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2019 Abstracts

G.01

Granzyme B Exacerbates Atopic Dermatitis Through Filaggrin, E-cadherin And Desmoglein Cleavage And Disruption Of Barrier Function
Christopher Turner1, Stephanie Santacruz1, Matthew Zeglinski1, Christine Wang1, Katlyn Richardson1, Sho Hiroyasu1, Hongyan Zhao1, Yue Shen1, Gail Gauvreau2, Hermenio Lima2, Roma Sehmi2, David Granville1
1University of British Columbia, Vancouver, BC, Canada, 2McMaster University, Hamilton, ON, Canada
GRANZYME B EXACERBATES ATOPIC DERMATITIS THROUGH FILAGGRIN, E-CADHERIN AND DESMOGLEIN CLEAVAGE AND DISRUPTION OF BARRIER FUNCTION

Christopher Turner1, Stephanie Santacruz1, Matthew Zeglinski1, Christine Wang1, Katlyn Richardson1, Sho Hiroyasu1, Hongyan Zhao1, Yue Shen1, Gail Gauvreau2, Hermenio Lima2, Roma Sehmi2, David Granville1
1University of British Columbia, Vancouver, BC, Canada, 2McMaster University, Hamilton, ON, Canada

Granzyme B (GzmB) is a serine protease minimally expressed in normal skin but drastically elevated in multiple chronic inflammatory skin diseases. Extracellular GzmB cleaves multiple substrates within the extracellular matrix and dermal-epidermal junction, contributing to impaired wound healing. We hypothesize GzmB contributes to development, severity, and impaired healing in atopic dermatitis (AD). To evaluate GzmB expression, excised human AD skin was examined histologically. GzmB’s role was assessed in a murine model of AD, comparing GzmB-/- and wild-type (WT) mice. Macroscopic severity scores, wound morphometry, inflammation, and barrier protein expression were assessed. To identify additional GzmB substrates, in vitro cleavage assays were performed. To assess mechanism, cultured keratinocytes were exposed to GzmB with barrier function, barrier protein expression, and cell viability assessed. GzmB was significantly elevated in human and mouse AD compared to healthy skin. A significant reduction in inflammation, lesion size, epidermal thickness, and AD severity scores were observed in GzmB-/- compared to WT mice. Topical administration of a small molecule GzmB inhibitor to WT mice also reduced AD severity compared to vehicle-treated controls. Cultured keratinocyte monolayers exposed to GzmB showed a dose-dependent increase in barrier function impairment. In vitro, GzmB effectively cleaved Filaggrin, E-cadherin, desmoglein-1, and -3. Keratinocyte monolayers exposed to GzmB showed disordered E-cadherin, desmoglein-1, and -3 expression. As expected, epidermal expression of Filaggrin (stratum corneum only), E-cadherin, desmoglein-1, and -3 were all decreased in WT AD-affected mice compared to healthy skin controls. However, GzmB-/- AD-affected mice displayed a significantly greater expression of each of these proteins than WT AD-affected ears. GzmB is elevated in AD and contributes to disease severity. Mechanistically, GzmB disrupts barrier function, cleaving key proteins linked to the pathogenesis of AD. GzmB inhibition provides a potential therapeutic option.

G.02

EPIDERMAL STEM CELL DERIVED EXOSOMES IMPROVE DIABETIC WOUND HEALING BY MODULATING FIBROBLAST AND MACROPHAGE FUNCTIONS
Georgios Theocharidis, Peng Wang, Antonio Lobao, Biaoliang Wu, Aristidis Veves
Beth Israel Deaconess Medical Center, Boston, MA, USA
EPIDERMAL STEM CELL DERIVED EXOSOMES IMPROVE DIABETIC WOUND HEALING BY MODULATING FIBROBLAST AND MACROPHAGE FUNCTIONS

Georgios Theocharidis, Peng Wang, Antonio Lobao, Biaoliang Wu, Aristidis Veves
Beth Israel Deaconess Medical Center, Boston, MA, USA

Background: Despite advancements in regenerative medicine, healing of diabetic foot ulcers (DFU) still remains a challenging clinical problem. Exosomes are nanosized vesicles released by different cells and implicated in numerous physiological processes. Epidermal stem cells (ESC) reside in the basal epidermal layer and have been shown to contribute to cutaneous wound healing. However, whether they secrete exosomes which also influence the wound repair process has yet to be reported. Methods: To investigate the role of ESC derived exosomes in DFU healing, we first isolated ESC from telogen murine skin. Purity of ESC was demonstrated with Itga6/Cd71 double staining. Exosome isolation was achieved through ultracentrifugation of ESC conditioned media and their presence was confirmed via transmission electron microscopy imaging, nanoparticle tracking analysis and western blot detection of teraspanin exosomal markers CD63 and CD81. Results: RNA-seq analysis revealed the presence of 87 miRNAs in the ESC exosomal cargo. Exosomes were internalized by primary mouse fibroblasts and macrophages in vitro, as evidenced by PKH-67 dye labelling and increased cell proliferation measured with Ki67 staining and alamar blue assay in a dose-dependent manner. In addition, they accelerated wound closure in a diabetic fibroblast scratch assay model and induced alternative or M2 polarization of macrophages with elevated expression of Ym1, Cd206 and Arg1 markers. Strikingly, intradermal injection of ESC exosomes in the wound edges of db/db mice led to 49% improved wound closure (n=10, p<0.01). Histological evaluation revealed increased vascularization, cell proliferation and M2 macrophages presence. Conclusions: In summary, we report for the first time detailed characterization of ESC exosomes and demonstrate their in vivo efficacy in improving diabetic wound healing. They could therefore be an alternative to cell therapy and be employed as a novel treatment for DFU.

G.03

MECHANICAL TENSION INFLUENCES THE REGULATORY LANDSCAPE OF MSC-DERIVED EXOSOMES DURING WOUND HEALING
Hima Vangapandu1, Matthew Robertson1, Daniel Colchado1, Natalie Templeman1, Hui Li1, Yao Ning1, Alexander Blum1, Paul Bollyky2, Sundeep Keswani1, Cristian Coarfa1, Swathi Balaji1
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA
MECHANICAL TENSION INFLUENCES THE REGULATORY LANDSCAPE OF MSC-DERIVED EXOSOMES DURING WOUND HEALING

Hima Vangapandu1, Matthew Robertson1, Daniel Colchado1, Natalie Templeman1, Hui Li1, Yao Ning1, Alexander Blum1, Paul Bollyky2, Sundeep Keswani1, Cristian Coarfa1, Swathi Balaji1
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA

Mesenchymal stem cells (MSCs) have a huge therapeutic potential in wound healing. While it is known that the microenvironment affects the MSC secretome, the role of mechanical tension on the extracellular vesicles, namely exosomes, released by MSCs, is not known. We hypothesize that tension regulates MSC exosome production and influences wound healing via paracrine effects on dermal fibroblasts. Human MSCs were cultured on silicone membranes +/-10% tonic strain for 24h and analyzed for phenotypic changes (morphology; alpha-SMA; fibrosis PCR-array). Exosomes were isolated and their size (zetasizer), protein levels(BCA Assay), surface markers(CD63,HSP70,CD9), and genomic cargo(Next-Gen Sequencing(NGS)) were analyzed. Human dermal fibroblasts(hdFB) were treated with MSC-derived exosomes from +/- tension, and changes in gene expression and migration assay were evaluated. p-values by ANOVA;(n=3/group). Tension resulted in the loss of the characteristic morphology of the MSC spindle shape and increased alpha-SMA staining. There was a significant change in ~30/77 (>2-fold) fibrotic/inflammation genes with tension. There was an increase in exosomal size distribution and protein cargo under tension (p<0.05). NGS of the exosomes revealed that the abundance of their tRNA increased, whereas the miRNAs and lincRNAs were reduced under tension. KEGG gene pathway analysis of down regulated miRNAs showed enrichment of intracellular/extracellular wound healing processes. The top MSC exosomal-miRs that were downregulated in response to tension corresponded to Hippo(growth/homeostasis), Ras, PI3K(proliferation) and AMPK (energy sensing) signaling. Measurement of the gene expression implicated in wound healing within these pathways revealed that YAP1, GSK3B and PPP2R1A decreased in hdFB upon treatment with MSC-derived exosomes under tension. Interestingly, MSC-derived exosomes under tension increased hdFB migration in a scratch wound assay (p<0.05). Mechanical tension induces a fibrogenic and inflammatory phenotype in MSCs. Understanding how bioactive cargo in MSC exosomes can be regulated by tension and can influence fibroblast behavior will contribute to the development of exosome-based clinical therapies for wound healing and fibrosis.

G.04

POSSIBILITY OF INJECTING ADIPOSE-DERIVED STROMAL VASCULAR FRACTION CELLS TO ACCELERATE MICROCIRCULATION IN ISCHEMIC DIABETIC FEET
Seung-Kyu Han, Ji Won Son, Jae youn Kim, Sik Namgoong
Korea University Guro Hospital, Seoul, Korea, Republic of
POSSIBILITY OF INJECTING ADIPOSE-DERIVED STROMAL VASCULAR FRACTION CELLS TO ACCELERATE MICROCIRCULATION IN ISCHEMIC DIABETIC FEET

Seung-Kyu Han, Ji Won Son, Jae youn Kim, Sik Namgoong
Korea University Guro Hospital, Seoul, Korea, Republic of

Background: Beneficial effects of human adipose-derived stromal vascular fraction (SVF) cell injection on microcirculation have been recently reported in in vitro and in vivo studies. However, no clinical studies have reported its effect in diabetic patients who commonly experience compromised tissue perfusion, regardless of the status of intravascular blood flow. The present piloting study was designed to clinically examine the possibility of SVF cell injection to accelerate microcirculation, particularly in ischemic diabetic feet. Methods: Ten diabetic feet were included to receive subcutaneous injection of SVF cells around wounds. Transcutaneous partial oxygen pressure (TcPO2) and cutaneous microvascular blood flow were measured before and every four weeks after cell injection until the 12th week visit. Results: TcPO2 values increased from 31.3 ± 7.4 before injection to 46.4 ± 8.2 mmHg at 12 weeks after SVF injection (1.5-fold, P < 0.05). Cutaneous microvascular blood flow levels increased from 34.0 ± 21.1 before injection to 76.1 ± 32.5 perfusion unit at 12 weeks after SVF injection (2.2-fold, P < 0.05). There were no adverse events related to SVF cell injection. Conclusions: Results of this study demonstrate that adipose-derived SVF cell injection have the possibility to provide beneficial effects on microcirculation in ischemic diabetic feet.

G.05

PYOCYANIN INDUCES MITOCHONDRIAL DYSFUNCTION IN THE BIOFILM INFECTED CUTANEOUS WOUND THROUGH UPREGULATION OF MIR-146A
Karamjeet Kaur, Mithun Sinha, Sashwati Roy, Chandan K. Sen
Indiana University, Indianapolis, IN, USA
PYOCYANIN INDUCES MITOCHONDRIAL DYSFUNCTION IN THE BIOFILM INFECTED CUTANEOUS WOUND THROUGH UPREGULATION OF MIR-146A

Karamjeet Kaur, Mithun Sinha, Sashwati Roy, Chandan K. Sen
Indiana University, Indianapolis, IN, USA

Background: Pyocyanin is a redox active secondary metabolite secreted by Pseudomonas aeruginosa. In the biofilm infected wounded skin, it penetrates cell membrane and disrupts intracellular redox homeostasis via mitochondrial dysfunctioning. This work shows that wound-site pyocyanin causes mitochondrial dysfunction by upregulating a mito-miR, miR-146a. Methods: Human keratinocytes were treated with 10μM pyocyanin for 5d. Such levels have been detected in patient wound fluid. miR-146a levels were determined via qRT-PCR. Oxygen Consumption Rate (OCR), respiration, proton leak and ATP production was measured using Sea Horse Analyzer XFe96. Mitochondrial untranslated protein response (UPRmt) was analyzed. Results: QRT-PCR data showed that pyocyanin (10 μM) treatment caused two-fold induction of miR-146a in keratinocytes (n=4, p<0.05). Upregulated miR-146a targets mitochondrial transcripts such as SOD2 causing mitochondrial dysfunction. In order to study mechanism underlying such mitochondrial dysfunctioning, pyocyanin (10 μM) treated cells and miR-146a mimic transfected cells were analyzed on Sea Horse Analyzer XFe96. OCR (pmoles/min) was significantly reduced in both the conditions (pyocyanin treated and miR-146a transfected cells n=4, p<0.05). miR-146a compromised cellular respiration (non-mitochondrial, basal and maximal) in keratinocytes. Under the same conditions, proton leak was also observed. Such leak may be viewed as sequelae of distorted or leaky mito-membrane. ATP production dropped from 40 pmoles/min to 20 pmoles/min in the pyocyanin treated/miR-146a transfected cells. Pyocyanin treatment also disrupted mitochondrial proteostasis which stimulated UPRmt (n=4, p<0.05). Elevated UPRmt is known to upregulate mitochondrial folding capacity in response to stress. Such response is aimed at circumventing deleterious protein aggregation. In cells with elevated UPRmt, transcription factors NFκB and p53 were upregulated. Conclusion: Presence of pyocyanin in the wounded skin disrupts mitochondrial bioenergetics which complicates wound healing. The disrupted mitochondrial bioenergetics is contributed by miR-146a induced by pyocyanin. Elevated miR-146a, thus, represents a key mechanism in this paradigm.

G.06

HYPERSPECTRAL IMAGING IN BURN EVALUATION FROM BENCH TO BEDSIDE: A CASE REPORT
Garrick Gu, BA, Melissa J. McCarthy, MPH, Jorge Lujan-Hernandez, MD, Danielle Stamer, BS, Janice Lalikos, MD
University of Massachusetts Medical School, Worcester, MA, USA
HYPERSPECTRAL IMAGING IN BURN EVALUATION FROM BENCH TO BEDSIDE: A CASE REPORT

Melissa J. McCarthy, MPH, Garrick Gu, BA, Jorge Lujan-Hernandez, MD, Danielle Stamer, BS, Janice Lalikos, MD
University of Massachusetts Medical School, Worcester, MA, USA

Background: Predicting burn depth is a major goal of imaging technology over the last 30 years. Hyperspectral imaging (HSI) is a FDA-approved non-invasive method of evaluating soft tissue oxygenation and perfusion using visible light; previously, it demonstrated in mice to predict burn depth earlier than clinical assessment. We now describe the potential utility of HSI in evaluating burns in a patient. Methods: A patient sustained thermal burns at various depths. He was evaluated in the ED 6hr post-burn. Based on initial clinical exam, burns were termed second (2Deg) or third degree (3Deg). 2Deg was subdivided into superficial (2DegS) and deep (2DegD). HSI images were taken on arrival and on post-burn days (PBD) 3, 4, and 5 with clinical evaluation, using unburned skin HSI results from PBD1 as control. HSI provided arbitrary units for oxygenated (OxyHb), deoxygenated (DeoxyHb), and total hemoglobin (THb), reflecting tissue perfusion. Burn areas that deepened between PBD1 to PBD5 were surgically excised. Clinical impressions from PBD1 to PBD5 (intra-operative) correlated with HSI findings. Results: Findings at 6hr post-burn versus control: OxyHb: +124% (2DegS), +28% (2DegD) and -42% (3Deg). DeoxyHb: +8% (2DegS), -38% (2DegD), -64% (3Deg). THb: +60% (2DegS), -8% (2DegD) and -56% (3Deg). Findings at 40hr post-burn 2DegS versus 2DegD: +134% OxyHb, +97% DeoxyHb and +120% THb. These findings mirrored our animal model. Discussion: Our data suggests HSI readings correlate with burn depth, reflecting our animal model. Increased Thb and OxyHb in 2DegS, denoting hyperperfusion, could be from reactive erythema, which is blunted in 2DegD from deeper dermal plexus damage and destroyed in 3Deg, yielding negative values. HSI can detect trends in OxyHb and DeoxyHb, denoting tissue perfusion, that are consistent with deepening burns. This data can help determine 2nd degree burn depth; a vital surgical delineation. If further validated, this could represent an application of HSI for predicting burn depth earlier, potentially improving patient outcomes.

G.07

TARGETING CD109 IN DUPUYTREN'S DISEASE
Yasser Almadani1, Ana Maria Pena Diaz2, Kenneth Finnson1, David O’Gorman2, Anie Philip1
1McGill University, Montreal, QC, Canada, 2Western University, London, ON, Canada
TARGETING CD109 IN DUPUYTREN’S DISEASE

Yasser Almadani1, Ana Maria Pena Diaz2, Kenneth Finnson1, David O’Gorman2, Anie Philip1
1McGill University, Montreal, QC, Canada, 2Western University, London, ON, Canada

Background and Rationale: Persistent accumulation of extracellular matrix (ECM) proteins, represents the clinical hallmark of several fibrotic disorders including Dupuytren’s disease. Aberrant activation of TGF-β signaling has been strongly implicated in Dupuytren’s disease fibrosis (DD). We have previously identified CD109, a membrane-anchored protein, as a potent inhibitor of TGF-β signaling and fibrotic responses in vitro and in vivo. Our objectives were to determine whether CD109 regulates the fibrotic process in DD and to understand the underlying mechanisms involved. Methods and Results: We examined CD109 expression and function in primary human DD fibroblasts (DDF) as compared to phenotypically normal fibroblasts from adjacent palmar fascia (DPF), or anatomically-matched normal fibroblasts (control) from carpal tunnel syndrome patients (CTF), using immunofluorescent staining, Western blot, qPCR or siRNA mediated deletion. Our results show that CD109 protein levels are decreased in DDF and adjacent normal DPF when compared to control CTF, while CD109 mRNA expression levels are similar in DD, PF and CT fibroblasts. Our results also suggest that CD109 protein released from the cell surface are similar in all three types of fibroblasts implying that CD109 degradation is likely enhanced in DDF. In addition, the diminished CD109 protein levels in DDF and DPF are associated with increased alpha-smooth muscle expression in those cells, which is consistent with the known function of CD109 to decrease fibrotic responses in several cell types. Furthermore, manipulation of CD109 protein levels leads to a differential regulation of ALK5/Smad2/3 signaling versus ALK1/Smad1/5 signaling with increased CD109 levels promoting the anti-fibrotic ALK1/Smad1/5 signaling. Conclusion: Our finding that CD109 protein levels are decreased in DD fibroblasts and adjacent PF fibroblasts as compared to normal CT fibroblasts, provides a mechanistic explanation for the increased fibrotic response in DD. This together with our finding that CD109 promotes the anti-fibrotic Smad1/5 pathway over the pro-fibrotic Smad2/3 pathway in these cells, suggests that CD109 plays an important role in DD fibrosis and thus may represent a molecular target for therapeutic intervention in DD.

G.08

EPIGENETIC MAPPING OF WOUND EDGE FROM CHRONIC WOUND PATIENTS USING NEXT GENERATION SEQUENCING
Kanhaiya Singh1, Sashwati Roy1, Durba Pal2, Subhadip Ghatak1, Dolly Khona1, Saba Tabasum1, Shomita Steiner1, Devleena Das1, Pearlly Yan2, Ralf Bundschuh2, Savita Khanna1, Chandan K. Sen1
1Indiana University School of Medicine, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA
EPIGENETIC MAPPING OF WOUND EDGE FROM CHRONIC WOUND PATIENTS USING NEXT GENERATION SEQUENCING

Kanhaiya Singh1, Sashwati Roy1, Durba Pal2, Subhadip Ghatak1, Dolly Khona1, Saba Tabasum1, Shomita Steiner1, Devleena Das1, Pearlly Yan2, Ralf Bundschuh2, Savita Khanna1, Chandan K. Sen1
1Indiana University School of Medicine, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA

Background: The loud biochemical microenvironment of the chronic wound sharply departs from that of the skin under homeostatic conditions and is likely to induce epigenetic changes thus influencing wound healing outcomes. Methods: Unbiased whole-genome DNA methylation (methylome) was studied in normal skin (NS) and in wound edge tissue of chronic wound (CW) patients. DNA (1 μg) isolated from CW or NS was enriched for the study of methylated DNA. Single-end 50 bp sequencing was performed using Illumina HiSeq 2500. Methylation status of proximal promoter (1 Kb) was calculated using MethylCap-Seq data analysis and PrEMeR-CG analyses. Whole genome RNA sequencing and microRNA nanoString analysis over CW and NS samples was performed to qualify the methylation data. Results: In proximal promoter, genes involved in epithelial to mesenchymal transition (EMT) were hypermethylated in CW compared to NS (n=3, p<0.0001). Bisulfite sequencing was used to validate hypermethylation of predicted upstream regulators (TP53 and BRCA1). microRNA promoters were differentially methylated in CW compared to NS (71 hyper methylated; 20 hypomethylated in CW, n = 3, p<0.05). A sum total of 1281 genes were found to be differentially expressed in CW compared to NS (n=3, p<0.05). Comparison between hypermethylated and downregulated expressed genes identified common candidate genes of EMT pathway like ADAM17, TWIST1 and SMURF1 obtained through two independent global screening approaches. Topical application of classical DNA demethylation agent 5'-azacytidine successfully rescued ischemic wounds of mice by increasing the expression of ADAM17. Lentiviral overexpression of ADAM17 alone rescued ischemic wounds by enabling the P53-ADAM17 cascade. Conclusion: Epigenetic activity is high at the chronic wound site. Such activity has significant impact on gene expression at the wound-edge and is therefore likely to influence healing outcomes.

G.09

ETRS WINNER SYSTEMIC DELIVERY OF ANTI-INTEGRIN ANTIBODIES REDUCE EARLY MACROPHAGE RECRUITMENT, INFLAMMATION AND SCAR FORMATION IN MURINE BURN WOUNDS
Xanthe L. Strudwick1, Damian H. Adams1, Natasha T. Pyne2, Michael S. Samuel2, Rachael Z. Murray3, Allison J. Cowin1
1Future Industries Institute, University of South Australia, Adelaide, Australia, 2Centre for Cancer Biology, University of South Australia, Adelaide, Australia, 3Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
SYSTEMIC DELIVERY OF ANTI-INTEGRIN αL ANTIBODIES REDUCE EARLY MACROPHAGE RECRUITMENT, INFLAMMATION AND SCAR FORMATION IN MURINE BURN WOUNDS

Xanthe L. Strudwick1, Damian H. Adams1, Natasha T. Pyne2, Michael S. Samuel2, Rachael Z. Murray3, Allison J. Cowin1
1Future Industries Institute, University of South Australia, Adelaide SA 5001, Australia, 2Centre for Cancer Biology, University of South Australia, Adelaide SA 5001, Australia, 3Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, 4001, Australia

Background: Macrophages present in early stages of the repair process play a key role in regulating wound fibrosis and scar formation. Burns are prone to increased macrophage recruitment leading to an excessive inflammatory response associated with elevated fibrosis and scarring. This recruitment relies upon integrins on the surface of monocytes that regulate their migration and extravasation from the circulation into the wound site, where they then differentiate into macrophages. High levels of key immune specific integrin αL, part of the lymphocyte function-associated antigen-1, were found expressed in circulating monocytes purified from the blood of burns patients compared to burn wound associated macrophages, from the same patient (n=7), suggesting a role in monocyte extravasation. Methods: Integrin αL was targeted in mice (n=8) one day post scald burn injury with 50μg intravenous delivery of neutralising antibody. Results: Burn wound associated macrophages were reduced by 54.7% at day 3 (p=0.030), with levels returning to normal by day 7. The early stage reduction in macrophages led to a concomitant reduction in pro-inflammatory tumour necrosis factor alpha (p=0.030) and pro-scarring transforming growth factor beta (p=0.022) levels. This reduced inflammatory response was associated with reduced alpha smooth muscle actin expression during the proliferative phase of healing (day 7, p=0.016) and an overall trend towards reduced scar formation. Wounds appeared more similar to unwounded skin, with a lower collagen I/III ratio (p=0.048) and improved scar appearance (p=0.041) 28 days following burn injury in mice treated with anti-integrin αL compared to control treated mice. Conclusion: These results suggest targeted integrin αL therapy reduces macrophage infiltration into burn wounds leading to a reduced early inflammatory response and reduced scar formation following burn injury.

H1.01

BIOMIMETIC ADIPIOSE STEM CELL DRESSING FOR SKIN REGENERATION
Artem Trotsyuk, Clark A. Bonham, Melanie Rodrigues, Paul Mittermiller, Jayakumar Rajadas, Mohammed Inayathullah, Geoffrey Gurtner
Stanford University, Stanford University, CA, USA
BIOMIMETIC ADIPIOSE STEM CELL DRESSING FOR SKIN REGENERATION

Artem Trotsyuk, Clark A. Bonham, Melanie Rodrigues, Paul Mittermiller, Jayakumar Rajadas, Mohammed Inayathullah, Geoffrey Gurtner
Stanford University, Stanford University, CA, USA

Background: Burns have important functional and psychosocial implications for patients. Decades of wound healing research have demonstrated a critical window within the first 24 hours after wounding during which there is a “switch” from scarless wound healing to scarring. Recently, cell-based therapies have been proposed as an option for improving healing and reducing scar formation in burn wounds. Here, we identify an optimal stem cell population to stimulate angiogenesis, modulate inflammation and improve wound healing. We then optimize a delivery platform for the identified stem cell population and test the efficacy in a murine contact burn model. Methods: Commercially-available and primarily isolated stem and progenitor cell populations were evaluated at a single cell level to determine which of the cells most closely resembled a fetal gene expression signature. Microfluidic single-cell qPCR and advanced bioinformatics was used to identify a potent subpopulation adipose-derived stromal cells (ASCs) that is found in both human and murine samples (CD26+/CD55+). Next, a hydrogel microenvironment was optimized for the identified ASC subpopulation. Finally, the cell-hydrogel combination was validated in a pre-clinical setting. Results: Single-cell gene transcriptional analysis of cultured ASCs demonstrated a considerable increase the genes favorable to wound healing. Cells seeded with dual-capillary force on the pullulan-collagen hydrogel exhibited improved viability. Wounds treated with CD26+/CD55+ cells on a pullulan-collagen hydrogel showed improved wound healing and neovascularization in a murine contact burn model. These results suggest the regenerative potential of the identified ASC subpopulation for future clinical application. Conclusion: The Gurtner laboratory has developed an ASC-hydrogel therapy for treating burns, with demonstrated pro-angiogenic, fibromodulatory and immunomodulatory effects. Further work is being done to validate the cell-hydrogel combination in a pre-clinical setting.

H1.02

LOW COST OXYGEN-SENSING FILMS FOR CLINICAL WOUND HEALING MONITORING
Daniel Naveed Tavakol, Samantha Schwager, Lindsay Jeffries, Anthony Bruce, Christopher DeRosa, Cassandra Fraser, Shayn M. Peirce, Patrick S. Cottler
University of Virginia, Charlottesville, VA, USA
LOW COST OXYGEN-SENSING FILMS FOR CLINICAL WOUND HEALING MONITORING

D. Naveed Tavako, Samantha Schwager, Lindsay Jeffries, Anthony Bruce, Christopher DeRosa, Cassandra Fraser, Shayn M. Peirce, Patrick S. Cottler
University of Virginia, Charlottesville, VA, USA

Background: Oxygen delivery is essential for wound healing, therefore, a non-invasive and inexpensive tool to map wound oxygen has clinical significance. Assessment of healing typically relies on qualitative and subjective analysis, while oxygen monitoring can be expensive, highly variable, or only provide indirect measurements. We have developed an easy-to-use, thin film capable of monitoring wound oxygenation. Methods: The flexible dual-layer film utilizes novel boron oxygen-sensing nanoparticles (BNPs) capable of monitoring changes in wound oxygen via the oxygen-permeable layer (chitosan-polycaprolactone with BNPs), while isolating the wound through an oxygen-impermeable layer (alginate). The BNPs are a dual emissive difluoroboron β-diketonate dye incorporated into a poly(lactic acid) matrix (BF2bdkPLA) that demonstrate emissions of a standard fluorescent peak and a phosphorescence peak that quenches with increasing oxygen. The ratio of fluorescence to phosphorescence (F/P) provides normalized values directly proportional to oxygen. Results: in vitro utility of the films was demonstrated through F/P analysis documenting the film’s capability to isolate a hypoxic environment from ambient oxygen through the oxygen-permeable and impermeable layers (p<0.05, n=3). The oxygen sensitivity of the BNPs in films was also shown to be equivalent to BNPs in solution in a mouse wounds (p=0.43, n=3). Additionally, wound related angiogenesis led to F/P ratios increasing by 22%, while total wound area decreased 45% during the initial 3 days of healing. Conclusions: BNPs in solution have documented utility, but present application and removal challenges. These results present a promising solution to directly map oxygen levels in wounds in a clinically relevant film formulation. Clinical use of the films has the potential to monitor chronic wounds healing trajectory, guiding care decisions.

H1.03

REAL-TIME TRACKING AND QUANTIFICATION OF CHRONIC WOUND BACTERIA VIA NANOFIBER BIOSENSORS
Craig Miller, Mark Livingstone, Jordon Gilmore
Clemson University, Clemson, SC, USA
REAL-TIME TRACKING AND QUANTIFICATION OF CHRONIC WOUND BACTERIA VIA NANOFIBER BIOSENSORS

Craig Miller, Mark Livingstone, Jordon Gilmore
Clemson University, Clemson, SC, USA

Background: There is a need to improve current assessment procedures used to track and diagnose the bacterial colonization of chronic, non-healing wounds towards infection. Real-time, rapid detection of antimicrobial susceptibility has the potential to significantly reduce healing times and morbidity in patients with chronic wound injuries. Current infection detection methods rely heavily on clinician experience and lab culturing. These methods are highly subjective and time consuming. Methods: In this work we developed a point-of-care device that can detect not only the presence of bacteria, but also metabolic activity towards the release of virulence factors related to the spread of infection. We used impedance-based sensing to provide clinicians with a quantitative tool to detect the onset of increased bacterial growth or antimicrobial susceptibility. Nonwoven nanofiber mats were fabricated by solution blow spinning of a poly-l-lactide and multi-walled carbon nanotube composite. Results: Conductivity values up to 474 S/cm were generated to detect the increased bacterial growth and antimicrobial susceptibility of Pseudomonas Putida (P. putida). Bacteria were cultured in the presence of glucose supplemented media and the impedance spectra were recorded over a 24-hour period. Functions for continuous and endpoint bacterial growth were derived from impedance data with respect to bacterial concentration and nanofiber composition. Additionally, AgNO3 (10, 100, 1000 mM) was used to determine the antimicrobial susceptibility of P. Putida to include a therapeutic effect along with the diagnostic sensor. Zone of inhibition testing was employed to determine the Antimicrobial efficacy of various AgNO3 concentrations. Conclusions: Further development of this technology has the potential to provide clinicians with a tool for rapid bacteria quantification and therapeutic decision making.

H1.04

CUSTOMIZABLE ELECTROSPUN SCAFFOLDS FOR SUSTAINED AND LOCAL DELIVERY OF SIRNA IN DIABETIC FOOT ULCERS
Amy C. Kauffman1, Alexandra S. Piotrowski-Daspit1, Themis Kyriakides2, W. Mark Saltzman1
1Yale University, New Haven, CT, USA, 2Yale School of Medicine, New Haven, CT, USA
CUSTOMIZABLE ELECTROSPUN SCAFFOLDS FOR SUSTAINED AND LOCAL DELIVERY OF SIRNA IN DIABETIC FOOT ULCERS

Amy C. Kauffman1, Alexandra S. Piotrowski-Daspit1, Themis Kyriakides2, W. Mark Saltzman1
1Yale University, New Haven, CT, USA, 2Yale School of Medicine, New Haven, CT, USA

Background: Foot ulceration is the leading cause of diabetic related amputations and up to 25% of diabetic patients will develop a foot ulcer during their treatment course. Many of these ulcers fail to heal due to molecular imbalances that can be traced back to inappropriate gene expression promoted by a hyperglycemic state. Thus, treatment should be targeted to correct these local genetic imbalances at the source. Overexpression of thrombospondin-2 (THBS-2) is commonly associated with Type II diabetes. Previous studies have demonstrated accelerated wound healing in THBS-2 total knockout mice compared to control. Our aim is to deliver siRNA targeted for THBS-2 silencing via a bioresorbable scaffold at to the wound site and observe the downstream effects and accelerate healing. Methods: Scaffolds were fabricated via electrospinning and composed of polycaprolactone (PCL) blended with highly customizable mildly cationic terpolymers known as poly(amine-co-esters) (PACEs). Physiochemical properties (mechanical, thermal, morphological) were assessed as well as biocompatibility, siRNA loading and efficacy, and degradation. Results: Three PCL/PACE electrospun scaffold designs demonstrated appropriate tensile strength (Young modulus range 0.7-1.0 MPa), surface hydrophilicity (contact angle <30°), and thermal stability to function as a synthetic skin. All three scaffold designs supported viability of primary human dermal fibroblasts greater than 85%, loading of ~2.5 pmol of siRNA per scaffold (ø6mm, 50 μm thick) and sustained release of siRNA over more than 3 days. Conclusions: This work demonstrated the feasibility of use of novel PCL/PACE electrospun scaffolds for sustain local delivery of siRNA in diabetic foot ulcers. Future studies will investigate in-vivo performance in both wild-type and diabetic mouse wound model.

H1.05

DEHYDRATED AMNION CHORION MEMBRANES INDUCE BROAD-SCALE CHANGES IN KINASE ACTIVITY OF ENDOTHELIAL CELLS
Miranda Burnette, John McQuilling, MaryRose Kammer, Kelly Kimmerling, Katie Mowry
Organogenesis Inc, Birmingham, AL, USA
DEHYDRATED AMNION CHORION MEMBRANES INDUCE BROAD-SCALE CHANGES IN KINASE ACTIVITY OF ENDOTHELIAL CELLS

Miranda Burnette, John McQuilling, MaryRose Kammer, Kelly Kimmerling, Katie Mowry
Organogenesis Inc, Birmingham, AL, USA

Angiogenesis is critical for successful wound healing; low oxygen levels and decreased perfusion in chronic wounds increase risk of impaired healing and infection. While a variety of approaches have been used to promote healing through acute oxygenation of wound beds with hyperbaric oxygen or negative pressure therapy, promotion of endogenous vasculature by releasing proangiogenic growth factors to the wound environment remains a promising alternative. Placental membranes have been shown to promote wound repair responses in vitro through a variety of mechanisms including promotion of angiogenesis. In the current study, we have evaluated pro-angiogenic responses in endothelial cells. To identify the signaling-pathways through which dACM-derived factors induce these responses, we performed a global analysis of both serine/threonine and tyrosine kinase activity using a high-throughput commercial platform (PamGene). Human microvascular endothelial cells (HMVECs) were treated for 30 minutes with either assay media or dACM conditioned media (CM). CM was obtained by incubating dACM grafts in basal media for 3-5 days at 4°C at a ratio of 1 cm2 dACM to 1 mL media. Cell lysate was run on the “kinomics” platform to directly detect phosphorylation of ~290 peptide probes by active kinases in each sample. dACM-derived factors were found to drive gross changes in kinase activity, with ERK1, ERK2, TEX14, and PDK1 most significantly upregulated in dACM-treated cells. Pathways functionally enriched in kinases activated by dACM include MAPK signaling, FC-epsilon receptor signaling, and toll-like receptor (TLR) 5 signaling pathway. The activation of VEGFR-2 by pro-angiogenic VEGF-A has been shown to preferentially signal through the PLCj-PKC-MAPK pathway. While generally associated with pathogen recognition and innate immunity, TLR4 signaling has been implicated in angiogenesis through an inflammatory pathway. This work provides an unbiased identification of pathways important for induction of angiogenesis by dACM in vitro.

H1.06

MID-INFRARED LASER-ACTIVATED RAPID SOFT TISSUE REPAIR AND REGENERATION
Inam Ridha1, Deepanjan Ghosh1, Ali Basiri1, Jung Keun Lee2, Jacquelyn Kilbourne1, Yu Yao1, Kaushal Rege1
1Arizona State University, Tempe, AZ, USA, 2Midwestern University, Glendale, AZ, USA
MID-INFRARED LASER-ACTIVATED RAPID SOFT TISSUE REPAIR AND REGENERATION

Deepanjan Ghosh1, Inam Ridha1, Ali Basiri1, Jung Keun Lee2, Jacquelyn Kilbourne1, Yu Yao1, Kaushal Rege1
1Arizona State University, Tempe, AZ, USA, 2Midwestern University, Glendale, AZ, USA

Sutures, staple, and conventional glues are commonly used to approximate tissue edges in surgery and wound healing. However, poor strength, infection, dehiscence, leakage, and/or acute inflammation are common complications associated with these methods. Laser-activated tissue sealing, in which, laser light energy is used to facilitate biomaterial incorporation with the tissue, provides an alternative approach for wound closure. Traditionally, light-absorbing chromophores and nanoparticles have been employed for converting near infrared (NIR) laser light to heat, resulting in the photothermal fusion of the sealant biomaterial with soft tissues. We now demonstrate a novel approach for sealing tissues without the need for chromophores using mid infrared (midIR) laser light. We characterized the absorption of midIR light by several different biomaterials and investigated the rise in local temperature at different laser powers. Optimal operating conditions were employed for midIR based photothermal sealing of incised/ruptured tissue ex vivo and using different skin surgical models in live mice. Recovery of mechanical properties including tensile strength and burst and leak pressures, in concert with histopathology analyses, were employed to determine the efficacy of the seal. The effect of midIR light on cell and tissue viability was also determined. Our results demonstrate that midIR lasers can be used for rapid sealing of soft tissues using conventional biomaterials without the need for chromophores or nanoparticles, which is a significant advantage for rapidly translating this technology in the clinic.

H2.01

COLLAGEN-PRODUCING MACROPHAGES CONTRIBUTE TO FIBROSIS
Britta A. Kuehlmann, Clark A. Bonham, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA
COLLAGEN-PRODUCING MACROPHAGES CONTRIBUTE TO FIBROSIS

Britta A. Kuehlmann, Clark A. Bonham, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA

Background: Implants stimulate collagen deposition resulting in a fibrotic capsule. The most common complication following breast implants is capsular fibrosis (CF). We analyze fibrotic human samples, and use animal models to clarify the mechanisms underlying this fibrotic process. Methods: We have established the largest tissue bank worldwide for breast capsular tissues. This registry provides the opportunity to analyze mechanisms underlying fibrosis. Using RNA analysis with a 2,559-gene probeset, the gene expression from 40 patients was analyzed. 20 human samples expressing the mildest form of CF (Baker I) were compared to 20 human specimens of the most severe form of CF (Baker IV). To recreate CF within an animal model, implants were placed in Bl6-mice. We used vav-reporter-mice for lineage tracking. Cells from the capsule were isolated and characterized by FACS, qPCR and single cell RNA sequencing to determine the genetic profiles of those most responsible for fibrotic development. Results: 1,543 genes were upregulated and 1,016 were downregulated in Baker IV vs Baker I. Genes regulating macrophage activation and macrophages surface marker expression were among the most highly expressed in Baker IV. Lineage tracking identified the presence of cells with distinct characteristics of macrophages, confirming their existence within the chronology of CF. In the murine capsules of Bl6-mice we found the predominant cells were myeloid cells, not fibroblasts. Using FACS, we confirmed that these macrophages produce ECM at the protein level with >80% of macrophages expressing collagen 1. Conclusions: For the first time, we demonstrate that collagen-depositing macrophages are responsible for fibrosis around implants by analyzing patient samples and murine models. Our findings have promising implications for the treatment of capsular fibrosis.

H2.02

OPEN ABDOMENS WITH ONGOING INTRAABDOMINAL PATHOLOGIES SUCCESSFULLY CLOSED USING A DYNAMIC TISSUE SYSTEM AND BIOLOGIC XENOGRAFT: A CASE SERIES
Beatrice Caballero, Yana Puckett, Michelle Estrada, Shirley McReynolds, Robyn Richmond, Catherine Ronaghan
Texas Tech University Health Sciences, Lubbock, TX, USA
OPEN ABDOMENS WITH ONGOING INTRAABDOMINAL PATHOLOGIES SUCCESSFULLY CLOSED USING A DYNAMIC TISSUE SYSTEM AND BIOLOGIC XENOGRAFT: A CASE SERIES

Beatrice Caballero, Yana Puckett, Michelle Estrada, Shirley McReynolds, Robyn Richmond, Catherine Ronaghan
Texas Tech University Health Sciences, Lubbock, TX, USA

Background: Closure of catastrophic open abdomens after damage control laparotomy presents many challenges, particularly in the case of patients with complex pathologies that make achieving myofascial closure exceedingly difficult. The implantation of Porcine Urinary Bladder Matrix (PUBM) allowed for primary skin closure of contaminated wounds. This case series presents an alternative approach for definitive myofascial closure and accelerated wound healing in the setting of open abdomens with ongoing intraabdominal pathology. Methods: We present 5 patients managed with the ABRA Dynamic Tissue System (DTS) in combination with a PUBM xenograft. PUBM particulate is implanted directly on the myofascial closure. A PUBM 2-layer sheet is then placed subcutaneously utilizing a sutureless technique followed by definitive skin closure. Data was collected on the mechanism of injury, patient presentation, surgical management and patient outcomes via retrospective chart review. All 5 patients presented to our tertiary referral center with emergency general surgery issues or penetrating traumatic injuries. These patients had ongoing complex intraabdominal pathology, including a duodenal stump blowout, anastomotic failures (ileocolonic, colocolonic and hepaticojejunostomy/jejunojejunostomy) and a pancreaticoatmospheric fistula associated with multiple intraabdominal injuries sustained following an abdominal gunshot wound. Results: Average maximum myofascial gap was 22.8 cm (range: 11cm – 29cm). Average visceral extrusion was 9.2 cm (range: 4cm – 13cm). The DTS remained in place an average of 11.6 days (range: 8-14 days). Delayed primary myofascial closure was achieved in 5/5 patients (100%) with no fascial dehiscence or surgical site infections (SSIs) observed. Conclusion: In these heavily contaminated wounds, there were 0% SSIs after implantation of PUBM and primary skin closure. This technique essentially eliminated the need for negative pressure wound therapy (NPWT) postoperatively. We achieved 100% total wound healing which appears accelerated compared to historic controls. Utilization of DTS in conjunction with a xenograft combines both mechanical and biologic advantages in definitive closure and complete wound healing of the complex open abdomen.

H2.03

TARGETING WNT SIGNALING TO REDUCE FIBROSIS
Britta A. Kuehlmann, Clark A. Bonham, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA
TARGETING WNT SIGNALING TO REDUCE FIBROSIS

Britta A. Kuehlmann, Clark A. Bonham, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA

Background: Wnt signaling plays a central role in various forms of fibrotic diseases, including skin fibrosis. Activation of the Wnt pathway increases excessive collagen deposition. Foreign body responses around implants mediate remodeling of the extracellular matrix (ECM) resulting in a surrounding capsule of fibrotic tissue. Methods: Fibrosis was induced by inserting implants in Bl6-mice and Wnt11-knockout mice. H&E staining was done to qualitatively assess the morphology and density of the fibrotic tissues. Cells were isolated and characterized by fluorescence-activated cell scanning (FACS), single cell quantitative polymerase chain reaction (qPCR) and single cell RNA sequencing to determine their impact on fibrotic responses. Additionally, SEM, TEM and 3D confocal imaging were performed. Results: FACS and immunohistochemistry stains revealed that collagen-depositing macrophages are responsible for the development of implant fibrosis. qPCR revealed that the majority of cells in the fibrotic tissue expressed macrophage genes and were found to significantly increase Col1a1 and Wnt11. Therefore, we looked at an intervention to reduce these macrophages by using Wnt11-knockout mice and subsequent induction of fibrosis. Using transcriptional analysis we found distinct markers to differentiate three macrophage subgroups (Group 1=Cd36, Group 2=Cd209, Group 3=Ccr2). FACS revealed that Cd36 and Ccr2 were significantly reduced in the fibrotic tissue of Wnt11-knockout mice compared to Bl6-mice. Wnt11-knockout mice displayed significantly thinner capsules with loosely arranged fibers after 3 months. Conclusions: For the first time, we identify macrophage subgroups that are essential to fibrotic development around implants and we use a methodology to decrease fibrosis in an animal model. These findings have promising therapeutic implications for the treatment of fibrosis around implants.

H2.04

EVIDENCE THAT FETUIN-A CONTRIBUTES TO CUTANEOUS SCAR FORMATION
Nicholas K. Pappa, Brian C. Wulff, Traci A. Wilgus
Ohio State University, Columbus, OH, USA
EVIDENCE THAT FETUIN-A CONTRIBUTES TO CUTANEOUS SCAR FORMATION

Nicholas K. Pappa, Brian C. Wulff, Traci A. Wilgus
Ohio State University, Columbus, OH, USA

The formation of scar tissue is an unwanted consequence of successful healing in mature skin. Unfortunately, effective therapies to limit scar formation are lacking, so there is a need to better understand the mechanisms controlling this process. Comparing scarless and fibrotic fetal wounds is one strategy that has been used successfully to identify novel regulators of dermal scar formation. Our lab performed a proteomics study comparing dermal fibroblasts isolated from embryonic day 15 (E15) skin, which heals scarlessly, and embryonic day 18 (E18) skin, which heals with a scar. One of the top differentially expressed proteins was Fetuin-A (Fet-A), which was more abundant in E18 fibroblasts. Fet-A is a serum protein that modulates calcification. Studies have suggested that Fet-A is a ligand for toll-like receptor 4 and that it promotes inflammation. Given that inflammation typically stimulates scar formation and that Fet-A was more highly expressed in fibroblasts from scar-forming E18 skin, we hypothesized that Fet-A may play a role in scar formation. in vitro studies using cultured murine fibroblasts showed that treatment with Fet A significantly increased collagen production compared to untreated controls. A role for Fet-A in scar tissue production was also tested in vivo using two different murine models. First, recombinant Fet-A was injected into E15 fetal wounds, which normally heal without a scar. A significant number of E15 wounds injected with Fet-A healed with a scar, whereas control wounds that were not exposed to Fet-A healed scarlessly. In addition, scar formation was compared in adult wild-type and Fet-A knockout mice after incisional wounding. Scar area was significantly reduced in Fet-A knockout mice compared to wild-type mice 14 days post-wounding. Together, the data suggest that Fet-A may promote collagen production and scar formation. Further studies are now being conducted to evaluate mechanisms by which Fet-A promotes scar formation.

H2.05

FIBROSIS AROUND IMPLANTS IS DRIVEN BY THREE UNIQUE SUBGROUPS OF MACROPHAGES
Britta A. Kuehlmann, Clark A. Bonham, Geoffrey C. Gurtner
Stanford, Stanford, CA, USA
FIBROSIS AROUND IMPLANTS IS DRIVEN BY THREE UNIQUE SUBGROUPS OF MACROPHAGES

Britta A. Kuehlmann, Clark A. Bonham, Geoffrey C. Gurtner
Stanford, Stanford, CA, USA

Background: Implants are used to restore and maintain bodily function in response to a wide variety of diseases. The body’s immune system attempts to destroy these foreign bodies through the foreign body response (FBR) and fibrotic tissue forms around the implant. These capsules cause complications and often require corrective surgery. Methods: We have attempted to discover the major cellular constituents that drive fibrotic development. Using a novel murine model, we placed small silicone implants in Bl6-mice. After 90 days, we harvested the fibrotic tissue surrounding the implants and isolated the cells. Cells were subjected to fluorescence-activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR) and single cell RNA sequencing to determine their genetic profiles and function. Results: FACS yielded distinct cell populations within the fibrotic tissue, with CD45+/CD11b+ macrophages being among the most prevalent. They appeared to be depositing collagen. We subjected the tissue at day 90 to single cell RNA-sequencing without FACS separation. Macrophages were found to be the predominant cell type in the capsule, with three subsets identified. All subsets expressed Cd45, Cd11b and Cd14. The first macrophage subset uniquely expressed Cd36 known to promote fibrogenic pathways. Subgroup 1 is characterized by a highly inflammatory genetic profile, regulating cell signal production associated with acute inflammation. The second macrophage subset distinctly expressed Cd209, which is essential to recognizing foreign bodies and inciting an inflammatory response. Macrophages in group 2 display genes that contribute to acute inflammation. Subgroups 1 and 2 both expressed Adgre1, which encodes F4/80. The third macrophage subset maintained a genetic profile based on its expression of Ccr2. The 3 subgroups seem to contribute to the induction of inflammation through pro-fibrotic and phagocytic activity and signaling. Conclusions: According to current literature, fibroblasts are the source of collagen deposition in the FBR. We found three macrophage subgroups that drive fibrosis and deposit collagen. Our findings have promising therapeutic implications for the treatment of skin fibrosis.

H2.06

MELANOCYTE RE-POPULATION AND CUTANEOUS RE-INNERVATION IN TEMPORAL WOUND HEALING AND SCAR FORMATION IN HUMAN SKIN
Sara Ud-Din1, Philip Foden2, Katie Stocking2, Douglas McGeorge3, Ardeshir Bayat1
1University of Manchester, Manchester, United Kingdom, 2Manchester University Foundation Trust, Manchester, United Kingdom, 3Nuffield Grosvenor Hospital, Chester, United Kingdom
MELANOCYTE RE-POPULATION AND CUTANEOUS RE-INNERVATION IN TEMPORAL WOUND HEALING AND SCAR FORMATION IN HUMAN SKIN

Sara Ud-Din1, Philip Foden2, Katie Stocking2, Douglas McGeorge3, Ardeshir Bayat1
1University of Manchester, Manchester, United Kingdom, 2Manchester University Foundation Trust, Manchester, United Kingdom, 3Nuffield Grosvenor Hospital, Chester, United Kingdom

Background: Communication between the nervous system and epidermal melanocytes in cutaneous healing remains underexplored. Therefore, the aim here was to quantitatively investigate melanocyte re-population and re-innervation in skin scarring created in human volunteers with similar skin-types over a sequential weekly time period upto eight weeks (8W) using a skin-biopsy model. Methods: Objective devices, which quantified melanin (SIAscopy), erythema (colorimeter), blood-flow (full-field laser perfusion imaging (FLPI), dynamic-optical coherence tomography (D-OCT) were used to monitor the scars weekly. Immunohistochemical analysis of melanogenesis, angiogenesis and innervation were performed. Results: SIAscopy and colorimeter showed a significant increase in melanin from uninjured intact skin (UIS) to W8 (210Au to 235Au:p=0.002; 20Au to 35Au: p=0.003 respectively). Masson Fontana, Melan-A and C-Kit analysis showed a 62% reduction at W1, as melanocytes were only evident at the wound edges and a 28% increase to W8 (p=0.04, p=0.04, p=0.03 respectively). The percentage of melanin in the epidermis of scar tissue remained less than that of surrounding UIS as pigment was absent from the centre and gradually migrated in from the periphery. Innervation analysis by PGP9.5, NGF and SP correlated with melanocyte analysis by showing an increased expression from UIS to W8 (p=0.01, p=0.02, p=0.02 respectively) and found to be greatest expression in the centre of the wound in the dermis but no expression in the basal layer of the epidermis at early time points. Erythema increased to W1 and reduced to W8 (p<0.001). Blood flow peaked at W1 (394.9Pu, 0.161Au respectively) (p<0.001) and decreased to W8 (130Pu, 0.107Au respectively) (p<0.001). CD105 demonstrated the same trend (p<0.02). Conclusions: We suggest a close relationship between intraepidermal innervation and melanocyte re-population with direct relevance to re-pigmentation post-scarring. These findings enhance our understanding and may lead to the development of objective classification of melanocytic re-population and skin re-innervation in response to scar therapies.

H3.01

COMPARISON OF PSEUDOMONAS AERUGINOSA BIOFILM INFECTION IN PARTIAL- AND FULL-THICKNESS BURN WOUNDS
Kenneth S. Brandenburg, Alan J. Weaver, Jr, Liwu Qian, Tao You, Ping Chen, SL Rajasekhar Karna, Shaina L. Van Stryk, Uzziel Pineda, Kai P. Leung
US Army Institute of Surgical Research, JBSA FT Sam Houston, TX, USA
COMPARISON OF PSEUDOMONAS AERUGINOSA BIOFILM INFECTION IN PARTIAL- AND FULL-THICKNESS BURN WOUNDS

Kenneth S. Brandenburg, Alan J. Weaver, Jr, Liwu Qian, Tao You, Ping Chen, SL Rajasekhar Karna, Shaina L. Van Stryk, Uzziel Pineda, Kai P. Leung
US Army Institute of Surgical Research, JBSA FT Sam Houston, TX, USA

Background: From 2001-2011, more than 10% (419) of soldiers evacuated from combat theatres with craniomaxillofacial battle injuries presented with burns. Due to its inherent virulence, antimicrobial resistance, and ability to form biofilms, the opportunistic pathogen Pseudomonas aeruginosa represents one of the greatest challenges in burn care. Methods: In this study, we compared biofilm formation by P. aeruginosa in both deep partial- and full-thickness burns. Scald wounds, ~10% of the total body surface area, were created in anesthetized male Sprague-Dawley rats (350-450g; n=144). Immediately post-burn, clinical P. aeruginosa strain 12-4-4(59) was spread over the burned skin. Animals were euthanized and tissue collected for CFU counts, biofilm gene expression, and host response to burn infection on days 1, 3, 7, and 11 post-burn. Results: P. aeruginosa developed robust wound infections (~1×10^9 CFU/g tissue) in both types of burn wounds. Expression of biofilm matrix genes and virulence genes indicated formation of mature P. aeruginosa biofilms within the burned skin regardless of burn depth. However, neutrophil activity was diminished in full-thickness as compared to partial-thickness burn infections. Conclusions: Development of biofilms within burn wounds, coupled with lack of easily deployable anti-biofilm therapies, remains a serious threat to the injured soldier. These new in vivo biofilm-infection models provide a clinically relevant vehicle for testing novel antimicrobial treatments. 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.

H3.02

STAPHYLOCOCCUS EPIDERMIS AND STAPHYLOCOCCUS AUREUS DIFFERENTIALY MODULATE ANTIMICROBIAL MOLECULE PERFORIN-2 DURING WOUND HEALING
Vivien Chen, Irena Pastar, Cheyanne R. Head, Katelyn O’Neill, Jamie Burgess, Natasa Strbo, Marjana Tomic-Canic
University of Miami Miller School of Medicine, Miami, FL, USA
STAPHYLOCOCCUS EPIDERMIS AND STAPHYLOCOCCUS AUREUS DIFFERENTIALY MODULATE ANTIMICROBIAL MOLECULE PERFORIN-2 DURING WOUND HEALING

Vivien Chen, Irena Pastar, Cheyanne R. Head, Katelyn O’Neill, Jamie Burgess, Natasa Strbo, Marjana Tomic-Canic
University of Miami Miller School of Medicine, Miami, FL, USA

Perforin-2 (P-2) is an antimicrobial protein with unique properties to kill intracellular bacteria, however its role in innate immune response to cutaneous pathogenic or beneficial microorganisms is largely unknown. We investigated P-2 expression pattern and cellular distribution in human skin, acute wound healing and in response to pathogenic Staphylococcus aureus or skin commensal S. epidermis. For this purpose we have established and utilized a novel approach for the measurement of P-2 mRNA within individual skin cells using an amplified fluorescence in situ hybridization (FISH) technique. The unique aspect of this approach is simultaneous detection of P-2 mRNA with immune-phenotyping for cell surface proteins. P-2 transcript was detected in both CD45+ and CD45- cutaneous cell populations. The highest level of P-2 was found in basal keratinocytes and gamma delta (GD) T cells. Utilizing human ex vivo wound model we show that wounding induces P-2 with the pick of expression at 48 h post wounding. In order to evaluate P-2 response to commensal and pathogenic bacteria, we developed human ex vivo wound infection model. The human tissue was sterilized to remove existing microflora, wounded and infected with either S. aureus or S. epidermidis. Infection of human wounds by S. aureus resulted in P-2 suppression predominantly in CD104+ keratinocytes, while in vitro P-2 overexpression resulted in reduction of intracellular bacterial load. In contrast to suppression by pathogenic bacteria, infection of human skin with commensal S. epidermis led to upregulation of P-2 and increase of the GDT cells frequency. Our findings reveal a novel P-2 mediated mechanism by which skin commensal bacteria may exert their beneficial role in modulating host innate immune response. In contrast to commensal bacteria, skin pathogens may escape cutaneous immunity through suppression of P-2 to cause persistent wound infections. Our ongoing studies focus on modulation of cutaneous immunity by commensal bacteria to prevent skin and wound infections.

H3.03

SELF-LOCOMOTIVE MICROBUBBLER FOR ACTIVE BIOFILM REMOVAL
Hyunjoon Kong, Yongbeom Seo, Yu-Heng Deng, Yu-Tong Hong
University of Illinois at Urbana-Champaign, Urbana, IL, USA
SELF-LOCOMOTIVE MICROBUBBLER FOR ACTIVE BIOFILM REMOVAL

Hyunjoon Kong, Yongbeom Seo, Yu-Heng Deng, Yu-Tong Hong
University of Illinois at Urbana-Champaign, Urbana, IL, USA

Bacterial cells form a biofilm on and within any materials and living tissues, negatively impacting human health and sustainability. Tremendous efforts have been conducted to remove biofilms from the substrates using various antibiotics and disinfectants. However, bacterial cells residing in biofilms are deemed 100 to 1,000 times more resistant to antibiotics and disinfecting agents than planktonic cells because the extracellular polymeric substances (EPS) matrix limits the transport of the anti-microbial agents and neutralizes them chemically. Therefore, removing biofilm remains a grand challenge despite tremendous efforts made so far, particularly when they are formed in confined spaces. To overcome this challenge, we present a bubbling microparticle that can actively penetrate and rupture biofilm matrix in confined spaces by generating, fusing, and bursting microbubbles. The bubbling microparticle was fabricated by doping manganese oxide (MnO2) nanosheets onto porous diatom silica particles. In antiseptic hydrogen peroxide (H2O2) solution, the MnO2 nanosheet-doped diatom became self-motile by generating oxygen gas bubbles. Subsequently, the diatom microbubblers infiltrated the biofilm structure. Within the EPS matrix, the diatom bubblers keep producing micro-sized bubbles that merged and, in turn, converted surface energy to mechanical energy high enough to fracture the biofilm. As a result, H2O2 molecules efficiently diffused into the biofilm and killed bacterial cells.

H3.04

N-ACETYL CYSTEINE TREATMENT DISMANTLES CHRONIC WOUND BIOFILM
Xin Cathy Li, Jane Kim, Jiabin Wu, Yinsheng Wang, Manuela Martins-Green
University of California Riverside, Riverside, CA, USA
N-ACETYL CYSTEINE TREATMENT DISMANTLES CHRONIC WOUND BIOFILM

Xin Cathy Li, Jane Kim, Jiabin Wu, Yinsheng Wang, Manuela Martins-Green
University of California Riverside, Riverside, CA, USA

Background: Chronic wounds affect many people worldwide. In developed countries, 1-2% of the population experience chronic wounds causing a significant financial and psychological burden to individuals and economic burden to the society. Persistent biofilm in chronic wounds leads to microbiome antimicrobial resistance and significantly delays wound healing. Despite the existence of many different treatments, none are effective in dismantling biofilm and simultaneously killing the bacteria. Method: We have previously shown that N-acetyl-cysteine (NAC) can reverse chronic wound biofilm formation in vivo. We hypothesize that NAC creates a microenvironment that affects bacterial survival and interferes with the integrity of the extracellular polymeric substances (EPS) of the biofilm. To test this hypothesis, we developed an in vitro biofilm system using microbiome taken directly from chronic wounds, which primarily contains Pseudomonas aeruginosa, a bacterium that is commonly found in human chronic wounds. Results: We show that treatment of the biofilm with NAC at concentrations with a pH

H3.05

Ossabaw Pigs As A Natural Preclinical Model Of Metabolic Syndrome Mediated Impaired Wound Healing
Mithun Sinha1, Nandini Ghosh2, Shomita Steiner1, Amitava Das1, Savita Khanna1, Daniel J. Wozniak2, Sashwati Roy1, Chandan K. Sen1.
1Indiana University, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA
OSSABAW PIGS AS A NATURAL PRECLINICAL MODEL OF METABOLIC SYNDROME MEDIATED IMPAIRED WOUND HEALING

Mithun Sinha1, Nandini Ghosh2, Shomita Steiner1, Amitava Das1, Savita Khanna1, Daniel J. Wozniak2, Sashwati Roy1, Chandan K. Sen1
1Indiana University, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA

Background: Infected chronic wounds in the metabolic syndrome (MetSyn) patients represent a major public health burden. The current study recognizes Ossabaw swine as a powerful pre-clinical experimental model to study mechanisms of impaired wound healing under conditions of metabolic syndrome. Objective: To study wound healing in high fat diet induced MetSyn model of Ossabaw pigs. Methods: Ossabaw pigs (n=20) were fed either high fat diet (HFD; n=15) or standard chow (n=5) for 8 months. Following 8 months of diet, full thickness (2″x2″) excisional wounds were done on the dorsum of the pigs that were infected with 10^8 cfu/ml of mixed species of Pseudomonas aeruginosa PA01, Staphylococcus aureus USA300 and Acinetobacter baumannii 19606 strains. The infected wounds were followed up to day 31 post-wounding. Results: The HFD Ossabaw pigs developed pre-MetSyn symptoms including dyslipidemia, abdominal obesity and high blood pressure. In HFD pigs, the wounds associated void space was filled with adipose tissue. Further, they showed increased expression of adipocyte marker perilipin in the granulation tissue. Pigs on HFD showed exaggerated and persistent inflammation in their wounds. HFD pigs showed lower abundance of endothelial cells in the granulation tissue pointing towards impaired vascularization (n=5, p<0.05). Masson's trichrome staining of the wound bed showed reduced collagen levels in HFD pigs. Reduced levels of fibroblast marker, FSP1, in HFD pigs is characteristics of disorganized granulation tissue. The length of rete ridge were significantly reduced in the repaired skin of HFD pigs (n=5, p<0.05) predicting compromised biomechanical properties of the repaired skin. Conclusion: HFD induced MetSyn in Ossabaw pigs with characteristics comparable to clinical outcomes. Specific mechanisms of cutaneous wound healing were compromised in HFD Ossabaw pigs. Thus this model offers an outstanding opportunity to study the cellular and molecular bases of healing outcomes in metabolic syndrome like obesity.

H3.06

BIOFILM INFECTION DYSREGULATES CERAMIDE METABOLISM COMPROMISING FUNCTIONAL WOUND CLOSURE OF THE SKIN
Nandini Ghosh1, Mithun Sinha, PhD2, Shomita Steiner, PhD2, Dayanjan S. Wijesinghe, PhD3, Savita Khanna, PhD2, Daniel J. Wozniak, PhD1, Gayle M. Gordillo, MD2, Sashwati Roy, PhD2, Chandan K. Sen, PhD2
1The Ohio State University, Columbus, OH, USA, 2Indiana University, Indianapolis, IN, USA, 3Virginia Commonwealth University, Richmond, VA, USA
BIOFILM INFECTION DYSREGULATES CERAMIDE METABOLISM COMPROMISING FUNCTIONAL WOUND CLOSURE OF THE SKIN

Nandini Ghosh1, Mithun Sinha, Ph.D2, Shomita Steiner, Ph.D2, Dayanjan S. Wijesinghe, Ph.D3, Savita Khanna, Ph.D2, Daniel J. Wozniak, Ph.D1, Gayle M. Gordillo, M.D.2, Sashwati Roy, Ph.D2, Chandan K. Sen, Ph.D2
1The Ohio State University, Columbus, OH, USA, 2Indiana University, Indianapolis, IN, USA, 3Virginia Commonwealth University, Richmond, VA, USA

Background: Cutaneous lipids, 50% of which are ceramides (Cer), have structural and signaling roles in skin. The current study is based on our previous finding that biofilm infected wounds may appear visually closed but remain functionally open because of lack of barrier function of the repaired skin. Such defectively closed wounds display high trans-epidermal water loss (TEWL). The objective of this study was to test whether cutaneous ceramide depletion following wound biofilm infection compromises skin barrier function. Methods: Full thickness burn wounds (2″×2″) were created on pigs and followed up to 56 days post-wounding with or without infection of Pseudomonas aeruginosa or Pseudomonas ceramidase mutant strains. Analyses of wound closure (digital planimetry), skin barrier function (TEWL), ceramide levels (lipidomics), PPARδ, ABCA12, expression (IHC, quantitative RT-PCR) was performed. Results: Bacterial ceramidases were over expressed (~500 fold, n=4, p<0.05) in biofilm-infected pig wound tissues. Immunohistochemical studies demonstrated that biofilm-infection caused focal erosion of ceramides in the porcine skin. Lipidomic analyses showed that long chain ceramides were depleted in biofilm-infected wounds. Such depletion of ceramide was however absent in biofilm-infection caused by ceramidase mutant of Pseudomonas. Depletion of cutaneous ceramides downregulated the transcription factor PPARδ resulting in compromised transport of lipid molecules to stratum corneum by ABCA12, a lipid transporter. We further observed that such depletion of ceramide could be rescued by bacterial ceramidase inhibitor ceramidastin (n=5, p<0.05). Conclusion: Excessive microbial ceramidases in biofilm infected wounds deplete host skin ceramides. Such disruption of ceramide homeostasis in the skin causes compromised barrier function of the skin. Considering that two-thirds of all chronic wounds are estimated to be biofilm infected, this study provides a mechanistic insight to the observation that biofilm infected wounds remain functionally open.

H4.01

A MICROBIOME AND METABOLOMIC SIGNATURE OF PHASES OF CUTANEOUS HEALING IDENTIFIED BY PROFILING SEQUENTIAL ACUTE WOUNDS OF HUMAN SKIN
Mohammed Ashrafi, Yun Xu, Howbeer Muhamadali, Iain White, Max Wilkinson, Katherine Holloywood, Mohamed Baguneid, Royston Goodacre, Ardeshir Bayat
The University of Manchester, Manchester, United Kingdom
A MICROBIOME AND METABOLOMIC SIGNATURE OF PHASES OF CUTANEOUS HEALING IDENTIFIED BY PROFILING SEQUENTIAL ACUTE WOUNDS OF HUMAN SKIN

Mohammed Ashrafi, Yun Xu, Howbeer Muhamadali, Iain White, Max Wilkinson, Katherine Holloywood, Mohamed Baguneid, Royston Goodacre, Ardeshir Bayat
The University of Manchester, Manchester, United Kingdom

Background: Profiling skin microbiome and metabolome has been utilised to gain further insight into wound healing processes, although knowledge of the metabolic profile of wounds and their role in wound healing processes is limited. The aims of this multi-part temporal study in 11 volunteers were to analytically profile the dynamic wound tissue and headspace metabolome and sequence microbial communities in acute wound healing at days 0, 7, 14, 21 and 28, and to investigate their relationship to wound healing, using non-invasive quantitative devices. Methods: Subjects had four 5-mm diameter skin biopsies to their arms. Metabolites were obtained using tissue extraction, sorbent and polydimethylsiloxane patches and underwent thermal desorption and were then separated by gas chromatography and detected by mass spectrometry. Metabolites were tentatively identified using the National Institute of Standards and Technology and Golm libraries. Participants wound tissue samples underwent DNA extraction and 16S rDNA sequencing. Spectrophotometric intracutaneous analysis, full-field laser perfusion imaging, Dermalab system and dynamic optical coherence tomography provided non-invasive quantitative measurements of melanin, haemoglobin, collagen, blood flow, transepidermal water loss, hydration and erythema. Results: Principal component analysis of wound tissue metabolome clearly separated time points with 10 metabolites of 346 being involved in separation. Analysis of variance-simultaneous component analysis identified a difference between the wound headspace metabolome, sites (P=0.0024) and time points (P<0.0001), with 10 metabolites of 129 measured were associated with separation between sites and time points. A reciprocal relationship between Staphylococcus spp. and Propionibacterium spp. was observed at day 21 (P<0.05) with a correlation between collagen and Propionibacterium (r=0.417; P=0.038) and Staphylococcus (r=-0.434; P=0.03). Procrustes analysis showed a significant similarity between wound headspace and tissue metabolome with non-invasive wound devices. Conclusions: This study demonstrates the temporal and dynamic nature of acute wound metabolome and microbiome presenting a novel class of biomarkers that correspond to wound healing.

H4.02

PILOT CLINICAL STUDY TO DEMONSTRATE THE FEASIBILITY OF MONITORING SUBCUTANEOUS BLOOD FLOW AMONG PERI-OPERATIVE SURGERY PATIENTS
Michael Neidrauer, Alec Lafontant, Vaishali Purohit, Elizabeth Mintz, Kaitlin Kelly, Dhriti Dedhia, Nataly Bingham, Brendan K. McCracken, Leonid Zubkov, Peter A. Lewin, Rose Ann DiMaria-Ghalili, Michael S. Weingarten
Drexel University, Philadelphia, PA, USA
PILOT CLINICAL STUDY TO DEMONSTRATE THE FEASIBILITY OF MONITORING SUBCUTANEOUS BLOOD FLOW AMONG PERI-OPERATIVE SURGERY PATIENTS

Michael Neidrauer, Alec Lafontant, Vaishali Purohit, Elizabeth Mintz, Kaitlin Kelly, Dhriti Dedhia, Nataly Bingham, Brendan K. McCracken, Leonid Zubkov, Peter A. Lewin, Rose Ann DiMaria-Ghalili, Michael S. Weingarten
Drexel University, Philadelphia, PA, USA

Purpose: The purpose of this study was to demonstrate the feasibility of using non-invasive optical measurements of local microcirculatory blood flow as a screening tool for pressure injury (PI) among peri-operative surgery patients. Background: Surgery is a risk factor for PI, and an estimated 9-12% of PIs originate as deep tissue pressure injury (DTPI) beneath the skin’s surface. DTI is not clinically apparent until it spreads through subcutaneous tissue and into the skin. By this time, the damage may be too extensive to avoid advanced ulceration. We previously reported that blood flow in dermal and subcutaneous tissue, measured using non-invasive optical diffuse correlation spectroscopy (DCS), could predict the development of advanced (Stage 2-4 or unstageable) sacral PIs prior to clinical appearance in a rehabilitation hospital setting. Methods: We enrolled 8 surgical patients (3 male, 5 female, 37-70 years of age) and measured subcutaneous blood flow from the sacral, ischial, and scapular regions immediately before and after surgery. Duration of surgical procedure, duration of anesthesia, patient positioning, medications, and other clinical, nutritional, and metabolic data were gathered. Results: Changes in pre-operative vs. post-operative sacral blood flow for subjects whose duration of surgery was greater than three hours (n=4) were significantly greater than blood flow changes in subjects in surgery for <3 hours (p<0.01). Conclusions: This study demonstrated the feasibility of using DCS to monitor changes in microcirculatory blood flow before and after surgery, and will inform the design of a larger clinical study of the effectiveness of DCS for early detection of DTPI among peri-operative patients. Acknowledgements: This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, under Award No. W81XWH-14-1-0614. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Department of Defense.

H4.03

PERIVASCULAR ADIPOSE TISSUE CONTROLS VASODILATATION AND TISSUE PERFUSION IN VIVO THROUGH ADIPOMUSCULAR MICROVASCULAR ANASTOMOSES
Alexander Turaihi, Carla Molthoff, Jasper Koning, Marie Jose Goumans, Connie Jimenez, John Yudkin, Yvo Smulders, Victor van Hinsbergh, Ed Eringa
Amsterdam University Medical Centers, Amsterdam, Netherlands
PERIVASCULAR ADIPOSE TISSUE CONTROLS VASODILATATION AND TISSUE PERFUSION IN VIVO THROUGH ADIPOMUSCULAR MICROVASCULAR ANASTOMOSES

Alexander Turaihi, Carla Molthoff, Jasper Koning, Marie Jose Goumans, Connie Jimenez, John Yudkin, Yvo Smulders, Victor van Hinsbergh, Ed Eringa
Amsterdam University Medical Centers, Amsterdam, Netherlands

Background: Vasodilation regulates delivery of substrates to tissues undergoing regeneration and growth. Perivascular adipose tissue (PVAT) controls vascular function through outside-to-inside communication and through vessel-to-vessel, or “vasocrine” signaling. We studied this hypothesis in mice by examining effects of surgical removal of local intramuscular PVAT on muscle blood flow (MBF) and glucose uptake. Methods: In lean C57/Bl6 mice we removed PVAT from the gracilis and femoral arteries. Mice underwent combined contrast-enhanced ultrasonography (CEUS) and Intravital Microscopy (IVM) to measure MBF and arteriolar diameter. PVAT vasodilator capacity was examined ex vivo using pressure myography. Local muscle glucose uptake was examined using positron emission tomography in vivo. Further, we used proteomic and microscopy experiments to understand the nature of the interaction between PVAT and the vessels. Results: Local PVAT removal reduces muscle glucose uptake by 50 percent. Ex vivo, gracilis artery diameter increased in the presence of PVAT (26.2%±25%; p=0.03) but not in absence of PVAT. CEUS and IVM of gracilis artery showed that PVAT removal abolishes increases in arterial diameter (2.0%±7% vs. 14.5%±6%) and abrogated insulin-stimulated increase in muscle blood volume (microvascular recruitment or IMVR; -4.8%±7% vs. 35.7%±31%). The effect of PVAT on IMVR was mediated by distinct microvessels or anastomoses, which we showed using lightsheet microscopy of mice expressing mCherry in endothelial cells. Proteomics analysis revealed that PVAT removal significantly alters expression of 109 of 1719 detected proteins in muscle. Observed changes in protein expression included reduction of a mitochondrial protein cluster and of vesicle-associated membrane protein 5 (Vamp5), involved in Glut4 trafficking. Conclusion: We have found that PVAT within muscle regulates muscle perfusion, glucose uptake and muscle protein expression, communicating with the distal microcirculation via microvascular anastomoses. These data highlight the importance of PVAT in vascular and metabolic physiology, relevant for tissue regeneration and wound healing.

H4.04

PROINFLAMMATORY ROLES OF FIBROBLASTS UNDER DEHYDRATION IN OPEN WOUNDS
Ping Xie, David Dolivo, Seok Hong, Robert D. Galiano, Thomas A. Mustoe
Northwestern university, Chicago, IL, USA
PROINFLAMMATORY ROLES OF FIBROBLASTS UNDER DEHYDRATION IN OPEN WOUNDS

Ping Xie, David Dolivo, Seok Hong, Robert D. Galiano, Thomas A. Mustoe
Northwestern University, Chicago, IL, USA

Backgrounds: Wound healing is a complex multicellular process involving a series of overlapping phases, including coagulation, inflammation, granulation tissue formation, and remodeling. The presence of a prolonged inflammatory phase has been demonstrated during wound repair under dry conditions, concomitant with a delay in the wound healing process. Disruption of dermal integrity leads to evaporative water loss, which subsequently results in an increase in extracellular sodium concentration. We have previously demonstrated that water loss or high salt treatment promotes an inflammatory responses in keratinocytes, which is partially mediated through the sodium channel NaX. In addition to their role in the synthesis of extracellular matrix, fibroblasts, as the predominant resident cell type in wounds, are able to produce and release various pro-inflammatory cytokines when they encounter external stimuli. In this study we investigated whether fibroblasts are involved in the inflammatory response under dry condition during wound healing. Methods and Results: Here we show that exposure to air (dehydration) induced upregulation of pro-inflammatory cytokines IL-8 and Cox-2 in non-epithelialized wounds on rabbit ears, concomitant with increased infiltration of neutrophils. Ex vivo, the granulation tissue containing numerous fibroblasts was excised from wounds and exposed to a 10% increase in sodium within the culture media (high salt treatment), under which the expression of IL8 and PTGS2 (the gene encoding COX-2) was upregulated. In vitro, high expression of NaX was detected in fibroblasts. Exposure of fibroblasts to high salt culture media led to an increase of expression of IL8 and PTGS2, which was able to be blocked by knockdown of NaX using shRNA. Conclusions: These data suggest that the local dehydration stimulus to open wounds increased inflammation in fibroblasts via NaX activation and signaling.

H4.05

SILK FIBROIN-HISTAMINE DRESSING ACCELERATES HEALING IN FULL THICKNESS MICE WOUNDS
Deepanjan Ghosh1, Sudhakar Godeshala1, Jacquelyn Kilbourne1, David J. Dicaudo2, Kaushal Rege1
1Arizona State University, Tempe, AZ, USA, 2Mayo Clinic, Scottsdale, AZ, USA
SILK FIBROIN-HISTAMINE DRESSING ACCELERATES HEALING IN FULL THICKNESS MICE WOUNDS

Deepanjan Ghosh1, Sudhakar Godeshala1, Jacquelyn Kilbourne1, David J. Dicaudo2, Kaushal Rege1
1Arizona State University, Tempe, AZ, USA, 2Mayo Clinic, Scottsdale, AZ, USA

Wound healing typically follows a programmed sequence which involves hemostasis, inflammation, proliferation and remodeling. In this work, we show local delivery of histamine, an immune modulator in combination with silk fibroin (SF) film resulting in faster closure of wounds in BALB/c mice when compared to wounds closed with conventional wound dressing (Tegaderm). Histamine was applied topically on 3.5 mm full thickness wounds and covered with either Tegaderm or a SF-gold nanorods (GNRs) film and irradiated with 800 nm near infrared laser (NIR) laser. Significant reduction in wound area and improved tissue biomechanical properties was observed in histamine treated wounds. SF-GNRs film-histamine treated wounds showed complete wound closure (p-value <0.0001, n=10) and higher tissue strength (p-value <0.01, n=6) compared to Tegaderm-histamine treated wounds at day 7 post-wounding. Immunohistological analyses also showed that SF-GNRs film-histamine treatment promoted angiogenesis (CD31+ cells, p-value <0.05, n=4) and myofibroblast mediated wound contraction (aSMA+, p-value <0.05, n=4). This also resulted in the increased epidermal thickness (Pan-cytokeratin, p-value <0.01, n=4) and reduced proliferation (Ki67+ cells, p-value <0.01, n=4) compared to Tegaderm-histamine treated wounds. IL-1beta and IL-6 levels in serum samples showed significant increased at day 1 post wounding and reduced to basal level at day 3 in SF-GNRs-histamine treated wounds showing a robust early inflammatory response to set the stage for subsequent proliferation and remodeling. We conclude that SF-GNRs film was effective in promoting wound healing and in combination with single histamine dose it outperformed clinically approved polyurethane wound dressing.

H4.06

CLINICAL EVALUATION OF MICROBIAL AND METABOLITE CHANGES IN MINOR WOUND HEALING
Latrisha K. Petersen, InSeok Seo, Erin Zaleski, Parneet Kaur, Kimberly Capone, Melinda Cettina, Paul Zhang, Robert J. Gambogi, Amisha Parikh-Das
Johnson & Johnson, Skillman, NJ, USA
CLINICAL EVALUATION OF MICROBIAL AND METABOLITE CHANGES IN MINOR WOUND HEALING

Latrisha K. Petersen, InSeok Seo, Erin Zaleski, Parneet Kaur, Kimberly Capone, Melinda Cettina, Paul Zhang, Robert J. Gambogi, Amisha Parikh-Das
Johnson & Johnson, Skillman, NJ, USA

Wound healing research has primarily focused on chronic or surgical wounds. There is a need for greater understanding of the minor wound and changes that occur with the biophysical and biological environments during its healing cycle. These environmental changes may impact the likelihood of infection, time of healing, tensile strength of healed wounds, scarring, and overall skin health. This clinical study was conducted to better understand biophysical and metabolite changes of the minor wound using novel methods in wound research. This single center, 15-day, clinical trial enrolled 35 healthy subjects. Each subject had 5 tape-stripped minor wounds created on their backs; each wound was randomized to coverage with one of 5 different marketed adhesive bandages or was left uncovered. On average, wounds covered with Band-Aid® Brand Adhesive Bandages resulted in significantly less water loss and faster recovery of redness (oxyhemoglobin level) in the wound as compared to uncovered wounds. This correlated very well with the metabolomics analysis which revealed that covered wounds had decreased metabolites associated with oxidative stress, lipid inflammation, cellular dehydration and collagen turnover. These improved biophysical and metabolite outcomes resulted in most Band-Aid® Brand Adhesive Bandage-covered wounds trending towards faster wound closure than uncovered wounds. In summary, this study identified key differences in wound healing of covered vs. uncovered minor wounds including cellular metabolites and environmental factors such as pH, water loss and oxyhemoglobin levels. These findings provide new insight into our understanding of the minor wound and highlight key features that could be targeted to provide a more optimal healing environment.

K1.01

ADIPOSE STEM CELL TISSUE SOURCE IS ASSOCIATED WITH DIFFERENTIAL EXPRESSION PROFILES IN CELLULAR MRNA AND EXOSOMAL MIRNA
Lauren Mangum1, Randolph Stone, II1, Shanmugasundaram Natesan1, Barbara Christy1, John Clifford2, Raina Kumar2, Andrew Cap1, Rasha Hammamieh2, Robert Christy1
1US Army Institute of Surgical Research, Fort Sam Houston, TX, USA, 2US Army Center for Environmental Health Research, Fort Detrick, MD, USA
ADIPOSE STEM CELL TISSUE SOURCE IS ASSOCIATED WITH DIFFERENTIAL EXPRESSION PROFILES IN CELLULAR MRNA AND EXOSOMAL MIRNA

Lauren Mangum1, Randolph Stone, II1, Shanmugasundaram Natesan1, Barbara Christy1, John Clifford2, Raina Kumar2, Andrew Cap1, Rasha Hammamieh2, Robert Christy1
1US Army Institute of Surgical Research, Fort Sam Houston, TX, USA, 2US Army Center for Environmental Health Research, Fort Detrick, MD, USA

Background: Severe traumatic injuries, including large total body surface area burns, limit the availability of autologous cells for wound repair. Adipose derived stem cells (ASCs) represent a potential source of autologous cells for use in reconstructive surgery and tissue engineering. We sought to better understand the role of ASC source on differential expression patterns of mRNA and miRNA to identify alterations to major signaling pathways. Methods: ASCs from subcutaneous adipose tissue associated with burned human (BH) or human abdominoplasty (HAP) skin were isolated under a protocol reviewed and approved by the Institutional Review Board. Commercially available ASCs were purchased from RoosterBio (RB). Cellular mRNA and exosomal miRNA were isolated from cell monolayers and culture media, respectively. Samples were submitted for sequencing and bioinformatics analysis. Significant differences in mRNA and miRNA expression was reported in log fold change at a threshold of p<0.05. Results: Comparing the cellular mRNA expression patterns, 202, 361, and 598 unique genes were identified for HAP, BH, and RB, respectively. When compared to RB, TGF-B, Beta-estradiol, and APP were identified as top upstream regulators for both HAP and BH. Analysis of exosomal contents revealed a small subset of differentially expressed miRNAs (6.5%) between HAP and BH groups. A larger subset of differentially expressed miRNAs was found between BH or HAP compared to RB ASCs (8.68% and 8.53%, respectively). Conclusions: Sequencing of mRNA and miRNAs from ASCs reveals source-dependent differences in several thousand genes governing diverse processes including cell cycle and proliferation. Analysis of mRNA and miRNA profiles indicated HAP and BH were more similar to each other than to RB. Preliminary analysis indicates tissue source, isolation procedures, and culturing conditions should be considered when developing stem cells or stem cell derived products for use in wound healing.

K1.02

WOUND HEALING POTENTIAL OF HUMAN IPSC-DERIVED VASCULAR SMOOTH MUSCLE CELLS
Henry Hsia, Biraja Dash, Ocean Setia, Hassan Peyvandi, Lara Lopes, James Nie, Alan Dardik
Yale School of Medicine, New Haven, CT, USA
WOUND HEALING POTENTIAL OF HUMAN IPSC-DERIVED VASCULAR SMOOTH MUSCLE CELLS

Henry Hsia, Biraja Dash, Ocean Setia, Hassan Peyvandi, Lara Lopes, James Nie, Alan Dardik
Yale School of Medicine, New Haven, CT, USA

Background: While proangiogenic cell therapy has already been shown to promote healing, their lack of availability in abundance is a major limitation. In this current study, we investigated the wound healing potential of naïve vascular smooth muscle cells (VSMCs) derived from human induced pluripotent stem cells (hiPSC) embedded within collagen scaffolds with the aim of using iPSC-derived VSMCs to enhance angiogenesis and promote healing. Methods: VSMCs were efficiently differentiated from hiPSCs using an embryoid body method. Highly pure and functional hiPSC-VSMCs were embedded in a hypoxia-inducing rolled scaffold made using type I collagen. Unrolled flat scaffolds were also created as normoxic controls. Scaffolds were assessed for growth factor production using biochemical assays and also implanted onto a nude mouse splinted back acute wound model. Results: The hypoxia activated VSMCs in the scaffolds, demonstrating cell viability and maintenance of their phenotype. The VSMC scaffold released significantly greater levels of proangiogenic growth factors such as VEGF and IL-8 compared to control flat, normoxic VSMC scaffolds. Both hypoxic and normoxic scaffolds released paracrine factors such as bFGF, SDF-1α and Ang-1. In an in vivo splinted back acute wound model in nude mice, the rolled VSMC scaffolds showed enhanced endogenous vascularization and formation of chimeric vessels as well as better re-epithelialization and dermis regeneration compared to control scaffolds. Conclusions: These data suggest that hypoxic rolled VSMC scaffolds may be useful in promoting accelerated wound closure and warrant further investigation into their translational potential for promoting regenerative healing.

K1.03

METABOLIC ORCHESTRATION OF EPIDERMAL STEM CELLS AND ADIPOCYTES PROMOTES HEALING IN OBESITY WOUND
Ji LIN1, Xiao-ning GAO2
1Institute of Basic Medicine, Chinese PLA General Hospital, Beijing, China, 2Department of Hematology, Chinese PLA General Hospital, Beijing, China
METABOLIC ORCHESTRATION OF EPIDERMAL STEM CELLS AND ADIPOCYTES PROMOTES HEALING IN OBESITY WOUND

Ji Lin1, Xiao-ning GAO2
1Institute of Basic Medicine, Chinese PLA General Hospital, Beijing, China, 2Department of Hematology, Chinese PLA General Hospital, Beijing, China

Background: Wound healing is a world-wide health problem and the prominent obesity has made it profoundly difficult, for the unclear mechanism of obesity-impeded healing. As epidermal stem cells (EpiSCs) and adipocytes are vital elements in the structural, metabolic and healing process of skin, we hypothesize that the potent associations between them and the subsequent intervention may influence healing prognosis in the obesity. Methods: A model of high fat diet-induced obesity using Sprague-Dawley rats was established, including groups of sham-injury (Sham), injury without cell transplantation (Injury), and injury plus EpiSCs 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 EpiSCs rats, as equivalent 1×PBS was injected similarly in Sham and Injury rats. Skin wounds and peripheral blood samples were harvested after injury, and subjected to histological investigations and colorimetric detections. Results: At 1, 3 and 7 days after injury, significantly reduced scar, improved contraction, promoted re-epithelialization, repopulated perilipin A-positive adipocytes, and orderly increased collagen deposition in the wound bed were found in EpiSCs rats (P<0.05, vs Injury). Meanwhile, a down-and-up fluctuation of serum FABP5 and FABP4, two fatty acid binding protein subtypes secreted by epidermal cells and adipocytes respectively, was identified in EpiSCs rats at 1 and 3 days after injury, as serum FABP5:FABP4 ratios in EpiSCs rats were higher at 7 and 14 days after injury (P<0.05, vs Sham or Injury). Moreover, related alterations of metabolic markers such as serum lactate, pyruvate, lactate:pyruvate ratios, NAD+, NADH, NAD+:NADH ratios, insulin, triglycerides and total cholesterol, as well as immunohistochemical expressions of FABP5 and FABP4 in the wounds were identified. Conclusions: EpiSCs interact with adipocytes and evoke subsequent rehabilitation in obesity wound, indicating an underlying metabolic perspective for therapy.

K1.04

RETRO-ORBITAL INFUSION OF HUMAN MESENCHYMAL STROMAL CELLS ACCELERATES WOUND HEALING THROUGH SYSTEMIC EFFECTS
Nina Kosaric, Wai Srifa, Harriet Kiwanuka, Matthew Porteus, Geoffrey Gurtner
Stanford University, Stanford, CA, USA
RETRO-ORBITAL INFUSION OF HUMAN MESENCHYMAL STROMAL CELLS ACCELERATES WOUND HEALING THROUGH SYSTEMIC EFFECTS

Nina Kosaric, Wai Srifa, Harriet Kiwanuka, Matthew Porteus, Geoffrey Gurtner
Stanford University, Stanford, CA, USA

Background: Mesenchymal stromal cells (MSCs) are a promising therapeutic agent due to their pro-regenerative and immunomodulatory properties. Intravenous infusion of MSCs is desirable due to the minimal invasiveness of the procedure. A major obstacle of intravenous infusion is entrapment of MSCs in pulmonary capillaries after delivery, limiting the number of cells that reach their target site. Methods: Here, we use a murine excisional wound healing model to examine whether intravenous infusion of human fetal bone marrow-derived MSCs can accelerate wound healing. 1×10^6 MSCs or PBS were administered retro-orbitally on the day of wounding. Biodistribution of administered MSCs was evaluated in the lung, wound, blood, and spleen using FACS, and the rate of healing was quantified until full wound closure. Wounds were harvested 2 days following wounding and subjected to single cell RNA-Seq. Results: MSCs were significantly present in the lung 1 day following infusion (0.094 % ± 0.03 of live cells, n=3, p < 0.05), but undetectable in the wound, blood, and spleen during the first 3 days following infusion. Treated mice healed 3.8 days earlier (12.0 ± 0.80 days) than control mice (15.8 ± 0.44 days, n=5; p<0.001). We identified four transcriptionally distinct F4/80+ macrophage subpopulations within the wound. Distribution of clusters varied by treatment group: MSC-treated mice exhibited depletion of a cluster highly expressing pro-inflammatory genes (Cxcl10, Ifit2, Ly6c2), and enrichment of a cluster highly expressing genes involved in T cell suppression (Vsig4), B cell recruitment (Cxcl13), and genes expressed by activated macrophages (Arg1, Saa3). Conclusions: Despite a lack of evidence that retro-orbitally administered MSCs migrate beyond the lung, infusion of MSCs significantly accelerated wound healing, suggesting that they are able to potentiate a systemic therapeutic effect. This effect may be due to a shift from inflammatory to regenerative macrophages in the wound.

K1.05

FIBROBLASTS: CHRONICALLY BAD BUT NOT SO DOWN IN THE MOUTH
Phil Stephens
Cardiff University, Cardiff, United Kingdom
FIBROBLASTS: CHRONICALLY BAD BUT NOT SO DOWN IN THE MOUTH

Phil Stephens, PhD
Cardiff University, Cardiff, United Kingdom

Background: Chronic non-healing wounds are a major health problem within our ageing society. Such wounds are characterized by persistent inflammation, failed re-epithelialisation, a lack of granulation tissue formation and they are exacerbated by high levels of bacterial infection. Despite some clinical advances, 10-20% of chronic wounds still do not heal and thus novel therapeutic approaches are required. Our investigations have focussed on whether age-related functional changes in dermal fibroblasts may contribute to this dysfunctional healing phenotype. Methods & Results: Microarray analysis of chronic wound fibroblasts (CWFs) and normal skin fibroblasts (NFs) demonstrated altered regulation of numerous genes including molecules involved in the protection against oxidative stress. CWFs proliferate more slowly than NFs and premature senescence of CWFs was confirmed by increased SA beta-Gal activity and their larger, polygonal morphology. Analysis of telomere lengths revealed that whilst senescence in some CWFs was telomere-dependent in others it was telomere-independent. This premature senescence significantly decreased their abilities to carry out key wound healing activities. This is however, in direct contrast to fibroblasts isolated from wounds that heal in a scarless manner (oral mucosal fibroblasts; OMFs) that demonstrate differential gene analysis, increased proliferation, lower levels of senescence (longer telomeres) and accelerated wound healing responses. From our microarray data we have now described a transcriptional signature for this ‘wound healing continuum’ which may assist in the therapeutic assessment/treatment of a patient’s wound. Our recent findings however, suggest it is the presence of a progenitor cell sub-population resident within the heterogeneous oral stromal population that is key to the preferential healing. Such oral mucosal lamina propria-progenitor cells (OMLP-PCs) can be rapidly and clonally expanded in vitro, are neural crest-derived and are multipotent. Furthermore, they are potently immunosuppressive and have distinct antibacterial activities. Such activities may be delivered by a distinct population of OMLP-PC exosomes which are currently under investigation. Conclusions: We postulate that OMLP-PCs could be an efficacious cell-based therapy for the future amelioration of chronic wounds.

K1.06

THE EFFECT OF AN AUTOLOGOUS KERATINOCYTE SPRAY ON HUMAN SKIN WOUND HEALING TRANSCRIPTOME
Kristo Nuutila1, Antti Siltanen2, Matti Korhonen3, Johanna Nystedt3, Jussi Valtonen4, Andrew Lindford4, Jyrki Vuola4, Esko Kankuri2
1Brigham and Women’s Hospital, Boston, MA, USA, 2University of Helsinki, Helsinki, Finland, 3Finnish Red Cross Blood Service, Helsinki, Finland, 4Helsinki Burn Center, Helsinki, Finland
THE EFFECT OF AN AUTOLOGOUS KERATINOCYTE SPRAY ON HUMAN SKIN WOUND HEALING TRANSCRIPTOME

Kristo Nuutila1, Antti Siltanen2, Matti Korhonen3, Johanna Nystedt3, Jussi Valtonen4, Andrew Lindford4, Jyrki Vuola4, Esko Kankuri2
1Brigham and Women’s Hospital, Boston, MA, USA, 2University of Helsinki, Helsinki, Finland, 3Finnish Red Cross Blood Service, Helsinki, Finland, 4Helsinki Burn Center, Helsinki, Finland

Background: Most of the molecular-level wound-healing data have been obtained from various animal models. This may lead to misinformation and affect the translation of results to patient care. To overcome this issue, we have utilized the split-thickness skin graft donor site as a clinical wound model. The donor site wound offers an excellent opportunity for studying the mechanisms of wound healing because they are standardized, non-infected and clean. Importantly, serial small biopsy samples can be harvested without major ethical issues. The purpose of this study was to investigate the effect of an autologous keratinocyte cell therapy on wound healing transcriptome. Methods: Small skin biopsies were collected from burn patients undergoing split-thickness skin grafting, in Helsinki Burn Center. The biopsies were immediately delivered to Finnish Red Cross Blood Service where keratinocytes were isolated under GMP conditions. During the same operation, the cell suspension was delivered back to the burn center and sprayed on the donor site wound. Biopsies were collected on the intact skin and on days 7, 14 and 21 postoperatively with a 3-mm biopsy punch. Non-treated donor site wounds acted as controls. The harvested samples were processed for human full genome microarray and subsequently gene ontology enrichment analyses were performed. Results: The results demonstrated significant differences in gene profiles between different time points and treatments. E.g. when comparing the day 14 treated and non-treated donor sites the analyses showed that in total 19 gene pathways were significantly upregulated in cell spray treated wounds. These pathways included keratinization, keratinocyte differentiation, skin development, epidermis development, epidermal cell differentiation, cornification ja establishment of skin barrier, demonstrating on a molecular level that the cell therapy is promoting healing. Conclusions: This study provides the first objective molecular level information on the effect of cell therapy on human wound healing over time.

K2.01

A THERMOGELLING HYALURONIC ACID VAGINAL STENT TO REDUCE POSTOPERATIVE VAGINAL SCARRING
Omar Wyman1, Swathi Balaji2, Sundeep Keswani2, Julie Hakim2
1University of Oklahoma, Norman, OK, USA, 2Texas Children’s Hospital, Houston, TX, USA
A THERMOGELLING HYALURONIC ACID VAGINAL STENT TO REDUCE POSTOPERATIVE VAGINAL SCARRING

Omar Wyman1, Swathi Balaji2, Sundeep Keswani2, Julie Hakim2
1University of Oklahoma, Norman, OK, USA, 2Texas Children’s Hospital, Houston, TX, USA

Background: Vaginal fibrosis is a debilitating problem effecting 50,000 girls and 213,000 adult women yearly in the USA, who require vaginal reconstructive surgery with 73% experiencing postoperative fibrotic sequelae. Standard-of-care includes local delivery of conjugated estrogen (CEE) creams in combination with vaginal stents, which are ineffective. Previous work in our lab has shown that hyaluronic acid (HA)-based therapeutics reduce fibrosis and promote regenerative wound healing. The goal of this study is to determine the mechanisms of how hyaluronic (HA) and β-estradiol (E2)-based therapies regulate post-injury vaginal tissue repair. Methods: First time development of a minimally-invasive murine intra-vaginal model to study full-thickness vaginal wounds in pre-estrus (4wk) CD1 female mice. We compared the effect of daily local estrogen delivery with CEE cream to sustained estrogen delivery with a novel E2-eluding Norb-HA hydrogel on vaginal wound healing. Wounds were harvested at 6, 12, 18, 24, 48, 72, & 120h post-injury(n=6/group), analyzed for wound healing by morphometric analysis(H&E) and modified Vaginal Scar Scoring(VSS), inflammation and angiogenesis by Macrophage inhibiting factor 1(MIF1), TGFβ1 and VEGF (qRT-PCR), and ECM by qRT-PCR for Col1, Col3, MMP2 and MMP9. p-values by ANOVA. Results: The kinetics of murine vaginal wound healing demonstrated an accelerated healing profile with resolution of mucosal integrity by 48h with decrease in proximal acute inflammation assessed by VSS (p<0.01). A statistically significant decrease in MIF1 expression was found in wounds treated with our novel HA-hydrogel estrogen (Norb-HA/E2) delivery vehicle compared to CEE cream at day 10. Norb-HA/E2 treatment also increased TGFβ1, TIMP1 and COL3/COL1 ratio (p<0.01) by day 10, and decreased MMP9 by day 5 (p<0.01). Conclusions: It appears that murine mucosal vaginal tissue heals with an accelerated timeline. Our novel vaginal HA-based hydrogel with sustained E2 release improves both anti-inflammatory and indirect pro-angiogenic effects of exogenous E2, enhances vaginal tissue healing compared to local exogenous estrogen cream. Further development of this platform may provide substantial increase in efficacy of E2 delivery to vaginal tissue and reduce post-operative vaginal fibrosis.

K2.02

PIRFENIDONE ATTENUATES THE PROFIBROTIC CONTRACTILE PHENOTYPE OF HUMAN DERMAL MYOFIBROBLASTS
Adrienne R. Wells, Kai P. Leung
US Army Institute of Surgical Research, Fort Sam Houston, TX, USA
PIRFENIDONE ATTENUATES THE PROFIBROTIC CONTRACTILE PHENOTYPE OF HUMAN DERMAL MYOFIBROBLASTS

Adrienne R. Wells, Kai P. Leung
US Army Institute of Surgical Research, Fort Sam Houston, TX, USA

Background: Burn injury often results in thick, inflexible hypertrophic scarring with debilitating contractures if occurring across craniofacial regions and joints. This scarring develops from dysregulated wound healing with reduced extracellular matrix turnover, increased matrix production, and the persistence of profibrotic contractile myofibroblasts. These cells are transformed from normal fibroblasts by exposure to transforming growth factor beta 1 (TGF-β1). F-actin stress fibers populated with alpha smooth muscle actin (α-SMA) confer substantial contractile properties to differentiated myofibroblasts. Pirfenidone (Pf), an FDA-approved antifibrotic, significantly reduces the transdifferentiation of normal human dermal fibroblasts (NHDF) into myofibroblasts as shown in vitro with concurrent TGF-β1/Pf treatment. Here we tested the in vitro effects of Pf on the profibrotic phenotype and contractility of established myofibroblasts in 3D collagen lattices and 2D culture conditions. Methods: NHDF were differentiated into myofibroblasts by 3-5 day TGF-β1 treatment. A single dose of Pf was administered and samples were collected over time for immunocytochemistry, Western blot and qRT-PCR. F-actin stress fibers and α-SMA expression were quantified in confocal images. Cell contractility was assessed by treating stressed myofibroblast populated collagen lattices with Pf and measuring the area after release. Results: Pf treatment of established myofibroblasts reduced F-actin and α-SMA in contractile stress fibers. Pf treatment also returned to normal the reduced levels of matrix metalloproteinase gene expression (MMP1) found in myofibroblasts. Finally, Pf treatment reduced collagen lattice contraction. Conclusions: Pf is a potent antifibrotic with the potential to mitigate excess ECM accumulation and contraction in scars by modulating the structure and function of myofibroblast cells. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. 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).

K2.03

TOPICAL FOCAL ADHESION KINASE INHIBITOR PROMOTES SKIN REGENERATION AND SCAR PREVENTION IN A PRECLINICAL PORCINE MODEL
Sun Hyung Kwon
Stanford University, Stanford, CA, USA
TOPICAL FOCAL ADHESION KINASE INHIBITOR PROMOTES SKIN REGENERATION AND SCAR PREVENTION IN A PRECLINICAL PORCINE MODEL

Sun Hyung Kwon1, Britta Kuehlmann1, Teruyuki Dohi1, Artem A. Trotsyuk1, Michael S. Hu1, Mohammed Inayathullah2, Jayakumar Rajadas2, Michael T. Longaker1, Geoffrey C. Gurtner1
1Department of Surgery, Stanford University, Stanford, CA, 2Biomaterials and Advanced Drug Delivery Center, Stanford University, Stanford, CA

Background: Pharmacological inhibition of FAK attenuates hypertrophic scar (HTS) formation following deep dermal injury, however, clinical translation of FAK inhibitor (FAKI) therapy has been challenging. Here we evaluated the efficacy and safety of topical FAKI hydrogel therapy in a preclinical large animal model. Methods: Red Duroc pigs were used to create 8 deep partial-thickness wounds of 25 cm2 per animal. Animals received either standard dressings, blank hydrogel alone (placebo), or FAKI-releasing hydrogel dressings. Dressings and hydrogels were changed every 2 days until the wounds closed and then every 4 days thereafter. Wound closure rate, HTS formation, and regrowth of hair follicles and skin appendages were evaluated over 6 months. Results: FAKI-treated wounds closed significantly faster on day 14±2.3 vs. day 24±1.6 for control wounds vs. 24±0.8 for placebo wounds (N=8, p<0.01). Scars were dramatically reduced with FAKI hydrogel treatment compared to untreated control or placebo wounds. Lower Visual Analog Scale scores indicated improved scar appearance evaluated at Day 90 and at Day 180 (control VAS=100 vs. placebo 93±4.6 vs. FAKI 58±6.5, N=8, p<0.001). FAKI-treated wounds regenerated skin appendages including hair follicles and eccrine glands. Lastly, the collagen architecture of FAKI-treated wound was very similar to the unwounded pig skin while collagen organization of untreated control and placebo wounds appeared randomly disorganized. Conclusions: Topically delivered FAKI was effective in improving wound healing and promoting regeneration in a large animal, and holds great potential for wound and scar management of large and deep dermal wounds.

K2.04

KERATINOCYTE PROLIFERATION CORRELATES WITH THE EXTENT OF SCAR FORMATION
Hui Li1, Swathi Balaji1, Xinyi Wang1, Paul Bollyky2, Manish Butte3, Sundeep Keswani1
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA, 3UCLA, Los Angeles, CA, USA
KERATINOCYTE PROLIFERATION CORRELATES WITH THE EXTENT OF SCAR FORMATION

Hui Li1, Swathi Balaji1, Xinyi Wang1, Paul Bollyky2, Manish Butte3, Sundeep Keswani1
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA, 3UCLA, Los Angeles, CA, USA

Background: Postnatal wound healing leads to scar formation, however, there is variability in scarring extent among individuals after similar injuries. Underlying mechanisms that determine the scar phenotype in healthy adults remain unknown. We determined cellular differences between healthy patients exhibiting low or high scar-forming responses. Methods: We obtained skin samples from female patients undergoing abdominoplasty to remove cesarean scars and compared keratinocytes from scar tissue against those from matched normal skin. The Vancouver Scar Scale was used to classify patients into low (score 1-3) and high (score 6-9) scar-forming groups. Formalin fixed paraffin embedded tissue sections were analyzed by immunohistochemistry using the cell proliferation marker Ki67. Moreover, keratinocytes were dissected from frozen tissue sections by Laser Microdissection (LMD) to quantitate Ki67 mRNA by qRT-PCR. Results: We analyzed a pilot sample of n=3 low scar and n=3 high scar-forming tissues from our biobank. Immunohistochemical analysis showed that low scar-forming patients had 3.8-fold more proliferating keratinocytes in their normal skin than high scar patients (12%+/-2.6 vs. 3.17%+/-2.1 Ki67+ cells in the epidermis). qRT-PCR analysis showed that Ki67 expression in control skin of high scar-forming patients was 0.54-fold lower than low scar-forming patients. Comparing scar keratinocytes versus matched control skin keratinocytes, we observed a 0.45- and 0.43-fold decrease in Ki67 expression in low and high scar-forming patients, respectively. Conclusions: These data reveal differences in keratinocyte proliferation among patients with different scarring phenotypes, suggesting that keratinocyte proliferation may inversely affect the individual propensity for scar formation, mediated through their roles in wound closure and ECM production during wound healing.

K2.05

SERUM AND WOUND ENVIRONMENT C- REACTIVE PROTEIN, VIABLE BACTERIA AND SICKNESS BEHAVIOR SYMPTOMS IN NON-HEALING WOUNDS
Joyce Stechmiller, RN PhD, Debra Lyon, RN PhD, Daniel Gibson, PhD, Michael Weaver, RN PhD, Joie Whitney, RN PhD, Jung Kim, RN PhD, Gregory Schultz, PhD
University of Florida, Gainesville, FL, USA

K2.06

EXOSOMES DERIVED FROM DERMAL FIBROBLASTS DEFINE THE INTRINSIC VARIABILITY IN WOUND HEALING RESPONSES
Hima Vangapandu1, Natalie Templeman1, Daniel Colchado1, Alexander Blum1, Hui Li1, Xinyi Wang1, Emily Steen1, Paul Bollyky2, Sundeep Keswani1, Matthew Robertson1, Cristian Coarfa1, Swathi Balaji1
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA
EXOSOMES DERIVED FROM DERMAL FIBROBLASTS DEFINE THE INTRINSIC VARIABILITY IN WOUND HEALING RESPONSES

Hima Vangapandu1, Natalie Templeman1, Daniel Colchado1, Alexander Blum1, Hui Li1, Xinyi Wang1, Emily Steen1, Paul Bollyky2, Sundeep Keswani1, Matthew Robertson1, Cristian Coarfa1, Swathi Balaji1
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA

Wound responses involve fibroblast(FB)-mediated scar formation potentiated by mechanical tension. The heterogeneity observed in fibrosis in different individuals in response to similar injuries is yet to be accounted for. Exosomes play a pivotal role in mediating cell communication which governs scarring. We hypothesize that FB-derived exosomes drive differential scar forming abilities in response to tension, which contributes to the human heterogeneity in scarring responses. Skin and paired scar tissue was obtained from women with C-section scars who underwent abdominoplasty. Patient-scars were classified as ‘high’ and ‘low’ based on VSS. FBs isolated from scars and corresponding normal skin were cultured on silicone membranes +/-10% static strain (24hrs). Changes in proliferation(Ki67) and fibrogenic potential(fibrosis array) among low (n=3) and high scarrers (n=3) were determined. Exosomes isolated from these different FBs were evaluated using next generation sequencing(NGS), and adoptively transferred into SCID murine skin-wounds, and wound repair was evaluated. p-values by ANOVA. Scar FBs from high scarrers had higher proliferation than their normal counterparts, and conversely, normal FBs from low scarrers proliferated faster than their scar FBs (p<0.05). Expression of several profibrotic genes COL3A1, TGFb3, MYC and PDGFA were upregulated in high scarring normal FBs as compared to low scarrers. NGS revealed significant differences between the FB exosomal-miRNA from normal skin and scar of high and low scarrers, which were further pronounced under mechanical tension. More miRNAs were downregulated (n=54) in low scarring normal FBs than upregulated (n=11) in response to tension, whereas as similar numbers of miRNA were downregulated (n=41) as upregulated (n=31) in high scarrers. Several miRNAs changed in opposite directions in high versus low scarring normal-FBs under tension. Low and high-scar FB-derived exosomes induced a corollary severity in scarring in SCID wounds. Our results demonstrate that there are intrinsic differences in different individuals and how they respond to tension mediated by exosomes that could influence fibrogenic phenotype.

K3.01

RESULTS OF THE OPINION SURVEY FROM PEOPLE WITH WOUNDS
Lisa Gould1, Vickie Driver2, Jing Liu3, Peggy Dotson4
1South Shore Health Center for Wound Healing, Weymouth, MA, USA, 2Novartis Institute for Biomedical Research, Cambridge, MA, USA, 3Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 4Health Care Reimbursement Strategy, Wilmington, NC, USA
RESULTS OF THE ‘OPINION SURVEY FROM PEOPLE WITH WOUNDS’

Lisa Gould1, Vickie Driver2, Jing Liu3, Peggy Dotson4
1South Shore Health Center for Wound Healing, Weymouth, MA, USA, 2Novartis Institute for Biomedical Research, Cambridge, MA, USA, 3Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 4Health Care Reimbursement Strategy, Wilmington, NC, USA

Background: The 2006 FDA ‘Guidance for Industry Chronic Cutaneous Wounds and Burns – Developing Products for Treatment’ emphasizes wound closure as the primary outcome for clinical trials. Wound-care professionals understand that complete wound healing is not always appropriate or achievable when evaluating new, innovative treatments. AAWC and WHS are working collaboratively with FDA to identify scientifically achievable, clinically relevant, patient-centered endpoints with sufficient support to serve as primary outcomes for clinical trials. The recently published survey of 628 wound-care professionals identified and content-validated 15 potential primary endpoints. The ‘Opinion Survey from People with Wounds’ addresses an important gap: the correlation between clinicians’ perception and the patient perspective regarding clinically meaningful endpoints for wound care. Methods: The survey, adapted from the clinician survey with adjustment for health literacy, was pilot tested and revised based on a limited number of patients in a single clinic. After central IRB approval, the on-line survey was administered in English and Spanish and submitted anonymously to a secure server with cooperation of multiple wound clinics and societies. Results: 451 patients and caregivers from across the US responded over a 10-month period. The most valuable clinical endpoints were reduced infection, recurrence, and amputation. The most valuable quality of life outcomes were increased independence, reduced social isolation and pain. The top five endpoints in terms of usefulness for measuring clinical trial success were faster healing, reduced size, infection, recurrence and pain. Free-text responses emphasized inability to perform activities of daily living and pain as major factors that impact the daily lives of patients with wounds. Conclusions: Engagement of patients in clinical trials and evaluating future treatments is critical to the success of wound care. This survey provides insight into the needs of wound-care patients and how to structure future clinical trials to better meet those needs and improve patient care.

K3.02

PERSONALIZED PRESSURE INJURY PREVENTION PLANNING: CLINICAL PRACTICE GUIDELINES, THE ELECTRONIC HEALTH RECORD AND BIG DATA
Kath M. Bogie1, M Kristi Henzel2, GQ Zhang3, Steven Roggenkamp3, Jiayang Sun1, Arielle Bloostein1, Jacinta M. Seton2, Youjun Li1, Mary Ann Richmond2, Monique Washington2
1Case Western Reserve University, Cleveland, OH, USA, 2Louis Stokes Cleveland VA Medical Center, Cleveland, OH, USA, 3University of Kentucky, Louisville, KY, USA
PERSONALIZED PRESSURE INJURY PREVENTION PLANNING: CLINICAL PRACTICE GUIDELINES, THE ELECTRONIC HEALTH RECORD AND BIG DATA

Kath M. Bogie1, M. Kristi Henzel2, GQ Zhang3, Steven Roggenkamp3, Jiayang Sun1, Arielle Bloostein1, Jacinta M. Seton2, Youjun Li1, Mary Ann Richmond2, Monique Washington2
1Case Western Reserve University, Cleveland, OH, USA, 2Louis Stokes Cleveland VA Medical Center, Cleveland, OH, USA, 3University of Kentucky, Louisville, KY, USA

Background: Clinical practice guidelines (CPG) aid clinicians in pressure injury (PrI) prevention. However, there is limited guidance on prioritization. This can be overwhelming and impractical to address, negatively impacting care and intervention planning. Effective clinical tools to prioritize the multiple recommendations in the CPG has been identified as a need by experts in the field. Methods: Bioinformatics enables data extraction, storage, and analysis for clinical decision support and user-interface development for complex clinical challenges such as PrI. Electronic health records (EHR) provide a rich information resource. Over 200 ICD-9 codes related to PrI risk factors were identified. EHR data over a 5 year period was collated for 36,626 Veterans with spinal cord injury (SCI). Natural language processing (NLP) was used to parse and analyze clinical narratives. Results: Hierarchical clustering and network analysis indicate that neurogenic bowel and bladder are the comorbidities most strongly associated with PrI development. Smoking and diabetes are lower tier risk comorbidities. Over 6 million text notes were analyzed using NLP. Many risk factors were recorded in the clinical notes but not coded. Conclusion: Understanding the relative impact of comorbidities is important for primary and secondary PrI planning for persons with SCI. The clinical notes provide a rich alternative source of clinical information related to PrI risk complementing ICD-9 coding. Systemic analysis of PrI risk factors recording in the EHR by ICD coding and clinical notes enable development of a personalized care planning tool. Each individual’s risk factor profile can provide the basis for adaptive personalized PrI prevention care planning based on CPG prioritization.

K3.03

RISK FACTORS FOR MAJOR AMPUTATION ON HINDFOOT ULCERS IN HOSPITALIZED DIABETIC PATIENTS
Seung Kyu Han, Jae youn Kim, Ji Won Son, Sik Namgoong
Korea University Guro Hospital, seoul, Korea, Republic of
RISK FACTORS FOR MAJOR AMPUTATION ON HINDFOOT ULCERS IN HOSPITALIZED DIABETIC PATIENTS

Seung Kyu Han, Jae youn Kim, Ji Won Son, Sik Namgoong
Korea University Guro Hospital, Seoul, Korea, Republic of

Approach: Between January 2003 and October 2017, a total of 1,657 diabetic patients were admitted to the diabetic wound center of Korea University Guro Hospital for foot ulcers. Among them, 117 diabetic patients with hindfoot ulcers were included in this study. One hundred and four patients (89%) healed without major amputation while 13 patients (11%) healed with major amputation. Data of 88 potential risk factors including demographics, ulcer condition, vascularity, bioburden, neurology, and serology were collected from patients in these two groups and compared. Results: Among these 88 potential risk factors, 15 risk factors showed statistically significant differences between the two groups. In univariate analysis carried out for 88 potential risk factors, statistically significant differences were observed for nine risk factors. In stepwise multiple logistic analysis, three of these nine risk factors remained statistically significant. Multivariate-adjusted odds ratios for pulmonary disorders, erythrocyte sedimentation rate (ESR) levels, and total iron-binding capacity (TIBC) levels were 38.525, 1.047, and 0.976, respectively. Innovation: Diabetic foot ulcers located at the hindfoot are closely associated with major amputation because of their proximity. However, there are only few studies on large cohorts that specifically discuss outcomes and characteristics of hindfoot ulcers. Conclusion: Risk factors for major amputation in diabetic hindfoot patients were pulmonary disorders, high levels of ESR, and decreased TIBC levels.

K3.04

MONITORING MICROCIRCULATORY BLOOD FLOW IN CHRONIC DIABETIC AND VENOUS ULCERS WITH DIFFUSE CORRELATION SPECTROSCOPY
Michael Neidrauer1, Michael S. Weingarten1, Alec Lafontant1, Keyanna Bynum1, Christine Wojciechowicz1, Elizabeth Mintz1, Leonid Zubkov1, David Margolis2, Rose Ann DiMaria-Ghalili1, Peter A. Lewin1
1Drexel University, Philadelphia, PA, USA, 2University of Pennsylvania, Philadelphia, PA, USA
MONITORING MICROCIRCULATORY BLOOD FLOW IN CHRONIC DIABETIC AND VENOUS ULCERS WITH DIFFUSE CORRELATION SPECTROSCOPY

Michael Neidrauer1, Michael S. Weingarten1, Alec Lafontant1, Keyanna Bynum1, Christine Wojciechowicz1, Elizabeth Mintz1, Leonid Zubkov1, David Margolis2, Rose Ann DiMaria-Ghalili1, Peter A. Lewin1
1Drexel University, Philadelphia, PA, USA, 2University of Pennsylvania, Philadelphia, PA, USA

Purpose: This report presents a preliminary analysis of an ongoing study to determine whether changes in microcirculatory blood flow in diabetic ulcers (DU) and venous ulcers (VU) correlate to wound healing outcomes. Background: The standard method for assessing the efficacy of chronic wound treatments is reduction of wound size during the first four weeks of treatment, but the positive predictive value of this method is reported below 60%. A more reliable method of assessing wound healing trajectory would allow clinicians to discontinue ineffective treatments and may lead to reduced morbidity and healthcare costs. Methods: Diffuse Correlation Spectroscopy (DCS) is a non-invasive optical technology that provides information about aggregate motion of blood cells in the microvasculature of dermal and subcutaneous tissue. We developed a DCS device that determines a blood flow index (BFI) at tissue depths ranging from 2-10mm. Patients with VUs (n=60) or DUs (n=60) are being recruited for an ongoing double-blinded randomized controlled trial to determine the effects of low frequency (20kHz), low intensity (<100 mW/cm2) ultrasound on chronic wound healing and quality of life. Each subject was seen weekly for 16 weeks or until complete wound closure. DCS was used to measure blood flow each week in wound tissue and non-wound control tissue. Results: Here, we analyze DCS data from the first 28 enrolled subjects. In DUs (n=6) and VUs (n=22), blood flow in wound tissue was significantly higher (p<0.001) than in non-wound tissue. Blood flow values decreased systematically in healing DUs and VUs as values approach those of non-wound tissue. Conclusions: This preliminary analysis suggests that microcirculatory blood flow may serve as an indicator of healing trajectory for chronic wounds. Acknowledgements: Supported by NIH-NINR grant 5R01NR015995. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH-NINR.

K3.05

PHASE 2 TRIAL OF HUMAN UMBILICAL CORD FOR COMPLEX NON-HEALING DIABETIC FOOT ULCERS
Herbert B. Slade, Tommy D. Lee, Nick McCoy, Scheffer Tseng
Tissuetech Inc, Fort Worth, TX, USA
PHASE 2 TRIAL OF HUMAN UMBILICAL CORD FOR COMPLEX NON-HEALING DIABETIC FOOT ULCERS

Herbert B. Slade, Tommy D. Lee, Nick McCoy, Scheffer Tseng
Tissuetech Inc, Fort Worth, TX, USA

Background: This open label Ph2 study was carried out in adults with complex DFU, defined as positive probe to bone and radiographic evidence of osteomyelitis. Methods: The study engaged 11 sites to enroll 32 of 30 planned adult diabetics (HbA1c <12%) with an ulcer on the foot excluding the dorsum, of 1.0 - 10.0 cm2 initial area at screening, with ABI ≥0.7 to ≤1.3. See NCT03230175 for full criteria. Aggressive surgical debridement included removal of infected/devitalized bone and tissue, with bone sent for histopath confirmation. Systemic antibiotics were to be used for 6 weeks. TTAX01 human umbilical tissue was sutured or stapled to the wound bed following the initial procedure. Secondary dressings were at the Investigator's discretion. All subjects were instructed to offload their wounds. Repeat application of TTAX01 was permitted at a minimum of 4 week intervals only for wounds that were not progressing towards healing. Results: One subject withdrew consent early, one was unable to receive all applications due to supply interruption. >50% of the remaining 30 achieved initial closure in 16 weeks, including all cases presenting with gangrene (n=3) and 75% of cases >12 m duration (n=4). Only 2 subjects experienced recurrent osteomyelitis. An average of ~ 1.5 applications were used in subjects achieving closure. Mean time to heal was 10.4 weeks (range 6 – 16). No adverse events were attributed to the TTAX01. Conclusions: Closure rates are encouraging in this population with very difficult to treat wounds. The protocol for a planned Ph3 confirmatory trial will be adjusted based on learnings in Ph2.

K3.06

TOWARD AN UNDERSTANDING OF PRESSURE INJURY RISK AND LOCAL AND CIRCULATORY BIOMARKERS
Kath M. Bogie1, M Kristi Henzel2, Mary Ann Richmond2, Nannette Alvarado2, Jen Zindle2, Katie A. Schwartz2, David Lemmer2, Jacinta M. Seton2, Youjun Li1, Jiayang Sun1
1Case Western Reserve University, Cleveland, OH, USA, 2Louis Stokes Cleveland VA Medical Center, Cleveland, OH, USA
TOWARD AN UNDERSTANDING OF PRESSURE INJURY RISK AND LOCAL AND CIRCULATORY BIOMARKERS

Kath M. Bogie1, M Kristi Henzel2, Mary Ann Richmond2, Nannette Alvarado2, Jen Zindle2, Katie A. Schwartz2, David Lemmer2, Jacinta M. Seton2, Youjun Li1, Jiayang Sun1
1Case Western Reserve University, Cleveland, OH, USA, 2Louis Stokes Cleveland VA Medical Center, Cleveland, OH, USA

Background: Despite the best standards of care, pressure injures (PrI) prevention remains challenging for many people with spinal cord injury (SCI). The purpose of this study was to identify the more susceptible individuals within this high-risk population. Methods: A repeated measures study of 38 persons with SCI was carried out. Seated interface pressures and muscle composition were assessed at baseline and repeated annually. Local and circulatory biomarkers of muscle composition and inflammatory status were assessed in gluteal muscle biopsy and whole blood samples. Primary fatty metabolite biomarkers of interest were FABP4(adipose) and FABP3 (skeletal muscle). Results: Mean ischial region pressure did not vary between people with or without a PrI history. Higher levels of gluteal intramuscular fat (IMAT) were significantly related to having a history of severe/recurrent PrI (p<0.001). Circulatory FABP3 and FAPB4 were observed in persons with greater than 20% IMAT. Inflammatory biomarker expression was several fold lower than either FABP3 or FABP4. Macrophage migration inhibitory factor (MMIF), colony stimulating factors 1 and 3 (CSF-1, CSF-3) and VEGF-a were significantly related to having a history of severe/recurrent PrI. Conclusion: Pressure mapping alone is not a useful measure of PrI risk. Muscle microstructure impacts the overall biomechanical response of the muscle to applied load and the uniformity of intramuscular load distribution. Loaded muscle with high IMAT infiltration acts like a mixed composite material. Changes in muscle composition, specifically increased IMAT, provide clinically significant risk factors for PrI. Circulatory biomarkers also vary with muscle composition. Levels of inflammatory biomarker expression may too low to be of routine clinical significance, e.g. indicative of infection, but may provide an indicator of underlying sub-clinical differences in inflammatory status and PrI risk.

K4.01

LOW MICRORNA-21 IN DIABETIC WOUND MACROPHAGES IMPAIR RESOLUTION OF INFLAMMATION
Amitava Das, Mithun Sinha, Pradipta Banerjee, Nirupam Biswas, Savita Khanna, Chandan K. Sen, Sashwati Roy
Indiana University School of Medicine, Indianapolis, IN, USA
LOW MICRORNA-21 IN DIABETIC WOUND MACROPHAGES IMPAIR RESOLUTION OF INFLAMMATION

Amitava Das, Mithun Sinha, Pradipta Banerjee, Nirupam Biswas, Savita Khanna, Chandan K. Sen, Sashwati Roy
Indiana University School of Medicine, Indianapolis, IN, USA

Background: Under conditions of diabetes, wound macrophages (wmφ) suffer from compromised efferocytosis which impairs resolution of inflammation. Our previous work demonstrated that delivery of miR-21 to human monocyte-derived macrophages (MDM) enhances efferocytosis and helps resolve inflammation. Here we test the hypothesis that diabetic wound macrophages are deficient in miR-21 which causes persistent inflammation. Methods: wmϕ were isolated from clinically presented chronic wounds using a standardized process involving processing of negative-pressure wound therapy (NPWT) dressings. In mice, subcutaneously implanted PVA sponges were used to isolate wmφ. Results: Both human and murine diabetic wmϕ showed significantly lower miR-21 levels compared to non-diabetics (p< 0.05; n=4). To determine the significance of lower miR-21 in wmϕ fate and resolution of inflammation, a myeloid cell-specific miR-21 knockdown mice (miR-21mϕΔ/Δ) was generated by crossbreeding mice carrying floxed miR-21 allele (miR-21fl/fl) with LysM-Cre mice. In wmϕ of miR-21mϕΔ/Δ, expression of pro-inflammatory markers were significantly higher, and those representing reparative phenotype were attenuated (p<0.05; n=4). Excisional wounds of miR-21mϕΔ/Δ mice showed increased abundance of neutrophils and elevated levels of inflammation related cytokines (p<0.05; n=4). Bioinformatics analyses predicted the neutrophil chemoattractant GRO-α to be a direct target of miR-21. Using 3'-UTR firefly luciferase reporter, this work validated GRO-α as a direct target of miR-21 (p<0.05; n = 4). Lower miR-21 in diabetic and miR-21mϕΔ/Δ wmϕ desilenced GRO-α (p<0.05; n=4) causing marked increase in neutrophil recruitment to the wound-site. Conclusion: This work recognizes a novel miR-21-GRO-α mechanism underlying diabetes related persistent inflammation.

K4.02

PIRFENIDONE REGULATES LPS MEDIATED HYPER ACTIVATION OF NEUTROPHILS
Shankar J. Evani, Kai P. Leung
USAISR, Fort Sam Houston, TX, USA
PIRFENIDONE REGULATES LPS MEDIATED HYPER ACTIVATION OF NEUTROPHILS

Shankar J. Evani, Kai P. Leung
USAISR, Fort Sam Houston, TX, USA

Excessive inflammation or abrogating inflammation may result in improper tissue recovery during infection/injury. Neutrophils are among the first respondents of innate immune system to arrive at the injury site and have been implicated in exacerbated inflammation during their fight against invading pathogens or tissue debridement for wound repair. Drugs with potential anti-inflammatory and anti-fibrotic effects are of promise in tissue recovery. Of interest, pirfenidone (Pf) an FDA approved anti-inflammatory/anti-fibrotic drug used in treating idiopathic pulmonary fibrosis was shown to ameliorate inflammation in some animal models. We have shown that Pf lessens the profibrotic phenotype of TGF-β1-stimulated human dermal myofibroblasts in vitro and also reduces pro-inflammatory cytokines in mouse burn wounds. However, there is a lack of understanding of Pf effect on neutrophils. Here, we examined the effect of Pf (0.1, 0.5 & 1 mg/ml) on neutrophils treated with LPS (20ng/ml) as a model of non-sterile inflammation. Neutrophils from healthy donors were used (n = 6; Stats: One/two-way ANOVA) and we showed that 0.5mg/ml Pf at 4/16h post LPS treatment, lowered oxidative burst (MPO, ROS), degranulation (MMP8, MMP9, CD35, CD45) and inflammation (TNFα, IL-1β, IL-8, MCP-1, MIP-1α) without abrogating phagocytosis and NETosis. Pf decreased neutrophil chemotaxis to chemoattractant. We also showed that Pf acted on LPS – TLR axis by down regulating Kinases. In conclusion, the results support the notion that Pf could modulate neutrophil functions in vitro. Funding source: 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).

K4.03

DIFFERENTIAL EXPRESSION OF GRANZYME B IN MACROPHAGES
Matthew R. Zeglinski, Christopher T. Turner, David J. Granville
University of British Columbia, Vancouver, BC, Canada
DIFFERENTIAL EXPRESSION OF GRANZYME B IN MACROPHAGES

Matthew R. Zeglinski, Christopher T. Turner, David J. Granville
University of British Columbia, Vancouver, BC, Canada

Background: Granzyme B (GzmB) is a serine protease widely recognized for its intracellular apoptotic role and its extracellular role in matrix remodeling. Although GzmB is most commonly associated with cytotoxic T-lymphocytes, other immune cells, including macrophages, express GzmB. Although macrophage expression of GzmB has been reported, it is unknown what subtype(s) of macrophages express GzmB and whether GzmB is retained within the cell or secreted into the extracellular environment. Therefore, we set out to determine the expression profile and spatial distribution of GzmB in differentially polarized macrophages. Methods: THP-1 monocytes were differentiated and polarized into M0, M1, M2a, and M2c subtypes. Gene expression was determined by qPCR. GzmB concentrations in cell lysates and culture supernatants was determined by ELISA. Cellular localization of GzmB was determined by confocal microscopy. Results: GzmB was expressed by all subtypes as determined by qPCR and ELISA. M2a macrophages expressed 6-fold more GzmB mRNA compared to M1 and M0 subtypes. M0 and M1 subtypes retained GzmB whereas M2a and M2c subtypes secreted the majority of GzmB. Confocal imaging demonstrated peri-nuclear GzmB staining in M0 and M1 subtypes, whereas GzmB was widely distributed in M2a cells. SerpinB9 was expressed by all subtypes with the highest levels observed in M1 cells. Perforin (Pfn) was also expressed by all subtypes with M2a cells expressing 4-fold more Pfn mRNA compared to M0 and M1 subtypes. Conclusion: Secretion of GzmB by M2a cells may contribute to matrix remodeling in chronic inflammatory disease.

K4.04

A MODIFIED COLLAGEN DRESSING INDUCES TRANSITION OF INFLAMMATORY TO REPARATIVE PHENOTYPE OF WOUND MACROPHAGES
Amitava Das1, Motaz Abas2, Nirupam Biswas1, Pradipta Banerjee1, Nandini Ghosh2, Savita Khanna1, Edward Stout3, Sashwati Roy1, Chandan K. Sen1
1Indiana University School of Medicine, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA, 3Southwest technologies Inc, Kansas City, MO, USA
A MODIFIED COLLAGEN DRESSING INDUCES TRANSITION OF INFLAMMATORY TO REPARATIVE PHENOTYPE OF WOUND MACROPHAGES

Amitava Das1, Motaz Abas2, Nirupam Biswas1, Pradipta Banerjee1, Nandini Ghosh2, Savita Khanna1, Edward Stout3, Sashwati Roy1, Chandan K. Sen1
1Indiana University School of Medicine, Indianapolis, IN, USA, 2The Ohio State University, Columbus, OH, USA, 3Southwest technologies Inc, Kansas City, MO, USA

Background: Collagen based dressings are widely used in wound care. However understanding of their mechanism of action is scanty. Previous studies using a modified collagen gel (MCG) dressing demonstrated robust vascularization of ischemic wounds and improved healing outcomes. Wound macrophages play a critical role in enabling wound angiogenesis and timely healing. Methods: In this work, we sought to investigate the direct action of MCG dressing on wound macrophage phenotype and function using a established murine PVA sponge model. Results: MCG increased macrophage recruitment to the wound site and attenuated pro-inflammatory (mϕinf) macrophage polarization (p<0.05; n=3). Decreased mϕinf polarization was associated with increased production of anti-inflammatory cytokine IL-10 and proangiogenic VEGF (p<0.05; n=6) indicative of a direct action of MCG in supporting resolution of inflammation and improving angiogenesis in wounds. Impaired clearance of apoptotic cell bioburden at wound-site enables chronic inflammation. Previous studies in our laboratory reported that engulfment of apoptotic cells by macrophages (efferocytosis) drives polarization of macrophages to reparative phenotype via a miR-21-PDCD4-IL-10 pathway. Elevated efferocytosis index (p<0.05; n=4) was noted in macrophages from MCG treated wounds. Such favorable outcome resulted in a significant induction (p<0.05; n=4) of miR-21 expression. Interestingly, MCG-mediated induction of IL-10 was blunted under conditions of miR-21 knockdown (p<0.05; n=4) by miR-21-zip pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK in macrophages resulted in attenuated IL-10 (p<0.05; n=4) production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 release by wound macrophages. Conclusion: This work presents direct evidence demonstrating that a collagen based wound care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes.

K4.05

THE EFFICACY OF A SYNTHETIC BIORESORBABLE ANTIMICROBIAL MATRIX AS AN IMPLANTABLE MATERIAL FOR AT-RISK SURGICAL WOUNDS
Ryan Chatelain
ETSU Quillen Physicians, Johnson City, TN, USA
THE EFFICACY OF A SYNTHETIC BIORESORBABLE ANTIMICROBIAL MATRIX AS AN IMPLANTABLE MATERIAL FOR AT-RISK SURGICAL WOUNDS

Ryan Chatelain
ETSU Quillen Physicians, Johnson City, TN, USA

Background: Surgery performed on diabetics is not without complication as increased serum glucose levels, neuropathy, and presence of an open wound potentiate post-operative infection risk. Implantation of a resorbable antimicrobial matrix aimed at reducing infection risk would prove a valuable addition to treatment protocol with surgery in this high-risk population. Methods: Diabetic patients undergoing non-emergent or elective surgery and recognized as at-risk were included in the study. Patients identified as at-risk had one or more of the following: neuropathy, infection (treated), open wound, history of recurrent infection, nonhealing wound, or peripheral vascular disease (treated). Patients were educated on potential risks, benefits and complications of implanting the bioabsorbable silver containing matrix. Once informed consent was obtained, patients underwent a foot or ankle surgical procedure specific to their pathology. After meticulous hemostasis and subcutaneous closure if applicable, the bioabsorbable antimicrobial matrix was implanted just deep to incision and the incision primarily closed. Nonadherent cover dressing was applied and patient was scheduled for 3-5 day routine follow up. Results: Signs of infection at the first post-operative appointment (between 3-5 days) were absent in all 20 patients and all patients went on to heal. 18 of the 20 healed at typical rate specific for the respective procedure. Two patients that took longer to heal did so secondary to weightbearing dehiscence. 100% of patients had diabetic peripheral neuropathy, 73% had open wounds and 67% had treated infection at time of surgery. For all patients, the first post-op appointment showed no post-op infection or other complication. Eighteen patients healed uneventfully. Only 2 patients experienced weight-bearing dehiscence with wounds resolving after revisional surgery, use of the matrix and appropriate wound care. Conclusions: Application of the synthetic bioabsorbable antimicrobial matrix prior to primary closure is efficacious in prevention of post-operative infection in this high-risk diabetic population.

K4.06

CAVEOLIN-1 PROMOTES BACTERIAL COLONIZATION AND INHIBITION OF WOUND CLOSURE IN AGED HUMAN SKIN
Ivan Jozic, Irena Pastar, Cheyanne R. Head, Robert S. Kirsner, Marjana Tomic-Canic
University of Miami Miller School of Medicine, Miami, FL, USA
CAVEOLIN-1 PROMOTES BACTERIAL COLONIZATION AND INHIBITION OF WOUND CLOSURE IN AGED HUMAN SKIN

Ivan Jozic, Irena Pastar, Cheyanne R. Head, Robert S. Kirsner, Marjana Tomic-Canic
University of Miami Miller School of Medicine, Miami, FL, USA

Aging, together with age-related co-morbidities, such as weakening of the immune system, diabetes and peripheral vascular disease, compromise skin integrity resulting in skin breakdown and increased susceptibility to infections, thus leading to development of non-healing chronic wounds. Regardless of clinical signs of infection, every chronic wound is colonized by opportunistic pathogens (both intra- and extra-cellular), which contributes to compromised ability to heal a wound and restore barrier. We have recently identified a structural membrane protein caveolin-1 (cav1) as a potential therapeutic target of non-healing chronic wounds commonly observed in the elderly population. Cav1 plays important roles in multiple cellular process fundamental for wound healing by: 1) being linked with age-associated immunity resulting in increased production of pro-inflammatory cytokines, i.e. chronic inflammation; 2) being associated with cellular senescence; 3) playing a role in facilitating evasion of lysosomal degradation of a common colonizer of chronic wounds Pseudomonas aeruginosa, thus allowing persistence inside the host cells. Using human tissue biopsies, we observed induction of cav1 expression in elderly skin as well as in chronic non-healing wounds on both mRNA and protein levels. Furthermore, we found that upregulation of cav1 in human keratinocytes regulates colonization of Staphylococcus aureus (a common chronic wound colonizer) in vitro, which can be reversed by CRISPR/Cas9-mediated knockdown of cav1 or perturbation membrane cholesterol by MβCD. Additionally, using wound scratch assays, we found that disruption of cav1 in primary human keratinocytes isolated from aged human skin rescues the retarded directional cell migration observed in these cells due to reorganization of cytoskeletal machinery and promoted wound closure in 3D organotypic skin equivalents assembled using aged keratinocytes. Together, our data provide evidence for a novel molecular mechanism by which upregulation of cav1 expression observed in elderly individuals inhibits wound re-epithelialization by targeting two processes considered as “hallmarks” of non-healing: 1) promotion of bacterial colonization/persistence and 2) inhibition of directional cell migration necessary for wound epithelialization.

M1.01

GENE EXPRESSION ANALYSIS OF DIABETIC FOOT ULCER DEBRIDED TISSUE FOR PREDICTING RESPONSIVENESS TO TREATMENTS
Jessica M. Eager1, Michael S. Weingarten, MD2, Will Dampier, PhD3, Kara L. Spiller, PhD1
1School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, USA, 2Department of Surgery, Drexel University College of Medicine, Philadelphia, PA, USA, 3Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA
GENE EXPRESSION ANALYSIS OF DIABETIC FOOT ULCER DEBRIDED TISSUE FOR PREDICTING RESPONSIVENESS TO TREATMENTS

Jessica M. Eager1, Michael S. Weingarten, MD2, Will Dampier, PhD3, Kara L. Spiller, PhD1
1School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, USA, 2Department of Surgery, Drexel University College of Medicine, Philadelphia, PA, USA, 3Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA

Background: Chronically open wounds are a common complication for diabetic patients. These ulcers (DFUs) are notoriously difficult to treat for a variety of reasons, but the major challenge stems from the mystery as to why some wounds respond to treatment and others do not. The goal of our study is to create a diagnostic assay that will determine the likelihood a DFU wound will close by 12 weeks in response to the standard of care. Methods: We used Nanostring™ for gene expression analysis of 227 wound healing and macrophage phenotype related genes in debrided tissue from the first visit to the clinic of 23 patients. The top 10 most highly expressed genes were used to train a neural network algorithm to classify healing outcome at 12 weeks as fully closed, remained open, or required amputation. 10-fold cross validation was performed to determine the best number of hidden units. The dataset was then divided into training and testing cohorts using a 60/40 split. Results: After training, the algorithm correctly classified the outcome for 7 of the 10 patients, resulting in 80% specificity and 60% sensitivity when the outcomes are binarized by combining the remained open and required amputation groups. Conclusions: These results, while preliminary, suggest that gene expression analysis of debrided tissue from the first visit can be used as a method of predicting healing outcome for DFUs. Because this method relies solely on the first visit, it has the potential to guide patients and clinicians toward treatment options better suited to the wound environment and more likely to facilitate healing.

M1.02

A PLASMA-ALGINATE COMPOSITE MATERIAL PROVIDES IMPROVED MECHANICAL SUPPORT FOR STEM CELL GROWTH AND DELIVERY
Nicholas E. Clay, Carissa Villanueva, Nicole Wrice, Andrew C. Kowalczewski, Shanmugasundaram Natesan, Robert J. Christy
US Army Institute of Surgical Research, Fort Sam Houston, TX, USA
A PLASMA-ALGINATE COMPOSITE MATERIAL PROVIDES IMPROVED MECHANICAL SUPPORT FOR STEM CELL GROWTH AND DELIVERY

Nicholas E. Clay, Carissa Villanueva, Nicole Wrice, Andrew C. Kowalczewski, Shanmugasundaram Natesan, Robert J. Christy
US Army Institute of Surgical Research, Fort Sam Houston, TX, USA

Background: Stem cell-based therapies are a promising option for treating skin wounds. Blood plasma-based gels are commonly used as a vehicle for delivering stem cells to wound beds. However, plasma-based gels often have poor mechanical properties which limit their use in demanding clinical settings. Therefore, simpler methods to create sturdier plasma/fibrin-based materials are needed. To this end, we hypothesized that mixing alginate and plasma together will create a plasma-alginate composite (PAC) material with improved mechanical and biological properties. Methods: Plasma and alginate were mixed together to create PAC gels with unique formulations. The stiffness and degradation kinetics of the PAC gels were assessed using rheology and a tissue plasminogen activator (tPA)-based degradation assay, respectively. Stem cells were cultured in the PAC gels for 8 days to assess in vitro cell viability and phenotype. An alginate-free plasma gel was used as a control throughout. Results: Our results demonstrated that PAC gels with specific compositions of alginate and plasma were 10-times stiffer than the plasma-only gels. Interestingly, tPA-mediated gel lysis rates were independent of alginate concentration. The stiffer PAC gels enabled stem cell proliferation and maintained cell stemness over 8 days in vitro. The presence of alginate within the PAC gel also helped maintain the gel shape and size in culture. Conclusions: In sum, we envision this PAC gel system will extend the use of plasma-based therapies for tissue engineering and wound healing applications. The improved mechanical properties of the PAC gel system will be useful in demanding clinical settings, such as austere trauma environments or mass casualty scenarios.

M1.03

NOTCH ACTIVATION STIMULATES WOUND HEALING
Liz Quintero, Timothy W. King
University of Alabama At Birmingham, Birmingham, AL, USA
NOTCH ACTIVATION STIMULATES WOUND HEALING

Liz Quintero, Timothy W. King
University of Alabama At Birmingham, Birmingham, AL, USA

Introduction: We are interested in improving wound healing therapies through the understanding of potential cellular and molecular mechanisms, especially those influenced by the Notch signaling cascade. We have previously shown that inhibiting Notch inhibits wound healing. Based upon our previous work, our hypothesis is that topical application of the Notch ligand, JAG1, to ex vivo excisional wounds of mice increases wound healing rates as compared to untreated wounds. We also wanted to determine if age and gender influence the wound healing rates. Methods: Skin biopsies 6-mm in diameter with another wound of 3-mm in the center were cultured ex vivo from 10-12 week-old (young) and at least one-year-old (aged) mice. We included both male and female mice from control and skin specific (K14) knockouts of Notch1 and Notch2. Topical application of JAG1 (1 μg/ml, 7 nM), vehicle (PBS), PBS+DAPT (gamma secretase/Notch signaling inhibitor) or JAG1+DAPT were applied every two days. Photomicrographs were taken and histological analysis performed. The tissue growth was calculated as the difference of the area at each timepoint minus the area at time=0. Significance was defined as p<0.05 using two-way ANOVA. Results: Significant tissue growth was seen in the biopsies from control mice, young and aged, males and females; compared to K14-Notch1 or K14-Notch2 knockouts. Treatment with DAPT inhibited the growth in mice where JAG1 had an effect. Conclusions: Notch activation by JAG1 increases the rate of tissue growth of cutaneous wounds in an ex vivo murine wound-healing model, regardless age/gender, but not in Notch1 or Notch2 knockouts, indicating that Notch signaling is crucial in wound healing. Further study of Notch in wound healing should be conducted which may then lead to better therapeutics.

M1.04

TOPICAL CROMOLYN SODIUM REDUCES POST-BURN HYPERTROPHIC SCARS IN FEMALE RED DUROC PIGS
Jayson W. Jay, Anesh Prasai, Amina El Ayadi, Christian Sommerhalder, Daniel Popp, Evan Ross, Elizabeth Blears, Guillermo Foncerrada, Christian Tapking, David N. Herndon, Celeste C. Finnerty
University of Texas Medical Branch, Galveston, TX, USA
TOPICAL CROMOLYN SODIUM REDUCES POST-BURN HYPERTROPHIC SCARS IN FEMALE RED DUROC PIGS

Jayson W. Jay, Anesh Prasai, Amina El Ayadi, Christian Sommerhalder, Daniel Popp, Evan Ross, Elizabeth Blears, Guillermo Foncerrada, Christian Tapking, David N. Herndon, Celeste C. Finnerty
University of Texas Medical Branch, Galveston, TX, USA

Background: Painful, motion-limiting hypertrophic scars (HTS) form subsequent to protracted wound healing in patients with severe full-thickness burns and pose difficult treatment challenges. Newer evidence points to mast cells as important regulators of intricate signaling cascades during the initiation and progression of post-burn scars. Previous investigations have demonstrated increased mast cell densities in burn wounds and during the formation of HTS. Mast cell proteases contribute directly to myofibroblast differentiation and excessive proliferation in burn wounds. Methods: Cromolyn sodium (CS) is an FDA-approved mast cell stabilizer known to inhibit degranulation and has been successfully used to relieve detrimental symptoms associated with mast cell activation. Here, we directly applied a 4% topical emulsification of CS to the post-burn HTS of red Duroc pigs (n=6). 3D images and scar biopsies were obtained monthly. Results: Toluidine blue stained scar tissue showed that CS significantly reduced mast cell density (p<0.05) over time. Histologically, epidermal thickness was also significantly reduced in CS-treated wounds. 3D analysis demonstrated that CS reduced scar height and volume while scar perimeter increased (p<0.05), flattening the scar over time compared to vehicle treatment alone or autologous split-thickness skin grafts. Additionally, immunohistochemical analysis showed that CS treatment significantly minimized dermal fibroblast expression of the protease-activated receptor-2 (PAR2, p<0.05) further indicating that fibrotic phenotype may be driven by mast cell tryptase activation of PAR2. Conclusion: Together, this evidence suggests that localized mast cell stabilization may be an effective approach to reduce pathologic scarring following a severe burn.

M1.05

EFFECTS OF A PEER-LED SELF-MANAGEMENT PROGRAM ON PATIENTS WITH DIABETIC FOOT ULCERS
Ye-Na Lee
College of Nursing, Korea University, Goyang-si, Korea, Republic of
EFFECTS OF A PEER-LED SELF-MANAGEMENT PROGRAM ON PATIENTS WITH DIABETIC FOOT ULCERS

Ye-Na Lee
College of Nursing, Korea University, Goyang-si, Korea, Republic of

Background: Most diabetes-related lower limb amputations can be attributed to the infection of foot ulcers. To reduce the risk of infection and subsequent amputation, not only medical care but also patients’ self-management is necessary during treatment periods. Therefore, it is important that nurses encourage patients to improve their self-management abilities. Using peers to lead health programs helps build a high level of rapport among individuals with similar age and life experiences. The present study aimed to examine the effects of a peer-led self-management program for patients with diabetic foot ulcers. Methods: Participants included 60 patients with diabetic foot ulcers (30 in the experimental group and 30 in the control group). In the experimental group, the designed intervention was implemented by a peer leader, while the control group received “usual care,” which included some education materials. Outcome variables, including self-efficacy, quality of life (QoL), and wound state (Wagner grade and size) were measured at the baseline and again four weeks later. ANOVA/ANCOVA were used for data analysis. Results: The peer-led group reported higher improvements in self-efficacy and QoL following the program as compared to the control group (F=4.36, p=0.04; F=4.94, p=0.03, respectively). Changes in wound state (Wagner grade and size) did not differ significantly between the two groups (F=0.10, p=0.76; F=0.05, p=0.83, respectively); however, an increasing trend was observed in the experimental group. Conclusions: The peer-led self-management program was effective in increasing self-efficacy and QoL in patients with diabetic foot ulcers.

M1.06

IMMUNE CELL ACTIVITY IN HUMAN CUTANEOUS WOUND HEALING AND SKIN SCARRING: A MULTIPLE TIME POINT IMMUNOHISTOCHEMICAL STUDY
Sara Ud-Din1, Douglas McGeorge2, Ardeshir Bayat1
1University of Manchester, Manchester, United Kingdom, 2Nuffield Grosvenor Hospital, Chester, United Kingdom
IMMUNE CELL ACTIVITY IN HUMAN CUTANEOUS WOUND HEALING AND SKIN SCARRING: A MULTIPLE TIME POINT IMMUNOHISTOCHEMICAL STUDY

Sara Ud-Din1, Douglas McGeorge2, Ardeshir Bayat1
1University of Manchester, Manchester, United Kingdom, 2Nuffield Grosvenor Hospital, Chester, United Kingdom

Background: Immune cells are involved in all aspects of the wound healing repair process including the initial haemostasis phase and their function in driving scar formation. It is thought that various immune cells including macrophages and T-cells, play a role in aberrant wound healing and the fibrotic process seen in scar formation in adult skin. Their link to abnormal healing has led to increased attention in the context of cutaneous repair. High numbers of immune cells have been found in hypertophic and keloid scars compared to normotrophic scars. Nevertheless, there is a paucity of evidence of their role and activity over time in acute cutaneous wound healing and normal skin scarring in humans. Methods: We utilised 5mm skin-biopsies taken from the upper arms of 62 healthy volunteers performed at multiple time points; at initial day 0 and at weekly intervals thereafter (weeks (W) 1,2,3,4,5,6,8). Detailed immunohistochemistry (IHC) was undertaken to evaluate the time course of immune cell recruitment in this human wound healing model. Results: Double staining for M2 macrophages using CD68/CD206 demonstrated a 127% increase in cell count from day 0 (uninjured intact skin) to week 5 (110, 250 respectively; p=0.02), with a subsequent 50% reduction to values closer to baseline by week 8 (125). We performed analysis on CD8 T cells and demonstrated a 82% increase in number from uninjured intact skin (64) to week 5 (117; p=0.03) where this plateaued thereafter to week 8 (109). The number of intraepidermal langerin+ Langerhans cells increased from uninjured intact skin (18) to week 4 (54) and reduced slightly by week 8 (42). Conclusions: These findings could lead to further studies on mechanisms of immune cell localisation and may aid the discovery of novel therapeutic agents aimed at reducing immune cell numbers in pathological skin conditions.

M1.07

SMAD7 AMELIORATES TGF?-MEDIATED SKIN INFLAMMATION AND ASSOCIATED WOUND HEALING DEFECTS
Li BIAN, Fulun Li, Shunsuke Iriyama, Zhe Jian, Bin Fan, Jing Jing Luo, Dongyan D Wang, Christian D. Young, Gangwen Han, Xiao-Jing Wang
Univisity Of Colorado Anschutz Medical Campus, Aurora, CO, USA
SMAD7 AMELIORATES TGFβ-MEDIATED SKIN INFLAMMATION AND ASSOCIATED WOUND HEALING DEFECTS

Li Bian*, Fulun Li*, Shunsuke Iriyama*, Zhe Jian, Bin Fan, Jing Jing Luo, Dongyan D. Wang, Christian D. Young, Gangwen Han, Xiao-Jing Wang
Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

We assessed roles of Smad7 in skin inflammation and wound healing using genetic and pharmacological approaches. In K5.TGFβ1/K5.Smad7 bigenic (double transgenic) mice, Smad7 transgene expression reversed TGFβ1 transgene-induced inflammation, fibrosis and subsequent epidermal hyperplasia, and molecularly abolished TGFβ and NF-κB activation. Next, we produced recombinant human Smad7 protein with a Tat-tag (Tat-Smad7) that rapidly permeates cells. Tat-Smad7 subcutaneous injection attenuated infiltrated F4/80+ and CD11b+ leukocytes and αSMA+ fibroblasts prior to attenuating epidermal hyperplasia in K5.TGFβ1 skin. Further, topically applied Tat-Smad7 on wounds of K5.TGFβ1 skin accelerated wound closure with improved re-epithelialization and reductions in inflammation and fibrotic response. A short treatment with Tat-Smad7 was also sufficient to reduce TGFβ and NF-κB signaling in K5.TGFβ1 skin and wounds. Relevant to clinic, we found that human diabetic wounds had elevated TGFβ and NF-κB signaling compared to normal skin. To assess the oncogenic risk of a potential Smad7-based therapy, we exposed K5.Smad7 skin to chemical carcinogenesis and found reductions in myeloid leukocyte infiltration in tumors but not accelerated carcinogenesis compared to wildtype littermates. Our study suggests the feasibility of using exogenous Smad7 below an oncogenic level to alleviate skin inflammation and wound healing defects associated with excessive activation of TGFβ and NF-κB.

M1.08

KNOWLEDGE, ATTITUDES, BELIEFS, AND BEHAVIORS ABOUT ANTIBIOTICS BY PERSONS WITH WOUNDS
Barbara Pieper1, Joanne Sobeck1, Linda Kaljee2, Thomas N. Templin1
1Wayne State University, Detroit, MI, USA, 2Henry Ford Health System, Global Health Initiative, Detroit, MI, USA
KNOWLEDGE, ATTITUDES, BELIEFS, AND BEHAVIORS ABOUT ANTIBIOTICS BY PERSONS WITH WOUNDS

Barbara Pieper1, Joanne Sobeck1, Linda Kaljee2, Thomas N. Templin1
1Wayne State University, Detroit, MI, USA, 2Henry Ford Health System, Global Health Initiative, Detroit, MI, USA

Background: This study examined knowledge, attitudes, beliefs, and behaviors about antibiotic use by adults who had a wound within the past year. Methods: The parent study was descriptive, cross-sectional that enrolled adults (N=505) across community-based (library, senior center, community recreation center, art center) and outpatient and hospital clinic sites; 26 of the participants reported a wound within the past year and are the focus of this report. Participants with wounds were 57.7% women, African American (42.3%) or Caucasian (30.8%) and had some college education (65.4%). Participants responded to an antibiotic knowledge, attitudes, beliefs, and behavior questionnaire. Hierarchical agglomerative cluster analysis of variables was used to find clusters of items on the attitude, beliefs, and behavior questions. Descriptive statistics and Spearman’s rho correlation were also examined. Results: The mean antibiotic knowledge score was 69%. Higher antibiotic knowledge was significantly related to higher education (rs=.71, p < .001) and higher rating of health (rs=.43, p=.028). There were 2 attitude and beliefs clusters: most participants (>85%) recognized the need for medical supervision of antibiotic use (cluster 1); belief about the need for antibiotics to prevent illness or treat wounds varied from 27% to 62% (cluster 2). There were 4 behavior clusters: almost all filled and took the antibiotic if prescribed (96%, cluster 1); disagreed with unapproved methods of obtaining antibiotics (>71%, cluster 2); and used prescribed antibiotics correctly (>87%, cluster 3). Participants heard about antibiotic resistance through TV or radio (36%) or internet (40%), cluster 4. Conclusion: Knowledge about antibiotics was low, while attitudes were positive. These findings identify the need for non-commercial information on the role of antibiotics in wound care.

N1.01

3D PATIENT SPECIFIC IMPLANT FOR CRANIOFACIAL RECONSTRUCTION
JongWon Rhie1, Hyunho Han2, Jin Hyung Shim3, Dong Woo Cho4, Chung Hwan Baek5
1The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, Korea, Republic of, 2Seoul Asan Medical Center, Seoul, Korea, Republic of, 3Korea Polytechnic University, Seoul, Korea, Republic of, 4Pohang University of Science and Technology, Pohang, Kyungbuk, Korea, Republic of, 5Seoul Samsung Medical Center, Seoul, Korea, Republic of
3D PATIENT SPECIFIC IMPLANT FOR CRANIOFACIAL RECONSTRUCTION

JongWon Rhie1, Hyunho Han2, Jin Hyung Shim3, Dong Woo Cho4, Chung Hwan Baek5 1The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, Korea, Republic of, 2Seoul Asan Medical Center, Seoul, Korea, Republic of, 3Korea Polytechnic University, Seoul, Korea, Republic of, 4Pohang University of Science and Technology, Pohang, Kyungbuk, Korea, Republic of, 5Seoul Samsung Medical Center, Seoul, Korea, Republic of

Background: Management of significant facial bone defects or asymmetry are challenging. Conventional options include autogenous bone grafts or alloplastic implants. However, bone grafts are limited by donor morbidity, unpredictable results and long operation time. Alloplastic implants are mainly non-absorbable and may cause foreign body reaction. We present the application of a 3D-printed patient specific absorbable mesh implant for reconstruction of bone defect caused by tumor resection. Methods: A customized polycaprolactone (PCL) scaffold was designed and fabricated for each patient. For this purpose, we used computer-aided design and manufacturing combined with 3D printing technology. The patients implanted with the PCL scaffolds were followed up for up to 2 years with careful evaluation of morphological changes in the face. We have experienced 5 more clinical case of craniofacial reconstruction with patient specific 3 D printed implant. Results: We confirmed that the patient-specific 3D-printed PCL scaffold effectively filled the maxillary defect and promoted regeneration of the deficient tissue while remaining stable in the body for a relatively long period of time. Conclusions: Employing customized tissue-engineered scaffolds built using the patient’s CT data and an extrusion-based 3D printing system is safe and clinically feasible, helping create and maintain improved morphological features of the face, which represents the most important aspect from the perspective of the patients.

N1.02

THE USE OF ACELLULAR DERMAL MATRIX PASTE FOR TREATMENT OF DIABETIC FOOT ULCER
Donghyeok Shin
Konkuk University Medical Center, Seoul, Korea, Republic of
THE USE OF ACELLULAR DERMAL MATRIX PASTE FOR TREATMENT OF DIABETIC FOOT ULCER

Donghyeok Shin Konkuk University Medical Center, Seoul, Korea, Republic of

Backgroud: Management of diabetic foot ulcer can often present challenging and complex problems. The purpose of this clinical trial study was to evaluate efficacy and safety of a paste-type acellular dermal matrix (ADM) product (CG paste, CGBio, Korea) in the treatment of diabetic foot ulcer. Method: This was a randomized, controlled, single-center study in which patients with diabetic foot ulcers. 49 patients with diabetic foot ulcers were randomized to either the ADM paste treatment group (n = 23) or the control group treated with simple dressing (n = 26). Before, all ulcers were subjected to debridement and irrigation to control infection. Then, the products were applied to the wounds and compressed gently to fill the wound cavities. Foam dressings were done to absorb the discharge. After 3 to 5 days, the dressings were removed and the wounds were inspected. Then the products were applied more times in serial. All procedures were done in the operating room. Results: Ulcer healing was achieved in 70.17+-28.35(5), 83.25+-19.92(%), mean wound area and depth, of the treatment group and 45.82+-30.46(%), 40.96+-28.97 (%) of the control group (p<0.05). The median times to complete closure were 17+-9.64 and 21.25+-5.85 days for the ADM paste and control groups, respectively. There were no complications after applying the ADM paste. Conclusion: Well grown granulation tissue is very important step for wound healing. The shortening the covering time with healthy granulation tissue leads to rapid wound healing. Our experience shows that the paste-type ADM product is very useful for enhancing the proliferation of granulation tissue.

N1.03

IMPACT OF EXTRACORPOREAL SHOCK WAVE THERAPY ON MICROCIRCULATION IN DIABETIC FEET: A PILOT STUDY
Seung Kyu Han, Jae youn Kim,, Ji Won Son, sik Namgoong
Korea University Guro Hospital, seoul, Korea, Republic of
IMPACT OF EXTRACORPOREAL SHOCK WAVE THERAPY ON MICROCIRCULATION IN DIABETIC FEET: A PILOT STUDY

Seung Kyu Han, Jae youn Kim, Ji Won Son, Sik Namgoong Korea University Guro Hospital, Seoul, Korea, Republic of

Objective: Patients with diabetic foot commonly experience vascular insufficiency and compromised tissue perfusion. Extracorporeal shockwave therapy (ESWT) reportedly promotes wound healing and angiogenesis. However, clinical studies on the effect of ESWT on angiogenesis are scarce and the exact mechanism of ESWT remains unclear. This study investigated the effect of ESWT on cutaneous microcirculation in diabetic feet. Methods: Ten patients with diabetic feet received ESWT twice weekly for a total of six sessions. Transcutaneous partial oxygen pressure (TcPO2) and cutaneous blood flow were measured before and after ESWT. Main Results: The treated feet had significant improvement in mean TcPO2 (P < 0.01) and cutaneous blood flow (P < 0.05) level compared to that of the control feet respectively. On the treated feet, the TcPO2 increased from 41.4±9.9 to 49.5±8.7 mmHg after therapy, an increase of 19.6%. In the opposite (control) feet, the TcPO2 decreased from 39.5±14.0 mmHg to 34.9±14.5 mmHg, a decrease of 11.6%. The average cutaneous blood flow level on the treated feet before ESWT was 36.9±25.6 and increased to 48.3±32.4 AU after ESWT, an increase of 30.9%. In the control feet, the cutaneous blood flow level decreased from 80.5±36.7 to 60.4±38.8 AU after ESWT, a decrease of 25.0%. Conclusion: These results demonstrate that ESWT may have beneficial effects on microcirculation in diabetic feet.

N1.04

TREATMENT OF FULL-THICKNESS WOUNDS WITH MICROFRAGMENTED ADIPOSE TISSUE AND PLASMA-BASED HYDROGELS
David Larson, Randolph Stone, II, John Wall, Shanmugasundaram Natesan, Robert Christy
US Army Institute of Surgical Research, Fort Sam Houston, TX, USA
TREATMENT OF FULL-THICKNESS WOUNDS WITH MICROFRAGMENTED ADIPOSE TISSUE AND PLASMA-BASED HYDROGELS

David Larson, Randolph Stone, II, John Wall, Shanmugasundaram Natesan, Robert Christy US Army Institute of Surgical Research, Fort Sam Houston, TX, USA

Background: Full-thickness skin wounds often incur severe damage to the subcutaneous adipose tissue. Surgical intervention involves autograft/allograft application and does not replace adipose tissue which often leads to wound contraction and scarring. We hypothesize that reconstruction of hypodermis using purified microfragmented adipose tissue (“Lipogems”) as a stem cell alternative combined with blood plasma-based hydrogels will improve graft-take, perfusion, contraction, and scar appearance in a porcine full-thickness excisional wound. Methods: Lipogems were isolated from porcine adipose tissue using a point-of-care non-enzymatic medical device. Lipogems-plasma hydrogels were prepared by suspending Lipogems in a polyethylene glycol-platelet free plasma (PEG-PFP) liquid mixture and cross-linking with thrombin. Full-thickness 6 cm diameter excisional wounds were created on the dorsum of anesthetized Yorkshire pigs and divided among four treatment groups: Lipogems, PEG-PFP hydrogels, Lipogems+PEG-PFP hydrogels, and no treatment. A positive control group was created by tangential excision to the top of the hypodermis (fat). All wounds were autografted with a meshed split-thickness skin graft (mSTSG). Results: Successful graft-take was observed on day 7 for all wounds. The perfusion measured on day 7 in wounds treated with Lipogems+PEG-PFP hydrogels was not different from the positive control group, indicating a beneficial effect of PEG-PFP+Lipogems. The overall contraction measured at day 60 of wounds treated were significantly higher in comparison to the positive control group. No difference in melanin content occurred between any of the treatment groups. Wounds treated with Lipogems±PEG-PFP did not show a significant difference in erythema compared to the positive control, indicating synergistic treatment with hydrogel and Lipogems in reducing wound erythema. Conclusions: Mechanically processed adipose tissue retains viability and essential factors necessary for active healing to take place. The Lipogems+PEG-PFP hydrogels described in this study provide a ‘point-of-care’ treatment option to mitigate scarring that may be translated to burn wounds. The entire protocol can be carried out in a clinical suite; thus requiring less sophisticated laboratory equipment to isolate and deliver stem cells.

N1.05

PRESERVING THE LENGTH OF A DIABETIC LIMB BY MICROSURGICAL TECHNIQUE
Hyunsuk Peter Suh, Professor
Korea University Medical Center, Seoul, South Korea
PRESERVING THE LENGTH OF A DIABETIC LIMB BY MICROSURGICAL TECHNIQUE

Hyunsuk Peter Suh, Professor Korea University Medical Center, Seoul, South Korea

Background: Diabetic foot ulcers can occur because of a combination of neuropathy, microvascular angiopathy, mechanical stress, and uncontrolled blood glucose levels. In addition to causing severe morbidities, they are the most common nontraumatic cause of amputations. Since microsurgery for diabetic foot reconstruction is no longer considered contraindicated, the use of free flaps for toe resurfacing after diabetic ulcers or reconstruction of defects secondary to toes amputation is an effective approach to maintain the length of the foot and preserve the ankle power generation in normal mechanics of gait. We propose that through this approach we avoid transmetatarsal amputation to ensure closure of the defect and decrease the healing time of patients in which secondary intention closure is the proposed option. Methods: Retrospective data of 43 patients were evaluated according to their surgical method. When required, toes amputations were performed by the orthopedic surgeon, sparing healthy surrounding tissue and metatarsal heads. Metatarsophalangeal joints were preserved when was possible. Results: Preoperative percutaneous transluminal angioplasty (PTA) was performed in 10 patients (23.25%) of which in 9 cases were successful. Toes amputations were performed in 26 cases; big toe only in 8 cases (30.76 percent), big and lesser toes in 1 case (3.8 percent), single lesser toe in 10 cases (38.46 percent), multiple lesser toes in 7 cases (26.92 percent). Among big toe amputation cases, 6 cases were at metatarso-phalangeal level (66.7 percent) and 3 cases were distal phalangeal amputation (33.3 percent). Microsurgical reconstruction for toe resurfacing and after toes amputation was performed in 43 patients; big toe only in 22 cases (51.16 percent), big and lesser toes in 3 cases (6.97 percent), single lesser toe in 10 cases (23.25 percent), multiple lesser toes in 8 cases (18.6 percent). Major reamputation rate after microsurgical flap reconstruction was 6.97 percent (3 cases) with 35.42 months of free major amputation time (range, 0.66 to 54.8). Minor amputation after reconstruction was performed in 6 cases (13.9 percent). Limb salvage rate was 93.03% with a mean follow-up of 1029.32 days (range, 25 to 3330 days). Conclusions: We strongly believe that avoiding transmetatarsal amputation by doing free flap reconstruction, allows maintaining the normal mechanics of gait and preventing new episodes of ulceration and therefore reamputation.

N2.01

COMPROMISED ANGIOGENESIS, INCREASED VESSEL PERMEABILITY, AND DECREASED PERICYTE COVERAGE IN IMPAIRED DIABETIC SKIN WOUNDS
Uzoagu A. Okonkwo1, Lin Chen1, Da Ma2, Veronica A. Haywood1, May Barakat1, Norifumi Urao1, Luisa A. DiPietro1
1University of Illinois at Chicago, Chicago, IL, USA, 2Guanghua School of Stomatology, SunYat-sen University, Guangzhou, China
COMPROMISED ANGIOGENESIS, INCREASED VESSEL PERMEABILITY, AND DECREASED PERICYTE COVERAGE IN IMPAIRED DIABETIC SKIN WOUNDS

Uzoagu A. Okonkwo1, Lin Chen1, Da Ma2, Veronica A. Haywood1, May Barakat1, Norifumi Urao1, Luisa A. DiPietro1 1University of Illinois at Chicago, Chicago, IL, USA, 2Guanghua School of Stomatology, SunYat-sen University, Guangzhou, China

Vascular deficits are recognized as a fundamental contributing factor of diabetes-associated diseases. Although previous studies have demonstrated that the pro-angiogenic phase of wound healing is largely blunted in diabetes, a comprehensive understanding of the mechanisms that regulate skin revascularization and capillary stabilization in diabetic wounds is lacking. Using a mouse model of diabetic wound healing, we confirmed that diabetic db/db mice demonstrated significantly slower rates of closure and impaired angiogenesis during wound healing when compared to wild type mice. MicroCT analysis of the 3-dimensional architecture of the capillary bed showed that vessel surface area, branch junction number, total vessel length, and total branch number were significantly decreased in wounds of diabetic mice as compared to WT mice. Diabetic mouse wounds also had significantly increased capillary permeability and decreased capillary pericyte coverage. We further examined the expression of a large group of known factors that influence capillary recruitment, maturation, and stability, along with markers that are expressed in dermal pericytes. Diabetic wounds had significant perturbations in factors that affect vascular regrowth, maturation and stability. Specifically, VEGF-A, SPRY2, PEDF, LRP6, TSP1, CXCL10, CXCR3, PDGFR-β, HB-EGF, EGFR, TGFβ-1, Sema3a, NRP1, Ang2, NG2, and RGS5 expression was down-regulated in diabetic wounds when compared to WT wounds. These findings were congruent with existing genomic data from human diabetic foot ulcers (GEO- GSE80178). Together, these studies provide novel information about the complex perturbations that occur during angiogenesis and vessel maturation in diabetic wounds. Therapeutic approaches that target factors responsible for vessel regression, maturation, and pruning, as well those that affect pericyte recruitment, maturation, and stability may have the potential to improve diabetic skin wound healing.

N2.02

IN VIVO EVALUATION OF ANGIOGENIC PROPERTIES OF A DEHYDRATED AMNION CHORION MEMBRANE
John McQuilling1, MaryRose Kammer2, Kelly Kimmerling1, Katie Mowry1
1Organogenesis Inc, Birmingham, AL, USA, 2Organogenesis, Birmingham, AL, USA
IN VIVO EVALUATION OF ANGIOGENIC PROPERTIES OF A DEHYDRATED AMNION CHORION MEMBRANE

John McQuilling1, MaryRose Kammer2, Kelly Kimmerling1, Katie Mowry1 1Organogenesis Inc, Birmingham, AL, USA, 2Organogenesis, Birmingham, AL, USA

Angiogenesis is an essential part of wound healing, and placental membranes have been shown to have angiogenic properties both in vitro and in vivo. The purpose of this study was to evaluate the angiogenic properties of a commercially available dehydrated Amnion/Chorion membrane (dACM) ° in vivo. Gelatin sponges soaked with conditioned media (CM) from dACM were implanted subcutaneously into Sprague Dawley rats and retrieved at 7 and 14 days and evaluated via histology and gene expression. CM was obtained by incubating dACM grafts in basal media for 5 days at 4°C at a ratio of 1 cm2 dACM to 1 mL media. The protocol for this study was reviewed and approved by the Bridge PTS IACUC (protocol 17-02). Control groups consisted of sponges soaked with basal media. At 7- and 14-days, implants were retrieved with half placed in 10% neutral buffered formalin for histological evaluation, and half placed in RNAzol for PCR evaluation for angiogenesis related targets. Fixed samples were then embedded and sections were then stained with CD31 and αSMA with hematoxylin counterstaining. By 7 days there were significant increases in several pro-angiogenic genes in the dACM CM group including FN1, EFNA1, TGFB3, VEGFC, TYMP, THBS1, and SERPINE1. ANGPT2, an antagonist of angiopoietin 1, was significantly downregulated at 7 days. At 7 days SMA quantification showed small increases in staining within the dACM implant compared to negative controls, but no differences in αSMA staining on the outside of the implant. By 14 days, αSMA staining outside the implant was increased in dACM CM groups compared to controls. These results demonstrate that dACM contains a number of growth factors and cytokines that promote a pro-angiogenic response in vivo.

N2.03

MIR-193B-3P SUPPRESSES CELLULAR MIGRATION AND CONTRIBUTES TO IMPAIRED WOUND CLOSURE IN DIABETIC FOOT ULCERS
Irena Pastar1, Ivan Jozic1, Horacio A. Ramirez1, Nkemcho Ojeh2, Rivka Stone1, Tongyu Cao Wikramanayake1, Robert S. Kirsner1, Marjana Tomic-Canic1
1University of Miami Miller School of Medicine, Miami Beach, FL, USA, 2Faculty of Medical Sciences, the University of the West Indies, Bridgetown, Barbados
MIR-193B-3P SUPPRESSES CELLULAR MIGRATION AND CONTRIBUTES TO IMPAIRED WOUND CLOSURE IN DIABETIC FOOT ULCERS

Irena Pastar1, Ivan Jozic1, Horacio A. Ramirez1, Nkemcho Ojeh2, Rivka Stone1, Tongyu Cao Wikramanayake1, Robert S. Kirsner1, Marjana Tomic-Canic1 1University of Miami Miller School of Medicine, Miami Beach, FL, USA, 2Faculty of Medical Sciences, the University of the West Indies, Bridgetown, Barbados

Regulation of keratinocyte migration is indispensable for successful wound healing. We aimed to identify cellular functions that contribute to pathophysiology and lack of epidermal migration in diabetic foot ulcers (DFU) by utilizing comparative mRNA and miR profiling of DFUs and healing acute wounds (AW). Ingenuity pathway analysis of paired mRNA/miR profiles identified suppression of cellular migration as a uniquely enriched entity in DFUs and revealed miR-193b-3p as a potential master regulator of downregulated genes in DFU, but not AW. We isolated epidermal miR from DFUs and AW using laser captured microdissection and confirmed induction of miR-193b-3p in DFUs, and not in the AW. Next we validated miR-193b-3p downstream target genes (KRAS, MAP3K1, TNS1, SOS2, JAK2 and ARHGEF6) by qRT-PCR or 3’-UTR luciferase reporter assays. Functional conformation of miR-193b-3p mediated inhibition of healing was performed by miR overexpression studies in vitro and in vivo. To examine the role of miR-193b-3p in cell migration, we performed wound scratch assays and confirmed that miR-193b-3p inhibits migration, without affecting proliferation. Importantly miR-193b-3p exhibited a dominant inhibitory effect on keratinocyte migration, it suppressed wound closure in vitro even in the presence of previously established pro-migratory miRs. To validate miR-193b-3p function in vivo, we injected control or miR-193b-3p mimics into murine excisional wounds, and confirmed delayed wound closure due to miR-193b-3p overexpression. To elucidate the mechanism of miR-193b-3p mediated inhibition of cellular migration we selectively induced filopodia, lamellipodia and stress fiber formation in keratinocytes over-expressing miR-193b-3p and visualized actin cytoskeleton by phalloidin-rhodamine staining. MiR-193b-3p inhibited formation of stress fibers in keratinocytes, which is essential for proper directional cell migration. In summary, we identified miR-193b-3p as a potential therapeutic target and major regulator responsible for healing impairment in DFUs through suppression of keratinocyte migration via inhibition of stress fiber formation.

N2.04

COLLAGEN MATRICES AND AUTOLOGOUS CELLS SUSPENSION DIFFERENTIALLY INFLUENCE RE-EPITHELIALIZATION AT 2 WEEKS IN A PORCINE WOUND MODEL
J Genevieve Park, PhD, MD1, Sita M. Damaraju, PhD2, Benjamin R. Mintz, PhD2, Ankur S. Gandhi, PhD2, Sunil Saini, PhD2, Ronald Ingram, PhD2, Joseph A. Molnar, PhD, MD1
1Wake Forest University School of Medicine, Winston-Salem, NC, USA, 2Integra Lifesciences, Plainsboro, NJ, USA
COLLAGEN MATRICES AND AUTOLOGOUS CELLS SUSPENSION DIFFERENTIALLY INFLUENCE RE-EPITHELIALIZATION AT 2 WEEKS IN A PORCINE WOUND MODEL

J. Genevieve Park, PhD, MD1, Sita M. Damaraju, PhD2, Benjamin R. Mintz, PhD2, Ankur S. Gandhi, PhD2, Sunil Saini, PhD2, Ronald Ingram, PhD2, Joseph A. Molnar, PhD, MD1 1Wake Forest University School of Medicine, Winston-Salem, NC, USA, 2Integra Lifesciences, Plainsboro, NJ, USA

The objective of this study was to evaluate if a single-stage procedure can be achieved by combining autologous cells suspension with dermal substitutes (DS) in the treatment of full thickness wounds in a porcine model. Split thickness skin grafts obtained from the pig were processed using an autologous cell harvesting device for preparation of an autologous cell suspension. Cells were seeded onto a commercially available bio-engineered matrix (DS1) or a decellularized dermal matrix (DS2). A total of twelve (4x4cm) full thickness wounds were created on the dorsum of each Yorkshire pig (n=12) and treated with: cells alone, cells+DS1, cells +DS2, DS1 alone, DS2 alone, or empty. Wound measurements and biopsies were taken on days 9, 14, 28 and 42. Histologic analyses included a combination of semi-quantitative grading scale and histomorphometry. Re-epithelialization at day 14 was significantly greater (p<.01 ANOVA, Tukey) in cells+DS1 and cells+DS2 compared to, DS1 and DS2, cells alone and the empty groups. On day 14, levels of epithelial hyperplasia significantly (p<.01 Mann-Whitney) increased in the pooled groups of cells+DS1 and cells+DS2 groups when compared to the DS1, DS2, and cells alone groups. Day 14 myofibroblast activity was markedly increased (p<.01, Mann Whitney) in the empty and cells only groups compared to DS groups and DS groups with cells.Results suggest the combination of autologous cells with DS may be promising for single-stage procedures for dermal reconstruction with marked increase in early re-epithelialization. However, additional effects of combination therapies with cells and matrices on epithelial hyperplasia emphasize the need for further investigation. Remodeling may require well over 42 days to resolve rendering possibility that longer-term studies are required to adequately assess the combined impact of autologous cells with DS on ultimate healed tissue quality.

N2.05

MIR-210 ENCAPSULATED EXTRACELLULAR VESICLES CONTRIBUTES TO ISCHEMIC INJURY OF THE SKIN
Ayan Biswas, Subhadip Ghatak, Mohamed El Masry, Savita Khanna, Sashwati Roy, Chandan K. Sen
Indiana University, Indianapolis, IN, USA
MIR-210 ENCAPSULATED EXTRACELLULAR VESICLES CONTRIBUTES TO ISCHEMIC INJURY OF THE SKIN

Ayan Biswas, Subhadip Ghatak, Mohamed El Masry, Savita Khanna, Sashwati Roy, Chandan K. Sen Indiana University, Indianapolis, IN, USA

Background: Chronic wounds are commonly associated with peripheral vasculopathies causing wound ischemia. Limitations in the ability of the vasculature to deliver O2-rich blood to the wound tissue leads to hypoxia. It has been reported that hypoxia induces release of extracellular vesicles (EV). The significance of such release in wound repair remain unclear. Methods: To determine the significance of keratinocyte-specific miR-210 during ischemic injury, animal model with keratinocyte specific knockout of miR-210 (K14cremiR-210Δ/Δ) was developed. Keratinocytes were captured using laser capture microdissection (LCM) from the skin. Mitochondrial respiration was measured through Seahorse XF96. Results: A significant knockdown (~50%) of miR-210 was noted in the skin epithelium of the K14cremiR-210Δ/Δ mice compared to their wildtype littermates. Using a mono-pedicle ischemic flap model, in C57BL/6 mice, it was noted that at day 3 postsurgery, miR-210 was induced commensurate with graded levels of hypoxia (n=5, p<0.05). The extent of hypoxia was categorically characterized by dividing the flap into three parts (proximal, intermediate and distal). The level of hypoxia gradually increased from the proximal to the distal part. Consistent findings were observed for miR-210 expression from different region of flap tissue (n=5, p<0.05). Interestingly, under similar condition, K14cremiR-210Δ/Δ mice showed increased perfusion resulting in better flap survival 3 days post-surgery (n=6, p<0.05). Furthermore, it was observed that the EV production from ischemic tissue was proportionate to the extent of hypoxia. Such influence of hypoxia on EV production was also verified in vitro in keratinocytes (n=4, p<0.05). qRT-PCR analysis of such EV showed high abundance of miR-210. Treatment with such miR-210-rich EV showed decreased mitochondrial respiration in normoxic keratinocytes. Conclusions: miR-210-rich EV may be viewed as paracrine mediator that minimizes oxygen cost by economizing metabolism of the cell. Inhibition of keratinocyte specific miR-210 may be an effective strategy to improve EV mediated ischemic chronic wound outcomes.

N2.06

NOVEL IMPLANTABLE BIOSENSORS FOR LIFETIME-BASED MONITORING OF OXYGENATION TO PREDICT WOUND HEALING
Preet Patel1, Mahmoud Ibrahim2, Ryan Schweller2, Bruce Klitzman2, Mohamid Ibrahim2
1Duke University School of Medicine, Durham, NC, USA, 2Duke University Department of Surgery, Duke University Medical Center, Durham, NC, USA
NOVEL IMPLANTABLE BIOSENSORS FOR LIFETIME-BASED MONITORING OF OXYGENATION TO PREDICT WOUND HEALING

Preet Patel1, Mahmoud Ibrahim2, Ryan Schweller2, Bruce Klitzman2, Mohamid Ibrahim2 1Duke University School of Medicine, Durham, NC, USA, 2Duke University Department of Surgery, Duke University Medical Center, Durham, NC, USA

Purpose: Free-tissue-transfer requires close postoperative-monitoring to rapidly identify vascular-occlusion in order to improve salvage. It is estimated that 6%- 25% of skin flaps require a secondary surgical exploration and ~10% of flaps fail. Currently, all monitoring methods have limitations and impose a significant delay between time of vessel-occlusion and its detection. The scope of this study is to introduce a long-term, real-time method of vascular-occlusion through the observation of sensor-modulation in response to vascular-manipulation. Methods: Experimental sensors were made by incorporating benzo-porphyrin dye into a matrix of Poly-Ethylene-Glycol-Diacrylate hydrogel, with each measuring 3 mm-long,1.5 mm-wide,0.5mm-thick. Using Sprague-Dawley rats (n=8), superficialinferior-epigastric-artery myocutaneous flaps on the ventral abdomen and modified caudal MacFarlane-type random-flaps were surgically-elevated. Experimental oxygen sensors were intradermally implanted at the tip, middle and base of the flap-site. Tissue oxygen tension (TOT) readings were obtained from implanted sensors both at baseline and during vascular-clamping of feeding blood vessels. FiO modulation with sensor reading, perfusion analysis using sodium-fluorescein, gross-flap-analysis and statistical-analysis utilizing ANOVA were performed to evaluate sensor-efficacy across surgical-flaps. Results: TOT was quantified and expressed after normalizing the oxygen to a reference arm. TOT measurements from the sensors were observed to modulate as anticipated by a magnitude that reflected the changes in inspired oxygen levels. Clinical observation of the flaps did not show any significant change in color and temperature of the flaps during or immediately after clamping of the feeding blood vessels. Real-time analysis of the sensors implanted in the myocutaneous flaps has demonstrated that acute vascular-clamping of feeding blood-vessels in the pedicle were immediately detected within 70-seconds.(*p<0.05) Conclusion: Oxygen-monitoring in tissues is highly sensitive and can be specific for the detection of acute vascular-occlusion/wound-healing. This approach is superior to clinical observation, faster than current standard of care methods and offers a cost-effective, accurate means of monitoring free-tissue-transfers.

N3.01

OVEREXPRESSION OF LNCRNA LETHE ENHANCES DIABETIC WOUND HEALING
Junwang Xu, Junyi Hu, Carlos Zgheib, Kenneth Liechty
University of Colorado, Aurora, CO, USA
OVEREXPRESSION OF LNCRNA LETHE ENHANCES DIABETIC WOUND HEALING

Junwang Xu, Junyi Hu, Carlos Zgheib, Kenneth Liechty University of Colorado, Aurora, CO, USA

Background: Impaired diabetic wound healing is associated with abnormal long non-coding RNA Lethe and chronic inflammation. We have previously shown that lncRNA Lethe is down-regulated in diabetic wounds and is involved in the regulation of Reactive oxygen species (ROS) production through modulation of NOX2 gene expression, indicating a potential role for Lethe in the pathogenesis of diabetic wounds. We hypothesized that overexpression of Lethe would reduce NOX2 expression and ROS production and enhance the woundhealing process. Methods: Db/Db diabetic mice and Db/+ non-diabetic mice were wounded with an 8-mm punch biopsy and the wounds treated with a lentiviral vector containing either the green fluorescent protein (GFP) or Lethe transgene. Computerized planimetry was used to measure wound size daily. Gain function of Lethe in RAW macrophage was achieved by transfection. Real-time PCR was applied to measure genes expression. Results: Overexpression of Lethe resulted in a significant increase in the rate of diabetic wound healing, (71% vs 99% in GFP-treated wounds at day 8 after injury; P < 0.02), and also improved the early phase of non-diabetic wound healing. Lethe overexpression resulted in significantly decreased NOX2 and proinflammatory cytokines including TNF-α, IL6 and MIP2, and ROS production in RAW macrophage. Discussion: The relative level of lncRNA Lethe in the wound plays a key role in the wound healing response. Alterations in the wound level of Lethe by overexpression, enhance healing by potentially decreasing ROS production and inflammatory cytokines in diabetic wounds.

N3.02

COMPARATIVE GLYCOMICS OF DIABETIC AND NON- DIABETIC SKIN REVEAL SIGNIFICANT DIFFERENCESDURING WOUND HEALING
Veronica R. Haywood1, Sylvain D. Lehoux2, Emily E. Rouse2, Karen Colley1, Luisa A. DiPietro1
1UIC, Chicago, IL, USA, 2Beth Israel Deaconess, Boston, MA, USA
COMPARATIVE GLYCOMICS OF DIABETIC AND NON- DIABETIC SKIN REVEAL SIGNIFICANT DIFFERENCES DURING WOUND HEALING

Veronica R. Haywood1, Sylvain D. Lehoux2, Emily E. Rouse2, Karen Colley1, Luisa A. DiPietro1 1UIC, Chicago, IL, USA, 2Beth Israel Deaconess, Boston, MA, USA

In 2017, Diabetes Mellitus (DM) and its comorbidities placed a 327 billion-dollar burden on the US economy. Approximately 33% of this burden can be attributed to diabetic lower extremity ulcers (DLEU’s/DFU’s). As the economic cost of DM continues to rise, it is imperative that biomarkers are identified to predict ulcer development and healing. Previous studies in our lab identified several glycosylation-related genes that are differentially regulated between diabetic and non-diabetic skin and wounds. To determine whether DM impacts downstream changes in skin and wound glycosylation, we compared the N- and O-linked glycan profiles of C57BL (WT) and db/dbLepr (DM) mouse skin and excisional wounds using MALDI-TOF mass spectrometry. Skin samples were collected at baseline and days 1, 3, and 10 post wounding to reflect the early inflammatory, mid- inflammatory, and proliferative phases of wound healing. Glycan profiling identified a total of 175 N-linked and 25 O-linked glycans in WT and DM skin and wounds. Among the glycans observed, only 25.7% of the N-linked glycans have been identified in mouse serum. When examined individually, 11 N-linked and 10 O-linked glycans were differentially regulated between WT and DM mice during wound healing (p<0.05). When examined by structural features, diabetic wounds demonstrated a 2 to 4-fold increase in highly fucosylated, complex N-glycans (p<0.05) during the inflammatory phase of wound healing. Additionally, diabetic wounds displayed increased core 2 fucosylation and blunted sialylation of core 1 and core 2 Olinked glycans (p<0.05) between the inflammatory and proliferative phases of wound healing. Overall, this data suggests that there may be a metabolic preference for fucosylation over sialylation of N- and O-linked glycans during the inflammatory phase of wound healing in diabetic mice. This change may contribute to the altered immune response observed in DM.

N3.03

Myostatin-regulated Muscle Regeneration In Compressed Rat Skin
Takeo Minematsu, Sanai Tsunokuni, Ayano Nakai, Tamae Urai, Toshihiro Tsukatani, Hiromi Sanada
The University of Tokyo, Tokyo, Japan
MYOSTATIN-REGULATED MUSCLE REGENERATION IN COMPRESSED RAT SKIN

Takeo Minematsu, Sanai Tsunokuni, Ayano Nakai, Tamae Urai, Toshihiro Tsukatani, Hiromi Sanada The University of Tokyo, Tokyo, Japan

Background: Skeletal muscle underlying skin is a vulnerable tissue with high regenerative activity. Its degeneration and regeneration might contribute to the development and healing of pressure ulcers. Myostatin (MSTN) is a major myokine that is constantly expressed in skeletal muscle to inhibit the overgrowth of muscle tissue. When muscle tissue is damaged, MSTN expression is inhibited, which promotes muscle regeneration. However, no studies have shown the contribution of MSTN for the development and healing of pressure ulcers. In this study, we investigated how pressure loading in rat skin influenced MSTN expression. Methods: The study protocols were approved by the Animal Research Committee of The University of Tokyo. We used 6-month-old male Sprague-Dawley rats. A metal plate was inserted subperitoneally and the overlying skin and muscle tissue were compressed using an indenter at 10 kg/3 cm2 for 6 hours under anesthesia. MSTN expression was examined using real-time RT-PCR, western blotting, and immunohistochemistry in the compressed and control skin tissue. Results: mRNA Mstn expression was detected in the control skin but not in the compressed skin. Western blotting and immunohistochemistry indicated the decreased expression of MSTN due to pressure loading. Conclusion: These results suggest MSTN’s contribution in pressure ulcer development. Further studies to show the effects of the overexpression of MSTN in compressed skin are required.

N3.04

IN VITRO BIOACTIVITY OF TRI-LAYER PLACENTAL ALLOGRAFT MEMBRANE
Sita M. Damaraju, Paul Bonvallet, Heli Modi, Morakot Likhitpanichkul, Ankur Gandhi, Sunil Saini
Integra Lifesciences, Corp, Plainsboro, NJ, USA
IN VITRO BIOACTIVITY OF TRI-LAYER PLACENTAL ALLOGRAFT MEMBRANE

Sita M. Damaraju, Paul Bonvallet, Heli Modi, Morakot Likhitpanichkul, Ankur Gandhi, Sunil Saini Integra Lifesciences, Corp, Plainsboro, NJ, USA

Application of placental tissue allografts has the potential to be an efficacious treatment for chronic wounds due to its inherent reservoir of growth factors, cytokines and extracellular matrix molecules. Processing of these placental tissues is essential for long term storage and “off the shelf “availability but without compromising the functionality of inherent bioactive factors. In this study, the bioactivity of a novel, dehydrated tri-layer placental allograft membrane (TPAM), consisting of amnion, chorion and amnion, was evaluated in vitro. TPAM was homogenized and extracted in media overnight at 4oC. Bioactivity of TPAM extracts was evaluated in a wound healing scratch assay with adult fibroblasts to assess cell migration. Chemotactic migration assay was performed with human mesenchymal stem cells (hMSCs) in a 24 well Trans-well plate setup. Inflammation assay was performed with lipopolysaccharide activated peripheral blood mononuclear cells (PBMCs) to determine whether TPAM could reduce the expression of pro-inflammatory cytokines. Adult fibroblasts were seeded on TPAM to evaluate cell attachment and proliferation over 7 days in culture. TPAM promoted enhanced adult fibroblasts and hMSCs migration, which was significantly higher than controls (n=4 per group; p<0.05). Addition of TPAM extracts to PBMCs significantly reduced the expression of pro-inflammatory mediators by more than 90% for TNF-alpha, IL-1beta, and IL-6, and by 40% for IL-8 (n=4 per group; p<0.05). In addition, TPAM promoted cell attachment and proliferation with adult fibroblasts in culture over a 7-day period. These results confirm that TPAM contains factors which are bioactive and could aid in providing the appropriate environment to close chronic wounds.

N3.05

MATRIX CHANNELS IMPROVE AUTOGRAFT TAKE IN A SINGLE STAGE PROCEDURE
Angana Kharge, PhD, Ankur Gandhi, PhD, Sunil Saini, PhD
Integra Lifesciences, Plainsboro, NJ, USA
MATRIX CHANNELS IMPROVE AUTOGRAFT TAKE IN A SINGLE STAGE PROCEDURE

Angana Kharge, PhD, Ankur Gandhi, PhD, Sunil Saini, PhD Integra Lifesciences, Plainsboro, NJ, USA

Background: Soft tissue defects treated with dermal substitutes often require a two-stage procedure to achieve epidermal closure. The purpose of the first stage is to achieve an appropriate dermal bed which can support a thin autologous split thickness skin graft (STSG) applied in a second stage, typically 2-3 weeks after the initial stage. A single-stage procedure, in contrast, will save the patient a second operative procedure, minimize hospital stay and thereby reduce overall cost of care. However, single-stage procedures could result in graft loss due to lack of contact between the graft and wound bed leading to insufficient nutrient support, and therefore additional procedures are required to achieve epidermal closure. We hypothesize that creation of micro channels in the dermal substitute may facilitate rapid cellular migration and formation of well vascularized granulation tissue to support graft viability. Methods: We investigate the effect of introducing channels into a collagen-chondroitin-6-sulfate (C6S) matrix on STSG take and compare findings to a collagen—C6S matrix without channels. In a pilot study, 4x4cm full-thickness wounds were created on the dorsum of a Yorkshire pig. A pattern of channels was mapped with a 2mm biopsy punch.STSG was applied to thewounds directly over the matrix with and without channels. Dressing changes were performed at 3 to 4 day intervals up to sacrifice at Day 14. Results: In non-fenestrated group, the autograft became necrotic and sloughed by Day 11. In contrast, the autograft was well integrated in the channel group. At 14 days, histology showed that granulation tissue formed within the channels to provide sufficient blood supply and nutrients to the overlying STSG, thereby enhancing graft survival. Conclusion: In this pilot single stage study, channels in the collagen–chondroitin6-sulfate matrix based matrix increased autograft take. This feature is expected to prevent the added morbidity associated with prolonged multi-staged reconstructions.

N3.06

WOUND HEALING ACTIVITY OF HUMAN UMBILICAL CORD BLOOD-DERIVED MESENCHYMAL STEM CELLS ON DIABETIC WOUND
Jae-A Jung, MD, PhD, Kyung-Chul Moon, PhD
Korea University Medical Center, Seoul, South Korea
WOUND HEALING ACTIVITY OF HUMAN UMBILICAL CORD BLOOD-DERIVED MESENCHYMAL STEM CELLS ON DIABETIC WOUND

Jae-A Jung, MD, PhD, Kyung-Chul Moon, PhD Korea University Medical Center, Seoul, South Korea

Recently, there has been increasing interest in the use of cell-based therapies for diabetic wounds. These therapies are designed to modulate the levels of biologic molecules, including growth factors and extracellular matrices that may promote wound healing. Various types of skin substitutes composed of either allogeneic or autologous fibroblasts and/or keratinocytes have been commercialized. Although allogeneic cells, derived from healthy donors, are already available, they may carry the risk of immunologic reactions or cross-infections. Autologous cells are free from these drawbacks. However, they require a long culture time, and, in diabetic patients, autologous cells may not have sufficient capacity to stimulate wound healing. Mesenchymal stem cells (MSCs) may be a good alternative for treating diabetic wounds because they have the advantage of containing both allogeneic and autologous cells. MSCs demonstrate low levels of immunity-assisted rejection and can divide without apoptosis. It was demonstrated that even after 20 or 30 cycles of cell doubling in culture, they still retain initial stem cell properties. Accordingly, MSCs have attracted much interest in the bioengineering field. Bone marrow (BM) stroma is one of the main sources of MSCs. Previous studies performed by our group demonstrated that BM-derived MSCs (BM-MSCs) synthesize higher amounts of collagen, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in vitro, as compared with dermal fibroblasts. However, the number of MSCs in the BM decreases with aging and procurement is relatively invasive. In addition, because of issues regarding approval by the Food and Drug Administration (FDA), it is difficult for a clinician to use cultured BM-MSCs for wound healing these days. In the meantime, a novel commercial drug that uses human umbilical cord bloodderived MSCs (hUCB-MSCs) has been developed and achieved recognition by the Korean FDA for helping the regeneration of knee cartilage. Human UCB-MSCs can be obtained in massive quantities without significant ethical issues. Besides, cord blood stem cells are more immature than adult MSCs and can proliferate easily in vitro. Because immune system of newborn is relatively immature than that of adult, cord blood-derived MSCs have been successfully transplanted without causing rejection. Today I will show previous studies performed by our group demonstrated that hUCB-MSCs have superior wound healing activities when compared with both healthy and diabetic fibroblasts not only in vitro but also in vivo studies.

N4.01

OPTIMIZATION OF DEBRIDEMENT DEPTH THAT RESULTS IN COMPLETE GRAFT TAKE FOR PORCINE BURN WOUNDS
Randolph Stone, II, David Larson, John Wall, Christine Kowalczewski, Shanmugasundaram Natesan, Robert Christy
US Army Institute of Surgical Research, Fort Sam Houston, TX, USA
OPTIMIZATION OF DEBRIDEMENT DEPTH THAT RESULTS IN COMPLETE GRAFT TAKE FOR PORCINE BURN WOUNDS

Randolph Stone, II, David Larson, John Wall, Christine Kowalczewski, Shanmugasundaram Natesan, Robert Christy US Army Institute of Surgical Research, Fort Sam Houston, TX, USA

Background: The current standard of care for burn wound management involves removal of necrotic tissue to a bleeding wound bed then application of a skin graft depending on the depth of injury. However, the debridement procedure is difficult, time consuming, and can result in hypertrophic scarring if too much tissue is removed thus resulting in a deeper wound. The purpose of this study was to determine the optimal amount of debridement necessary to result in successful graft take on porcine burn wounds. Methods: Deep partial and full thickness 5×5 cm burn wounds were created on the dorsum of six anesthetized Yorkshire pigs using appropriate pain control methods. After 4 days, the necrotic eschar was debrided via a dermatome to three depths (0.030”, 0.060”, 0.090”) and a meshed split thickness skin graft was applied. Graft success (defined as >70% graft take) was assessed on days 7 and 14. Results: Approximately 65% of wounds with the least debridement amount resulted in graft failure. No differences in bacterial counts were detected in the post-debrided wounds. Laser speckle imaging indicated no differences in the burn contact times immediatelypost-burn but significantdifferences weredetected at thepre-debridement time point when comparing the burn depths. After combining all wounds into either graft success vs. failure, laser speckle imaging detected a significantly higher blood flow in post-debrided wounds for thewound beds that resulted ingraft success. Conclusions: This study allowed for the creation of a model to duplicate the clinical situation of inadequate debridement in burn wounds resulting in graft failure. Other potential reasons such as infection and mechanical shearing were ruled out as contributors in the observed graft failure. Most intriguing, laser speckle imaging was able to differentiate between the wounds that were adequately debrided and those where necrotic tissue remained; thereby, providing clinicians with a non-invasive technique that could help determine when to graft.

N4.02

TRANSCRIPTOMIC ASSESSMENT OF THE P53 SIGNALING PATHWAY IN THE BURN WOUND ZONE OF STASIS
Gaurav Garg, Abdulnaser Alkhalil, Bonnie Carney, Robert Smith, Kyle Monger, Lauren Moffatt, Jeffrey Shupp
The Burn Center, MedStar Washington Hospital Center, Washington, DC, USA
TRANSCRIPTOMIC ASSESSMENT OF THE P53 SIGNALING PATHWAY IN THE BURN WOUND ZONE OF STASIS

Gaurav Garg, Abdulnaser Alkhalil, Bonnie Carney, Robert Smith, Kyle Monger, Lauren Moffatt, Jeffrey Shupp The Burn Center, MedStar Washington Hospital Center, Washington, DC, USA

This study was designed to characterize transcriptomic changes induced in the p53 pathway by burn injury to understand burn wound conversion and guide development of therapies to improve tissue salvage and reduce the need for excision and grafting. Brass combs heated to 100°C were used to generate 4 burns intercalated with 3 unburned interspaces on the dorsum of 10 rats. Contact time was varied to create different wound depths. Interspaces were biopsied at 3, 6, 12, 24, and 48 hours post-injury for comparison to pre-burn skin. Laser doppler imaging (LDI) was used to identify burn wound conversion. Following mRNA isolation, p53 pathway PCR arrays were used to capture transcript-level changes in 84 genes in the interspace tissues over time. The fold change in expression of each gene was determined, and a Z-score was calculated to assess overall pathway regulation. Analysis of deep partial-thickness wounds revealed induction of the p53 pathway at hour 6 (Z-score 0.853, p<0.0001) followed by downregulation at hour 48 (Z-score -1.606, p<0.0001). LDI confirmed conversion in full-thickness wounds, which had less perfusion compared to superficial partial-thickness wounds (baseline: p>0.05, all other timepoints: p<0.05). Superficial partial-thickness wounds showed downregulation and upregulation of Esr1 and IL-6, respectively, from hours 6-24, followed by upregulation of genes for cell survival (Birc5), cell cycle progression (Ccnb1, Ccne1, Cdk4), and DNA repair (Brca1, Chek1/2, Msh2, Pcna). In full-thickness wounds, multiple genes differentially regulated at hour 6 remained so until hour 48. Upregulation of Rprm and Cdkn1a/Cdkn2a with downregulation of Ppm1d indicated cell cycle arrest. This coincided with induction of pro-apoptotic Tnf and Tnfrsf10b and downregulation of DNA repair (Lig4). Downregulation of Stat1 and Egfr suggested a decrease in downstream transcription of numerous factors impacting cellular viability. Following this first examination of alterations in the p53 pathway in the burn wound zone of stasis, further investigation will focus on proteomic changes to identify potential therapeutic targets.

N4.03

INCREASED EXPRESSION OF PRO-INFLAMMATORY CYTOKINES AND PERIOSTIN IN BURN ESCHAR PERICYTES
Alexander Evdokiou1, Stephanie Gierek1, Onur Kanisicak2, Richard Bodnar3, Latha Satish1
1Shriners Hospitals for Children-Cincinnati, Cincinnati, OH, USA, 2Department of Pathology and Laboratory Medicine, University of Cincinnati, College of Medicine, Cincinnati, OH, USA, 3Veterans Affairs Medical Center, Pittsburgh, PA, USA
INCREASED EXPRESSION OF PRO-INFLAMMATORY CYTOKINES AND PERIOSTIN IN BURN ESCHAR PERICYTES

Alexander Evdokiou1, Stephanie Gierek1, Onur Kanisicak2, Richard Bodnar3, Latha Satish1 1Shriners Hospitals for Children-Cincinnati, Cincinnati, OH, USA, 2Department of Pathology and Laboratory Medicine, University of Cincinnati, College of Medicine, Cincinnati, OH, USA, 3Veterans Affairs Medical Center, Pittsburgh, PA, USA

Background: Pericytes exhibit mesenchymal stem cell-like properties and have a key role in various fibrotic disease by contributing to the myofibroblast formation. Periostin is shown to exacerbate fibrosis in various organs. In this study, the influence of burn wound environment on the inflammatory status of pericytes and their effect on periostin deposition in skin wound stroma is determined. Methods: Pericytes were isolated from discarded burn eschar and normal skin obtained from patients undergoing elective plastic surgery. RNA was isolated and subjected to RNA-seq and real-time RT-PCR analyses. Changes in protein levels were determined by immunohistochemistry, western blots, and ELISA. Data analyses were done using ANOVA and p<0.05 was considered statistically significant. Results: We identified 443 statistically significant differentially expressed genes between normal- and burn eschar-derived pericytes. Five of the pro-inflammatory genes, namely IL-6, IL-1β, IL-1α, IL-8, and TNFαIP3, were upregulated and one pro-inflammatory gene (CXCL-14) was downregulated in burn pericytes. Two anti-inflammatory genes (NFkBIZ and NFkBIα) were upregulated in burn pericytes. Western blots showed increased basal expression of NFkB in burn pericytes. ELISA confirmed upregulation of IL-6 and IL-8 at the basal level in burn eschar pericytes compared to normal skin pericytes (p<0.05). An increase in periostin in burn eschar tissues and pericytes was determined both at the mRNA and protein levels compared to normal skin and pericytes. Conclusions: We report for the first time that burn eschar pericytes are inflammatory and may contribute to burn scar contractures and fibrosis by increasing the expression of periostin, a protein produced by myofibroblasts in cardiac fibrosis.

N4.04

TOPICALLY APPLIED MORPHINE-LOADED KERATIN HYDROGELS ON WOUND HEALING IN A PORCINE BURN MODEL
Christine Kowalczewski1, Nicholas Clay1, Nicole Wrice1, Bopaiah Cheppudira1, John Clifford2, Robert Christy1
1USAISR, Fort Sam Houston, TX, USA, 2USACEHR, Fort Detrick, MD, USA
TOPICALLY APPLIED MORPHINE-LOADED KERATIN HYDROGELS ON WOUND HEALING IN A PORCINE BURN MODEL

Christine Kowalczewski1, Nicholas Clay1, Nicole Wrice1, Bopaiah Cheppudira1, John Clifford2, Robert Christy1 1USAISR, Fort Sam Houston, TX, USA, 2USACEHR, Fort Detrick, MD, USA

Currently burn wound pain management relies heavily on systemic administration of opioids which often result in a number of adverse side effects such as addiction. Therefore, topical administration of opioids directly to the wound site would reduce the total opioid usage. Recent studies have suggested that topical opioids may play a beneficial role in wound healing. In order to provide local delivery of morphine, the opioid can be loaded into a biomaterial such a keratin, a filamentous protein found in human hair. The objective of this study is to assess the effects of topically administered morphine-loaded keratin hydrogels on burn wound healing. Ten partialthickness burns were created using a 100°C heated 5 cm square brass block applied to the dorsum of an anesthetized Yorkshire swine (N=5). Two days post injury, pigs were re-anesthetized, necrotic tissue was debrided, bleeding was stopped, and respective treatments were applied and dressed. Keratin hydrogels loaded with 0, 1, 5, or 10mg/mL of morphine were compared to no treatment and non-injured physiological controls. Biopsy and blood samples were collected and processed for histology and a cytokine panel analysis. Histomorphologic scores were determined by a trained veterinary pathologist. A Luminex assay was used to measure local inflammatory markers. Findings show a dose-dependent increase in the rate of wound reepilelialization with morphine-loaded keratin hydrogels. Granulation tissue thickness and cytokines were not exacerbated by treatment groups. This study has been conducted in compliance with the Animal Welfare Act, Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals.

N4.05

USE OF AUTOLOGOUS SKIN COLUMNS IN PORCINE DEEP-PARTIAL THICKNESS BURN WOUND HEALING
Anders Carlsson, Tyler Everett, Kai Leung, Rodney Chan
US Army Institute of Surgical Research, San Antonio, TX, USA
USE OF AUTOLOGOUS SKIN COLUMNS IN PORCINE DEEP-PARTIAL THICKNESS BURN WOUND HEALING

Maria Batchinsky, BS, Anders H. Carlsson, PhD, Everett TR, Kai P. Leung, PhD, Rodney K. Chan, MD Dental and Craniofacial Trauma Research Directorate, United States Army Institute of Surgical Research, JBSA For Sam Houston, USA Burn wounds and injuries present a series of issues for both military and civilian casualties. This study compares the use of full-thickness skin columns (FTSC) to current therapy of split-thickness skin grafts (STSG) in deep partial thickness burn (DPTB) porcine wound models. STSG results in hypertrophic scarring, wound contracture, absent adnexal structures, and skin re-harvest from donor sites. We have demonstrated that FTSCs of 1.5 mm in diameter can be easily harvested with minimal donor site morbidity and can cover areas up to 10x larger than the harvest site. We hypothesized FTSCs will exhibit improved healing and scar outcomes in donor and wound sites of a porcine DPTB wound model. Five animals received ten thermal DPTBs of 58 cm2 each that were induced with a thermocoupled burn device (100°C) on the dorsal skin of anesthetized pigs. The wounds were debrided seven days later and FTSCs were harvested at the neck region. Wound area tracing, perfusion, histologic data, and POSAS was collected over the 90 day experimental time frame. This work was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the USAISR. Improvement was demonstrated in re-epithelialization and perfusion of the wound sites. FTSCs can be used successfully to improve healing. The FTSC donor sites heal quicker than traditional STSG donor sites and without the complications of traditional dermatome harvesting. STSG are limited in re-harvest potential as donor site skin availability lessens. This porcine study highlights the value in transplanting skin microcolumns as an alternative treatment with reduced sequela.

N4.06

PLGF-1 CONTAINED IN NORMAL WOUND MYOFIBROBLAST-DERIVED MICROVESICLES STIMULATES COLLAGEN PRODUCTION BY DERMAL FIBROBLASTS
Syrine Arif
Université Laval, Québec, QC, Canada
PLGF-1 CONTAINED IN NORMAL WOUND MYOFIBROBLAST-DERIVED MICROVESICLES STIMULATES COLLAGEN PRODUCTION BY DERMAL FIBROBLASTS

Syrine Arif, Sébastien Larochelle, Véronique J. Moulin

Centre de recherche en organogénèse expérimentale de l’Université Laval/ LOEX – Centre de recherche du CHU de Québec-Université Laval, QC, Canada Myofibroblasts are differentiated cells from fibroblasts that appear during the last stages of healing. They can communicate with other cells using secreted factors but also using microvesicles (MVs). Our aim is to evaluate the effect of MVs produced by myofibroblasts from normal wound (Wmyo) on skin fibroblasts and to determine the signaling molecule responsible for these effects. To model a cell to cell communication between Wmyo and fibroblasts, we studied fibroblasts uptake of fluorescent Wmyo-derived MVs by flow cytometry. We analyzed cytokines distribution in MV samples by a multiplex ELISA detecting 45 cytokines. Fibroblasts cells were then cultured in presence of different concentration of MVs or a selected cytokine for 5 days. Following the treatments, parameters linked to the ECM were studied including pro-collagen I production assayed by ELISA. Finally, fibroblasts were treated with MVs preincubated with a neutralizing antibody for PLGF-1 before to evaluate collagen production. In the presence of fluorescent MVs, a large portion of fibroblast cells demonstrated fluorescence shift with a fold change of 2.96 ± 0.31 versus untreated fibroblasts (N= 2). Cytokine array analysis showed that MVs samples contained a high amount of PLGF-1 (57.53 pg/ug proteins in MVs ± 13.11, N= 6). Cutaneous fibroblasts treated with MVs (10μg protein/ml) or PLGF-1 (10ng/ml) significantly simulated pro-collagen I level production with a fold change of 1.80±0.18 for MVs and, 2.07±0.18 for PLGF-1 (N=3) versus untreated cells. Finally, the neutralization of PLGF-1 in MVs samples effectively neutralized the production of procollagen I (N=3) by fibroblasts. Wmyo derived-MVs stimulate fibroblast collagen production during healing possibly through PLGF-1 signaling.