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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. C 2015 by the Wound Healing Society 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. C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 A1–A37 V 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 C 2015 by the Wound Healing Society 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. References 1. Zweier JL, Talukder MA. The role of oxidants and free radicals in reperfusion injury. Cardiovasc Res 2006; 70: 181-90. 2. Reeder BJ, Wilson MT. Hemoglobin and myoglobin associated oxidative stress: from molecular mechanisms to disease States. Curr Med Chem 2005; 12: 2741-51. 3. Makhsous M, Lin F, Pandya A, Pandya MS, Chadwick CC. Elevation in the serum and urine concentration of injury-related molecules after the formation of deep tissue injury in a rat spinal cord injury pressure ulcer model. PM R 2010; 2: 1063-5. 4. Gefen A, Cornelissen LH, Gawlitta D, Bader DL, Oomens CW. The free diffusion of macromolecules in tissue-engineered skeletal muscle subjected to large compression strains. J Biomech 2008; 41: 845-53. 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 C 2015 by the Wound Healing Society 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