
since the 1990s prompted an increased interest in the involved biological mechanisms. Apart from the debriding activity, maggot’s excretions/secretions also
have activities against microorganisms and biofilms, are anti-inflammatory and
induce granulation tissue formation. Peptides and enzymes attributed to the beneficial effects have been identified, patented, and envisioned as adjunctive novel
bio-actives for either direct application or incorporation into medical devices to
aid healing of chronic wounds. Despite this, none have so far made it to the
clinic or have been incorporated into commercial products. The focus of this
presentation will be to highlight the pitfalls and drawbacks that may be keeping
the biochemical potentials from maggots from becoming airborne.
OUTCOMES OF DIABETIC FOOT INFECTIONS—WHERE ARE WE
NOW?
J. Apelqvist1
Diabetic Foot Centre, University Hospital of Malm€
o, Malm€
o, Sweden
1
The diabetic foot can be defined as infection, ulceration, and/or destruction of deep
tissues associated with neurological abnormalities and various degree of peripheral
vascular disease in the lower limb. The complexity of diabetic foot ulcers necessitates an intrinsic knowledge of underlying pathophysiology and a multifactorial
approach in which aggressive management of infection is of major importance. An
infection in the diabetic foot is a limb threatening condition and has been the immeC 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
diate cause for amputation in 25–50% in subjects with diabetes in European studies
and is considered the most common cause for amputations in developing countries.
In almost 50% of cases there is a combination of infection and ischemia. Following
revascularization of neuroischemic feet in individuals with type I diabetes 26% of
amputations are related to infection. Control of infection in diabetic foot ulcers is a
tremendous challenge since a substantial number of infections do not react with substantial signs of inflammatory reaction and in chronic ulcers infection is usually polymicrobial. The absence of these symptoms and signs may be explained by factors
like impaired immunodefense, decreased peripheral perfusion, and poor metabolic
control. The basic concept in management is damage control—removal of
destroyed/devitalised tissue—improvement of tissue perfusion and wound management. An acute deep foot infection has to be considered as an emergency case usually requiring prompt empiric antibiotic therapy and foot surgery—incision and
debridement—to save a leg. There is a tremendous need for studies with regard to
diabetic foot infection. Present knowledge is hampered by the lack of prospective
studies with regard to microbiology, characteristics, management, and outcome.
ADVANCED SCAFFOLD FOR ADIPOSE TISSUE RECONSTRUCTION
J. Appelt1,2, T. Metcalfe1,2, G. Phillips2, Y. Martin1,2
Blond Mcindoe Research Foundation, East Grinstead, United Kingdom; 2The
Brighton Centre for Regenerative Medicine, School of Pharmacy &
Biomolecular Sciences, University of Brighton, Brighton, United Kingdom
1
Introduction: The loss of subcutaneous adipose tissue due to removal of tumors,
congenital malformations, deep burns, or trauma can have a severe disfiguring
impact on the normal body contour. This can leave patients distressed both physically
and emotionally. Current clinical treatment methods applied to replace lost adipose
tissue often fail to restore the natural body contour. We present a gelatin scaffold
combined with an extracellular matrix environment which supports adipogenesis.
Methods: Gelatin scaffolds were created using particulate leaching of alginate
beads as templates for the macroporous structure. The microporous structure
within the gelatin walls was altered through the application of 2808C and 2208C
freezing and freeze drying. The viability and distribution of adipose-derived stem
cells (ADSCs) within the scaffolds was assessed. Type I collagen and laminin
were combined to create an extracellular matrix environment within the scaffold.
After culture, adipogenesis was investigated by assessing gene expression.
Results: The scaffolds supported ADSC viability and displayed different cell distribution within the porous scaffold. Thus, the freezing techniques can be used to control
the scaffold architecture and, therefore, cell distribution. ADSCs delivered in the scaffold with a collagen I/laminin hydrogel showed increased adipogenic gene expression.
Conclusions: The integration of a natural adipose environment within the scaffold resulted in a composite scaffold with features that can support adipose tissue reconstruction.
FIBROBLAST-SPECIFIC STAT3 SIGNALING OF IL-10 MEDIATES
REGENERATIVE WOUND HEALING
S. Balaji1, N. Han1, C. Moles1, S. Bhattacharya1, P. Bollyky2,
T. Crombleholme3, S. Keswani4
1
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH USA; 2Stanford
School of Medicine, Palo Alto, CA USA; 3Children’s Hospital Colorado,
Aurora, CO USA; 4Texas Children’s Hospital, Houston, TX USA
Introduction: Lentiviral-mediated interleukin (IL)-10 overexpression results in
fetal-type regenerative repair in postnatal wounds via a STAT3-dependent
increase in hyaluronan (HA). We hypothesized that IL-10’s regenerative effects
are dependent on fibroblast-specific STAT3 signaling. We further tested if
delivery of IL-10 in a clinically translatable HA-based hydrogel (HH10) can
result in regenerative healing in postnatal wounds.
Methods: Inducible STAT3 knockdown murine models which were fibroblastspecific (Col1a2-Cre), keratinocyte-specific (Krt14-Cre) and skin-specific (UBC-Cre)
received IL-10 (lentiviral-CMV-IL-10;106 T.U.), and 4 mm wounds were created and
evaluated at 28 days. In a gain-of-function experiment, syngeneic fibroblasts expressing IL-10 or GFP were transplanted into the wounds of skin-specific STAT3 knockout
mice. Efficacy of HH10 made of 2:1:1 (HA:collagen I:crosslinker) and IL-10 (800
ng/wound) was tested. A quantifiable histologic scar scoring scale was developed to
evaluate scar formation. p values were calculated by ANOVA/t-test.
Results: In C57BL/6J controls, lenti-IL-10 resulted in a significant attenuation
of scar compared to phosphate-buffered saline (p < 0.0001). Skin-specific
STAT3 knockdown resulted in loss of IL-10 effects on scar attenuation, suggesting that IL-10 effects are mediated via STAT3 signaling. Keratinocytespecific STAT3 knockout resulted in a partial attenuation of IL-10 effects
(p < 0.01), but fibroblast-specific STAT3 knockout abrogated IL-10 effects
(p 5 ns), suggesting that fibroblasts are primary mediators of IL-10’s effects.
Syngeneic transplant of fibroblasts overexpressing IL-10 resulted in a significant
scar reduction (p < 0.01), albeit less than the effect of lenti-IL-10 treatment.
HH10 was equally potent as viral over-expression of IL-10 in achieving scar
attenuation in C57BL/6J wounds.
A2
Abstracts
Conclusions: IL-10 effects on attenuating scar formation are primarily mediated
via STAT3 signaling in dermal fibroblasts. HH10 obviates some of the translatable
concerns with IL-10 gene therapy. The therapeutic benefits extend beyond the cosmetic benefit, and may apply to any disease characterized by excessive fibroplasia.
NUTRIENTS AND COLLAGEN SYNTHESIS
A. Barbul1
Division of General Surgery, Vanderbilt University School of Medicine,
Medical Center North, Nashville, TN USA
1
The close relationship between nutrition and nutrients and wound healing has
been appreciated since biblical times. Most commonly the deleterious effects of
nutritional deficiencies were recognized for their negative impact on the healing
process. More recently, it has become recognized that provision of certain
nutrients above and beyond levels required for nutritional sufficiency can have
a positive impact on wound healing, in particular collagen synthesis. Amino
acids such as arginine, ornithine, and possibly leucine; derivatives of amino
acids such as b-hydroxy-b-methyl butyrate; perhaps some trace minerals; and
antioxidants—all have been shown singly or in combination to have a positive
influence of collagen synthesis. A review of these nutrients and sources of such
nutrients will be provided.
WOUND FLUID ANALYSIS DEMONSTRATES SIGNIFICANT
ALTERATIONS IN PROTEIN ADHESION FINGERPRINTS ON
IMPLANT SURFACES
S. Barr1, E. Hill2, A. Bayat3
1
Plastic and Reconstructive Surgery Research, University of Manchester,
Manchester, United Kingdom; 2Computer Sciences, University of Manchester,
Manchester, United Kingdom; 3Institute of Inflammation and Repair, University
of Manchester, Manchester, United Kingdom
Introduction: Increasing numbers of women undergo breast implantation for
cosmetic and reconstructive (congenital and neoplastic) purposes. Contracture
of the fibrous capsule, which encases the implant, remains the most common
complication of implantation. Capsular contracture leads to significant pain,
poor cosmesis, and reoperation. The initial coating of proteins on implant surfaces has been shown to be an important determinant of implant biocompatibility.
The affinity of proteins to silicone implants is poorly understood.
Methods: Wound drain fluid was collected postsurgical mammoplasty. This fluid
was incubated on the surface of 13 commercially available implant surfaces.
Adsorbed protein was digested and eluted using an in situ technique, before they
were run on an LTQ-Orbitrap mass spectrometer. Proteins were identified using
the mascot platform and statistical analysis performed using Progenesis software.
Cell spreading and adhesion of breast-derived fibroblasts was investigated using
scanning electron microscopy and immunocytochemistry of actin and vinculin.
Results: 640 proteins were identified in the isolated wound drain fluid. A total
of 274 proteins were identified adsorbed onto implant surfaces and of these
46 proteins were found to be significantly (p ! 0.05) altered between implant
surfaces. Fibronectin and vitronectin demonstrated significant alterations in
abundance on certain implant surfaces. Both fibronectin and vitronectin play
major roles in regulating biocompatibility, through their action on cell spreading
and adhesion via focal adhesion formation. Cell spreading and focal adhesion
formation of breast-derived fibroblasts on implant surfaces corroborated these
results. In addition to the above finding, complement factors D, H, and C3 preferentially adsorbed to different implant surfaces (p ! 0.05), demonstrating an
innate potential for these implants to foster a pro-inflammatory environment.
Conclusions: We present for the first time a protein-based assay of breast implant/
wound fluid biocompatibility, offering a unique insight into the biological influence that breast implant surfaces can have on the in vitro wound healing response.
HUMAN SKIN MICROORGANISMS ARE DISTRIBUTED IN BIOFILM
AGGREGATES AT WOUND EDGES
L. Bay1, M. S. Ågren2,3, S. S. Poulsen4, K. S. A. Ghathian5, H. Calum5,
T. Bjarnsholt1,6
1
Costerton Biofilm Centre, Department of Immunology and Microbiology,
University of Copenhagen, Copenhagen N, Denmark; 2Copenhagen Wound
Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen
NV, Denmark; 3Digestive Disease Center, Bispebjerg Hospital, University of
Copenhagen, Copenhagen NV, Denmark; 4Endocrinology, Department of
Biomedical Science, University of Copenhagen, Copenhagen N, Denmark;
5
Department of Clinical Microbiology, Hvidovre Hospital, University of
Copenhagen, Hvidovre, Denmark; 6Department of Clinical Microbiology,
Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Introduction: The distribution of bacterial habitats on human skin has medical
relevance concerning infection and healing after skin injury. We hypothesized,
that microorganisms are heterogeneously distributed in the vicinity of wounds.
A3
The conventional method of detecting microorganisms is by cultivation of swab
samples which does not provide information of the local distribution. We have,
therefore, studied the distribution of microorganisms in biopsies from standardized human epidermal wounds and adjoining uninjured skin by fluorescence
microscopy.
Methods: Suction blisters (10 mm) were raised and de-roofed on the buttock of
24 healthy volunteers. Wounds were treated daily with sterile water and covered
with impervious polyurethane film dressing with central pad. On post-wounding
day 4, full-thickness skin biopsies (12 mm 3 3 mm) were obtained under local
anesthesia and fixed in 4% paraformaldehyde. Four micrometer sections were
cut of the paraffin-embedded tissue, and stained with hematoxylin-eosin in addition to universal and specific coagulase-negative staphylococci (CNS) peptide
nucleic acid (PNA)-fluorescence in situ hybridization (FISH) probes and diamidinophenylindole. The samples were examined using confocal laser scanning
microscopy (CLSM). Additionally, the imaging results were compared with
swab samples cultured from the 4-day-old wounds and adjacent skin.
Results: CNS was detected in 18 of 24 biopsies by PNA-FISH and CLSM. No
bacteria were detected by direct imaging in the remaining six biopsies. These
results represented 94% sensitivity and 83% specificity when compared with
the culture findings. The microorganisms were heterogeneously distributed as
biofilm aggregates in small niches of epidermis near the wound edges. In the
wounds, no bacteria were detected by direct imaging.
Conclusions: The microorganisms were distributed as biofilm aggregates in epidermis near the wound edges.
KELOID DISEASE: A CHALLENGING ENIGMATIC DISORDER WITH
UNRESOLVED LINKS BETWEEN EXAGGERATED REPAIR AND
QUASI-NEOPLASTIC TENDENCIES IN THE HUMAN SKIN
A. Bayat1
Institute of Inflammation and Repair, University of Manchester, Manchester,
United Kingdom
1
Some scars become symptomatic, disfiguring, and difficult to treat. These
abnormal raised dermal scars known as keloid scars are physically and psychosocially disabling. Their management is complex, costly, and long-term as therapy remains ill defined. Virtually, all clinical treatment options are plagued by
a high risk of recurrence. Keloids have a multifactorial origin involving an intricate interaction between both the genetic influence and the environment. Unlike
other scar types, keloids have quasi-neoplastic behavior and tendencies. Keloid
tissue has a characteristic hypoxic microenvironment, not too dissimilar to
many neoplastic lesions. Keloid fibroblasts demonstrate a highly proliferative,
invasive behavior, thought to resemble cancer cell bioenergetics. Keloids never
metastasize but tend to continue to remain locally infiltrative, which seems clinically aggressive as the lesion often invades healthy unaffected surrounding
skin. Keloids are unique to humans resulting in the absence of a useful animal
model. Hence, there is difficulty in better understanding the underlying mechanisms leading to the development of this enigmatic disorder. This talk will be
focus on the presenter’s extensive experience in clinical and scientific understanding of keloid pathobiology in relation to neoplasia.
DEVELOPMENT OF AN EX VIVO BURN METHOD TO STUDY
WOUND HEALING IN BURNS INFLICTED BY A NOVEL BURNING
DEVICE
K. Blom1, A. Persson1
Medibiome AB, M€
olndal, Sweden
1
Introduction: To establish a reproducible ex vivo burn model in explanted
human living skin using a novel burning device to evaluate wound healing in
burns up to 10 days.
Methods: Different burning devices were evaluated and prototypes were built.
These were connected to a soldering iron and a temperature regulator. Also a
temperature sensor was connected to the burning device to assess the actual
temperature by which the skin was exposed to. Burns were inflicted during 1 s
without any pressure. Macroscopic and histological evaluations were done to
assess the burns and the reproducibility of their creation in human living skin.
In addition, a wound healing study was performed and analyzed by histology.
Results: A new burning device in solid aluminum with the burning edge
0.5 mm wide and 11 cm long was developed and connected to a soldering iron
and temperature regulator. The burning device was designed to enable measurements of the exact temperature at the interface between the device and the skin
when burning. The skin surface temperature could be maintained at 150 6 18C.
Also, the burns could be inflicted reproducibly indicated by macroscopic and
microscopic assessments as well as by temperature measurements. Furthermore,
histological analyses revealed that blisters were created and that the wound
could be healed.
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Abstracts
Conclusions: This study demonstrates that reproducible burns can be inflicted
by a novel burning device and that wound healing processes can be evaluated
in burnt human explanted skin.
NEW PLASMA DEVICES FOR TREATING CONTAMINATED
WOUNDS
B. Boekema1, A. Sobota2, M. Vlig1, D. Guijt1, P. Smits2, G. Pemen2,
G. Kroesen2, M. Ulrich1,3, E. Middelkoop1,4
1
Association of Dutch Burns Centres, Beverwijk, The Netherlands; 2Technical
University Eindhoven, Eindhoven, The Netherlands; 3VU University Medical
Center, Amsterdam, The Netherlands; 4Department of Plastic, Reconstructive
and Hand Surgery, VU University Medical Center, Amsterdam, The
Netherlands
Introduction: Patients with extensive burns are susceptible to pathogens due to
their large open wounds and compromized immune system. Use of available
antimicrobials is hampered by poor tissue penetration or bacterial resistance.
Cold plasma might provide an alternative antimicrobial treatment. Cold plasma
is an ionized gas at atmospheric pressure and generates active, bactericidal particles, which can be well-controlled at a safe level for physiological application.
Methods: We developed and tested two configurations of cold plasma devices:
an Argon jet and a flexible dielectric barrier discharge (DBD), 2.5 cm in diameter, which can be applied as a plaster. Tests were conducted on bacteria, cultured cells and ex vivo human skin. Surviving bacteria were quantified, cellular
activity was measured, and epidermal outgrowth was measured microscopically.
Results: The plasma jet is highly efficient when used on bacteria in non-buffered
solutions. Maximum reduction was reached within 2 minutes of treatment for
both Pseudomonas aeruginosa and Staphylococcus aureus. Plasma treatment of
bacteria on intact skin was far less efficient. Treatment of in vitro burn wound
models for 8 minutes with plasma jet did not affect epithelialization, but bacterial
reduction was limited. DBD plasma treatment of bacteria on agar resulted in log
reductions of up to 8 in 1 minute. Treatment of bacteria in collagen matrix or on
intact skin resulted in high log reductions in 2 minutes. When using burn wound
models, high bacterial reductions were obtained with colonizing or biofilm phase
bacteria, after 6 minutes of treatment. DBD plasma did not affect fibroblast viability or the metabolic activity of skin biopsies. The effect of DBD plasma on
wound healing in the burn wound model is under investigation.
Conclusions: With the flexible plasma strip, we were able to quickly kill high
numbers of bacteria in skin, without affecting the viability of skin cells.
EXOME SEQUENCING STUDY OF KELOID DISORDER
A. Bowcock1, M. Tirgan2
1
National Heart and Lung Institute, Imperial College, London, United
Kingdom; 2Keloid Research Foundation, St. Luke’s Roosevelt Hospital Center,
Rockefeller University Hospital, New York, NY USA
Introduction: Keloid disorder (KD) is a chronic genetic skin disorder with a
very diverse phenotype. Keloidal lesions form in individuals who are genetically susceptible to the illness and they vary in shape, size, and location. KD
often runs in families. We hypothesized that the underlying genetics of KD may
be linked to mutations in one or more genes.
Methods: This Institutional Review Board-approved study was undertaken to
conduct exome sequencing on DNA obtained from circulating nucleated cells in
peripheral blood in 65 subjects among 34 families.
Results: Numerous mutations were detected among several subjects, and some
found among subjects from different families.
TYPE III COLLAGEN REGULATES STROMAL RESPONSE IN
WOUND HEALING AND TUMOR MICROENVIRONMENTS
THROUGH ITS EFFECT ON MECHANOTRANSDUCTION
B. Brisson1, J. D. H. Han2, R. Kent III2, R. Wells3, S. Volk1
University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA
USA; 2University of Pennsylvania School of Engineering and Applied Science,
Philadelphia, PA USA; 3University of Pennsylvania School of Medicine,
Philadelphia, PA USA
1
Introduction: Collagen deposition and remodeling plays a key role in wound
healing, chronic fibrosis, and cancer development and progression. We previously identified roles for type III collagen (Col3) in promoting a regenerative
wound healing response and in suppressing a tumor permissive microenvironment. We hypothesized that Col3 suppresses development of a pathologic
matrix in vivo and that this effect is due in part to its ability to disrupt longrange force transmission between cells.
Methods: Orthotopic injection of mammary carcinoma cells (murine 4T1 and
human MDA-MB-231), in the presence and absence of exogenous Col3 (2 mg/
mL), was performed in syngeneic BalbC or Nod-Scid-Gamma mice. Primary
tumor growth was assessed two to three times weekly and collagen deposition
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Wound Rep Reg (2015) 23 A1–A37 V
and organization was assessed in histological samples. Long-range force transmission in collagen matrices was analyzed using thick collagen gels, with collagen alignment and compaction visualized by collagen second harmonics
generation (SHG) imaging.
Results: Primary tumor growth in orthotopic mouse models was attenuated in
the presence of exogenous Col3 (p < 0.05). Addition of Col3 to MDA-MB-231
cells at the time of injection reduced collagen deposition in tumors to approximately 50% of control tumors (cells injected with vehicle alone) and caused a
30% reduction in the frequency of Tumor Associated Collagen Signature-3
(TAC-3), a collagen organization signature associated with metastasis. In the
SHG in vitro studies, we found that NIH3T3 fibroblast aggregates cultured on
1.5 mg/mL gels of type I collagen (Col1) or Col1:Col3 mixtures for 24 hours,
then imaged for SHG, showed a significant decrease in collagen compaction
and alignment between aggregates and a decrease in compaction and directionality of collagen in the presence of Col3 plus Col1 compared to Col1 alone,
regardless of aggregate size.
Conclusions: Col3 plays a critical role in matrix deposition and organization.
Modulation of Col3 levels in both wound healing and tumor microenvironments
may provide novel therapies to reduce scar formation and cancer progression.
INVESTIGATION OF THE BACTERIAL DIVERSITY AND
THERAPEUTIC PROPERTIES OF LACTIC ACID BACTERIAL
SYMBIONTS IN VENOUS INSUFFICIENCY ULCERS
E. Butler1, C. Lindholm2, T. Olofsson1, R. Oien3, R. Jelnes4, H. Andersson5,
B. Nilson6, A. Vasquez1
1
Department of Laboratory Medicine Lund, Section of Medical Microbiology,
Lund University, Lund, Sweden; 2Sophiahemmet University, Stockholm,
Sweden; 3Blekinge Wound Healing Centre, Blekinge Hospital, Karlshamn,
Sweden; 4Wound Healing Clinic, Sygehus Sønderjylland, Sønderborg,
Denmark; 5Infektionskliniken, Danderyds Sjukhus, Danderyd, Sweden; 6Clinical
Microbiology, Labmedicin, Region Skåne, Lund University, Lund, Sweden
Introduction: Venous insufficiency ulcers are a significant burden on the
healthcare system across the world and account for over 50% of diagnosed leg
ulcers in the US alone. The discovery of safe ecological treatments for the management of these ulcers has become import in the research. A novel symbiotic
flora was discovered within the honey stomach of the honeybee that seems to
be effective on human and animal wound pathogens including some antibiotic
resistant strains. These symbionts composed of 13 species of Lactobacillus and
Bifidobacterium (LAB) are involved in protection of the honeybee and are present in fresh honey. These symbionts produce a wide variety of proteins and
antimicrobial substances including organic acids and hydrogen peroxide which
contribute to their activity on pathogens. The purpose of this study was to
investigate the healing effect of a standardized topical mixture of Swedish
heather honey and the viable 13 LAB symbionts on venous leg ulcers.
Methods: Fifteen patients with venous leg ulcers from Swedish and Danish
wound healing centers were studied over a 6-week period on the diversity of
bacteria in the wounds.
Results: We detected that this LAB and honey treatment had reduced the wound
areas, increased epithelialization and decreased the bacterial load in some patients.
Through culture dependent and molecular techniques including DNA sequencing
and mass spectrometry, we identified the most common wound bacteria in the
patients. Staphylococcus was the most common genus. We observed that bacterial
diversity is patient specific and each patient had a unique wound flora.
Conclusions: We believe that with further studies this novel mixture of symbionts could be a standardized topical treatment for patients with hard-to-heal
venous leg ulcers.
ISOLATION AND DIFFERENTIATION OF SATELLITE CELLS FROM
HEAD MUSCLES
P. C. Monroy1, F. Wagener1, J. Von den Hoff1
1
Orthodontic and Craniofacial Biology, Radboud Institute for Molecular Life
Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
Introduction: Surgical closure of a soft palate cleft often fails to allow normal
speech development. This is mainly due to fibrosis in the reconstructed muscles
of the levator veli palatini (LVP). Fibrosis may be prevented by the implantation of satellite cells (SCs) in the surgical wounds. The aim of this study was to
isolate SCs from three different head muscles and compare their functional differentiation in vitro.
Methods: SCs are isolated from the masseter (Ms), digastricus (Dig), and LVP
muscles of 5-week-old Wistar rats by enzymatic digestion. SCs (1,250) are
seeded onto 2-mm spot coatings of Matrigel. The SCs are cultured for 5, 7, and
10 days to monitor differentiation by immune staining (Pax7, MyoD, MyoG)
and myofiber formation. Data are analyzed by one-way ANOVA.
Results: At day 5, about 95% of all cells contain the SC marker Pax7 and the
activation marker MyoD. The fraction of Ms-SCs containing the differentiation
A4
Abstracts
marker MyoG is larger than that of the Dig-SCs and LVP-SCs (30% versus
3%) but increases to about 60% at day 7. At the end of culture (day 10), only
10% of all cells are Pax7 and MyoD positive. MyoG expression is also strongly
decreased. About 40% of the culture area is covered with mature myofibers for
all three cell types. Some of the myofibers show spontaneous contractions.
Conclusions: Highly potent SCs can be isolated from all three head muscles
and form functional myofibers in vitro. The Ms cells seem to differentiate earlier that the others but in the end all SCs form similar amounts of myofibers.
Ms-SCs might be most suitable for cleft palate therapy since biopsies from the
masseter muscle can be easily obtained. Further research focuses on suitable
hydrogels as a carrier for implantation of the SCs.
Reference
1. Carvajal Monroy PL, Grefte S, Kuijpers-Jagtman AM, Wagener FA, Von
den Hoff JW. Strategies to improve regeneration of the soft palate muscles after
cleft palate repair. Tissue Eng Part B Rev 2012; 18: 468-77.
EXPRESSION OF VISUAL AND NON-VISUAL PHOTOACCEPTORS IN
HUMAN DERMAL FIBROBLASTS: IMPLICATIONS FOR LIGHTBASED WOUND HEALING THERAPIES
I. Castellano1, N. E. Uzunbajakava2, O. Kamala1, B. Raafs2, G. Westgate1, V. A.
Botchkarev1, M. J. Thornton1
1
Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom;
2
Philips Research, High Tech Campus, Eindhoven, The Netherlands
Introduction: Light-based wound healing therapies have been developed, but
with inconsistent results. Until now, cytochrome c oxidase was proposed to be
the main photo-acceptor responsible for phototherapeutic effects of light on
skin. Although other photoreceptors, e.g., visual and non-visual opsins (OPN)
and circadian clock cryptochromes have been identified in several cell types
(1), their presence and function in human skin is not clear.
Methods: Characterization of photo-acceptor mRNA transcripts in female dermal fibroblasts (breast n 5 3; scalp n 5 2; face n 5 2; abdomen n 5 2) and
donor-matched facial keratinocytes was determined by qRT-PCR. Fibroblast
monolayers were scratched and changes in expression were compared between
non-wounded and wounded cultures. Protein expression was confirmed by
immunocytochemistry. Response of human skin to light was determined with an
ex vivo wound-healing model using female abdominal skin. Wounded skin samples (n 5 12) were cultured at the air–liquid interface and exposed to red or
blue light daily for 3–6 days.
Results: Human dermal fibroblasts all expressed cryptochromes 1 (CRY1) and 2
(CRY2); opsin 1-short wavelength (OPN1-SW), a cone opsin activated by blueviolet light; opsin 1-middle/long wavelength (OPN1-MLW); and the non-visual
opsin OPN3 (encephalopsin), which is highly expressed in brain. OPN2 (Rhodopsin [RHO]), absorbing green-blue light, was only expressed in dermal fibroblasts,
while OPN5, a UV sensitive photoreceptor, was only expressed in keratinocytes.
OPN4 (melanopsin) was not detected in either cell type. Scratched assays demonstrated that expression of RHO was immediately down regulated (after 15
minutes) following wounding and remained low for 36 hours (2). In the ex vivo
wound healing assay, exposure to light accelerated wound closure.
Conclusions: Cultured primary keratinocytes and fibroblasts express both visual
and non-visual photo-acceptors. Understanding the function and relationship of
these photo-acceptors will lead to the development of reliable light-based
wound healing therapies.
References
1. Haltaufderhyde K, Ozdeslik RN, Wicks NL, Najera JA, Oancea E. Opsin
expression in human epidermal skin. Photochem Photobiol 2015; 91: 117-23.
2. Pomari E, Dalla Valle L, Pertile P, Colombo L, Thornton MJ. Intracrine
sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts. FASEB J 2015; 29: 508-24.
REVASCULARIZATION OF AN IMMATURE TOOTH USING
PLATELET RICH FIBRIN—A CASE REPORT WITH 2 YEARS
FOLLOW-UP
A. Chandra1, C. Rathinavel1, A. Tikku1, P. Verma1
1
Faculty of Dental Sciences, K.G’s Medical University, Lucknow, India
Introduction: Trauma to anterior permanent teeth commonly occurs between
the age group of 8 and 12 years. This is one of the major causes for necrosis of
pulp leaving thin canal walls and open apex. Complete debridement of the canal
is difficult as the mechanical shaping makes the fragile wall susceptible to fracture. Achieving proper apical seal is most challenging due to the open apex (1).
Methods: The case describes revascularization of pulp in an immature tooth using
platelet rich fibrin (PRF). A 16-year-old female patient was presented with immature central incisor. Access cavity was prepared and the canal was disinfected with
triple antibiotic paste. PRF prepared from the patient’s blood, was filled in the canal
and mineral trioxide aggregate plug was given. The access cavity was sealed.
A5
Results: The lesion gradually reduced in size. This emphasized that the healing
of the apical periodontitis occurs if the canal is thoroughly instrumented and
disinfected (2). After 2 years of follow-up, the lesion healed completely.
Conclusions: The probable result may be repair rather than regeneration, but eventually the replacement of the pulp by vital tissue is better than replacement with biomaterials, in an immature permanent tooth. Long-term follow-up studies will make
regenerative procedure as a successful treatment option in the near future.
References
1. Gutmann JL, Saunders WP, Saunders EM, Nguyen L. A assessment of
the plastic Thermafil obturation technique. Part 1. Radiographic evaluation of
adaptation and placement. Int Endod J 1981; 14: 173-8.
2. Fabricius L, Dahl"en G, Sundqvist G, Happonen RP, M€
oller AJ. Influence of
residual bacteria on periapical tissue healing after chemomechanical treatment and root
filling of experimentally infected monkey teeth. Eur J Oral Sci 2006; 114: 278-85.
EPIGENETIC DYSREGULATION OF IGF2 EXPRESSION IS A
COMMON FEATURE OF PALMAR FASCIA FIBROSIS
D. Charles1, C. Raykha1, D. O’Gorman2
Roth Mcfarlane Hand and Upper Limb Centre, University of Western Ontario,
London, Canada; 2Lawson Health Research Institute, University of Western
Ontario, St. Joseph’s Hospital, London, Canada
1
Introduction: Palmar fibromatosis, or Dupuytren’s disease (DD), is the result
of abnormal palmar fascia repair. In addition to exhibiting biomarkers of benign
fibrosis, primary fibroblasts derived from fibrotic fascia exhibit biomarkers of
malignant tumors, such as increased expression of IGF2 (1), a gene that frequently exhibits loss of parent-of-origin specific allelic expression (genomic
imprinting) in tumors (2). We assessed the imprinted expression of IGF2 in
fibroblasts derived from patients with “informative” IGF2 alleles (i.e., where
paternally derived and maternally derived alleles can be distinguished) to determine if loss of imprinting (LOI) of IGF2 was evident in palmar fascia fibrosis.
Methods: Primary fibroblasts (DD cells) were derived from fibrotic palmar fascia of eight informative patients, adjacent, macroscopically uninvolved palmar
fascia in these patients (PF cells), and normal palmar fascia from three informative patients with normal palmar fascia (CT cells). Mono or bi-allelic expression
of IGF2 was correlated with total IGF2 transcript levels and with the expression
levels of H19 and WT1, syntenic genes with imprinted expression in normal
fibroblasts, using restriction mapping and quantitative PCR.
Results: Seven of eight DD cell cultures exhibited bi-allelic IGF2 expression,
indicating LOI, whereas all three CT cell cultures exhibited normal monoallelic (imprinted) IGF2 expression. Six of eight PF cell cultures exhibited LOI
of IGF2. LOI of IGF2 was directly correlated with increased IGF2 expression
and inversely correlated with H19 expression in DD cells, but not in PF cells.
The detectable expression of WT1 was consistently restricted to DD cells.
Conclusions: These findings imply links between abnormal palmar fascia repair,
LOI of IGF2 and dysregulated expression of IGF2, H19, and WT1. These findings are consistent with the pathogenesis of palmar fascia fibrosis and malignant
tumors sharing similar epigenetic mechanisms.
References
1. Raykha C, Crawford J, Gan BS, Fu P, Bach LA, O’Gorman DB. IGF-II
and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren’s disease. Biochim Biophys Acta 2013; 1832: 1511-9.
2. Cui H. Loss of imprinting of IGF2 as an epigenetic marker for the risk of
human cancer. Dis Markers 2007; 23: 105-12.
CHANGING PERSPECTIVES OF THE CLINICAL IMPACT OF BUGS
ON WOUNDS
R. Cooper1
Cardiff Metropolitan University, Cardiff, Wales, United Kingdom
1
The routine microbiological investigation of clinical samples collected from
wounds has been achieved by traditional culturing techniques for more than a century. It has provided valuable information about the presence of wound pathogens
and their antibiotic susceptibilities, but limited information about their impact on
wound healing. Within the past 10 years, sophisticated molecular approaches have
started to yield insight into the diversity of microbial species, their communal
associations, their distribution patterns and their metabolic activity within the
vicinity of the wound. The relevance of the wound microbiomes to clinical outcomes is starting to be unravelled. The potential of these newer advances will be
illustrated by reviewing studies that explore how healing in chronic wounds is
affected by microbes and how clinical practice might be influenced.
URGOK2 A NEW FORM OF COMPRESSION AND SORBACT AN
ADVANCED WOUND DRESSING IN VENOUS ULCER TREATMENT
T. Coppin1, L. Libert1, M. Humez1, H. Fortey1, A. Pieronne1
Centre Hospitalier de Douai Route de Cambrai, Douai cedex, France
1
A venous ulcer is an open skin lesion affected by venous hypertension. The
treatment is debridement, compression and primarily treatment of venous
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
hypertension. Noninfected venous ulcers are usually colonized by multiple
micro-organisms and have to be treated without the routine use of topical
antimicrobial-containing dressing. We present our experience with a new antibacterial dressing and a new form of compression for venous leg ulcers. Sorbact
action is the production of hydrophobic bond between fatty acid of the compress and bacteria. When the dressing is changed it reduces the level of bacteria
and improves the healing. UrgoK2 has been used for therapeutic pressure
because compression is important to heal a venous ulcer. Two women with
large venous leg ulcers have been treated by Sorbact and UrgoK2. The two
patients presented venous hypertension due to saphenous reflux, but they
refused to be operated. Different types of dressing such as foams, alginates and
specialty dressings have been used without any ulcer improvement. We use Sorbact and UrgoK2 for venous leg ulcers with good results. The synergy between
the dressing and the compression has improved the healing of venous ulcer.
ASPIRIN TOPIC TREATMENT IMPROVES CUTANEOUS WOUND
HEALING IN DIABETIC MICE THROUGH LIPOXYGENASEDEPENDENT PRODUCTION OF PRO-RESOLVING LIPID MEDIATOR
patients; group B and C patients had 1 of 24 (4.1 7%) erythema. In control
group patients 6/25(24%) developed erythema.
Conclusions: We evaluated the effectiveness of the hydrocolloid dressings and
ceramide containing dressings in which ceramide containing dressings showed
effective prevention and water holding capacity. Our study highlights that ceramide containing dressings found to be more effective in reducing erythema
and improving the healing of PUs.
MODULATORY EFFECTS OF NOVEL EPOXY-TIGLIANE
PHARMACEUTICALS ON DERMAL FIBROBLAST-MYOFIBROBLAST
WOUND HEALING RESPONSES MEDIATE THEIR ENHANCED ANTISCARRING PROPERTIES
J. Dally1, R. Moses1, A. Midgley1, R. Howard-Jones1, R. Errington 2,
P. Reddell3, R. Steadman1, R. Moseley1
1
Cardiff Institute of Tissue Engineering and Repair; Cardiff University, Cardiff,
United Kingdom; 2Institute of Cancer & Genetics; Cardiff University, Cardiff,
United Kingdom; 3Qbiotics Limited, Yungaburra, Australia
Introduction: Healing disorders are one of the leading causes of morbidity and
mortality of diabetic patients. The understanding of the deregulation of cellular
and molecular mechanisms of the wound healing process is a key issue for the
development of new therapeutic strategies. Delayed healing in diabetic patients
is due to dysregulations of the inflammatory process. The arrest of inflammatory phase, which involves the recruitment of polarized M2 macrophages, is a
key step in the resolution of inflammation and tissue repair. Our hypothesis was
that deregulation of diabetic wound healing was the result of an impairment in
the polarization of macrophages.
Methods: We developed a new full-thickness wound model in type 2 diabetic
mice (in high fat diet or db/db mice) associated with the development of an
innovative device to treat the wound, follow its evolution and characterized
phenotypically and functionally cells within the exsudates.
Results: We demonstrated that the severe impairment of healing in diabetic
mice is correlated to a strong neutrophil and inflammatory macrophage M1
infiltration, a lack of influx of anti-inflammatory macrophages M2, leading to a
failure in the inflammatory cells apoptosis and efferocytosis. We established a
decrease of lipoxin A4 (LXA4), a bioactive lipid from arachidonic acid (AA)
metabolism involved in the resolution of inflammation, and an increase of
potent chemotactic lipid leukotriene B4 (LTB4). We demonstrated that topical
daily application of acetylsalicylic acid on the wound, 3 days after the skin
lesion, orients the AA metabolism towards the synthesis of LXA4 at the
expense of LTB4, triggers the recruitment of M2-polarized macrophages, and
promotes apoptosis of neutrophils and their efferocytosis.
Conclusions: The resolution of inflammation by an original mechanism toward
the 12/15-LOX induction regardless of the COX-2 expression allows us to
envisage the development of a new therapeutic strategy to promote diabetic
wound healing.
Introduction: The novel epoxy-tiglianes, 12-tigloyl-13-(2-methylbutanoyl)-6,7epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (EBC-46) and 12-tigloyl-13-(2methylbutanoyl)-5,6-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (EBC-211),
occur within seeds of the Fontain’s Blushwood tree (Fontainea picrosperma), indigenous to Queensland’s tropical rainforest. In clinical studies, EBC-46 stimulates
exceptional dermal wound healing responses following tumor destruction, including
minimal scar formation. As transforming growth factor (TGF)-b1-driven, fibroblastmyofibroblast differentiation is pivotal to wound closure, contraction and scarring,
fibroblasts and myofibroblasts represent viable targets for the anti-scarring properties
of epoxy-tiglianes. Therefore, this study examined the effects of EBC-46 and EBC211 on dermal fibroblast proliferation, migration and TGF-b1-driven, myofibroblast
differentiation.
Methods: Human dermal fibroblasts were cultured in DMEM with EBC-46 or
EBC-211 (0 mg/mL or 0.001-100 mg/mL). Fibroblast proliferation and cell cycle
analysis were analysed by MTT assay and Draq5/FACS. Migration was assessed
using in vitro scratch wounds, time-lapse microscopy and ImageJ analysis.
TGF-b1-driven, fibroblast-myofibroblast differentiation was examined by the
immuno-cytochemical and qRT-PCR detection of a-smooth muscle actin (aSMA) expression and stress fibre formation.
Results: EBC-46 and EBC-211 induced significant fibroblast cytotoxicity at 100
mg/mL (p < 0.001). EBC-46 significantly retarded fibroblast proliferation at 24
hours (1 mg/mL), 120 hours (0.001 mg/mL and 1-10 mg/mL) and 168 hours
(0.001–10 mg/mL). EBC-211 also inhibited fibroblast proliferation at 24 hours
(10 mg/mL), 72 hours (0.1 mg/mL and 1–10 mg/mL), 120 hours (0.1 mg/mL and
1-10 mg/mL) and 168 hours (0.001 mg/mL and 0.1-10 mg/mL). Neither epoxytigliane exerted any effects on fibroblast migratory responses (p > 0.05).
Although EBC-46 had no effects on a-SMA expression, stress fibre organization
and myofibroblast formation at 0.001-0.01 mg/mL or 1-10 mg/mL, EBC-46 significantly inhibited a-SMA expression and stress fibre formation at 0.1 mg/mL,
with cells retaining normal fibroblastic morphologies. EBC-211 induced similar
myofibroblast inhibitory effects at 10 mg/mL.
Conclusions: These findings suggest that epoxy-tiglianes attenuate fibroblast
proliferation and TGF-b1-driven, myofibroblast differentiation, explaining the
enhanced anti-scarring responses observed in epoxy-tigliane-treated skin.
EVALUATION OF EFFECTIVENESS OF HYDROCOLLOID DRESSING
VS CERAMIDE CONTAINING DRESSING AGAINST PRESSURE
ULCERS
PYODERMA-LIKE CUTANEOUS ULCERATION: AN
UNDERESTIMATED SIDE-EFFECT OF TYROSINE KINASE
INHIBITORS
H. Daifeng1, G. Feng1, W. Chu1, Z. Chen1, S. Li1
1
Department of Wound Repair Center, Burn & Plastic Surgery, First Affiliated
Hospital of Chinese PLA General Hospital, Beijing, China
E. De Keyser1, H. Beele1
1
Department of Dermatology, Ghent University, Ghent, Belgium
C. Dardenne1,2, L. Lefevre1, E. Meunier1, M. A. Eddine1, J. Bernad1,
M. Bouschbacher2, A. Coste1, B. Pipy1,3
1
UPS, UMR 152 (PHARMA-DEV), Universit"e de Toulouse, Toulouse, France;
2
Laboratoires Urgo, Chenove, France; 3Universit"e Toulouse III Paul-Sabatier,
Toulouse, France
Introduction: Pressure ulcers (PUs) are often an indication of quality of nursing
care as it remains as severe manifestation of the integrity of impaired skin. Our
study designed to evaluate the effectiveness of hydrocolloid dressings and
ceramide dressings in healing of PU.
Methods: The study was done at Chinese hospital between February 2014 and
November 2014. Seventy-two study group patients and 25 control group
patients were included in the study. The study group patients were divided into
Group A with 24 patients receiving only ordinary hydrocolloid dressings, Group
B comprised 24 patients who received ceramide containing hydrocolloid dressings and Group C with 24 patients receiving both hydrocolloid and ceramide
dressings. Dressings were applied for 4 weeks. The dressings were changed
every 10 days. Erythema, pH, and hydration of the skin were measured.
Results: Among 72 study population 48 (66.7%) were male and 24 (33.3%)
were females. Group A comprised 18 (75%) males and 6 (25%) females, group
B 20 (83.3%) males and 4 (16.7%) females, and group C 10 (41.7%) males and
14 (58.3%) females. Twenty-five control group patients with 8 (32%) males and
16 (68%) females. Erythema was observed in 4 of 24 (16.67%) group A
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Introduction: We report the case of three patients who developed painful refractory leg ulceration(s) after the start of a tyrosine kinase inhibitor (TKI) for metastatic renal cell carcinoma. Two patients had been treated with sunitinib and the
third with pazopanib. The lesions improved after TKI discontinuation. The clinical aspect as well as the biopsy results (available for one patient) were suggestive
for pyoderma gangrenosum (PG). A systematic literature review was performed
to investigate the prevalence, etiopathogenesis and possible treatments of PG-like
cutaneous ulcerations during TKI treatment.
Methods: Pubmed and MEDLINE databases were searched between January 1,
2006, and April 1, 2015, for studies and case reports describing cutaneous ulceration and/or PG in people treated with TKIs.
Results: The search yielded thirteen case reports describing PG-like ulcerations in
patients treated with a TKI (sunitinib, n 5 8; pazopanib, n 5 1; imatinib, n 5 1; gefitinib, n 5 1; sorafenib, n 5 2). The majority of the patients developed ulceration(s)
on the lower limbs. The ulcerations appeared after a variable period of time after
the initiation of TKI therapy (ranging from 1 week to 18 months). All ulcers
improved or even disappeared after discontinuation of the TKI. In some cases, oral
and/or topical corticoids were administered.
A6
Abstracts
Conclusions: PG-like cutaneous ulceration is a probably underestimated side
effect of TKI therapy. The exact mechanism is not fully understood, but possibly due to the anti-angiogenic effect of vascular endothelial growth factor
receptor (VEGFR) blockade (in sunitinib, pazopanib, and sorafenib), there
might be a decreased blood supply to the skin, resulting in ischemia with subsequent infiltration of neutrophils and ulceration of the skin. Early diagnosis and
adequate treatment of this complication, especially in patients with advanced
cancer, is mandatory.
References
1. Ueharaguchi Y, Kabashima K, Sasahashi M, Matsuda A, Matubara K,
Matsui M. A case of pyoderma gangrenosum possibly associated with sunitinib
treatment. Int J Dermatol 2013; 52: 634-6.
2.Usui S, Otsuka A, Kaku Y, Dainichi T, Kabashima K. Pyoderma gangrenosum of the penis possibly associated with pazopanib treatment. J Eur Acad
Dermatol Venereol 2015 [Epub ahead of print].
SYNTHESIS AND CHARACTERIZATION OF FIBRIN AND
SYNTHETIC POLYMER BIODEGRADABLE BIOMATERIALS FOR
WOUND REPAIR.
M. Deneufchatel1, O. Fichet2, V. L. Garde3
1
Laboratoire de Physicochimie des Polymères (LPPI), Cergy Pontoise, France;
2
Equipe de Recherche sur les Relations Matrice Extracellulaire Cellules
(Errmece), University of Cergy-Pontoise, Cergy-Pontoise, France
Introduction: Innovative biomaterials created for wound healing have been
developed. These materials are based on fibrin gel, the poor mechanical properties of which are reinforced by using an interpenetrating polymer network (IPN)
architecture. A synthetic polymer, polyethylene oxide (PEO) is chosen as the
partner network. It is copolymerized with modified serum albumin to obtain
fully degradable materials.
Methods: Materials are synthetized as previously described (1). Mechanical characterization is performed by rheology. The biodegradability is verified toward
thermolysin. Cytocompatibility of human foreskin fibroblasts (CRL 2522) is
V
checked by a Live/Dead assay at different culture times. The cell morphology
and the extracellular matrix proteins are observed after immunostaining.
Results: IPNs are synthesized by mixing all reactants in the appropriate proportions and the mixture is placed at 378C for 1 hour. The gel times measured by
rheology are about 10 minutes or less. The materials are homogeneous, and
transparent. They exhibit sufficient mechanical properties (storage moduli G0 is
above 1.2 kPa) since they are self-supported and can easily be handled. Confocal microscopy analysis confirms that the fibrin network morphology is similar
in the IPN to physiological conditions. This morphology depends on the proportion of polymer. Biodegradability assessed by immersing the materials in a thermolysin solution shows that the materials can be either completely or partially
degraded. Materials’ biocompatibility was assessed by cultivating fibroblasts on
top of the materials. After 3 weeks, the viability was above 90% and a dense
extracellular matrix was produced.
Conclusions: We synthetized innovative self-supported biomaterials based on a
fibrin gel, which is a crucial actor in the wound healing process. The fibrin gel
was associated with PEO copolymerized with an enzymatically degradable protein, serum albumin. Such materials are biodegradable and biocompatible: the
cells are viable and retain their ability of producing an extracellular matrix.
Reference
1. Bidault L, Deneufchatel M, Hindi"e M, Vancaeyzeele C, Fichet O, Larreta-Garde V. Fibrin-based interpenetrating polymer network biomaterials with
tunable biodegradability. Polymer 2015; 62: 19-27.
BIOPHOTONIC THERAPY MINIMIZES SCARRING IN HUMAN SKIN
HEALING IN VIVO USING THE DERMAL FIBROTIC MOUSE MODEL
J. Ding1, Z. Ma1, Z. Zhu1, S. Alrobaie1, E. Devemy2, E. Tredget1
University of Alberta, Edmonton, Canada; 2KLOX Technologies Inc, Laval,
Quebec, Canada
1
Introduction: KLOX technology is a biophotonic-based therapy designed to
speed wound healing in chronic wounds, which uses a combination of medical
devices, a specific wavelength LED light with either a photo converter wound
gel or membrane both containing a fluorescent chromophore. We hypothesized
that the biophotonic therapy may promote wound healing and minimize scarring
in dermal fibrotic mouse model, in which the split thickness human skin transplanted to full thickness excision wounds on the back of nude mouse developed
a thickened, raised, contracted scar resembling human hypertrophic scars.
Methods: After 1 week postoperatively, wounds were treated with light alone,
or gel plus light, or membrane plus light twice a week and sham treatment. The
wounds were monitored by digital photography weekly before the animals were
euthanized 4 weeks after treatment and the excised xenografts were examined.
Results: Morphologically, there were no visible significant differences between
the groups grossly or in wound contraction measured by planimetery. By 4
weeks post-engraftment, significant reductions in scar thickness were measured
histologically in the gel plus light and membrane plus light treatment group as
compared to both the control and light-only treated groups, (1.35 6 0.07 mm,
1.35 6 0.08 mm versus 1.69 6 0.13 mm, 2.07 6 0.08 mm; p < 0.05) with
improvements in re-epithelialization. Morphological improvements in collagen
fiber bundles and orientation on Masson-trichrome staining were associated
with accelerated collagen remodeling in the gel plus LED lights and membrane
plus LED light treated groups versus the control and light-only groups (collagen
orientation index: 0.18 6 0.04, 0.21 6 0.06 versus 0.50 6 0.08, 0.52 6 0.08;
p < 0.05).
Conclusions: Biophotonic therapy appears to provide a therapeutic potential for
acceleration of wound healing and reduction of fibrosis in human fibroproliferative disorders such as hypertrophic scarring.
R
NORMAL AND PATHOLOGICAL SCARRING MECHANISMS
A. Desmoulière1
1
Department of Physiology, Faculty of Pharmacy, University of Limoges,
France
Normal tissue repair includes a number of overlapping phases. After the early
inflammatory step characterized by hemorrhage and clotting, during the development of the granulation tissue, fibroblasts invade the wound and commence
replacing the provisional matrix with a more mature wound matrix. As the
granulation tissue phase proceeds, fibroblasts begin showing a new phenotype
with prominent microfilament bundles. These typical myofibroblasts develop a
smooth muscle-like phenotype, and are responsible for wound contraction.
Lastly, in the resolution phase of healing, there is considerable loss of various
cell types including myofibroblasts, by apoptosis. The signal for this cell death
is unknown but may be related to modification in the mechanical properties of
the microenvironment. Interestingly, neurogenic inflammation may be
involved in excessive scarring. In addition, age-related modifications of
peripheral innervation could affect wound healing in elderly patients. This
underlines the roles of innervation during normal and pathological cutaneous
repair processes.
A7
ACHILLES TENDON HEALING, HOW DOES IT HEAL AND CAN WE
IMPROVE IT?
P. Eliasson1
1
Institute of Sports Medicine Copenhagen, University of Copenhagen,
Copenhagen, Denmark
Tendon healing after rupture is believed to occur through three overlapping
phases, similar to wound healing. The injury causes bleeding, platelet activation,
haematoma formation, and infiltration of inflammatory cells. The first initial
inflammatory response is followed by a proliferatory response with an increased
protein synthesis. The last phase is dominated by remodeling of the tissue
where a poor quality matrix is replaced by more organized, better quality
matrix, mainly type I collagen. But what do we actually know about Achilles
tendon healing in detail? Which cells are involved in the process and for how
long is this whole healing process going on? When is an Achilles tendon healed
after a rupture in patients? Finally, animal studies have shown that tendons can
be manipulated to heal faster, by adding different drugs, growth factors, loading
and microtrauma but how much has been shown to improve Achilles tendon
healing in patients?
DIRECT ADHESION OF MACROPHAGES PROMOTES
MYOFIBROBLAST DIFFERENTIATION BY ESTABLISHING A NICHE
OF ACTIVE TGF-b1
C. Elizabeth1, L. Monika1P. Pardis1, B. Stellar1, A. Kjetil1, B. Hinz1
Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group,
Faculty of Dentistry, University of Toronto, Toronto, Canada
1
Introduction: Lung fibrosis is characterized by the chronic co-existence of
macrophages (M/), which produce transforming growth factor (TGF)-b1, and
myofibroblasts, which are activated by TGF-b1 from different precursor cells.
Myofibroblasts secrete and contract collagenous ECM and apoptose once normal tissue repair is completed. Chronic activation of myofibroblasts leads to
accumulation of stiff scar tissue that obliterates organ function. The key mechanisms turning beneficial repair into destructive fibrosis are unclear. We propose
that direct binding of M/ to myofibroblasts retains both cell types and contributes to their persistent activation in fibrosis.
Methods: We generated populations of primary fibroblasts and myofibroblasts
from mouse lung explants and M/ with different polarizations from mouse
bone marrow.
Results: Among the different polarization types, pro-fibrotic M/2a produced
highest amounts of latent but not active TGF-b1. Direct co-culture of M/ and
fibroblasts induced active TGF-b1 production compared to segregated cultures
demonstrating the importance of close proximity of these two cell types in
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
cultivating a profibrotic environment. In attachment assays, M/2a always
adhered faster and stronger to myofibroblasts than to fibroblasts, indicating specific interaction of pro-fibrotic cell populations. We identified the adherens
junction protein cadherin-11 to be upregulated in both cell types during fibrogenesis and to mediate homotypic heterocellular attachment. In samples from a
mouse model of lung fibrosis induced by bleomycin and human fibrotic lung,
we demonstrate increasing formation of cadherin-11 junctions between M/ and
myofibroblasts in fibrotic foci with increasing severity of fibrosis.
Conclusions: Our results suggest that cadherin-11-mediated adhesion keeps
M/2a and myofibroblasts in close proximity to establish a pro-fibrotic microenvironment rich in active TGF-b1.
DYNAMICS AND CONSEQUENCES OF THE INFLAMMATORY
RESPONSE IN TISSUE REPAIR AND REGENERATION
S. A. Eming1
1
Department of Dermatology, University of Cologne, Cologne, Germany
Evidence in different model organisms indicates that the immune system is of
primary importance in determining the outcome of the repair response, including the extent of scarring as well as the restoration of organ structure and function. The functional relationship between repair, regeneration and the immune
response is complex and not completely understood. Indeed, there is evidence
for both negative and positive roles. To unravel the dual role of the immune
response in diverse repair mechanisms, we developed novel mouse models that
allowed conditional depletion of macrophages or myeloid cell-specific genes
during the sequential stages of the wound healing response. The presentation
will provide novel insights in mechanisms how macrophages exert distinct functions during the diverse phases of skin repair, which are critical for a timely
healing response. Our findings might be relevant for the development of novel
monocyte-based therapies in tissue repair.
ANTI-INFLAMMATORY EFFECT OF MILK FAT GLOBULES IN
BURNED SKIN
K. K. Fardoussi1, S. Alwasmi1, A. H. Mokresh1, S. Dibsi2
Department of Pathology, Al Baath University, Hama, Syria, Arab Republic;
2
Independent, M€onchengladbach, Germany
1
Introduction: Skin thermal burns are one of the most severe, long-term injuries
known in medicine which cause high morbidity and mortality rates among the
affected patients. Cells burn under the influence of heat and it leads to inflammation of the skin injury. Burn injuries produce both local effects at and around
the site of the injury as well as general responses involving the whole body.
The aim of this study was to evaluate whether topically applied milk fat globules (MFG) can inhibit the release of arachidonic acid metabolites after fullthickness skin burn.
Methods: A randomized, controlled study was conducted with 30 rabbits by following the guidelines and recommendations of the Institutional Animal Care
and Use Committee. Each rabbit was injured with one full-thickness burn of an
area of 3.8 cm2. The wounds (n 5 30) were randomized and topically treated
with 25% containing MFG cream (group 1, n 5 10), with saline as control
(group 2, n 5 10) and with a vehicle control cream base without MFG as placebo (group 3, n 5 10). Two hours after applying the treatment 2 cm2 skin biopsies were taken including dermis and epidermis. Enzyme-linked immunosorbent
assay was used to measure the arachidonic acid metabolites prostaglandin E2
(PGE2), thromboxane B2 (TXB2) and leukotriene B4 (LTB4).
Results: In the MFG group 1 the PGE2 accumulation fell roughly by half and
TXB2 and LTB4 accumulation were reduced by less than half compared to the
control group 2 and placebo group 3.
Conclusions: Topical treatment with MFG indicated inhibition of arachidonic
acid metabolites release such as PGE2, TXB2, and LTB4 in burned skin in
comparison to the control and placebo groups. Based on this study, MFG seems
to be a potential treatment for burn victims with anti-inflammatory effect.
MILK FAT GLOBULES THE TOPICAL TREATMENT OF DEEP BURN
WOUNDS
K. K. Fardoussi1, S. Alwasmi1, A. H. Mokresh1, N. Mansour1, S. Dibsi2
Department of Pathology, Al Baath University, Hama, Syria, Arab Republic;
2
Independent, M€onchengladbach, Germany
n 5 10) and with a vehicle control cream base without MFG as placebo (group 3,
n 5 10). Macroscopic and histologic analyses were performed.
Results: In the course of treatment scabbing occurred in all groups after seven
days which loosened up during further treatment. After 14 days areas of epithelialization were formed completely in group 2 and 3 after 28 days, whereas in
group 1 wound healing was caused by positive contraction. After 42 days of
treatment the wound surface in group 1 was reduced to 5-11% of the original
size, while this reduction was lower in group 2 (24–38%) and group 3 (28–
45%). The histological evaluation was performed after 42 days. The wounds
treated with MFG could restore the normal skin structure and granulation tissue
was less formed versus control and placebo group. In contrast, granulation tissue with densely packed fibroblasts, more basophilic and cellular structures was
clearly observed in group 2 and 3 compared to the treated MFG group 1.
Conclusions: As compared to silver sulfadiazine and placebo MFG showed a
faster and better wound closure. MFG inhibited hypertrophic scar formation and
showed progressive wound healing by contraction. Therefore, MFG might represent a useful treatment for patients with deep burn wounds.
ABERRANT FIBROBLAST DIFFERENTIATION TOWARDS
CARTILAGE AND BONE UNDERLIES HUMAN KELOIDS
nas1,2, N. Kunjravia1,
J. Fuentes-Duculan1, K. Bonifacio1, M. Su"arez-Fari~
M.Tirgan1,3, J. G. Krueger1
1
The Rockefeller University, New York, NY USA; 2Icahn School of Medicine
at Mount Sinai, New York, NY USA; 3Mount Sinai St Luke’s Roosevelt
Hospital, New York, NY USA
Introduction: Keloids are common, benign fibroproliferative tumors that occur
more frequently among African Americans with an incidence rate of 6-16%. To
date, the etiopathogenesis of keloid is not fully understood.
Methods: To better understand the pathogenesis of keloids, we performed transcriptional profiling of biopsies from extensive keloid lesion, adjacent nonlesional
skin (n 5 3) and newly formed keloid lesion of African Americans diagnosed
with keloids using Affymetrix HGU133 2.0 plus arrays. We also performed
immunohistochemistry staining to confirm protein expression of selected genes.
Results: We identified 1,202 upregulated and 961 downregulated differentially
expressed genes (DEG) between lesional keloid and nonlesional skin. There
were 1,819 upregulated and 1,867 down-regulated DEGs between newly formed
keloid and nonlesional skin, while 492 up-regulated and 775 downregulated
DEGs were identified between lesional skin and newly formed keloids (fold
change > 2, false discovery rate < 0.05). Most of the top up-regulated DEGs
between lesional and nonlesional skin are involved in bone/cartilage formation,
which includes FBN2 (fibrillin 2), COL10A1 (collagen type X a1), ASPN
(asporin), CILP2 (cartilage intermediate layer protein 2), CDH11 (cadherin 11),
and BMP1 (bone morphogenic protein 1). Many upregulated DEGs from the
comparison between the newly formed keloid lesion and nonlesional skin are
also related to cartilage or bone, i.e., COL10A1, SPP1 (Secreted phosphoprotein
1), FBN 2, ASPN, CDH11, RUNX2 (runt-related transcription factor 2), BMP1;
and nerve, i.e., NRP2 (neuropilin 2), NEFH (neurofilament). By Immunohistochemistry staining, we found increased protein expression of COL10, FBN2,
ASPN, CDH11, BMP1, SPP1, RUNX2, on lesional and newly formed keloid
skin compared to nonlesional skin.
Conclusions: Keloids are not simply “excess” scar tissue formed by increased
extracellular matrix molecules in the dermis. Keloids represent a dysplasia of
connective tissue towards immature cartilage/bone differentiation. The phenotype is potentially regulated by overexpression of RUNX2 or other transcription
factors. This knowledge may give insights to guide the development of better
treatment for the disease in the future.
EVIDENCE-BASED RECOMMENDATIONS FOR BURN SCAR
ASSESSMENT
U. Gankande1, J. Duke1, F. Wood1,2, P. L. Danielsen3, H. Wallace1
1
Burn Injury Research Unit, School of Surgery, University of Western
Australia, Perth, Australia; 2Burns Service of Western Australia, Fiona Stanley
Hospital and Princess Margaret Hospital for Children, Australia; 3Department of
Dermatology, Bispebjerg Hospital, University of Copenhagen, Copenhagen NV,
Denmark
1
Introduction: The aim of this study was to evaluate whether topically applied
milk fat globules (MFG) can improve wound healing and scar formation following deep burn trauma.
Methods: A randomized, controlled study was conducted with 15 pigs by following the guidelines and recommendations of the Institutional Animal Care and Use
Committee. Each pig was injured with 2 dorsal second-degree burns with an area
of 50 cm2. The wounds (n 5 30) were randomized and topically treated with 25%
containing MFG cream (group 1, n 5 10), with silver sulfadiazine (group 2,
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Introduction: Clinical burn scar assessment is hampered by a lack of evidence
to inform practice and the use of subjective assessment scales. We have published three clinical studies (1–3) generating evidence on the subjective modiR
fied Vancouver Scar Scale (mVSS) and the objective DermaLab ComboV
device (Cortex Technologies, Hadsund, Denmark) for measuring height, pliability vascularity and pigmentation of burn scars. The aim of this study was to
synthesize this evidence to formulate recommendations for clinical burn scar
assessment.
Methods: A critical synthesis of the evidence on inter-rater reliability (intraclass correlation coefficients [ICC] and weighted kappa statistic), test-retest
A8
Abstracts
reliability (ICC) and exploration of validity (classification of DermaLab
R scores based on mVSS reference standard) was undertaken. MeasureComboV
ment issues were also taken into consideration.
R measurements was
Results: Inter-rater reliability of the DermaLab ComboV
superior to most mVSS scar measurements (pigmentation, vascularity, height/
thickness) but test-retest reliability of vascularity was poor. Clinical interpretaR measurements is limited at present and some meastion of DermaLab ComboV
urements were not technically feasible in all scars.
Conclusions and recommendations: (1) A hybrid method of scar assessment
incorporating the most reliable and feasible measurements should be used. Based
R ; pliability—mVSS;
on current evidence: height—mVSS and DermaLab ComboV
R . (2) Individvascularity—mVSS; pigmentation—mVSS and DermaLab ComboV
ual scar parameter scores should be used rather than ‘total’ scores as they provide
information on underlying physiological processes. (3) Standardized scar assessment protocols should be established within burns facilities and utilized for training new scar assessors and supporting quality assurance programs. (4) Global
standardized burn scar assessment protocol(s) should be established for current
and emerging burn scar assessment methods to ensure compatibility of data for
R
clinical management and research. (5) Further testing of the DermaLab ComboV
in burn scar assessment using robust experimental design is essential to facilitate
translation into clinical burn scar assessment.
References
1. Gankande TU, Wood FM, Edgar DW, Duke JM, DeJong HM, Henderson
AE, Wallace HJ. A modified Vancouver Scar Scale linked with TBSA (mVSSTBSA): Inter-rater reliability of an innovative burn scar assessment method.
Burns 2013; 39: 1142-9.
2. Gankande TU, Duke JM, Danielsen PL, DeJong HM, Wood FM, Wallace
HJ. Reliability of scar assessments performed with an integrated skin testing
R . Burns 2014; 40: 1521-9.
device - the DermaLab ComboV
3. Gankande TU, Duke JM, Wood FM, Wallace HJ. Interpretation of the
R pigmentation and vascularity measurements in burn scar
DermaLab ComboV
assessment: an exploratory analysis. Burns 2015 [Epub ahead of print].
COMPLICATIONS 15 YEARS AFTER BREAST AUGMENTATION
WITH POLYACRYLAMIDE
H. Ghasemi1, T. Damsgaard1, L. B. Stolle2, B. A. O. Chistensen1
Department of Plastic Surgery, Aarhus University Hospital, Aarhus, Denmark;
2
Department of Plastic Surgery, Odense University Hospital, Odense, Denmark
1
Introduction: Polyacylamide hydrogel (PAAG) may be potentially dangerous
filler for breast augmentation, causing substantial irreversible damage to the
breast in healthy women. Methods and
Results: A 44-year-old, Ukrainian woman presented deformed, asymmetric and
painful left breast due to swelling, migration of PAAG, subcutaneous nodules
and fistulas. Fifteen years earlier, she had been treated with the permanent filler
PAAG for bilateral breast augmentation.
Conclusions: With the increasing interest and availability of fillers for cosmetic
use, it is to be expected, that complications of fillers will occur more often.
Therefore, it is important to gather all possible information about these serious
complications and their possible treatment.
References
Cheng NX, Wang YL, Wang JH, Zhang XM, Zhong H. Complications of
breast augmentation with injected hydrophilic polyacrylamide gel. Aesthetic
Plast Surg 2002; 26: 375-82.
Narins RS, Coleman WP 3rd, Rohrich R, Monheit G, Glogau R, Brandt F,
Bruce S, Colen L, Dayan S, Jackson I, Maas C, Rivkin A, Sclafani A, Spivak
JC. 12-Month controlled study in the United States of the safety and efficacy of
a permanent 2.5% polyacrylamide hydrogel soft-tissue filler. Dermatol Surg
2010; 36(3 Suppl): 1819-29.
Pallua N, Wolter TP. A 5-year assessment of safety and aesthetic results after
facial soft-tissue augmentation with polyacrylamide hydrogel (Aquamid): a prospective multicenter study of 251 patients. Plast Reconstr Surg 2010; 125: 1797-804.
Patlazhan G, Unukovych D, Pshenisnov K. Breast reconstruction and treatment algorithm for patients with complications after polyacrylamide gel injections: a 10-year experience. Aesthetic Plast Surg 2013; 37: 312-20.
Rauso R, Freda N, Parlato V, Gherardini G, Amore R, Tartaro G. Polyacrylamide gel injection for treatment of human immunodeficiency virus-associated
facial lipoatrophy: 18 months follow-up. Dermatol Surg 2011; 37: 1584-9.
BREAST TISSUE ENGINEERING
G. Giatsidis1, E. D. Venezia1, D. De Stefani2, R. Rizzuto2, F. Bassetto2
1
Clinic of Plastic Surgery, University of Padova, Padova, Italy; 2Department of
Biomedical Sciences, University of Padova and CNR Neuroscience Institute,
Padova, Italy
Introduction: Decellularization of tissues provides inductive extracellular matrices (ECM) for effective organ reconstruction: this promising approach has not
A9
been translated to breast reconstruction yet. We investigated effectiveness of
different decellularization protocols of porcine mammary glands with the purpose of prospective breast tissue engineering.
Methods: Porcine mammary glands were cut in homogeneous samples (10 3 10
3 2 cm) and processed according to three different decellularization protocols (A,
B, C) via multiple chemical treatments (A: 0.02% trypsin, 0.05% ethylenediaminetetraacetic acid [EDTA], 3% Triton X-100 and 4% deoxycholic acid; B: collagenase [3 mg/g], 0.02% trypsin, 0.05% EDTA and 10 U/mL lipase; C: collagenase
[3 mg/g], 0.05% EDTA, 4% sodium deoxycholate, 1% sodium dodecyl sulfate and
Tris-buffered saline [0.9% NaCl] with protease inhibitors). Obtained specimens
were analyzed by macroscopic (morphologic) and microscopic methods (hematoxylin-eosin, immunofluorescent labeling with 40 ,6-diamidino-2-phenylindole [DAPI],
quantitative measurement of DNA and DNA fragment size).
Results: Glands could be molded to required shape and adjacent glands could be
harvested together (up to 700 g). Size varied (average: 20 3 40 3 3 cm). Blood
supply was based on reliable vascular pedicles. Decellularization protocols had
variable effectiveness: all samples showed macroscopic evidence of decellularization preserving original morphology. DAPI, quantitative measurement of DNA
(below 50 ng/mg dry tissue weight) and of DNA fragment size (below 200 base
pairs) showed effective reduction of immunogenic components in each protocol.
Histological analysis showed that the morphology was more preserved using protocol A and the decellularized tissue resembled more closely the native architecture of ECM and vascular/ductal networks. After treatment according to protocols
B and C the tissues were slightly damaged and the structure was altered, defined
by the histological criteria.
Conclusions: Decellularization of porcine mammary tissue represents a novel
and reliable preliminary approach for breast tissue engineering.
THE USE OF MINIPIGS IN WOUND HEALING RESEARCH
P. Glerup1, C. Skytte1
CiToxLAB Scantox A/S, Lille Skensved, Denmark
1
Introduction: The minipig is considered the preferred animal species for testing
pharmaceuticals for dermal application due to its skin’s similarity with the
human skin with respect to structure and physiology. Minipigs are non-furred,
and their skin is firmly attached to the underlying structures and selectivelypermeable with a well-vascularized dermis, similar epidermal thickness and rete
ridge structure to human skin. In addition, the sensitivity of the pig skin is similar to human skin, whereas exacerbated reactions are often observed in the rabbit. In drug development, regulatory health authorities require that the most
appropriate animal species is used for non-clinical safety testing. Therefore, the
minipig is increasingly used as for dermatotoxicology testing including wound
healing studies and is accepted for this purpose by the authorities.
Methods: This presentation reviews our more than 20 years of experience on
the use of the minipig in wound healing research for efficacy and safety testing
and considers the requirements of specific guidelines. Comparisons are made to
other animal species that may be used in non-clinical wound healing studies.
The review will also cover scientific, technical, and practical aspects of wounding, wound treatment and bandaging procedures for split-thickness and fullthickness wounds, healing processes and parameters/methods used for evaluating wound healing.
Results: Wound healing studies in the minipig are mostly carried out to assess
tissue compatibility and effects on healing rates of different medical devices
and pharmaceuticals. In particular, wound contraction differs from loose skinned
animals such as the rat and mice.
Conclusions: The minipig constitutes many advantages compared to other animal species since the physiology and pathology closely resemble the healing
phases in humans.
BUGS IN WOUNDS: WHAT ARE THE PROBLEMS?
F. Gottrup1
1
Copenhagen Wound Healing Center, Department of Dermatology, Bispebjerg
Hospital, University of Copenhagen, Copenhagen, Denmark
Barriers for healing are bacteria, necrotic tissue, exudate, molecular, environment, cellular dysfunction (senescent, aged, and non-migrating cells). Bacteria
and infection are probably the most important determinants for delayed or nonhealing wounds (1). All chronic wounds are contaminated with microorganisms;
many are also colonized while only a small fraction of chronic wounds become
clinically infected. This depends on a balance between on one side microbial
factors (type, number, virulence, and resistance of bacteria and biofilm) and on
the other side the host defence mechanisms. The importance of the bacterial
load is demonstrated by the fact that low levels of bacteria (101 to 102 CFU/g
tissue) actually accelerate wound healing, while higher levels (105 to 108 CFU/
g tissue) delay or even block healing. Antimicrobial agents are common in
wound treatment but inappropriate use of them, especially antibiotics, creates an
environment for the selection of resistance. Future perspectives should
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
essentially focus to the following topics: What mechanisms control bugs in
wounds? What are the most relevant bug features especially in relation to development of resistance by misuse of antimicrobials? What is the impact of biofilms on wound healing?
Reference
1. Gottrup F, Apelqvist J, Bjarnsholt T, Cooper R, Moore Z, Peters EJ,
Probst S. EWMA document: Antimicrobials and non-healing wounds. Evidence,
controversies and suggestions. J Wound Care 2013; 22 (5 Suppl): S1-S89.
SURGICAL SITE INFECTION (SSI): MECHANISMS AND
RESPONSIBLE FACTORS
F. Gottrup1
1
Copenhagen Wound Healing Center, Department of Dermatology, Bispebjerg
Hospital, University of Copenhagen, Copenhagen, Denmark
Surgical site infection (SSI) is divided into: superficial, deep, and organ space.
Superficial SSI is defined according to Centre for Disease Control and Prevention
as: Occurring within 30 days; only involving skin or subcutaneous tissue; and
purulent drainage, isolation of microorganisms or clinical signs of infection. SSI
results from a change in the balance between on one side microbial conditions
and on the other side the host resistance mechanisms. Prevention of SSI primarily
includes: Preoperatively: prophylactic antibiotics, supplementary oxygen, avoid
smoking 6 weeks before surgery and optimal hygienic precautions; Peroperatively: correct surgical incision, minimize the duration of surgery, optimal suture
technique and use of surgical materials such as sutures, drains and dressings;
Postoperatively: optimal dressing selection, use of surgical materials and avoiding
wound fluid and hematoma. SSI is a major barrier for healing of surgical wounds.
Factors like tissue oxygen tension (1, 2) and smoking (3, 4) are important.
References
1. Gottrup F. Prevention of surgical-wound infections. N Engl J Med 2000;
342: 202-4.
2. Gottrup F. Oxygen in wound healing and infection. World J Surg 2004;
28: 312-15.
3. Sorensen LT, Karlsmark T, Gottrup F. Abstinence from smoking reduces
incisional wound infection: a randomized controlled trial. Ann Surg 2003; 238: 1-5.
4. Sørensen LT. Wound healing and infection in surgery: the pathophysiological impact of smoking, smoking cessation, and nicotine replacement therapy: a systematic review. Ann Surg 2012; 255: 1069-79.
WHAT IS THE DILEMMA BETWEEN SCIENCE AND CLINICAL
PRACTICE IN WOUND HEALING?
F. Gottrup1
Copenhagen Wound Healing Center, Department of Dermatology, Bispebjerg
Hospital, University of Copenhagen, Copenhagen, Denmark
1
Many of the procedures, methods, materials, and technologies used in wound
management are not evidence-based by randomized clinical trials and metaanalyses (1–3). Although basic science constantly provides us with new and exciting knowledge on wound healing biology very few of these discoveries are translated into clinical practice. This is a critical issue and the gap ought to be
reduced by increasing the exchange of knowledge and understanding of the working procedures in basic science and clinical life. Establishing evidence should be
a priority before implementatiing any new tecknology into the clinic.
References
1. Gottrup F, Apelqvist J, Price P; European Wound Management Association Patient Outcome Group. Outcomes in controlled and comparative studies
on non-healing wounds: recommendations to improve the quality of evidence in
wound management. J Wound Care 2010; 19: 237-68.
2. Gottrup F. Controversies in wound healing. Int J Low Extrem Wounds
2010; 9: 9.
3. Gottrup F, Apelqvist J. The challenge of using randomized trials in wound
healing. Br J Surg 2010; 97: 303-4.
COMPETENCE OF RETINAL PIGMENTED EPITHELIAL CELLS FOR
REPROGRAMMING IN NEURONAL DIRECTION IN THE PROCESS
OF RETINAL TISSUE REGENERATION IN THE NEWT
E. N. Grigoryan1
1
Koltzov Institute of Developmental Biology, RAS, Moscow, Russian
Federation
Introduction: Retinal pigment epithelial (RPE) cells of the adult newt are capable of reprogramming to all types of retinal neurons and glial cells, and that is
a basis of structurally and physiologically complete regeneration of that tissue.
Knowledge of molecular and cellular fundamentals of the process could help us
to understand how to induce it or at least its initial steps in mammals and
human for treatment of retinal degeneration of any kind.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Methods: We summarized our own and literature data on many features
of RPE cell biology which could be associated with their unique ability of
cell type conversion. Approaches of the studies included microsurgery,
morphological, immunohistochemical, PCR, BrdU, PCNA, radioautography,
and in vitro assays.
Results: As a result a combination of specialization (final differentiation) traits
with signs of low-differentiated cells was found for mature native RPE of the
newt. In particular, in RPE of that animal an expression of transcription factors,
signal molecules, and protein markers of progenitor cells were observed; and
vise versa—RPE cells making first steps on the way of reprogramming still
consisted of the key markers of specialization: melanin granules, RPE65 protein, and Otx2 gene expression. Besides, persistent low proliferative activity and
cytoskeleton specific protein replacement as well as “weak” cell contacts could
also contribute to successful reprogramming to neural differentiation. All
described factors of the competence to reprogramming can be found in animals
whose RPE cells are not capable of phenotype change to neural one in vivo,
but the range of them based on the permissive epigenetic state, possibly, is the
inner characteristic of the newt RPE.
Conclusions: We suggested it could be the result of evolutional fate of tailed
amphibians that underwent paedomorphosis and as a result kept expressed some
juvenile traits being mature.
Acknowledgment
This work was partially supported by Russian Fund for Foundation Research
(grant #14-04-00184a).
ALLEVIATING ISCHEMIA IN A MURINE MODEL OF DMD USING
VEGF AND ANG1
K. Gutpell1,2, J. Hadway2, L. Desjardins2, F. Su3, T.-Y. Lee2,3, L. Hoffman1,2
Western University, London, Canada; 2Lawson Health Research Institute,
London, Canada; 3Robarts Research Institute, London, Canada
1
Introduction: There is significant muscle ischemia and fibrosis in Duchenne
muscular dystrophy (DMD) patients, creating a microenvironment that is not
conducive to either endogenous muscle repair or regenerative strategies. Angiogenic therapy may improve the regenerative “niche” by alleviating these effects
(1). In the present study we sought to determine whether vascular endothelial
growth factor (VEGF), a potent inducer of angiogenesis, induces a fibrotic
response in fibroblasts derived from a murine model of DMD, as it has been
shown to do in a model of scleroderma (2). Second, this study investigates
whether VEGF alone or in combination with angiopoeitin-1 (Ang1) can enhance
muscle perfusion.
Methods: Quantitative polymerase chain reaction was employed to assess
changes in transcript expression of type 1 collagen (Col1a1) and connective tissue growth factor (Ctgf) following VEGF treatment in fibroblasts derived from
a murine model of DMD (mdx/utrn1/2 mice). Dynamic contrast-enhanced
computed tomography (DCE-CT) was used to determine if localized delivery of
VEGF of a combination of VEGF/Ang1 results in changes in blood flow and
blood volume. Sham-injected contralateral limbs were used as a control.
Results: Col1a1 and Ctgf levels did not increase following treatment with
VEGF in diaphragm or gastrocnemius fibroblasts derived from mdx/utrn1/2
mice. DCE-CT results indicate that blood flow and blood volume were significantly higher in mice that received VEGF alone versus mice that received a
combination of VEGF and Ang1 (p < 0.05). There was no significant difference
between right and left hind limbs in either the VEGF only group or the VEGF/
Ang1 group, suggesting that growth factor delivery was systemic rather than
localized.
Conclusions: The findings from this study support the use of VEGF as a treatment for DMD since it does not appear to stimulate a fibrotic response in fibroblasts derived from a murine model of DMD. Further, DCE-CT results suggest
a potential anti-inflammatory role for Ang1 that warrants further investigation
in future studies.
References
1. Ennen JP, Verma M, Asakura A. Vascular-targeted therapies for Duchenne muscular dystrophy. Skelet Muscle 2013; 3:9.
2. Maurer B, Distler A, Suliman YA, Gay RE, Michel BA, Gay S, Distler
JH, Distler O. Vascular endothelial growth factor aggravates fibrosis and vasculopathy in experimental models of systemic sclerosis. Ann Rheum Dis 2014;
73: 1880-7.
HOW TO MANAGE SCARS WITH LASERS
M. Haedersdal1
1
Department of Dermatology, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark
Patients with different types of scars raise a common concern in dermatology daily
practice. Advances in laser technology have led to substantial clinical improvement
and increasing body of evidence supports the use of light-based techniques in
A10
Abstracts
clinical management of scars. Several techniques are available to remodel skin texture, including pulsed dye laser, non-ablative and ablative fractional lasers, as well
as full ablative lasers. One of the new evolving areas in scar management is lightbased interference with the early wound healing process to minimize scar formation
and potentially induce scar prevention. This session will bring an update on available treatment techniques for different types of scars, focusing on the most recent
advances, which are achieved in the field of fractional lasers.
TOPICAL ERYTHROPOIETIN TREATMENT ACCELERATES THE
HEALING OF CUTANEOUS BURN WOUNDS IN DIABETIC PIGS
THROUGH AN AQUAPORIN-3 DEPENDENT MECHANISM
S. Hamed1, P. Liu2
1
Technion, Nazareth Illit, Israel; 2Department of Plastic Surgery, Rhode Island
Hospital/Brown Medical School, Providence, RI USA
Introduction: In diabetes mellitus, cutaneous wound healing is delayed due to
impaired angiogenesis and reduced cutaneous cellular activity. We have previously reported that the topical application of erythropoietin (EPO) to cutaneous
wounds in rats and mice with experimentally induced diabetes accelerates
wound healing by stimulating angiogenesis, epithelialization, and collagen deposition, and suppressing the inflammatory response and apoptosis. Aquaporin-3
(AQP3) is an integral membrane transporter of water in cutaneous cells and
facilitates epidermal cell migration and proliferation. We hypothesized that EPO
stimulates wound healing through AQP3 in diabetes.
Methods: Partial-thickness skin burns in pigs with experimentally induced type1 diabetes were treated with or without EPO.
Results: We found that topical EPO treatment of the burn wounds in the diabetic pigs accelerated their healing through an AQP3-dependent mechanism by
stimulating angiogenesis and ECM production. We also found that incorporating
fibronectin, a crucial constituent of the extracellular matrix, into the EPOcontaining topical gel, can potentiate the accelerating effect of EPO on the healing of burns in the diabetic pigs.
Conclusions: These findings indicate that EPO-induced acceleration of wound
healing in diabetic pigs is mediated by AQP3-dependent mechanism(s) that activate(s) angiogenesis by endothelial cells, collagen, and hyaluronic acid synthesis
by fibroblasts and epithelialization by keratinocytes.
A BIOACTIVE FRACTION, ISOLATED FROM AUSTRALIAN NATIVE
STINGLESS BEE (TETRAGONULA CARBONARIA) CERUMEN,
MODULATES DERMAL FIBROBLAST PROLIFERATION,
MIGRATION, AND DIFFERENTIATION IN VITRO
K. Hamilton1, R. Moseley2, R. Steadman2, P. Brooks3, S. Ogbourne3, F. Russell1
Inflammation and Healing Research Cluster, University of the Sunshine Coast,
Maroochydore DC, Australia; 2Cardiff Institute of Tissue Engineering and
Repair, Cardiff University, Cardiff, United Kingdom; 3Genecology Research
Centre; University of the Sunshine Coast, Maroochydore, Australia
1
Introduction: Cerumen produced by Tetragonula carbonaria represents a novel
Australian source of this plant-derived bee product. Following bioactivityguided fractionation, we isolated a fraction of T. carbonaria cerumen that scavenged free radicals and inhibited the pro-inflammatory 5-lipoxygenase signaling
pathway in vitro (1). Using cultured human fibroblasts derived from healthy
dermis (NF) and chronic wounds (CWF), we investigated additional bioactivities of the fraction on cellular responses implicated in normal and pathological
wound healing.
Methods: The bioactive fraction was collected from a methanol-water extract of
cerumen, obtained from 40 T. carbonaria hives located in South-East Queensland, Australia. The effects of the fraction on NF and CWF proliferation were
investigated using MTT assays. In vitro scratch assays and automated timelapse microscopy were used to examine NF migration over 48 hours. Gene and
protein expression of a-smooth muscle actin (a-SMA) in transforming growth
factor (TGF)-b1-stimulated NFs (10 ng/mL; 72 hours) were measured by quantitative reverse transcription polymerase chain reaction and immunocytochemistry, respectively.
Results: The cerumen fraction time- and dose-dependently stimulated NF
(214.6 6 26.4% versus dimethyl sulfoxide control; 3 mg/mL) and CWF proliferation (134.8 6 5.7% versus dimethyl sulfoxide control; 3 mg/mL) over 120 hours
(p < 0.05). NF migration was significantly increased after 48 hours exposure to
1 mg/mL fraction (p < 0.05). Fraction concentrations of 3–5 mg/mL inhibited NF
migration, and TGF-b1-induced a-SMA gene and protein expression (p < 0.05).
Conclusions: Our in vitro findings suggest that a bioactive fraction of T. carbonaria cerumen may promote the closure of acute and chronic wounds, by stimulating fibroblast proliferation during the early phases of wound healing. Its
inhibitory effects on fibroblast-myofibroblast differentiation may additionally
resolve the late, wound maturation phase of healing and prevent pathological
scarring. Chemical analyses of the bioactive fraction, to elucidate the structures
of its constituents, are on going.
A11
Reference
1. Hamilton KD, Russell FD, Brooks PR. Bioactivity-guided fractionation of
Australian native stingless bee (Tetragonula carbonaria) propolis extracts, based
on in vitro free radical-scavenging and 5-lipoxygenase activities. pA2 Online
2013; 11(3): 047P.
THE THROMBIN-DERIVED HOST DEFENSE PEPTIDE GKY25
INHIBITS ENDOTOXIN-INDUCED RESPONSES THROUGH
INTERACTIONS WITH LIPOPOLYSACCHARIDE AND
MACROPHAGES/MONOCYTES
omdahl1, M. Malmsten2,
F. Hansen1, M. Kalle1, M. van der Plas1, A.-C. Str€
M. M€
orgelin3, A. Schmidtchen1,4
1
Division of Dermatology and Venereology, Department of Clinical Sciences,
Lund University, Lund, Sweden; 2Department of Pharmacy, Uppsala University,
Uppsala, Sweden; 3Division of Infection Medicine, Department of Clinical
Sciences, Lund University, Lund, Sweden; 4LKC Medicine, Dermatology,
Nanyang Technological University, Singapore
Introduction: Host defense peptides have recently gained much interest as
novel anti-infectives owing to their ability to kill bacteria and simultaneously
modulate host cell responses. The cationic host defense peptide GKY25
(GKYGFYTHVFRLKKWIQKVIDQFGE), derived from the C terminus of
human thrombin, inhibits pro-inflammatory responses in vitro and in vivo, but
the mode of action is unclear. In this study, we show that GKY25, apart from
binding bacterial lipopolysaccharide (LPS), also interacts directly with monocytes and macrophages in vitro, ex vivo, and in vivo.
Methods: The effect of GKY25 on LPS-induced NF-jB activation was analyzed using specific reporter cell lines. To test whether GKY25 prevents Tolllike receptor (TLR)24 dimerization during LPS stimulation we performed
experiments using electron microscopy and flow cytometry analysis. The binding of GKY25 to monocytic cells in vitro, ex vivo and in vivo was visualized
by confocal microscopy and flow cytometry.
Results: GKY25 inhibits TLR-4 and TLR-2-induced NF-jB activation in
response to several microbe-derived agonists including LPS, LTA, zymosan and
peptidoglycan. Furthermore, GKY25 reduces LPS-induced phosphorylation of
MAPKs p38a and JNK1/2/3. Flow cytometry and microscopy analyses showed
that GKY25 interferes with TLR-4/myeloid differentiation protein-2 dimerization and binds to monocytic cells in vitro, ex vivo and in vivo.
Conclusions: The results demonstrate a previously undisclosed activity of the
host defense peptide GKY25, based on combined LPS and cell interactions
leading to inhibition of TLR-4 dimerization and subsequent reduction of NF-jB
activity and pro-inflammatory cytokine production in monocytes and macrophages. Thus, host defense peptides of thrombin may be interesting therapeutic
candidates for reduction of excessive inflammation and infection in various
clinical settings.
A BIO-HYBRID INJECTABLE SCAFFOLD IMPROVES HEALING
OUTCOME IN BOTH FIBROTIC AND NON-HEALING WOUND
ANIMAL MODELS
R. Hartwell1, R. Jalili1, M. Poormasjedi-Meibod1, B. Chan1, A. Ghahary1
1
University of British Columbia, Vancouver, Canada
Introduction: Burns and chronic wounds comprise nearly two-thirds of the
advanced wound care sector, which amounts to nearly $14 billion worldwide.
Rapid biological wound coverage can greatly benefit wound care, but as of yet,
has failed to overcome key tissue engineering hurdles, such as preparation time,
ease of use, and integration with the recipient tissue. Using previously approved
polymers our goal was to establish a biomimetic network that could function
with simple biochemistry in order to expedite the regulatory process, reduce treatment cost and easily gel within the wound, ultimately improving many unmet
needs of burn and wound care. Our hypothesis is that a hydrogel-containing scaffold will be able to rapidly integrate with the wound surface and provide a means
in which transplanted cells can remodel the environment.
Methods: In situ gelling scaffolds were fabricated by combining collagen with a
pH-sensitive hydrogel. For most treatments, collagen scaffolds with and without
hydrogels were compared against a solid support. Rabbits received full thickness
punch wounds in the ear and were filled with an in situ gelling scaffold, with and
without cells. Mice received a full thickness wound that was splinted to prevent
contracture. Scaffolds containing allogeneic cells expressed an immunomodulatory enzyme (Indoleamine-2,3-dioxygenase [IDO]).
Results: In situ gelling scaffolds, containing hydrogels, were non-toxic, exhibited significantly faster fibril formation than controls (p < 0.05). Hydrogel scaffolds demonstrated a greater mechanical strength, as well as resistance to
contracture and degradation (p < 0.05). Wounds treated with composite gel scaffolds in the presence and absence of IDO expressing cells showed a significant
reduction in scarring and increase in both innervation and vessel-like structures
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
compared to controls. Splinted wounds demonstrated a significantly faster time
to wound closure over controls (p < 0.05).
Conclusions: Collectively our data suggest that the hydrogel containing scaffolds showed to be a promising method to provide rapid, integrative wound
coverage that may improve treatment outcome. Ultimately, this method may
prove to be useful in resolving unmet needs within cellular transplantation of
other tissues.
SALMON-ROE DERIVED BIOLOGIC ACTIVES ACCELERATE
WOUND HEALING IN BURNS INFLICTED IN HUMAN EXPLANTED
SKIN
M. Heldrup1, G. Loenne1, D. Andrys2, C. Clemm1, K. Blom3, H. Bysell4,
H. Lund1
1
Regenics AS, Oslo, Norway; 2Department of Biotechnology, West Pomeranian
olndal,
University of Technology, Szczecin, Poland; 3Medibiome AB, M€
Sweden; 4SP Technical Institute, Stockholm, Sweden
Introduction: This study assessed the efficacy of two different formulations of
salmon roe extract (Solution V and H) on epithelialization in human ex vivo
wound models.
Methods: Two types of wounds, excisional and burn wounds, were made in donor
skin from one patient who had undergone abdominal reduction surgery. The excisional wounds (n 5 36) were inflicted using a sterile punch biopsy (3 mm diameter)
through epidermis to mid-dermis. Burn wounds (n 5 36) were inflicted using a aluminum device (0.5 mm wide) in contact with the skin for 1 second at a skin interface
temperature of 1508C. The wounded skin explants were excised with a 6 mm punch
biopsy knife and incubated (378C, 10% CO2/air) in medium containing 2% or 10%
fetal bovine serum (FBS) in the presence or absence of the two different salmon roe
extract formulations. Media with or without the two salmon roe extracts were
replaced every second day. All treatments were done in triplicate. After 5 and 10 days
of incubation, skin explants (three in each group) were fixed in formalin, paraffinembedded and tissue sections stained with hematoxylin eosin. Epithelialization was
assessed by quantitative light microscopy by blinded investigator.
Results: Both formulations accelerated wound healing of the burn wounds compared to 2% and 10% FBS alone. These effects were detected at both 5 and 10
days. Solution H appeared to be superior to Solution V. Complete epithelialization was only observed with added salmon roe extract. For the excisional
wounds, the effect of the two formulations on re-epithelialization was similar to
the medium alone controls.
Conclusions: The results of our study suggest that salmon roe extracts may
stimulate wound healing of second-degree burns. We cultivated the explants in
2% FBS to mimic the suboptimal conditions of hard-to-heal wounds. The
salmon roe extracts accelerated epithelialization under these conditions as well
as under the normal wound healing conditions represented by 10% FBS.
ALTERED COLLAGEN TURNOVER IN PATIENTS WITH HERNIAS
N. A. Henriksen1,2
1
Department of Gastroenterology, Koege Hospital, Koege, Denmark; 2Digestive
Disease Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen
NV, Denmark
Hernia formation is a multifactorial disease that involves endogenous factors
such as male gender, older age, anatomic variations and inheritance. Smoking is
an important exogenous factor associated with the formation of incisional
hernias and hernia recurrences. In tissue biopsies from hernia patients, the architecture of the collagen seems to be altered. In both fascia and skin, the ratio
between type I collagen and type III collagen is decreased in hernia patients
compared with hernia-free patients. It has been suggested that these collagen
alterations are linked to increased activity of matrix metalloproteinases.
Recently, serologic markers for type IV collagen were increased in patients
with hernias, whereas type V collagen turnover was decreased. Furthermore,
patients with rare connective tissue disorders and patients that are operated on
for abdominal aortic aneurysms have a high frequency of abdominal wall hernias than others. These findings suggest that the hernia disease may be systemic. Primary inguinal hernias appear to be linked to a systemic predisposition
to altered connective tissue, whereas impaired healing affects the formation of
incisional hernias and hernia recurrences.
COLLAGEN DEPOSTION AND MATRIX METALLOPROTEINASE-2
ACTIVITY DURING EARLY WOUND HEALING IN HERNIA PATIENTS
N. A. Henriksen1, L. T. Sørensen1, L. N. Jorgensen1, M. S. Ågren1,2
Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark; 2Copenhagen Wound Healing Center, Bispebjerg
Hospital, University of Copenhagen, Copenhagen NV, Denmark
1
Introduction: Altered collagen metabolism and impaired wound healing seem
to be associated with hernia formation. Matrix metalloproteinases (MMPs) are
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
important in remodeling of collagen during wound healing. In particular, MMP2 has been linked to the hernia disease. We have studied collagen and MMP-2
levels in early granulation tissue formed subcutaneously in patients with hernias
of three different etiologies and in control patients without hernias. Granulation
tissue was induced by the implantation of an empty porous (90-120 mm)
expanded polytetrafluoroethylene (ePTFE) tube that allowed for ingrowth of
cells synthesizing extracellular matrix components and proteinases.
Methods: Patients with primary inguinal hernia (n 5 15), three or more different
hernias (n 5 20) or an incisional hernia (n 5 23) were included. The hernia-fee
control group comprised patients subjected to laparoscopic elective surgery for
cholecystic stones (n 5 14). The 72 patients, 47 males and 25 postmenopausal
females, were median 63 years old (range: 40–92 years). During hernia repair or
cholecystectomy, one 8-cm long ePTFE tube was implanted subcutaneously in the
groin region and removed 10 days later. The level of hydroxyproline, as an indicator of collagen, was measured in delipidated and lyophilized ePTFE tube by standard colormetric assay. MMP-2 was analyzed in tissue extracts (0.5 mg proteins) of
R reagent per g wet weight) and rhMMP-2 (50 pg) by
ePTFE tube (20 mL T-PERV
gelatin zymography and subsequent densitometry (ImageJ).
Results: The ePTFE tubes contained 256 6 22 mg (geometric mean 6 SEM)
hydroxyproline and 350 6 42 ng MMP-2 per g ePTFE tube. There were no
significant differences in the hydroxyproline (p 5 0.717, one-way ANOVA) or
MMP-2 levels (p 5 0.690) in the ePTFE tubes among the four groups.
Conclusions: Collagen and MMP-2 levels in deposited granulation tissue did
not appear to be influenced by the hernia disease during early wound healing.
NEW LANDMARKS IN BASIC WOUND HEALING—A BRIEF
OVERVIEW
B. Hinz1
1
Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group,
Faculty of Dentistry, University of Toronto, Toronto, Canada
Cutaneous wound healing is the physiological response to skin injury and pathological healing continues to be one of the leading causes of morbidity and mortality. Patients suffering from diabetes, physical disablement, or vascular diseases
are prone to exhibit poor healing and develop chronic wounds. Conversely, if tissue remodeling continues after successful healing, it develops into excessive connective tissue deformations that are visible as keloids and hypertrophic scars. Both
myeloid cells and resident stromal cells are central effectors in normal, insufficient, and excessive wound healing. Good communication between these different cell types contributes to the re-establishment of tissue extracellular matrix
and homeostasis whereas communication breakdown is detrimental. The local
wound environment profoundly influences the healing success by controlling the
activity of all cells that are involved in the repair process. Hence, pharmacological as well as mechanical modulation of this environment bears significant
potential to improve the outcome of wound healing. I will briefly introduce the
factors leading to excessive healing and tissue contracture and provide an overview on possible strategies on how to improve the wound healing process.
Emphasis will be placed on molecular targets and/or agents that have been
shown to improve tissue repair in cell culture and animal models and that have
entered clinical trials.
LASER CAPTURE MICRODISSECTION AND MICROARRAY
ANALYSIS OF ACUTE SEQUENTIAL HUMAN CUTANEOUS WOUNDS
ENABLES DERMAL/EPIDERMAL PROFILING OF CHEMOKINE
EXPRESSION AND INNATE LYMPHOID CELL LOCALISATION
T. Hodgkinson1, N. Jumper2, M. Coles3, A. Bayat4
Manchester Institute of Biotechnology, University of Manchester, Manchester,
United Kingdom; 2Plastic and Reconstructive Surgery Research, University of
Manchester, Manchester, United Kingdom; 3University of York, York, United
Kingdom; 4Institute of Inflammation and Repair, University of Manchester,
Manchester, United Kingdom
1
Introduction: The complex and evolving nature of wound healing means that
the determination and localization of cell signaling remains challenging. Here,
we combined laser capture microdissection (LCM) with sequential human cutaneous wound biopsies to provide previously inaccessible epidermal/dermal gene
expression information. Using this, we aimed to focus on recently identified
RORC1 innate lymphoid cells (ILCs) and the cytokines associated with their
function through the healing process.
Methods: Healthy volunteers received two upper arm biopsies (5 mm) which
were re-biopsied at days 3, 7, 10, and 14 (7 mm). Tissue was snap frozen for
cry sectioning and microarray analysis. Wound cross-sections were immunohistologically (IHC) examined, focusing on ILC3 profiles. For microarray, timepoint cross-sections were separated into dermis and epidermis by LCM and
RNA extracted. Data was analyzed using Array Studio v7.2 (using criteria
p < 0.05; q < 0.05; fold change > 2) and enriched with Ingenuity Pathway
Analysis.
A12
Abstracts
Results: Microarrays showed significant differences between time-points and
dermal/epidermal expression. Dermally, chemokine analysis identified a significant late rise in interferon-c inducible chemokines, which could be linked to
wound resolution. Increases in the expression of ILC-linked recruitment factors
(CCL20, interleukin [IL]-23A) were observed at day 3 and subsequently
decreased. ILC3-linked effector expression showed initial upregulations of IL17F, IL-17A, IL-22, and CCL20 (p < 0.0001; q < 0.05) in the dermis, but no
corresponding epidermal expression. Depots of RORC1 ILCs were not located
in uninjured skin, with only rare cells observed. IHC showed populations
increased significantly at dermal wound peripheries, peaking at days 3-7 postwounding, corresponding with the expression of recruitment and effector chemokine expression profiles.
Conclusions: LCM allowed separation of dermal and epidermal gene expression
in a healing wound. Using LCM, ILC3-related gene expression and related factors
were found to be tightly regulated. This corresponded with increases in observed
RORC1 cells, indicating a role in skin healing with therapeutic potential.
FIBRIN MATRIX IMPROVES ANGIOGENESIS IN DIABETIC WOUND
HEALING IN A RAT MODEL
T. Hoppenbrouwers1, H. van Neck1, M. de Maat1, B. Tuk1, E. Fijneman1
Erasmus MC, Rotterdam, The Netherlands
1
Introduction: Fibrin is an important determinant of progression of the first
phase of wound healing. Fibrin enables migration of inflammatory cells, fibroblasts and endothelial cells to clear and restore the damaged tissue. The aim of
this study was to improve impaired wound healing in diabetic rats by applying
a fibrin matrix.
Methods: Fifty-four female WAG/RijCrl rats received an intraperitoneal injection of streptozotocin. After a diabetic state of 5 weeks, the animals received
two dorsal surgical full-thickness wounds. One wound was treated with a fibrin
matrix that was created by mixing 2 mg/mL human fibrinogen with 1 U/mL
human thrombin. The other wound was treated with phosphate-buffered saline.
After 7, 21, and 42 days the rats were killed. At days 7, 21, and 42 we performed wound area measurements. Also, at days 21 and 42, histology (hematoxylin-eosin stain), immunohistochemistry (CD68 scoring) and breaking strength
measurements of the wound area were carried out. Additionally, perfusion
measurements (02C and Laser Doppler) were performed weekly to day 42.
Results: Fibrin-treated wounds showed a significantly higher perfusion
(p < 0.05) compared to the control wounds on day 28 and day 35 based on 02C
measurements, suggesting improved angiogenesis. Moreover, oxygen saturation
and relative hemoglobin levels were significantly improved due to the addition
of fibrin over time. Vessel capacity measured by Laser Doppler perfusion did
not differ between control and treated groups. Wound area was significantly
smaller in fibrin-treated wounds on day 7. Also, the mean epidermal thickness
of fibrin-treated wounds was lower, although this difference did not reach statistical significance. CD68 scores and breaking strength of the wounded skin did
not show significant differences.
Conclusions: The application of a fibrin matrix improved perfusion and
decreased the wound area at early time points. CD68 immunostaining revealed
no additional immune response with a fibrin matrix.
HOST DEFENSE PEPTIDE REDUCES INFECTION AND
INFLAMMATION IN BIOMATERIALS
omdahl1, A. Schmidtchen1,2
L. Ignatowicz1, A.-C. Str€
Division of Dermatology and Venereology, Department of Clinical Sciences,
Lund University, Lund, Sweden; 2LKC Medicine, Dermatology, Nanyang
Technological University, Singapore
1
Introduction: There is a great and unmet need for new innovative products that
will reduce infection and inflammation in patients with burns or post-surgical
wounds, or in other situations when biomaterials or medical devices are used.
Current treatment concepts focus on materials or formulations with added components, such as antibiotics, silver, or various polymers. Today’s solutions
address only one of the issues at a time (such as infection), and there are no
products available that simultaneously tackle excessive infection-inflammation.
Lack of control over both of these aspects leads to various negative outcomes
in patients, such as delayed healing and risk of invasive infection and sepsis.
Methods: Methodologies used to study effects of biomaterial functionalization
include microbiological methods (radial diffusion assay, solution assays), inflammation models (NF-jB activation in human monocytic cells), in vitro release studies,
imaging studies (employing scanning electron microscopy and fluorescence microscopy), as well as in vivo models utilizing classical readouts as bacteria and cytokines,
but also IVIS bioimaging for tracking of NF-jB activation and bacterial spread.
Results: Our results demonstrate that coating with, or addition of a multifunctional host defense peptide to various medical products counteracts infectioninflammation in vitro and in vivo. Thus, such peptides should be of interest in
the further development of new biomaterials with combined antimicrobial and
anti-endotoxic functions for use in surgery and wound treatment.
A13
Conclusions: Endogenous host defense peptides that possess antibacterial
effects, in combination with reduction of inflammation via blocking responses
to endotoxins, as well as coagulation control, represent a novel platform for
development of new treatment concepts targeting infection-inflammation. Utilization of these peptides as a functional additive to implantable biomaterials and
wound dressings present a novel method of addressing both bacterial contamination and local excessive inflammation.
COMPARATIVE EFFECTIVENESS RESEARCH FOR CHRONIC
WOUND THERAPIES
R. Isseroff1
1
University of California-Davis and Department of Veterans Affairs Northern
California Health Care System, Sacramento, CA USA
The fundamental question that comparative effectiveness research (CER) aims
to answer, is: what treatment works best for a particular patient population, and
under what conditions. Currently, there is no consensus opinion on how to
design comparative studies for wound healing drugs, devices or technologies to
provide the most useful evidence for decision-makers. This presentation will
review current CER on chronic wound healing technologies, compare the roles
of the two different methods for CER: evidence synthesis versus evidence generation, analyze the need for real-world use effectiveness studies versus highly
controlled efficacy studies, discuss the potential for variability in specific elements of study design and generate discussion on how to best disseminate and
implement research conclusions.
CHARACTERIZATION OF INTERACTIONS BETWEEN A NOVEL
NANOCELLULOSE WOUND DRESSING AND THE WOUND
PATHOGENS STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS
AERUGINOSA
A. Jack1, H. Nordli2, L. Powell1, K. Hill1, G. Chinga-Carrasco3, B. Pukstad4, D.
Thomas1
1
Advanced Therapies Group, Cardiff School of Dentistry, Cardiff, United
Kingdom; 2Norges Teknisk-Naturvitenskapelige Universitet, Trondheim,
Norway; 3Paper and Fibre Research Institute, Trondheim, Norway; 4Trondheim
University Hospital, Trondheim, Norway
Introduction: Novel materials composed of nanocellulose wood-pulp fibers
derived from Pinus radiata, can be developed with a variety of physical properties
(tensile strength, ductility and absorbance) for utility in wound healing applications. This study characterizes microbial growth on nanocellulose dispersion films
and aerogels (TEMPO-mediated oxidized and homogenized, 33) with the common wound isolates Pseudomonas aeruginosa and Staphylococcus aureus.
Methods: Overnight microbial cultures were adjusted to OD600 0.08 in Mueller
Hinton broth and growth rates of P. aeruginosa PAO1 and S. aureus 1061A
monitored for 24 hours in nanocellulose suspensions. Films (air dried) or aerogels (freeze dried) incorporating nanocellulose (20 g/m2) were fabricated and
sterilized by c-irradiation. Establishment of wound biofilms on nanocellulose
materials was monitored using confocal and scanning electron microscopy and
quantified using COMSTAT image-analysis software. Production of P. aeruginosa PAO1 virulence factors (pyocyanin, rhamnolipids, elastase and protease)
in the presence of nanocellulose (2 3 2 cm) was also measured over 24 hours.
Results: Nanocellulose suspensions did not support the growth of either P. aeruginosa PAO1 or S. aureus in liquid culture. Instead, whilst no inhibition was
observed for P. aeruginosa PAO1, growth of S. aureus in vitro was reduced
compared to the broth control. Whilst biofilm formation was evident on all
materials tested, production of P. aeruginosa virulence factors was unaffected
by the nanocellulose materials (p > 0.05).
Conclusions: This study demonstrates the prospective utility of nanocellulose as
a wound dressing material due to its failure to support bacterial growth. As the
physical and chemical composition of nanocellulose is highly malleable, the
development of biocomposite dressings based on nanocellulose offers significant
clinical potential.
A COMPUTATIONAL MODEL OF THE DYNAMICS BETWEEN CELL
DAMAGE AND CELL REPAIR, IN THE PRESENCE OF OXIDATIVE
STRESS AND MECHANICAL DEFORMATION
N. S. Jagannathan1, A. Gefen2, L. Tucker-Kellogg1
Duke-Nus Medical School, Singapore; 2Department of Biomedical Engineering
Tel Aviv University, Tel Aviv, Israel
1
Introduction: During pressure ulcer formation, the amount and duration of
mechanical deformation necessary to kill cells is dependent on cell state. Meanwhile, oxidative stress may arise due to ischemia-reperfusion (1) or due to
necrotic debris (such as myoglobin from muscle cells (2) released in the extracellular environment (3). Our aim is to simulate system-level implications of
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
small changes in oxidative stress versus mechanical stress, in a system where
damage and repair are in dynamic competition.
Methods: We used computational modeling to perform a qualitative simulation of
the dynamics of cell stress, cell repair, and cell death among muscle cells. The following phenomena were explicitly represented: deformation increased cell permeability (4); repair decreased cell permeability; permeability caused myoglobin
release (3); ischemia-reperfusion caused oxidative stress (1); extracellular myoglobin caused oxidative stress (2); and oxidative stress decreased the rate of repair (5).
Results: After deformation was applied, time-course simulations showed a variable time of latency, with gradual accumulation of non-lethal cell stress, followed
by a rapid increase in cell death. The spatial spread of oxidative stress depended
on permeabilization of cells that were damaged but not killed by deformation.
When we included the ability of oxidative stress to slow the speed of cellular
repair, the result was a significant increase in vulnerability to mechanical deformation and greatly increased regions of cell death. The spatial propagation of cell
death could be prevented by rapid clearance of oxidative stress.
Conclusions: We conclude that the effects of oxidative stress and mechanical
injury can be greater than the sum of their parts. Furthermore, an ongoing race
between damage and repair allows small differences in cellular capacity to be
amplified over space and time.
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3. Makhsous M, Lin F, Pandya A, Pandya MS, Chadwick CC. Elevation in
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5. Howard AC, McNeil AK, McNeil PL. Promotion of plasma membrane
repair by vitamin E. Nat Commun 2011; 2: 597.
EPIDERMAL FRACTIONAL SKIN GRAFTING: AN EXPERIENCE IN
10 PATIENTS WITH TYPICAL AND ATYPICAL SKIN WOUNDS
A. Janowska1, V. Dini1, M. Macchia1, M. Romanelli1
1
Department of Dermatology, University of Pisa, Pisa, Italy
Introduction: The epidermal fractional skin grafting is a new treatment using a
harvesting automated system (1). The indications for this type of graft are acute
and chronic wounds presenting with a small amount of exudate, a good granulation tissue and a partial-thickness staging. This report is presenting our clinical
experience of 10 patients with hard-toheal wounds.
Methods: Ten patients (six females and four males) with a mean age 65.5 years
(range: 49–89 years) with typical and atypical skin wounds were treated with
the epidermal fractional skin grafting technique. The grafts were obtained with
a harvesting system that combines vacuum and heat and results in thin sections,
with constant orientation of epidermal skin from dermal-epidermal junction.
The donor area on the inner thigh was first cleansed with isopropyl alcohol and
then the vacuum head and harvester were applied for 30 minutes. The microdomes were harvested into micrografts through a non-adherent dressing, transferred to the recipient site and covered with an absorbent foam dressing.
Results: The follow-up was performed on days 7, 14, 21, and 28. Between days
7 and 14 we observed engraftment of the micrografts. At day 28, wound healing
was observed in 6 out of 10 patients (60%). Furthermore, a reduction in the
wound area of >50% was observed in 4 out of 10 patients (40%). Ten out of
10 ulcers (100%) showed an improved wound bed at 28 days.
Conclusions: Clinical improvement, the absence of pain during the procedure,
minimum amount of scarring on donor site and the simplicity of the technique
have shown that epidermal fractional skin grafting technique can provide a valid
alternative to manage hard to heal wounds.
Reference
1. Romanelli M, Dini V. Fractional epidermal skin grafting. Br J Dermatol
2015; 172: 853-4.
healing have been suggested. The purpose of this study was to assess the impact
of gender on cellular and biochemical processes during wound healing.
Methods: Healthy non-smoking volunteers (23 men and 23 premenopausal
women) and aged 18–40 years were included. In a suction blister model, blisters
(12 mm in diameter) were raised and the roof removed. The transepidermal
water loss (TEWL) was measured 2, 4, and 7 days after wounding. In a fullthickness punch biopsy model, the wound was excised after one week of healing and fixed in 10% formalin. Tissue was paraffin-embedded and sections
stained conventionally (hematoxilin eosin and Alcian blue) and immunohistochemically (angiogenesis [CD31], macrophages [CD68], procollagen-I [PINP]).
Two independent histopathologists measured wound dimensions and scored cellularity in the wound bed and periphery semi-quantitatively.
Results: Seven days after wounding the mean TEWL was 12.0 (range: 5.9–
36.1) g/m2/h in women and 14.7 (6.2-39.1) g/m2/h in men (non-significant).
The diameter of the biopsy wounds at day 7 was 3.52 6 0.78 mm (mean 6 SD)
in women and 3.72 6 0.54 mm in men (p 5 0.01). The corresponding wound
depths were 1.41 6 0.57 mm and 1.39 6 0.61 mm (non-significant). The histological assessment disclosed significant more macrophage cellularity in men’s
wound bed and significant more inflammation in men’s wound periphery. No
difference was found in angiogenesis, fibroblasts and procollagen-I.
Conclusions: After seven days of healing, men compared to premenopausal women
had wider wounds and more wound bed macrophage cellularity and more wound
periphery inflammation. Wound angiogenesis, fibroblasts and collagen synthesis as
well as TEWL indicating epidermal regeneration did not differ. These findings may
indicate different gender healing capacities.
INFECTION IN TRAUMA PATIENTS: PROGRESS IN OUTCOME
G. N. Jukema1
1
University Hospital Z€
urich, Z€
urich, Switzerland
Although today modern algorithms in trauma surgery are applied in patients
with wounds and soft tissue injuries, these soft tissue injuries in combination
with (open) fractures are still challenging to achieve a favorable clinical outcome. Many patients are still suffering from late consequences after severe
trauma for example osteomyelitis. Patients’ risk for infection after trauma is
related to several factors. First, the injury by itself will influence the risk of
infection (primarily open versus closed fractures). Also the body region of the
injury contributes to the infection risk profile. For example, fractures to the distal lower leg, such as pilon or calcar bone fractures, are associated with an
increased risk of infection where the bones are covered with less soft tissue.
Surgery on the pelvis is more at risk for infectious complications. Furthermore
co-morbidities will influence the outcome of fracture healing and soft tissue
regeneration. Diabetes, immunosuppressive medication, bleeding disorders and
older patients have a higher risk for unfavorable outcome on soft tissue regeneration and fracture healing. For improved wound healing of serious soft tissue
injuries, modern treatment with collagen matrices can support a faster and better
clinical outcome. Deep bone infections (osteomyelitis) can be faster and more
effective treated by a combination of debridement surgery with negative pressure wound therapy in combination with instillation technique of the foams
with an antiseptic agent (e.g., polyhexamethylene biguanide hydrochloride solution). Modern concepts in trauma surgery for open fractures and complicated
wounds can reduce long-term infectious complications and reduce the risk for
recurrence of posttraumatic osteomyelitis.
References
1. Osterhoff G, Zwolak P, Kr€
uger C, Wilzeck V, Simmen HP, Jukema GN.
Risk factors for prolonged treatment and hospital readmission in 280 cases of negative-pressure wound therapy. J Plast Reconstr Aesthet Surg 2014; 67: 629-33.
2. Timmers MS, Graafland N, Bernards AT, Nelissen RG, van Dissel JT,
Jukema GN. Negative pressure wound treatment with polyvinyl alcohol foam
and polyhexanide antiseptic solution instillation in posttraumatic osteomyelitis.
Wound Repair Regen 2009; 17: 278-86.
AN INNOVATIVE APPROACH TO DISSECTING KELOID DISEASE
LEADING TO IDENTIFICATION OF THE RETINOIC ACID PATHWAY
AS A POTENTIAL THERAPEUTIC TARGET
N. Jumper1, Y. Har-Shai4, G. Arscott5, R. Paus2,3, A. Bayat1,2
Plastic and Reconstructive Surgery Research, Manchester Institute of
Biotechnology, University of Manchester, Manchester, United Kingdom;
2
Institute of Inflammation and Repair, University of Manchester, Manchester,
United Kingdom; 3Laboratory for Hair Research and Regenerative Medicine,
Department of Dermatology, University of M€
unster, M€
unster, Germany; 4The
Unit of Plastic Surgery, Carmel Medical Center, Haifa, Israel; 5Department of
Plastic and Reconstructive Surgery, The University of West Indies, Kingston,
Jamaica
1
WIDER WOUNDS AND MORE INFLAMMATION AFTER SEVEN DAYS
OF HEALING IN MEN THAN PREMENOPAUSAL WOMEN
E. M. B. Jessen1, S. Ladelund2, L. T. Sørensen1
Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark; 2Clinical Research Centre, Hvidovre Hospital,
Hvidovre; University of Copenhagen, Copenhagen NV, Denmark
1
Introduction: Wound healing in men is attenuated compared to premenopausal
women and men have more postoperative wound complications. The mechanisms are unclear, but different properties by sex steroids to stimulate wound
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Introduction: Despite extensive research into keloid scarring etiopathogenesis, none
of the involved biomarkers has yielded an effective therapy to date. Retinoic acid
A14
Abstracts
(RA) has been implicated in key functions including cell cycle regulation, apoptosis,
cell differentiation and shown to affect pro-fibrotic factors, including transforming
growth factor-b. The aim here was to develop and test new hypotheses underlying
keloids by taking the novel approach of examining this entity from both site-specific
and dermo-epidermal perspectives, leading to targeted potential therapies.
Methods: Normal skin biopsies and keloid biopsies, harvested from the centre and
margin of the lesion, were cryosectioned and laser capture micro-dissected, where
the epidermis and dermis were separately collected and microarrayed (whole
genome array). The data was analyzed using Array Studio v7.2 and filtered using
the following criteria: p < 0.05, q < 0.05 and fold change " 2. The resultant list of
genes was enriched using Ingenuity Pathway Analysis and key pathways identified
and then validated using a combination of qPCR and Western blot analysis.
Results: The filtered, enriched data highlighted several members of the RA biosynthesis pathway as significantly dysregulated in keloid compared with normal skin.
The keloid epidermis revealed the reductase enzyme AKR1B10 to be significantly
upregulated (p 5 1.19E-06, fold change 75), a result confirmed with qRT-PCR and
western blot. Several other pathway members including ALDH1a1 (p 5 0.0008),
ADH7 (p 5 0.0011), CYP26B1 (p 5 0.0002), CRABP1 (p 5 0.0255), and CRABP2
(p 5 0.0279) were also dysregulated. AKR1B10 overexpression through transfection of normal keratinocytes demonstrates keloid-like expression in the RA pathway
and subsequent increased fibrotic gene expression in fibroblasts.
Conclusions: The manipulation of AKR1B10 to ascertain its effect on RA
activity and the consequent downstream events in keloid with regard to regulation of fibrosis represents compelling therapeutic potential.
DIFFERENCES IN HUMAN DERMAL FIBROBLASTS DERIVED FROM
DONOR-MATCHED TERMINAL AND VELLUS HAIR-BEARING SKIN:
IMPLICATIONS FOR WOUND HEALING
O. Kamala1, A. Graham1, M. J. Thornton1
1
Centre for Skin Sciences, School of Medical Sciences, University of Bradford,
Bradford, United Kingdom
Introduction: Superior healing occurs in skin containing terminal (T) growing
(anagen) hair follicles; but most human skin contains small vellus (V) resting
(telogen) follicles, following apoptotic-driven regression. Although inhibition of
X-linked inhibitor of apoptosis protein (XIAP) delays murine wound healing
(1), the role of specific IAPs in human skin is unknown. We compared IAP
expression in human scalp and adjacent facial skin of the same donors; matching dermal fibroblast (DF) cultures (DF[T]; DF[V]) were also established. Since
estradiol (E2) also improves wound healing (2), we sought to establish whether
it modulates IAP expression.
Methods: IAP expression was confirmed by qRT-PCR and immunohisto/cytochemistry. Cell size, complexity, proliferation, viability, migration, and caspase3 activity compared in matching DFs (same passage). Immunohistochemistry of
murine skin was included.
Results: All IAPs were expressed in human and murine epidermis, but only in
human dermis. In human skin expression was lower in vellus epidermis, which
was also thinner. Dermal T and V expression was similar. Overall IAP mRNA and
protein expression was higher in DF(V) cells. Mechanical scratching decreased
mRNA for cIAP2 and Apollon and while it decreased XIAP in DF(T), expression
was increased in DF(V). DF(V) cells were smaller, with a more granular cytoplasm and proliferated faster, but there was no difference in migration. Embelin, a
XIAP inhibitor reduced cell viability in scratched cells, which was abolished by
E2 in DF(T); E2 also reduced caspase 3 activity in DF(T). After 24 hours E2
increased expression of all IAPs in scratched DF(T) cells.
Conclusions: In contrast to mice, the IAPs XIAP, cIAP2, Apollon, and NIAP
are all expressed in the human dermis. DFs cultured from terminal or vellus
hair bearing skin of the same donors exhibit significant differences in vitro. E2
may modulate dermal healing via XIAP. Further studies are required to understand the relationship between E2 and IAPs in human cutaneous healing.
References
1. Fuchs Y, Brown S, Gorenc T, Rodriguez J, Fuchs E, Steller H. Sept4/ARTS
regulates stem cell apoptosis and skin regeneration. Science 2013; 341: 286-9.
2. Thornton MJ. Estrogens and aging skin. Dermatoendocrinology 2013; 5:
264-70.
HOW CAN SCIENCE BE INTEGRATED INTO CLINICAL PRACTICE:
COMPARATIVE EFFECTIVENESS RESEARCH
R. Kirsner1
1
University of Miami Hospital Wound Center, University of Miami Miller
School of Medicine, Miami, FL USA
Ideas from science drive clinical care. Often time to validate clinically applicable idea, efficacy trials are carried out to test whether an idea such as an intervention works in an idealized setting like a clinical trial. What may be more
important is if products are effective, that is do they work in real life practice.
Effectiveness research is probably more important to payers than efficacy
A15
research as it answers the question ‘does’ an intervention work as opposed to
‘can’ an intervention work. When two products are compared in a real life setting this is called comparative effectiveness research. Trial design often differs
in effectiveness research often using clinical databases or pragmatic designs.
Comparative effectiveness data may help determine which intervention works
for which patient and when or help clinicians any payers decide which product
is superior in real life. Recently advanced therapies have undergone both efficacy and effectiveness studies and will be highlighted in this session.
IMMUNOGENICITY OF ACELLULAR FISH SKIN AND PIG URINARY
BLADDER: PRELIMINARY FINDINGS
H. Kjartansson1, B. T. Baldursson2, M. S. Ågren3,4, G. F. Sigurjonsson5
Department of Emergency Medicine, 1National University Hospital Iceland,
Reykjavik, Iceland; 2Department of Dermatology, National University Hospital
of Iceland, Reykjavik, Iceland; 3Digestive Disease Center, Bispebjerg Hospital,
University of Copenhagen, Copenhagen NV, Denmark; 4Copenhagen Wound
Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen
NV, Denmark; 5Kerecis, Isafjordur, Iceland
1
Introduction: Acellular animal tissues are used for wound management and
implantation although their immunogenicity is poorly established. Here, the
immunogenic response of extracts of a novel fish skin product and a porcine
urinary bladder matrix as well as bovine type II collagen was assessed in mice
by serological tests and clinical outcome.
Methods: Four groups, each comprising seven to eight of 2-month-old male
mice (DBA/1J) were immunized by injecting subcutaneously 200 mg collagen
extracted from piscine skin or porcine urinary bladder emulsified in Freund’s
adjuvant, 200 mg bovine type II collagen (Chondrex, Redmond, WA) in
Freund’s adjuvant or Freund’s adjuvant alone (control) at start and after 3
weeks. Type I and II collagen antibody levels in serum were measured after 8
weeks by enzyme-linked immunosorbent assay (Chondrex). The type II collagen
specific-arthritic score was assessed weekly.
Results: Antibodies were undetectable in non-immunized mice. Type I collagen
IgG levels were 6.1 6 1.3 mg/mL (mean 6 SD) in the piscine group, 3.3 6 2.2
mg/mL in the porcine group and 2.5 6 2.2 mg/mL in the group injected with
type II bovine collagen compared with 5.0 6 1.4 mg/mL for the control group.
Anti-type II collagen serum levels exceeded the maximum of the assay ("1.86
mg/mL) in the type II bovine collagen group. The porcine group also developed
type II collagen antibodies (0.88 6 0.66 mg/mL) while they were below the
detection level in the piscine and control groups. These biochemical findings
correlated with the clinical outcome as the type II collagen exposed mice all
had arthritic paws within 8 weeks while none of the other mice did.
Conclusions: Our preliminary data indicate that the examined acellular piscine
skin and acellular porcine urinary bladder elicited no systemic autoimmunity
against type I or type II collagens in mice although these data should be confirmed in larger trials.
FISH SKIN ACELLULAR DERMAL GRAFT FACILITATES CELLULAR
INGROWTH
H. Kjartansson1, S. Magnusson2, B. T. Baldursson3, G. F. Sigurjonsson2
1
Department of Emergency Medicine, National University Hospital Iceland,
Reykjavik, Iceland; 2Kerecis, Isafjordur, Iceland; 3Department of Dermatology,
National University Hospital of Iceland, Reykjavik, Iceland
Introduction: Acellular dermal grafts (ADGs) are used in the treatment of
chronic wounds. ADGs support cell migration and regeneration of tissue.
Chronic wounds are characterized by attenuated fibroblast migration in the
wound bed (1). We have studied the effect of an ADG from fish skin on fibroblast ingrowth by immunofluorescence and histology.
Methods: The structure of the fish skin ADG was examined with scanning electron microscopy (SEM). Further experiments focused on the interaction of cells
with the fish derived ADG. NIH 3T3 fibroblasts were seeded onto ADG pieces
anchored in 96-well tissue culture plates. Fish skin ADG was viewed with confocal microscopy after fluorescent labeling with actin and nuclear markers. In
addition, cultured fish skin ADGs were fixed, embedded into paraffin, and sections cut and stained with hematoxylin-eosin and examined with light
microscopy.
Results: SEM images suggested that the structure of the fish skin ADG supports
cell ingrowth. The experiments using light and confocal microscopy revealed
that NIH 3T3 fibroblasts can migrate into and grow within the fish skin ADG
as well as on its surface.
Conclusions: The ability of fish skin ADG to support cell ingrowth indicates its
suitability for the treatment of chronic wounds and tissue repair.
Reference
1. Lerman OZ, Galiano RD, Armour M, Levine JP, Gurtner GC. Cellular
dysfunction in the diabetic fibroblast: impairment in migration, vascular
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
endothelial growth factor production, and response to hypoxia. Am J Pathol
2003; 162: 303-12.
LOCAL AND SYSTEMIC INFLAMMATION RELATED TO
COMPLEMENT ACTIVATION IN A PIG BURN WOUND MODEL
H. I. Korkmaz1, M. Ulrich1,2, P. Krijnen1, R. Emmens1, M. Vlig1,2, K. Meyer1,
P. Sinnige1, P. van Zuijlen1,3, H. W. M. Niessen4
1
VU University Medical Center, Amsterdam, The Netherlands; 2Association of
Dutch Burn Centres, Beverwijk, The Netherlands; 3Red Cross Hospital,
Beverwijk, The Netherlands; 4Department of Pathology, VUMC, The
Netherlands
Introduction: In patients with burn wounds a massive inflammatory response is
induced that not only negatively affects healing of the burn wound, but also
exerts negative systemic effects in different organs, including the heart. An
important factor herein is the acute phase response, in which complement is
playing a central role (1). It is known that these acute phase proteins are elevated up to months after burn injury in the blood. The aim of this study was to
analyze the timeframe of post-burn complement activation in the blood and the
burn wound and the infiltration of inflammatory cells in the burn wound and in
the heart, in a pig burn wound model.
Methods: In pigs, full thickness burn wounds (2% total body surface area
[TBSA]) were created using a heated copper stamp. Biopsies from the center of
the burn wound and venous blood were collected at different time points up to
60 days post-burn. The hearts were collected at 30 or 60 days post-burn. Complement levels were determined in the blood and in the burn wound. In addition, infiltration of neutrophils, macrophages and lymphocytes were quantified
in the burn wound and in the heart.
Results: In the blood, complement C3 levels were increased from 3 to 60 days
post-burn. In the burn wound, complement C4 and neutrophils were significantly
increased from day 3 until 30 days and macrophages until 60 days post-burn. Complement C3 and lymphocyte infiltration increased from 14 until at least 30 days
post-burn (lymphocytes until 60 days post-burn). In the heart, both at 30 and 60
days post-burn infiltration of neutrophils and macrophages was observed. Of note,
at both time points extensive infiltration of lymphocytes was observed, which was
most pronounced in the right side of the heart, in particular in the right atrium.
Conclusions: In pigs, there is prolonged complement activity both locally in the burn
wound as well as in the blood, even at a low TBSA of 2%. Moreover, this coincides
with increased inflammatory cell infiltration in the burn wound and in the heart.
Reference
1. van de Goot F, Krijnen PA, Begieneman MP, Ulrich MM, Middelkoop E,
Niessen HW. Acute inflammation is persistent locally in burn wounds: a pivotal
role for complement and C-reactive protein. J Burn Care Res 2009; 30: 274-80.
MASSIVE WEIGHT LOSS AND WOUND HEALING
V. Koudahl1
1
Department of Plastic Surgery, Aarhus University Hospital, Aarhus, Denmark
Obesity rates continue to rise in Europe and the US as well as in many other
parts of the world. Obesity has a negative effect on health and increases the
risk of various diseases, particularly type 2 diabetes. Bariatric surgery is an
effective treatment for morbid obesity with a positive effect on diabetes and
cardiovascular risk factors. Recent guidelines suggest that patients with body
mass index > 30 kg/m2 and comorbidities are candidates for bariatric surgery
and now even teenagers are treated with bariatric surgery. The weight loss
leaves the patients with redundant skin, which causes functional as well as aesthetic problems and the need for post bariatric surgery is increasing. Postbariatric surgery has a high risk of complications including wound healing problems. The patients have experienced a very fast weight loss and many are in
state of malnutrition making diet and timing of surgery crucial.
THE INSOLUBLE COLLAGEN FRACTION OF ANASTOMOTIC
WOUNDS IN LEFT COLON IS RESPONSIBLE FOR THEIR
LONGITUDINAL STRENGTH FOLLOWING ACUTE OBSTRUCTION
P.-M. Krarup1, L. N. Jorgensen1, M. B. Hansen1, M. S. Ågren1,2
Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark; 2Copenhagen Wound Healing Center, University of
Copenhagen, Copenhagen NV, Denmark
1
Introduction: Breaking strength is a common surrogate outcome of anastomotic
leakage in research on gastrointestinal anastomoses. It is unknown whether the
strength is due to the quantity and/or the quality of the collagen. In this study, we
examined the relation between the biomechanical strength and the composition of
collagen after chemical fractionation of the collagens present in anastomotic wounds.
Methods: Obstruction of left colon was induced laparoscopically in 13 male
Sprague-Dawley rats (226–269 g). After 12 hours, end-to-end single layer anastomoses were constructed 30 mm from the peritoneal reflection with nine interC 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
rupted 6/0 polyamide sutures. On postoperative day 3, anastomoses with sutures in
situ were stretched in the longitudinal direction at 10 mm/minutes until rupture.
Wound tissue, extending about 2 mm from the suture line, was excised and stored
R for 10 seconds
at 2808C. Tissues were dispersed by high-speed T10 Ultra-TurraxV
in 5 mL 0.5 N acetic acid containing 1 mg pepsin (P7012, Sigma-Aldrich) per
10 mg wet tissue. The homogenate was incubated with constant stirring for
24 hours at 48C and centrifuged at 16,000 rcf for 10 minutes. Soluble collagen in
supernatant was measured by SirCol (Biocolor, Carrickfergus, UK) and the insoluble collagens by the hydrodroxyproline content of hydrolyzed pellets.
Results: The total collagen concentration in the anastomotic wounds was
16.1 6 1.5 (mean 6 SD) mg collagen/mg tissue composed of 5.1 6 1.5 mg insoluble collagen/mg tissue and 11.0 6 0.5 mg soluble collagen/mg tissue. We found
significant correlations between anastomotic breaking strength and total collagen (rP 5 0.670, p 5 0.012) and insoluble collagens (rP 5 0.727, p 5 0.005). In
contrast, no significant correlation was observed between soluble collagen species and breaking strength (rP 5 20.094, p 5 0.760).
Conclusions: Our data indicate that early breaking strength of experimental
colonic anastomoses is due to insoluble, cross-linked collagens.
LOCAL HYPERGLYCEMIA IN FULL-THICKNESS WOUNDS IN
EUGLYCEMIC RATS IMPAIRS WOUND HEALING IN A
CONCENTRATION DEPENDENT MANNER
C. Kruse1,2, K. Nuutila1, E. Eriksson1, J. A. Sørensen2
1
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA USA;
2
Department of Plastic and Reconstructive Surgery, Odense University Hospital,
Odense, Denmark
Introduction: The purpose of this study was to investigate the effect of local
hyperglycemia in vitro and in vivo in a rat wound model.
Methods: Human primary keratinocytes and fibroblasts were cultured in the
presence of different concentrations of glucose (0–100 mM). Cell viability, proliferation and migration were studied. Supernatant from the cell cultures was
collected for matrix metalloproteinase (MMP)21 ELISA measurements. The
effect of topical glucose was also investigated in a rat full-thickness wound
model. Four 2-mm punch biopsy wounds were created on the dorsum of female
Wistar rats. After wound creation custom-made titanium chambers enclosed the
wound, creating an isolated, well-controlled environment. The glucose concentration inside the chambers was modified by adding 500 ll of keratinocyte
serum free media with glucose concentration ranging from 0 to 100 mM.
Wound healing was followed over time; wound fluid samples and tissue biopsies were collected on days 4 and 8 postoperatively.
Results: In vitro experiments showed that glucose concentrations at ! 26 mM
did not have an effect on cell viability while at " 26 mM glucose the cells died.
Glucose at " 5.6 mM promoted fibroblast proliferation but did not impact keratinocyte proliferation. In both keratinocytes and fibroblasts glucose at " 5.6 mM
prevented cell migration. Our results also showed that glucose at 23 mM
increased MMP-1 production in fibroblasts up to 20-fold. In vivo experiments
demonstrated that topical application of 5.6 mM and 26 mM glucose solutions
delayed wound closure and at 100 mM glucose blocked healing completely.
Conclusions: Hyperglycemia impaired migration of cultured primary keratinocytes and fibroblasts, but increased proliferation and MMP-1 production in fibroblasts. Local hyperglycemia impeded wound healing in full-thickness wounds in a
concentration dependent manner.
ROLE OF SENSORY INNERVATION ON THE CUTANEOUS HEALING
PROCESS AFTER BURN
B. Laverdet1, D. Girard1, N. Bordeau1, C. Egles2, L. Misery3, B. Coulomb4,
J.-J. Lataillade5, A. Desmoulière1
1
EA 6309 “Myelin Maintenance and Peripheral Neuropathies”, Faculties of
Medicine and Pharmacy, University of Limoges, Limoges, France; 2CNRS
UMR 7338, Universit"e de Technologie de Compiègne, Compiègne, France; 3EA
4685, University of Brest, Brest, France; 4UMRS 1197 INSERM, Paris Sud
University, Paris, France; 5Unit"e de Th"erapie Cellulaire Et R"eparation
Tissulaire, H^
opital Percy, Clamart, France
Introduction: Damage to the peripheral nervous system influences wound healing. After a deep burn injury, cutaneous nerve regeneration can occur but this
process is imperfect, resulting in the alteration of skin sensation. A translational
research program NERVAL, for “R"einNERVAtion après br^
uLures,” was initiated to appreciate the role of innervation during cutaneous repair and to investigate why innervation is not correctly restored after burn. A deep second-degree
burn model was developed in rats combined with the use of resiniferatoxin
(RTX) known to promote sensory neuropathy affecting small fibers.
Methods: Anesthetized rats were injected intraperitoneally either with 185 mg/
kg RTX or vehicle. Mechanical and thermal sensory tests were performed one
day after injection and weekly afterward. The structural integrity of the sciatic
nerve was investigated using transmission electron microcopy. Seven days after
RTX injection, thermal burns were performed on the dorsal skin, under
A16
Abstracts
anesthesia, using a 7 mm 3 12 mm 708C or 908C heated template applied for
45 seconds under a pressure of 0.6 N. Following debridement, wound closure
was monitored and samples were collected for histological analysis, immunohistochemistry and immunoblotting for neuronal (e.g., PGP 9.5, NF 200) and activated fibroblast (a-smooth muscle actin) markers.
Results: RTX promoted both tactile perception deficit and thermal hypoalgesia
up to 14 and 28 days post injection, respectively. This transient RTX-mediated
sensory deficit occurred without damaging the nerve fibers. Wound closure rates
were similar in both groups, but the kinetics of granulation tissue remodeling
seemed to be delayed with RTX treatment. Immunohistochemistry and immunoblotting studies are ongoing.
Conclusions: This work aims to establish the roles of innervation during skin
healing process and to provide new insights into mechanisms responsible for
defective axonal regrowth following cutaneous burn with the objective to establish new strategies to improve patients’ quality of life.
INCIDENCE OF HOSPITAL ACQUIRED PRESSURE ULCERS IN 2014
AT A FACILITY LOCATED IN SOUTHWEST UNITED STATES
E. Lew1, D. Betcher1
1
Mayo Clinic, Phoenix, AZ USA
Introduction: Hospital-acquired pressure ulcers (HAPU) lead to poor outcomes
for patients including pain, increased length of stay and time to recovery (1).
Additionally, HAPU lead to increased costs for hospitals as the average cost for
treating a pressure ulcer exceeds $40,000 in the US (2).
Methods: We recently conducted a retrospective study of HAPU incidence for
2014 at an academic hospital located in the Southwest region of the United
States.
Results: In 2014, there were 13347 inpatient admissions. 140 patients were
identified with HAPU. Of these, 98 (70%) were male and 108 (76%) were older
than 60 years of age. 63 (45%) patients had undergone surgery lasting at least 2
hours and 107 (76%) had a length of stay of more than 7 days. Common medical comorbidities included diabetes (34%) and coronary artery disease (41%).
Overall, 58 (41%) had scored 19 or higher on the Braden Scale on admission,
indicating low risk for developing pressure ulcers. Approximately one-third of
the diabetic and cardiac HAPU patients were also misclassified using the
screening method.
Conclusions: Currently, we used the Braden Scale to assess inpatients for risk
of pressure ulcer development where a score of 18 or less indicating high risk.
In our study we saw a substantial number of HAPU patients with coronary
artery disease or diabetes, and the negative effect of prolonged surgery on the
development of HAPU. The Braden Scale also gave low risk scores to 41% of
HAPU patients. In addition to using the Braden Scale, performing detailed history and physical examination, ensuring accurate handoff, and having a keen
awareness of a patient’s risk factors outside of what are assessed via the Braden
Scale are necessary in the prevention of HAPU.
References
1. Theisen S, Drabik A, Stock S. Pressure ulcers in older hospitalised
patients and its impact on length of stay: a retrospective observational study. J
Clin Nurs 2012; 21: 380-7.
2. Cooper KL. Evidence-based prevention of pressure ulcers in the intensive
care unit. Crit Care Nurs 2013; 33: 57-6.
SKELETAL MUSCLE INJURY AND TISSUE HEALING: ROLE OF
MUSCLE STEM CELLS
A. L. Mackey1, M. Kjaer1
1
Institute of Sports Medicine, Department of Orthopaedic Surgery M, Bispebjerg
Hospital and Department of Biomedical Sciences, Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen, Denmark
The presence of resident stem cells (satellite cells) in skeletal muscle affords a
potential to regenerate fully after injury and yet the incidence of injury recurrence suggests that muscle repair is often complete. Also it is known that ageing is associated with a decline the satellite cell content of muscle together with
an increase in resistance to activation. A better understanding of the activity of
different cell types involved in muscle repair following injury, or muscle growth
with exercise training, could contribute to the development of interventions
capable of improving repair after injury. The focus of this presentation will be
on the regeneration of young and old human skeletal muscle including confocal
and transmission electron microscopy to describe in three dimensions human
muscle fibres undergoing regeneration, with particular focus on the distribution
and activity of satellite cells and macrophages.
A17
THE USE OF THERMO-RESPONSIVE SURFACES TO HARVEST
POLARIZED AND RESTING STATE MACROPHAGES
V. Malheiro1, Y. Elbs-Glatz1, M. Obarzanek-Fojt1, A. Bruinink1
Empa, St. Gallen, Switzerland
1
Introduction: The medical applicability of scaffolds may be determined by their
effect on the polarizing state of contacting macrophages. One characteristic of macrophages is their very strong adhesion to the substratum. As a result they are difficult to harvest. This limits the type of investigations addressing interaction of
resting state M0, and polarized M1 and M2 (with its subtypes a to d) macrophages
especially in the context of biomaterials for tissue repair. More than two decades
ago Yamada and coworkers (1) introduced a new enzyme-free method to recover
cultured cells using a tissue culture treated polystyrene (TP) substrate coated with
the thermoresponsive poly(N-isopropylacryl-amide) (PP). Comparing monocyte
activation and subsequent macrophage isolation using TP and PP, the aim of the
present study was to answer the following questions in this regard with the THP-1
monocyte cell line as tool: (i) Is monocyte activation towards M0 macrophages and
number of adhering cells affected by the substratum? (ii) Is the polarization potential modified by the substratum? (iii) Are cells differently affected by the isolation
procedure in terms of yield and viability of M0, M1 and M2a? (iv) Does the harvesting procedure of the cells affects cell reseeding in respect to yield of cell attachment, and M0, M1 and M2a characteristics stability? Results and
Conclusions: Before harvesting THP-1 cells behave similarly on PP and TP
surfaces. PP surfaces showed to be superior over TP substratum as cells could
be harvested with higher cell yield, less cell death and, if reseeded onto TP,
higher yield of cell re-attachment.
Reference
1. Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y.
Thermo-responsive polymeric surfaces; control of attachment and detachment of
cultured cells. Makromol Chem Rapid Commun 1990. 11: 571-6.
TISSUE REMODELING OF BONE, CARTILAGE AND SYNOVIUM
MEASURED BY UNIQUE BIOMARKERS OF TISSUE FORMATION
AND DEGRADATION
T. Manon-Jensen1, C. S. Thudium1, A. S. Siebuhr1, C. F. Kjelgaard-Petersen1,
Y. He1, N. S. Gudmann1, K. Henriksen1, T. Christiansen1, A.-C. Bay-Jensen1,
M. A. Karsdal1
1
Nordic Bioscience A/S, Herlev, Denmark
Introduction: Tissue remodeling is a complex process of tissue degradation and
formation which is tightly controlled in healthy individuals. During pathologies,
this balance is perturbed, resulting in more tissue degradation or formation, leading
to an altered extracellular matrix (ECM), altered tissue quality and eventually organ
failure. Osteoarthritis (OA) is a joint disease that results in the breakdown of the
entire joint; synovium, articular cartilage and subchondral bone. Each tissue consists
of a specific ECM, which is remodeled as part of the pathogenesis of OA. The aim
of the study was to develop and characterize the turnover of human synovial membrane, cartilage and bone by tissue-specific biomarkers in ex vivo culture models.
Methods: Synovial membrane explants from OA patients undergoing total knee
replacement were cultured with TNF-a, IL-1b, or TGF-b2. Supernatants were
analyzed on the matrix metalloproteinase (MMP)-mediated type I and III collagen
degradation biomarkers C1M and C3M, and active MMP-3. Cartilage explants
were cultured for 3 weeks with TNF-a, oncostatin M or control. The aggrecanasemediated aggrecan degradation marker AGNx2 and the MMP-mediated type II
collagen marker C2M were investigated. Mature human osteoclasts were cultured
on bone slices in the presence of M-CSF and RANKL. Bone resorption was
assessed by the biomarker CTX-I reflecting cathepsin K-degraded type I collagen.
Results: In cultured synovial membrane, C1M (10-fold, p < 0.05) and C3M
(100-fold, p < 0.0001) were increased day 7 in response to TNF-a compared to
no TNF-a addition. IL-1b showed similar pattern. Active MMP-3 was increased
(p < 0.0001) in synovial membrane explants treated with TNF-a or IL-1b
throughout the study compared with no cytokine additions. In cartilage, AGNx1
was significantly increased day 7 (p < 0.0001) and day 14 (p < 0.01) in response
to catabolic activation by TNF-a and oncostatin M, while a trend towards
increased C2M was observed. The bone resorption marker CTX-I was increased
<1000% (p < 0.001) in response to RANKL and inhibited by bisphosphonates.
Conclusions: We have characterized three primary tissue models with corresponding unique biomarkers of tissue remodeling in synovium, cartilage and bone.
TREATMENT OF CAPSULAR CONTRACTION ON BREAST
IMPLANTS
R. Mares1
1
Unidad de Quemados Y Cirugia Pl"astica, Servicios de Salud del Estado de
Puebla, Puebla, Mexico
Introduction: Capsular contracture is the most common postoperative complication in breast augmentation that may require revisionary breast surgery, with
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
a high probability of recurrence. Total capsulectomy is the gold standard treatment, but it is a traumatic method. Our objective was to propose a surgical
technique for correction of capsular contracture, breast implant replacement and
changing surgical plane.
Methods: Ten patients that suffered from capsular contracture were investigated
from the year of 2008 to 2012. Clinical and psychological data were recorded.
Before the revisionary surgery, all patients had implant"s distortion, breast"s distortion, palpable firmness or rigidity, sensation of tension and hardening, pain,
and visible and palpable contracture.
Results: The mean age of the patients was 30 years (range: 23-37 years).
Follow-up ranged from 18 to 24 months. Six patients suffered from capsular
contracture Baker grade III and four patients grade IV. Eight patients had their
implants in subglandular position and two in subpectoral, six had periareolar
approach and four inflamammary. The average volume of inserted implants was
360 mL (range: 280-410 mL), round texturized implants filled with highly cohesive gel. There was only one case of recurrence.
Conclusions: Good aesthetic results and patient satisfaction was achieved with
this new, effective, and less traumatic technique to correct capsular contracture.
It reduces the risk of recurrence by inserting the new implant in a novel pocket.
LENGTH-PRESERVING TECHNIQUE OF THE AFFECTED
AMPUTATED LIMBS WITH NEGATIVE PRESSURE WOUND
THERAPY IN ELECTRICAL BURN INJURIES
R. Mares1
1
Unidad de Quemados Y Cirugia Pl"astica, Servicios de Salud del Estado de
Puebla, Puebla, Mexico
Amputation is considered the last resort when limb salvage is impossible. The
decision to amputate one or more limbs is always difficult but reduces morbidity and increases patient survival rate. The preservation of the function is the
primary concern. Therefore amputation should be performed in the most distal
possible level. The more upward the level of amputation, the greater the metabolic demand. The goal of treatment in these cases is to cover with skin to prevent infection and allow early mobilization. Technological advances that exist
in the field of prosthetics are plentful; sadly the developing countries fail to
appreciate their benefits. Two cases of electrical burns received negative pressure wound therapy as part of the art of preserving the amputated limb length.
The length preserving technique presented in this paper is innovative and superior to other techniques.
CULTIVATED IN VITRO EPIDERMAL ALLOGRAFT:
A MEXICAN PRODUCT
R. Mares1
Unidad de Quemados Y Cirugia Pl"astica, Servicios de Salud del Estado de
Puebla, Puebla, Mexico
1
Introduction: Cultivated keratinocyte allografts have been shown in controlled
clinical studies, if applied early to increase epithelialization of burns by more
than 40% compared with conventional treatments. The grafts can be refrigerated without loss of activity. They reduce repair time, complications, number
of surgeries, the cost of care and hospital stay time; situations that result in
more cost-effectiveness.
Methods: Cultivated in vitro epidermal allograft is prepared from a biopsy of
about 1 cm2 of healthy skin. Epidermis is separated from the dermis with the
aim of obtaining growing keratinocytes and fibroblasts.
Results: The treatment results are described according to their integration,
appearance, functionality, and physical recovery by clinical photographs.
Conclusions: Cultivated in vitro epidermal allograft accelerates epithelialization
of superficial and deep second-degree burns by releasing growth factors that
stimulate migration and proliferation of keratinocytes.
USE OF CULTIVATED IN VITRO EPIDERMAL ALLOGRAFT ON
FACIAL BURNS
R. Mares1
1
Unidad de Quemados Y Cirugia Pl"astica, Servicios de Salud del Estado de
Puebla, Puebla, Mexico
Introduction: Cultivated in vitro epidermal allografts accelerate epithelialization of superficial and deep second-degree burns.
Methods: The burns were covered for the first 5 days with cultivated in vitro
epidermal allograft.
Results: The application of cultivated in vitro epidermal allograft reduced repair
time and contributed to better cosmetic results leading to less time for functional and psychological impairments of the patients with facial burns.
Conclusions: Cultivated in vitro epidermal allograft accelerates wound healing
by stimulating granulation tissue formation and epithelialization by producing
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
growth factors such as epidermal growth factor, fibroblast growth factors, transforming growth factor-a, and vascular endothelial growth factor.
INCIDENCE OF PATIENTS SEEN IN THE BURN UNIT OF THE
NORTH ZONE GENERAL HOSPITAL OF PUEBLA FROM 2009
TO 2014
R. Mares1
Servicios de Salud del Estado de Puebla, Unidad de Quemados Y Cirugia
Pl"astica, Puebla, Mexico
1
Burn injuries are a national public health issue that cause severe physical, psychological, social, and labor impairment. Not only are burn injuries responsible
for facial and corporal deformities they account for major economic expenses
due to their high rehabilitation and treatment costs. Burn injuries are one of the
most complicated issues to treat; they require 24/7 direct attention and monitoring, as well as very specific medication and equipment. In Mexico, The
National Health Information System (SINAIS), a juridically sustained organization by the General Health Law, published its last results on national epidemiology on burn patients in 2006. The lack of information available and updated
studies has obscured and underestimated this as a national public health issue.
Therefore we have chosen to shed light on this matter in order to increase its
relevance as a national health issue but also improve the quality and treatment
of burned patients lives. The national average for hospital stay lies within
20 days compared with 14–17 days for our unit. The infection rate is zero, due
to the strict attachment to the hospital policies, to the Clinical Practice Guides
and to the Treatment Reference Guides from the National Health Organization.
Currently the number of patients hospitalized due to burn injuries in specialized
units nation wide is unknown, which shows why burned patients are not given
the essential care and attention needed. Conducting a deep and thorough investigation of this nature is not only necessary but also detrimental to the development and continuous improvement of treatment. This study will also allow us to
release information on the average hospital stay, infection rate and how the
implementation of new technologies—such as placing synthetic and biological
dressings, placing and taking grafts—significantly improving the burn victim’s
outcome. This way we can establish continuous improvement programs resulting in better treatment.
BART SYNDROME DYSTROPHIC EPIDERMOLYSIS BULLOSA AND
APLASIA CUTIS: A CASE REPORT
R. Mares1
1
Servicios de Salud del Estado de Puebla, Unidad de Quemados Y Cirugia
Pl"astica, Puebla, Mexico
This rare (1:1,000,000) syndrome was described in a large family in 1966 and
consists of any one or a combination of the following three characteristics: congenital absence of skin, blistering and associated nail abnormalities. Ultrastructurally, poorly formed anchoring fibrils and cleavage below the lamina densa
were found in Bart’s kindred (1). Genetic linkage of the inheritance of the disease points to the region of chromosome 3 near the collagen, type VII, a1 gene
(COL7A1). The blister fluid is negative for IgG, IgA, C1q, C3c, kappa, and
lambda. We treat the denuded skin and blisters for 5 days with cultivated in
vitro epidermal allografts, which stimulated granulation tissue formation and
epithelialization.
Reference
1. Zelickson B, et al. Zelickson B, Matsumura K, Kist D, Epstein EH Jr,
Bart BJ.Bart’s syndrome. Ultrastructure and genetic linkage. Arch Dermatol
1995; 131: 663-8.
DEATH AND AMPUTATION
D. J. Margolis1, O. Hoffstad1, J. Walsh1, N. Mitra1
1
Department of Dermatology and Department of Biostatistics and
Epidemiology, University of Pennsylvania, Philadelphia, PA USA
Introduction: Individuals with diabetes and lower extremity amputation (LEA)
are known to be at higher risk of death then those with diabetes without an
LEA. The goal of the study was to determine if complications of diabetes wellknown to be associated with death such as cardiovascular disease and renal failure fully that are also risk factors for LEA explain the higher rate of death in
those who have undergone an LEA.
Methods: A longitudinal cohort study of patients cared for in The Health
Improvement Network. LEA was the primary exposure and the outcome was
death. “Risk factor variables” included a history of cardiovascular disease (a
history of myocardial infarctions, cerebrovascular accident, and peripheral vascular disease-arterial insufficiency), Charlson index, and a history of chronic
kidney disease. We estimated the effect of LEA on death using proportional
hazards models. Factors were evaluated as confounders, surrogates on the causal
pathway, and as predictors.
A18
Abstracts
Results: The hazard ratio (HR) for death after a LEA was 3.02 (95% confidence
interval: 2.90-3.14). The fully adjusted (all risk variables) LEA HR was diminished only about 22% to 2.37 (2.27-2.48). Causal statistical models and structural equation models that included all “risk factor” variables did not
significantly alter the strong association of LEA with death. LEA had an area
under the receiver operating curve (AUC) of 0.51, which is poorly predictive,
and the full model AUC was 0.77, which is fairly predictive. Sensitivity analysis revealed that it is unlikely that there exits an unmeasured confounder to
fully explain the association of LEA with death.
Conclusions: Individuals with diabetes and an LEA are more likely to die at
any given point in time than those who have diabetes but no LEA. While some
of this variation can be explained by known complications of diabetes, there
remains a large amount of unexplained variation.
CLINICAL TRIAL DESIGNS WITH REAL WORLD ENDPOINTS
D. J. Margolis1
1
Department of Dermatology and Department of Biostatistics and
Epidemiology, University of Pennsylvania, Philadelphia, PA USA
In 2006, the Food and Drug Administration of the United States published a
guidance document on developing products for the treatment of chronic wounds
and burns. This guidance document is still frequently referenced when comparative efficacy studies are designed. The efficacy endpoints described in this
document were grouped into two categories: improved healing and improved
wound care. Many outcome measures were described in this document. This
document has been generally interpreted to indicate that wound closure by at a
minimum of 3 months should be the primary outcome of interest. This presentation will discuss this outcome and others that could be considered clinically
important endpoints for evaluation in clinical trials of chronic wound therapies.
A SERUM-FREE AND FEEDER-FREE METHOD FOR EXPANDING
HUMAN KERATINOCYTES ON MICROCARRIERS FOR THE
TREATMENT OF SEVERE BURN INJURIES
Y. Martin1, T. Metcalfe1
Blond Mcindoe Research Foundation, The Brighton Centre for Regenerative
Medicine, School of Pharmacy & Biomolecular Sciences, University of
Brighton, East Grinstead, United Kingdom
1
Introduction: Autologous keratinocytes are used in the treatment of severe
burns to augment wound healing. Cells are commonly expanded in serumcontaining medium in the presence of lethally irradiated mouse fibroblast feeder
cells. Subsequent application to the wound bed in single cell suspension can
lead to significant cell damage and cell loss. We previously demonstrated
improved wound healing outcomes in the porcine model of wound repair when
cells were delivered using biodegradable gelatin micro carriers instead of as cell
spray. We present here an improved method of culturing human keratinocytes
on biodegradable micro carriers under serum-free and feeder-free conditions.
Methods: Keratinocytes were isolated from discarded human skin and cultured
in a serum-free and feeder-free, defined medium until sub-confluent (5–6 days).
Porous gelatin microcarriers were seeded with 5 3 106 cells and cultured for a
further 4 days in a stirring glass bioreactor. Cell phenotype was assessed by
microscopy and qRT-PCR.
Results: We obtained gelatin microcarriers with sub-confluent keratinocytes
after 4 days in culture. Cell phenotype was comparable to cells expanded on
tissue culture plastic.
Conclusions: Severe burns are often treated with autologous keratinocytes
within 10-20 days of hospital admission. Using the culture protocol reported
here, highly proliferative keratinocytes are available for transplantation within
10 days. Our approach of using microcarriers for transplantation ensures that
the cells are not damaged by enzymatic digestion during removal from tissue
culture flasks. Using a serum-free and feeder-free medium system would further
limit the risk to patients associated with use of xenobiotic substances.
A PILOT STUDY ON THE USE OF MEDICAL GRADE HONEY IN THE
MANAGEMENT OF CANINE OTITIS EXTERNA
E. Maruhashi1, B. S~ao Braz2, T. Nunes1, A. M. Lourenço1
1
University of Lisbon Faculty of Veterinary Medicine, Lisbon, Portugal;
2
Avenida Da Universidade Tecnica, Lisbon, Portugal
Introduction: This study’s objective included assessment of medical-grade
honey (MGH) in the management of bacterial and/or fungal canine otitis
externa and development of an effective alternative to conventional treatments.
The treatment"s aim includes auricular skin barrier restoration and otic environment amelioration through attenuation of clinical signs and infection resolution,
whilst simultaneously contributing to the reduction in use of antibiotics and prevention of resistance.
A19
Methods: Client-owned dogs (n 5 15) with a confirmed diagnosis of otitis
externa were enrolled. Dogs were prescribed MGH (1 mL daily per ear) until
pre-established cure or during a maximum of 21 days. Evaluation was based on
weekly clinical score, which included erythema, edema/swelling and erosion/
ulceration, as well as cytological progression and owner assessment of pruritus
through a visual analogue scale (VAS). Swab samples were sent for culture and
susceptibility testing, as well as for biocidal activity on behalf of the MGH.
Results: MGH yielded rapid clinical progress, with 70% of dogs achieving clinical cure between days 7 to 14 and over 90% on day 21. There was a decrease
in clinical scores throughout trial duration (p < 0.001) with owner VAS scores
also decreasing (p < 0.05). Methicillin-resistant strains of Staphylococcus pseudintermedius (MRSP), among other resistant bacterial strains were present and
in vitro results revealed biocidal activity of MGH towards all bacterial agents.
Conclusions: MGH was particularly effective in the rapid relief and resolution
of clinical signs, including the restoration of the protective skin barrier inside
the ear, whilst successfully eliminating infection, including cases involving
resistant bacterial strains.
This study therefore supports the growing research addressing the potential
of MGH as a therapeutic alternative which can be extrapolated for use in the
difficult scenario that is chronic wound management and infection control.
References
1. Nuttall T, Bensignor E. A pilot study to develop an objective clinical
score for canine otitis externa. Vet Dermatol 2014; 25: 530-7, e91-2.
2. Satarupta R, Subha G. Physical, chemical and antioxidant properties of
honey: a review. Asian J Chem Pharmaceutical Res 2014; 2: 96-9.
COLD ATMOSPHERIC PRESSURE PLASMAS FOR WOUND HEALING
K. Masur1, S. Hasse1
1
Leibniz Institute for Plasma Science and Technology, Greifswald, Germany
Introduction: A new promising tool for successful treatment of chronic wounds
is cold atmospheric pressure plasma. Plasma consists of partially ionized gas
and contains a range of reactive oxygen and nitrogen species (ROS/RNS) combined with UV radiation. For a decade or so it has been known that plasma
exhibits bactericidal effects. Here we demonstrate that plasma treatment of
eukaryotic cells and human skin tissue can stimulate cellular activities resulting
in a short term activation of cell proliferation. The aim of this study was to
investigate the impact of cold atmospheric pressure plasma on human skin cells
both in vitro and in situ in order to disentangle the underlying mechanisms of
plasma cell interactions.
Methods: Human skin cell lines as well as skin tissue (biopsies) were treated
with the atmospheric pressure plasma jet (kiNPen). Cellular activities were
measured by either analyzing their metabolic activity via resazurin conversion
(Alamar Blue Assay), detection of proliferation (Ki-67) or of apoptotic events
(Annexin V or TUNEL staining). Western blot analysis was applied to detect
the involvement of MAP kinases in mediating the plasma based cell activation.
Results: Besides a clear treatment time dependency of the plasma induced cellular reactions also cell type dependent effects could be detected by measuring
metabolic activities of the plasma-treated cells. Applying phospho-specific antibodies of MAP signaling cascade (e.g., p38MAPK, ERK1) the molecular mechanisms of cell activation could be evaluated. Further investigations on the
cytokine secretion indicated a treatment time dependent release of pro- and
anti-inflammatory interleukins, e.g., interleukin (IL)-6 and IL-8. Similar results
could be obtained from biopsy samples. Short-term plasma treatment led to a
stimulation of basal keratinocytes as indicated by Ki-67 staining.
Conclusions: These results underline the great potential of cold plasmas to support wound healing and give first insights in the underlying mechanisms.
IN SILICO MODELING OF THE SENESCENCE ASSOCIATED
SECRETORY PHENOTYPE IN A WOUND HEALING ENVIRONMENT
ussel2, P. Maity1, K. Singh1, M. Wlaschek1, H. A. Kestler2,
P. Meyer1, C. M€
K. U. Scharffetter-Kochanek1
1
Department of Dermatology and Allergic Diseases, University of Ulm, Ulm,
Germany; 2Medical Systems Biology, Ulm, Germany
Introduction: Permanent cell-cycle arrest or senescence is a protection mechanism that helps cells recover from damage. Cellular senescence can be accompanied by a senescence associated secretory phenotype (SASP) that causes
chronic inflammation and paracrine senescence. While senescence in general
seems to be beneficial for wound healing, the SASP is not and can cause temporary or chronic wound healing disorders. There are indications that senescence is causal for chronic venous leg ulcers, explaining why the severity and
occurrence is higher in aged individuals. We additionally propose that it is not
only the amount of pre-existing senescent cells but also the developing SASP
that determines the onset of a chronic wound and the outcome of wound
healing.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
Methods: Here we present a core gene regulatory network of the development and
maintenance of senescence and the SASP incorporating published gene expression
and interaction data of different signaling pathways like interleukin (IL)-1, IL-6,
p53, and NF-jB under the assumption of DNA damage or oncogenic stress.
Results: The modeled simulations correspond to published data on cellular
senescence and the SASP. Furthermore we can predict different in silico knockouts that prevent key SASP-players, like IL-1, IL-6, and IL-8, from getting activated upon cell cycle arrest. In a first screening we found different gene knockouts and knock-out combinations that prevent the activation of IL-6 signaling,
factors that among others seem to be responsible for spreading and retaining the
SASP. In this way we could single out the NFjB Essential Modifier (NEMO)
as a target. Under the assumption of DNA damage, a NEMO-knock-out was
enough to prevent the activation of IL-6 and IL-8 in silico.
Conclusions: Consequently, this gives us the power to create in vitro and in vivo
models that might help understand the dynamics of the SASP and can be used to
broaden our understanding of highly important wound healing mechanisms.
RECEPTOR SYNDECAN-1 PLAYS A PIVOTAL ROLE IN LAMININ 332
OR CYTOKINE INDUCED MMP-9 EXPRESSION DURING
KERATINOCYTE MIGRATION
A. Michopoulou1, D. Guila1, P. Rousselle1
1
Laboratoire de Biologie Tissulaire Et Ing"enierie Th"erapeutique - CNRS UMR
5305, Lyon, France
lar surface tension in preterm infants to ease breathing. Phospholipid films with
surfactant proteins regulate the shape and activity of alveolar macrophages by
controlling surface tension. Here, results of the effect of topical bovine lung surR ; Lyomark Pharma GmbH, Oberhaching, Germany) on
factant (AlveofactV
wound healing and fibrosis in a murine full-thickness wound model are presented. In contrast to the robust fibrotic response observed in the Vaseline
gauze controls, a thin epidermal layer and fluffy dermis was found without
pathologic scarring after surfactant treatment. These effects were accompanied
by reduced levels of tumor necrosis factor (TNF)-a converting enzyme, TNF-a,
interleuikin-1b and matrix metalloproteinase (MMP)-9. Furthermore, the surfactant down-regulated transforming growth factor (TGF)-b2, TGF-bRI, MMP-3,
a-smooth muscle actin, angiotensinII-R2, and connective tissue growth factor
expression. Lung surfactant seems to have anti-inflammatory and anti-fibrotic
effects on skin wound healing. These findings are novel and hitherto not
described. By wound treatment with lung surfactant, local inflammation may be
decreased and thereby wound closure enhanced and scar formation reduced.
This implies an innovative treatment option for skin wounds and for the prevention of excessive scarring.
PRO-INFLAMMATORY MATRIX METALLOPROTEINASE-3 IS KEY
EFFECTOR OF TNF-ALPHA-INDUCED TYPE I COLLAGEN
DEGRADATION
U. Mirastschijski1,2, A. J. Caliani2, D. Wedekind3, L. McCawley4, M. S. Ågren5
Department of Plastic, Reconstructive and Aesthetic Surgery, Klinikum
Bremen-Mitte, Bremen, Germany; 2Center for Biomolecular Interactions
Bremen, Department of Chemistry and Biology, University of Bremen, Bremen,
Germany; 3Institute for Laboratory Animal Science, Hannover Medical School,
Hannover, Germany; 4Department of Cancer Biology, Vanderbilt University,
Nashville, TN USA; 5Digestive Disease Center and Copenhagen Wound
Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen
NV, Denmark
1
Introduction: During skin repair, the epithelialization phase occurs by an orderly
series of events whereby keratinocytes migrate, proliferate, and differentiate to restore
the barrier function. Keratinocyte migration determines the efficiency of the overall
wound repair process. The migratory behavior is governed at extracellular and intracellular levels and depends on the balanced dynamic interactions of the cells with
extracellular matrix (ECM) components, growth factors and cytokines. Among ECM
proteins, laminin 332 was shown to contribute to skin epithelialization through its a3
chain C-terminal domains LG45. These domains induce keratinocyte migration, an
event that relies on the involvement of the pro-migratory matrix metalloproteinases
(MMP)-1 and MMP-9, two MMPs known to play a role in epithelialization. As findings from our laboratory have reported that LG45 domains participate in cytoskeleton
dynamic and cell movement through binding of the heparan sulphate proteoglycans
syndecan-1 and syndecan-4, we analyzed their potential involvement in this process.
Methods: We altered syndecan-1 and syndecan-4 binding site in a recombinant
LG45 by site-directed mutagenesis. We knocked-down syndecans with siRNAs or
overexpressed them. We used PCR and zymography to analyze MMP-9 expression.
Results: Our PCR analysis and zymography results revealed that depending on
the mutated proteins, the MMP activation profile was different in keratinocytes,
suggesting that syndecan-1 plays a role in LG45-induced MMP-9 expression
and activation. We confirmed these results by knocking down syndecans expression. We revealed that this phenomenon also occurred when cells were treated
with tumor necrosis factor-a or interleukin-1b, two cytokines known to upregulate MMP-9 expression.
Conclusions: Taken together, our data demonstrate for the first time that
syndecan-1 plays a pivotal role in MMP-9 expression. Our results further suggest that the LG45 module, when released from laminin 332, has the ability to
impact the MMP-9 expression balance during keratinocyte migration.
References
1. Carulli S, Beck K, Dayan G, Boulesteix S, Lortat-Jacob H, Rousselle P.
Cell surface proteoglycans syndecan-1 and -4 bind overlapping but distinct sites
in laminin a3 LG45 protein domain. J Biol Chem 2012; 287: 12204-16.
2. Momota Y, Suzuki N, Kasuya Y, Kobayashi T, Mizoguchi M, Yokoyama
F, Nomizu M, Shinkai H, Iwasaki T, Utani A. Laminin a3 LG4 module induces
keratinocyte migration: involvement of matrix metalloproteinase-9. J Recept
Signal Transduct Res 2005; 25: 1-17.
3. Okamoto O, Bachy S, Odenthal U, Bernaud J, Rigal D, Lortat-Jacob H,
Smyth N, Rousselle P. Normal human keratinocytes bind to the alpha3LG4/5
domain of unprocessed laminin-5 through the receptor syndecan-1. J Biol Chem
2003; 278: 44168-77.
4. Rousselle P, Beck K. Laminin 332 processing impacts cellular behavior.
Cell Adh Migr 2013; 7: 122-134.
Introduction: Inflammatory conditions associated with increased tumor necrosis
factor (TNF)-a result in increased collagen degradation due to upregulation of
matrix metalloproteinases (MMPs). Previous studies using organ-cultured
human skin explants indicated the involvement of MMP-3 in these collagen catabolic processes (1). We have studied the specific impact of MMP-3 with or
without inflammatory stimulus on collagen turn-over using a murine skin organ
culture model.
Methods: Full-thickness skin biopsies (8 mm) were excised from 3-month-old
MMP-3 knock-out (KO) male mice and transgene (TRANS) male mice overexpressing MMP-3 in the skin and their respective wild-type (WT) counterparts.
The skin explants from 5 animals in each group were incubated submerged
over 8 days without or with 10 ng/mL rmTNF-a under serum-free conditions.
Results: Collagen degradation, assessed by the release of hydroxyprolinecontaining peptides into conditioned media (1), was significantly (p < 0.00004)
reduced in skin explants from MMP-3 KO compared to WT over the 8-day culture period. Significantly more pro-MMP-9 was observed in MMP-3 KO conditioned media (p 5 0.02) while MMP-2, MMP-8, and MMP-14 tissue levels were
similar in the KO and WT-cultured skin explants. TNF-a treatment increased
collagen turn-over significantly in WT (p 5 0.005) and in KO (p 5 0.004) skin
explants. Concomitantly, TNF-a increased MMP-3 tissue levels in WT but not
in KO skin explants. MMP-3 overexpression in TRANS skin explants showed
similar collagen degradation compared to WT but responded to TNF-a with significantly increased (p 5 0.008) release of cleaved collagen compared to no
TNF-a addition.
Conclusions: TNF-a increased collagen degradation via up-regulation of MMP3 in normal but not in MMP-3 deficient murine skin. Intrinsic cutaneous overexpression of MMP-3 augments collagen metabolism only after inflammatory
challenge.
Reference
1. Ågren MS, Schnabel R, Christensen LH, Mirastschijski U. Tumor necrosis
factor-alpha-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo. Eur
J Cell Biol 2015; 94: 12-21.
ANTI-INFLAMMATORY EFFECT OF BOVINE LUNG SURFACTANT
IN CUTANEOUS WOUND HEALING
ACELLULAR DERMAL MATRICES IN VENTRAL HERNIA REPAIR:
CLINICAL EXPERIENCE OVER 6 YEARS
uhlbier2, P. Lindner3, F. Stahl3
U. Mirastschijski1, A. J. Caliani1, U. Zier1, A. F€
1
Center for Biomolecular Interactions Bremen, Department of Chemistry and
Biology, University of Bremen, Germany; 2Department of Plastic,
Reconstructive and Hand Surgery, Hannover Medical School, Hannover,
Germany; 3Institute for Technical Chemistry, Leibniz University, Hannover,
Germany
U. Mirastschijski1,2, C. Cedidi1
1
Department of Plastic, Reconstructive and Aesthetic Surgery, Klinikum
Bremen-Mitte, Bremen, Germany; 2Center for Biomolecular Interactions
Bremen, Department of Chemistry and Biology, University of Bremen, Bremen,
Germany
Aberrant skin wound healing is thought to derive, in part, from persistent
inflammation. Lung surfactants are used as standard therapy for reducing alveoC 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Introduction: After abdominal surgery or trauma, the incidence of ventral hernia formation is about 30%. The recurrence rate after herniotomy is even higher
with incidence up to 50%. Treatment with synthetic meshes is associated with
A20
Abstracts
an infection risk of 12%. This risk increases to 41% in cases of previous infection and the use of synthetic meshes is contraindicated under contaminated conditions. Therefore, biological materials are alternative treatments. Currently,
acellular dermal matrices (ADM) of porcine or human origin are used predominantly in plastic-reconstructive surgery.
Methods: In this retrospective study spanning 6 years, 18 patients with abdominal wall herniation after surgery (eight patients had hernia recurrence and eight
patients had been treated with synthetic mesh) underwent abdominal wall reconstruction by either sublay or sandwich technique. Cross-linked porcine (n 5 4;
PermacolTM), non-cross-linked porcine (n 5 13; StratticeTM) or non-cross-linked
human (n 5 2; EpiflexTM) ADM were used. Patients were examined clinically
and by ultrasound. Quality of life was assessed by the SF-12 questionnaire.
Results: One hernia recurrence was noted after cross-linked porcine ADM and
one with non-cross-linked porcine ADM. The latter ADM was replaced by
human ADM. One non-cross-linked porcine ADM was lost due to postoperative
wound infection and replaced by free microvascular autologous graft tissue
transfer. By ultrasound, matrices were visible for 3 to 6 months postoperatively;
thereafter the continuous dorsal rectus fascia was indistinguishable from the previously implanted ADM. Patients were highly satisfied with the postoperative
results, and were physically active and mobile.
Conclusions: ADM are perfectly suited for abdominal wall reconstruction after
tissue loss or for hernia repair and are revascularized and remodeled into autologous tissue. ADM can be used in contaminated conditions and are therefore
excellent alternatives to synthetic meshes. Hernia recurrence rates are low with
ADM. The major draw-back is the high unit cost of ADM.
BIGLYCAN AND ELASTIN EXTRACELLULAR MATRIX
BIOMARKERS IN HERNIAS
J. H. Mortensen1, L. Lorentzen2, N. A. Henriksen2, L. N. Jorgensen2,
M. S. Ågren2,3, M. A. Karsdal1, A.-C. B. Jensen1
1
Nordic Bioscience A/S, Herlev, Denmark; 2Digestive Disease Center,
Bispebjerg Hospital, University of Copenhagen, Copenhagen NV, Denmark;
3
Copenhagen Wound Healing Center, Bispebjerg Hospital, University of
Copenhagen, Copenhagen NV, Denmark
Introduction: Biglycan is an extracellular (ECM) protein that has a supportive
role for a functional ECM structure and regulates TGF-b and BMP-4, but is
also involved in elastin fiber formation. Elastin is a major component of elastic
fibers of the ECM which contribute to the elasticity of tissues. Biglycan has
never been studied in hernia patients and only a few studies have reported elastin alterations. The purpose of this study was to elucidate two ECM biomarkers
in relation to hernias in a pre-surgery hernia cohort and a follow-up cohort.
Methods: The two biomarkers BGM and EL-NE were measured in serum by
ELISA from a total of 81 samples, obtained from patients diagnosed with inguinal hernia (n 5 17), multiple hernia (n 5 21), incisional hernia (n 5 25), and in
patients without hernia (n 5 18). Venous blood samples were collected prior to
surgery and after a median of 3.7 years after surgery. The biomarker levels
were log-transformed. One-way ANOVA was applied.
Results: The serum levels of the biomarker BGM was significantly lower in
patients with inguinal hernias compared to healthy controls (p < 0.01). Postsurgery follow-up revealed that that BGM serum levels was normalized to the
serum levels of the healthy controls in patients with multiple and incisional hernias. Patient with inguinal hernias (p < 0.01) had a continuous significantly
decreased serum level of BGM. The serum levels of the biomarker EL-NE was
significantly elevated in the multiple hernias (p < 0.05) compared to healthy
hernia-free control patients. The post-surgery serum levels of EL-NE had normalized in patients with multiple hernias.
Conclusions: BGM and EL-NE were decreased in patients with inguinal hernias
compared to controls; both before and after surgery. This may indicate that persistent decreased serum levels of BGM and EL-NE can be contributing factor
to hernia development.
IDENTIFICATION OF GENE EXPRESSION PROFILES UNDERLYING
PREFERENTIALLY STIMULATED KERATINOCYTE WOUND
HEALING RESPONSES AND RE-EPITHELIALIZATION BY NOVEL
EPOXY-TIGLIANE PHARMACEUTICALS
R. Moses1, G. Boyle2, P. Reddell3, R. Steadman1, R. Moseley1
1
Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff,
United Kingdom; 2QIMR Berghofer Medical Research Institute, Brisbane,
Australia; 3QBiotics Limited, Yungaburra, Australia
Introduction: The novel epoxy-tiglianes, 12-tigloyl-13-(2-methylbutanoyl)-6,7epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (EBC-46) and 12-tigloyl-13-(2methylbutanoyl)-5,6-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (EBC-211),
occur within seeds of the Fontain’s Blushwood tree (Fontainea picrosperma), indigenous to Queensland’s tropical rainforest. EBC-46 is currently being developed by
QBiotics, as a human and veterinary anti-cancer pharmaceutical. In clinical studies,
A21
EBC-46 stimulates exceptional dermal wound healing responses following tumor
destruction. Our previous studies have demonstrated that EBC-46 and EBC-211
stimulate significant keratinocyte proliferative and migratory responses in vitro, supporting the enhanced epithelialization observed in treated skin. This study examined
the key genes differentially expressed following epoxy-tigliane treatment to elucidate
the underlying mechanisms of action responsible for these preferentially stimulated
keratinocyte responses.
Methods: Immortalized human epidermal keratinocytes (HaCaT) were cultured in
DMEM/1% fetal calf serum with EBC-46 or EBC-211 (0, 0.001, 0.1, or 10 lg/
mL), for 24 or 48 hours. Total RNA was extracted/purified and biotinylated, amplified antisense RNA (cRNA) obtained. Global gene expression analyses were performed by Microarrays (Human HT-12 v4 Expression BeadChips). Statistical
differences in gene expression were identified between epoxy-tigliane-treated and
untreated control cultures using GeneSpring, with Ingenuity Pathway Analysis elucidating the key pathways involved. Differentially expressed genes were confirmed
by protein analyses (Western blotting, ELISAs, and activity assays).
Results: Microarray analyses identified key genes differentially expressed in
EBC-46/EBC-211-treated HaCaT, which contribute to their stimulatory effects
on keratinocyte proliferation and migration. These differentially expressed genes
were verified using protein assays. Up-regulated genes included certain keratins
(KRT9, KRT13, KRT15, KRT81), positive cell cycle/proliferation regulatory
genes (CCNB2, CDKN3, CDCA7, GINS2, KIAA0101), and proteinases (matrix
metalloproteinase [MMP]-1, MMP-7, MMP-10). EBC-46/EBC-211 treatments
also downregulated other keratins (KRT6B, KRT16, KRT17) and many cytokine/growth factor/chemokine-related genes.
Conclusions: This study has identified the genes principally involved in mediating
enhanced keratinocyte proliferative and migratory responses following epoxytigliane treatment, highlighting the mechanisms of action of these novel pharmaceuticals and their potential as therapies for impaired wound healing situations.
Reference
1. Moses R, Reddell P, Steadman R, Moseley R. Preferential stimulation of
keratinocyte proliferation and migratory responses by novel tigliane pharmaceuticals contribute to enhanced wound reepithelialization. Wound Repair Regen
2014; 22: A92.
METALLOPROTEINASES, WOUND HEALING AND AGING
H. Nagase1
Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics,
Rheumatology and Musculoskeletal Sciences, University of Oxford, London,
United Kingdom
1
The timely degradation of extracellular matrix (ECM) is essential for morphogenesis and tissue remodeling as demonstrated by Gross and Lapière’s discovery of collagenase in tadpole undergoing metamorphosis. Injurious cutaneous damage
initiates blood coagulation, degranulation of platelets and inflammation, followed
by epithelialization, granulation tissue formation, and matrix remodeling. These
processes are highly orchestrated, involving activation of a series of cytokines,
growth factors, and ECM turnover accompanied by timely cell migration, proliferation and differentiation. The major proteolytic enzymes involved are serine proteases such as the plasminogen activator-plasmin system, the matrix
metalloproteinases (MMPs), the adamalysin-like metalloproteinases (ADAMs),
and the ADAMs with thrombospondin motifs (ADAMTSs). Upon aging, wound
healing process is impaired due to a decrease in cellular function and regenerative
response of the tissue. Several factors are altered: e.g., decreased immunity, epithelial cell migration and proliferation, collagen synthesis, and increased inflammation, reactive oxygen species (ROS), and MMP activities. The matricellular protein
CCN1 (also known as cysteine-rich protein 61) is elevated in aged and replicative
senescent dermal fibroblasts and increases ROS and MMP-1 production. Acute
hypoxia caused by blood clotting during wounding of skin induces keratinocytes to
secrete heat shock protein 90a (Hsp90a) that then interacts with LDL receptorrelated protein-1 (LRP1). This interaction stimulates the migration of keratinocytes, fibroblasts, and microvascular endothelial cells, and promotes wound closure. LRP1 is an endocytic receptor of many ligands including plasminogen
activators, MMPs and ADAMTSs and regulates ECM turnover. It may therefore be
an important receptor in cutaneous wound healing.
THE EFFECT OF A SYNTHETIC HEPARAN SULFATE COMPOUND
ON BREAKING STRENGTH OF COLONIC ANASTOMOSES AND
LAPAROTOMY WOUNDS: A RANDOMIZED AND BLINDED STUDY
IN RATS
M. Nerstrøm1, P.-M. Krarup1, L. N. Jorgensen1, M. S. Ågren1,2
Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark; 2Copenhagen Wound Healing Center, Copenhagen
NV, Denmark
1
Introduction: Wound dehiscence is a common postoperative complication in
gastrointestinal surgery. Heparin binding growth factors (HBGFs) stimulate cell
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
migration, proliferation and differentiation during wound healing. Synthetic
polymers derived from dextran including OTR4120 (OTR3, Paris, France) have
been developed to protect HBGFs from proteolytic degradation in injured tissues. These compounds have previously shown healing properties in different
tissues including colon and skin. Our aim was to investigate the effect of locally
applied OTR4120 on early healing of colonic anastomosis and laparotomy
wounds.
Methods: Fifty male Sprague-Dawley rats, weighing 220-319 g, were randomized to receive OTR4120 (n 5 25) or placebo (n 5 25). The peritoneal cavity
was accessed by a 40 mm midline incision and 10 mm of the left colon was
resected. Sterile OTR4120 (1 mg/linear mm) in saline or saline alone (placebo)
was applied locally to each colonic end and an end-to-end single layer anastomosis was constructed using eight interrupted 6/0 polyamide sutures. The
abdominal wall was closed by a midline suture, and OTR4120/placebo applied
to the incision, that was closed by 8 metal clips. On postoperative day 3, breaking strengths were determined of the anastomoses with sutures in situ and of
two 15-mm wide strips cut from the incisional wounds.
Results: Neither the breaking strength of the anastomoses (p 5 0.622, t-test) nor
the laparotomy wounds (p 5 0.737) differed significantly between the two
groups. The mean anastomotic breaking strength of the OTR4120 group was
1.52 N (95% confidence interval: 1.40-1.63 N) and of the placebo group 1.47 N
(1.34-1.60 N). The corresponding values for the laparotomy wounds were 0.67
N (0.55-0.78 N) and 0.64 N (0.58-0.71 N).
Conclusions: Single local application of the mimetic heparan sulfate compound,
OTR4120, did not increase the biomechanical strength of colonic anastomoses
or laparotomy wounds on postoperative day 3 in rats.
EARLY INFLAMMATORY DIFFERENCES BETWEEN
NORMOTROPHIC AND HYPERTROPHIC SCARS IN HUMANS
F. Niessen1
1
VUMC, Haarlem, The Netherlands
Introduction: Hypertrophic scar formation is a result of adverse cutaneous
wound healing. The pathogenesis of hypertrophic scar formation is still poorly
understood. A problem next to the lack of suitable animal models, is that most
studies compare normal skin to hypertrophic scar samples and rarely to normotrphic scar samples. Also often only one time point after wounding is
studied.
Methods: In this study we performed a microarray analysis on material of
human normotrophic scars (n 5 6) and hypertrophic scars (n 5 5) at six different
time points (before wounding and up to 52 weeks after wounding).
Results: RNA levels for several factors involved in different phases of cutaneous wound healing, especially in the inflammation and proliferation phase (e.g.,
interleukin [IL]-1a, CCL2, fibroblst growth factor [FGF]-2) were decreased in
hypertrophic scars compared to normotrophic scars. Gene levels were only
increased in hypertrophic scar compared to normotrophic scars for factors
involved in matrix production, remodelling or degradation (Col3A1, THBS1,
PLAU, TGFb3). The RNA data were confirmed by immunofluorescence protein
staining of tissue sections for the biomarkers CCL2, FGF-2 and TGF-b3.
Conclusions: Hypertrophic scar formation is claimed to be associated with an
exaggerated inflammation. In contrast, our data suggests that hypertrophic scar
formation is related to an extended down regulation of inflammatory and mitogenic genes whereas genes involved in matrix production remain up-regulated
thus resulting in an overproduction of extracellular matrix. This study has
enabled us to identify typical biomarkers characteristic for hypertrophic scar
formation.
MATRIX METALLOPROTEINASE EXPRESSION IN SIMPLE VERSUS
COMPLEX ANOCUTANEOUS FISTULAS
A. Nordholm-Carstensen1, L. N. Jorgensen1, M. S. Ågren1,2
1
Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark; 2Copenhagen Wound Healing Center, Bispebjerg
Hospital, University of Copenhagen, Copenhagen NV, Denmark
Introduction: Fistula-in-ano is characterized by a complex and not fully understood wound healing deficiency. Management strategies remain difficult and
often include the risk of fecal incontinence. Further clarification of the underlying molecular abnormalities could hold the potential of improved classification
of fistulas and future novel interventions. This study aimed to analyze potential
differences in MMP-2 and MMP-9 expression and activity between simple (SF)
and complex (CF) anocutaneous fistulas based on anatomical classification.
Methods: Fistula tissue was collected during elective surgery by pulling a cytobrush through the fistula tract. Patients had stable fistula-in-ano disease without
acute abscess formation. Tissues were washed in saline and extracted (10 ml
CNTZ buffer/mg tissue) for 24 hours at 48C. Tissue extracts were loaded at 1
mg total protein/lane together with 50 pg of MMP-2/MMP-9 and electrophoC 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
resed on 10% zymogram gel with 0.1% gelatin. MMP-2 and MMP-9 contents
were determined by densitometry.
Results: Ten patients, seven males, with a median age of 46 years were
included. Six patients had CF (high trans/suprasphincteric) and four SF (intersphincteric). There was a trend (p 5 0.056, t-test) of higher MMP-9 expression
in CF (216.2 6 44.1 pg/mg total protein, mean 6 SEM) than in SF (76.5 6 37.1
pg/mg). No statistically significant differences were detected regarding MMP-2
expression (CF: 9.5 6 3.3 pg/mg, SF: 13.1 6 7.7 pg/mg; p 5 0.627) or percentage
of active MMP-2 (CF: 33.1 6 6.9%, SF: 32.2 6 6.7%; p 5 0.937).
Conclusions: This study suggests that MMP-9 may be upregulated in CF compared to SF, whereas MMP-2 showed similar expression irrespective of the
complexity of the fistula. Larger patient series are needed for further clarification of MMPs role in the severity of fistula-in-ano. Finally, the use of a cytobrush instead of more invasive fistulectomy is a feasible and easy method to
obtain fistula tissue for molecular analysis.
A NOVEL ABSORPTIVE DRESSING CONTAINING OXYGEN
SIGNIFICANTLY INCREASES HEALING RATE IN PORCINE
DERMAL WOUNDS
J. Ollerenshaw1, L. K. S. Parnell2, D. F. Roe1, V. L. Young3
1
Halyard Health, Alpharetta, GA USA; 2Precision Consulting, Missouri City,
TX USA; 3Mercy Health Research, Washington, MO USA
Introduction: The importance of an adequate continuous oxygen supply to the
site of injury is well established in wound healing and for preventing infection.
Many wounds heal poorly due to co-morbidities resulting in inadequate tissue
perfusion and hypoxia, and clinical tools to help enrich hypoxic wound sites
with oxygen are lacking. This is a significant clinical problem for an increasing
number of patients at risk for severe complications from slowly healing or nonhealing wounds. We have examined the ability of a novel absorptive wound
dressing that contains oxygen to accelerate healing of dermal wounds in healthy
pigs, which is an established model of dermal wound healing in humans.
Methods: Ten juvenile farm pigs were each subjected to eight 6.25 cm2 fullthickness dermal wounds on the dorsal surface. Following surgery, 40 wounds
were treated with a novel absorptive oxygen-rich wound dressing, and the
remaining 40 with a control gauze dressing. Dressings were changed daily and
images collected for monitoring wound areas prior to euthanasia of all animals
at 28 days.
Results: Treatment with oxygen-rich wound dressing resulted in wound sizes
significantly smaller than for control gauze dressing treatment on all days measured after day 0 (p < 0.05 days 7 and 21; p < 0.01 days 14 and 28). The
oxygen-rich wound dressing treated sites were 23% of original wound size versus 40% for control at day 14, and 2% versus 13% at day 28.
Conclusions: These data show a significant difference in the reduction of
wound size following use of the oxygen-rich wound dressing compared to gauze
control dressing in the first 7 days using this predictive model. Results suggest
a potential reduction in treatment time and an improvement in quality of life for
clinical populations with dermal wounds.
A NOVEL ABSORPTIVE DRESSING CONTAINING OXYGEN
SIGNIFICANTLY INCREASES HEALING RATE IN AGED DIABETIC
PORCINE DERMAL WOUNDS
J. Ollerenshaw1, L. K. S. Parnell2, D. F. Roe1, V. L. Young3
1
Halyard Health, Alpharetta, GA USA; 2Precision Consulting, Missouri City,
TX USA; 3Mercy Health Research, Washington, MO USA
Introduction: Diabetic wounds are a serious healthcare problem associated
with delayed healing, higher complication rates, and increased cost of treatment.
These wounds heal poorly due to diabetes and create conditions that result in
inadequate tissue perfusion and hypoxia. The importance of oxygen is well
established in wound healing and for preventing infection. Clinical tools to help
enrich hypoxic wound sites with oxygen are lacking. We have examined the
ability of a novel absorptive wound dressing that contains oxygen to accelerate
healing of dermal wounds in aged diabetic pigs, as a model of dermal wound
healing in similarly conditions in humans.
Methods: Two aged diabetic Yucatan pigs were each subjected to eight
6.25 cm2 full-thickness dermal wounds on the dorsal surface. Diabetes was
induced at a young age in the animals through alloxan treatment to remove pancreatic beta cells. Following surgery, 8 wounds were treated with the novel
absorptive oxygen-rich wound dressing (4 per animal), and the remaining 8
wounds (4 per animal) were treated with a control gauze dressing. Dressings
were changed daily and images collected from monitoring wound areas prior to
euthanasia of all animals at 52 days.
Results: Treatment with the oxygen-rich wound dressing resulted in smaller
wound sizes than for control gauze dressing treatment on days measured after
day 0. The oxygen-rich wound dressing treated sites were 22% of the original
wound size versus 43% for control at day 25 and 3% versus 13% at day 52.
A22
Abstracts
Conclusions: These data show a substantial difference in the reduction of
wound size following use of the oxygen-rich wound dressing compared to gauze
control dressing particularly in the first 25 days using this predictive model.
Results suggest a potential reduction in treatment time and an improvement in
quality of life for diabetic and aged populations with dermal wounds.
ANTICANCER TREATMENTS PROMOTE PREMATURE
SENESCENCE OF MESENCHYMAL STEM CELLS: IMPLICATIONS
FOR TISSUE HOMEOSTASIS
A. Papadopoulou1, D. Kletsas1
1
Laboratory of Cell Proliferation and Ageing, IB-A, NCSR Demokritos, Athens,
Greece
Introduction: Mesenchymal stem cells (MSCs) play a major role in tissue
regeneration. As other normal somatic cells, they can undergo replicative- or
stress-induced senescence (SIPS). One side-effect of various anticancer treatments is the induction of premature senescence of somatic cells. However,
the long-term effect of these treatments on MSCs has not been studied in
detail.
Methods: Human bone marrow and adipose-derived mesenchymal stem cells
were isolated and characterized for surface marker expression by FACS
analysis. They were serially exposed to non-cytotoxic doses (estimated with
the MTT assay) of ionizing radiation and chemotherapeutic drugs (taxol,
cisplatin, doxorubicin) and, after subculture, the cells were tested for the
induction of senescence (measured by SA-b-Gal staining and BrdU incorporation). Expression of specific markers and signaling pathways were evaluated by Western analysis and qRT-PCR. Finally, the differentiation
capacity of treated MSCs was estimated by the appropriate staining procedures and the expression of specific transcription factors for each differentiation lineage.
Results: Serial exposure of MSCs to non-cytotoxic doses of ionizing radiation
and chemotherapeutic drugs provokes premature senescence. Interestingly,
although in most cases this was the effect of the activation of the DNA
damage response (DDR) observed by the activation of the Chk2-p53p21WAF1-pRb axis, SIPS was also provoked by taxol which does not activate
a DDR. Prematurely senescent MSCs, similarly to senescent somatic cells,
express also a catabolic and pro-inflammatory phenotype. Furthermore, senescent MSCs have a reduced ability to differentiate towards various lineages
(adipogenic, chondrogenic and osteogenic) and a reduced expression of the
specific transcription factors PPARc, Sox9, and Runx2, respectively. The paracrine effect of media conditioned by senescent MSCs on skin fibroblasts will
also be discussed.
Conclusions: Our data indicate that several treatment regimes may have as a
side effect the induction of premature senescence of MSCs, most probably
reducing their regenerative capacity.
MESENCHYMAL STROMAL CELL IMMUNE MODULATION:
INTEREST IN BURN TREATMENT?
J. Peltzer1, S. L. Burel1, F. Montespan1, C. Thepenier1, G. Uzan2, J.-J.
Lataillade1
1
Unit"e de Th"erapie Cellulaire Et R"eparation Tissulaire, H^
opital Percy, Clamart,
France; 2Inserm U1197, Villejuif, France
Introduction: Mesenchymal stem cells (MSCs) have been tested for many
years in several clinical trials for their modulation properties of migration, proliferation, functional activation, or apoptosis. These properties could be of great
interest for regenerative medicine and especially for burn treatment. Potential
interest in MSCs for burn treatment is not limited to skin wound repair (1).
Indeed, because of their immunomodulatory properties, one could contemplate
using MSCs to modulate the massive lymphocyte apoptosis occurring in severe
burns and=or their frequent septic complications. Besides, MSC immune modulatory properties have recently gained attention from the sepsis community. For
example, it has been shown that MSCs enhance septic mice survival partly by
inducing macrophage release of interleukin-10, thereby reducing inflammation
(2). MSC efficacy is variable and related to cell sources and donors. It seems
possible to deeply modify the secreted factor profiles of MSCs with different
cytokine stimuli to license MSCs for a better paracrine potential. Therefore, our
work focuses on the identification and selection of the most effective combination of bone marrow-MSC donors and cell-licensing procedures in lymphocyte
apotosis/dysfunction inducing models.
Methods: Human peripheral blood mononuclear cells were collected from
whole blood samples and intoxicated or not by lipopolysaccharide for 24 hours
and then activated by phytohemagglutinin (PHA). We evaluated the effect of
various donors of naive or primed MSCs on lymphocyte proliferation restoration. Results and
Conclusions: Lymphocyte intoxication induced a reduction in response to PHA.
This response could be partially restored when lymphocytes were co-cultured
A23
with MSCs. We obtained contrasted effects of cytokine licensing procedure
probably due to an important between-donor variability.
References
1. Leclerc T, Thepenier C, Jault P, Bey E, Peltzer J, Trouillas M, Duhamel
P, Bargues L, Prat M, Bonderriter M, Lataillade JJ. Cell therapy of burns. Cell
Prolif 2011; 44 Suppl 1: 48-54.
2. N"emeth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K,
Robey PG, Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star
RA, Mezey E. Bone marrow stromal cells attenuate sepsis via prostaglandin
E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 2009; 15: 42-9.
ROLE OF EXTRACELLULAR MATRIX COMPONENTS IN THE
PROLIFERATIVE EFFECTS OF TGF-BETA ON FETAL VERSUS
ADULT HUMAN SKIN FIBROBLASTS
H. Pratsinis1, A. Armatas1, E. Mavrogonatou1, M. Angelopoulou1,
A. Kouroumalis1, D. Kletsas1
1
Laboratory of Cell Proliferation and Ageing, IB-A, NCSR Demokritos, Athens,
Greece
Introduction: Based on our previous observations, transforming growth factor
(TGF)-b regulates the proliferation of normal human skin fibroblasts according
to their developmental origin. More specifically, TGF-b inhibits fetal fibroblasts
via the activation of protein kinase A (PKA) and the induction of cyclindependent kinase inhibitors p21CIP1/WAF1 and p15INK4B, while it stimulates
the proliferation of adult ones by the release of fibroblast growth factor (FGF)-2
and the subsequent activation of the MEK-ERK pathway. Aim of our current
work is to delineate the role of various extracellular matrix (ECM) components
in this differential proliferative response of fibroblasts to TGF-b.
Methods: Human fetal and adult skin fibroblasts were cultured in the presence
of fibronectin or type I collagen or hyaluronic acid (HA), as well as, on top or
within three-dimensional matrices of polymerized collagen. Alternatively, the
cells were cultured in the presence of hyaluronidase, to digest endogenously
produced HA. Their proliferative response to TGF-b was studied using tritiated
thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors.
Results: Fetal skin fibroblast-proliferation was inhibited by TGF-b, while that
of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was influenced through the autocrine
activation of FGF receptor and the MEK–ERK pathway, in line with our previous reports for cultures in the absence of exogenous ECM components. On the
other hand, in the presence of hyaluronidase, the inhibitory effect of TGF-b on
fetal fibroblasts was abrogated.
Conclusions: The differential response of fetal versus adult skin fibroblasts to
TGF-b seems to be a general phenomenon, persisting also in three-dimensional
cultures. As it is HA-dependent, it probably reflects the different repair strategies followed in these developmental stages
USE OF QUALITY OF LIFE DATA TO INFORM CLINICAL TRIAL
OUTCOMES
P. Price1
1
Health Science, VCO Cardiff University, Cardiff, United Kingdom
Patient reported outcomes evaluate the risks and benefits of therapy from the
patient’s perspective. Initially considered subjective, efforts to develop valid and
reliable instruments have resulted in recommendations on standard tools and methods for patient reported outcomes in clinical trials across a range of health states,
particularly chronic and degenerating conditions. Physiological measures are important to clinicians, but can be of limited interest to patients particularly as they often
correlate poorly with functional capacity and well-being. In the past 20 years there
has been an increase in methodological rigour, an emphasis on analytic approaches,
interpretation of scale scores, cultural and language issues, as well as the development of shorter measures. This presentation covers the important role quality of life
data can play in establishing the evidence base for therapies.
CHARACTERISATION AND QUANTITATIVE IMAGE ANALYSIS OF
IN VITRO INTERACTIONS OF THE ALGINATE OLIGOMER
(OLIGOG CF-5/20) WITH PSEUDOMONAL BIOFILMS
M. Pritchard1, E. Ferguson1, L. Powell1, E. Onsøyen2, P. Rye2, K. Hill1,
D. Thomas1
1
Advanced Therapies Group; Cardiff School of Dentistry, Cardiff, United
Kingdom; 2AlgiPharma AS, Sandvika, Norway
Introduction: OligoG CF-5/20 is a novel alginate oligosaccharide which has been
previously shown to potentiate the effect of antibiotics against a range of multi-drug
resistant bacteria including Pseudomonas, Burkholderia and Acinetobacter spp. in vitro.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
Initial studies demonstrated the ability of OligoG to alter charge of the bacterial cell surface. In these studies, we sought to characterize and quantify the anti-biofilm effects of
OligoG in vitro, employing both OligoG and a fluorescently labeled derivative.
Methods: The ability of OligoG to penetrate and disrupt biofilms of the mucoid
Pseudomonas aeruginosa isolate (NH53788A) was studied in vitro. OligoG was
R (TxRd) cadaverine. Unestablished
conjugated to the fluorophore Texas-RedV
and 24-hour established biofilms were tested with labeled- and unlabeledR and SYTO-9V
R confocal laser scanning microscopy was
OligoG. LIVE/DEADV
employed to study the disruption of biofilm formation. Controls to test for
inherent anti-biofilm properties of TxRd and possible dissociation of the conjugate were also performed. Disruption of the biofilm was evaluated using COMSTAT image analysis software to quantify alterations in biofilm formation.
Results: Dose-dependent effects on NH53788A biofilm formation were evident
when treated with OligoG, demonstrating inhibition of growth, as well as disV
ruption of established biofilm. TxRd -labeled OligoG demonstrated similar in
vitro efficacy. COMSTAT analysis revealed a significant decrease in biofilm
volume/height with increase in the dose of labeled OligoG (0.5-6%; p < 0.05)
establishing that TxRd-labeled OligoG diffused through the entire biofilm depth.
TxRd alone showed no evidence of biofilm disruption or dissociation.
Conclusions: These studies support the previously reported in vitro anti-biofilm
properties of OligoG and quantify, for the first time, the extent of this disruption. The imaging studies using TxRd, provided an insight into the interaction
of OligoG with the biofilms’ extracellular polymeric substance, visualizing the
diffusion of OligoG in a pseudomonal biofilm.
R
TREATMENT OF SKIN CHRONIC WOUNDS IN ELDERLY PATIENTS
WITH TOPICAL PHARMACEUTICAL COMPOSITION CONTAINING
AS ACTIVE INGREDIENT A MIXTURE OF POLLEN EXTRACT AND
VEGETABLE OIL UNSAPONIFIABLES: PRELIMINARY DATA ON A
PERSONAL SERIES
A. Ragno1, D. Marsili1, L. S. M. Martin1, A. Silvestri1, G. L. Limiti1,
D. Pierangeli1, A. E. Catucci1
1
Unit of Internal Medicine “Regina Apostolorum” Hospital, Albano Laziale
(Rome), Italy
Introduction: Chronic skin wounds (CSW) are frequent in clinical practice.
The impact of age and accompanying multi-morbidity on the effectiveness of
existing and emerging treatment approaches for CSW is unknown (1). Pollen
extracts and unsaponifiables fractions of vegetable oils are trophic, moisturizing
and anti-inflammatory for skin (2).
Methods: We included 5 males and 4 females (79.9 6 9.2 years, mean 6 SD) suffering
from chronic venous insufficiency (n 5 3), diabetic foot ulcers (n 5 2) and CSW of
other etiologies (n 5 4). The CSW and surrounding healthy skin were treated topically
with the original pharmaceutical composition (3) twice daily. The pain was evaluated
during formulation application by visual analogue scale (VAS) and time to complete
closure was recorded. Adjuvant systemic therapy was administered when indicated.
Results: The pain VAS scores were 7.4 6 0.7 before initiation of the local therapy and 3.3 6 0.9 at the final visit. The time to complete healing of the CSW
was 25.3 6 4.9 days. Antihypertensive therapy, antibiotics, diuretics, acetylsalicylic acid, low molecular weight heparin, oral anticoagulant, and albumin were
given during the study period.
Conclusions: Treatment of CSW in elderly patients with the topical pharmaceutical composition was effective and quickly resolved the skin lesions. Controlled
studies are needed to confirm our preliminary findings.
References
1. Gould L, Abadir P, Brem H, Carter M, Conner-Kerr T, Davidson J,
DiPietro L, Falanga V, Fife C, Gardner S, Grice E, Harmon J, Hazzard WR,
High KP, Houghton P, Jacobson N, Kirsner RS, Kovacs EJ, Margolis D,
McFarland Horne F, Reed MJ, Sullivan DH, Thom S, Tomic-Canic M, Walston
J, Whitney JA, Williams J, Zieman S, Schmader K. Chronic wound repair and
healing in older adults: current status and future research. J Am Geriatr Soc
2015; 63: 427-38.
2. Aburjai T, Natsheh FM. Plants used in cosmetics. Phytother Res 2003;
17: 987-1000.
3. De Gregorio C. U.S. Patent No.: 6,342,255 B1; 1/2002.
BETA CATENIN ASSOCIATES WITH CANCER-RELATED GENES IN
DUPUYTREN’S DISEASE
C. Raykha1, G. Gloor2, D. O’Gorman3
1
Roth Mcfarlane Hand and Upper Limb Centre, University of Western Ontario,
London, Canada; 2Department of Biochemistry, University of Western Ontario,
London, Canada; 3Lawson Health Research Institute, University of Western
Ontario; St. Joseph’s Hospital, London, Canada
Introduction: Dupuytren’s disease (DD) is a benign fibrosis of the palmar fascia.
Like other fibro-proliferative diseases including malignant tumors (1), increased
levels of beta catenin, a trans-regulator of gene expression, and dysregulated gene
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
transcripts are evident in primary fibroblasts derived from DD tissues relative to
controls (2, 3). The identities of the genes that associate with, and are potentially
trans-regulated by, beta catenin in DD have not been reported. We have performed
chromatin immunoprecipitation sequencing (ChIP-seq) to identify these genes with
the aim of detecting novel therapeutic targets for attenuating DD development.
Methods: Primary fibroblasts were derived from the fibrotic palmar fascia of three
patients (DD cells), the adjacent, macroscopically uninvolved palmar fascia in these
patients (PF cells), and normal palmar fascia from three patients (CT cells). ChIP
experiments were performed with antibodies to beta catenin, histone H3 (positive control) and with non-specific IgG (negative control). Peaks of beta catenin interaction
with the genome were identified by sequencing and Model-based Analysis of ChIPseq. Ingenuity Pathway Analyses (IPA) were utilized to identify disease and biological
function associations between the genes identified in the ChIP Seq analysis.
Results: A total of 11, 528 unique peaks of beta catenin association with the
genome were identified in all three DD cell cultures, compared to 2,312 unique
peaks of beta catenin association in all three PF cell cultures and 4,535 unique
peaks of beta catenin association in all three CT cell cultures. IPA analyses
revealed a robust and significant increase in associations between beta catenin
and cancer-related genes in DD cells relative to both PF and CT cells.
Conclusions: These findings implicate roles for beta catenin-mediated trans-regulation of gene expression in DD, and suggest that DD and malignant fibroproliferative diseases share some common pathogenic mechanisms.
References
1. Bhattacharya B, Dilworth HP, Iacobuzio-Donahue C, Ricci F, Weber K,
Furlong MA, Fisher C, Montgomery E. Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and
myofibroblastic lesions. Am J Surg Pathol 2005; 29: 653-9.
2. Varallo VM, Gan BS, Seney S, Ross DC, Roth JH, Richards RS, McFarlane RM, Alman B, Howard JC. Beta-catenin expression in Dupuytren’s disease: potential role for cell-matrix interactions in modulating beta-catenin levels
in vivo and in vitro. Oncogene 2003; 22: 3680-4.
3. Satish L, LaFramboise WA, Johnson S, Vi L, Njarlangattil A, Raykha C,
Krill-Burger JM, Gallo PH, O’Gorman DB, Gan BS, Baratz ME, Ehrlich GD,
Kathju S. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren’s Contracture. BMC Med Genomics 2012; 5: 15.
FIBRIN AS A DELIVERY TOOL
H. Redl1,2
1
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology/
AUVA Research Center, Vienna, Austria; 2Austrian Cluster for Tissue
Regeneration, Vienna, Austria
Fibrin is the end result of blood clotting. It forms hydrogel with polymerized network of fibrin fibres by the action of thrombin on fibrinogen monomers. Since the
1970s commercially prepared fibrin sealants consisting of human plasma derived
concentrated fibrinogen and thrombin have been used in clinics for tissue sealing
and hemostasis. We have been involved in the development of appropriate application tools and preclinical models. Due to its additional properties such as support of
wound healing and its complete in vivo resorption fibrin has also been used as a
potent biomaterial. By adaption of fibrinogen and thrombin concentration, addition
of fibrinolysis inhibitors and using linking technologies we will show the use of
fibrin as a hydrogel to “train” cells in bioreactors, to deliver cells or growth factors.
In addition, we have used fibrin as a gene activated matrix for non-viral gene therapy. Such an “old drug” has a lot of future in the tissue repair/regeneration area.
THE INFLUENCE OF HUMAN ACUTE WOUND FLUID ON THE
ANTIMICROBIAL PERFORMANCE OF SILVER AND
POLYHEXAMETHYLENE BIGUANIDE CONTAINING FOAM
DRESSINGS – AN IN-VITRO ASSESSMENT
ohm1, N. Sch€afer1, E. K. St€
urmer1
J.-D. Rembe1, C. Fromm-Dornieden1, J. B€
Institute for Research in Operative Medicine, University of Witten/Herdecke,
Campus Cologne-Merheim, Cologne, Germany
1
Introduction: Treating chronic and/or infected wounds still represents a major
challenge. A targeted and individualized therapeutic regimen according to
wound condition is desirable and systemic antibiotic therapy should remain a
last resort. Various interactions of antiseptic dressings with wound environments
regarding antimicrobial potency remain unclear and comparative analyses are
lacking. Therefore, this work aimed to investigate the antimicrobial efficacy of
different antiseptic foam dressings in vitro against the typical wound flora challenged with human acute wound fluid (AWF).
Methods: Eight antiseptic polyurethane foam dressings containing either a silver
formulation or polyhexamethylene biguanide (PHMB) were assessed regarding
their antimicrobial potency against Staphylococcus aureus, Escherichia coli and
Pseudomonas aeruginosa using a modified time-kill-assay based on ISO EN
A24
Abstracts
20743. The antiseptic efficacy was evaluated standardly as well as under the
influence of human AWF after 2, 4, 6, and 24 hours.
Results: The specific chemical formulation and concentration of the antiseptic substance (ionic silver, silver sulfadiazine, nanocrystalline silver, PHMB 0.1% and
PHMB 0.5%) embedded within the dressings seemed to play a key role. P. aeruginosa showed a significantly higher survival rate than S. aureus or E. coli in vitro.
The overall survival rate was remarkably low under the influence of AWF compared
to unchallenged test series, regardless of the investigated bacterial strain. Unchallenged the efficacy of PHMB was comparable to silver against P. aeruginosa and
even significantly superior against S. aureus and E. coli, while challenged with AWF
the reduction rates for silver even exceeded those of PHMB for P. aeruginosa.
Conclusions: Within a challenging wound environment, silver surpasses PHMB
in terms of bacterial reduction. Regarding the presented in vitro results, the biomolecular interactions of antiseptic wound dressings with the human wound
environment should be part of more extensive investigations, considering varying factors such as bacterial species and wound microenvironment to develop
targeted therapeutic regimens for the individual patient.
COMPARISON OF HEMOSTATIC WOUND PADS USING A NEW
SPECTROPHOTOMETRIC COAGULATION ASSAY
J.-D. Rembe1, J. B€ohm1, C. Fromm-Dornieden1, N. Sch€afer1, M. Maegele2, M.
Fr€ohlich2, E. K. St€urmer1
1
Institute for Research in Operative Medicine, University of Witten/Herdecke,
Campus Cologne-Merheim, Cologne, Germany; 2Department of Traumatology,
Orthopaedic Surgery and Sports Traumatology, Cologne-Merheim Medical
Centre (CMMC), Witten/Herdecke University, Campus Cologne-Merheim,
Cologne, Germany
Introduction: The number of elderly patients depending on oral anticoagulation
is expected to rise prospectively due to demographical changes. Prolonged
bleeding times after traumatic injuries represent the drawback of these medications. This is the case not only in major trauma, but also in trivial situations.
Therefore, plasters capable to accelerate coagulation onset and shorten bleeding
times are desirable for these patients.
Methods: The hemostatic potential and physical properties of different types of
wound pads (standard wound pad, two alginates, chitosan, collagen, oxidized
cellulose, and kaolin-impregnated gauze) were assessed in vitro. For this purpose the clotting times of blood from healthy volunteers were compared with
those of patients treated with coumarin derivatives or acetylsalicylic acid by
using a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity.
Results: The fastest coagulation onset was observed for oxidized cellulose (Ø
2.47 minutes), Lantor alginate (Ø 2.50 minutes) and the kaolin-impregnated
gauze (Ø 3.01 minutes). Chitosan (Ø 5.32 minutes) and the collagen (Ø 7.59
minutes) induced clotting comparatively late. Regarding physical parameters,
the kaolin-impregnated gauze showed the lowest while chitosan and both alginates achieved the highest absorption capacity and speed. Oxidized cellulose
performed well regarding clotting, but revealed weaknesses in terms of absorption capacity.
Conclusions: All tested specimens seem to induce clotting independently from
the administered type of oral anticoagulant. In this regard, the kaolinimpregnated gauze, oxidized cellulose and Lantor alginate were superior to chitosan and collagen. Due to its additional well-known positive effect on wound
healing Lantor alginate should be considered for further investigations.
POLYAMINOPROPYL BIGUANIDE AS AN ALTERNATIVE FOR
POLYHEXAMETHYLENE BIGUANIDE FOR BACTERIAL
ERADICATION IN WOUND HEALING
1
1
1
1
1
ohm , N. Sch€afer , E. K. St€
urmer
J.-D. Rembe , C. Fromm-Dornieden , J. B€
Institute for Research in Operative Medicine, University of Witten/Herdecke,
Campus Cologne-Merheim, Cologne, Germany
1
Introduction: In this study, the efficacy of polyaminopropyl biguanide (PAPB)
as an alternative antiseptic agent compared to the molecular closely related polyhexamethylene biguanide (PHMB) was evaluated in vitro.
Methods: Cytotoxicity of both substances against human keratinocytes (HaCaT)
and murine fibroblasts (L929) was determined according to ISO EN 10993-5.
Tests of antimicrobial efficacy were performed via identification of the minimal
bactericidal concentration (MBC), quantitative suspension method for substances and investigation of two dressings containing either PAPB or PHMB against
Staphylococcus aureus, Eschericia coli and Pseudomonas aeruginosa, according
to international standards.
Results: PHMB was highly cytotoxic even in low concentrations for both tested
cell lines, but showed a high antimicrobial efficacy against S. aureus and E.
coli. In case of PAPB, no or only low cytotoxic impact was detected after 72
hours, while similar antibacterial features like PHMB could not be confirmed,
as PAPB showed no relevant antimicrobial effects.
A25
Conclusions: Even though chemically closely related, PAPB is not as effective
as PHMB regarding bacterial eradication. It could not be evaluated as an equivalent alternative, whilst PHMB has proven to be highly effective. With bacterial
resistances further rising and established substances like PHMB being in discussion of carcinogenicity, the discovery of effective alternative antiseptic agents is
mandatory. Nevertheless, it needs to be emphasized that agents like PHMB and
PAPB which are chemically closely related do not necessarily exhibit the same
efficacy or effects and need to be carefully distinguished and assessed prior to
using them in different indications.
THE SURFACE Q-SNARE COMPLEX STX4/SNAP23 REGULATES
DELIVERY OF MMP-14 TO THE PLASMA MEMBRANE IN
MACROPHAGES
J. R€
ohl1, A. Zaharia1, R. Z. Murray1
Institute of Health and Biomedical Innovation, School of Biomedical Sciences,
Faculty of Health, Queensland University of Technology, Brisbane, Australia
1
Introduction: Elevated levels of matrix metalloproteinases (MMPs) contribute
to excessive tissue damage and prolong inflammation. MMPs also enable macrophages to infiltrate the wound tissue. Increased macrophage numbers amplify
inflammation by secreting large amounts of MMPs and pro-inflammatory cytokines. Treatments for chronic wounds targeting secreted MMPs have not proven
to be very effective. Preventing MMPs reaching the cell surface where they
access their substrates in the extracellular milieu could lead to better therapeutics. Methods/Results: We have shown that in unactivated macrophages soluble
MMP-9 and membrane-anchored MMP-14 are expressed at low levels. Upon
activation of macrophages with the bacterial cell wall component lipopolysaccharide these enzymes are then dramatically upregulated and secreted comparable to what is observed in the chronic wound environment. Disruption of the
Golgi complex using brefeldin A showed that the newly synthesized pools of
MMP-9 and MMP-14 are trafficked through this organelle en route to the cell
surface. To identify the responsible trafficking machinery proteins, we performed siRNA knockdown of different SNARE proteins that mediate distinct
fusion events of vesicles with organelles and ultimately the plasma membrane.
Fusion-competent SNARE complexes consist of one R-SNARE and two/three
Q-SNAREs. We have shown that the Golgi R-SNARE VAMP4 and the late
endosome/lysosome R-SNAREs VAMP7 and VAMP8 play a role in trafficking
of MMP-14. It was also found that the surface Q-SNARE complex Stx4/
SNAP23 regulates the final step in the delivery of newly synthesized MMP-14
to the cell surface. Furthermore, when disrupting the Stx4/SNAP23 complex
macrophages lose the ability of gelatin matrix degradation due to the decreased
MMP-14 surface levels.
Conclusions: MMP-9 and MMP-14 are transported through the Golgi complex
to the plasma membrane. The MMP-14 pathway is regulated by Stx4/SNAP23
and also VAMP4, VAMP7, and VAMP8. Targeting these identified SNARE
proteins could prevent MMP surface delivery, reduce macrophages numbers and
improve wound healing.
DEVELOPMENT AND VALIDATION OF A SIMPLE AND COMMON
METHOD FOR THE EXTRACTION OF EPIDERMAL CELLS
G. Rolin1, Y. Wang2, C. Viennet2, M. Tissot2, P. Muret2, P. G. Humbert1,3
Cutaneous Engineering and Biology Laboratory, Inserm UMR 1098, University
of Franche-Comt"e, Besançon, France; 2Clinical Investigation Centre, Inserm
1431, University Hospital, Besançon, France; 3Dermatological Department,
University Hospital, Besançon, France
1
Introduction: Cell extraction is an inevitable and critical step in the development and production of advanced therapy medicinal products (ATMP) as for
the establishment of primary cell bank. Concerning cells derived from human
skin, such as keratinocytes and melanocytes, enzymatic techniques described in
literature are usually based on trypsin, alone or in combination with dispase.
Such enzymes are used in order to separate dermis from epidermis and subsequently provide a suspension of epidermal cells. The implementation of such
protocols is often operator-dependent, not suitable for small samples and
requires animal derived products (not compatible with clinical approach). The
objective of this work was to define and validate an epidermal cell extraction
method being the simplest, the most effective and applicable to smaller samples
and avoiding animal reagents.
Methods: A new animal-free product (TrypLETM, Life Technologies) has been
tested on skin biopsies. The extraction efficiency was judged on the following
criteria: separation epidermis/dermis, cultured melanocytes and keratinocytes,
functionality of the extracted cells.
Results: Data obtained showed the ease of separation between the dermis and
epidermis on the one hand and between the epidermal cells on the other. A
minimum size of skin sample was defined to allow the extraction of functional
cells (i.e., melanocytes and keratinocytes).
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
Conclusions: This optimal method opens new perspectives for establishment of
optimal epidermal cell lines suitable for cutaneous pathophysiology research
and production of ATMP.
CORRELATION BETWEEN CHRONIC WOUND BED AND
SURROUNDING SKIN TEMPERATURE WITH A CLINICAL WOUND
BED SCORE
M. Romanelli1, P. Salvo2, V. Dini1, F. Di Francesco2, M. Macchia1, S. Panduri1,
A. Janowska
1
Department of Dermatology, University of Pisa, Pisa, Italy; 2Department of
Chemistry, University of Pisa, Pisa, Italy
Introduction: Chronic wounds are characterized by defective remodeling of the
extracellular matrix and prolonged inflammation. To obtain biochemical and
physical information about the wound bed and the surrounding skin different
options of non-invasive and objective measurements have been developed. Noninvasive temperature assessment of wound bed and perilesional skin could be a
reliable parameter of inflammation and infection.
Methods: We included 22 patients affected by venous leg ulcers. The chronic
wounds were evaluated by two physicians blinded to each other. The first investigator measured the temperature of the wound bed by a handheld infrared thermometer (Fluke Ti9). The second investigator evaluated the wounds clinically
using a wound bed score validated in 2006.
Results: The correlation of data was determined following the scatter plot for
the wound bed score and the temperature of the skin. The coefficient of determination (r2 5 0.65) indicated that about 65% of the variations could be
explained by the linear regression model. Therefore, instead of linearity, we
benchmarked the monotonicity of data by the Spearman’s rank-order coeffiR . The coefficient, q, is 0.805
cient. Calculations were performed using MatlabV
with a p-value of 2#1026 (confidence level 95%). This result proves a monotonic increasing relationship between the wound-bed score and the temperature of the skin. The data set composed by the wound-bed score and the
temperature of the perilesional skin returned q 5 0.55 and p 5 0.005 (confidence level 95%).
Conclusions: Although in this case the monotonicity is weak, the result suggests that this correlation is worth being further investigated with a larger data
set.
EPIDERMAL FRACTIONAL SKIN GRAFTING AND NEGATIVE
PRESSURE WOUND THERAPY IN A SELF-INDUCED BREAST
WOUND
M. Romanelli1, A. Janowska1, V. Dini1, S. Panduri1, M. Macchia1
1
Department of Dermatology, University of Pisa, Pisa, Italy
Introduction: Factitious wounds represent a challenging medical aspect in
terms of diagnosis, treatment and patient compliance.
Methods: In April 2015, a 34-year-old woman was attending our clinic
because of an injury due to toothpaste injection into her right breast. The
patient reported a history of bipolar disorder since long time, type II diabetes
and episodes of self-harm. Clinically, the self induced area was presenting
with a necrotic wound, with most of the right breast lower quadrants heavily
damaged and two fistulae. The necrotic wound had been treated with surgical
debridement, IV antibiotic therapy and a partial mastectomy. The wound had
improved and patient started with a 2-week of negative pressure wound therapy (NPWT). During treatment the lesion has decreased significantly in size
and the patient reported reduction of level of pain. To facilitate final wound
closure the epidermal fractional grafting technique was performed as the last
approach.
Results: The grafts obtained with an epidermal fractional system in combination
with NPWT and warmth, results in thin skin sections, with constant orientation
of epidermal skin from dermal-epidermal junction. The first follow-up was performed after 3 days and then at days 7 and 14.
Conclusions: We observed the complete wound healing with a good cosmetic
result thanks to the combination of these two tissue repair techniques.
DECELLULARIZED HUMAN DERMAL MATRICES PRODUCTION
FOR THE TREATMENT OF BURN PATIENTS: DEVELOPMENT OF
QUALITY CONTROL METHODS
T. Rose1, J. P. Draye1, G. Verween 1, G. Verbeken 1, S. Jennes 1, J. P. Pirnay 1,
D. Devos 1, M. Boone2, R. Ekila1,3, S. Delcave 1,3, S. Efesotti1,3,
V. del Marmol2, G. B. E. Jemec4
1
Burn Wound Center, Queen Astrid Military Hospital, Brussels, Belgium;
2
Hopitale Erasme, Dermatology, Brussels, Belgium; 3LabMCT, Brussels,
Belgium; 4Roskilde Hospital, Dermatology, Copenhagen, Denmark
Introduction: For patients with severe burn injuries, timely removal of
necrotic tissue and early skin grafting is critical. For those patients with very
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
restricted donor sites, availability of dermal substitutes could be beneficial.
The purpose of this work was the development of quality control methods for
a skin decellularisation process allowing the preparation of sufficient amount
of Decellularized human dermal matrices (DHDM) to treat patients with large
burns. Cryopreserved allogeneic human skin (0.3-0.4 mm thick), obtained
from deceased human donors after ethics committee approval, was used to
prepare DHDM.
Methods: A two-steps decellularization method was developed to prepare the
DHDM. The epidermis of allogeneic skin samples was removed after a first
incubation in the presence of NaCl (1 M) at 378C for 24 hours. The resulting
dermal samples were subsequently incubated in 0.5% Triton X-100 for 24h at
room temperature with continuous agitation for the removal of cell debris. After
this incubation, the dermal samples were washed several times in phosphatebuffered saline to remove the detergent and thereafter were cryopreserved In
addition to bacteriological/mycological testing and histological evaluation, MTT
viability testing and high-definition optical coherence tomography (HD-OCT)
methods were developed and used to evaluate the quality of the resulting dermal matrices. Furthermore, a test to evaluate the removal of residual detergent
(Triton X-100) was developed.
Results: Results showed that MTT test and histology were useful to evaluate
the removal of cell and cell debris and HD-OCT images were beneficial to
evaluate the three dimensional architecture of the DHDM (dermal papillae and
vascular spaces). Repeated washes (n 5 6) were found to be necessary to
decrease the concentration of Triton X-100 to about 1 ppm in the washing
solution.
Conclusions: The selected DHDM production process and the quality control
methods used were found to be appropriate to prepare sufficient amount of
DHDMs to treat burn patients.
MACROPHAGE - FIBROBLAST INTERACTIONS REGULATE
MATRIX REMODELING IN ACUTE SKELETAL MUSCLE
REGENERATION AND FIBROSIS DURING MUSCLE DISEASE
M. Saclier1, G. Juban1, R. Mounier1, B. Chazaud1
Centre de G"en"etique et de Physiologie Mol"eculaire et Cellulaire, Universit"e
Claude Bernard Lyon 1, CNRS UMR 5534, Villeurbanne, France
1
Macrophages are important regulators of tissue homeostasis and acute inflammation. They play important roles in both the mounting of the inflammatory
response after tissue damage and in the subsequent phase of resolution of
inflammation required for proper tissue repair. They exert pleiotropic roles on
cells in the tissue including fibroblasts, vessel cells and parenchymal cells. We
aimed at comparing the effects of macrophages on fibroblasts in acute regeneration versus chronic disease. We evidenced opposite roles of macrophage populations sorted from normal regenerating muscle versus degenerating myopathic
muscle on proliferation, apoptosis, differentiation and collagen synthesis of/by
fibroblasts. Pharmacological treatments aiming at skewing macrophage phenotype towards an anti-inflammatory status reduces fibrosis and is beneficial for
the tissue in muscle dystrophy.
EXTRACELLULAR MATRIX TURNOVER PROFILE OF CHRONIC
OBSTRUCTIVE PULMONARY DISEASE PATIENTS WITH
EXACERBATIONS
J. Sand1, A. Knox2, P. Lange3, S. Sun1, J. H. Kristensen1, M. A. Karsdal1, D.
Leeming1, C. Bolton2, S. R. Johnson2
1
Nordic Bioscience A/S, Herlev, Denmark; 2University of Nottingham,
Nottingham, United Kingdom; 3University of Copenhagen, Copenhagen,
Denmark
Introduction: Exacerbations of chronic obstructive pulmonary disease (COPD)
are acute events resulting in progressive loss of lung function. Episodes of exacerbations are associated with increases in inflammatory cells and proteolytic
activity in the lungs. Collagens and elastin constitute the main structural proteins of the ECM, upholding tissue structure and function. We hypothesized
that the increase in proteolytic activity associated with exacerbations resulted in
an accelerated turnover rate of collagens and elastin, leading to increased
release of protein fragments into the systemic circulation. Therefore, we undertook this study to investigate the ECM turnover in patients with COPD undergoing exacerbations.
Methods: Sixty-nine patients with COPD hospitalized for an exacerbation were
recruited at admission and returned for a 4 week follow-up visit. Protein degradation was assessed by competitive ELISAs measuring circulating levels of
MMP generated protein fragments of type III collagen (C3M), type IV collagen
(C4M), and type VI collagen (C6M), and elastin fragments generated by MMP7 (ELM7) and neutrophil elastase (EL-NE). Collagen formation was assessed
by competitive ELISAs measuring circulating levels of the prodomain of type
III collagen (Pro-C3), the 7S domain of type IV collagen (P4NP 7S), and the
prodomain of type VI collagen (Pro-C6).
A26
Abstracts
Results: At time of exacerbation, the collagen degradation biomarkers C3M,
C4M and C6M, and the elastin degradation markers ELM7 and EL-NE were
significantly elevated as compared to 4 week follow-up. The level of Pro-C3
was not altered by exacerbations, while P4NP 7S was significantly elevated and
Pro-C6 was significantly decreased at time of exacerbation as compared to follow-up.
Conclusions: Elevated levels of circulating fragments of collagens and elastin
suggest that patients with COPD have accelerated ECM degradation during
exacerbations. This may contribute significantly to disease progression.
THROMBIN-DERIVED HOST DEFENSE PEPTIDES—FROM BASIC
CONCEPTS TO THERAPY
A. Schmidtchen1,2
1
Division of Dermatology and Venereology, Department of Clinical Sciences,
Lund University, Lund, Sweden; 2LKC Medicine, Dermatology, Nanyang
Technological University, Singapore
Introduction: Our innate immune system is rapidly activated during injury and
infection, leading to a fast response in order to control microbial invasion and inflammation. Dysfunctional inflammation, involving excessive cytokine and coagulation
responses, underlie many infectious and inflammatory processes, as seen in wound
infections, invasive infections and sepsis, and during use of biomaterials. In all these
cases, unresolved infection-inflammation leads to destructive acute or chronic disease, which in current treatment regimens is not fully addressed.
Methods: The studies encompass multiple methodologies spanning from
advanced in silico analyses, in vitro analyses (biochemistry, microbiology, cell
biology and molecular biology), to ex vivo and in vivo infection models utilizing “classical” infection-inflammation models, as well as state of the art technologies utilizing in vivo bioimaging.
Results: Our research on the innate immune response activated during wounding,
has led to the discovery of new classes of bioactive peptides, derived from the Cterminal end of thrombin, having dual antimicrobial and anti-inflammatory effects.
Results from experimental models of infection and inflammation show that treatment
with such thrombin-derived host defense peptides (HDP) not only reduces bacterial
loads during infection but also inhibit local as well as systemic inflammation.
Conclusions: Thrombin, after fulfilling its primary function by generating a first
line of defense, the fibrin clot, serves an additional role by the generation of HDPs.
Our results illustrate that our innate immunity expands also to the coagulation system, a fundamental system activated during wounding and infection. Utilizing
HDPs as modulators not only at the bacterial level but also with effects on immune
responses constitutes an effective “natural” strategy of targeting both bacteria as
well as dysregulated inflammation in infective-inflammatory diseases. This modulatory approach has advantages compared with classical approaches based on antibiotics, which only target bacteria. In addition to this, the approach is more
sustainable with respect to development of resistance.
References
1. Hansen FC, Kalle-Brune M, van der Plas MJ, Str€
omdahl AC, Malmsten
M, M€orgelin M, Schmidtchen A. The thrombin-derived host defense peptide
GKY25 inhibits endotoxin-induced responses through interactions with lipopolysaccharide and macrophages/monocytes. J Immunol 2015; 194: 5397-406.
2. Kalle M, Papareddy P, Kasetty G, M€
orgelin M, van der Plas MJ, Rydengård V, Malmsten M, Albiger B, Schmidtchen A. Host defense peptides of
thrombin modulate inflammation and coagulation in endotoxin-mediated shock
and Pseudomonas aeruginosa sepsis. PLoS One, 2012; 7: e51313.
3. Kasetty G, Papareddy P, Kalle M, Rydengård V, M€
orgelin M, Albiger B,
Malmsten M, Schmidtchen A. Structure-activity studies and therapeutic potential of host defense peptides of human thrombin. Antimicrob Agents Chemother
2011; 55: 2880-90.
4. Kasetty G, Kalle M, M€
orgelin M, Brune JC, Schmidtchen A. Anti-endotoxic and antibacterial effects of a dermal substitute coated with host defense
peptides. Biomaterials 2015; 53: 415-25.
5. Merza M, Rahman M, Zhang S, Hwaiz R, Regner S, Schmidtchen A,
Thorlacius H. Human thrombin-derived host defense peptides inhibit neutrophil
recruitment and tissue injury in severe acute pancreatitis. Am J Physiol Gastrointest Liver Physiol 2014; 307: G914-21.
6. Papareddy P, Rydengård V, Pasupuleti M, Walse B, M€
orgelin M, Chalupka A, Malmsten M, Schmidtchen A. Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog 2010; 6: e1000857.
BASIC SCIENCE: WHAT CONTROLS BUGS IN WOUNDS?
A. Schmidtchen1,2
1
Division of Dermatology and Venereology, Department of Clinical Sciences,
Lund University, Lund, Sweden; 2LKC Medicine, Dermatology, Nanyang
Technological University, Singapore
In normal wound healing, various host defense systems control both bacteria
and inflammation. Recently, new classes of bioactive host defense peptides
A27
(HDP) with dual antimicrobial and anti-inflammatory activities have been
discovered. For example, proteins involved in the coagulation system, an
evolutionary old and significant part of the innate immune system, have
been shown to participate in formation of multiple HDPs during wounding.
Dysfunctional host responses underlie many infectious-inflammatory processes, as seen in wound infections and non-healing ulcers. In all these cases,
unresolved infection-inflammation leads to destructive acute or chronic disease, which in current treatment regimens is not fully addressed. Today,
there is an unmet need for new therapeutic concepts targeting not only bacteria but also the following inflammatory processes. In my talk, I will
review the fascinating area of HDPs, and also discuss how these molecules
can be utilised in the development on novel anti-infective and antiinflammatory therapies.
(BIO)SENSOR SYSTEMS EMBEDDED IN WOUND DRESSINGS FOR
CHRONIC WOUND MONITORING
B. Schyrr1, J. Mosig1, F. Sorin1, H. Vuagnat2, G. Voirin3
Laboratory of Photonic materials and fiber devices, Ecole Polytechnique
F"ed"erale de Lausanne (EPFL), Lausanne, Switzerland; 2Centre plaies et
cicatrisation, H^
opitaux Universitaires de Genève (HUG), Geneva, Switzerland;
3
Centre Suisse d’Electronique et de Microtechnique SA (CSEM), Neuch^atel,
Switzerland
1
Chronic wounds are a major public health and economical threat, aggravated
both by population ageing and the concurrent increase in comorbidities such as
diabetes, obesity and cardiovascular diseases. Despite the proliferation of guidelines and new treatments for chronic wounds, their proof of effectiveness has
been limited by the lack of standardized and quantitative assessment methods to
guide their implementation. Hence, further progresses in advanced wound management require an improvement in the availability of diagnostic tests for key
biomarkers to support wound management decision and enable personalized
medicine. In this context, we propose a new wound monitoring system to enable
evidence-based wound management decisions. Our approach consists in using
highly flexible and miniature fiber-optic sensors integrated in conventional
wound dressings to measure, in real-time, critical parameters of the healing process. Here we report the development of two sensors, for pH and matrix metalloproteinase (MMP) activity. The performance of the sensors was characterized
in human serum as a model biological fluid, highlighting their suitability for
clinical use. The device communicates the data to an external platform for visuR , thus opening new perspectives for telealization and analysis via BluetoothV
medicine. At a later stage, the integration of additional sensors will allow the
detection of parameters related to established wound management methods,
such as pressure sensors for compression therapy, oxygen sensors for hyperbaric therapy and hypoxia detection, and humidity sensors for moisture balance of dressings. This approach circumvents several limitations of current
wound assessment strategies. By offering early detection of wound complications such as infections in an ambulatory fashion, this smart wound dressing
system could enable both a decrease in medical consultations/hospitalization
and in painful dressing changes, while supporting treatment decisions with
continuous biochemical and physical data. Altogether, this device could play a
significant role in improving the efficiency and sustainability of chronic
wound management.
UP-REGULATED NEURAL DIFFERENTIATION INDUCED BY
ELECTRICAL STIMULATION IN HUMAN SEQUENTIAL
CUTANEOUS WOUNDS ACCELERATES RE-INNERVATION AND
REPAIR
A. Sebastian1, P. Halai1, J. Colthurst2, A. Bayat1
Institute of Inflammation and Repair, University of Manchester, Manchester,
United Kingdom; 2Oxford Bioelectronics, Innovation Centre, 99 Park Drive,
Abingdon, Oxon, United Kingdom
1
Introduction: Electrical stimulation (ES) accelerates human cutaneous wound
healing by increasing epithelialization and angiogenesis, down-regulating
inflammation and advancing remodeling. There is no report of neural regeneration in ES treated skin wounds. Therefore, we investigated role of ES in neural
differentiation and re-innervation of acute wounds in vivo, and further validated
by in vitro experiments.
Methods: In a randomized study of humans (n 5 40), skin wound biopsies were
either treated with ES or left to heal. Whole genome microarray of sequential
wounds was performed on days (D) 3, 7, 10, and 14. qRT-PCR, Western blotting (WB) and quantitative immunohistochemistry (IHC) of re-innervation and
neural differentiation were performed. SHSY5Y human neuroblastoma cells
were subjected to a novel in vitro ES methodology for cell differentiation.
Here, FIG4 gene was knocked down by siRNA transfection and further gene/
protein analyses of FIG4-involved phosphatidylinositol (3,4,5)-triphosphate
(PIP3) pathway performed.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
Results: Bioinformatic analysis of microarray data identified neural
differentiation-related transcripts, class III b-tubulin (TUBB3) and factorinduced gene 4 (FIG4) as highly up-regulated (p < 0.05) in D14 ES-treated
wounds (ES14), further confirmed with qRT-PCR and WB. Quantitative IHC
showed up-regulation (p < 0.05) of TUBB31 and FIG41 cells, in addition to
PGP9.51 nerve fibers and substance P1 cells in ES14 wounds. Moreover, number of melanocytes (gp1001) and melanogenesis (Masson Fontana staining) was
upregulated (p < 0.05) in ES-treated wounds. ES also increased CK201 Merkel
cells, CK201/TUBB31 and CK201/PGP9.51 cells, plus, nerve growth factor
expression in wounds, showing enhanced differentiation and re-innervation. To
further probe differentiation, FIG4 was knocked down in SHSY5Y cells resulting in late endosomes formation and vacuologenesis, with decreased TUBB3
expression and cell degeneration. ES treatment post FIG4-siRNA transfection
showed decreased vacuologenesis, enhanced differentiation, cell integrity, and
upregulation in PIP3 pathway proteins.
Conclusions: ES upregulates neural differentiation biomarkers which accelerates
re-innervation, leading to enhanced rate of cutaneous repair.
THE INFLUENCE OF AN ALTERED INFLAMMATORY LANDSCAPE
ON KELOID FIBROBLAST DIFFERENTIATION
T. Shaw1
1
Division of Basic Medical Sciences, St George’s, University of London,
London, United Kingdom
The fibrotic response to skin wounding can proceed out of control in a subset
of the population, resulting in disfiguring and painful keloid scars. Currently,
there are no successful treatments that prevent or eliminate keloids, but an
improved understanding of their etiology is anticipated to inform future therapeutic approaches. The inflammatory response to tissue injury or infection is
established to exacerbate fibrosis; accordingly, we are working to define the
inflammatory cell infiltrate in keloids and its effects on dermal fibroblasts. Our
data confirm that the abundance and characteristics of macrophages are altered
in a subset of lesions. Excitingly, in vitro experiments demonstrate that this
change in inflammatory milieu can contribute to the inappropriate pro-fibrotic
cell differentiation that underlies keloidogenesis.
SUCCESSFUL TREATMENT OF CHRONIC LEG ULCERS WITH
ORAL IRON CHELATORS IN A PATIENT WITH SYSTEMIC IRON
OVERLOAD
MATRIX METALLOPROTEINASE ACTIVITY AND
EXTRACELLULAR MATRIX PROTEINS STATE AT ASCENDING
AORTA ANEURYSM OF DIFFERENT GENESIS
L. Smagina1, A. Malashicheva2, O. Irtyuga3, O. Moiseeva3, I. Voronkina4
Institute of Cytology, Russian Academy of Sciences, Sank-Petersburg, Russian
Federation; 2Institute of Molecular Biology and Genetics, Almazov Federal
Heart, Blood and Endocrinology Center, Saint Petersburg, Russian Federation;
3
Federal Center of Heart, Blood and Endocrinology by Almazov, SankPetersburg, Russian Federation; 4Russian Academy of Sciences, SankPetersburg, Russian Federation
1
Introduction: Abnormal matrix metalloproteinase (MMP) activity is involved
in formation of abdominal aortic aneurysms. Recent studies show that abnormal
MMP activity may also be associated with the formation of aneurysm of the
thoracic aorta. Bicuspid aortic valve (BAV) is associated with the inner aortic
pathology, which predisposes to formation of aneurysm, but that do not occur at
tricuspid aortic valve (TAV). The aim of this work was to evaluate the contribution of disorders of extracellular matrix (ECM) proteins balance and MMP
activity in the development of ascending aorta aneurysm of various geneses.
Methods: The study included 38 patients with ascending aorta aneurysm with
BAV and TAV and a control group comprising 17 patients without aortic
pathology. Tissue biopsies were obtained during surgery. Type I collagen, elastin and fibrillin were evaluated by immunoblotting. The forms and amount of
MMP-2 and MMP-9 were analyzed by gelatin zymography.
Results: Maximum content of type I collagen was found in control aortic samples. Type I collagen content was higher at BAV than at TAV. The elastin content was minimal in the control group; in the BAV group elastin was
significantly lower than in TAV group. The content of fibrillin in aortic tissue
was minimal at BAV, and did not differ significantly from the control and TAV
groups. The degree of fragmentation of the studied proteins in aortic tissue was
also different for BAV and TAV groups. Increased amount of latent MMP-2
and MMP-9 was detected in a subset of patients with TAV as compared with
control group. In patients with BAV the raise of latent and active forms of
MMP-9 was found in comparison with the control group.
Conclusions: Differences in amount and activity of MMPs, as well as increased
ratio of collagen/elastin can explain the features of ascending aorta aneurysm
formation in patients with BAV and TAV.
TUBULINS CLASS BETA MODULATE WOUND HEALING
PROPERTIES IN HUMAN MICROVASCULAR ENDOTHELIAL CELLS
offer1, T. Peters1,
M.-A. Sindrilaru1, L. Schriever1, M. Huber1, K. Geth€
L. A. Schneider1, K. U. Scharffetter-Kochanek1
1
Department of Dermatology and Allergic Diseases, University of Ulm, Ulm,
Germany
K. Sobierajska1, M. Wawro1, W. M. Ciszewski1, K. Wieczorek1,2, I. SacewiczHofman1, M. Wiktorska1, J. Niewiarowska1
1
Department of Cell Molecular Mechanisms, Medical University of Lodz, Lodz,
Poland; 2Department of Endocrinology and Metabolic Diseases, Medical
University of Lodz, Lodz, Poland
Introduction: Macrophages are potent regulators of all phases of adult wound
healing. There is growing evidence that a vast diversity of macrophage phenotypes induced by wound-specific cues may drive physiologic and pathologic
wound healing. We have recently identified iron-overloaded and over-activated
macrophages as novel phenotypic subset responsible for the non-healing status
in chronic venous leg ulcers (CVU). These iron-overloaded, pro-inflammatory
cytokine-producing macrophages are hardly therapeutically considered, even by
modern strategies.
Methods: We here report the successful treatment of CVU with an oral iron
chelator in a patient with systemic iron-overload syndrome.
Results: A 42-year-old female patient with CVU on both legs was presented.
The CVU occurred at the age of 20 years and had never healed since despite
adequate wound treatment. At the age of 4 years she was diagnosed with rare
congenital dyserythropoietic anemia due to the SEC23B gene mutation. This
resulted in secondary hemosiderosis and an 8-times increased tissue ironoverload determined by FerriScan analysis of liver tissue. Immunostainings for
iron and macrophage markers revealed CD681 macrophages heavily loaded
with iron and expressing tumor necrosis factor (TNF)-a, which abundantly infiltrated the patient’s wound margins, but not her unaffected skin. We hypothesized that these iron-loaded macrophages at the patient’s wound margins are
responsible for an unrestrained, macrophage-driven inflammation and impaired
wound healing, and treated the patient with the oral iron chelator deferasirox
along with adequate wound management. This therapy lead to complete healing
of the CVU within 16 months and was paralleled by a remarkable decrease of
the systemic iron overload and of the iron content in wound macrophages and
their consistently reduced expression of the pro-inflammatory TNF-a.
Conclusions: As iron-overloaded and over-activated macrophages also underlie
the non-healing status in CVU, this is the first clinical evidence that therapeutic
scavenging of iron in wound-associated macrophages reduces inflammation and
may promote wound healing in this difficult-to-cure condition.
Introduction: Increasing number of reports demonstrated that cells can have a
remarkable plasticity. Endothelial-to-mesenchymal transition (EndMT) is one of
the processes underlying the cellular flexibility. EndMT is essential during
embryonic development and tissue regeneration but it is also involved in pathological stages induction like fibrotic diseases. Among other processes associated
with EndMT is cytoskeleton reorganization involving microtubules. The main
goal of our study was to analyze the role of tubulins in wound healing process.
Methods: EndMT was induced in a human microvascular endothelial cell line
(HMEC-1) by tumor growth factor (TGF)-b stimulation. Expression of tubulins
isoform was studied by real-time PCR, Western-blot and immunocytochemicals
methods. Next, the role of tubulins in cellular behavior was investigated by
wound healing process.
Results: We observed changes in the expression of two classes of beta tubulins
(tubulin b3 and tubulin b4) during the induction of primary stages of the
EndMT. Interestingly, these changes were associated with translocation of studied proteins within the cell. Stimulation of the cells with TGF-b caused translocation of both tubulins in the nucleus and close to the cell membrane area
associated with focal adhesion. After TGF-b stimulation cell migration was
increased in a wound healing assay, whereas silencing of each tubulin caused
reduction of cell migration.
Conclusions: Our results suggest the participation of selected tubulins in fibrotic
formation. These preliminary data require further analysis which may contribute
to the more thorough understanding of the molecular mechanisms of EndMT
development and especially in age-related fibrosis.
Acknowledgments
This project has received funding from the Polish-Norwegian Research Program (MOMENTO Pol-Nor/209521/5/2013) and European Union’s Seventh
Framework Program for research technological development and demonstration
under grant agreement no 316300 Financial support (HARC FP7-REG-POT2012-2013-1).
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
A28
Abstracts
CHRONICALLY BAD FIBROBLASTS ARE NOT SO DOWN
IN THE MOUTH
P. Stephens1
Wound Biology Group, Cardiff Institute of Tissue Engineering and Repair,
Tissue Repair and Reparative Dentistry, School of Dentistry, College of
Biomedical and Life Sciences, Cardiff University, Cardiff, Wales, United
Kingdom
1
Chronic non-healing wounds are a major health problem within our ageing
society. Such wounds are characterized by persistent inflammation, failed epithelialization, a lack of granulation tissue formation and they are exacerbated
by high levels of bacterial infection. Despite some clinical advances, 10-20%
of chronic wounds still do not heal and thus novel therapeutic approaches are
required. Our investigations have focussed on whether age-related functional
changes in dermal fibroblasts may contribute to this dysfunctional healing
phenotype. Microarray analysis of chronic wound fibroblasts (CWFs) and normal skin fibroblasts (NFs) demonstrated altered regulation of numerous genes
including molecules involved in the protection against oxidative stress. CWFs
proliferate more slowly than NFs and premature senescence of CWFs was
confirmed by increased SA-b-Gal activity and their larger, polygonal morphology. Analysis of telomere lengths revealed that whilst senescence in some
CWFs was telomere-dependent in others it was telomere independent. This
premature senescence significantly decreased their abilities to carry out key
wound healing activities. This was however, in direct contrast to fibroblasts
isolated from wounds that heal in a scarless manner (oral mucosal fibroblasts;
OMFs) that demonstrate differential gene analysis, increased proliferation,
lower levels of senescence (longer telomeres) and accelerated wound healing
responses. From our microarray data, we have now described a transcriptional
signature for this “wound healing continuum” which may assist in the therapeutic assessment/treatment of a patient’s wound. Our recent findings however, suggest it is the presence of a progenitor cell sub-population resident
within the heterogeneous oral stromal population that is key to the preferential
healing. Such oral mucosal lamina propria-progenitor cells (OMLP-PCs) can
be rapidly and clonally expanded in vitro, are neural crest-derived and are
multipotent. Furthermore they are potently immunosuppressive and have distinct antibacterial activities. We, therefore, postulate that OMLP-PCs could be
an efficacious cell-based therapy for the future amelioration of chronic
wounds.
IN VIVO CHARACTERIZATION OF THE ALGINATE-POVIDONE
IODINE FILM IN THE WOUND HEALING MODEL IN MICE
M. Summa1, I. Bayer2, I. Liakos2, T. Bandiera1, A. Athanassiou2, R. Bertorelli1
Istituto Italiano DI Tecnologia, Pharmachemistry Facility, Department of Drug
Discovery and Development, Genova, Italy; 2Istituto Italiano DI Tecnologia,
Smart Materials Department of Nanophysics, Genova, Italy
1
Introduction: The aim of this study was to characterize the efficacy of biodegradable polymeric materials based on alginate and povidone iodine (PVPI)
complex in a mouse model of wound healing. The new materials combine the
excellent wound healing properties of alginates with the bactericidal and fungicidal properties of PVPI (1).
Methods: Male C57BL/6J mice, weighing 22-24 g were used. Mice were anesthetized, and the dorsal surface shaved and rinsed with an alcohol swab. A
full-thickness excisional wound of 1 cm2 was induced in each animal. Mice
(n 5 5 per group) were dressed with alginate-PVPI film, and then covered
with polyurethane film dressing (only for the first 3 days). After 3 days, the
experimental film dressing was applied once a day for two consecutive days
and then left in place up to the end of experiments, while control-wounded
animals were left uncovered. Animal wounds were photographed regularly for
at least 12 days.
Results: Wound closure was analyzed as the percentage of the reduction in
wounded area at day 3, 7, 9, and 12. The film-treated animals showed significantly more wound closure than untreated-wounded animals at each time points
considered. Specifically, the size of the wound decreased more rapidly in the
film-treated group compared to the untreated group (p < 0.01 at days 3, 7 and 9
and p < 0.01 at day 12; Two-way ANOVA and Bonferroni post-tests). Interestingly, wound closure was achieved within 12 days only in the film-treated group,
indicating that the full-thickness wounds more rapidly healed in these animals.
Conclusions: The results demonstrate that the alginate-PVPI system in the form
of film membrane is biocompatible, devoid of toxicity and possesses healing
properties accelerating the wound closure.
Reference
1. Liakosa I, Rizzelloa L, Bayera IS, Pompaa PP, Cingolanib R, Athanassioua A. Controlled antiseptic release by alginate polymer films and beads. Carbohydrate Polymers 2013; 92: 1176-83.
A29
SMOKING IMPAIRS HEALING—ARE THE MECHANISMS
REVERSIBLE?
L. T. Sørensen1
Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen,
Copenhagen NV, Denmark
1
Smokers have a higher incidence of adverse healing events than non-smokers.
These events embrace tissue necrosis, dehiscence and rupture of sutured tissue,
lack of healing and wound infection. After smoking cessation wound infections
decrease whereas other adverse healing events appear to be unaffected. Studies
of the mechanisms involved show that smoking decreases tissue oxygenation
and aerobe metabolism temporarily. The inflammatory healing response is attenuated by a reduced inflammatory cell chemotactic responsiveness, migratory
function, and oxidative bactericidal mechanisms. In addition, the release of proteolytic enzymes and inhibitors is imbalanced. The fibrotic response is impaired
by a reduced fibroblast migration and proliferation in addition to a downregulated collagen synthesis and deposition. Smoking cessation restores tissue
oxygenation and metabolism rapidly. Inflammatory cell response is reversed in
part within 4 weeks, whereas the proliferative response remains impaired.
DEVELOPMENT OF AN IN VITRO WOUND CONTAMINATION
MODEL TO TEST THE EFFICACY OF ANTIMICROBIAL DRESSINGS
FOR INFECTION PREVENTION
F. Taherinejad1, M. Werth"en1, L. Sandberg2
M€
olnlycke Health Care, G€
oteborg, Sweden; 2SCA, G€
oteborg, Sweden
1
Introduction: Antimicrobial wound dressings are today commonly used for infection prevention and management in many different wound types. For evaluation of
dressing efficacy they are often tested in vitro using models with little regard to the
intended use, such as protection from microbial contamination, or elimination of
bioburden in an already infected wound. Product design and development therefore
need new preclinical models, simulating relevant wound environments. The aim of
this study was to develop a more challenging in vitro model, in comparison to the
often used zone of inhibition (ZOI) method, to evaluate the ability of an antimicrobial dressing to inhibit bacterial growth in a wound contaminated with Pseudomonas
aeruginosa, relevant for example for burns or other traumatic wounds.
Methods: To mimic wound tissue, a circular piece of a porcine dermal matrix
(EZ Derm), was placed on a blood agar plate containing extra serum. A small
volume of a P. aeruginosa (PAO1) culture containing 100 to 1,000 colonyforming units (CFU) was inoculated on dermal matrix. A dressing piece, with a
larger diameter was then applied. The samples were incubated at 358C for 24
hours and the results were evaluated by visual inspection and by plate count of
CFU extracted from the dermal matrix.
Results: The described test model allows for visual evaluation of the antimicrobial effect of the concept in a more challenging environment than a ZOI agar
plate. Even though the inoculation concentration is lower than common ZOI
inoculations, the model is more challenging since “diffusion” of the bacteria in
the test environment is more pronounced.
Conclusions: The wound contamination model allows for evaluation of the ability of an antimicrobial dressing to prevent spreading of P. aeruginosa from a
contaminated site, in a wound like environment favorable for the bacteria, and
it therefore provides a more clinically relevant test method.
THE ROLE OF HUMAN DERMAL FIBROBLASTS IN REGULATING
AVAILABILITY OF BIOLOGICALLY ACTIVE VITAMIN D:
IMPLICATIONS FOR CUTANEOUS WOUND HEALING
J. Q. Tay1, O. Kamala2, A. Graham3, A. Mahajan1, M. J. Thornton2
1
Plastic Surgery and Burns Research Unit, University of Bradford, Bradford,
United Kingdom; 2Centre for Skin Sciences, University of Bradford, Bradford,
United Kingdom; 3Centre for Skin Sciences, School of Medical Sciences,
University of Bradford, Bradford, United Kingdom
Introduction: Keratinocytes are a target and source of vitamin D. They convert
cholecalciferol to the active metabolite 1a,25-dihydroxyvitaminD3 via 25hydroxylase (CYP2R1) and then 1a-hydroxylase (CYP27B1) (1). Another enzyme
24-hydroxylase (CYP24A1), is important in regulating local levels by inactivating
1a,25-dihydroxyvitaminD3. Recent studies reported that absence of vitamin D
receptor (VDR) or 1a,25-dihydroxyvitaminD3 impairs granulation tissue formation
in murine wound healing (2), but little is known about the paracrine and intracrine
regulation of vitamin D in human skin following cutaneous injury.
Methods: We used qRT-PCR to compare the relative expression of VDR,
CYP2R1, CYP27B1 and CYP24A1 in donor-matched primary cultures of
human dermal fibroblasts and keratinocytes. We also determined whether incubation with cholecalciferol or 1a,25-dihydroxyvitaminD3 (1–100 nM) modulated fibroblast migration in a scratch-wound assay, and after 24 hours if there
was any change in mRNA expression modulated by incubation with cholecalciferol or 1a,25-dihydroxyvitaminD3.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
Results: The relative expression of VDR was higher in dermal fibroblasts than
donor-matched keratinocytes, while expression of CYP27B1 and CYP24A1 was
lower, under both wounded and non-wounded conditions. 1a,25-dihydroxyvitaminD3 inhibited fibroblast migration as early as 4 hours and up to 24 hours.
Cholecalciferol only inhibited migration at 100 nM after 24 hours. 1a,25-dihydroxyvitaminD3 reduced VDR and CYP2R1 expression in wounded fibroblasts,
while CYP24A1 expression increased; there was no change in CYP27B1
expression. In contrast, cholecalciferol increased expression of VDR, CYP2R1,
CYP27B1 and CYP24A1 in wounded fibroblasts.
Conclusions: This study has confirmed that human dermal fibroblasts express
the necessary enzymes for autocrine production of 1a,25-dihydroxyvitaminD3.
Higher VDR expression suggests they may be more responsive than keratinocytes. Changes in the metabolism of vitamin D and VDR levels in the presence
of cholecalciferol or 1a,25-dihydroxyvitaminD3 highlight fine-tuning of vitamin
D availability in the dermis after wounding. Although migration was inhibited,
delineating the molecular mechanisms may lead to improved therapies for
chronic wounds.
References
1. Slominski A, Kim TK, Zmijewski MA, Janjetovic Z, Li W, Chen J, Kusniatsova EI, Semak I, Postlethwaite A, Miller DD, Zjawiony JK, Tuckey RC.
Novel vitamin D photoproducts and their precursors in the skin. Dermato-endocrinology 2013; 5: 7-19.
2. Luderer HF, Nazarian RM, Zhu ED, Demay MB. Ligand-dependent
actions of the vitamin D receptor are required for activation of TGF-b signaling
during the inflammatory response to cutaneous injury. Endocrinology 2013;
154: 16-24.
CAN HUMAN PLATELET LYSATE IN COMBINATION WITH THREE
DIMENSIONAL SCAFFOLDS IMPROVE THE WOUND HEALING
PROPERTIES OF HUMAN KERATINOCYTES AND FIBROBLASTS?
J. Thompson1, M. J. Thornton1, W. Roberts1
1
Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom
Introduction: The burden of chronic wounds is a growing issue in the UK.
This project investigated the capabilities of platelet lysate coated on electrospun
scaffolds to promote keratinocyte and fibroblast functional responses. Platelets
are the first cells to arrive at the site of injury, where they quickly release
growth factors and chemokines that regulate skin cell responses (1). Electrospun
scaffolds mimic the native three-dimensional extracellular matrix and have successfully been used to model wound restoration and to promote wound closure
(2). We hypothesized that platelet lysate in combination with electrospun scaffolds would strongly promote healing due to the complex array of cytokines
and growth factors present.
Methods: Cytokine content of human platelet lysate (HPL) was evaluated by
proteome profiler assay kits (n 5 4 donors). Electrospun scaffolds were coated
with HPL prior to seeding with primary human fibroblasts or keratinocytes.
Cell adhesion and proliferation were measured luminescently using cell titre
glow. The ability of coated scaffolds to induce migration was determined by the
use of Boyden chamber assays.
Results: Proteome profiler assay kits showed that HPL was a rich source of a
range of cytokines and growth factors including interleukin (IL)21a, IL-2 and
epidermal growth factor, platelet-derived growth factors and insulin-like growth
factors. Coating scaffolds with HPL increased the adhesion of primary fibroblast
by 240 6 4% (p < 0.001), proliferation by 326 6 14% (p < 0.05), and increased
the migration of fibroblasts onto the scaffolds by 320 6 24% (p < 0.05). Interestingly HPL did not increase the ability of scaffolds to support adhesion or
proliferation of keratinocytes, but increased migration by 174 6 16% (p < 0.05).
Conclusions: These data show HPL in combination with electrospun scaffolds
strongly promotes fibroblast and keratinocyte responses.
References
1. Ranzato E, Mazzucco L, Patrone M, Burlando B. Platelet lysate promotes
in vitro wound scratch closure of human dermal fibroblasts: different roles of
cell calcium, P38, ERK and PI3K/AKT. J Cell Mol Med 2009; 13: 2030-8.
2. Sun T, Mai S, Norton D, Haycock JW, Ryan AJ, MacNeil S. Self-organization of skin cells in three-dimensional electrospun polystyrene scaffolds. Tissue Eng 2005; 11: 1023-33.
MAINTAINED BURST AND PHAGOCYTOSIS EFFECTS OF
LEUCOPATCHES MAY TERMINATE BACTERIAL BIOFILMS IN
CHRONIC WOUNDS
K. Thomsen1, H. Trøstrup1, L. Christophersen1, R. Lundquist2, N. Høiby1, C.
Moser1
1
Department of Clinical Microbiology, Rigshospitalet, University of
Copenhagen, Copenhagen, Denmark; 2Reapplix ApS, Birkerød, Denmark
Introduction: Polymorphonuclear neutrophils (PMNs) in autologous plateletrich fibrin patches (leucopathes) possibly promote the termination of biofilms in
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
recalcitrant wounds. The present study examined the phagocytic competence of
PMNs in leucopatches.
Methods: Leucopathes were prepared from donor blood of seven healthy volunteers (four males) between 30 and 50 years of age. Luminol chemiluminescence
determined the reactive oxygen species (ROS) production by challenging leucopatches with Pseudomonas aeruginosa (PA), phorbol myristate acetate and
zymosan. The phagocytic bacterial killing was evaluated by challenging leucopatches with planktonic PA and subsequently alginate embedded bacteria,
resembling biofilm. The chemotactic properties of the PMNs in leucopatches
were evaluated by their ability to migrate towards the chemoattractants fMLP
and PA in a membrane separated two-phase system. The interaction between
leucopatches and PA were performed by peptide nucleic acid-fluorescence in
situ hybridization technology and fluorescence microscopy, visualizing the
localization of PA in close contact with the subjacent layer in the leucopatches
and accordingly the accessibility to PMN-mediated phagocytosis.
Results: The production of ROS was sustained in PMNs residing in leucopatches, thus all stimulants facilitated a prominent chemiluminescense response.
The bactericidal activity of leucopatches was apparent as the viability of planktonic PA was reduced by 89% after 20 minutes of phagocytosis compared to
non-leucopatch buffer control (p < 0.02). When leucopatches were challenged
with PA embedded in alginate, mimicking biofilm, the bacterial load was
reduced by 70% after 2 hours of phagocytosis compared to non-leucopatch
buffer control (p < 0.005). The chemotaxis assay showed that PMNs were able
to detach their fixation in the leucopatches and migrate towards chemotactically
active factors.
Conclusions: Our results showed preserved oxidative burst as well as phagocytic activity and subsequent bacterial killing by leucopatches that may accelerate healing of chronic wounds.
CHALLENGES IN KELOID DISORDER – CLINICAL PRESENTATION
AND PSYCHO-SOCIAL IMPACT
M. Tirgan1
1
Keloid Research Foundation, St. Luke’s Roosevelt Hospital Center,
Rockefeller University Hospital, New York, NY USA
Keloid is a fibro-proliferative disease of cutaneous connective tissue, thought to
be caused by dysregulation in various skin repair processes in the individuals
who are genetically predisposed to this disorder. Keloid disorder (KD) is characterized by excessive collagen and/or glycoprotein deposits in the dermis. KD
has a very diverse phenotype and presents itself for most part during childhood
and teenage years. Several cases will be presented to emphasize the variety of
size, shape and location of keloidal lesions among subjects of various ethnic
back ground. Although benign, KD can cause aesthetic and occasional functional problems in patients thus producing negative impact on the individual’s
quality of life. Psychosocial impact of KD will be discussed in the context of
cases what will be presented.
TREATING KELOID DISORDER – WHERE ARE WE IN 2015?
M. Tirgan1
Keloid Research Foundation, St. Luke’s Roosevelt Hospital Center,
Rockefeller University Hospital, New York, NY USA
1
Keloid disorder (KD) is relatively resistant and often refractory to treatment,
with very high rate of recurrence to any single modality treatment. After intralesional steroid injections, surgery is the second most commonly used treatment
modality for KD. KD often results in formation of benign skin tumors of variable size, especially among Africans and dark colored individuals. Surgery
alone results is near 100% recurrence rate, often resulting in lesions that will be
larger than before and worse in their clinical behavior. Limited data, especially
from Asia supports surgery as an intervention in KD of ear only. Lack of efficacy and worsening of KD after surgery have to be discussed with patients prior
to any surgical intervention. Role of cryotherapy, intralesional steroid, radiation
therapy and chemotherapy will be discussed. The need for large scale retrospective and well controlled prospective studies to assess the role of surgery in treatment of KD will be discussed.
BIOMARKERS AND MOLECULAR MIDPOINT MARKERS AS
STRATEGIC TARGETS
M. Tomic-Canic1
Wound Healing and Regenerative Medicine Research Program, University of
Miami Miller School of Medicine, Miami, FL USA
1
One of the major obstacles to prompt and complete healing of chronic wounds
is the inability to predict which wounds are not likely to respond to standard
treatment protocols and will require alternative interventions. The current
evidence-based protocols are based on early recognition and comprehensive
treatment, including treatment of the wound and associated infection, off-
A30
Abstracts
loading or compression (depending on the type of the ulcer), weekly objective
measurement of wound size. Despite these efforts, more than 50% of patients
fail to heal with standard care. Rapid recognition of patients that will heal with
standard care and patients that will require advanced care is critical to improving outcomes. Various strategies are being developed for feasible quantitative
approaches to identify biomarkers including bacterial bioburden, matrix metalloproteinases and tissue-specific biomarkers and will be discussed. The development of objective quantitative biomarkers should facilitate wound healing,
increase overall treatment efficiency and reduce healthcare costs for patients.
References
1. Trøstrup H, Lundquist R, Christensen LH, Jorgensen LN, Karlsmark T,
Haab BB, Ågren MS. S100A8/A9 deficiency in nonhealing venous leg ulcers
uncovered by multiplexed antibody microarray profiling. Br J Dermatol 2011;
165: 292-301.
2. Trøstrup H, Thomsen K, Christophersen LJ, Hougen HP, Bjarnsholt T,
Jensen PØ, Kirkby N, Calum H, Høiby N, Moser C. Pseudomonas aeruginosa
biofilm aggravates skin inflammatory response in BALB/c mice in a novel
chronic wound model. Wound Repair Regen 2014; 21: 292-9.
IMPROVING FLAP SURVIVAL WITH FRESH AND CULTUREEXPANDED ADIPOSE-DERIVED STROMAL CELLS IN A
XENOTRANSPLANTATION RAT MODEL
S100A8/A9 WOUND FLUID LEVELS ARE REDUCED IN NONHEALING VENOUS LEG ULCERS COMPARED WITH NEUROPATHIC
FOOT ULCERS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS
N. Toyserkani1, C. H. Jensen2, S. P. Sheikh2, J. A. Sørensen1
Department of Plastic and Reconstructive Surgery, Odense University Hospital,
Odense, Denmark; 2Department of Clinical Biochemistry and Pharmacology,
Odense University Hospital, Odense, Denmark
H. Trøstrup1, A. C. Laursen2, P. Holstein2, L. Christophersen1, B. Jørgensen2, T.
Karlsmark2, N. Høiby1,3, C. Moser1, M. S. Ågren2,4
1
Department of Clinical Microbiology, Rigshospitalet, University of
Copenhagen, Copenhagen, Denmark; 2Copenhagen Wound Healing Center,
Bispebjerg Hospital, University of Copenhagen, Copenhasgen NV, Denmark;
3
Costerton Biofilm Center, Department of Immunology and Microbiology,
Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen, Denmark; 4Digestive Disease Center, Bispebjerg Hospital,
University of Copenhagen, Copenhagen NV, Denmark
1
Introduction: Flap necrosis due to limited vascular supply is an ongoing challenge in plastic surgery. In the last decade especially adipose-derived stromal
cells have emerged as a possible solution to improve flap survival. The cells
can be used freshly isolated as the stromal vascular fraction (SVF) or after
culture-expansion of the adipose-derived stromal cells (ASCs). The aim of this
study was to compare the efficacy of human SVF and ASCs for improving flap
survival in a rat model.
Methods: Lipoaspirates from three human female donors were used for isolation
of SVF. Thirty-six male Sprague-Dawley rats were randomized to three groups.
A caudally based random flap measuring 2 3 7 cm including a triangular area
at the tip was elevated on the dorsum of each rat. The flaps were injected with
5 3 106 SVF cells, 5 3 106 ASCs or phosphate-buffered saline (PBS). Seven
days later flap survival was analyzed with standardized photography. Flap skin
was prepared for histological examination for capillary density and stem cell
retention.
Results: The mean survival rates 6 SD were 55.0 6 7.2% for the SVF,
50.4 6 9.1% for the ASC and 45.7 6 9.5% for the PBS control group. The SVF
group was better (p < 0.05) than control group.
Conclusions: Flap survival was most improved with SVF treatment. The clinical effect was modest and could represent the physiologic limits of stem cell
induced angiogenesis in an acute setting. Future research should focus on which
cellular subpopulation of SVF that is responsible for this effect.
PSEUDOMONAS AERUGINOSA BIOFILM INFECTION SUPPRESSES
LOCAL HOST RESPONSE IN BURN WOUNDS
H. Trøstrup1, C. J. Lerche1, L. Christophersen1, K. Thomsen1, P. Ø. Jensen1, N.
Høiby1,2, C. Moser1
1
Department of Clinical Microbiology, Rigshospitalet, University of
Copenhagen, Copenhagen, Denmark; 2Institute for International Health,
Immunology and Microbiology, University of Copenhagen, Copenhagen,
Denmark
Introduction: Bacterial biofilms in chronic wounds have received growing
attention. S100A8/A9 levels are reduced in non-healing venous leg ulcers compared to healing wounds (1). We hypothesized that the S100A8/A9 levels would
be reduced in the presence of Pseudomonas aeruginosa (PA) biofilm due to its
suppressive effect on the innate host response. The aim was to study the impact
of PA biofilm infection on local and systemic host response in the two mouse
strains BALB/c and C3H/HeN.
Methods: One full-thickness burn wound (4.4 cm2) was inflicted in 27 mice of
each strain (2). Two days later, a PA biofilm solution (106 colony-forming
units) was injected subcutaneously in 18 mice per strain. Groups of 6 PAinfected and 3 non-infected mice were killed 4, 7 and 10 days after administration of PA biofilm. Wound tissue was analyzed for PA, S100A8/A9, interleukin
(IL)-1b and keratinocyte-derived chemokine (KC) contents. Systemically, white
blood cell (WBC) and polymorphonuclear (PMN) counts were determined in
peripheral blood days 4, 7 and 10.
Results: PA growth in the wounds did not change appreciably in either mouse
strain over the 10-day observation period. Reduced S100A8/A9 and KC but
increased IL-1b tissue levels were found days 4, 7 and 10 in PA-infected
wounds compared to wounds without PA biofilm. S100A8/A9 but not IL-1b or
KC levels were increased in infected wounds in BALB/c compared to infected
wounds in C3H/HeN mice day 7 (p ! 0.0007) and day 10 (p ! 0.012). Elevated
WBC and PMN blood counts in animals with PA-infected wounds were prolonged (p < 0.03) to day 7 in the BALB/c mice while they dropped (p < 0.05)
from days 4 to 7 in the C3H/HeN mice.
Conclusions: PA biofilm suppressed S100A8/A9 wound tissue levels in BALB/
c and C3H/HeN mice. This may contribute to the persistence of PA infections
in wounds due to the antimicrobial activity of S100A8/A9.
A31
Introduction: Recently we reported reduced levels of the immunomodulating
and antimicrobial S100A8/A9 in non-healing venous leg ulcers (VLUs) while
another study found increased S100A8/A9 in diabetic foot ulcers (DFUs) (1, 2).
To clarify these apparently contradictory findings, we compared S100A8/A9 as
well as an inducer, lipopolysaccharide (LPS) and selected innate immune
response mediators in wound fluids from non-healing DFUs and VLUs with
healing wounds.
Methods: Wound fluids were collected over a 24-h period from neuropathic
DFUs (n 5 6) and VLUs (n 5 9) of median 2 years’ duration, and splitthickness skin graft donor site wounds (n 5 10) by standardized method using
hydrophobic foam covered with impervious polyurethane film dressing (3).
None of the patients had ischemic extremities or clinically infected wounds.
LPS was determined by limulus amoebocyte lysate test, and granulocyte-colony
stimulating factor (G-CSF), interleukin (IL)210 and vascular endothelial growth
factor (VEGF) by immunospecific quantitative assays.
Results: LPS levels were median 8.7 (5.4-21.2, interquartile range) ng/mL in
DFUs compared with 121 (22 to 2,000) ng/mL in VLUs. S100A8/A9 was
higher (p 5 0.020) in DFUs (718 [634-811] mg/mL) than in VLUs (303 [252533] mg/mL). Neither G-CSF nor IL-10 wound fluid levels differed significantly
between the chronic wound groups. VEGF levels correlated with LPS
(r 5 0.758, p 5 0.011, n 5 10) and were higher (p 5 0.024) in VLU wound fluids. LPS (p < 0.0001), S100A8/A9 (p 5 0.005), G-CSF (p 5 0.003), IL-10
(p 5 0.003) and VEGF (p 5 0.005) were increased in chronic wound fluids combined compared with the sterile donor site wound fluids. The protein alterations
in the wounds were not reflected in the patients’ sera.
Conclusions: Low S100A8/A9 levels may contribute to poor wound healing in
colonized chronic VLUs.
References
1. Trøstrup H, Lundquist R, Christensen LH, Jorgensen LN, Karlsmark T,
Haab BB, Ågren MS (2011) S100A8/A9 deficiency in nonhealing venous leg
ulcers uncovered by multiplexed antibody microarray profiling. Br J Dermatol
2011; 165: 292-301.
2. Krisp C, Jacobsen F, McKay MJ, Molloy MP, Steinstraesser L, Wolters
DA. Proteome analysis reveals antiangiogenic environments in chronic wounds
of diabetes mellitus type 2 patients. Proteomics 2013; 13: 2670-81.
3. Zillmer R, Trøstrup H, Karlsmark T, Ifversen P, Ågren MS. Duration of
wound fluid secretion from chronic venous leg ulcers is critical for interleukin1a, interleukin-1b, interleukin-8 levels and fibroblast activation. Arch Dermatol
Res 2011; 303: 601-6.
EVIDENCE TO SUPPORT WHAT IS TAKING PLACE IN SCIENCE
AND CLINICAL PRACTICE
D. Ubbink1
1
Academic Medical Center, Surgery, Amsterdam, The Netherlands
In this presentation, the current highlights in wound care and wound healing are
sketched in a nutshell to set the scene for some of the sessions during this conference. Recent evidence will be briefly covered in the form of Cochrane
reviews on pressure ulcers and surgical site infections, the effectiveness of skin
grafts and tissue replacements in diabetic foot ulcers, hyperbaric oxygen, and
ischaemic ulcers. Also, diagnostic studies on common wound classification
scales and the progress in terms of state-of-the-art wearables (e.g., wireless
pedobarography, corneal glucometry) will be illustrated. Available prognostic
studies and guidelines will be presented on, for example, the prediction of the
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
healing of chronic and acute wounds, and the influence of lifestyle and aging.
Finally, the current practice and organisation of wound care in wound expertise
centres and web-based clinical management systems are discussed. We should
underpin our clinical practice with evidence rather than by experience only.
ments demonstrated an increase in melanin at day 7 and slight reduction to day 14,
whilst melanogenesis increased by 46.7% from day 0 to day 14.
Conclusions: These findings demonstrate the utility of non-invasive objective
devices in the quantitative evaluation of wound healing parameters in human
skin.
WHY NOT COVER UP DIABETIC FOOT ULCERS? A SYSTEMATIC
REVIEW
D. Ubbink1, P. Poyck1, K. Santema1
Department of Surgery, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
1
Introduction: Despite the many strategies available to treat diabetic foot ulcers,
not all ulcers achieve complete healing. Additional treatments with skin grafts
and tissue replacement products have been developed that aim to promote complete wound closure by covering the skin defect (1–3). We systematically
reviewed the effectiveness of tissue replacements in addition to standard care
for diabetic foot ulcers.
Methods: In our Cochrane systematic review, we included all available evidence from randomized clinical trials performed worldwide. Study selection,
data extraction and quality assessment was undertaken by two review authors
independently.
Results: Fifteen of the 265 identified publications were eligible, comprising
1,488 randomized participants. Twelve trials compared a skin graft or tissue
replacement with placebo or standard care. The remaining three trials compared
two types of tissue replacement products. Most trials assessed the effectiveness
of a cultured human dermal replacement consisting of dermal matrix proteins
and fibroblasts. Pooled results showed that, compared to standard care, tissue
replacements increased ulcer healing rate (relative risk: 1.50 [95% confidence
interval: 1.30–1.73]; risk difference: 0.23 [0.14–0.32]; number needed to treat: 6
[5–9]). Three studies directly compared two types of tissue replacement products. None of these showed significant differences in healing rates, time to complete ulcer healing or recurrence.
Conclusions: Current best available evidence shows that tissue replacements
can increase the healing rate of diabetic foot ulcers when used in conjunction
with standard care. Long-term results are not yet available and their costeffectiveness is uncertain.
References
1. DiDomenico L, Landsman AR, Emch KJ, Landsman A. A prospective
comparison of diabetic foot ulcers treated with either a cryopreserved skin allograft or a bioengineered skin substitute. Wounds 2011; 23: 184-9.
2. Landsman A, Roukis TS, DeFronzo DJ, Agnew P, Petranto RD, Surprenant M. Living cells or collagen matrix: which is more beneficial in the treatment of diabetic foot ulcers? Wounds 2008; 20: 111-6.
3. Puttirutvong P. Meshed skin graft versus split thickness skin graft in diabetic ulcer coverage. J Med Assoc Thai 2004; 87: 66-72.
QUANTITATIVE VALIDATED ASSESSMENT OF PROGRESSION OF
ACUTE WOUND HEALING IN HUMAN SKIN
S. Ud-Din1, N. Greaves2, A. Sebastian2, M. Baguneid3, A. Bayat1
1
Institute of Inflammation and Repair, Manchester Institute of Biotechnology,
University of Manchester, Manchester, United Kingdom; 2Plastic and
Reconstructive Surgery Research, Institute of Inflammation and Repair,
Manchester Institute of Biotechnology, University of Manchester, Manchester,
United Kingdom; 3Vascular Surgery, University Hospital of South Manchester
NHS Foundation Trust, Manchester, United Kingdom
Introduction: The use of non-invasive devices has important implications for
diagnosis and monitoring treatment efficacy. There is a lack of validation in the
use of certain devices, where objective measurements taken by non-invasive
tools have been corroborated by immunohistochemical analysis. Thus, the aim
was to analyze data from three acute wound healing studies using three noninvasive devices with validation by immunohistochemistry.
Methods: One hundred and ten healthy volunteers had 5 mm diameter skin
biopsies to their arms. Spectrophotometric intracutaneous analysis (SIAscopy),
full-field laser perfusion imaging (FLPI) and three-dimensional (3D) imaging
provided quantitative measurements of melanin, hemoglobin, collagen, blood
flow and wound size; all of which were supported by immunohistochemistry.
Results: FLPI showed blood flow increased to day 7 and decreased by 40% to day
14. SIAscopy showed that haemoglobin increased to day 7 and reduced to day 14.
CD31 analysis corroborated this by showing a 76% increase in blood vessel density
to day 7 and a reduction by 14% to day 14. 3D imaging demonstrated wound surface
area reduced by 82% from day 0 to day 7 and wound volume reduced by 63% from
day 0 to day 3. The staining of the myofibroblast marker a-smooth muscle actine
supported these trends by showing increased levels by 72% from day 0 to day 14
(corresponding to wound contraction). Collagen, measured by SIAscopy, decreased
to day 7 and increased to day 14, which was validated by collagen III analysis.
Additionally, collagen I increased by 14% from day 0 to day 14. SIAscopy measureC 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
ARE PAIN AND PSYCHOLOGICAL STRESS REFLECTED IN THE
INFLAMMATORY CYTOKINE PROFILE OF BURN PATIENTS?
M. Ulrich1,2, T. Rose3, N. E. E. van Loey1, M. Vlig1, W. Talhout1,4, R. H. J.
Beelen4, H. Hofland1,5, E. Vandermeulen3
1
Association of Dutch Burns Centres, Beverwijk, The Netherlands; 2Department
of Plastic, Reconstructive and Hand Surgery, VU University Medical Center,
Amsterdam, The Netherlands; 3Burn Wound Center, Queen Astrid Military
Hospital, Brussels, Belgium; 4Molecular Cell Biology and Immunology, VU
University Medical Center, Amsterdam, The Netherlands; 5Burn Centre,
Maasstad Hospital, Rotterdam, The Netherlands
Objective: Pain is a complex experience of which physiologic and psychological
components constitute an important component and are assumed to interact. It
is assumed that changes in the endocrine and immune systems are key mediators. The aim of this study was to investigate the possible correlation between
the pro-inflammatory cytokines interleukin (IL)-6, IL-1, tumor necrosis factor
(TNF)-a, the neurohormones cortisol and oxytocin, and the wound pain and
traumatic stress symptoms.
Methods: We included 53 patients with burns in this study. Patients were
requested to provide pain scores the day before surgery and to complete the
Impact of Event Scale (IES) during the first or second week of hospitalization.
During surgery eschar was collected. IL-6, IL-1, TNF-a, cortisol, and oxytocin
were determined by luminex-assay. Pearson correlations and a point biseral
coefficient for pain > 4 were calculated.
Results: Significant positive correlations appeared between pain scores IL-6 and
IL-1. There was a negative correlation between pain and oxytocin but it did not
reach statistical significance. Traumatic stress symptoms were negatively correlated with oxytocin.
Conclusions: Cytokines in the eschar of burns were associated with higher selfreported pain scores and traumatic stress symptoms were associated with lower
oxytocin levels in eschar. Psychological stress is suggested to affect oxytocin
levels at wound site and may contribute to higher pain levels.
PSEUDOMONAS AERUGINOSA ELASTASE CLEAVES A CTERMINAL PEPTIDE FROM HUMAN THROMBIN THAT INHIBITS
HOST INFLAMMATORY RESPONSES
om2, H. Siller1, G. Kasetty1, M.
M. van der Plas1, R. Bhongir1, S. Kjellstr€
M€
orgelin3, A. Schmidtchen1,4
1
Division of Dermatology and Venereology, Department of Clinical Sciences,
Lund University, Lund, Sweden; 2Department of Biochemistry and Structural
Biology, Center for Molecular Protein Science, Institute for Chemistry and
Chemical Engineering, Lund University, Lund, Sweden; 3Department of
Clinical Sciences, Division of Infection Medicine, Lund University, Lund,
Sweden; 4LKC Medicine, Dermatology, Nanyang Technological University,
Singapore
Introduction: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen notoriously persistent in chronic infective conditions such as non-healing
leg ulcers. This species is known for its immune evasive abilities amongst
others by degradation of a large variety of host proteins. However, it has never
been investigated whether protein degradation by P. aeruginosa enzymes may
lead to the direct formation of bioactive peptides exerting novel functions.
Therefore, the aim of this study was to investigate whether P. aeruginosa can
generate peptides that modulate host responses.
Methods: Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, HPLC, and N- and C-terminal sequencing we studied thrombin degradation. Analysis of peptide function was done using various in vitro and in vivo
inflammation assays, in vitro killing assays, slot blot and electron microscopy.
Results: We found that P. aeruginosa elastase cleaves a C-terminal derived
peptide from thrombin, which inhibits pro-inflammatory responses to several
pathogen-associated molecular patterns in vitro and in vivo by preventing receptor dimerization and subsequent activation of down-stream signaling pathways.
Interestingly, small C-terminal derived peptides were found in chronic wound
fluids and on leukocytes derived from chronic wound fluids.
Conclusions: Taken together, P. aeruginosa elastase cleaves thrombin, resulting in
the formation of a peptide that dampens inflammation. These findings constitute a
novel concept of pathogen-host interactions, where bacteria mimic an endogenous
anti-inflammatory mechanism that can aid in circumvention of host responses.
A32
Abstracts
PSEUDOMONAS AERUGINOSA MODULATES HOST
INFLAMMATION BY ABATING CYTOKINE FUNCTION
M. van der Plas1, A. Brinkåker1, A. Schmidtchen1,2
Division of Dermatology and Venereology, Department of Clinical Sciences,
Lund University, Lund, Sweden; 2LKC Medicine, Dermatology, Nanyang
Technological University, Singapore
1
Introduction: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen notoriously persistent in chronic infective conditions such as non-healing
leg ulcers. The presence of this species may result in wounds that lack visible
signs of inflammation.
Methods: Immunomodulating activities of bacterial derived conditioned medium
or purified elastase and lipopolysaccharide were studied in vitro and in vivo by
analysis of transcription factor activation, and intracellular and extracellular cytokine and chemokine levels. Furthermore, digestion of cytokines and chemokines
was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Results: We show that P. aeruginosa elastase degrades cytokines in the extracellular milieu. No differences were found between strain PAO1 as compared to
lasB mutant PAOB1 in activation of transcription factors NFjB and AP1, and
subsequent intracellular cytokine production in monocytes. Interestingly, we
found neither difference in the percentage of elastase producing strains nor in
elastase activity between P. aeruginosa isolates from chronic ulcers as compared to those from sepsis patients.
Conclusions: Taken together, P. aeruginosa may modulate host inflammation
by abating cytokine function.
IDENTITY AND PHENOTYPE OF CULTURED HUMAN GINGIVAL
AND SKIN FIBROBLASTS
C. Viennet1, M. Tissot1, C. Lallam1, P. Muret1, S. Robin2, G. Rolin3, P. G.
Humbert4
1
Cutaneous Engineering and Biology Laboratory, Inserm UMR 1098, University
of Franche-Comt"e, Besançon, France; 2Bioexigence, Besançon, France;
3
Clinical Investigation Centre, Inserm 1431, University Hospital, Besançon,
France; 4Dermatological Department, University Hospital, Besançon, France
Introduction: Differences between gingival and cutaneous wound healing have been
described, including a faster remodeling capacity following injury and a scarless tissue
repair for gingiva. Fibroblasts play a central role in wound healing and myofibroblast
differentiation is a crucial event that is linked to connective tissue remodeling and
scar formation. Understanding why gingiva and skin behave differently in response to
tissue injury should be a viable approach to controlling scarring. This work compared
the phenotype of human gingival fibroblasts (GFs) and skin fibroblasts (SFs) cultured
in monolayers and three-dimensional tensed dermis equivalent.
Methods: Cell proliferation, morphology, a-smooth muscle actin (a-SMA)
expression, migration and extracellular matrix (ECM) deposition were analyzed
by real-time PCR, enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunocytochemical methods. The contractile forces generated by
fibroblasts were also quantified using the Glasbox device.
Results: GFs displayed morphologically distinct organization with an elongated shape
and a-sma stress fibers, and proliferated faster than SFs. GFs developed highly contractile forces and migrated more rapidly as compared with SFs. GFs expressed elevated levels of molecules involved in ECM remodeling (matrix metalloproteinase-1) while SFs
showed significantly higher expression of type I collagen and type III collagen.
Conclusions: GFs display an intrinsic activation state characteristic of myofibroblastic phenotype, conducive for fast ECM contraction and remodeling,
while SFs have a profibrotic phenotype. Faster wound healing and less scar formation in gingiva could be explain by an appropriate balance of ECM synthesis
and degradation, more rapid migration and contractile potency, compared to
normal skin. The present study identifies GFs as a potential source of cell-based
therapies within the cutaneous environment.
PATIENT-REPORTED OUTCOMES AND PERCEPTIONS FOLLOWING
USE OF COMPOUNDED SCAR/BURN CREAM: INTERIM RESULTS
FROM AN OBSERVATIONAL SURVEY STUDY
H. J. Visser1, E. Harris2, P. Hurwitz3, D. Dietze4, C. Viereck4
1
Mid-West Podiatry and Associates, LLC, Creve Coeur, MO USA; 2Safe
Harbor Compliance and Clinical Services, LLC, Austin, TX USA; 3Clarity
Research and Consulting, LLC, Narragansett, RI USA; 4Metrics for Learning,
LLC, Queen Creek, AZ USA
Introduction: In addition to aesthetic implications, scar tissue can cause symptoms including pain, itching, tenderness, physical deformities, and psychological
effects, and can interfere with daily activities (1, 2). Therefore, collection of
patient-reported outcomes and perceptions is important in the study of scar
treatments. This pre-planned interim analysis of an observational survey study
(institutional review board-approved) involving 21 sites, evaluated patient-
A33
reported outcomes and perceptions regarding the use and effects of treatment
with compounded scar/burn creams over 4 months.
Methods: Adult patients with scar/burn tissue "1-month old, healed, closed, and not
infected and using one of two formulations of a compounded scar tissue treatment
(collagenase 200 U/g, Naltrexone 10 mg/g and Aloe Vera freeze-dried 1:200 3 mg/g
in Pracasil Plus gel or Naltrexone 10 mg/g, EGCG 1%, dimethyl sulfone 5%, caffeine
1% in anhydrous gel) were enrolled. Patient surveys were administered at each visit.
Results: Interim results (data collected 2014/2015) report on paired analyses
(n 5 69, 52 females/17 males) from baseline to visit 3, separated by a mean of
129 6 51 days. 59% (40/68) of patients reported reduced scar/burn size after treatment. Itching ratings decreased 48% (2.1 to 1.1/10; p 5 0.006, n 5 66). Scar/burn
interference with mood and daily activities (BPI short form interference questions)
decreased 47% (1.7 to 0.9/10, p 5 0.001, n 5 69). Patients taking medication for
pain decreased 50% (82% to 41%, p < 0.001, n 5 67). Adverse events were
reported by 26% (18/69); other—not specified (10), dryness (5), rash or redness at
scar site (3). No serious AEs were reported. 90% (62/69) indicated the creams
helped/improved scar appearance. 48% (32/67) felt their emotional well-being
positively changed since using the scar medication. At visit 3, a higher proportion
of patients with scar < 1 year (72.4%, 21/29) indicated scar size reduction compared to patients with scar >1 year (48.7%, 19/39, p 5 0.050).
Conclusions: Interim analysis demonstrated that the treatments were safe and
well-tolerated. Other results suggest that the compounded study creams may
reduce scar size, itching ratings, and mood/daily living interference scores in
adults. Trial continuation justified.
References
1. Ahn ST, Monafo WW, Mustoe TA. Topical silicone gel: a new treatment
for hypertrophic scars. Surgery 1989; 106: 781-7.
2. Ahn ST, Monafo WW, Mustoe TA. Topical silicone gel for the prevention
and treatment of hypertrophic scar. Arch Surg 1991; 126: 499-504.
THE INTERACTION OF CULTURED MESENCHYMAL
STROMAL CELLS WITH THREE-DIMENSIONAL
POLYLACTIDE MATRIX
I. Voronkina1,2, L. Smagina2, N. Yudintseva2, P. Nikonov3, E. Ivanova4,
Y. Nashchekina2, 5
1
Russian Academy of Sciences, Sankt-Petersburg, Russian Federation; 2Institute of
Cytology, Russian Academy of Sciences, Sankt-Petersburg, Russian Federation;
3
Sankt-Petersburg Polytechnic University, Sankt-Petersburg, Russian Federation;
4
Sankt Petersburg State Technological Institute, Sankt-Petersburg, Russian
Federation; 5Sankt-Petersburg State University, Sankt-Petersburg, Russian Federation
Introduction: The purpose of this research was to study the interaction of mesenchymal stromal cells (MSCs) with three-dimensional (3D) polylactide matrices and their synthesis of extracellular matrix (ECM) proteins.
Methods: Rabbit MSCs were cultured in 3D polylactide matrices with collagen
gels for 9 days. The following variants were compared: i) collagen gel with different densities; ii) polylactide matrices; and iii) polylactide matrices in collagen
gels of different densities. The deposition of type III collagen, type IV collagen,
laminin and fibronectin was determined by immunocytochemistry. The amount
of ECM proteins synthesized by MSCs and activity of matrix metalloproteinases
(MMP) MMP-1, MMP-2, MMP-3 and MMP-8 were determined by Western
blot and zymography at day 1, 5 and 9.
Results: For cell culture the polylactide surface required to be treated with
type I collagen. The density of collagen gel embedded into 3D polylactide
matrix can vary in certain range depending on the purpose. The experiments
showed that the rate of ECM synthesis depends on collagen density and on
mechanical strength. The protein composition of ECM synthesized by MSCs
cultured in 3D polylactide matrices showed differences in spatial organization
and composition. MMP activity depends on density of collagen gel embedded
into matrices.
Acknowledgment
This work was supported by RFBR (grant 13-04-12077-ofi-m).
COMPARATIVE ANALYSIS OF EXTRACELLULAR MATRIX
SYNTHESIS BY HUMAN FIBROBLASTS CULTURED ON SURFACES
WITH DIFFERENT ADHESIVE PROPERTIES
I. Voronkina1, O. Milenina2,3, L. Smagina3, E. Ivanova2,3, N. Yudintseva3, Y.
Nashchekina3,4
1
Russian Academy of Sciences, Sankt-Petersburg, Russian Federation; 2StPetersburg State Technology Institute (Technical University), Sankt-Petersburg,
Russian Federation; 3Institute of Cytology, Russian Academy of Sciences, RAS,
Sankt-Petersburg, Russian Federation; 4Sankt-Petersburg State University,
Sankt-Petersburg, Russian Federation
Introduction: Dermal cells and their complexes with extracellular matrix
(ECM) components are used as skin substitutes for transplantations. For this
purpose studies on the composition and structure of ECM produced by different
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
fibroblasts are important in regenerative medicine. The aim of this study was to
investigate differences in composition and structure of ECM synthesized by
human fibroblasts of different origins.
Methods: We used human dermal fibroblasts from adult normal skin and scar
tissues as well as embryonic fibroblasts. Fibroblasts were cultured on common
culture plastic (normal situation) and on the fluorocarbon perfluorodecaline
(minimal adhesion) for 24 and 72 hours. ECM deposition was analyzed by
Western blot for laminin, fibronectin, type I collagen and type IV collagen.
ECM structure was determined by immunocytochemical staining of cells for
type I collagen, laminin, fibronectin, hyaluronic acid, chondroitin sulfate,
decorin and versican. The immunostained cells were visualized by confocal
microscopy. The activity of matrix metalloproteinases (MMPs) was studied by
zymography.
Results: Our results showed that scar ECM contains a variety of smaller protein components compared to ECM of normal fibroblasts. Immunocytochemically, the spatial distribution of studied proteins in the microenvironment of
dermal fibroblasts varied for normal and scar tissue. Embryonic and scar
fibroblasts were synthetically more active than normal fibroblasts. Embryonic
fibroblasts had the highest MMP activity on both substrates. On plastic MMP2 activity for all cells was higher than on the nonadhesive surface (perfluorodecaline) and on non-adhesive surface additional bands of activated MMP-9
exist.
Conclusions: In both cases—common plastic and non-adhesive surface—the
composition of ECM was changed not qualitatively but quantitatively and significantly. The observed changes depended on the origin of fibroblasts and on
the adhesive properties of the substrate.
Acknowledgment
Work was supported by RFBR (grant 13-04-12077-ofi-m).
SCAR ETIOLOGY: AN INTEGRATIVE GENOMIC APPROACH
H. Wallace1
1
Burn Injury Research Unit, School of Surgery, University of Western
Australia, Crawley, Australia
Scarring after burn injury is a significant health problem. While there are surgical and non-surgical interventions to manipulate the healing process and influence scar outcome, therapies that address specific molecular pathways in scar
formation are not yet available due to gaps in knowledge about the biological
mechanism of scarring. A program of research is underway using an integrated
genomic approach, incorporating multiple levels of information to understand
the biology of skin fibrosis after burn wound healing. Bioinformatic approaches
are used to examine the intersection of genomic, transcriptional (gene expression), epigenomic (DNA methylation), and clinical datasets. In particular, we
leverage the information contained in the transcriptome and epigenome of burn
scars to prioritize gene regions for analysis of DNA variation in relation to scar
phenotype. Validation of the genetic variants using the innovative “Scar-in-ajar” model is a concrete first step toward development of novel anti-fibrotic
strategies.
HUMAN FETAL FIBROBLASTS HAVE REDUCED ADHESION BUT
IMPROVED MIGRATION
M. Walraven1, R. H. J. Beelen2, M. van Egmond1, M. Ulrich1,3
VU University Medical Center, Amsterdam, The Netherlands; 2Molecular Cell
Biology and Immunology, VU University Medical Center, Amsterdam, The
Netherlands; 3Association of Dutch Burn Centres, Beverwijk, The Netherlands
1
Introduction: Fibroblast migration and adhesion affect scarring and wound
healing. Fibrotic fibroblasts are reported to have enhanced adhesive signaling
and improved adhesive capacities (1). The aim of this study was to compare the
adhesive and migratory capacities of adult fibroblasts with those of fetal fibroblasts, which are derived from a scarless environment.
Methods: Human fibroblasts were isolated from fetal and adult dermis (n 5 6)
and cultured up till passage six. Quantitative RT-PCR, cell adhesion and cell
migration assays were performed and a commercially available a/b Integrinmediated cell adhesion assay was used to determine integrin expression patterns
of the a1, a2 a3, a4, a5, and a6 subunit integrins, the b1, b2, b3, b4, and b6
subunit integrins and the a5b1, aVb3, and aVb5 integrin complexes.
Results: Fetal fibroblasts had lower mRNA expression of several pro-adhesive
genes, i.e., focal adhesion kinase (FAK), paxillin, Ras-related C3 botulinum
toxin substrate 1 (Rac1), Ras homolog gene family member A (RhoA) and
RhoE. Fetal fibroblasts also showed reduced adhesion after 30 minutes at 378C
to uncoated plastic (43.1 6 7.1% versus 66.8 6 4.5% for adult fibroblasts) and
to type I collagen (69.6 6 2.8% versus 93.3 6 4.5%) or fibronectin (71.9 6
3.8% versus 94.6 6 4.5%) coated plastic. Fetal fibroblasts migrated faster compared to adult fibroblasts on plastic (30.6 6 2.8 mm/hour versus 16.3 6 1.6
mm/hour) or coated plastic. Furthermore, significant (p < 0.05) lower levels of
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Wound Rep Reg (2015) 23 A1–A37 V
the a1, a3 and a4 subunits and the a5b1 and the aVb5 complexes were measured in fetal fibroblasts.
Conclusions: This study shows that fetal fibroblasts have reduced adhesion but
improved migration compared to adult fibroblasts. This migratory phenotype
might be due to differential integrin expression patterns with low levels of the
integrin subunits a1, a3 and a4, and the a5b1 and aVb5 heterodimers. Blocking
these integrins in adult fibroblasts might, therefore, result in reduced adhesion
or improved migration. Since fibrotic fibroblasts have an adhesive phenotype
and fetal fibroblasts have a migratory phenotype, stimulating fibroblast migration might positively affect wound healing.
Reference
1. Chen Y, Shi-Wen X, van Beek J, Kennedy L, McLeod M, Renzoni EA,
Bou-Gharios G, Wilcox-Adelman S, Goetinck PF, Eastwood M, Black CM,
Abraham DJ, Leask A. Matrix contraction by dermal fibroblasts requires transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing
proteoglycans, and MEK/ERK: insights into pathological scarring in chronic
fibrotic disease. Am J Pathol 2005; 167: 1699-1711.
INVESTIGATION OF THE MECHANISM OF INCREASED HEALING
TENDON STRENGTH AFTER BASIC-FIBROBLAST GROWTH
FACTOR OR VASCULAR ENDOTHELIAL GROWTH FACTOR GENE
THERAPY BY ADENO-ASSOCIATED VIRUS2 VECTORS
X. T. Wang1, Y. F. Wu2, Y. L. Zhou2, J. B. Tang1,2, P. Y. Liu1
1
Department of Plastic Surgery, Rhode Island Hospital/Brown Medical School,
Providence, RI USA; 2Department of Hand Surgery, Affiliated Hospital of
Nantong University, Nantong, Jiangsu, China
Introduction: Tendon healing is innately weak. Previously, we demonstrated
that bFGF or VEGF gene therapy using AAV2 vectors significantly increased
healing in a chicken flexor tendon model (145-220% of control). Here we
investigated the possible mechanism for this result—how endogenous tendon
healing-related genes were activated and the differences between epitenon and
endotenon using laser capture microdissection (LCM).
Methods: Eighteen digital flexor tendons from the big toes of 9 chickens were
completely transected and divided into three groups: AAV-bFGF, AAV-VEGF
and no-treatment control. After AAV injection, the tendons were surgically
repaired. Four weeks after surgery, the tendons were formalin-fixed, paraffinembedded and sectioned. LCM was used to dissect epitenon and endotenon separately. The tissue was analyzed for gene expression of collagen type I alpha 1
(COL1A1), collagen type III alpha 1 (COL3A1), proliferating cell nuclear antigen (PCNA), fibronectin 1 (FN1), scleraxis (SCX) and tenascin C (TNC) using
RT-qPCR analysis.
Results: The gene expression of FN1, PCNA, SCX, COL1A1 and COL3A1 was
significantly increased (p < 0.05 or p < 0.01) in AAV-VEGF and AAV-bFGF
groups. Increases in TNC gene expression trended to statistical significance;
TNC gene was highly expressed in endotenon (10-fold greater than in the epitenon) in AAV-VEGF injected tendon. TNC in the endotenon of AAV-VEGFinjected tendon was raised by 55-fold compared with that of the non-injected
tendon.
Conclusions: There were significant increases in expression of studied genes
specific to tendon growth and regeneration after AAV2-bFGF or AAV2-VEGF
gene therapy. We also found precisely that epitenon and endotenon are equally
responsive to the gene therapy. This study provides evidence to support the
effective changes of the gene expression by the gene therapy, which may be
key mechanistic findings to support the use of these gene therapies. An IND
has been filed with the FDA to test this in humans.
ENHANCING SURVIVAL OF TRANSPLANTED CELLS IN THE
WOUND BED
A. Wells1
1
Department of Pathology, Pittsburgh, PA USA
Introduction: Transplantation of cells to augment the intrinsic regenerative
capacity is a potential therapy for non-healing wounds. Unfortunately, such
approaches have been less than successful, as the majority of introduced cells
are lost within days due to cell death from the harsh wound environment that
lacks nutrients and presents apoptosis-inducing cytokines. A cellular engineering
approach to enhanced survival could overcome this obstacle.
Methods: Activation of the EGF receptor at the cell surface, but not from internalized receptors in the endosomes, promotes cell survival in the face of cellular starvation, death cytokine signaling, and toxic agents. The signaling cascade involves
tonic low-level activation of Erk MAPK and PI3K/Akt pathways. To achieve this
mode of signaling the triggering ligand must be retained in or attached to the substratum or matrix. This can be accomplished by either tethering classical EGF
receptor ligands to the substratum, or activating the EGF receptor with cryptic
ultralow affinity matrikines present in tenascin-C and laminin V.
A34
Abstracts
Results: We have created matrices with (or without) tethered EGF or tenascinC. These were introduced into acute full-thickness wounds in mice, internal soft
tissue in mice (subfascial), and critical cortical bone defects in dogs. In all three
instances wound healing and vascularization was significantly increased in the
presence of EGF receptor ligand. Xenotransplanted cells were retained up to
one month in immunocompetent mice, compared to less than a week without
the tEGF or tenascin-C. The presence of these cells with the tenascin-C limited
scarring in a hypertrophic scar model, even 6 months after wounding, when all
the transplanted cells have been rejected.
Conclusions: The extended survival of these transplanted cells allows them to
educate the local wound environment, and turn the healing toward regeneration
and away from nonfunctional scarring or failure to heal. This represents a new
approach to cellular support for dysrepair.
THE BRIGHT AND THE DARK SIDES OF REACTIVE OXYGEN
SPECIES IN WOUND REPAIR
S. Werner1
1
Institute of Molecular Health Sciences, ETH Zurich, Switzerland
A large percentage of the population suffers from chronic non-healing wounds, and
their incidence is continuously increasing in an aging society. Therefore, it is essential
to develop novel strategies for the improvement of wound healing and this requires a
thorough understanding of the underlying molecular and cellular mechanisms. In
recent years, reactive oxygen species (ROS) have been identified as key players in
normal and impaired wound healing. Thus, low levels of ROS are required for efficient cellular signaling, for attraction of immune cells to the site of injury and for the
repair process itself. On the other hand, excessive levels of ROS cause oxidative
stress, which was identified as an important pathogenic factor in the development of
chronic, non-healing ulcers and which is also involved in skin aging. Therefore, an
efficient cellular antioxidant defense system is required for normal wound healing. I
will provide an overview on the role of ROS in wound healing and present recent
results from our group that demonstrate a key role of the Nrf2 transcription factor and
its cytoprotective target genes in skin homeostasis and wound repair.
DEVELOPMENT OF AN IN VITRO SOFT TISSUE INFECTION MODEL
RELATED TO PERCUTANEOUS MEDICAL DEVICES
M. Werth"en1, F. Taherinejad1
M€olnlycke Health Care, G€
oteborg, Sweden
1
Introduction: Biomaterial-associated infection is a common and serious complication in long-term tissue-biomaterial interactions. Established infections are often persistent and hard to fight with antibiotics. Recent studies question the strong focus on
surface adhered biofilm as the key factor of these infections, and are instead suggesting
that biofilm formation in the tissue surrounding the device may be equally or more
important. That in turn requires relevant in vitro models when testing novel antimicrobial treatments, to reduce the distance to in vivo models. The aim of this study was to
develop an in vitro model simulating soft tissue contamination and infection related to
skin penetrating devices, with possibilities to analyze bacterial colonization of both the
biomaterial surface and the surrounding medium mimicking the soft tissue.
Methods: The model comprises a collagen matrix in which a contaminated
model device is incubated. Segments of silver-coated and non-coated silicon
urinary catheters were here used as model biomaterials and Staphylococcus aureus and Pseudomonas aeruginosa were used as model bacteria. To mimic a perioperative contamination, the catheter segments were submerged for a short
time in a highly diluted bacterial culture, before insertion in the collagen matrix.
Bacterial quantification was performed with plate count method.
Results: Even the smallest inocula/contaminations resulted in bacterial growth both
on the model biomaterial and in the surrounding collagen matrix. Lower amount of
P. aeruginosa was only found on the silver coated material, whereas in the surrounding collagen matrix, bacterial growth was similar for both model biomaterials.
Conclusions: With a soft tissue infection model which does not discard any bacteria
during the experimental steps, it is possible to quantify and observe where the bacteria are located, both on the biomaterial surface and in the surrounding medium. Such
model better mimics the challenges for biomaterials in the clinical situation.
PYOCYANIN FROM PSEUDOMONAS AERUGINOSA BINDS TO
POLYURETHANE FOAM DRESSINS – A POSSIBLE INDICATION OF
INFECTION AND PROTECTION FROM TOXIC EFFECTS
M. Werth"en1, A. Persson2, A. Dahlberg1
1
M€olnlycke Health Care, G€
oteborg, Sweden; 2Medibiome AB, M€
olndal,
Sweden
Introduction: Pseudomonas aeruginosa is one of the most problematic wound
pathogens. The blue-green pigment pyocyanin is one of its many virulence factors, with toxic effects on the wound tissue. When P. aeruginosa is present in a
wound, the wound dressing may be visibly stained by pyocyanin, and the question has been raised as to whether this green coloration indicates that the dress-
A35
ing material promotes bacterial growth. The aim of this study was to
investigate, in vitro, any correlation between P. aeruginosa growth in the presence of a dressing and pyocyanin staining of the dressing material.
Methods: The growth and pyocyanin production by P. aeruginosa (PAO1) cultures were studied in the presence of commercially available wound dressings
of different materials and brands. Bacterial numbers were quantified by viable
plate counts, and pyocyanin was measured by absorbance spectrophotometry.
Results: No difference in bacterial growth was observed in the presence of any
dressing. The amount of pyocyanin increased over time in all cultures. The cultures with dressings always resulted in lower pyocyanin concentrations than the
control without dressing. The lowest levels were found in cultures surrounding
the polyurethane foam dressings in which pyocyanin was found to accumulate.
Conclusions: There is no evidence that any of the examined materials promote
growth of P. aeruginosa. Instead one may speculate that in the clinical setting,
the more pyocyanin that is found in the dressing, the less of this harmful virulence factor will be left in the wound tissue. Also, the more visible the pyocyanin, the easier infection could be detected.
TREATMENT AND PREVENTION OF WOUND INFECTIONS FROM A
PRECLINICAL PRODUCT DEVELOPMENT PERSPECTIVE
M. Werth"en1, K. Hamberg1
1
M€
olnlycke Health Care, G€
oteborg, Sweden
Introduction: Wound infections as well as biomaterial-associated infection
involve bacteria growing as biofilm, which plays a crucial role in the persistence of these soft tissue infections. There is a need for novel products/strategies
to prevent and treat biofilm infections, and that in turn requires relevant in vitro
models to reduce the distance to in vivo models during product development.
The aim of this study is to highlight the need for relevant preclinical test models during design and development of antimicrobial wound dressings.
Methods: Antimicrobial effect of four different antimicrobial dressings on Pseudomonas aeruginosa, was tested in three different test models, with increasing
challenges, such as nutrients and growth phase. The Direct contact test is a log
phase test in low nutrient levels and relatively dry conditions. The Two compartment model provides higher serum proteins in growth medium and a moist
environment. The Biofilm test model starts with an established biofilm in a collagen matrix. All tests involve 24 hours of incubation in 358C. The number of
viable bacteria in the cell culture was determined with plate count. Data are
presented as mean values 6 SD (n 5 3).
Results: It is clearly shown that different test models, with increasing challenges
for the dressings regarding nutrients for the bacteria and their growth phase, often
generate very different results for the same antimicrobial wound dressing.
Conclusions: The present study highlights the fact that choice of test method
matters for the dressing performance, and there is a need for relevant in vitro
models, mimicking the environment of the actual treatment site, when testing
antimicrobial dressings. It is recognized that in vitro data cannot be directly
extrapolated to clinical outcomes. Nevertheless, in vitro models may be beneficial to enhance our understanding of how topical antimicrobial agents and
wound dressings may facilitate wound management.
REGULATION OF DIFFERENTIATION AND CHROMATIN
REMODELLING IN MYELOID CELLS DURING WOUND HEALING IN
THE DIABETIC ENVIRONMENT
K. Wicks1, H. Alsadoun1, T. Torbica1, S. Alrdahe1, K. Mace2
University of Manchester, Manchester, United Kingdom; 2The Healing
Foundation Centre, Faculty of Life Sciences, Manchester, United Kingdom
1
Introduction: The number of patients suffering from chronic wounds is nearing
epidemic proportions as the prevalence of diabetes continues to rise dramatically (1-6). Fifteen per cent of diabetic patients develop chronic, non-healing
wounds, 84% of which result in lower limb amputation. During the last two
decades, deregulated inflammation has been implicated in chronic wound development although the underlying mechanisms remain elusive.
Methods: Bioinformatic analyses of our RNA-seq data from non-diabetic (ndb)
and diabetic (db)-derived myeloid cells was used to identify differentially
expressed genes. Quantitative RT-PCR, protein expression analyses, and chromatin immunoprecipitation were used to validate and investigate the functions
of differentially expressed intrinsic factors. Hoxa3 protein transduction of ndb
and db-derived macrophages was used to assess its capacity to rescue the diabetic phenotype.
Results: We found that defects in diabetic mouse myeloid cell function are
linked to defects in differentiation caused by aberrant epigenetic chromatin
marks. Similar differences were found in diabetic patients. These changes are
associated with hyper-polarization of myeloid cells towards a pro-inflammatory
phenotype that contributes to chronic inflammation in diabetic wounds. Protein
transduction of the transcription factor Hoxa3 suppresses inflammation and promotes wound healing, in part through rescuing differentiation defects. Data will
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Abstracts
be presented that elucidate the molecular mechanisms underlying myeloid
deregulation in diabetes and how it may be therapeutically reversed.
Conclusions: These findings have important implications, as epigenetic changes
constitute a “cellular memory” that may influence therapeutic treatments. Thus,
it is critical to determine whether epigenetic changes persist after resolution of
diabetic symptoms (e.g., high blood sugar), and how these changes impact
potential therapies.
References
1. Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing
in diabetes. J Clin Invest 2007; 117: 1219-22.
2. Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global
burden of diabetic foot disease. Lancet 2005; 366: 1719-24.
3. Boulton AJ. The diabetic foot: grand overview, epidemiology and pathogenesis. Diabetes Metab Res Rev 2008; 24 Suppl 1: S3-6.
4. Mace KA, Restivo TE, Rinn JL, Paquet AC, Chang HY, Young DM,
Boudreau NJ. HOXA3 modulates injury-induced mobilization and recruitment
of bone marrow-derived cells. Stem Cells 2009; 27 1654-65.
5. Mace KA, Hansen SL, Myers C, Young DM, Boudreau N. HOXA3 induces cell migration in endothelial and epithelial cells promoting angiogenesis and
wound repair. J Cell Sci 2005; 118: 2567-77.
6. Mahdipour E, Charnock JC, Mace KA. Hoxa3 promotes the differentiation of hematopoietic progenitor cells into proangiogenic Gr-1+CD11b+ myeloid cells. Blood 2011; 117: 815-26.
IS ASPIRIN SAFE AND EFFECTIVE AS AN ADJUNCT TO
COMPRESSION THERAPY FOR VENOUS LEG ULCERS?
G. Wiesner1, A. Barker1, I. Darby2, T. Haines1, J. McNeil1, M. Underwood3, S.
Ward1, C. Weller1
1
Monash University, Melbourne, Australia; 2Royal Melbourne Institute of
Technology, Bundoora, Australia; 3Warwick University, Coventry, United
Kingdom
Introduction: An estimated 400,000 Australians suffer from venous leg ulceration (VLU) costing $2–3 billion per year (2010 data). Moreover, the burden of
disease is expected to rise with an ageing population and the growing diabetes
and obesity epidemics. Although current best practice involves compression
therapy 30–50% of VLUs remain unhealed after 2 years and recurrence is common. Two small studies previously suggested that aspirin can improve healing
rates and decreases recurrence. Hence the Aspirin in Venous Leg Ulcer
(ASPiVLU) Study (ACTRN12614000293662) will evaluate the therapeutic
value of adding aspirin to compression therapy via a robust, double-blind,
randomized controlled trial.
Methods: The ASPiVLU intervention consists of 12 weeks of standardized,
weekly compression therapy in combination with 12 months of daily study drug
(Aspirin 300 mg or placebo). Participants must be 401 years old, not taking
routine aspirin, with an ulcer that has existed for at least 6 weeks in the presence of chronic venous insufficiency. The primary endpoint is time to healing.
Secondary endpoints include recurrence rate, quality of life (EQ-5D-5L and
wound pain), various biomarkers of inflammation and platelet activation,
adverse events, and intervention adherence.
Results: The study commenced in March 2015 and will be completed by
December 2017. It aims to recruit 268 participants from six hospital wound
clinics from around Australia.
Conclusions: Aspirin is a widely used drug that has several actions potentially
capable of influencing the progression of VLUs (1). Our findings may improve
the quality of life and reduce treatment costs for a large number of people in
the future. If proved safe and effective, the low cost of aspirin would make it
an affordable adjunct to compression for people with VLUs world-wide.
Reference
1. Varatharajan L, Thapar A, Davies AH. Adjunctive aspirin for venous
ulcer healing. Phlebology 2014; 29: 336-7.
RESETTING MIR-21 IMPROVES THE TISSUE REPAIR POTENTIAL
OF MSCS BY ERASING FIBROTIC MECHANICAL MEMORY
ACQUIRED IN CELL CULTURE
L. C. Xi1, T. Nilesh1, B. Stellar1, B. Jenna2, B. Hinz1
University of Toronto, Toronto, Canada; 2Yale University School of Medicine,
New Haven, CT USA
1
Introduction: Due to a mechanical memory imprinted during conventional stiff
cell culture expansion, mesenchymal stem cells (MSCs) acquire pro-fibrotic
myofibroblast features that are robust against subsequent environmental
changes. Continued pro-fibrotic behavior after tissue transplantation jeopardizes
the success of MSC therapy in wound healing applications. We set out to elucidate and exploit the molecular mechanism preserving mechanical memory to
improve the repair potential of MSCs.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V
Methods: Using continued culture on physiologically soft silicone culture substrates, we show that soft-priming suppresses myofibroblast development and
de-sensitizes MSCs against subsequent mechanical activation.
Results: Transplantation of soft-primed MSCs dramatically reduces scar features
in a rat model of excessive wound healing whereas transplantation of stiffprimed MSCs amplifies scarring. We identify the micro RNA miR-21 as a
novel mechano-sensitive regulator of the fibrogenic program and memory
keeper in MSCs. Cellular miR-21 levels always adjust to the levels of substrate
stiffness existing during priming and persist after exposure to new mechanical
conditions. Importantly, transient knock-down of miR-21 by the end of the stiffpriming period is sufficient to erase—or reset—mechanical memory and de
novo sensitizes MSCs to subsequent exposure to soft substrates. Such stiffprimed but memory-erased MSCs reduce wound scarring after implantation similar to soft-primed MSCs.
Conclusions: Hence, both soft-priming and erasing mechanical memory during
the cell culture period will protect MSCs from fibrogenesis in the host wound
environment and increase the chances of MSC therapy success in tissue repair
applications.
QUANTITATIVE ASSESSMENT OF SCAR STIFFNESS USING
ULTRASONOGRAPHY
S. Yamawaki1, R. Aya2, Y. Katayama2, T. Enoshiri2, S. Saitoh2, M. Naitoh2, S.
Suzuki2
1
Department of Plastic and Reconstructive Surgery, Takeda General Hospital,
Kyoto, Japan; 2Department of Plastic and Reconstructive Surgery, Graduate
School of Medicine, Kyoto University, Kyoto, Japan
Introduction: Keloid and hypertrophic scars are fibroproliferative diseases and
these diseases occurred after the important or trivial trauma. The steroid injection is one of the most effective treatments for the scars. After steroid injections, scars become soft and flat. We assessed the treatment courses of scars
after steroid injections using ultrasonography.
Methods: Six keloids of five patients and three hypertrophic scars of two
patients were enrolled in this experimental study. We used HI VISION Avius
ultrasound scanner with real-time elastography and the L74 linear (5–13 MHz)
probe (Hitachi Medical Corporation, Tokyo, Japan). We measured the stiffness
of keloid lesions (Strain Ratio; SR) by real time tissue elastography, and the
thickness of scars by B-mode scan.
Results: Almost all scar lesions have become softer and thinner gradually, and
it showed as the decrease of the numerical data. One lesion that occurred after
the resection of the pigmented nevus on her chest worsened her clinical findings
in spite of regular steroid injections and it was showed as the increase SR and
thickness.
Conclusions: The improvement of clinical findings of scars was indicated as
the decrease of SR and thickness. Ultrasonography is noninvasive and convenient. Moreover, using real time tissue elastography, we can obtain the stiffness
as numerical data. Real time tissue elastography was developed in early 2000s
by Matsumura et al. The device is used for the diagnosis of the breast cancer,
thyroid cancer and liver fibrosis which are characteristics of stiffness. This
device seems very promising tool for the scar assessment.
EFFECT OF NEGATIVE PRESSURE WOUND THERAPY WITH
INSTILLATION ON BIOBURDEN IN CHRONICALLY INFECTED
WOUNDS
C. Yang1, S. Goss1, S. Alcantara1, Q. Yang2, G. Schultz2, J. Lantis1
Surgery, Mount Sinai St. Luke’s Hospital and Mount Sinai Roosevelt Hospital,
New York, NY USA; 2Institute for Wound Research, University of Florida,
Gainesville, FL USA
1
Introduction: Infection and subsequent delayed healing are common complications of chronic wounds, resulting in enormous burden to those affected. Treating infection and reducing microbial colonization aids in wound healing. The
use of negative pressure wound therapy (NPWT) with topical irrigation solution
has shown promise as an adjunct to sharp debridement for sterilization of
wounds. The goal of this study was to evaluate the effectiveness of NPWT with
instillation on biofilm-protected and planktonic bacteria in chronic wounds.
Methods: A prospective, randomized trial was conducted. Following sharp
debridement, 20 patients with chronic wounds were randomized to 1 week of
either NPWT with 0.125% sodium hypochlorite solution instillation (n 5 11) or
NPWT without instillation (n 5 9). Serial wound biopsy were performed predebridement and post-debridement at week 0 and week 1. Biopsies were analyzed for quantitative biofilm-protected and planktonic bacteria.
Results: There was no difference in wound size between the two groups at 1
week. Within group analysis showed a statistically significant 42% reduction in
biofilm-protected bacteria at 1 week in the NPWT-instillation group (p < 0.05)
and a non-statistically significant 24% increase in biofilm-protected bacteria in
the NPWT without instillation group (p > 0.05). No between group differences
A36
Abstracts
were found for the NPWT and NPWT-instillation groups on any biopsy for
biofilm-protected and planktonic bacteria.
Conclusions: Consistent with previous studies, we demonstrate that NPWT with
instillation sodium hypochlorite solution is effective at reducing bioburden of
chronically infected wounds. This wound management therapy provides both
antimicrobial and NPWT benefits and may be a tool for the preparation of
infected wound beds prior to definitive closure.
HAIR FOLLICLE TRANSPLANTATION AS A NOVEL APPROACH TO
HEALING CHRONIC WOUNDS IN THE PORCINE MODEL
C. Yang1, S. Goss1, S. Alcantara1, J. Lantis1
Surgery, Mount Sinai St. Luke’s Hospital and Mount Sinai Roosevelt Hospital,
New York, NY USA
1
Introduction: Chronic wounds have been demonstrated to lack functional dermis
by which to protect and regenerate epidermis. Stem cells in the hair follicle bulge
region are a potential source of new functional dermis to the chronic wound bed
while affecting only a limited area of the donor site. The goal of this translational
study is to regenerate full thickness dermis in a porcine chronic wound model by
autologous transplant of stem cells in hair follicle bulge regions.
Methods: A diabetic state was induced in four pigs with streptozotocin
(150 mg/kg). Four dorsal wounds were then created on each pig. Wounds were
maintained with hydrogel for a period of 7 days at which point wounds on each
pig received either low density hair follicle transplant, high density fair follicle
transplant, sham, or full-thickness skin graft (FSTG). Wounds were maintained
for an additional 7 days before biopsied for histological analysis.
Results: Both low density and high density hair follicle graft treatment showed
histological evidence of dermal regeneration, namely density, depth, and
adnexal structures that were comparable to the FSTG group but not seen in the
sham group. The high density hair follicle group had additional evidence of
capillary ingrowth and connective tissue formation comparable to the FSTG
group. Inflammatory cells were present in all wounds.
Conclusions: Hair follicle transplantation in a diabetic porcine wound model
results in dermal regeneration comparable to FSTG. These results serve as a
proof of concept and framework for further studies into hair follicle transplantation as a tool to heal chronic wounds. Future direction will entail applying this
treatment approach to a pilot study in human subjects.
EFFECT OF DEBRIDEMENT AND SECONDARY DRESSINGS ON
BIOBURDEN IN CHRONICALLY INFECTED ULCERS
C. Yang1, S. Goss1, S. Alcantara1, C. Gendics1, Q. Yang2, D. Gibson2, G.
Schultz2, J. Lantis1
1
Surgery, Mount Sinai St. Luke’s Hospital and Mount Sinai Roosevelt Hospital,
New York, NY USA; 2Institute for Wound Research, University of Florida,
Gainesville, FL USA
Introduction: Infection and subsequent delayed healing are common complications of chronic ulcers, resulting in enormous burden to those affected. Treating
infection and reducing bioburden aids in wound healing. The goal of this study
is to evaluate the effect of debridement alone and in conjunction with secondary
dressings on both biofilm-protected and planktonic bacteria.
Methods: A retrospective review was conducted. Eighty patients with chronic
ulcers were identified who underwent debridement with pre- and postdebridement ulcer biopsies analyzed for quantitative biofilm-protected and
planktonic bacteria. Patients subsequently underwent secondary treatment with
either topical medical honey (n 5 20), topical cadexomer iodine gel (n 5 30), or
negative pressure wound therapy with instillation (NPWTi) (n 5 30). Ulcer
biopsies were taken pre- and post-secondary treatment at fixed intervals.
Results: Debridement alone resulted in a reduction of 78% (p < 0.05) in planktonic bacteria and 23% (p > 0.05) reduction in biofilm-protected bacteria. Topical medical honey reduced planktonic bacteria by 30% (p < 0.05) but did not
result in a significant change in biofilm-protected bacteria. Topical cadexomer
iodine gel reduced planktonic bacteria by 34% (p > 0.05) and biofilm-protected
bacteria by 24% (p < 0.05). NWPTi increased planktonic bacteria by 51%
(p > 0.05) and decreased biofilm-protected bacteria by 42% (p < 0.05).
Conclusions: Consistent with previous studies, we demonstrate debridement is
effective for reducing planktonic bacteria but ineffective for biofilm-protected
bacteria. Secondary treatments produced promising although mixed results. Further investigation into treatments for the elimination of biofilm-protected bacteria is needed.
A37
EFFECT OF TOPICAL CADEXOMER IODINE GEL ON BIOFILM IN
CHRONIC DIABETIC FOOT ULCERS
C. Yang1, C. Gendics1, Q. Yang2, D. Gibson2, G. Schultz2, J. Lantis1
Surgery, Mount Sinai St. Luke’s Hospital and Mount Sinai Roosevelt Hospital,
New York, NY USA; 2Institute for Wound Research, University of Florida,
Gainesville, FL USA
1
Introduction: Infection and delayed healing are common complications of diabetic foot ulcers (DFUs), resulting in enormous burden to those affected.
Treating infection and reducing microbial colonization aids in wound healing.
Topical cadexomer iodine gel has been demonstrated to have an antimicrobial
effect on planktonic bacteria. The goal of this study is to evaluate the effect
of cadexomer iodine gel on non-planktonic, or biofilm-protected bacteria.
Methods: A 4-week prospective, randomized trial was conducted. Twenty
patients with chronic diabetic foot ulcers were randomized to either topical
cadexomer iodine gel with foam dressing (n 5 10) or hydrogel gel with foam
dressing (n 5 10). Wounds were assessed weekly for size and evidence of infection and were debrided sharply. Serial wound biopsies were performed to test
for quantitative biofilm prior to debridement.
Results: There was no difference in change in wound size between the two
groups. At 4 weeks the cadexomer group had a 24% decrease in quantitative
biofilm whereas the hydrogel group had a 15% increase (p < 0.05). Patient
reported differences between the two groups include reduced odor and pain in
the cadexomer group. Observed differences between the two groups include
increased wound surface exudate and surrounding erythema in the hydrogel
group.
Conclusions: Cadexomer iodine gel has known anti-microbial action against
planktonic bacteria. We now demonstrate that cadexomer iodine gel is effective
in reducing the number of biofilm-protect bacteria in DFUs after 4 weeks of
treatment compared to control. No significant change in wound size as was
expected. This represents one of the first in vivo assays of effectiveness of a
topical antimicrobial agent on biofilm presence.
THE NATURAL BEHAVIOR OF MONONUCLEAR PHAGOCYTES IN
HYPERTROPHIC SCAR FORMATION
Z. Zhu1, J. Ding1, Z. Ma1, E. Tredget1
1
University of Alberta, Edmonton, Canada
Introduction: Hypertrophic scars (HTS) are caused by trauma or burn injuries
to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and
M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are pro-fibrotic and might contribute to HTS formation. Our laboratory
has established human HTS-like nude mouse model, in which the grafted
human skin develops red, raised and firm scar resembling human HTS. In this
study, we observed the natural behavior of mononuclear phargocytes in the
human HTS-like nude mouse model at multiple time points.
Methods: Thirty athymic nude mice received human skin grafts and thirty mice
received mouse skin grafts as controls. The grafted skin and blood were harvested
at one, two, three, four and eight weeks. Wound area, thickness, collagen, the cell
number of myofibroblasts, M1 and M2 macrophages in the grafted skin, as well as
monocyte fraction in the blood were investigated at each time points.
Results: The mice in the human skin grafted group developed obvious contracted
and thickened scars. The grafted skin four weeks post-grafting resembled human
HTS by showing fibrotic orientation of collagen bundles, increased thickness
(171.3 6 15.84% versus 100 6 8.17%) and myofibroblast number (115 6 1.76 versus 5 6 0.55) compared to controls. In human skin grafted group, monocytes dramatically decreased at one week post-grafting compared to controls
(2.3 6 0.11% versus 9 6 0.37%) and gradually returned back to normal in the
following weeks. M1 macrophages were found predominantly in one or two
weeks post-grafting compared to M2 macrophages (110 6 1.82, 65 6 1.14 versus
19 6 0.86 versus 37 6 1.08) whereas, M2 macrophages were abundant at three to
four weeks post-grafting compared to M1 macrophages (61 6 0.86, 54 6 1.11
versus 30 6 1.58, 19 6 0.86).
Conclusions: M1 macrophages involve in early phases while M2 macrophage
involve in late phases of HTS formation, which suggests that macrophage
depletion in the subacute phases of wound healing might reduce or prevent
HTS formation.
C 2015 by the Wound Healing Society
Wound Rep Reg (2015) 23 A1–A37 V