Dates
Location
Anita Roberts Award
George Glinos
Excellence in Translational Regenitive Science Award
Subhadip Ghatak
The Ohio State University, Columbus, OH
Excellence in Translational Regenitive Science Award
Leslie Gold
New York University School of Medicine, New York, NY
Industrial Research & Development Poster Award
Cornelia Weigand
University Hospital Jena, Jena, Germany
Industrial Research & Development Poster Award
Ping Xie
Northwestern university, Chicago, IL
Industrial Research & Development Poster Award
Randolph Stone
US Army Institute of Surgical Research, Fort Sam Houston, TX
Industrial Research & Development Poster Award
Kristo Nuutila
Applied Tissue Technologies, Hingham, MA
Industrial Research & Development Poster Award
Amitava Das
University Wexner Medical Center, Columbus, OH, USA.
Industrial Research & Development Poster Award
Yong Mao
Rutgers University, Piscataway, NJ
Industrial Research & Development Poster Award
Rusty L. Montgomery
miRagen Therapeutics, Boulder, CO
Industrial Research & Development Poster Award
Sandeep Dhall
Osiris Therapeutics Inc., Columbia, MD
Junior Faculty Award
Yixuan Zhao
Shanghai Ninth People’s Hospital,Shanghai Jiaotong University
SAWC Scholarship Award
Bojie Dai
Travel Trainee Award presented by The Wound Healing Society
Mohammed Ashrafi
The University of Manchester, Manchester, United Kingdom
Travel Trainee Award presented by The Wound Healing Society
Christopher T. Turner
University of British Columbia, Vancouver, BC, Canada
Travel Trainee Award presented by The Wound Healing Society
Clark A. Bonham
Stanford, Stanford, CA
Travel Trainee Award presented by The Wound Healing Society
John Luo
University of Western Ontario, London, ON, Canada
Travel Trainee Award presented by The Wound Healing Foundation
Michelle S. Bach
Stanford University, Stanford, CA
Travel Trainee Award presented by The Wound Healing Foundation
Marielle Walraven
University of Toronto, Toronto, ON, Canada
Travel Trainee Award presented by The Wound Healing Foundation
Kanhaiya Singh
Ohio State University, columbus, OH
Travel Trainee Award presented by Wiley
Mithun Sinha
The Ohio State University, Columbus, OH
Travel Trainee Award presented by Wiley
Vikram G. Mookerjee
Rhode Island Hospital, Providence, RI
Travel Trainee Award presented by Wiley
Ayan Biswas
Ohio State University, columbus, OH
Travel Trainee Award presented by Wiley
Mohammed Ashrafi
The University of Manchester, Manchester, United Kingdom
Young Investigator Award
Amitava Das
University Wexner Medical Center, Columbus, OH
Young Investigator Award
Sara Ud-Din
University of Manchester, Manchester, United Kingdom
Young Investigator Award
Ivan Jozic
University of Miami Miller School of Medicine, Miami, FL
Young Investigator Award
Rubinder Basson
University of Manchester, Manchester, United Kingdom
Young Investigator Award
Georgios Thercharidis
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
Young Investigator Award
Michael Hu
Stanford, Stanford, CA
Young Investigator Award
Xinyi Wang
BCM, Houston, TX
Young Investigator Award
Roel Op't Veld
Radboud University Medical Centre, Gelderland, Netherlands
Lifetime Achievement Award
William H. Eaglestien
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M1.04.
Background- While at low concentrations reactive oxygen species (ROS) are known to drive cell signaling towards wound healing, excessive ROS such as during chronic inflammation and diabetes stall wound repair. This work shows that biofilm infection may add to the burden of oxidative stress at the wound site by redox cycling of pyocyanin. Pyocyanin (5-methyl-1-hydroxyphenazine) is a secretory product of Pseudomonas aeruginosa. Methods- Engineered human skin was treated with 10µM pyocyanin for 3d. Electron Paramagnetic Resonance (EPR) studies of the treated skin was conducted. The change in reducing equivalents (NADH, NADPH and GSH) pool was also determined in 10µM pyocyanin treated human HaCaT keratinocytes. Results- EPR spectrum of 10µM pyocyanin treated engineered human skin showed the presence of reduced pyocyanin radical which following UV exposure for 30 minutes generated superoxide and hydroxyl radicals. Thus, exposure to direct sunlight can worsen the already damaged biofilm infected wound site. A decline in NADH, NADPH and GSH levels was observed in 10µM pyocyanin treated keratinocytes as compared to untreated cells. Depletion of reducing equivalents was caused by the transfer of electrons from reducing equivalents to pyocyanin which helps bacteria to modulate their intracellular redox state. Modulation of intracellular redox state is crucial for survival of bacteria at high cell densities when there is electron-acceptor limitation, so that they can form thick biofilm. As this is achieved, there is more pyocyanin production, more ROS generation, continuous depletion of reducing equivalents pool and increased oxidative stress which does not allow the biofilm infected wound site to recover. Conclusion- Pyocyanin is cytotoxic to human cells due to its redox-active nature. Such toxicity markedly magnifies upon UV exposure. Pyocyanin oxidizes cellular reducing equivalents, induces oxidative stress and is therefore likely to complicate wound healing.
M1.05.
Background: Oral mucosal wounds heal more rapidly with significantly less inflammation, faster re-epithelialization, more refined angiogenesis, and less scar formation than skin wounds. Tight junctions (TJ) are intercellular junctions between adjacent cells that play pivotal roles in barrier function and cell polarity as well as in innate immunity. Methods: Using microarray, we compared the expression of TJ related genes in mouse skin and tongue wounds at 6, 12, and 24 hours and day 3, 5, 7, and 10 post-wounding. Using qPCR, the expression of occludin, claudins 1 and 4, ZO-1, and JAM-1 was compared between a wounded human skin keratinocyte cell line, HACAT and an immortalized human gingival epithelial cell line, TIGK. Results: The gene expression of multiple TJ molecules including occludin, claudins 1, 3, 4, 7, 10-12, 14, and 23, and ZOs 1-3, and JAMs 1-3 was found to change significantly over the course of healing process in skin wounds. By comparison, tongue wounds showed an overlapping but different pattern of expression that included occludin, claudins 1, 2, 4, 5, 10-13, 15, 18, 19, and 23, and ZO-2 and JAMs 1-3 (p<0.05, One-way Anova analysis). Since epithelial cells are one of the primary cell types to express TJ, we next investigated the in vitro gene expression of a subset of the TJ molecules that were differentially expressed in skin and tongue wounds. The expression of occludin and claudin 1 was significantly higher in HACAT than in TIGK after injury; Claudin 4 and JAM-1 were significantly higher in TIGK than in HACAT. No significant difference was observed in ZO-1 expression between these two cell lines after injury. Conclusions: The results suggest that certain TJ molecules are differentially expressed in skin and oral tissues. This differential expression may contribute to the distinct healing phenotypes seen between skin and oral mucosal wounds.
P.AW01.
BACKGROUND. Patients with chronic kidney disease develop a multitude of skin changes associated with their renal failure. Causative comorbidities such as peripheral vascular disease and diabetes directly impact healing. Poor healing contributes to prolonged hospital stays, susceptibility to infective complications and significant morbidity, including considerable negative psychological impact. Methods to assess wound healing in uremia will be valuable. METHODS. We developed a rodent model of excisional wound healing. Six week old male Wistar rats were fed a standard diet supplemented with 0.75% adenine for 3 weeks to establish uremia. Healthy controls were fed standard chow. Survival, growth rate and well-being were monitored. Bilateral 5mm full thickness dorsal punch biopsies were made. At day 3 and 7 measurements of the wounds were taken. Blood samples obtained by cardiac puncture were centrifuged for plasma and organs were harvested for analysis. Wounds were excised and bisected, one semicircle section was stored in formalin, the other snap frozen under liquid nitrogen. Experiments were conducted under our UK Home Office license after institutional approval. RESULTS. This model successfully established a uremic state with no premature deaths, excess bleeding or infected wounds observed. Serum urea was significantly higher in the uremic group at both day 3 and 7 (p<0.01) as was serum creatinine (p<0.02). Percentage of the wound area healed compared to day 0 was significantly greater in the control group at day 3 and day 7 (p<0.02 and p<0.001). Sample size n= 5 per group.
CONCLUSIONS. We developed a rodent model with low complication rates to study factors contributing to delayed wound healing in uremia. Moreover, we have demonstrated a delay in healing in uraemia.
P.AW02.
Background: Although wound models have been described, most in vitro or in vivo models do not reflect conditions in full-thickness human skin. Our aim was to develop an ex vivo human skin model to characterize products for promotion of wound healing (re-epithelialization). Our three-pronged approach employed assays for antimicrobial efficacy, cell toxicity, and effects on biomarkers associated with wound healing. To determine the utility of our model, we investigated Protosan wound gel in comparison to a series of proprietary formulations. Methods: Our model comprised 5-mm explants of full-thickness ex vivo human skin with 2- to 3-mm punch “wounds” that extended into the dermis. For antimicrobial studies: wounds were inoculated with methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, or Acetinobacter baumannii, and incubated at 37° for 2 h (simulate wound contamination) or up to 3 d to achieve biofilm. After incubation, explants (n=3 per treatment) were untreated or treated with Prontosan wound gel or proprietary formulations. For wound healing studies: wounds were untreated or treated with keratinocyte growth factor (KGF, positive control for re-epithelization), Prontosan wound gel or proprietary formulations. Following treatment, explants were incubated for 24h followed by (1) microbial enumeration, (2) cytotoxicity testing (MTT assay), or (3) biomarker concentrations (Human XL Cytokine Discovery Luminex® High Performance Assay, R&D Systems). Results: We identified solutions with optimal wound-healing efficacy, determined by cytotoxicity (≥50% viability) and antibiofilm efficacy (≥2-log reduction in microbial burden compared to untreated explants). We further believe that these formulations are associated with changes in concentrations of wound-healing biomarkers predictive of re-epithelialization. Conclusion: Our model identified biofilm-preventing/wound-healing formulations associated with low cytotoxicity, reduced microbial burden, and changes in concentrations of known wound-healing biomarkers. Our wound-healing model may obviate animal testing and optimize product testing, with consequent faster progression to clinical trials involving acute and chronic wounds in patients.
P.AW03.
BACKGROUND – Volatile organic compounds (VOCs) emanate from human skin and their identification as biochemical markers in the process of wound healing is currently understudied. Therefore, the aim of this novel study was to identify the VOC profiles during the acute phase of cutaneous wound healing.
METHODS – Six healthy male subjects had four 5-mm diameter skin biopsies to their arms. VOC samples were obtained non-invasively from wounded and healthy skin as well as the ambient background at days 0, 7, 14, 21 and 28 using polydimethylsilicone membranes and underwent thermal desorption and were then separated by gas chromatography and detected by mass spectrometry. VOCs were tentatively identified using the National Institute of Standards and Technology library and relative abundances compared. Spectrophotometric intracutaneous analysis, full-field laser perfusion imaging, colorimetry and dynamic optical coherence tomography provided quantitative measurements of melanin, haemoglobin, collagen and blood flow.
RESULTS – Generalised estimating equation model showed significant differences in VOC profiles (P<0.05), obtained from wounded skin, healthy skin and background, followed a Gaussian distribution over time (D0-77;D7-150;D14-170;D21-151;D28-112 VOCs). Twenty-five VOCs allowed differentiation between sampling areas independent of sampling time point (P<0.05). Twenty-seven VOCs allowed discrimination between wounded from healthy skin at different time points (P<0.05). Two-hundred-and-nineteen VOCs significantly varied in wounded skin over the 28 day period (P<0.05) of which 41 showed no significant variation in healthy skin which allowed differentiation. Undecane, 3-carene, 1,3-dimethyl-benzene, acetoacetic-3(10)-caren-4-ol, 3,7-bicyclo[4.1.0]heptan-3-ol and 1-undecanol significantly correlated with blood flow (R>0.7;P<0.001). Hept-3-yl ester benzoic acid significantly correlated with melanin (R<-0.7;P<0.001) and also correlated consistently from D7-28 with both melanin and collagen when comparing across time points (R<-0.7;P>0.05).
CONCLUSIONS – For the first time, VOC profiles of the acute phases of wound healing of human skin as potential biomarkers have been identified. Their relationship to the metabolomic and microbiome profile of wounds is currently underway.
P.AW04.
BACKGROUND: Moist wound dressings support rapid healing but non-moist dressings are still commonly used. We evaluated efficacy and safety of a 3% povidone-iodine-containing polyurethane dressing (Betafoam®) for donor-site dressing, versus Allevyn® and PG.
METHODS: This prospective Phase 4 study (NCT02543034) was conducted between Mar-2016 and Apr-2017 at 8 sites in Korea. Consenting subjects (aged ≥19 years, scheduled for STSG) were randomized 1:1:1 to Betafoam®, Allevyn®, or PG dressings for up to 28 days after donor-site collection. We assessed time to complete epithelialization (CE), proportion with CE at Day 14, wound infection, inflammation, pain, treatment acceptability, modified Vancouver Scar Scale (mVSS) score at Day 28.
RESULTS: 98 subjects provided evaluable data (n=31 Betafoam®; n=33 Allevyn®; n=34 PG). Epithelialization time was shortest with Betafoam® (12.74±3.51 days), versus Allevyn® (16.61±4.45 days; p=0.0003, t-test), PG (15.06±4.26 days, p=0.0205). At Day 14, 83.87% of Betafoam® donor-sites had CE, versus 36.36% of Allevyn® donor-sites (p=0.0001, t-test), and 55.88% of PG donor-sites (p=0.0146). There were no wound infections. At Day 28, mVSS scores were 1.47±1.71 (Betafoam®), 2.71±0.37 (Allevyn®), and 3.61±3.54 (PG). Other measures did not differ significantly between groups. Adverse event (AE) incidence was comparable in Betafoam® (17.65%), Allevyn® (37.14%), and PG (29.41%) groups (χ2 p=0.1940), with no serious AEs; and no dressing-related or skin-related AEs with Betafoam®.
CONCLUSIONS: Betafoam® required less time to complete epithelialization and had a good safety profile.
DISCLOSURE: Abstract submitted in parallel to European Wound Management Association (EWMA) 2018 meeting
P.AW05.
Prolonged intake of high-fat diet leads to low grade chronic inflammation and plays an important role in obesity development. Previous studies showed that the long-term administration of a high-fat diet to rats delays cutaneous healing in both obesity-prone and obesity-resistant animals; suggesting that the diet composition was more important in promoting the delay of wound healing than the adipose tissue accumulation. It is also known that short-term administration of high-fat diet was capable of inducing pro-inflammatory pathways in heart, adipose and muscle tissues. However, the effects of a short-term administration of a high-fat diet on skin wound repair are not known. The aim of this study was to investigate the effects of short-term administration of a high-fat diet on cutaneous healing in mice. Mice (n=20) were divided into standard chow (10% of calories from fat) and high-fat chow (60% of calories from fat) groups. After 10 days of diet administration, an excisional lesion was performed and lesion was collected 10 days later. Neither average body weight, nor glucose metabolism were different in studied groups. The high fat chow group presented delayed wound contraction and increased cellular density (p<0.05) The high-fat chow group also presented increased neutrophils and macrophages 10 days after wounding (p<0.05). The high-fat diet group presented lower type I collagen protein expression; however, 10 days after wounding there was an increase in type III collagen protein expression (p<0.05). In conclusion, short-term administration of high-fat diet exerts negative effect on mice cutaneous healing, due to impairment of wound contraction and extracellular matrix deposition and prolongation of the inflammatory phase.
P.AW05.
Prolonged intake of high-fat diet leads to low grade chronic inflammation and plays an important role in obesity development. Previous studies showed that the long-term administration of a high-fat diet to rats delays cutaneous healing in both obesity-prone and obesity-resistant animals; suggesting that the diet composition was more important in promoting the delay of wound healing than the adipose tissue accumulation. It is also known that short-term administration of high-fat diet was capable of inducing pro-inflammatory pathways in heart, adipose and muscle tissues. However, the effects of a short-term administration of a high-fat diet on skin wound repair are not known. The aim of this study was to investigate the effects of short-term administration of a high-fat diet on cutaneous healing in mice. Mice (n=20) were divided into standard chow (10% of calories from fat) and high-fat chow (60% of calories from fat) groups. After 10 days of diet administration, an excisional lesion was performed and lesion was collected 10 days later. Neither average body weight, nor glucose metabolism were different in studied groups. The high fat chow group presented delayed wound contraction and increased cellular density (p<0.05) The high-fat chow group also presented increased neutrophils and macrophages 10 days after wounding (p<0.05). The high-fat diet group presented lower type I collagen protein expression; however, 10 days after wounding there was an increase in type III collagen protein expression (p<0.05). In conclusion, short-term administration of high-fat diet exerts negative effect on mice cutaneous healing, due to impairment of wound contraction and extracellular matrix deposition and prolongation of the inflammatory phase.
P.AW06.
Background:Traumatic soft tissue injury with tissue loss is a frequent and challenging problem, requiring operations that have long-term functional and cosmetic consequences. This, combined with painful dressing changes, prolonged wound healing, and increased resource utilization, prompted exploration of more effective solutions. ABRA dynamic tissue system (DTS) has been successfully used to facilitate open abdomen closure. There are both invasive and non-invasive variations of the device for closing soft tissue extremity and trunk defects. ACeLL Matristem is a porcine urinary bladder matrix (PUBM) which accelerates wound healing through constructive remodeling.
Objective:We describe a series of patients with combined mechanical and biological closure of complex extremity and trunk soft tissue wounds.
Methods:We identified 14 patients with large complex traumatic wounds and used DTS and PUBM to definitively heal these wounds. In most cases we used both DTS and PUBM and in select patients we used only PUBM. Detailed photographic documentation was performed of each wound.
Results:There was 100% healing of each wound without the need for skin grafting or tissue flaps. There were no surgical site infections (SSI) even in the most contaminated of wounds. In each case, wound healing was accelerated with excellent cosmetic and functional outcomes.
Conclusion:Complex traumatic wounds of the trunk and extremities are a challenging problem. Combined use of DTS and PUBM can be used to successfully heal these wounds.
P.AW06.
Background:Traumatic soft tissue injury with tissue loss is a frequent and challenging problem, requiring operations that have long-term functional and cosmetic consequences. This, combined with painful dressing changes, prolonged wound healing, and increased resource utilization, prompted exploration of more effective solutions. ABRA dynamic tissue system (DTS) has been successfully used to facilitate open abdomen closure. There are both invasive and non-invasive variations of the device for closing soft tissue extremity and trunk defects. ACeLL Matristem is a porcine urinary bladder matrix (PUBM) which accelerates wound healing through constructive remodeling.
Objective:We describe a series of patients with combined mechanical and biological closure of complex extremity and trunk soft tissue wounds.
Methods:We identified 14 patients with large complex traumatic wounds and used DTS and PUBM to definitively heal these wounds. In most cases we used both DTS and PUBM and in select patients we used only PUBM. Detailed photographic documentation was performed of each wound.
Results:There was 100% healing of each wound without the need for skin grafting or tissue flaps. There were no surgical site infections (SSI) even in the most contaminated of wounds. In each case, wound healing was accelerated with excellent cosmetic and functional outcomes.
Conclusion:Complex traumatic wounds of the trunk and extremities are a challenging problem. Combined use of DTS and PUBM can be used to successfully heal these wounds.
P.AW07.
BACKGROUND-Intracellular ATP delivery (ATP-vesicles) enhanced wound healing by reducing the traditional 3-6-day lag time, a phenomenon never seen or reported in the past, but the mechanisms are unclear. This study was designed to explore the cellular mechanism in the rapid tissue regeneration.
METHODS-Seventy rabbits were used and four wounds were created on each ear. Two were treated with ATP-vesicles (10 mM Mg-ATP) and the other two were treated with controls (normal saline or RegranexTM). They were sacrificed at 5h, 12h, and days 1, 2, 3, 4, 6, 9, and 15 post-surgery.
RESULTS-Immunohistochemical analysis of the ATP-vesicle-treated wounds showed increase of platelets, neutrophils and cytokines as early as 5-12h by CD61, neutrophil, MCP-1 and TNF-alpha antibodies. Massive macrophage accumulation detected by CD68, CD16 and Anti-Mac by 24h. Stem cells were detected using CD106, CD146 and CD44 antibodies. CD36, arginase and collagen type 1 immunoreactivity were detected as early as day two. Early neovascularization was also detected using CD105, CD34 and CD31 antibodies. Double staining with M1 and M2 macrophage markers showed massive early M2 macrophage polarization and double staining of macrophages with pre-collagen markers showed their active collagen synthesis as early as day 3. The control wounds treated by normal saline or RegranexTM did not show similar changes.
CONCLUSIONS-We conclude that intracellular ATP delivery causes rapid tissue regeneration by initiating the wound healing cascade as early as 5hr. All the cellular events associated with this healing process may provide a new strategy for wound healing.
P.AW08.
This study reviews our recent clinical experience using UBM-ECM (Urinary Bladder-Extracellular Matrix) to treat and achieve healing of clinically challenging acute plantar foot wounds ranging from a thru and thru heel pad wound to a total foot degloving wound from the level of the ankle.
Methods- We performed a retrospective review of the last 10 cases of acute plantar foot wounds treated with the use the UBM-ECM wound device. There were 8 males and 2 females with ages ranging from 15 – 66 years of age. There were varying degrees of tissue loss and depth of injury.
Results- Initial healing was achieved in all cases with 8 patients having either split or full thickness skin grafts used for final closure. NPWT was used while wounds drained 25 cc or more per day. Three heel pad patients developed secondary small wounds which were treated with additional UBM-ECM with 2 patients having achieved stable healing and the third patient with a recent opening responding well to treatment despite him standing at work over 60 hours a week. No patient required long-term narcotic use and all patients except the totally degloving foot patient have been able to resume all pre-injury activities. The total degloving wound patient had return of protective plantar sensation
We believe that the early use of UBM-ECM in the management of these potentially devastating plantar wound patients offers new hope to managing these complex wounds. Our present clinical treatment regimen and treatment outcomes will be presented. We strongly advocate early UBM-ECM use in managing these potentially devastating foot wounds.
P.AW09.
BACKGROUND – Hidradenitis suppurativa (HS) is a chronic, debilitating skin disease, characterized by recurrent inflammatory boils and abscesses, mainly located in the inverse body areas. Early wide surgical excision is important and effective in order to prevent complications, however, reconstruction is quite challenging, often requiring skin graft or skin flap. To evaluate the efficacy of secondary intention healing after wide excision for treating severe HS
METHODS – Over the last 10 years, 9 patients with severe HS (Hurley grade II and III) underwent surgical excision/ wide exteriorization with reconstruction using secondary intention healing and artificial collagen insertion. We evaluated and compared intraoperative and post-operative data, retrospectively.
RESULTS – Seven patients (77.77%) showed no recurrence after surgery. The mean treatment time to complete wound healing was 115±110days(range, 36-339days). The mean disease -free duration after treatment was 665±283 (range, 123-924 days).
CONCLUSIONS – Based on our experience, secondary intention healing after excision is an effective treatment option for patients with severe HS presenting multiple interconnected tracts and abscesses
P.AW10.
Backgroun:The objective of this study is to evaluate the efficacy of the negative pressure wound therapy after distal digit amputation for invasive subungal melanoma
Method: Retrospective review of 11 patients (3 males, 8 females) from December 2015 to April 2017 was performed.
Results / Discussion: Mean age of the patients was 54.6 years (32~75 years) and all patients were diagnosed with SUM with deep invasion (Breslow thickness range : 1.5 ~ 5.0 mm / mean thickness : 2.85 mm). Index finger was involved in 4 patients, thumb in 3 patients, great toe in 2 patients, third finger in 1 patient, and second toe in 1 patient. The open amputation site was covered with fillet flap utilizing the volar skin and NPWT was applied for an average of 7.63 days (range : 3~14 days). The wound dressing period was 21 days (range : 12 ~ 37 days) and the flap survived successfully in all but 1 case (survival rate : 90.9%). In one case, the NPWT was stopped after 2 days due to pain of the perioperative margin and the flap did not heal well.
Conclusion: Distal digit stump is a difficult area to heal and ulceration is not uncommon complication. NPWT can be used effectively for flap survival and thus to shorten the duration of treatment after distal digit amputation. Since the stump is covered with thick and robust flap pad, functional and aesthetic outcomes are excellent.
P.AS01.
BACKGROUND: This study aimed to provide basic data for the design of education program about diabetic foot care for nursing home care workers. The purpose of this study was to identify knowledge and practice of care workers toward the diabetic foot care and to evaluate the factors affecting practice of diabetic foot care. METHODS: The research design was a cross-sectional study using a structured questionnaire. The participants were 90 care workers who were working in three nursing homes in J city from September to November 2016. RESULTS: Mean score of knowledge for the participants was 8.77 (range 6-10) and mean score of practice was 28.17 (range 20-30). Positive correlation was observed between knowledge and practice (r=.33, p=.002). Practice was significantly predicted by knowledge (p=.017) and received education on diabetic foot care (p=<.001) which explained 20.4% of the variance in practice. CONCLUSIONS: Knowledge and received education on diabetic foot care were found to be very important factors associated with practice of diabetic foot care in nursing home care workers.
P.ANG01
BACKGROUND The concept of immediate versus delayed application of active topical formulations in order to optimize healing prior to scar formation is new. We previously evaluated the effects of an active versus placebo, post-scar formation. METHODS The aim here was to assess the application of an active versus placebo at multiple time-points pre-scar formation around the injury zone in 62 subjects (Group A:immediate application from Day (D) 0 around wound site, Group B:delayed application at D14 directly to scar) using quantitative non-invasive devices and immunohistochemical (IHC) analysis in a double-blind randomized-controlled trial. RESULTS Group A had greater hydration from D0 to W1 (148Au to 154.5Au) compared to placebo (174.4Au to 128.0Au) (p=0.002). TEWL reduced from D0 to W2 (6.3Au to 4.3Au) versus placebo (8.5Au to 16.1Au) (p=0.02). Group B showed increased hydration with active (21.0Au) compared to placebo (0.1Au) at W1 (p=0.028). IHC analysis established that hyaluronic acid (HA) proteins were located predominantly in the epidermis with weaker expression in the dermis. HA marker area was higher at all time-points in both groups. This increase was greatest at W1 with active (38%) versus placebo (21%) (p=0.03). Blood-flow measured by dynamic-OCT increased in Group A compared to placebo from D0 (0.009Au, 00.068Au respectively) to W1 (0.106Au, 0.086Au respectively) (p<0.001) and W2 (0.09Au, 0.081Au respectively) (p<0.001). Blood-flow was lower with active compared to placebo at all time-points (p=0.037). This was supported by VEGFA, which confirmed lower marker area with active (25.7%) versus placebo (46%) at W1 (p=0.02). CD31 analysis displayed lower vessel density with active (60vessels/mm2) at W1 than placebo (89vessels/mm2) (p=0.03). CONCLUSIONS These findings provide evidence, for the first time, of zonal conditioning for the immediate application of an active around the zone of injury in order to optimize healing outcome prior to scar formation and maturation.
P.ANG02.
Background: The angiogenic property of fibrocytes and phenotypic change of fibrocytes remains fully unknown in wound healing. We previously demonstrated the specific induction of angiogenic fibrocytes by basic fibroblast growth factor (bFGF) in rat skin wounds. We here examined change in marker expression of angiogenic fibrocytes during the course of angiogenesis in rat planter decubitus ulcers. Methods and Results: Double immunofluorescence staining in the normal region showed that capillary-like structures were almost composed of CD34+/procollagen I+ fibrocytes. However, in the ulcers, capillary-like structures composed of CD34 -positive/procollagen I-negative cells were markedly formed. The ulcers also showed enhanced expression of TGFβ1 and VEGF-A mRNA and reduced expression of bFGF mRNA, without any change in levels of PDGF mRNA expression, as detected by Real-time PCR. Conclusion: We previously showed that bFGF is required for vascular formation composed of the CD34+/procollagen I+ fibrocytes in wounds. Therefore, enhanced VEGF-A expression in the ulcers markedly inhibited the vascular formation composed of this type of fibrocytes through reduction of bFGF expression in the ulcers.
P.BIO01.
Dysregulation of a broad spectrum of proteases due to persistent inflammation is widely accepted as a feature of chronic dermal wounds. It has been proposed that excessive proteolytic activities in wound fluid may play a role in preventing wound closure, suggesting that wound dressings with antiproteolytic properties may be beneficial. An ovine-derived decellularized collagen extracellular matrix (cECM) comprising both collagen and non-collagen ECM components including elastin, fibronectin, laminin and glycosaminoglycans was utilized within this study. Here we describe an in vitro model designed to mimic the in situ use of this ovine dressing, whereby samples of ovine dressing were placed in microwell plates and real-time enzyme kinetics studies were performed for up to 6 days on 5 proteases commonly found in wound fluid: matrix metalloprotease-1 (MMP-1), MMP-2, MMP-8, MMP-9, and human neutrophil elastase (hNE); these were incubated in the presence or absence of ovine dressing, with a fluorogenic substrate (520 MMP FRET Substrate XIV; Anaspec). The activity of all 5 proteases tested was modulated by incubation with the ovine dressing, with inhibition levels reaching 80-90% after as little as 6 hours of incubation and maintained for at least 6 days for most enzymes. The inhibitory effect was perhaps more gradual for MMP-8 and MMP-9; and the incubation times beyond 1 day could not be explored for MMP-2 due to a relative instability of this enzyme. These data support the use of this ovine dressing to address the proteolytic conditions of chronic dermal wounds as well as set a new bar for demonstrating modulation of protease activity by collagen wound dressings.
| MMP-1 | MMP-2 | MMP-8 | MMP-9 | hNE | |
| 0 | 69.4 | 84.9 | 79.5 | -11.7 | 67.4 |
| (1h) | 74.6 | 88.0 | -20.8 | 31.9 | 75.2 |
| 6h | 92.3 | 95.3 | 61.2 | 78.5 | 97.5 |
| 1d | 91.8 | 84.5 | 79.2 | 88.0 | 97.3 |
| 2d | 91.6 | 53.5 | 82.0 | 85.2 | 98.7 |
| 4d | 94.6 | 50.2 | 85.2 | 81.3 | 98.1 |
| 6d | 92.3 | 21.4 | 93.9 | 78.5 | 98.5 |
P.BIO02.
Recently, a novel lyopreservation method for ambient storage of living tissue has been developed. In this study, the effect of the novel lyophilization on the components of the placental tissue was evaluated. Structural matrix, growth factors, and cell viability of amniotic (AM) and chorionic (CM) membranes, and umbilical tissue (UT) were evaluated using 4 placentas. AM, CM, and UT were divided into two parts to enable comparison between fresh and lyophilized tissue. Fresh AM, CM, and UT were analyzed immediately. Lyophilized samples were analyzed immediately post-lyophilization, and after 6 months of ambient storage. Histological analysis (Hematoxylin and eosin staining, Masson’s trichome staining) was used to assess tissue structure. Multiplex arrays and ELISAs were used for evaluation of growth factors, and viability of cells in the tissues was analyzed using a LIVE/DEAD viability/cytotoxicity kit. Histologically, the matrix of fresh tissue was retained in rehydrated lyopreserved AM, CM, and UT. There were no significant differences in the concentration of growth factors (basic Fibroblast Growth Factor, Platelet-Derived Growth Factor, Angiogenin-1, Stromal cell Derived Factor-1, Iinterleukin-1Receptor Antagonist) and cell viability between fresh and lyopreserved tissues. The matrix, growth factors and viable cells of fresh tissue were retained in lyophilized samples stored for 6 months. Results show that the new lyopreservation method is applicable for AM, CM and UT, reproducible and provides extended tissue shelf life. This preservation method for ambient storage of living tissues has tremendous practical significance for both scientific and clinical applications.
P.BIO03.
Background: Chronic wounds are a major clinical problem with an estimated 40 million people suffering from them worldwide, and one of the most costly healthcare problems today. ‘Intelligent’ dressings may be defined as materials that respond to specific changes in the wound environment to bring about a useful result. Considerable promise in the application of nanocellulosic hydrogels, aerogels, and nanocomposites as dressings has been demonstrated due to favorable properties that promote optimal moisture conditions. Here we present a series of modified nanocellulosic and cellulosic materials designed to, remove harmful proteases from a chronic wound while detecting protease levels. Methods: The biosensors were prepared by a method previously report (Edwards’s et al. J. Biomat. Appl., 2017). To start the sensor/protease reaction 50 μL of human neutrophil elastase at various concentrations ranging from 2 – 0.015 U/mL were utilized. Material protease sequestration evaluation was performed in a like manner using a 96-well format by incubating 2 milligrams of material in a protease solution for 1 hour. Results: A correlation between zeta potential values and the degree of protease sequestration imply that the greater the negative surface charge of the nanomaterials, the greater the sequestration of positively charged neutrophil proteases. The biosensors gave detection sensitivities of 0.015-0.13 units/ml, which are at detectable human neutrophil elastase levels present in chronic wound fluid. Conclusion: The sensor portion is a fluorescent peptide-cellulose conjugate interchangeable on the surface of different semi-occlusive dressing motifs and sensitive to protease levels found in chronic wounds. The protease modulation portion is based on the degree of surface zeta potential required on the material surface to remove excess wound protease levels. The physical and interactive biochemical properties of the nano-based biosensors are suitable for interfacing with protease sequestrant prototype wound dressings. A discussion of the relevance of protease sensors and cellulose nanomaterials to current chronic wound dressing design and technology is included.
P.BIO04.
BACKGROUND – Impaired skin healing is a significant and growing clinical concern, particularly in relation to diabetes, venous insufficiency and immobility. In previous research, we developed electrospun collagen based scaffolds for the delivery of periostin (POSTN) and connective tissue growth factor 2 (CCN2), matricellular proteins involved in the proliferative phase of healing. Addition of the scaffolds rescued delayed healing in db/db diabetic mice. The aim of this study was to investigate the effect of POSTN/CCN2 electrospun scaffolds on wound healing in a porcine model. METHODS – 2cm2 full thickness wounds were created bilaterally in the back of the pigs and scaffolds implanted. Healing was assessed through closure kinetics, hydroxyproline content, and blood vessel density at 28 days post-wounding (N=6). RESULTS – Wound closure was reduced in the presence of the three scaffold treatments (bovine serum albumin (BSA), POSTN/CCN2, and galectin-3 (GAL3)) when compared to empty control wounds. This effect was attenuated with POSTN/CCN2 and GAL3 scaffolds. Masson’s Trichrome staining showed that collagen content was reduced by BSA, POSTN/CCN2, and GAL3 scaffold treatments but was near unwounded tissue levels in the empty wound condition. Blood vessel density increased in healing wounds with similar values between all scaffolds and empty treatments. CONCLUSIONS – Delayed contraction indicates scaffold influence over fibroblast to myofibroblast transition, potentially reducing excessive collagen deposition. These results indicate no negative effect of the electrospun scaffolds on blood vessel density. Future research is necessary with a larger sample size to validate the current findings.
P.BIO04.
BACKGROUND – Impaired skin healing is a significant and growing clinical concern, particularly in relation to diabetes, venous insufficiency and immobility. In previous research, we developed electrospun collagen based scaffolds for the delivery of periostin (POSTN) and connective tissue growth factor 2 (CCN2), matricellular proteins involved in the proliferative phase of healing. Addition of the scaffolds rescued delayed healing in db/db diabetic mice. The aim of this study was to investigate the effect of POSTN/CCN2 electrospun scaffolds on wound healing in a porcine model. METHODS – 2cm2 full thickness wounds were created bilaterally in the back of the pigs and scaffolds implanted. Healing was assessed through closure kinetics, hydroxyproline content, and blood vessel density at 28 days post-wounding (N=6). RESULTS – Wound closure was reduced in the presence of the three scaffold treatments (bovine serum albumin (BSA), POSTN/CCN2, and galectin-3 (GAL3)) when compared to empty control wounds. This effect was attenuated with POSTN/CCN2 and GAL3 scaffolds. Masson’s Trichrome staining showed that collagen content was reduced by BSA, POSTN/CCN2, and GAL3 scaffold treatments but was near unwounded tissue levels in the empty wound condition. Blood vessel density increased in healing wounds with similar values between all scaffolds and empty treatments. CONCLUSIONS – Delayed contraction indicates scaffold influence over fibroblast to myofibroblast transition, potentially reducing excessive collagen deposition. These results indicate no negative effect of the electrospun scaffolds on blood vessel density. Future research is necessary with a larger sample size to validate the current findings.
P.BIO05.
Human amnion (hAM) has a long history of use for wound management. hAM is anti-inflammatory, anti-microbial and anti-fibrotic. It provides pain relief, keeps wound moist and supports angiogenesis, granulation tissue formation, and re-epithelialization. These beneficial properties are attributed to hAM components: structural matrix, growth factors, and viable cells. Advancement in preservation technologies led to hAM commercialization.Nevertheless, majority of the preservation methods destroy viable cells resulting in acellular or devitalized hAM. Cryopreservation is a method allowing retention of viable cells in hAM. However, data show that percent of viable cells in hAM post-thaw are not always consistent. Therefore, cell viability is an important test to ensure consistency and quality of cryopreserved hAM. Using fresh hAM samples of different sizes from multiple donors we evaluated several cell viability methods. We found that the LIVE/DEAD fluorescent staining method is the most appropriate for assessment of cell viability in tissues. However, high variability in number and distribution of viable cells across hAM grafts can lead to unreliable results when microscopic fields, a traditional approach, is used for estimation of cell viability percentage. We demonstrate that for accurate assessment of hAM cell viability scanning of large macroscopic tissue areas should be performed instead of microscopic field assessment. Then, we utilized the developed approach for assessment of cell viability in cryo- and lyopreserved hAM. We showed that there were no significant differences in cell viability between fresh, cryo- and lyopreserved hAMs. The developed approach gives accurate cell viability results and recommended for assessment of cell viability of the amniotic and other tissues for confirmation that applied tissue processing protocols do not compromise cell viability.
P.BIO06.
Background: Diabetes and its concurrent complications impact a significant proportion the US population and create a large financial burden on the American health care system. Maggot debridement therapy (MDT) is the FDA-approved application of sterile, laboratory-reared Lucilia sericata (green bottle fly) larvae to non-healing wounds, such as diabetic foot ulcers. Larvae secrete a mixture of excretions and secretions (ES) as part of extracorporeal digestion of dead wound tissue that inhibit bacterial growth and promote wound healing. Human growth factors stimulate cell proliferation and survival in the wound environment, promote wound healing, and have been investigated as a possible topical treatment for non-healing wounds. We have previously illustrated the characterization of an L. sericata strain that expresses and secretes the human growth factor PDGF-BB, under the control of an inducible system. Methods: To continue optimizing our expression system, we are currently characterizing gene expression in an exhaustive panel of larval digestive system tissues and isolating promoter candidates for driving tissue-specific expression. Results: Having identified a number of promising candidates, we are also currently broadening the applications of our expression system by engineering new effector candidates with utility in wound healing. Conclusions: Honing expression to specific tissues will result in a lowered fitness cost to the organism and enhance efficiency of exogenous factor secretion. Our system could potentially be used to deliver a variety of growth factors and anti-microbial peptides to the wound environment with the aim of enhancing wound healing. Further, rearing maggots is relatively inexpensive, and could provide a cost effective treatment to patients in lower income regions.
M1.01.
BACKGROUND: Hypertrophic scarring (HTS) following major burn injury remains a critical morbidity with limited treatment options that ultimately diminishes quality of life for burn victims. We and others have found that cutaneous mast cells (MC) are increased significantly in burn wounds and HTS. Tryptase, released by the stimulated MC, activates the protease-activated receptor-2 (PAR2) and is thought to play an important role in post-burn HTS pathophysiology. Here, we examined serum tryptase concentrations in a pediatric burn population. Additionally, in an in vitro model, we investigated the anti-fibrotic effects of PAR2 knockdown in primary post-burn HTS fibroblast cultures. METHODS: Serum was collected from age-matched non-burned and burned pediatric patients with greater than 20% of total body surface area burned in this IRB-approved study. Tryptase-β2 was measured by ELISA. In vitro, primary HTS fibroblasts were treated with siRNA to PAR2 or scrambled siRNA for 72 hours prior to 1-hour treatment with PAR2 activating peptide SLIGKV. After treatment, mRNA targets of PAR2 activation and subsequent fibrotic signaling were quantified by RT-qPCR in three independent experiments. RESULTS: Following severe burn injury, serum tryptase increased significantly during acute ICU admission (n=5, 6.104 ng/mL ± 0.93, p=0.0002) and remained elevated for at least 6 months post-burn (n=5, 5.105 ng/mL ± 1.793, p=0.0009) compared to non-burned controls (n=6, 0.7083 ng/mL). Furthermore, PAR2 siRNA knockdown significantly reduces mRNA transcript expression of fibrotic genes, including Collagens-1, and -3, MMPs-2, and -9, and α-SMA (p<0.05) compared to control-treated cells. Moreover, stimulation with SLIGKV confirmed complete PAR2 knockdown as there was no increase in the expression of the fibrotic genes. CONCLUSION: Together, these data show PAR2 activation intensifies the HTS fibrotic phenotype in burn HTS fibroblasts and further suggests that targeted PAR2 antagonism at wound sites may decrease MC tryptase’s proliferative effect and potentially limit detrimental fibrosis in post-burn HTS over time.
P.BW01.
BACKRGROUND: Pressure garments are commonly employed to reduce scarring following burn injury, with varying efficacy rates reported. The optimum magnitude of pressure necessary to achieve the greatest benefit is unknown, though it has been suggested that pressure should exceed capillary pressure, 25-30 mm Hg. Unfortunately, higher pressures are associated with increased risk of side effects such as deformation of skeletal features or constricted breathing. METHODS: To better understand the role of pressure magnitude on therapy efficacy, pressure garment therapy and scar development was studied in a porcine model. Full-thickness burns (1×1 in) were created on female, red Duroc pigs, excised, and autografted with split-thickness autografts. Adjustable pressure garments were applied 1 wk after grafting and maintained at either 10, 20, or 30 mm Hg for 11 weeks (n=16 scars/group). RESULTS: All pressure-treated groups were significantly less contracted than controls. Scars in the 30 mm Hg group were 56% larger than controls and 16% larger than the 20 mm Hg group at 11 weeks post-grafting (p<0.05). Pressure therapy, at all magnitudes, significantly improved scar elasticity and pliability, with the greatest improvements observed with 30 mm Hg. 30 mm Hg was the most effective at reducing scar contraction and improving biomechanical properties compared to 20 or 10 mm Hg. CONCLUSIONS: While pressure at a magnitude of 30 mm Hg resulted in the most significant benefit, the pressure is highly uncomfortable and would likely reduce the duration for which the patient can wear the garment. For greatest clinical efficacy, a balance between pressure magnitude and patient compliance must be achieved.
P.BW02.
BACKGROUND: The suggested duration of pressure garment therapy is commonly 1-2 years; however, maintaining patient compliance throughout this period is a major challenge. The goal of this study was to examine changes in scar properties after early cessation of pressure garment therapy. METHODS: Full thickness burns (1 x 1 in) were created on female Red Duroc pigs. Burns were excised and grafted with split-thickness autograft. Pressure garments were applied within 1 week and maintained at 20 + 2 mm Hg. Treatment groups included: continuous pressure group, which received pressure for 29 weeks, pressure release group, which received pressure for 17 weeks, then pressure was removed for an additional 12 weeks; and a control group that did not receive pressure (n=8 scars/group). RESULTS: Pressure garment therapy significantly reduced contraction and scar height versus controls at 17 weeks post-grafting. When garments were removed, scars in the pressure release group rapidly contracted, with scar area at the conclusion of the study 22% greater than controls in the pressure release group and 86% greater versus controls in the continuous pressure group (p < 0.001). Scar height also increased 2-fold after pressure release (p < 0.0) and collagen fiber reorientation was observed after therapy was ceased. Pressure garments reduced scar height and contraction after 4 months of use; however, when therapy was stopped, scars rapidly contracted and became thicker. CONCLUSIONS: Pressure garment therapy must be continued until full scar maturation. Investigations into the change in scar behavior (gene expression, extracellular matrix production, cytokine production) upon garment removal are underway to identify key regulators of scar suppression via pressure therapy.
P.BW03.
Background- Hypertrophic scarring (HTS) is characterized by firm, raised lesions, and excessive accumulation of collagen, and is one of the adverse outcomes of dermal burn injuries. HTS results in contractures that cause negative functional and aesthetic consequences while hindering full recovery and quality of patient’s life. The scarring is thought to be a result of excessive collagen accumulation and rearrangement and is one of the frequently used as the clinically relevant endpoints for the measurement of HTS in different animal burn models. However, the kinetics of collagen accumulation during HTS formation in burn wounds over time and different anatomical location are not well determined. Method- Using a red Duroc porcine HTS model, we studied the kinetics of collagen accumulation comparing deep partial-thickness (DPT) to shallow burns spatially and temporally. Additionally, we also compared collagen kinetics of these burn wounds in the Yorkshire pig, which is less prone for scarring. Collagen levels were determined by total collagen assay and expressed as µg collagen / gram of freeze dried wound tissue. Results- Compared to shallow burns we detected a decrease in total collagen in DPT burn wounds for the middle region of the back of the Yorkshire pig during the first 30 days after burn. Thereafter, by post-burn day 60, DPT wound collagen levels returned to levels similar to those of shallow wounds and normal skin. On-going studies will determine the collagen kinetics of burn wounds in red Duroc pigs. Conclusion- Based on our current work, collagen levels per gram of wound tissue may not be an optimal clinical endpoint for HTS in the porcine animal model. Disclaimer: The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
P.BW04.
Background: The paucity of donor sites in patients with burns involving large total body surface areas highlights the need for better cellular and tissue-based products (CTPs) that can achieve complete coverage without the need to graft. The purpose of this preclinical trial was to evaluate two xenogenic CTPs on deep partial thickness (DPT) burn wounds. Methods: DPT 5×5 cm burn wounds were created on the dorsum of anesthetized Yorkshire pigs using appropriate pain control methods. After 24 hours, wounds were excised to a viable wound bed and treated with fish skin graft (FSG, N=12) or fetal bovine dermis (FBD, N=12). FSG was reapplied after 7 days and all wounds were allowed to heal by secondary intentions for 60 days. Quantitative measurements include epithelialization, contraction rates, transepidermal water loss, hydration, and blood flow. Results: The wounds treated with FSG resulted in faster epithelialization (50.2% vs. 23.5% at day 14 and 81.7% vs. 62.3% at day 21, p<0.001). The FBD took longer to integrate into the wound bed than the FSG which was evident in higher hydration values at day 21 (2500.4 vs. 309.7 µS, p<0.0001) and lower blood flow measured at day 14 via laser speckle (3.3 vs. 5.1 fold change increase over normal skin, p<0.0001). Conclusions: Our results indicate that FSG integrated faster and allowed quicker wound closure without grafting while not increasing contraction of burn wounds. These results suggest that FSG could be an improved CTP that can promote healing of burn wounds while eliminating grafting requirements.
P.BW05.
BACKGROUND Thoracoabdominal injuries often occur in association with multiorgan injuries. Here we present a case of severe high-voltage electrical burn with thoracoabdominal injury and share some experience of clinical treatment. METHODS A 60-year-old man suffered a severe work-related electrical injury caused by high-voltage current (10,000V). On admission, the patient presented progressive dyspnea with left chest wall defect. The full-thickness burn was 15% of his body surface area. Thoracoscopy exploration performed immediately. Diaphragm injury didn’t found. The burn wounds were debridement, covered with artificial skin. Continuous negative pressure (125mmHg) was applied. On day 5, there was large amount of drainage in the chest tube. CT indication of colonic perforation. We detected perforation on splenic region of the colon and rupture of diaphragm. The gastrointestinal surgeons performed colostomy. The thoracic surgeons repaired the diaphragm injury with autologous dermis. On day 7, Splenectomy and partial pancreatectomy were performed because of splenic vein rupture and pancreatic tail necrosis. On day 16, high concentration amylase in intraperitoneal drainage fluid demonstrated pancreatic fistula. The burn wounds were cured by skin grafts and local flaps. Individualized negative pressure wound therapy helped the abdominal wound healing. RESULTS The patient discharged after 119 days. CT showed that the left lung was reexpansion and the fibrous adhesions formed diaphragm-like structure. Three months later, the colostomy closure was performed. After 6months of follow-up, the patient had no symptoms of discomfort. CONCLUSIONS Delayed necrosis of electrical burns is mostly unexpected. Physical examination combined with imaging methods and laboratory tests permit early diagnosis of severe injuries. Multidisciplinary treatment will reduce morbidity and mortality. Negative pressure wound therapy plays an important role in surgical treatment, especially in severe patients.
P.CW01.
Background: Non-healing chronic wounds can lead to morbidity and mortality in patients. Chronic human wound fibroblasts (CHWF) are known to have deficits in cell proliferation, migration, and contraction. Our previous studies have demonstrated that the myocardin-related transcription factors A and B (MRTF-A/B) are involved in myofibroblast formation and function during normal cutaneous wound healing. We hypothesize that MRTF-A/B activity is reduced in CHWFs and that ectopic expression of MRTF-A/B will promote myofibroblast function in CHWFs.
Methods: CHFWs were obtained from Coriell Institute for Medical Research and normal human dermal fibroblasts (NHDF) were obtained from Lonza. Whole cell lysates from CHWFs infected with lentiviral vectors to overexpress MRTF-A, MRTF-B, and LacZ and from NHDF infected with lentiviral vectors expressing either shRNA targeting MRTF-A/B or non-targeting shRNA were subjected to Western-immunoblotting for MRTF-A, MRTF-B, smooth muscle alpha actin (SMαA), SM22α, collagen type III, and GAPDH. Whole cell lysates of CHWFs and NHDFs were subjected to Western-immunoblotting for SMαA, MRTF-A, MRTF-B, SM22α, and GAPDH. Nuclear and cytoplasmic fractions from jasplakinolide (induces actin polymerization) treated CHWFs were subjected to Western-immunoblotting for MRTF-A, MRTF-B, Tubulin, and Lamin A/C. Lastly, contractile properties of MRTF-A/B depleted NHDF cells were analyzed in a stressed-relaxed collagen lattice contraction assay. Results: Ectopic expression of either MRTF-A or MRTF-B in CHWFs induced the expression of pro-contractile genes SMαA, SM22α. CHWFs expressed less SMαA and SM22α compared to NHDF. Jasplakinolide-induced actin polymerization in CHWFs promoted increased nuclear localization of both MRTF-A and MRTF-B. Depletion of MRTF-A/B in NHDFs reduced the expression of SMαA, SM22α, collagen type III and reduced stressed-relaxed collagen lattice contraction. Conclusions: These results suggest that decreases in MRTF-A/B activity results in a CHWF phenotype and that ectopic expression of MRTF-A/B in CHWFs can induce myofibroblast formation and function suggesting that MRTFs may be a therapeutic target to promote better wound healing in chronic wounds.
P.CW02.
Modified keystone flap with fortune cookie design: New “workhorse” flap in gluteal region ABSTRACT Background The reconstruction of extensive soft tissue defects in gluteal region is a great challenge. Here, we describe our surgical experience of using modified keystone flap. Methods We retrospectively reviewed the data of 13 consecutive patients who underwent soft tissue reconstruction with modified keystone flaps between March and December 2017. (figure.1) Results Soft tissue reconstruction was performed without any major complications in the follow-up period. The reconstructed defect had a mean width of 6.3 cm and mean length of 7.3 cm, whereas the flap had a mean width of 8.3 cm and mean length of 12.1 cm. We have not experienced any partial flap congestion or necrosis. All patients were satisfied with the aesthetic outcomes.(figure.2-3) Conclusions Considering its simplicity, versatility, and reliability, our technique of using modified keystone flaps could be a reasonable surgical option for soft tissue reconstruction in gluteal region.
P.CW03.
BACKGROUND – One major obstacle in chronic wound research is the lack of appropriate animal model with long ischemic time to recapitulate the primary pathophysiology of non-healing wounds. The purpose of this study was to create a significantly extended ischemic time in rabbits.METHODS – Twenty young adult New Zealand White rabbits were used. Employing the minimally invasive technique, three skin incisions were made on one ear base to ligate the central and cranial arteries, and a circumferential tunnel was created to disrupt the subcutaneous tissues. A medical grade silicone rod 3-5 mm in diameter was threaded through the tunnel but punch holes were made for the central and caudal veins. Tissue perfusion was measured daily for the first two weeks and then weekly using laser speckle Doppler imaging, TiVi8000 Tissue Viability Imaging system, a thermal imaging camera, skin temperature, and subcutaneous oxygen tension/saturation. RESULTS – Postoperative recovery was similar to the regular ischemic operation. In some rabbits, the ischemic ear showed edema for a few days. The implanted rod can be seen clearly on the ear base. Ear tissue perfusion was significantly reduced for more than 3 months in most rabbits with the longest one lasted for 10 months. Histology shows that, the implanted silicone rod occupies almost all the subcutaneous tunnel space, making it almost impossible for major collateral formation. No repeated surgeries were needed to maintain long-term ischemia. CONCLUSIONS – This new rabbit ear model provides a reliable extended ischemic period, closely resembling many types of human chronic wounds. It will be a valuable tool for chronic wound research.
P.CW04.
We investigated toxicity of bandages for wound healing with silver (three brands) or octenidine and hyaluronic acid (OCT-HA). The bandages were investigated in viability assays of keratinocytes and fibroblasts (NHEK, NHDF, respectively) and cell death flow-cytometry assay (all n=4). Ex vivo porcine skin was cultivated with the bandages on dermal side (n=4). Silver penetration was visualized with autometallography in porcine skin sections. γH2A.X was detected with immunofluorescence in the porcine skin and with WB in the skin homogenates. Gene expression was measured with qPCR (n=4). Patients (n=7) who gave a written consent were treated concomitantly on different parts of leg ulcers with silver bandage or OCT-HA. Biopsies were collected at the beginning, after 2 and 6 weeks and evaluated with histology – autometallography, trichrome stain and naphthol chloroacetate (granulocytes). Cell viability of both NHEK and NHDF treated with eluates from silver bandages was significantly decrased (p<0.05, t-test). NHDF died by necrosis after treatment with the eluates for 24 hours unlike the cells treated with OCT-HA eluate. Porcine skin incubated with silver bandages (27 or 51 hours) exhibited significantly higher DNA damage (elevated γH2A.X, p<0.05, t-test) and expression of stress genes HSPH1 and DNAJA1 (p<0.05, t-test). Silver penetrated deep into the ex vivo and in vivo tissues. Wounds treated with silver bandage had more granulocytes and inferior collagen maturation (both p<0.05) compared to OCT-HA after 2 weeks. High silver penetration, toxicity and no biodegradability impose unnecessary burden on the wounds. Therefore, use of silver bandages may slow down wound healing and one should consider safer alternatives.
P.CW05.
Chronic ulcers harbor a plethora of microorganisms that are resistant not only to conventional wound care but also to physical debridement, topical therapies and dressings in addition to multidisciplinary treatment strategies. The presence of biofilms in chronic wounds, present in over 80% of patients with infection, is a significant obstacle to wound closure. Current topical applications are not effective in treating bacterial biofilms in wounds.
To determine if disrupting chronic wound biofilm would be therapeutically efficacious, we studied the use of a novel topical agent for wound management, specifically targeting biofilms. 36 patients with chronic recalcitrant wounds were randomized to a 12-week treatment with a broad spectrum antimicrobial ointment or a biofilm disrupting wound gel. Wound healing rate was assessed by measuring wound size reduction and closure rates.
Wound size decreased significantly with a 71% reduction in wound area for wounds treated with the biofilm disrupting gel, compared to 24% for the control (p < 0.001). Wound closure was attained in more than half of the patients treated with the test product. 53% of these patients achieved closure by 12 weeks, as opposed to 17% for the control (p < 0.01). There were no adverse events related to the biofilm disrupting product while two adverse reactions occurred with the control.
The combination of the novel biofilm disrupting agent with wound debridement, significantly improves wound healing rates by disrupting the biofilm which protects multispecies bacteria within a chronic wound. Given the significant wound size reduction and closure rates observed in these long-term non-healing wounds, and a lack of related serious adverse events, the biofilm disrupting wound gel, in our setting and experience, is a safe and effective treatment for recalcitrant chronic wounds.
P.CW06.
Purpose: The perforator-based island flap is a popular option for defect coverage. In cases with deep cavities, however, the classical island flap may not be a suitable option. By de-epithelization of the peripheral portion of a perforator-based island flap, the distal part of the flap can be used to fill deep spaces, as the flap can be folded and inserted into the spaces. Methods: From June 2015 to April 2017, 21 cases of deep internal defects were reconstructed with perforator-based island flaps with peripheral de-epithelization. A fasciocutaneous flap was elevated and rotated with the pivot point on the perforator traced by hand-held Doppler. After measuring the size of internal space, de-epithelization was performed on the periphery of the flap. The de-epithelized portion of the flap was inserted and anchored into the internal defect (Figure 1 & 2). Demographic information about the patients, the size of the defects, the perforators that were used, and complications were recorded (Table 1). Results: During the follow-up period (mean, 14.2 months) of total 21 cases (16 pressure sores, 2 meningomyelocele defects, and 3 cutaneous fistulae), no major complications such as flap loss occurred. In 2 cases, a minor complication was observed. Temporary flap congestion was seen in 1 case, and was treated with a short period of leech therapy, and the other case was partial necrosis on the flap margin, which was cured with minimal debridement and conservative treatment. No major problems have occurred, especially on the de-epithelized part of the flap and in the occupied space. All deep cavities were completely reconstructed. Conclusion: A perforator-based island flap with partial de-epithelization can be a useful option for the surgical treatment of deep cavities. With performing safe procedure, the flap can be transferred safely without anxiety regarding the buried flap.
P.CW07.
Background – Chronic diabetic wounds impact over 3 million Americans per year and cost more than $9 billion to treat on an annual basis. With the concomitance of an increasingly aging population the deleterious effects of diabetic wounds are becoming even more dramatic in the elderly, as aging represents an independent, synergistic risk factor for impaired wound healing. This scenario points to the need to develop reliable preclinical models of aged diabetic wounds with the goal of studying the unique patho-physiology of this condition and the effectiveness of novel therapeutic approaches. Methods – Here we adopted an established murine model of type-2 diabetes (db/db) and investigated the varying biology of wound healing in young (10-weeks old) and old (12-months old) animals (n = 6 /group). A dorsal wound was performed on each animal and covered with a transparent dressing. Digital pictures and tissue specimens of wounds were obtained on day 0,5, and 10. Histological samples were used to measure granulation tissue formation, inflammation, cell proliferation and angiogenesis. Anova with post-hoc Bonferroni correction was used for statistical analyses. Results – In the db/db model aged animals showed a moderate by significant slower wound closure rate compared to younger mice (p=0.05). Histology confirmed significantly lower granulation tissue formation, pathologic collagen remodeling, and decreased angiogenesis in the older animals compared to their younger controls. Conclusions – The aged db/db mouse might represent a preclinical model to study the synergistic effects of aging and type-2 diabetes in chronic wound healing. This model should be further characterized and implemented to help design effective clinical therapies.
P.CW08.
Background: Wound healing is a multiple cellular processes including proliferation, migration, and angiogenesis, which are severely interfered under diabetes mellitus. Amnion was clinically demonstrated to enhance the healing of chronic diabetic foot ulcers as it contains numerous cytokines, growth factors and hormones well known to be potential to facilitate wound healing processes. However, the pathways through which these biological components of the placental tissue promote diabetic cutaneous wound healing are highly required to be elucidated. Utilizing diabetic splinted excisional wounds murine model established previously in our laboratory, the effect of dehydrated human amnion/chorion allografts (dHACAs) on diabetic wound healing was investigated in this study. Methods: Splinted, full thickness excisional wounds were created on the dorsal skin of db/db mice, and immediately applied with dHACAs topically or covered with dressing alone. The wounds was tracked and harvested on post operation day (POD) 7 and 14 for further measurement healing parameters including re-epithelialization, granulation tissue deposition, proliferation, angiogenesis and immune-inflammatory responses through histological analysis, immunohistochemistry and PCR array. Result: Compared to the primary dressing alone, dHACA notably promoted the re-epithelialization rate, and deposition of granulation tissue in wounds as showed in histological analysis. Immunohistochemistry staining displayed that the expression proliferation (Ki67) and angiogenesis (CD31) in the diabetic wounds were also enhanced by dHACA administration. The ratio of remodeling/wound healing M2 to proinflammatory M1 macrophages was also increased in the dHACA treated wounds. PCR array data indicated that the genes related to angiogenesis, keratinocyte migration, proliferation, and inflammatory response were regulated by the administration of dHACA. Conclusions: These data demonstrated that amnion/chorion membranes promoted diabetic wound healing through enhancing cellular proliferation, migration and angiogenesis, as well as regulating immune-inflammatory response of the host.
P.CW09.
Introduction Chronic wounds (CW) have constituted a very serious medical problem in recent years especially when critically colonized or infected, they need special preparation for future treatment, especially possibility of use of antiseptics which are sometimes controversial.
Study aim
P.CW10.
THE COMBINED EFFECT OF MESENCHYMAL STEM CELLS AND CHICKEN EMBRYO EXTRACT ON THE FLAP VIABILITY AND MAST CELLS IN RANDOM SKIN FLAPS 1.Mohammad Bayat, Ph D, 2.Farzaneh Chehelcheraghi, Ph D, 3. Sufan Chien, MD, 1, 3. Noveratech LLC of Louisville, KY, Price Institute of Surgical Research, Hiram C Polk Jr. MD KY, and Department of Surgery, University of Louisville, Louisville, KY, USA. Email:bayat_m.com 2. Anatomical Sciences Department, Lorestan University Medical Sciences, Khorramabad, Iran. The aim of present study was to investigate the effect of bone marrow mesenchymal stem cells (BMMSCs), and chicken embryo extract (CEE) on the viability, blood vessels number, and mast cells(MCs) number and degranulation of random skin flaps (RSFs). A 30×80 -mm RSF were made in dorsum of forty rats. They were divided into four groups as follow. Group 1 was served as control, group 2 received BMMSCs, group 3 received CEE+ BMMSCs, and group 4 received CEE. BMMSCs and CEE were injected to the flaps immediately after surgery. Seven days after surgery, survived part of flaps were measured, and blood vessels sections, mast cells number and degranulation were examined. We observed significant increase in survival area of flaps, and blood vessel sections of all treatment groups compared to control group. BMMSCs group showed significant decreases in survival part of flap compared to CEE and CEE+ BMMSCs groups. Moreover type two MCs (complete degranulation state of MCs) and total number of MCs in BMMSC group were significantly higher than other groups. The stimulatory effect of BMMSCs, CEE, and CEE+BMMSCs was presented by significant increase in survival part of flaps compared to the control group. And CEE group was more effective statistically compared to the BMMSCs, and BMMSCs+CEE groups. We hypothesized that MCs productions induced an inhibitory effect on survival of the RSFs.
P.CW11.
Chronic ulcers harbor a plethora of microorganisms that are resistant not only to conventional wound care but also to physical debridement, topical therapies and dressings but also to multidisciplinary treatment strategies. The presence of biofilms in chronic wounds, present in over 80% of patients with infection, is a significant obstacle to wound closure. Current topical applications are not effective in treating bacterial biofilms in wounds.
The use of a novel biofilm disruptive wash that is broad-spectrum with low to no toxicity to the host tissue is a very promising approach to solving the chronicity of diabetic wound ulcers. The objective of this 10-subject prospective pilot study is to assess the reduction in the bacterial load within the wound by DNA typing after the use of the biofilm disrupting wash post-debridement and the associated reduction in size or closure of chronic wounds over 12 weeks
In our study, we were able to quantify bacteria for all samples pre- and post-debridement. For all patients, there was a measurable decrease in the bacterial load within the wound after treatment with the lavage. The average wound size reduction for the ten patients over the first four weeks of treatment using the wash after debridement was 57%, with 6/10 patients achieve greater than a 50% reduction in the first four weeks of treatment.
These patients will need to be followed to determine the wound healing rate for and the correlation of bacterial burden to wound healing. A larger sample size study will need to be performed to achieve statistical significance of the correlation between the use of the biofilm disrupting wash and its effect on wound healing.
P.CW12.
Natural compounds have a significant potential to treat wounds caused by trauma, diabetes, ischemic syndromes and other pathological diseases. This is due to their antioxidant, anti-inflammatory and anti-bacterial characteristics. Wound healing complexity necessitates the use of multi-target drugs with immunomodulatory, pro-angiogenesis and pro-healing properties. In this context, polyphenols have received increasing attention due to their low toxicity and potential to alleviate symptoms and inhibit the development of various skin disorders, skin aging, and skin damage, including wounds and burns. In this work, we explored the potential of a flavonoid, myricetin-3-O-rhamnoside, and a phenolic compound, chlorogenic acid, both isolated from Parrotia persica leaves, to suppress inflammation, promote wound healing and accelerate angiogenesis. This was accomplished using in vitro scratch and tube formation assays utilizing human epidermal keratinocytes (NHEKs), human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs). We also investigated the influence of these compounds on driving pro-inflammatory environment to anti-inflammatory environment to accelerate wound closure of fibroblast using conditioned media of macrophages. The assessment of dose response of the compounds demonstrated no cell toxicity between the dosages of 1 µg/mL to 100 µg/mL. Wound closure as early as 6 hours, demonstrated approximately 40% of the gap to be closed with 10 µg/mL of chlorogenic acid. On the contrary, myricetin was most effective in promotion of wound closure in assays using NHDFs (3-fold increase) as compared to negative control. Both compounds were able to induce tube formation at an approximately 50% higher rate as compared to control groups. Altogether, our results demonstrate the potential of myricetin and chlorogenic acid to be used in combination in treatment of cutaneous wounds.
P.CW13.
Background: In the treatment of diabetic foot ulcers (DFU), there are many treatment options, however, to address the pathophysiology, the most important consideration is offloading. Negative pressure wound therapy (NPWT) is one option to enhance healing. In practice when electrical power NPWT is applied, this usually precludes offloading with total contact casting (TCC). In this limited case series, we share our approach in combining cellular tissue based products, NPWT and TCC in the treatment of DFU. Methods: Three patients with refractory DFU’s and multiple recurrences were treated using simultaneous application of a cellular tissue based product, mechanical power NPWT and TCC offloading. Results: These three patients demonstrated favorable clinical progress toward healing and were able tolerate this approach without significant difficulty or adverse event. Conclusion: Our preliminary impression is that combining these three treatment methods (cellular tissue based products, negative pressure wound therapy and TCC offloading) in the treatment of DFU is an extremely useful protocol. This multipronged approach simultaneously addresses several pathological features underlying the diabetic foot ulcer and could potentially lead to shorter healing times and less complications. These findings need to be replicated in other practices and at larger scale.
P.CW14.
Background: In the treatment of VLU, there are many treatment options, however, to address the pathophysiology, the most important consideration is compression therapy (1). Negative pressure wound therapy (NPWT) is one of the options to enhance healing. In practice when electrical power NPWT is used, usually it precludes compression therapy. In this limited case series we are sharing our approach in combining multiple treatment methods for VLU patients and to enhance benefit of cellular tissue products. Methods: Three patients with refractory VLU and multiple recurrences were treated with this approach, using simultaneously a tissue based product, mechanical power negative pressure wound therapy and compression therapy. Results: These three patients had very satisfactory clinical progress towards healing and were able to tolerate this approach without significant difficulty or adverse events. Conclusions: Our preliminary impression is that combining multiple treatments methods simultaneously (tissue based product, NPWT and compression) is an extremely useful treatment protocol. Potentially this can lead to shorter healing time and less complications. These finding need to be replicated in other practices and at larger scale.
P.CW15.
Background: The purpose of this pilot study was to demonstrate the feasibility of using non-invasive optic measurements of blood flow as a screening tool for pressure injury (PI) in the surgical intensive care unit (SICU). Frequently, these injuries originate as deep tissue injury (DTI) several millimeters beneath the skin’s surface. DTI is not clinically apparent until it spreads through subcutaneous tissue. By this time, the damage may be too extensive to avoid advanced ulceration.
Methods: Our long-term goal is to develop and validate a method for early detection of DTI by measuring blood flow in the dermis and subcutaneous tissue with a non-invasive optical method termed diffuse correlation spectroscopy (DCS).
Results: We recently completed a pilot study at a rehabilitation hospital suggesting that sacral blood flow measurements can detect sacral PI development prior to clinical appearance. For this study, we recruited 10 patients (60% male, 57.5 + 13.13 years of age) from a SICU and gathered data on blood flow, clinical, nutrition and metabolic characteristics. We compared the data obtained from patients who developed PIs (N=1) with those who did not (N=9). Although only one subject developed a sacral PI during this study, the data obtained from this subject’s sacral tissue suggested that an increase in local microcirculatory blood flow may have preceded ulceration.
Conclusions: These preliminary results and the logistic information obtained will be used in the design of a larger-scale clinical trial to determine the efficacy of DCS for early detection of PI in critical care populations.
Acknowledgements: Work supported by the Office of the Assistant Secretary of Defense for Health Affairs, under Award No. W81XWH-14-1-0614 and Drexel University’s Clinical Translational Research Institute Award.. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Department of Defense.
P.EP01.
P.EXT01.
Porcine urinary bladder extracellular matrix (UBM-ECM) with its formulations as a micronized powder and sheets of varying thickness both lyophilized and vacuum-pressed has allowed for the use of these devices to move from an adjunct to topical healing to use as a primary reconstructive modality. Methods- We performed a retrospective review of our series of 353 patients with 429 wounds treated with UBM-ECM and identified 14 significant wound cases in medically complex patients where the wound device was used as a primary reconstructive modality in lieu of standard flap procedures. There were 9 males and 5 females with an age range of 24 – 80 years. There were 6 acute and 8 chronic wounds treated. Nine wounds had significant bacterial colonization at the time of treatment. There were 2 head and neck, 2 upper extremity, and 8 lower extremity cases. Results- Multiple UBM-ECM formulations were used in all case. Nine patients required multiple operative device applications and/or office applications. Skin grafts both split and full thickness skin grafts were employed at a secondary procedure in 3 cases. Secondary wound care involved negative pressure wound therapy with wounds draining over 30 cc/day. The time to healing varied and roughly correlated with the size of the wound. Compared to standard flap operative procedures, the use of UBM-ECM wound devices took longer to achieve healing but avoid donor sites other than skin grafts. These devices performed well in the face of multiple medical co-morbidities and bacterial colonization. No patient failed ECM treatment and had to be “salvaged” with a reconstructive flap procedure. We believe the use of the UBM-ECM wound devices offer a reasonable treatment alternative for medically complex wound patients who desire less complex treatments and should at least be mentioned to patients when reviewing possible treatment options.
P.EXT02.
Osteomyelitis is an infectious process of the bone caused by prolonged immobility, bacterial dissemination through blood, immunosuppression, trauma, or vascular insufficiency. The diagnosis of osteomyelitis is made by multiple modalities. Some of these include bone cultures, bone biopsy, imaging, and clinical determination1. In this study we plan to chronicle patients with suspected osteomyelitis secondary to prolonged immobility resulting in pressure ulceration. Our aim is to find correlations between bone cultures and biopsy findings of the bone. The relationship between the two entities can help guide extended antibiotic therapy.
M1.03.
BACKGROUND The skin provides a barrier between the outside environment and the internal milieu of the body. Disruption of the stratum corneum which occurs in wounding leads to evaporative water loss and we hypothesize an increase in [Na+]. We have previously shown that when keratinocytes are exposed to a 10% increase in extracellular [Na+] – which could potentially occur in open wounds, the sodium channel NaX becomes permeable leading to the influx of Na+. The resultant increase in intracellular [Na+] in-turn activates signaling pathways leading to inflammation and scar formation. METHODS Fibroblasts are crucial to the wound healing process by stimulating the production of collagen for tissue repair. Fibroblasts may also serve as vital immunoregulatory cells.Me In this study we investigated whether exposure of fibroblasts to increases in extracellular [Na+] could modulate fibroblast function during wound healing. Here we show that under dehydrating conditions, in non-epithelized wounds where fibroblasts are the predominant cell type, the inflammatory mediators Cox-2 and Il-8 are markedly upregulated. RESULTS In vitro, NaX is highly expressed in fibroblasts and exposure of fibroblasts to a 10% increase in culture media [Na+] led to a rapid influx of Na+ and upregulated the expression of COX-2 and IL-8. Knock-down of NaX expression using shRNA blocked the induction of COX-2 and IL-8 in response to high NaCl. In the presence of increased NaCl collagen contraction and cell migration were not affected suggesting that high Na+ preferentially modulates the inflammatory signaling pathways within fibroblasts. CONCLUSIONS These data suggest that increases in local [Na+] such as those that would occur in an open wound stimulate inflammation in fibroblasts via NaX activation and signaling.
M1.06.
We evaluated the clinical efficacy of our -79 °C spray-type cryotherapy with molecular and pathologic evidence for the treatment of keloids. We evenly split each of ten keloid lesions into a non-treated (C-) and treated (C+) area; the C+ area was subjected to two freeze-thaw cycles of spray-type cryotherapy using -79 °C spray-type Cryotherapy. This treatment was repeated after an interval of two weeks. The proliferation and migration abilities of the fibroblasts isolated from the dermis under the cryotherapy-treated or untreated keloid tissues (at least 5 mm deep) were compared and pathologic findings of the full layer were evaluated. Molecular analysis revealed that the number of dermal fibroblasts was significantly higher in C+ group as compared with C- group. The dermal fibroblasts from C+ group showed more than two-fold increase in the migration ability as compared with the fibroblasts from C- group. The expression of matrix metallopeptidase 9 was increased by more than two-fold and a significant increase in transforming growth factor beta 1 expression and Smad2/3 phosphorylation level was observed in C+ group. C+ group showed more extensive lymphoplasmacytic infiltration with thicker fibrosis and occasional “proliferating core collagen” as compared with C- group. Thus, -79 °C spray-type cryotherapy is ineffective as a monotherapy and should be used in combination with intralesional corticosteroids or botulinum toxin A for favourable outcomes in the treatment of thick keloids.
M1.07.
BACKGROUND The goal of any topical formulation is efficient transdermal delivery of its actives. However, delivery of compounds can be problematic with penetration through layers of dermal scar tissue. Testing of the presence and depth of penetration of compounds can prove challenging. We propose a new combined approach to assessment of transdermal delivery of topicals; initially using an ex vivo human skin culture with high performance liquid chromatography (HPLC), and subsequently validated by Raman Spectroscopy (RS) of in-vivo human normal and scarred skin. METHODS Topicals were applied to ex-vivo skin organ culture, which were quantified by an optimised HPLC system. Longitudinal sections were analysed to differentiate presence of the topical between skin layers. In-vivo normal and scarred skin from sequential biopsies with application of both topicals were analysed with RS by acquiring static spectral point measurements from cross sections. RESULTS HPLC isolated peaks for 2 actives. One compound was identified in ex vivo ‘whole skin’ and within the papillary and reticular dermis. The presence of the topical was confirmed by RS in the epidermis and reticular dermis (98% and 99% specificity, 89% and 93% sensitivity, 96%, and 97% accuracy respectively). RS also demonstrated presence of this topical over sequential time points (day 0 to week 8 and 12). Alterations in the secondary structure conformation of protein peaks (α-helix to β-sheet) accounted for changes during remodelling. CONCLUSIONS This unique approach enables successful detection as well as measurement of exact depth of penetration of compounds following application of a topical formulation in cutaneous scar tissue in both ex-vivo and in-vivo models. Where there is uncertainty regarding discrimination of skin layers using longitudinal sections, the cross-sectional approach in RS can validate findings, allowing for the simultaneous evaluation of the effects of the compounds in healing wounds and scar maturation over time.
P.FS01.
Although it is known that fibroblasts promote scarring, differences in phenotypic variability and the signaling mechanisms between fibroblasts are yet to be defined. Here, we postulate that differential fibroblast responses to mechanical tension regulate exosome production.
We recruited patients who had given birth by caesarian section and were undergoing abdominoplasty procedures under IRB guidelines and pre-operatively evaluated them using the Vancouver scarring scale(VSS;n=8;29-55y.o. female;healthy and non-smoking). Skin and C-section scar samples were obtained from discarded tissues and evaluated by histological staining and immunohistochemistry(F4/80[macrophages]; CD3/CD69[T-cells]). Fibroblasts isolated from patients’ normal skin and C-section scars were cultured on silicone membranes ±10% static strain(24hrs). qRT-PCR evaluated expression of fibrotic markers(CD26/alpha-SMA/TGF-B) and genes encoding exosome synthesis(RAB27a-b;SMPD3). Exosomes were analyzed for size and quantity(Zetasizer). Qualitative analysis was performed by three independent blinded investigators; in vitro data: mean ±SD;p by ANOVA.
Patient scars were classified as “low” or “high” based on VSS(<3 vs. >6). Histological staining revealed thicker collagen bundles/whorls(H&E), elevated collagen turnover(Herovici), and increased presence of F4/80 and CD3-positive cells(p<0.05) in high-scar samples relative to low-scar and normal skin(n=3). Preliminary data from one patient’s normal skin and C-section scar-derived fibroblasts, as well as high/low-scar derived fibroblasts, showed that tension increases expression of CD26(~2.8-fold), a-SMA (~3-fold), and TGF-B1(~2.3 fold) in all four groups; p<0.05;3 passages per cell-line. Notably, high-scar fibroblasts significantly increase expression of these markers relative to other groups(p<0.05). In contrast, exosome synthesis genes(RAB27a-b, SMPD3) are down regulated (~2-fold; p<0.05) in all groups under tension. Exosome profiles are more variable: under static conditions, exosomes from normal skin and low-scar derived fibroblasts were similar, while those from high-scar derived fibroblasts were more abundant and larger in size(p<0.05).
Our findings suggest that fibroblasts’ differential response to tension includes changes in exosome production, which could influence fibrogenic phenotype. Elucidating the exosome profile/cargo could reveal fundamental biologic differences underlying heterogeneity in human scarring.
P.FS02.
BACKGROUND The ability to characterize cutaneous fibrosis is of clinical and scientific relevance, as this is a critical step in assessing scarring severity, healing potential, and determining response to therapies across all wound and scars. A number of non-invasive objective technologies have been used to assess cutaneous fibrosis. Optical coherence tomography (OCT) and high-frequency ultrasound (HFUS) are real-time, non-invasive techniques that are used effectively for measuring physiological changes in tissue structure and function. We have previously compared OCT to histological assessment of acute wound healing and identified a novel measure of dermal fibrosis and remodeling known as the mean grey value. METHODS Our aim was to generate normative data using OCT and HFUS for all phases of healing with multiple sequential biopsies (day 0 and weeks 1,2,3,4,5,6,8) created in 62 healthy volunteers supported by histological and immune-histochemical analysis. RESULTS The attenuation coefficient values from OCT were significantly reduced from baseline (2.11/mm) to week 5 (1.43/mm) (p<0.001). Herovici staining demonstrated that mature collagen was greatest in normal skin (59%) compared to week 8 (24%) (P=0.04), whilst immature collagen was greater at all subsequent time points compared to baseline (p<0.05). This was further supported by additional collagen I and III analysis. HFUS measurement of skin thickness increased from week 1 (1.28mm) and slightly increased to week 6 (1.62mm), which was also confirmed by OCT measurements and H&E analysis (p=0.004). Elastin was significantly greater in normal skin (49.1%) compared to scar tissue with lowest values at week 4 (23.0%,p=0.01) and a gradual return to values similar to baseline by week 8 (41.4%). Fibronectin intensity was highest at week 4 (0.28Au, p=0.02) and reduced to week 8 (0.16Au). CONCLUSIONS OCT and HFUS provide a quantitative baseline for the assessment of dermal fibrosis, which may assist in the development of new theranostic strategies throughout therapy.
P.FS03.
Background: Hypertrophic scar is an important clinical problem with limited therapeutic options. Topical treatment by a repurposed medication already in widespread clinical use would be an important addition to the therapeutic armamentarium. Statins are a class of commonly prescribed cholesterol-lowering medications, and have also been demonstrated to inhibit fibrosis. We have been investing for many years statin treatment which acts by reducing CTGF expression as an option for hypertrophic scar. However, the low dermal penetrative ability of statins limited their topical treatments for hypertrophic scars. The objective of this study is to develop novel statin formulations with liposome which can enhance the dermal penetrative ability, and evaluate their efficacy on hypertrophic scar formation utilizing our validated rabbit ear hypertrophic scar model. Methods: Simvastatin (hydrophobic) and pravastatin (hydrophilic) were encapsulated in liposome using a novel flexible liposomal formulation. Hypertrophic scars formed after the cutaneous wounds created on rabbit ears healed completely on post operation day (POD) 14, and were daily treated with either liposomal simvastatin or pravastatin at 6.5% concentration topically until POD 25. The contralateral ear received liposome vehicle alone served as vehicle control. All the scars were tracked and harvested on POD28 for the evaluation of scar elevation index as well as gene expression related to fibrosis. Results: Topical treatment with simvastatin and pravastatin in liposome significantly reduced scar elevate index by 31.1±9.0% (p<0.01) and 35.0±8% (p<0.01) separately. The mRNA level of CTGF, TGFβ-1, collagen I and III in the scar tissue were also decreased by the liposomal pravastatin treatment. Conclusion: The novel statins formulations encapsulated in liposome were successfully delivered through topical application, and significantly reduced hypertrophic scarring in a rabbit ear model.
P.FS04.
BACKGROUND-Discrete subaortic stenosis(DSS) is a congenital anomaly characterized by fibrosis in the left ventricular outflow tract(LVOT). Treatment is usually surgical intervention but is associated with significant recurrence. The pathogenesis of DSS is unknown because of a lack of appropriate models. It is postulated that altered shear stresses in the LVOT cause endothelial cell injury and a fibrotic cascade. Therefore, we have created a novel bioreactor to mimic intra-cardiac shear-forces and hypothesized that altered shear-stress is sensed by endocardial endothelial cells(EEC) resulting in inflammatory-mediated fibrotic tissue formation. METHODS-EEC and cardiac fibroblasts(CF) were isolated from porcine LVOT and subjected to normal and pathologic shear-forces based on DSS patient echocardiographic data. Using the parallel plate flow bioreactor, variable shear forces were applied to EEC monolayers and analyzed by immunocytochemistry(CD31;VE-Cadherin;ASMA) and PCR array. The conditioned media from the monolayers were then transferred to CF, and analyzed by PCR array. Human DSS tissues were obtained from the Texas Children’s patient tissue-repository(n=7), and compared to dermal scar specimens by immunohistochemistry and histology. RESULTS-High shear-stress mimicking DSS caused the de-localization of mechanosensitive CD31 from the VE-Cadherin-rich cellular junctions in EEC. PCR array analysis revealed up-regulation of pro-inflammatory CCL3, CCL4 and CSF1 genes under pathologic shear(26.11, 14.38 and 5.07 fold regulation versus normal) and a significant upregulation of ASMA, a marker of endoMT(6.2±3.3,p<0.05). Pathologic shear-conditioned EEC media resulted in upregulation of fibrosis and matrix remodeling genes(TGFb,col3a,Bmp7) in CF. Histology revealed phenotypic similarities between fibrosis in DSS tissues and dermal scars, including increased collagen remodeling(Herovici), disorganized collagen(Trichrome), and presence of activated fibroblasts(25% of resident cells, ASMA). CONCLUSIONS-Our data suggests altered shear forces affect mechanosensitive proteins in EEC and upregulate inflammatory and fibrosis related genes. Additionally, histologic characteristics of DSS mirror fibrosis in dermal scarring. Understanding the pathogenesis of DSS through mechanosensing may elucidate novel therapeutic targets for conditions characterized by fibrosis associated with altered mechanical forces.
P.FS05.
BACKGROUND – Pathologic dermal scarring such as hypertrophic scarring occurs when the normal wound healing processes of extracellular matrix deposition and remodeling become dysregulated. The profibrotic cytokine transforming growth factor beta 1 (TGF-β1) transforms fibroblasts in the wound bed into myofibroblasts with contractile F-actin stress fibers containing a smooth muscle actin isoform (α-SMA). Previous in vitro studies promoted pirfenidone as a potential dermal fibrosis prophylactic and established that concurrent treatment of normal human dermal fibroblasts with TGF- β1 and pirfenidone diminished the development of the contractile machinery elements of myofibroblasts. This in vitro study investigates the potential for pirfenidone treatment to mitigate an established myofibroblast phenotype.
METHODS – Normal human dermal fibroblasts were treated with pirfenidone after three-day stimulation by TGF- β1. Time course experiments were analyzed via immunocytochemistry and quantitative image analysis of F-actin stress fibers and α-SMA. Effects on cell contractility were assessed with fibroblast populated collagen lattice contraction assays.
RESULTS – Preliminary findings indicate that pirfenidone treatment of established myofibroblasts reduced F-actin and α-SMA in contractile stress fibers and reduced collagen gel contraction.
CONCLUSIONS – Pirfenidone is a potent antifibrotic capable of modulating the structure and function of the main effector cells of scarring, myofibroblasts.
This work is in part funded through the Congressionally Directed Medical Research Programs, U.S. Army Medical Research and Materiel Command W81XWH-15-2-0083 and the Naval Medical Research Center’s Advanced Medical Development program (MIPRN3239815MHX040). The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
P.FS06.
BACKGROUND: Excessive scar formation can be a devastating consequence of wound healing after injury from chemical or physical agent burns of the skin and other organs. There are few, if any, effective treatments to promote wound healing while effectively preventing scar formation. New treatments are needed. One option in topical wound healing is the use of temporary dressings that allow the natural healing process with minimal scar formation. METHODS: We evaluated PermeaDerm C (PDC), a biosynthetic variable porosity matrix that contains gelatin and Aloe Vera for chronic wounds, and PDC derivatives coated with the anti-scarring agent, salinomycin termed PermeaDerm A (PDA) for their ability to promote cell growth while preventing excessive scar-forming cell (myofibroblast) accumulation. Human and porcine fibroblasts and human stem cells grow favorably in vitro in the presence of PDC matrices. RESULTS: PDA attenuated TGFβ-induced scar forming myofibroblast formation of human and porcine cells. Additionally, both PermeaDerm matrices (PDC and PDA) significantly reduced the production of the inflammatory cytokines: IL-6, IL-8, MCP-1 and GRO. Specifically, PDC and PDA reduced IL-6 production by 50%, IL-8 by 20%, MCP-1 by 75% and GRO by 60% in human mesenchymal stem cells treated with TGFβ. CONCLUSIONS: These studies highlight the potential of PDC and PDA to allow effective wound healing while preventing excessive scar formation.
P.FS07.
Background- The high incidence of hypertrophic scar formation is one of the many challenges to treating deep partial-thickness burns. Treatment modalities for hypertrophic scars include laser therapy, pressure bandages, revision surgery and others, but are only marginally successful. For this reason, we evaluated prophylactic treatment of deep partial-thickness burns with an anti-fibrotic drug (pirfenidone) in C57BL/6 mice. Pirfenidone is an FDA-approved anti-fibrotic drug for systemic use in the treatment of idiopathic pulmonary fibrosis and other fibrotic disorders. Methods- Deep partial-thickness burns were induced in anesthetized mice with scalding water. Experimental groups (n=6/group), were treated twice during the inflammatory phase of wound healing with one of 4 topical ointment formulations containing different drug doses. Skin biopsies were taken at the inflammatory, proliferative and remodeling phases of wound healing and assessed for pirfenidone’s effect on αSMA expression, hydroxyproline deposition and reepithelialization. Results- Compared to vehicle ointment, 6.5% pirfenidone significantly reduced expression of αSMA 12 days after the induction of burns and modestly reduced hydroxyproline in the burn area 22 days after the induction of burns. Additionally, we determined that pirfenidone did not affect reepithelialization when mice were treated during the inflammatory stage of wound healing. Conclusion- The results from pirfenidone prophylactic treatment on mice with deep partial-thickness burns warrant further studies to validate its effectiveness in preventing hypertrophic scarring. Disclaimer- The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
P.FS08.
Background: Focal adhesion kinase (FAK) plays a pivotal role in transducing mechanical signals to elicit scar formation during wound repair. Clinical translation of FAK inhibitor (FAKI) therapy has been challenging, however, due to the lack of an effective drug delivery system especially for extensive burn and blast injuries and large excisional wounds. In a previous mouse study, pullulan-collagen hydrogel-mediated FAKI delivery to excisional and burn wounds accelerated healing and reduced scar formation. Here we evaluated the efficacy and safety of topical FAKI hydrogel therapy in a large animal model.
Methods: Four female Duroc swine were used to create multiple 25cm2 deep partial-thickness wounds. With multiple excisions, each wound depth was uniformly set to be 0.070 in. to create near full-thickness in the center and deep partial-thickness in the periphery. Animals received either standard dressings or FAKI-releasing hydrogels immediately after wounding, and dressings and hydrogels were changed every 2-3 days until wounds closed and then every 4 days thereafter. Wound closure rate and scar formation were evaluated over 90 days.
Results: Porcine excisional wound healing was accelerated with FAKI hydrogel treatment. Wounds treated with FAKI hydrogel closed significantly faster on day 14±2.0 vs. day 24±2.4 for control wounds (N=4 for each group, statistical difference at p<0.01). Visually inspected scars at Day 90 were substantially reduced with FAKI treatment and histological analysis demonstrated that fibrosis was substantially attenuated.
Conclusions: Biomaterial-based topical delivery of FAKI was effective in improving wound healing and reducing scar formation in a large animal model, and holds great potential as a novel therapeutic strategy for wound and scar management of large and deep dermal wounds.
P.FS09.
DETERMINATION OF tRNA-DERIVED FRAGMENTS AND tRNA HALVES LEVELS IN HUMAN KELOIDS Background: Small non-coding RNAs (ncRNAs) have been found processing various functions in human health and diseases while sequencing studies have suggested that transfer-RNAs (tRNAs) may serve as a major source of ncRNAs. tRNA-derived ncRNAs can be broadly classified into two groups: tRNA halves (tiRNAs) and tRNA-derived fragments (tRFs). However, the role of tRFs and tiRNAs expression in human keloids remains unknown. Methods: In this research, five keloid tissue samples and five normal skin (NS) samples were recruited to go through high-throughput sequencing study. The tRFs and tiRNAs expression levels were measured and normalized as tag counts per million of total aligned tRNA reads. Results: Venn diagram indicated 2660 genes expressed in both of the groups and 592 specific expressed genes in keloids. Pie plots were used to show the unique tRFs and tiRNAs of expressed level percentage of each subtype. tiRNA-5 and i-tRFs are major subtypes of tRFs/tiRNAs in most of the samples. 68 up-regulated genes and 5 down-regulated genes having fold changes ≥ 2 and p-values ≤ 0.05 are selected as the significantly differentially expressed genes by differential expression analysis of tRFs and tiRNAs. Conclusions: These results demonstrate a differential expression of tRFs/tiRNAs in human keloids as well as propel our understanding of this still vastly uncharted genomic territory. Further investigation on tRFs/tiRNAs with potentially important biological roles may provide new means for keloid mechanisms exploration and treatment.
P.FS10.
Introduction: Scar formation is the typical response to cutaneous injury in humans, but not loose-skinned animals. Human hypertrophic scars have significant levels of poorly organized collagen, loss of hair follicles, a raised appearance, and long-lived myofibroblasts. The only current method for producing human-like hypertrophic scars in loose-skinned animals requires the use of a palatal expander to add mechanical tension to an incisional wound. We hypothesize that using compression springs to apply dynamic circumferential mechanical tension to excisional wounds will result in human-like hypertrophic scarring. Methods: Compression springs were engineered to produce a range (1.5-9N) of forces. To apply circumferential mechanical tension on an excisional wound, the spring is first compressed by an internal suture and attached to the edges of a 6mm wound on the dorsum of mice with 6 simple interrupted sutures at 6 equidistant positions. The spring is then uncompressed by releasing the internal suture at day 3-5 post-wounding and removed 19 days post-wounding. Scars were photographed, fixed, and paraffin embedded at 19 days, 6 and 15 weeks post-wounding. Tissues were trichrome stained or immunostained for smooth muscle alpha actin (SMαA). Results: Springs below 4.5 N became distorted due to wound forces; therefore we used 9 N of force. An increase in SMαA positive myofibroblasts, scar area, absence of hair follicles, and an observable raised scar was observed 19 days post-wounding in mechanically loaded wounds compared to splinted wounds. At 6 weeks scar area and an observable raised scar were observed. 15 week post-wounding an observable raised scars was also observed. Determination of SMαA positive myofibroblasts and scar area for 6 and 15 weeks is ongoing. Conclusion: We report the use of compression springs to produce human-like hypertrophic scars in loose-skinned animals via mechanical tension. This method is valuable due to being inexpensive and reproducible. We anticipate this model to be useful for testing agents for the prevention of pathological scarring.
P.FS11.
Pathological scarring in wounds is a common medical problem with limited prognostic options. In this study, we present an approach to identify wound proteins that could serve as putative prognostic biomarkers of pathological scarring in traumatic skin wounds. We developed and validated a computational model that predicts temporal changes in the levels of 6 cell types and 21 molecular mediators, including collagen, during an injury-initiated wound healing response. By analyzing thousands of model-simulated wound-healing scenarios, we identified proteins whose levels were consistently elevated (or decreased) in wounds that resulted in pathological scarring (i.e., hypertrophic scars or keloids). We found that interleukin(IL)-10, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and fibronectin levels are consistently elevated as early as 2 and 3 weeks post-wounding in wounds that resulted in pathological scarring. Next, we quantified the predictive accuracy of these three protein markers using receiver operating characteristic curve analysis. We found that, using the levels of IL-10, TIMP-1, and fibronectin at 2 weeks post-wounding as predictors, we could predict the occurrence of a pathological scarring outcome at 6 weeks post-wounding with an 80% accuracy. Clinical validation of these model-predicted putative biomarkers could provide prognostic tools for objective clinical assessments of traumatic wounds. DISCLAIMER: The opinions and assertions contained herein are private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Army or of the U.S. Department of Defense. This abstract has been approved for public release with unlimited distribution.
P.GRO01.
Chronic wounds such as venous leg ulcers (VLU), pressure sores and diabetic foot ulcers are challenging clinical problems that affect a growing number of people due to the global expansion of the elderly, diabetic and obese. VLU affect approximately 1% of the adult US population, i.e., 2.3 million individuals. These wounds generally do not heal for over a month, can last years in severe cases and require advanced healing modalities, such as growth factors. However, the clinical results of using growth factors for treatment of chronic wounds have been very modest. Here, using chronic wound fluid from patients, we show that chronic wounds have high levels of proteases particularly neutrophil elastase. We report that neutrophil elastase degrades the clinically approved growth factor PDGF (platelet derived growth factor) in minutes, thereby rendering PDGF ineffective. We further report the development of novel PDGF formulations that extend the half-life of PDGF in the presence of neutrophil elastase. These formulations were tested in wound fluid derived from chronic wound patients (n=10), and we report that the PDGF was protected from degradation for up to 24 hours. Finally, we tested the formulation in a rodent chronic wound model with high neutrophil elastase and observed that the protected growth factor induced granulation and vascularization while the unprotected version displayed no effect (n=4 mice/group). Our results indicate that the developed PDGF formulation extends the life of the growth factor in chronic wounds by 24 hours. Therefore it is extremely promising for improving healing outcomes in chronic wounds.
P.GRO02.
BACKGROUND: Accumulation of scar tissue in fibrosis diminishes organ function. Fibrosis is characterized by the chronic co-existence of macrophages (MΦ), which produce pro-fibrotic growth factors and myofibroblasts (MFs), which secrete and contract collagen. Our recent data show that MΦ express latent TGF-β1, which is only activated in co-culture with MFs upon direct contact. We investigate the mechanism how MΦ present latent TGF-β1 to MFs for local activation. Regulatory T cells are known to present latent TGF-β1 using the transmembrane protein glycoprotein A repetitions predominant (GARP).
METHODS: To test whether tissue MΦ express GARP, we immuno-colocalized GARP with MΦ marker CD68 in normal, inflamed and fibrotic human lung. Primary cultures of human MΦ were tested for GARP expression by Western blotting, flow cytometry and RT-PCR.
RESULTS: Our results show that GARP is expressed in MΦ of normal (n=17) and inflamed lung (n=32) and on the surface of in vitro polarized pro-inflammatory MΦ (6%). Western blots performed with cultured MΦ under non-reducing conditions detect GARP, LAP and TGF-β1 at 250 kDa, identifying the GARP-LAP-TGF-β1 complex. To test whether MFs release latent TGF-β1 from MΦ-GARP, we measured active TGF-β1 levels in MF co-cultures with GARP-expressing and -depleted MΦ. The release mechanism is currently under investigation.
CONCLUSIONS: Collectively, our results indicate that GARP serves as novel surface anchor on MΦ to provide a local TGF-β1 source in inflammation and fibrosis. Interfering with local TGF-β1 presentation and/or activation are potential novel anti-fibrotic strategies.
P.IB01.
BACKGROUND: Biofilm, a community of bacteria, is tolerant to antimicrobial agents and host immune defenses due to their self-secreted protective extracellular polymeric substance. Biofilms are ubiquitous in chronic wounds, and the presence of biofilm correlates with non-healing. In wounds, biofilms reoccur within 24 hours post-debridement, therefore, prevention of biofilm formation is a desired property for wound care products. It has been shown that in a chronic DFU clinical trial that the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture condition. METHODS: In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on the biofilm formation of S.aureus and P.aeruginosa, the two most prominent pathogens associated with chronic wounds. The biofilm formation was measured using a high throughput method and an ex vivo porcine dermal tissue model. RESULTS: We demonstrated that the formation of biofilm on synthetic surfaces and porcine dermal tissue explants was significantly reduced in the presence of CVAM or CVUT-derived conditioned media compared to the control assay medium. CONCLUSIONS: This is the first study demonstrating that human cryopreserved viable placental tissues prevent biofilm formation. Anti-biofilm activity together with anti-inflammatory, anti-fibrotic and proangiogenic activities make placental tissue ideal for management of chronic wounds and should be considered as a desired biomaterial for engineering of new wound therapies.
P.IB02.
Background– Persisters such as small colony variant (SCV) of Pseudomonas aeruginosa belong to the variant of regular bacteria, often biofilm associated, which show tremendous antibiotic tolerance in the host tissue. Superiority in SCV infection is often linked due to its complex biofilm structure. Thus we aimed to explore the structural organization of a persister variety of Pseudomonas aeruginosa biofilm. Methods– P. aeruginosa (PAO1) and its SCV ΔwspF were studied. Scanning transmission electron microscopy (STEM) and STEM tomography were used to explore the three dimensional structure of PAO1 and ΔwspF biofilm. We performed Energy Dispersive X-ray Spectroscopy (EDS) for elemental analysis of different biofilm layers of ΔwspF. Besides, scanning electron microscopy enabled visualization of extracellular polymeric substance, extracellular DNA (eDNA) and biofilm thickness. Results– Both PAO1 and ΔwspF displayed presence of thread like structure of eDNA (DNAse sensitive) and extracellular vesicles like structures as visualized through STEM tomography. ΔwspF was thicker than that of PAO1. Ultrastructure of ΔwspF biofilm revealed more electron scattered bacteria in apical layers, which appeared white in dark field STEM images. Bacteria at the surface-anchoring basal layer appeared dark. Contrast differences, as determined by EDS, between apical and basal layers of bacteria was attributed to differences in mass. Apical white bacteria had more mass and were heavier than the basal dark bacteria. Elemental analysis showed more abundance of nitrogen and oxygen in the apical layer. Thus the basal layers were hypoxic and metabolically silent. In both PAO1 and ΔwspF biofilm, bacteria were most compact at the base having abundance of cell debris. Conclusions– Insights into the ultrastructure of bacterial biofilm is likely to provide cue on their pathogenicity and should prove to be helpful in developing countermeasure strategies.
P.IB03.
BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that frequently infects wounds, impairing their healing. We tested hypothesis that gene expression changes of P. aeruginosa as it adapts to the wound at early or late times (i.e., biofilm formation) are required to induce wound inflammation and impair healing, and that knocking out the genes that greatly change expression can cripple the bacterium’s pathogenicity. METHODS: To evaluate gene expression changes during infection, we inoculated P. aeruginosa into full-thickness dermal-excision wounds (rabbit ear model), and then used RNA-sequencing to generate the bacterium’s transcriptome profiles. To study global gene expression changes at early times of infection, the full-thickness wounds were infected on post-wounding day 3 with planktonic P. aeruginosa (106 CFU), and the bacteria were then harvested after 0, 2, 6, and 24 hrs. To study late-stage infection, the wounds were treated with topical antibiotics at 24 hrs post-infection, to kill planktonic cells, which resulted in biofilm-predominant infection; then the bacteria were harvested after 5 and 9 days RESULTS: During early infection, Pseudomonas up-regulated genes enriched most into Clusters of Orthologous Groups (COGs) categories ‘cell motility’, ‘inorganic ion transport and metabolism’, and ‘secretion and vesicular transport’. In contrast, during late-stage infection, three COG categories stood out as being most up-regulated: ‘defense mechanisms’, ‘secondary metabolites biosynthesis’, and ‘carbohydrate transport and catabolism’. Our transcriptomes of Pseudomonas infecting rabbit excision-wounds were also compared with previously published transcriptomes of Pseudomonas infecting the wounds of other species. CONCLUSIONS: Our P.aeruginosa gene expression data were more like that of human burn wounds than of that of mouse burn or excision wounds. Our ongoing studies use gene-knockouts to test for specific P. aeruginosa genes required for wound pathogenicity.
P.IB04.
Background: Over 400 soldiers evacuated from combat with craniomaxillofacial injuries suffered from burns from 2001-2011. Pseudomonas aeruginosa infection is the highest concern in burn care, due to its virulence, antimicrobial resistance, and propensity to form biofilms. Methods: In this study, we characterized P. aeruginosa biofilm formation in a deep partial-thickness burn wound model in rats. Deep partial-thickness burn wounds, ~10% of the total body surface area, were created in anesthetized male Sprague-Dawley rats (350-450g; n=84) using a modified Walker-Mason scald model. Immediately post-burn, 100μl of a clinical strain of P. aeruginosa, strain 1244 (1×103, 1×104, or 1×105 cells/wound) in PBS was spread over the burn surface. At 1, 3, 7, and 11 days post-burning, animals were euthanized and tissue was collected for CFU counts, biofilm gene expression, and scanning electron microscopy (SEM). Results: P. aeruginosa developed robust wound infections, plateauing at ~1×109CFU/g burn tissue within 7 days. Expression of alginate genes and other biofilm genes indicated formation of a mature P. aeruginosa biofilm within the burn. Additionally, SEM showed P. aeruginosa invaded 200-300µm into the burn eschar. Conclusion: The development of P. aeruginosa biofilms within deep partial-thickness burn wounds has not been demonstrated experimentally until now. This model is valuable for testing anti-biofilm agents in vivo to advance burn care. Disclaimer: The opinions or assertions contained herein are the private views of the author and not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
P.IB05.
Background: Bacterial infection accounts for 75% of fatalities in patients with a burn greater than 40% of their total body surface area (TBSA). In this study, we characterized the host response to Pseudomonas aeruginosa biofilm infection within deep partial-thickness burn wounds using a scald rat burn model. Methods: Deep partial-thickness burn wounds (~10% TBSA) were created in anesthetized male Sprague-Dawley rats using a modified Walker-Mason scald model (n=84). Immediately post-burn, the wound was inoculated with a clinical strain (strain 1244) of P. aeruginosa (1×103, 1×104, and 1×105 cells/wound). At 1, 3, 7, and 11 days post-burning, animals were euthanized and samples collected for blood and serum analysis, histology, and myeloperoxidase activity of the burn wound. Results: P. aeruginosa developed robust infections within the burn wound, plateauing at ~1×109 CFU/g of burn tissue within 7 days. Blood analysis revealed elevated monocytes and neutrophils, coupled with corresponding pro-inflammatory cytokines in the serum. H&E staining of the burn eschar showed massive influx of inflammatory cells into the burn wound, correlating with inoculum. Myeloperoxidase activity was significantly elevated in inoculated groups by day 11. Conclusions: A dynamic host response was seen in a novel model of P. aeruginosa biofilm infection of deep partial-thickness burns. These studies may provide insights into improved treatment methods for burn care. Disclaimer: The opinions or assertions contained herein are the private views of the author and not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
P.IB06.
Background: Bacteria can form a biofilm in the dressing of a wound. This allows the bacteria to continually reseed the wound and overwhelm the host defense. This study is on the use of a dressing material that blocks biofilm formation in the dressing. Methods: A dressing was prepared that contained an organo-selenium (OS) coating which can catalyze superoxide formation in the dressing. This dressing was placed upon a wound on the back of a mouse and then different bacteria were injected below the dressing. Staphylococcus aureus and Pseudomonas aeruginosa were tested. After five days of growth, the amounts of bacteria growing in the bandage and in the wound were determined by a colony forming unit (CFU) assay. The amount of biofilm in the bandage was also determined by Confocal Laser Scanning Microscopy (CLSM). Results: Using colony forming unit (CFU) assays, over 7 logs of inhibition (100%) was found for both of the bacterial strains on the material of the OS coated wound dressing when compared with the control (untreated) dressing. CLSM along with IVIS Spectrum In Vivo Imaging were used to confirm the CFU results. What was surprising was that in the tissue under the OS dressing, the results of the CFU assay again showed a 7-8 log (100%)reduction in bacteria, versus that of the control covered wound. All experiments were done at least in triplicate. Conclusion: The OS dressing inhibits biofilm formation in both the bandage and in the wound, while control dressing showed biofilm in the bandage and the wound. Since the organo-selenium coating does not leave the bandage, this implies that a bandage can serve as a reservoir for biofilm formation in a wound. Thus, a bandage that does not inhibit biofilm formation in the bandage, makes it harder for the wound to resist biofilm formation.
P.IB07.
Background
This study sought to understand how ChloraPrep induced changes in the skin microbiome may be related to surgical site infections.
Methods
Surgical site skin swabs were collected from consenting women 18 years or older undergoing breast reconstructive surgery over a 2-month period at Yale New Haven Hospital. Swabs were obtained immediately prior to and approximately 1 minute after skin sterilization with ChloraPrep. DNA was extracted using the Invitrogen Purelink Genomic DNA kit. PCR amplification of bacterial DNA was done using primers targeted to the 16S V1-V3 hypervariable regions. Genomic sequencing was performed with the Illumina MiSeq platform. A Wilcoxon Signed-Rank test was used to compare pre- and post-surgical preparation skin microbiome composition.
Results
36 samples (18 pre-prep and 18 post-prep) from 9 patients were sequenced. On average, 20 operational taxonomic units (SD±7.4) were found within each sample. In each pre-prep and post-prep sample, Streptococcus species comprised an average of 1.5% (SD±0.1) and 3.7% (SD±3.8) of the skin microbiome, respectively, and Enterobacteriaceae genera comprised an average of 4.2% (SD±5.7) and 14.1% (SD±12.6) of the skin microbiome, respectively. Post-prep proportions were statistically significantly higher than pre-prep proportions for Streptococcus species (S= 56, p=0.0063) and Enterobacteriaceae genera (S=79.5, p<0.0001). There was no statistically significant pre- and post-prep difference in the proportion of Staphylococcus and Corynebacterium species, which are also known to be components of normal skin flora.
Conclusions
ChloraPrep appears to have differential effects on certain microorganisms, leading to an increase in the proportion of the overall skin microbiome comprised by Streptococcus species and Enterobacteriaceae genera, both of which have been implicated in surgical site infections. Appropriate perioperative antibiotic dosing should be employed to potentially decrease the risk of such infections.
P.IB08.
Wound infections increase patient, economic and clinical burdens of care. International consolidated wound infection guideline (ICWIG) developers identified and content validated evidence-based ICWIG recommendations to harmonize multidisciplinary team practice, encourage timely referral and improve the consistency of care across specialties and settings. Purpose: Describe needs for wound infection education and research based on content validity and standardized levels of evidence supporting ICWIG recommendations. Methods: Nineteen volunteer wound experts used structured literature searches of PUBMED, Cochrane and CINAHL literature from 1986 through 2013 to develop evidence-based wound infection recommendations. Forty-two independent wound expert respondents to an online survey used judgment quantification to rate each recommendation as relevant (3-4 on a 1-4 rating scale) or conferring more benefit than harm (1 on a 0-1 rating scale). A recommendation was content validated as relevant if at least 75% of respondents rated it 3-4. Results: Most (88.8%) recommendations were rated relevant and beneficial to patients (summarized in checklist format). Twenty recommendations were rated irrelevant and harmful by most respondents. Recommendations with high content validity and low evidence (requiring more research) or with low content validity and ample research (opportunities for education) are summarized. Conclusion: Relevant, safe recommendations inform team wound infection management across specialties and settings, while highlighting research and education opportunities.
P.IB09.
BACKGROUND: Annual cost of treatment of chronic and burn wounds exceeds $25 billion in the U.S. Biofilms are a key factor in delayed healing in chronic wounds. Here we show that antibiofilm gallium (Ga3+) and antimicrobial silver (Ag+) ions work synergistically at non-cytotoxic concentrations to kill bacteria encased in biofilm. Ga3+ sensitizes bacteria in biofilm by substituting its Fe3+ uptake and Ag+ readily kills the sensitized bacteria.
METHODS: Biofilms containing 108 CFU of P. aeruginosa were established on a biologic dressing over 48h and rinsed with saline to remove planktonic bacteria. Moist biofilm samples were treated with dissolvable polymeric dressings which were fabricated as a composite of a 200 nm thick polyelectrolyte multilayer (PEM) nanofilm supported on a dissolvable 20 μm thick polyvinyl alcohol (PVA) layer. PVA layer dissolved leaving the nanofilm in intimate contact with biofilm where it released Ga3+ and Ag+ ions in a sustained manner in microenvironment of the biofilm.
RESULTS: 4Log10 CFU reduction and >90% biofilm mass dispersal was achieved from non-cytotoxic concentration of Ga3+ (4 μg/cm2) and Ag+ (10 μg/cm2), when these ions were delivered through a 200 nm thick PEM nanofilm. In contrast, only 1 Log10 CFU reduction was seen when (a) Ga3+ was incorporated in the PVA layer even at a level 20x of that in nanofilm, or (b) Ga3+ and Ag+ were formulated as topical solutions. This demonstrates that simultaneous and sustained release of Ga3+ and Ag+ ions in intimate contact with biofilm is required for effective antibiofilm activity. Furthermore, commercial silver-based antimicrobial dressings were not only ineffective against biofilm but also cytotoxic per MTT cytotoxicity assay.
CONCLUSIONS: Pairing of silver and gallium in a PEM nanofilm is suitable for assessment in the treatment of biofilms in chronic wounds.
P.II01.
The purpose of this study was to identify the mechanism of action (MOA) of ProgenaMartix™ which is composed of keratin and keratin associated protein (KAP) matrices in accelerating wound healing in chronic and acute wounds. Preliminary evidence in diabetic mice and acute wounds in porcine studies indicate that the wounds respond well immediately with these advanced tissue matrices but the mechanism of action (MOA) is poorly understood. We measured human neutrophil elastase activity in vitro by fluorescence resonance energy transfer (FRET) assays and in vivo using a commercially available point of care diagnostic to determine the elevated levels of host proteases. We also measured cytokine levels in the porcine model and neutrophil cell viability and the mobilization of macrophages in a DB mouse model system. Our findings indicate the KAPs in ProgenaMatrix™ are actively suppressing the protease activity of neutrophil elastase which is concomitant with the reduction of periwound redness. Furthermore, ProgenaMatrix™ enriched in KAPs seems to dramatically improve the viability of neutrophils and enhances the mobilization of type I and II macrophages.
P.II02.
BACKGROUND Macrophages exhibit functional heterogeneity and differentiate (M1 or M2 polarization) as a result of their microenvironment. The M1 phenotype is characterized by a pro-inflammatory state. The M2 phenotype is characterized by anti-imflammatory functions including tissue remodeling. Diabetic wounds demonstrate poor wound healing due to a variety of reasons, including dysregulated inflammation. We hypothesize that prolonged polarization of macrophages towards a M1 phenotype is involved in the persistent inflammatory response and poor wound healing seen in diabetic wounds.
METHODS A murine in-vitro macrophage model was used in this study. RAW 264.7 as well as murine bone marrow derived macrophages (BMM) were grown in cell culture, were treated with IFN/LPS or IL-4 to induce polarization (in RAW cells), and were harvested for PCR and Reactive Oxygen Species (ROS) Analysis. Statistical comparisons were made using Student’s t-test, with a p<0.05 considered statistically significant. RESULTS In the RAW cell model, cells stimulated with IFN and LPS demonstrated a M1 phenotype with increased expression of TNF, IL-6, and STAT-1 (p<0.05). Cells stimulated with IL-4 showed increased expression of M2 phenotypic markers such as MRC and IRF4 (p<0.05). The IFN/LPS stimulated cells also exhibited increased ROS production (p<0.05). Using this information for characterization of the macrophage phenotypes, BMM from heterozygote and diabetic mice were examined. Diabetic macrophages demonstrated increased expression of IL-6 and decreased expression of MRC, IRF4, and STAT-6 compared to heterozygote macrophages, suggestive of a M1 phenotype.
CONCLUSIONS Diabetic macrophages demonstrate a proinflammatory (M1) phenotype compared to non-diabetic macrophages which demonstrate a regenerative (M2) phenotype. This likely contributes to the poor wound healing noted in diabetic wounds and offers a future therapeutic target.
P.NOV01.
BACKGROUND Our lab has previously shown that local delivery using cerium oxide nanoparticles conjugated to miR-146a (CNP-miR146a) improves wound healing in a murine model through decreased inflammation and improved angiogenesis. The purpose of this study is to evaluate the effect of CNP-miR146a on wound closure in diabetic pig. METHODS Diabetes was induced in a domestic white pig with a single dose of streptozotocin and confirmed with blood glucose measurements 4 weeks after administration. Two sets of five full thickness excisional wounds (1inX1in) were created on the back of pigs with a depth of approximately 2cm to reach the subcutaneous fat in all wounds. Half the wounds were treated with CNP-miR146a while the other half were treated with PBS (control) only. All wounds were digitally photographed in the presence of a standard reference ruler. Wound area was calculated using the ImageJ software. RESULTS Five of ten wounds on the right side were treated with CNP-miR146a at day 0 as described. Five wounds were treated with PBS as control. Digital imaging of wound was performed on day 0, 3, 7, 10, 14 for measurement of wound area. The wound area as measured by digital planimetry was plotted graphically. Data expressed as Mean in square centimeters. CONCLUSIONS Our results indicate accelerated wound healing of CNP-miR146a treated diabetic pig wounds at day 10 and 14 post healing based on decreased wound size. This porcine model better represents human diabetic wounds and provides support for CNP-miR146a as a possible therapeutic to improve wound healing.
P.NOV02.
Electric field (EF) stimulation of tissue repair is a promising strategy for treating non-healing diabetic ulcers, but progress in developing EF-based therapies is hindered by a poor mechanistic understanding of the EF interactions with cells and tissues. Our recent theoretical and experimental studies reported discovery of a novel EF modality for non-contact stimulation of vascular cells that results in activation of angiogenic signaling under normal and diabetic conditions. This study tests the hypothesis that the novel wireless electrotherapy based on the recently discovered EF modality will improve wound healing in the porcine model, resulting in enhanced vascularization and faster healing, as compared to non-stimulated wounds. Full-thickness wounds (n=16/animal) were created on the dorsal side of adult Yorkshire pigs (n=3 animals) and filled with saline or hydrogel (n=8/group). Half of the wounds were treated using a custom set-up that delivered non-thermal, non-contact EF therapy for 1 hr/day, 5 days/wk, for 2 weeks. Effects of EF, including wound closure, morphology, granulation tissue area, wound vascularization and collagen deposition were quantified using blind assessment of wound images or paraffin-embedded and stained tissue sections. EF therapy resulted in improved healing phenotype, significantly enhanced wound re-epithelization at day 5 and increased granulation tissue formation and vascularization, with evidence of improved collagen organization and a reduced fibrosis, as compared to no-EF controls. Studies are ongoing to quantify wound strength and inflammation. In conclusion, these results demonstrate that the novel EF-based technology is promising for regenerative wound healing therapies to help accelerate closure of chronic wounds and decrease amputations in patients with diabetic and other chronic ulcers.
P.NOV03.
Background: Processed microvascular tissue (PMVT) human structural allograft is derived from lyophilized human tissue containing microcirculatory cellular components. Since PMVT serves as a source of ECM, growth factors, cytokines, and chemokines modulating angiogenesis, inflammation, apoptosis, and endogenous cell recruitment, we hypothesized its application will accelerate wound regeneration in a validated pressure ulcer (PU) model developed in C57BL/6 mice using two 24hr cycles of skin ischemia/reperfusion created by placement and removal of external magnets. Methods: Two identical PU injuries (n=50 female mice) were treated with (a) topical particulate PMVT, (b) injected rehydrated PMVT, or (c) saline control injection, and assessed daily for closure rates, scab formation/removal, and temperature. A baseline control cohort (n=5) was euthanized at day 0 and treatment group cohorts (n=5) were sacrificed at 3, 7, or 14 days post-injury. The PU injuries were collagenase-digested for flow cytometric analysis of inflammatory, reparative, and stem cell frequencies; and analyzed by H&E histology and immunofluorescence. Results: PVMT accelerated wound closure; most notably, topical PMVT significantly increased mean closure from d5 (13% vs. -9%) through d13 (92% vs. 38%) compared to PBS controls (p<0.05). PMVT also hastened scab formation/removal; significantly accelerated disappearance of inflammatory myeloid (CD11b+) cells while upregulating α-smooth muscle actin, VEGF-A, and PLGF; and raised skin temperature surrounding the PU site, consistent with increased blood flow. Conclusions: These results indicate that PMVT has potential as an advanced treatment for restoring normal tissue function in ischemic wounds and merits clinical study.
P.NOV04.
Microtubules (MT) are intracellular polymers that provide the cell structural rigidity and act as important regulators of many cellular processes, including cell migration. The dynamicity and function of the MT cytoskeleton are determined in large part by its regulatory proteins, including the recently discovered enzyme Fidgetin-like 2 (FL2). FL2 severs and depolymerizes microtubules specifically at the leading edge of polarized cells. The localized nature of FL2 activity enables it to significantly impact specific cellular functions, namely cell migration. In vitro, down-regulation of FL2 expression with siRNA results in a more than two-fold increase in cell migration rate, with a noted enhancement of directional movement. To investigate these findings in vivo, we utilized a platform consisting of concentrated poloxamer (CP) (PluroGel) to deliver collagen microparticles containing siRNA to the site of injury. Topical application of FL2-CP-siRNA to murine animal models of full-thickness excision wounds and full-thickness burn wounds resulted in significant improvements in both the clinical and histological characteristics of the wound zone compared to controls. Wound healing occurred more rapidly and with high fidelity, resulting in properly organized collagen substructure and decreased scarring. Taken together, these findings indicate that FL2 is a promising target for continued therapeutic development in wound healing and that concentrated poloxamers offer a promising delivery platform.
P.NOV05.
Background: Various guidelines for wound assessment have been suggested but none of them are widely accepted as a standardized method for various wounds. Therefore, there is a need for developing a standardized and practical wound assessment tool, which is easily applicable to a variety of wounds. Here, we propose a D.I.R.E.C.T. coding system to guide healthcare professionals involved in wound care as a simple but efficient wound evaluation tool. Methods: Eleven Korean specialists including plastic surgeons and wound, ostomy, and continence nurses participated in the development the D.I.R.E.C.T. coding system as a practical wound assessment tool and a guide to establish treatment regime. Results: The D.I.R.E.C.T. coding system classifies all types of wounds on the basis of the Debridement of necrosis, Infection control, Revascularization, Exudate control, Chronicity, and Top surface, which are abbreviated by the acronym ‘D.I.R.E.C.T.’ The system has several superior points compared to the other systems. First, the system is versatile and thus applicable to the wounds with various etiologies and occurring locations. Second, it provides detailed grading based on the wound status, enabling clinicians to track the healing progression or regression easily. Third, the system covers critical physiological points important for wound healing while the other systems focus on limited area. Finally, the theoretical basis of the system is easy and straightforward that makes its application user-friendly. Conclusions: As a practical wound assessment system, the D.I.R.E.C.T. coding will enable healthcare professionals responsible for wound care to grade and assess all kinds of wound with simplicity and systemicity. Because it is also very easy to learn and apply to real practice, we think it may be universal wound grading tool.
| D | Debridement |
| I | Ischemia |
| R | Revascularization |
| E | Exudate |
| C | Chronicity |
| T | Top surface |
P.OX01.
BACKGROUND: Dyspigmented hypertrophic scar following full-thickness wounds and burn injury has a negative psychological impact on patients. Despite a thorough understanding of normal pigment synthesis cascades, the cellular and molecular mechanisms leading to this specific dyspigmentation type remain unknown. The multitude of cell types and molecules involved in wound healing, that simultaneously induce variations in pigment synthesis, exacerbate the difficulty of investigating this health problem. METHODS: Dyspigmented scars were produced in a red Duroc pig scar model. Differences in gene expression between hyper- and hypo-pigmented scar biopsy samples were investigated using genome-wide microarrays. RESULTS: Differences in the abundance of scores of transcripts clearly separated hyper- and hypopigmented scars in both principal component and hierarchical clustering analyses. The list of top regulated genes included many pleiotropic genes potentially affecting melanin synthesis directly or indirectly. Pathway enrichment analysis of significantly (p-value < 0.01, LEF > 1.3) differentially regulated genes in hyper- and hypo-pigmented scars identified 10 pathways more significantly present in hypo- relative to hyper-pigmented scars (p-value difference < 1 log and > 2x in hyper-pigmented). Six out of these pathways, namely, the mitochondrial dysfunction, arginine degradation I and VI, nitric oxide signaling in the cardiovascular system, urea cycle, and the citrulline biosynthesis are central to maintaining redox homeostasis. Genes underlying the identification of several of these pathways include the mitochondrial NADH dehydrogenase II (MT-ND2), arginase 1 (ARG1), and ornithine carbamoyltransferase (OTC). CONCLUSIONS: Results of this work identify mechanisms involved in dyspigmentation pathogenesis and provide target candidates for prophylactic treatment and therapy.
P.OX02.
Background: Topical lidocaine is a traditional local anesthetic which improves the survival of random pattern skin flap in rats. Fractionated ablative carbon dioxide laser was also introduced as a new drug delivery-enhancement technique. The potential effect of fractionated ablative carbon dioxide laser and topical lidocaine spray (Xylocaine) on random pattern skin flap survival was investigated.
Methods: Forty eight male 2-month-old Sprague-Dawley rats were randomly divided into four groups, a control group, lidocaine group, fractionated ablative carbon dioxide laser group, and fractionated ablative carbon dioxide laser + lidocaine group (n=12 in each group). In all groups, caudally based McFarlane type random-pattern skin flaps were elevated. On postoperative day 7, the areas of necrotic flap were measured and percentages of flap survival were calculated. The number of vessels and neutrophil count was evaluated. Anti-rat VEGF antibody and CD31 antibody activity were measured.
Results: The survival area of the flap in fractionated ablative carbon dioxide laser + lidocaine group was significantly higher than that in the other groups. Mean neutrophil count in fractionated ablative carbon dioxide laser + lidocaine group was lower significantly than that of other groups. No statistically significance were observed between the groups in terms of the number of vessels. Anti-rat VEGF antibody and CD31 antibody activity was significantly higher in fractionated ablative carbon dioxide laser + lidocaine group than that in the other groups.
Conclusions: This study showed the positive effects of fractionated ablative carbon dioxide laser in the enhancement of random-pattern skin flap survival in rats with lidocaine. Fractionated ablative carbon dioxide laser treatment with lidocaine spray can represent a new therapeutic approach to enhance flap viability.
M1.08.
Background: Satellite cells are capable of completely replacing muscle fibers after injury, by producing a large number of myoblasts. However, the contribution of individual satellite cells is poorly understood in vivo.
Methods: To assess the relative contribution of individual satellite cells, we developed a mouse with inducible fluorescence in satellite cells [1]. After tamoxifen induction, the mice express one of four fluorescent colors in Pax7+ cells, while the remaining muscle tissue is non-fluorescent.
Results: Sixteen days after a cardiotoxin injury in healthy 6-month-old mice, up to ninety percent of muscle fibers in the injured area were fluorescent, indicating that fluorescent satellite cells contributed to regeneration of almost all fibers. Most strikingly, the four fluorescent colors of the confetti mouse appeared in regional patches, rather than uniformly distributed across the fibers. Using computational image analysis [2] and stochastic modeling, we analyzed the distribution of observed isochromatic patches, and concluded that the observed color patches were not statistically compatible with satellite cells contributing myonuclei to only a single fiber. Instead we infer that roughly a third of the fluorescent satellite cells must have contributed myonuclei toward multiple fibers.
Conclusions: These results have implications for our understanding of the muscle regeneration process, not only in normal circumstances, but also in pathologies such as muscular dystrophy, sarcopenia, and chronic ulcers. Furthermore, this confetti mouse will be useful for further studies of muscle regeneration, particularly for interrogating the ability of candidate interventions [3-4] to alter the regenerative function of endogenous satellite cells.
[1] Tucker-Kellogg et al., Wound Repair and Regeneration 24(2):A26, 2016.
[2] Nguyen et al., BMC Systems Biology 10(5):124, 2016.
[3] Heemskerk et al., Annals of the New York Academy of Sciences 1175(1):71, 2009.
[4] Jagannathan et al., Journal of Biomechanics 49(8):1311, 2016.
P.REG01.
Background: Volumetric muscle loss can result in devastating functional deficits. Decellularized extracellular matrix has been a promising candidate for muscle regeneration, however functionalization requires motor nerve innervation. We examined the myogenic and neurogenic effects through direct neurotization of decellularized muscle matrix. Methods: Bilateral 1 cm latissimus dorsi defects were surgically created in 12 Sprague-Dawley rats. 8 defects were left alone, 8 were implanted with decellularized muscle matrix, and 8 were implanted with matrix then neurotized. 3-5 peripheral motor nerves innervating the panniculus carnosus were transposed onto the ipsilateral decellularized implant based on anatomic feasibility. Tissue was harvested on post-operative days 90 and 180. Samples were assessed through gross appearance, immunohistochemistry, and western blot analysis for myogenesis, inflammation, neovascularity, and neural ingrowth. Results: Both neurotized and non-neurotized matrices appeared well integrated within the surrounding muscle, while the defect alone healed with a thin layer of fibrous connective tissue. Neurotized matrix demonstrated greater infiltration by mature myocytes compared to non-neurotized controls based on MHC staining. CD-31 staining revealed similar levels of microvascular networks within neurotized and non-neurotized matrices, both greater than defect alone. Cholinergic neural tissue ingrowth was only observed in neurotized matrix. Direct stimulation of the matrix implants with a peripheral nerve stimulator demonstrated contraction of both neurotized and non-neurotized matrices, however a difference could not be grossly appreciated. Conclusion: Direct neurotization of decellularized muscle matrix in a model of volumetric muscle loss leads to more robust myogenesis and neurogenesis within the implant as compared to non-neurotized matrix and defect alone. Further studies are needed to evaluate functional differences in more detail.
P.REG02.
The repair process of wounded tissue involves the coordinated activities of muscle precursor cells (MPCs) in response to local and systemic signals. Following tissue injury, the microenvironment, which is characterized by excessive production of reactive oxygen species (ROS), is attenuated. Here we studied the efficacy of an antioxidant, N-Acetyl-L-Cysteine (NAC), in vitro and in vivo to ameliorate the damage that results from trauma so that repair and regeneration of wounded muscle tissue is enhanced and functionality is improved. MPCs were isolated from rat tibialis anterior muscles and exposed to increasing concentrations of H2O2 in the presence or absence of NAC for the in vitro assays. MPC proliferation, differentiation, and fusion into myotubes was assessed by IncuCyte microscopy, live/dead viability assays, MTS proliferation assays, and myosin heavy chain immunohistochemistry. CellROX reagent was used to detect ROS. For in vivo assays, adult female Lewis rats were subjected to compartment syndrome injury by applying 120-140 mmHg compression for 3hrs. Half of the injured rats received NAC injected intramuscularly and the other half received equal volume of PBS at 24, 48, and 72hrs after injury. Myogenesis, angiogenesis, fibrosis, and function were determined using RT-PCR, western blot, Masson’s trichrome stain, and contractile force of the TA muscle as an isometric force (Hz), respectively In vitro, NAC administration resulted in a decrease in oxidative stress levels that was associated with significant survival benefit during oxidative damage. When used in vivo, NAC administration also showed decrease in tissue fibrosis, increase in myogenesis, angiogenesis and muscle function. These results suggest that treatment of skeletal muscle injuries with antioxidants may be a viable option for the prevention of long-term fibrosis and scar formation and improvement of recovery of muscle function.
P.REG03.
Background. It has been shown that epidermal stem cells that reside in the hair follicle (HF) bulge regions are important for wound healing. During the healing process these cells start differentiating and migrating to the wound bed thus contributing to wound closure. We have recently shown that wound healing using dermal grafts that contain HFs is comparable to split-thickness skin grafts (STSGs). Our analysis suggested that CD34 positive cells in the skin appendages contributed to wound closure by differentiating into basal keratinocytes during wound healing. The purpose of this study was to investigate HF cell behavior in a growth factor containing hyaluronic acid (HA) gel, use the gel as a vehicle to transplant HFs into rat full-thickness wounds and demonstrate that epidermal stem cells located in HF bulge regions can contribute to wound re-epithelialization. Methods. HFs were harvested from the rats by pulling the hair out by the root. 1.5% HA gel with defined keratinocyte medium was formulated and harvested rat HFs were cultured in the gel. Cell viability and proliferation were measured at days 1, 3 and 5. In addition, HFs in the gel were transplanted into rat full-thickness wounds. Wound closure and re-epithelialization were followed over time macroscopically and histologically and compared to STSG (n=6/time point/group). Results. The results indicated that when cultured in the HA gel with growth factors, cells started migrating from the HF to the gel, stayed viable and proliferated over time. In addition, it was shown that HFs in HA gel contributed to wound closure and re-epithelialization when transplanted to full-thickness wounds. Keratinocyte positive staining and wound re-epithelialization was observed on days 5 and 10 after transplantation. Conclusions. It is concluded that HA gel is a good vehicle for HF transplantation and this technique could potentially provide an autologous noninvasive alternative for STSG. This study was funded by the Wound Healing Foundation (Sponsored by Medline Industries).
P.REG04.
Background: Fetal wounds have been demonstrated to heal in a regenerative manner, however fetal extracellular matrix has not been well studied. We looked at the use of decellularized fetal soft tissue matrix for skeletal muscle regeneration. Methods: Composite soft tissue was harvested from the trunk of New Zealand White rabbit fetuses on gestational day 24. Bilateral 1 cm latissimus dorsi defects were surgically created in 12 rats. One defect was implanted with fetal decellularized matrix while the other was left untreated. Tissue was harvested on post-operative days 30 and 60. Samples were evaluated through gross appearance, immunohistochemistry, and western blot analysis for myogenesis, inflammation, neovascularity, and neural ingrowth within the explanted matrices and defects alone. Results: Scanning electron microscopy and picrosirius red staining revealed that fetal matrix is composed of a loose network of type III collagen reticular fibers. At the time of tissue harvest, the fetal matrix appeared well integrated within the surrounding native muscle as compared to defect alone, which healed with a thin layer of fibrous connective tissue. Fetal matrix played host to robust cell infiltration without evidence of immune rejection. Myosin heavy chain staining demonstrated substantial ingrowth of myocytes into the fetal matrix compared to defect alone. A dense microvascular network was appreciated within the implanted matrices. Furthermore, neuronal antibody staining revealed a time-dependent ingrowth of neural tissue into the fetal matrices as compared to the defects alone, which showed no evidence of ingrowth. Conclusion: Decellularized fetal soft tissue matrix is a promising scaffold for muscle regeneration, demonstrating significant myocyte proliferation and supporting microvascular as well as neural ingrowth.
M1.02.
Background: Mesenchymal stem cell (MSC) expansion is crucial to obtain sufficient cell numbers for tissue repair therapies. Traditional culture expansion on stiff surfaces reduces regenerative potential and jeopardizes therapeutic outcomes by inducing scar features in MSCs. In contrast, ‘priming’ on skin-soft culture surfaces preserves regenerative MSCs that improve the healing of rat wounds (Li et al. Nature Materials 2017). We hypothesize that mechanical priming in culture will alter secretory functions and thereby paracrine actions of MSCs in the wound environment. Methods: Human umbilical cord perivascular cells (HUCPVCs) from three donors were soft- or stiff-primed. Mechanical priming was verified using qRT-PCR and Western blotting for markers of stromal cell activation. Conditioned medium from primed HUCPVCs was analyzed using cytokine arrays. Primed HUCPVCs were transplanted to splinted rat full thickness wounds, traced for grafting success, and wound tissue was analyzed for cell and matrix composition. Results: Soft-primed HUCPVCs exhibited faster doubling times (3.0±0.4 vs 4.7±0.4 days) and reduced gene and protein expression of scar markers α-SMA and ED-A fibronectin compared to stiff-primed HUCPVCs. Of 79 cytokines detected in conditioned medium, 48 were down-regulated and 4 were up-regulated in all soft- versus stiff-primed HUCPVCs. Of 10 most differentially expressed cytokines, 7 were related to inflammation and 3 to cell cycle regulation. Soft- and stiff-primed HUCPVCs produced distinct healing outcomes compared to vehicle controls in a rat model of exacerbated wound healing. Conclusions: Soft-priming enhances the regenerative capacity of human MSCs by preserving cell proliferation, suppressing scarring features, and creating distinct paracrine profiles that persist in a wound environment.
P.ST01.
The aim of this study was to investigate the effect of human bone marrow mesenchymal stem cell-conditioned medium (hBM-MSC-CM) on the microbial flora and tensiometrical properties of an infected experimental model of type 1 diabetes mellitus (TIDM). TI DM rats were induced by streptozotocin (STZ) and held for thirty days. Under general anesthesia and aseptic conditions, one full–thickness excision wound was made on the back of each rat. Rats were divided into two groups. The experimental group (N=6) received hBM-MSCs-CM 4 times while the control group (N=6) received regular medium only. All wounds were infected with methicillin-resistant staphylococcal aureus (MRSA) immediately after surgery. On days 4, 7, and 15 microbiological examinations were performed. We counted the numbers of microbes per sample, as colony-forming units (CFUs). The animals were euthanized at day 15 and standardized rectangular skin specimens were extracted across each wound and the adjacent normal skin. The specimens were mounted in a material testing machine. Data were analyzed by student t-test. Our results showed that hBM-MSC-CM significantly increased tensiomerical properties of repaired wounds and significantly decreased colony-forming units (CFUs) compared to control wounds. In conclusion, application of hBM-MSC-CM accelerated wound healing process in an MRSA infected cutaneous wound model in TI DM rats, as demonstrated by the significant increase in tensiometrical properties, and antibacterial activity.
P.ST02.
THE EFFECTS OF COMBINED PHOTOBIOMODULATION AND HUMAN BONE MARROW MESENCHYMAL STEM CELL-CONDITIONED MEDIUM ON WOUND HEALING IN DIABETIC RATS Mohammad Bayat, Ph D,1-3; Ramin Pouriran, 1; Sufan Chien, MD, 2,3 1.Department of Biology and Anatomical sciences, School of Medicine, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2. Noveratech LLC of Louisville, KY. 3. Price Institute of Surgical Research, Hiram C Polk Jr. MD Department of Surgery, University of Louisville, Louisville, KY, USA We investigate the effects of Photobiomodulation and human bone marrow(hBM)-MSC-CM alone or in combination on histological, and stereological parameters, and the quantitative real-time polymerase chain reaction(RT-PCR) for the analysis of bFGF, HIF-1alfa, and SDF-1 α in the repairing wound in a chemical induced type one diabetes mellitus (TIDM) in rats. hBM-MSCs were isolated, and expanded. hBM-MSC -CM(CM) was prepared by culturing hBM-MSCs . TIDM was induced in 24 rats. For all animals, two incisions were made on their backs. Rats were divided into four groups . First group were considered as the control group. The second group received LASER; the third group received CM two times, the fourth group received LASER+ CM. On days 4, 7, and 15 skin samples were extracted for histology and stereology, and RT-PCR analyses of bFGF, HIF-1 α, and SDF-1 α gene expression.We observed that in proximal and distal wounds all treatment groups showed significantly better stereological results compared to control group. And in the most cases results of CM+Laser group was significantly better than other treatment groups. In addition stereological parameters respond ositively to CM, Laser, and CM+Laser treatments at inflammation, proliferation, and remodeling phases of wound healing process. In the most cases results of RT-PCR in both CM +Laser, and Laser groups were significantly better than CM and control groups.
P.LB01.
BACKGROUND Obesity is a prominent global health issue, but its impeded wound healing remain unresolved. As epidermal stem cells (EpSC) and adipocytes are essentials in the structural, metabolic and healing process of skin, their interactions may influence wound healing in the obesity. METHODS A rat model of diet-induced obesity was established with groups of sham-injury, injury without transplantation (Inju) and injury plus EpSC transplantation, then a 6-mm diameter full-thickness excision was developed on the dorsal skin. 1×10^5 epidermal basal stem cells isolated from neonatal mice skin and suspended in 30 μl 1×PBS were injected subcutaneously into each wound in EpSC group, as equivalent 1×PBS was injected in sham and Inju groups. Skin wounds and serum samples were harvested at 1, 3, 7 and 14 days post injury, and subjected to histological investigations and colorimetric detections. RESULTS At 1, 3 and 7 days post injury, promoted scar area, wound contraction, re-epithelialization, collagen deposition and adipocytes repopulation were found in EpSC group (P<0.05, vs Inju). Simultaneously, a “down then up” fluctuation of serum FABP5 and FABP4, two fatty acid binding protein subtypes secreted by epidermal cells and adipocytes, respectively, was investigated in EpSC group at 1 and 3 days post injury, while FABP5/FABP4 ratios in EpSC group were higher at 7 and 14 days post injury (P<0.05, vs sham or Inju). Moreover, altered glucose and lipid metabolism containing serum lactate/pyruvate ratios, NAD+/NADH ratios, insulin, triglycerides and total cholesterol were identified. CONCLUSIONS EpSC-promoted wound healing in the obesity associates with adipocytes, and the metabolic interaction will be a novel breakthrough for treating obesity wound.
P.LB02.
Background: The purpose of this study was to demonstrate the safety and reliability of combined preoperative angioplasty and free-flap transfer in patients with peripheral arterial occlusive disease (PAOD) by analyzing the surgical outcomes. Methods: Between September 2011 and October 2015, the patients who had undergone lower-extremity angiography and subsequent free-flap transfer were retrospectively reviewed. Data collected included demographics, perioperative data, and postoperative outcomes. The cases were divided into two groups; one group with microanastomosis performed on revascularized artery by balloon angioplasty and the other group performed on native artery. Multiple logistic regression model using propensity score and linear regression were computed to determine the association between preoperative angioplasty and the surgical outcomes. Results: A total of 62 lower limb reconstruction cases (19 angioplastied cases and 43 native cases) were included in the study. Complications occurred in 6 cases of the angioplastied group and in 11 cases of the control group. The overall limb salvage rate was 100 percent during the average follow-up of 35 months in the angioplastied group and that of non-angioplastied, control group was 97.7 percent during the average follow-up of 38 months. Preoperative angioplasty was not a significant predictor for increased complications and longer postoperative downtime in logistic and linear regression model, both in weighted and unweighted model. Conclusions: The combined approach of preoperative endovascular revascularization and free-flap transfer for limb salvage in PAOD patients can be performed safely and effectively with acceptable morbidity.
P.LB03.
BACKGROUND: The decellularized extracellular matrix biomaterial, Ovine Forestomach Matrix (OFM), is an established scaffold for use in wound healing and tissue repair indications. We have recently developed an antimicrobial variant of OFM containing ionic silver, termed OFM-Ag. This study sought to characterize the functional properties of OFM-Ag in relation to its use as an antimicrobial wound dressing material. METHODS: The antimicrobial effectiveness spectrum and wear time of OFM-Ag toward 11 microbial species encompassing clinically relevant bacteria, yeast and mold was determined using ISO20743 methodology. The cell compatibility of OFM-Ag was assessed via MEM elution cytotoxicity assay utilizing mammalian fibroblasts. Silver content and silver elution profile of OFM-Ag was quantified by atomic absorption spectroscopy, while retention of the native collagen architecture and matrix integrity was assessed via differential scanning calorimetry and scanning electron microscopy. RESULTS: OFM-Ag demonstrated >4 log10 reductions toward all species tested, indicating broad spectrum antimicrobial effectiveness. Furthermore, OFM-Ag retained this effectiveness over a period of 7-days, the maximum antimicrobial wear time period tested. OFM-Ag was determined to be non-toxic to mammalian cells. The dressing was characterized as containing 0.30% w/w silver and upon elution the silver remained bound to the matrix, retaining effective antimicrobial concentrations in the material over 7-days of elution. Silver functionalization had no detrimental effects toward the composition or integrity of the matrix, with calorimetric analysis showing preserved native collagen structure. CONCLUSIONS: The present work indicates OFM-Ag retains the intact extracellular matrix properties of OFM desirable for wound healing while providing sustained and potent broad spectrum antimicrobial effectiveness.
P.LB04.
Based on the established correlation of both wound duration and biofilm with wound chronicity, we set out to maintain and infect an acute wound in an attempt to develop a novel chronic wound model. Prior studies demonstrated a ~350% delay in healing of 5% Glutaraldehyde (GL) cross-linked wounds, which was correctable with surgical debridement. In the present study, we screened 3 organic cross-linkers for their effects on wound healing rate and bioburden. Twenty full-thickness 2cm dorsal wounds were created in two pigs. Eight wounds were treated with 5% GL and 8 were treated with D-ribose/3% hydrogen peroxide (R-HP). Eight wounds were treated with 1 g/mL N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) and 8 were treated with 11mM Genipin (GN). Four untreated wounds on each pig served as untreated controls. All wounds were inoculated with 106 CFU/mL Staphylococcus aureus, Pseudomonas aeruginosa and Fusibacterium. Wounds were covered with non-adherent dressings and healing was measured at day 1, 4, 7, 11 & 13. Wounds were biopsied and selectively cultured on days 1, 4 & 13. EDC-treated and GL wounds showed the greatest delay in wound healing, followed by GN. Treatment with R-HP did not result in healing rates different from untreated wounds. EDC-treated and GL wounds also showed the greatest bacteria, with total bioburden and Pseudomonas reaching >8log at days 4 and 13, while Staphylococcus showed 7log at day 4 with reduction to 5log at day 13. These data support the hypothesis that crosslinking and inoculation of the acute wound shows promise as a stable infected chronic wound model that may be useful for studies of debridement and other therapies.
P.LB05.
BACKGROUNDChronic wounds fail to heal when the overlapping phases of wound-healing become disarrayed, but many treatments only target a single phase. If wounds become recalcitrant, particularly diabetic ulcers, amputation rates and 5-year mortality escalate. We tested the use of a hyaluronic acid dressing after pre-application of a surfactant; both treatments are FDA-approved, but they have never been tested in combination. We aimed to investigate whether combination therapy targeting multiple phases of wound-healing could provide superior healing outcomes.
METHODSCombination dressing-surfactant therapy was administered once-weekly for 12 weeks to patients with wounds failing standard care and persisting ≥6 months. Volumetric analysis was performed to calculate interval changes in wound size (cm3) before, at initiation, and termination of therapy. Sizes pre- and post-combination therapy were compared by paired T-Test. Percent size reductions during standard care versus combination therapy were compared by Student’s T-Test.
RESULTSTotal 4 patients with 4 wounds: 2 diabetic ulcers, 2 venous ulcers. Wounds had average 1 year duration and had failed to heal under standard wound care regimens. Sizes were decreased post-combination therapy (p<0.05), and percent size reductions were greater (p<0.05) compared to the preceding 6-month standard care interval. No wounds closed completely. No adverse events occurred.
CONCLUSIONSIn patients with recalcitrant chronic wounds, combination therapies targeting different phases of wound-healing may synergize to provide a novel stimulus to the wound-healing apparatus. This may explain the salutary effects observed in patients treated with surfactant targeting the external wound macroenvironment and a hyaluronic acid dressing targeting the internal wound microenvironment.
P.LB06.
PREGNANCY IMPROVES SKIN WOUND HEALING IN MURINE MODEL Fan Yang1, Haiying Pan2, Yan Cui2, *Aiping Lu2,3, *Johnny Huard2,3 1.Department of Traumatic Surgery, Tongji Hospital, HUST, Wuhan, China.2. Department of Orthopaedic Surgery, UTH, Houston, TX; 77054, USA3. Steadman Philippon Research Institute, Vail, CO. 81657, USAAbstract:Wound healing is a complex regenerative process involving various cell types and also requires a series of growth factors and hormones that act in concert to restore the integrity of the injured tissue. Many studies have shown that exposure to factors present in the serum of young mice restores the regenerative potency of aged cells. One idea is that pregnancy could represent a unique biological model of a naturally-shared circulatory system between young and old. We investigated the wound healing process in pregnant and non-pregnant mice via a laceration injury model. The mice were sacrificed at 7 and 14 days after injury, and hematoxylin/eosin and immunofluorescence staining were performed. We observed that the granulation tissue and layers of regenerated epithelial cells in wounded area were thicker in pregnant mice than in non-pregnant mice. Results from immunostaining of smooth muscle actin and CD31 indicated the appearance of greater numbers of myofibroblasts and blood vessels in wounded area in pregnant mice than in non-pregnant mice. Our results suggest that pregnant mice show superior wound healing compared to non-pregnant mice, suggesting that circulating factors present during pregnancy may play an important role in wound healing by enhancing the function of progenitor cells in the skin and increasing angiogenesis. Identifying rejuvenating factors within blood circulation during pregnancy could have potential for the development of novel therapies for wound healing.
P.LB07.
Introduction: Suboptimal wound healing affects millions of patients annually. We are interested in novel strategies to enhance the wound healing process in diabetic patients. We have previously shown that inhibiting Notch inhibits wound healing. Based upon our previous work, we propose that upregulation of Notch would increase rates of wound healing. JAG1 is a known activator of Notch. Therefore, we hypothesized that applying topical JAG1 to ex vivo excisional wounds on the backs of mice would result in increased Notch activity, and thus an increased wound healing rate as compared to untreated wounds.
Methods: Skin biopsies from 12-week old, healthy mice, (1-cm2 full-thickness) were cultured ex vivo. A 3-mm wound was created in the center of the skin biopsy. Topical application of JAG1 (10 nM) or vehicle (PBS) was applied daily. Digital photographs were taken and histological and protein analysis were performed. Wound area was calculated as a percent area of the original wound size. Statistical significance was defined as p<0.05 using the students’ t-test.
Results: Partial to complete re-epithelialization was seen in the wounded tissues over the experimental period in both the control & JAG1 treated groups. The mouse skin treated with topical JAG1 had an increased rate of wound closure when compared to wounds treated with PBS
Conclusions: JAG1 increases the rate of re-epithelialization of cutaneous wounds in an ex vivo murine wound-healing model, indicating that Notch signaling plays a crucial role in wound healing in mice. Based upon our findings, further study of Notch in wound healing should be conducted which may then lead to better therapeutics for the wound healing process in patients.
P.LB08.
Introduction Objective measurement tools are of great value to provide reliable image of a variety of pathological conditions including burn, traumatic wounds, diabetes, aging and shock because they are associated with changes in local or systemic blood perfusion. The aim of this study was to compare various currently used techniques to assess their validity and reliability. Hypothesis We hypothesized that there are no quantitative/qualitative differences between microcirculation measurement techniques, to differentiating ischemia. Methods In a group of 20 young adult New Zealand White rabbits, we rendered one ear ischemic and local tissue perfusion was measured and compared using laser speckle contrast imaging (LSCI, Moor FLPI-2), tissue viability imager (Tivi8000micro), thermal imager (FLIR C3), tissue oxygenation monitor (MoorVMS-Oxy), transcutaneous pO2 and pCO2 (Radiometer TCM4), and several pulse oximeters (Bionet Vet and Ohmeda Biox 3740). Result All the perfusion measurement showed clear perfusion differences between the ischemic and non-ischemic ears. LSCI has shown the most consistent result. Tivi8000 showed similar results but sometimes was affected by skin color changes. Thermal imager camera also showed good results but the sensitivity was not the best. Both MoorVMS-Oxy and TCM4 had good results but required longer time to measure. Pulse oximeters had some difficulty when the main artery was ligated. Conclusion Our preliminary results show the most consistent measurement outcomes with the use of LSCI technique. A combination of two different methods can give us a better understanding of perfusion measurement.
P.LB09.
Hypertrophic scars are debilitating and decrease burn patients’ quality of life; surgical excision and laser ablation are the most effective therapies. Oxandrolone in combination with propranolol reduces post-burn scars by a still unknown mechanism. Here, we treated burn patient-derived hypertrophic scar fibroblasts for 7 and 14 days with propranolol, oxandrolone, or their combination, and assessed basal and TGF-β-induced fibrotic signaling via Western blot. Oxandrolone alone or in combination with propranolol decreased basal α-SMA protein expression by 7(p<0.05) and 14 days (p<0.001). TGF-β-induced α-SMA protein expression was reduced by oxandrolone alone or in combination with propranolol at 7 (p<0.05) but not 14 days. Oxandrolone plus propranolol decreased basal collagen-I protein expression at 7 and 14 days (p<0.05). N-cadherin protein levels decreased 7 days post-treatment with oxandrolone or oxandrolone plus propranolol (p<0.05). Oxandrolone alone or in combination with propranolol decreased basal fibronectin protein expression at day 7 (p<0.05) and TGF-β-induced fibronectin expression at day 14(p<0.05). Basal protein expression of vimentin and collagen III was not affected by any of these treatments, while TGF-β-induced protein expression of vimentin and collagen-III was decreased by propranolol at day 14 (p<0.05). CD44, Integrin B1, and B5 protein levels were not affected by any of these treatments, though basal HAS-2 protein levels were decreased by 7 and 14 days treatment with oxandrolone plus propranolol (p<0.05). Collectively, these data show that combined treatments that aim to modulate both the catecholamine and the cortisol surge post-burn are the most effective in reducing fibrotic phenotypes in burn patients hypertrophic scar fibroblasts.
P.LB10.
BACKGROUND: Measurements of molecular markers in wound fluid can provide valuable information for assessing wound status and monitoring wound healing. Currently, molecular analysis of wound fluid requires laboratory analysis, which is laborious, time-consuming and expensive. While point-of-care tests for measuring wound biomarkers have been demonstrated, they only provide qualitative results or require invasive sample collection which can disturb the natural wound healing process. To address these limitations, we demonstrate a novel dressing-based wireless biosensor for rapid measurements of wound biomarkers.
METHODS: The sensor is comprised of interdigitated carbon sensing electrodes with a silver coil antenna, which are screen-printed onto dressing. Measurements are wirelessly read using an impedance analyzer with a transmitter antenna.
RESULTS: We first investigated the sensor’s capability for wireless measurements by detecting uric acid spiked in simulated wound fluid (50% v/v serum and 50% v/v buffer) from 0 – 800 µM. A direct correlation between the uric acid concentration and the detection signal was observed with excellent linearity (R2 of 0.994) and responsivity (6.25kHz/μM) over the tested concentration range. We also studied the influence of a silver-containing antimicrobial wound dressing (AQUACEL® Ag EXTRA™) on the sensor response by placing the dressing on top of the sensor and found that it did not impede its performance. Lastly, we performed measurements of uric acid spiked in clinical wound fluids which showed that our sensor can accurately detect wound biomarkers in a complex extracellular matrix.
CONCLUSIONS: This wireless dressing-based biosensor offers a non-invasive method for quantifying wound biomarkers in situ, which can aid clinicians in monitoring wound status and predicting the healing process.
P.LB11.
Hypertrophic scarring(HS) is observed in more than 70% of the severe burn survivors. HS is mainly composed of myofibroblasts and macrophages that secrete a significant amount of extracellular matrix. Imbalance in the macrophage phenotype and function can lead aberrant repair, and eventually scar in different pathological conditions. We hypothesized that HS has different expression of macrophage phenotype compared to non-burned controls and macrophage phenotype controls scarring sequelae. Methods: Scar biopsies with greater than 30% total body surface area burns(n=12) were compared to the skin biopsies from non- a burned area from the burn patients. Tissue sections were stained with CD68 (M1marker) and CD206 (M2marker) and immunofluorescence was utilized to analyze the expression. While in-vitro THP-1 cells were stimulated with M1 and M2 phenotype then co-cultured with a scar and skin fibroblast to elucidate the interaction between two distinct types of cells. Results: Both scars and burn control skin samples stained for CD68 and there was no significant difference in the expression between the two groups. However, scar biopsies had a significantly higher expression of CD206 (M2 marker) compared to the burn controls skin biopsies (p<0.05). In-vitro studies showed that M2 induced macrophages significantly increases collagen-1A expression in scar and skin fibroblast compared to non-treated fibroblast or co-culture with M1 induced macrophages (p<0.05). Conclusion: M2 phenotype macrophage is associated with the production of anti-inflammatory but pro-fibrotic factors like TGF-b. Presence of significant amount of M2 macrophages in scar biopsies and increase in ECM component in skin and scar fibroblast following co-culture with M2 macrophage demonstrate that scar pathology has a different phenotype of macrophages expression.
