the 2007 Abstract Presentations - Wound Healing Society

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Abstracts 141 NITRIC OXIDE SYNTHASE INHIBITION REVERSES SUBSTANCE P MEDIATED ACCELERATION OF WOUND CLOSURE Cheng yu Xie MD, Qiang Wang MD, Lara Muffley BS, Carina Morningstar BA, Nicole S. Gibran MD University of Washington Department of Surgery Introduction: Substance P (SP), a proinflammatory neuropeptide released from sensory nerves modulates response to cutaneous injury. We have shown that topical SP shortens time to wound closure in congenitally diabetic (db/db) mice compared to NaCl. Since SP regulates cellular nitric oxide (NO) levels wanted to determine the role of NO in SP mediated responses to injury wound healing in db/db and db/ mice. Methods: Two full-thickness 6-mm punch biopsy wounds were created on the dorsal surface of 36 db/db mice and 36 heterozygous db/– mice following anesthesia. mice. Nitrate levels were measured in the uninjured skin using Greiss reagents. Wounds were randomized to receive daily (days 0–6) topical application of either normal saline (NS), SP (10 6 M), L-NAME (non-specific NOS inhibitor; 10 4 M) or SP (10 6 M) with L-NAME (10 4 M). Wounds were photographed on PODs 0, 3, 7 and 10 and were measured using computer assisted image analysis by two blinded observers. Results were analyzed using ANOVA. Significance was accepted at p o 0.05. Results: NO levels were lower in uninjured skin from the db/db mice (1.9 mM/ L/mg compared to the db/- mice (2.6 mM/L/mg; p o .001). At POD 10, SP treated db/- mice wounds (0.04 cm 2 ) were smaller than NaCl treated wounds (0.06; p o .05). Whereas L-NAME alone (0.08 cm 2 ) did not change wound closure compared to NaCl, L-NAME negated any benefit of SP on wound closure (0.07 cm 2 ; p = 0.01). At POD 10, db/db wound size was not altered with any treatment compared to NaCl. Conclusions: These data suggest that SP acceleration of wound closure in nondiabetic mice wounds at POD 10 may be mediated by nitric oxide. This work was supported by NIDDK R01DK58007 and NIGMS R01GM56483 143 DO HAIR FOLLICLE FIBROBLASTS HAVE A ROLE IN WOUND HEALING? S. Stevenson, D.T. Sharpe, M.J. Thornton Burns & Plastic Surgery Unit & Cutaneous Research, Medical Biosciences, School of Life Sciences, University of Bradford, UK Improved wound healing is seen in hairy skin, and it is thought that while hair follicle epithelial cells are important for re-epithelialisation, hair follicle fibroblasts may act as a mesenchymal reservoir for wound healing. Therefore we have compared the migration, proliferation, collagen and VEGF secretion by cultured dermal fibroblasts (DF) and hair follicle dermal papilla (DP) and dermal sheath (DS) cells using an in vitro wound healing assay. Primary cultures were established from the same biopsies of female scalp skin; DF (n = 7), DP (n = 5), DS (n = 4). Monolayers were scratched to create a mechanical wound and incubated serum. Migration was evaluated using a scratch-wound assay, DNA synthesis by 3 H-thymidine uptake, collagen secretion by the Sircol assay, and VEGF secretion by ELISA. Post-wounding, serum significantly increased migration at differential rates; the greatest effect was seen in DS (98%), then DF (85%), then DP (22%). Wounding produced a significant increase in DNA synthesis in all cells, but the greatest increase was seen in DS, then DF, then DP cells. Basal total collagen secretion was significantly higher in DP compared to DF and DS. Wounding significantly increased collagen secretion by DF and DS, but had no effect on DP cells. All cells secreted similar basal levels of VEGF. Whilst wounding significantly increased VEGF secretion by DF and DS, it had no effect on DP cells. These results demonstrate that hair follicle DS cells have similar characteristics to DF in vitro, which are distinct to the hair follicle DP cells. This provides further evidence that the hair follicle fibroblasts can play a role in wound healing. 142 RAPID AND EASY METHODS FOR DETECTION OF WOUND INFECTIONS U. Bhat 1 , N. Gul 1 , J.S. Graham 2 , and S.M. Milner 1 1 Johns Hopkins Burn Center/Michael D. Hendrix Burn Research Center, Baltimore, Maryland USA, 2 U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, USA Infection remains one of the major causes of delay in wound healing and development of sepsis. The rapid detection of pathogens in burns and other type of wounds are critical for ensuring the early treatment strategy to prevent such complications. Traditional methods to detect wound bacteria often rely on time-consuming growth in culture media, followed by isolation, biochemical identification, and sometimes serology. Therefore, we have surveyed current technology for available rapid and easy methods for detecting infections including miniaturized biochemical kits, antibody- and DNA-based tests, and assays that are modifications of conventional tests. With the exception of a few kits where results can be read in 15 minutes, most require 4–24 hrs. While DNAbased assay formats mainly use PCR techniques for detecting pathogens, the highly specific binding of antibody to antigen, especially monoclonal antibody has facilitated the design of a variety of antibody based assays. Almost all rapid methods are designed to detect a single target, which makes them ideal for quick screening for the presence of a particular pathogen or their products. But, in real sense, rapid methods still lack sufficient sensitivity and specificity for direct testing; hence, a positive result quite often needs to be confirmed by standard methods. Recent developments in lateral flow technology have been utilized by Binax, Inc. to detect Streptococcus pyogenes and Staphylococcus aureas, and Pocket Dignostics, UK to dectect sevreal plant pathogens. Analysis takes only 15 minutes, is easy to perform and is highly specific for these pathogens. We conclude that it is possible to develop similar devices for other pathogens and can be adapted for identification of wound pathogens. Such kits will have great advantage in the physician’s office where identification of infection can be made during a patient’s visit so that a suitable treatment strategy can be recommended. 144 TREATMENT OF ISCHEMIC WOUNDS WITH NONCONTACT LOW-FREQUENCY ULTRASOUND: THE MAYO CLINIC EXPERIENCE (2004–2006) S.J. Kavros, J.L. Miller, S.W. Hanna Mayo Clinic, Rochester, MN USA Background: Low frequency non-contact ultrasound (LFU) has been shown to be an effective method in the treatment of chronic foot ulcerations. The purpose of this study was to evaluate the clinical role of LFU in the treatment of nonhealing leg and foot ulcers associated with chronic critical limb ischemia. Methods: The study was performed in a multidisciplinary vascular wound healing clinic. The study comprises 2 groups: Group 1 consisted of 35 patients, median age 74 years, who received LFU as part of their standard of wound care. Group 2 consisted of 35 patients, median age 76 years, who did not receive LFU as part of their standard of wound care. Both treatment arms continued for 12 weeks or until fully healed. Outcomes were considered favorable if the wound healed or reduced in size by 50% from the index measurement. An unfavorable outcome was noted if the wound healing was less than 50% of the index measurement. Patients in the LFU group were treated 3 times per week, 5minutes per ultrasound treatment. Results: In the Control Group, 25 patients (72%) failed to achieve minimum baseline positive outcomes. Ten patients (28%) achieved the required minimum wound healing parameters. In the LFU group, 22 patients (63%) had complete healing or 4 50% reduction in size from the index measurement. Thirteen patients (37%) failed to meet minimum baseline healing requirements. Both wound healing and closure rates were significantly better in the LFU group (p o 0.001). The predictive value of the transcutaneous oxygen (TcPO2) values with respect to wound healing with the use of LFU is noted to be of benefit. Conclusion: Our study data reveals that patients with chronic critical limb ischemia with associated cutaneous foot/leg ulcerations improve significantly when LFU is part of their standard of wound care. A52 Wound Rep Reg (2007) 15 A14–A54 c 2007 by the Wound Healing Society
Abstracts 145 FAT-DERIVED STROMAL CELL-PROMOTED WOUND HEALING S.P. Schmidt 1,2 , M. Evancho-Chapman 1,2 , W.I. Horne 1 , E.D. Boland 3 1 BioMedical Research Associates, Inc., Akron, OH USA, 2 Summa Health System, Akron, OH USA, 3 Tissue Genesis, Inc., Honolulu, HI USA Transplantation of an inoculum of autologous cells involved in the orchestration of wound healing into a nonhealing chronic wound bed may jump-start the healing process. The objective of this study was to evaluate the use of adiposederived stromal cells for this purpose. Using a swine full thickness dermal wound healing model, standard parameters of wound healing were compared between (1) wounds treated with an inoculum of autologous adipose tissuederived stromal cells dispersed into the collagen portion of a collagen/cellulose primary dressing (Promogran) that was placed cell/collagen side down into the wound bed and then covered with Allevyn, (2) wounds treated with Promogran with no cells added covered with Allevyn and (3) wounds treated with Allevyn dressings alone. Wound healing progressed in an orderly manner in all wounds. There was no evidence of infection, erythema or edema in any of the wound beds at any time of evaluation either based upon gross or histological observations. Exudate volumes were normal in wounds from all study groups. Re-epithelialization was first observed at Day 11 in the cell treated group but by Day 16 re-epithelialization was evident in biopsies obtained from all treatment groups. Statistical analyses of ranks of collagen organization and neovascularity revealed no significant differences in organization between the three treatment groups at any time point of analysis. There was no evidence that the presence of the stromal-derived cells unduly influenced inflammatory processes. In conclusion, wounds dressed with Promogran containing an inoculum of adipose-derived stromal cells healed as well as wounds dressed with Promogran alone or Allevyn but in this model of wound healing in young, healthy swine, did not confer any clinically significant benefit. Supported by Tissue Genesis, Inc. 147 KERATINOCYTE AND FIBROBLAST CROSS TALKING MODULATES THE EXPRESSION OF MMPS IN FIBROBLASTS Ghahary A., Li Y., Kilani RT., Y. Lam and A. Ghaffari Department of Surgery, BC. Professional Firefighter Burn and Wound Healing Research Group, University of British Columbia, Vancouver, Canada, We have recently established a keratinocyte/ fibroblast co-culture system and demonstrated a potent keratinocyte derived anti-fibrogenic factor (KDAF) for dermal fibroblasts relative to that of control cells. Later experiments identified KDAF as being the keratinocytes releasable 14-3-3 sigma. It is also known as stratifin. In this study, we hypothesize that stratifin would modulate the expression of other MMPs and that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3 proteins in conditioned medium. We also suggested that fibroblasts are not the primary source of these proteins. To examine this hypothesis, a microarray system was used to evaluate the expression of MMP-1, 3, 8 and 24. The results showed a significant increase in expression of these MMPs in response to either stratifin or keratinocyte conditioned medium treated fibroblasts. Further, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in our test medium consist of 49% keratinocyte serum free medium (KSFM), 49% DMEM and 2% fetal bovine serum up to 24 days. When KCM was collected every other days and added to fibroblasts, the expression of collagenase mRNA was undetectable in cells receiving either fresh or 48 hr conditioned KSFM. However, the level of collagenase mRNA expression was markedly increased in fibroblasts receiving KCM collected at either early (day 2 and 4) or later (day 12–22) time points of replacing the KSFM with test medium. To examine whether fibroblasts also release different 14–3–3 isoforms, cultured conditioned media from both fibroblasts and keratinocytes were collected and evaluated for the presence of different isoforms of 14-3-3 proteins by western blot analysis. The results revealed that fibroblasts which are highly responsive to some of the 14-3-3 isoforms such as a/b and s forms, are barely able to express and release these factors into conditioned medium. This was in sharp contrast to a very high level of 14-3-3 proteins found in KCM. In conclusion, keratinocytes, but not fibroblasts, release different forms of 14-3-3 proteins which may function as MMP-1 stimulating factors for fibroblasts. Acknowledgment: This work was supported by the Canadian Institute of Health Research (CIHR). 146 WOUND HEALING POTENTIAL OF EMBLICA OFFICINALIS BY MODULATING COLLAGEN AND DECREASING REACTIVE OXYGEN SPECIES M. Sumitra 1 , P. Manikandan 1 , V.S. Gayathri 2 , and L. Suguna 3 1 Department of Carolina Cardiovascular Biology Centre, University of North Carolina, Chapel Hill, North Carolina-27599, USA, 2 Department of Chemistry, SSN College of Engineering, Kalavakkam, Chennai, 3 Department of Biochemistry, Central Leather Research Institute, Chennai- 600 020, E-mail: slouchiu@yahoo.co.uk During wound healing, the wound site is rich in oxidants, such as hydrogen peroxide, mostly contributed by neutrophils and macrophages. Ascorbic acid and tannins of low molecular weight, namely emblicanin A (2,3-di-O-galloyl- 4,6-(S)-hexahydroxydiphenoyl2-keto-glucono-d-lactone) and emblicanin B (2,3,4,6-bis-(S)hexahydroxydiphenoyl-2-ketoglucono-d-lactone) present in Emblica officinalis (Emblica), shown to exhibit a very strong antioxidant action. We proposed that antioxidants in the wound microenvironment support the repair process. The present investigation was undertaken to determine the efficacy of Emblica on dermal wound healing in vivo. Fullthickness excision wounds were made on the back of the rat and topical application of Emblica accelerated wound contraction and closure. Emblica increased cellular proliferation and cross linking of collagen at the wound site, as evidenced by increase in DNA, type III collagen, acid-soluble collagen, aldehyde content, shrinkage temperature and tensile strength. Tissue levels of ascorbic acid, a-tocopherol, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), results supported that Emblica application enhanced the anti-oxidizing environment at the wound site by scavenging DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. In summary, this study provides firm evidence to support that topical application of Emblica represents a feasible and productive approach to support dermal wound healing. We thank the Council for Scientific and Industrial Research for its financial assistance. 148 CXC RECEPTOR 3 LIGANDS MEDIATE VASCULAR REGRESSION DURING WOUND HEALING Richard J. Bodnar and Alan Wells Veterans Affairs Medical Center of Pittsburgh, and Department of Pathology, University of Pittsburgh, Pittsburgh, PA Angiogenesis plays a critical role wound healing. Recently, we have shown that CXCR3 inhibits endothelial migration and vessel formation through a PKAmediated pathway. Here we show that CXCR3 ligands can also induce the regression of newly formed vessels. To understand the physiologic role CXCR3 plays in vessel regression, we analyzed the ability of IP-10, a CXCR3 ligand, to induce vessel regression in vivo and in vitro. IP-10 treatment of newly formed endothelial tubes on Matrigel caused tube dissociation. A similar dissociation of vessels induced by IP-10 was observed in an in vivo subcutaneous Matrigel plug. This was due to the receptor CXCR3, as IP-10 did not cause vascular regression in CXCR3 / mice. To identify the signaling pathways mediating this dissociation, we looked at beta 3 integrin, the main adhesion molecule maintaining endothelial function. Activation of CXCR3 by IP-10 triggers mucalpainmediated proteolysis, which then is able to cleave beta 3 integrin. To verify the role mu-calpain plays in vessel dissociation, newly formed tubes were treated with A23187, calcium ionophore an activator of mu-calpain; this caused tube dissociation. The A23187 mediated tube dissociation was inhibited by CI- 1, a non-specific calpain inhibitor, but not calpain inhibitor IV, a m-calpain specific inhibitor. Furthermore, we show that treatment of endothelial caused a cleavage of beta 3 integrin. We also found that mucalpain activation caused endothelial cell apoptosis, concurrent with beta 3 cleavage. These data support a model in which CXCR3 induces mu-calpain cleavage of beta 3 integrin to accomplish vascular regression secondary to endothelial apoptosis. Thus, we have found a novel, endogenous signal for mediating vessel regression. Wound Rep Reg (2007) 15 A14–A54 c 2007 by the Wound Healing Society A53
Page 1 and 2: Wound Repair and Regeneration Wound
Page 3 and 4: Abstracts 3 FREEZING ALTERS THE VIS
Page 5 and 6: Abstracts 10 EASY AND OBJECTIVE SKI
Page 7 and 8: Abstracts 18 ISOLATION AND CHARACTE
Page 9 and 10: Abstracts 26 TRANILAST INHIBITS THE
Page 11 and 12: Abstracts 33 SILICONE GEL OCCLUSION
Page 13 and 14: Abstracts 41 BFGF GENE TRANSFER BY
Page 15 and 16: Abstracts 49 DEVELOPMENT OF A RAPID
Page 17 and 18: Abstracts 56 PREVENTING STAGE IV PR
Page 19 and 20: Abstracts 64 TGF-BETA1-STIMULATED P
Page 21 and 22: Abstracts 71 RADIATION IS A KEY REG
Page 23 and 24: Abstracts 78 INCREASED SUSCEPTIBILI
Page 25 and 26: Abstracts 86 EXPRESSION OF THE A-SM
Page 27 and 28: Abstracts 92 TREATMENT OF SEVERE HI
Page 29 and 30: Abstracts 100 COMPARISON OF CELL VI
Page 31 and 32: Abstracts 106 IMPAIRED NOTCH SIGNAL
Page 33 and 34: Abstracts 114 ROLE OF NEUROINFLAMMA
Page 35 and 36: Abstracts 122 SEQUENTIAL APPROACH O
Page 37 and 38: Abstracts 130 OXYGEN APPLICATION TO
Page 39: Abstracts 137 OCCLUSION DECREASES H

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