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2020 Abstracts

G.01

EPIDERMAL NRF2 ORCHESTRATES TISSUE REGENERATION THROUGH REGULATION OF CCL2
Alvaro Villarreal Ponce1, Melat Tiruneh-Worku2, Joshua David1, Chris Guerrero-Juarez3, Kristen Dammeyer1, Jasmine Lee1, Joe Kuhn1, Jennifer Kwong1, Piul Rabbani1, Daniel Ceradini1
1NYU Langone Health, New York, NY, USA, 2Rutgers Robert Wood Johnson School of Medicine, New Jersy, NJ, USA, 3UC Irvine, Irvine, CA, USA
Epidermal Nrf2 orchestrates tissue regeneration through regulation of Ccl2

Alvaro Villarreal Ponce, Melat Tiruneh-Worku, Joshua David, Chris Guerrero-Juarez, Kristen Dammeyer, Jasmine Lee, Joe Kuhn, Jennifer Kwong, Piul Rabbani, Daniel Ceradini | NYU Langone Health; Rutgers Robert Wood Johnson School of Medicine; UC Irvine

Background: This study focuses on uncovering the molecular mechanisms orchestrated by epidermal Nrf2 that are critical for initiating a regenerative response and are defective in diabetic wounds. Methods: Type 2 diabetic Lepr db/db mice and epidermal Nrf2-specific transgenic knockout mice (K14 CreER; Nrf2 fl/fl; cKO) were used to describe dysfunctional cellular and molecular programs that impair wound healing. We generated 10mm diameter, stented, full-thickness excisional wounds on the dorsum of 6-8-week-old WT, diabetic, and cKO mice. Cellular function of epidermal Nrf2 was analyzed by RNA-seq, ChIP, immunohistochemistry, qPCR/Western analysis, and functional assays. Results: Nuclear translocation of Nrf2 is dysfunctional in wound edge-associated keratinocytes of diabetic mice (WT:97.0+/-3.6% vs. diabetic:22.5+/-16.4%; p<0.05). Deletion of epidermal Nrf2 results in severe healing delay (WT:15.6+/-1.2days vs. cKO:34+/-1.7days vs. diabetic:30+/-0days; p<0.0001). Wound histology reveals this delay stems from impaired re-epithelialization (p<0.0001), neo-vascularization (p<0.05), and collagen maturation. RNA-seq and ChIP analysis uncovers epidermal Nrf2 as a direct regulator of oxidoreductase-related genes and those affecting paracrine-mediated regenerative programs. Reduced monocyte/macrophage trafficking at early repair (WT:130.3+/-15.0 cells/area vs. cKO:35+/-2.6 cells/area; p<0.05) results from downregulation of Ccl2, which has a Nrf2-binding motif. Nrf2 induction in primary keratinocytes results in elevated Ccl2 expression, sufficient for restoring physiologic tissue regeneration in cKO wounds (Vehicle:29.7+/-1.73days vs. +Ccl2:17.3+/-0.6days; p<0.0001). Conclusions: Epidermal Nrf2 is an indispensable regulator of paracrine crosstalk between keratinocytes and monocytes/macrophages via direct regulation of Ccl2 activation, promoting monocyte/macrophage trafficking to the wound site.

G.02

HUMAN NRF2-ACTIVE MULTIPOTENT STROMAL CELL EXOSOMES REVERSE PATHOLOGIC DIABETIC WOUND HEALING
Joseph Kuhn, Absara Hassan, Sonali Sharma, Jennifer Kwong, Montaha Rahman, Salma Adam, Alvaro Villarreal-Ponce, Jasmine Lee, Piul S. Rabbani
NYU Langone Health, New York, NY, USA
Human Nrf2-active multipotent stromal cell exosomes reverse pathologic diabetic wound healing

Joseph Kuhn, Absara Hassan, Sonali Sharma, Jennifer Kwong, Montaha Rahman, Salma Adam, Alvaro Villarreal-Ponce, Jasmine Lee, Piul S. Rabbani | NYU Langone Health, New York, NY

Background: Exosomes, nanosized extracellular vesicles, may be key to translating MSC therapy to the bedside. We previously found that Nrf2 regulates MSC multipotency and promotion of diabetic tissue repair. Here we explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates MSC effects on diabetic wound healing. Methods: MSCs were subcultured from whole bone marrow of human donors. Exosomes were harvested by differential ultracentrifugation. For Nrf2-active exosomes, MSCs were incubated with Nrf2 activator CDDO-Im. Exosomes were characterized by TEM, nanoparticle tracking analysis, and immunoblotting. Full-thickness stented wounds were created on adult Leprdb/db mice. Exosomes were injected circumferentially to the wound margin 1-day post-excision. Results: Nrf2-activated MSCs increase exosome secretion by 54% compared to Nrf2-baseline MSCs (p<0.05). Both Nrf2-baseline and Nrf2-active exosome treatment significantly reduced closure time (15.5 and 14 days respectively) compared to 29.8 days for vehicle-treated diabetic wounds (p<0.05). Exosome treatment of diabetic wounds eliminated the delay in healing compared to C57/B6 wounds (16.6 days; p>0.05). Histological analysis shows exosome-treated db/db wounds have significant decreases in epithelial gap, expanded granulation, and greater CD31+ vessel density. Conclusions: Activating Nrf2 in MSCs multiplies exosome yield. MSC exosome-based therapies hold tremendous promise to improve wound outcomes for patients with diabetes.

G.03

HUMAN TISSUE REPAIR AND REGENERATION - REGULATORY LANDSCAPES IN RESPONSE TO INJURY
Trevor R. Leonardo1, Lin Chen1, Phillip T. Marucha2, Kimberly Glass3, Luisa A. DiPietro1
1University of Illinois at Chicago, Chicago, IL, USA, 2Oregon Health Science University, Portland, OR, USA, 3Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
Human tissue repair and regeneration: Regulatory landscapes in response to injury

Trevor R. Leonardo, Lin Chen, Phillip T. Marucha, Kimberly Glass, Luisa A. DiPietro | University of Illinois at Chicago; Oregon Health Science University; Brigham and Women’s Hospital/Harvard Medical School

Background: Healing in the oral cavity is differentiated from cutaneous healing by faster re-epithelialization, shortened inflammation, brief angiogenic response, and minimal scar formation. We hypothesize that transcriptional gene regulatory networks (GRN) of the oral and cutaneous responses to injury are intrinsically different. Methods: We utilized a human microarray gene expression dataset from gingiva and skin spanning the first seven days of wound healing over five time points (n=96). This data, together with protein-protein interaction and transcription factor DNA-binding motif data, was incorporated to estimate sample-specific GRNs using the PANDA and LIONESS algorithms. GRNs from each time point were compared to their respective unwounded tissue networks using LIMMA. Results: GRN analyses show that the skin and oral network responses to injury are intrinsically different. Communities of TFs targeting specific biological processes were identified at each step in the injury response. In the gingiva, a subset of TFs target developmental processes as early as six hours after injury. In skin, TFs target largely proliferative gene sets in the first twenty-four hours and immune response processes at days three and seven. Conclusions: This is the first data-driven network-based approach to identify master regulators of the response to injury. We identified communities of TFs that target similar biological processes and developed an underlying map of the transcriptomic response to injury in human gingiva and skin.

G.04

COMMENSAL MICROBIOTA REGULATES SKIN BARRIER FUNCTION AND REPAIR VIA SIGNALING THROUGH THE ARYL HYDROCARBON RECEPTOR
Aayushi Uberoi1, Casey Bartow McKenny1, Qi Zheng1, Laurice Flowers1, Simon Knight1, Victoria Lovins1, Neal Chan1, Monica Wei1, Julia Bugayev1, Joseph Horwinski1, Charles Bradley1, Jason Meyer2, Debra Cumrine2, Peter Elias2, Elizabeth Mauldin1, Thomas R. Sutter3, Elizabeth Grice1
1University of Pennsylvania, Philadelphia, Philadelphia, PA, USA, 2University of California, San Fransisco, CA, USA, 3University of Memphis, Memphis, TN, USA
Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor

Aayushi Uberoi, Casey Bartow McKenny, Qi Zheng, Laurice Flowers, Simon Knight, Victoria Lovins, et al. | University of Pennsylvania; University of California San Francisco; University of Memphis

Background: Commensal microbes have important roles in maintaining skin homeostasis. However, their mechanisms of crosstalk with host epithelia during barrier disruption and repair remain poorly defined. Methods: RNAseq was used to compare skin epithelial transcriptomes of germ free (GF) vs. specific pathogen free (SPF) mice. Barrier repair was measured in vivo using tape-stripping and transepidermal water loss (TEWL). The aryl hydrocarbon receptor (AhR) pathway was identified as downregulated in GF epidermis. Results: Epithelial development, differentiation, and wound healing genes were downregulated in GF mice. GF mice were deficient in barrier repair compared to SPF mice (p<0.0001). A similar effect was observed after antibiotic depletion in SPF mice. Conditional knockout mice with AhR deleted in basal epithelia were also impaired in barrier repair (p<0.0001). Human commensal skin bacterial isolates that activate the AhR reporter were identified; colonization of GF mice with this consortium rescued barrier repair defects in vivo (p<0.001) and in vitro (p=0.0024). Conclusions: Commensal microbes engage in crosstalk with host epithelia via the AhR to promote skin integrity, revealing novel targets for microbiota-directed therapy for skin barrier disorders.

G.05

DIGIT TIP REGENERATION RELIES ON GERM LAYER RESTRICTED WNT AND HEDGEHOG SIGNALING
Janos Barrera, Zeshaan Maan, Yuval Rinkevich, Dominic Henn, Kellen Chen, Clark A. Bonham, Jagannath Padmanabhan, Michael Januszyk, Irving L. Weissman, Geoffrey C. Gurtner
Stanford, Stanford, CA, USA
Digit tip regeneration relies on germ layer restricted Wnt and Hedgehog signaling

Janos Barrera, Zeshaan Maan, Yuval Rinkevich, Dominic Henn, Kellen Chen, Clark A. Bonham, Jagannath Padmanabhan, Michael Januszyk, Irving L. Weissman, Geoffrey C. Gurtner | Stanford University

Background: Reports of spontaneous regeneration in human fingertips and advances using mouse digit tip regeneration models raise the possibility of regenerative approaches to traumatic hand injuries. The discrete signaling mechanisms governing this regenerative process remain poorly understood. Methods: Tissue from murine digit tips 7 days after amputation at the regenerative vs. non-regenerative plane were compared using microarray analysis. Viral knockdown of candidate genes was used to confirm functional roles in vivo. Tissue-specific expression of target genes was assessed using gene-specific reporter mice. Results: Ingenuity pathway analysis identified Wnt and Hedgehog (HH) signaling as the top associated gene networks differentially regulated in regenerating digit tips. Suppression of either Wnt or HH signaling significantly impaired digit tip regeneration. HH overexpression impaired epidermal closure (typically driven by Wnt), suggesting crosstalk between pathways. Wnt expression and responsiveness were restricted to the ectoderm, while HH expression originated in the ectoderm but responsiveness occurred in the mesoderm. Conclusions: Wnt and HH signaling play a central role in digit tip regeneration. Understanding the crosstalk between these pathways could enable regeneration in normally non-regenerative extremity injuries.

G.06

GRANZYME B CONTRIBUTES TO PRESSURE INJURY SEVERITY IN AGED SKIN
Christopher Turner, Matthew Zeglinski, Juliana Bolsoni, Hongyan Zhao, Anthony Papp, David Granville
University of British Columbia, Vancouver, BC, Canada
Granzyme B contributes to pressure injury severity in aged skin

Christopher Turner, Matthew Zeglinski, Juliana Bolsoni, Hongyan Zhao, Anthony Papp, David Granville | University of British Columbia, Vancouver, BC

Background: Pressure injuries (PIs) are on the rise due to our aging population. Granzyme B (GzmB) is a serine protease minimally expressed in normal skin but dramatically elevated in age-related chronic inflammatory skin diseases. We hypothesized GzmB contributes to PI development, severity, and impaired healing. Methods: GzmB expression was evaluated in human PI wound fluid (ELISA) and excised tissue (histology). A causative role was assessed in GzmB-/- and WT mice through ischemia-reperfusion injury. PIs were also induced in ApoE-/- and GzmB/ApoE double knockout (DKO) mice on high fat diet for 30 weeks to create an aged phenotype. Results: GzmB was dramatically elevated in PI patient wound fluid and excised tissue, positively correlating with disease severity. In aged mice, PI severity was significantly reduced in DKO compared to ApoE-/- mice, with improved wound closure, improved ECM remodeling via GzmB-mediated decorin cleavage, reduced fibrosis, and overall inflammation reduction. Reduced IL-16, IL-1beta, TIMP-1 and TREM-1 expression was identified in DKO mice. Conclusions: GzmB is elevated in PI and contributes to increased wound severity, providing a promising therapeutic target.

G.07

THEORETICAL-EXPERIMENTAL APPROACH TO DEVELOP ELECTROTHERAPY FOR DIABETIC WOUND HEALING
Nava P. Rijal1, Jeremy Lesas2, Caroline Meguerditchian2, Dmitrii Kruglov3, Sagar Mehta4, Jonathan Bath5, Andrei Kogan3, Daria Narmoneva1
1Department of Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA, 2Université de Bordeaux, France, France, 3Department of Physics, University of Cincinnati, Cincinnati, OH, USA, 4Vascular Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA, 5Vascular Surgery, University of Missouri School of Medicine, Columbia, MO, USA
Theoretical-experimental approach to develop electrotherapy for diabetic wound healing

Niva P. Rijal, Jeremy Lesas, Caroline Meguerditchian, et al. | University of Cincinnati; Universite de Bordeaux; University of Missouri School of Medicine

Background: Poor vascular supply is a significant factor in impaired healing of chronic diabetic wounds. Electric field (EF) stimulation is a promising strategy to enhance vascularization. We present a novel theoretical model describing interactions between applied wireless EF and underlying skin and wound tissues using numerical in-situ simulation (ANSOFT/HFSS). Methods: 2×2 cm dorsal wounds were created on STZ-diabetic or wild-type female Yorkshire pigs and treated with wireless electrotherapy (1hr/day, 3-5days/week) or controls (3-4 wounds/group/animal). Tissue was harvested at 4wks post-wounding. Results: The model demonstrates EF penetration throughout the wound from epidermis (2500V/m) to dermis (870V/m) to muscle layers (500V/m). EF stimulation resulted in significant reduction in diabetic wound area (0.64cm2 vs. 0.90cm2, day14; p<0.01) and significantly lower scar area (p<0.05). EF therapy led to 80-100% increase in blood vessel density in non-diabetic (p<0.0005) and diabetic wounds (p<0.005). ELISA showed significantly lower IL-6 (p<0.05) and higher IL-10 (p<0.05) and VEGF (p<0.05) in EF-treated diabetic wounds. EF treatment significantly improved repair tissue strength (p<0.01). Conclusions: Wireless electrotherapy enhances diabetic wound healing via deep stimulation of wound tissues, increasing vascularization and reducing inflammation.

G.08

CUTANEOUS TRPV1+ NOCICEPTORS MEDIATE THE INFLAMMATORY RESPONSE TO PSEUDOMONAS AERUGINOSA IN INFECTED WOUNDS
Michelle D. Bagood, MS1, Daniel J. Yoon2, Alan V. Nguyen1, Daniel R. Fregoso3, Andrea I. Medina Lopez3, Kevin J. Tracey4, William J. Murphy3, Athena Soulika3, R Rivkah Isseroff3
1Immunology Graduate Group, University of California, Davis, CA, USA, 2Dermatology Section, VA Northern California Health Care System, Sacramento, CA, USA, 3Department of Dermatology, UCD Health, Sacramento, CA, USA, 4Feinstein Institutes for Medical Research, Northwell Health, New York, NY, USA
Cutaneous TRPV1+ nociceptors mediate the inflammatory response to Pseudomonas aeruginosa in infected wounds

Michelle D. Bagood, Daniel J. Yoon, Alan V. Nguyen, Daniel R. Fregoso, Andrea I. Medina Lopez, Kevin J. Tracey, William J. Murphy, Athena Soulika, R. Rivkah Isseroff | University of California Davis; VA Northern California Health Care System; Feinstein Institutes for Medical Research, Northwell Health

Background: Recent work reveals interactions between the nervous, immune, and microbial systems in skin that impact healing. We hypothesized that TRPV1+ sensory nerves regulate the inflammatory phase of cutaneous wound healing, and that without these neuronal signals, persistent inflammation delays healing. Methods: We compared healing of full-thickness wounds in C57BL/6 wild-type mice and mice with TRPV1 channel genetically ablated (Trpv1-/-). Immunophenotyping was performed using flow cytometry to determine the impact of dysfunctional sensory nerves. Results: Uninfected wounds healed similarly in C57BL/6 and Trpv1-/- animals (n=10/group, p=0.585). Pseudomonas aeruginosa infection delayed healing in all wounds, but more so in the Trpv1-/- wounds (n=12/group, p=0.001). Infected, wounded Trpv1-/- animals exhibited abnormal myeloid immune cell frequencies, elevated proinflammatory cytokine transcription including IL-1beta, and atypical neuropeptide transcription. Conclusions: Lack of functioning cutaneous TRPV1+ sensory nerves results in elevated inflammation in response to wound infection, resulting in delayed healing.

G.09

ETRS WINNER BIOINSPIRED NANOMATERIALS FOR CELL-SELECTIVE ACTIVATION OF GROWTH FACTORS TO PROMOTE HEALING
N Oliva and B Almquist
Imperial College London, Bioengineering, London (UK)
Bioinspired nanomaterials for cell-selective activation of growth factors to promote healing

Nuria Oliva-Jorge, PhD, Benjamin Almquist | Imperial College London, Bioengineering

Background/Methods: Inspired by the naturally occurring process of activation of TGF-beta1 during healing, we developed an aptamer-based technology platform that harnesses cellular traction forces to unfold the aptamer and release active growth factors, called Traction Force-Activated Payloads (TrAPs). Aptamers were synthesized with a cell-adhesive peptide on one end (e.g., RGD) and a chemical group for scaffold conjugation on the other (e.g., thiol). We showed that TrAPs enable various cells (dermal fibroblasts, microvascular endothelial cells, smooth muscle cells) to mechanically unfold the aptamers and release active forms of PDGF and VEGF. Results: TrAPs technology is easy to integrate within a variety of cell culture systems and biomaterials spanning 2D glass coverslips, polyacrylamide gels, and 3D collagen scaffolds. This is the first demonstration of cell-selective activation of growth factors – e.g., activation by smooth muscle cells but not dermal fibroblasts – by tailoring cell-adhesive peptides to specific cell types (VAPG peptide to smooth muscle cells). Conclusions: TrAPs harness cellular traction forces as a biophysical trigger to activate and release therapeutics without passive or external triggers, enabling spatiotemporal, cell-selective activation of localized bioactivity for regenerative medicine.

H1.01

PRELIMINARY ANALYSIS OF A PHASE 3 OPEN-LABEL, CONTROLLED, RANDOMIZED TRIAL EVALUATING THE EFFICACY AND SAFETY OF A BIOENGINEERED REGENERATIVE SKIN CONSTRUCT IN PATIENTS WITH DEEP PARTIAL-THICKNESS THERMAL BURNS
James H. Holmes1, Jeffrey W. Shupp2, David Smith3, Victor Joe4, Joshua Carson5, Jeffrey Antell6, Steven Kahn7, Tracee Short8, Leopoldo Cancio9, Julie Rizzo10, Jeffrey Carter11, Kevin Foster12, Janice Smiell13, Angela Lf Gibson14
1Department of Surgery, Wake Forest University School of Medicine, Winston Salem, NC, USA, 2Department of Surgery, Medstar Washington Hospital Center, Washington DC, WA, USA, 3Department of Plastic Surgery, University of South Florida, Tampa, FL, USA, 4Department of Surgery, University of California at Irvine, Irvine, CA, USA, 5Department of Surgery, University of Florida, Gainesville, FL, USA, 6Department of Surgery, University of Missouri Health Care, Columbia, MT, USA, 7Department of Surgery, University of South Alabama Medical Center, Mobile, AL, USA, 8Department of Surgery, Baton Rouge General Medical Center, Baton Rouge, LA, USA, 9United States Army Institute of Surgical Research, Fort Sam Houston, TX, USA, 10United States Army Institute of Surgical Research, Joint Base, San Antonio, TX, USA, 11Department of Surgery, Louisiana State University School of Medicine, University Medical Center New Orleans, New Orleans, LA, USA, 12Department of Surgery, The Arizona Burn Center at MIHS, Phoenix, AZ, USA, 13Mallinckrodt Pharmaceuticals, Bedminster, NJ, USA, 14Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA

H1.02

KERATINOCYTE-DERIVED EXOSOMAL PACKAGING DRIVES CONVERSION OF INJURY-SITE MACROPHAGE TO FIBROBLAST-LIKE CELLS IN GRANULATION TISSUE OF MURINE WOUND
Subhadip Ghatak1, Xiaoju Zhou1, Amanda P. Siegel1, Brooke A. Brown2, Amitava Das1, Sashwati Roy1, Yi Xuan1, Mangilal Agarwal1, Robert J. Lee3, David E. Clemmer2, Chandan K. Sen1
1Indiana University, Indianapolis, IN, USA, 2Indiana University, Bloomington, IN, USA, 3Ohio State University, Columbus, OH, USA

H1.03

WOUND HEALING MACROPHAGES: THE INTERVENTION OF ATP TO STIMULATE PRO-WOUND HEALING PHENOTYPES
Harshini Sarojini, Sarah Eichenberger, Sufan Chien
University of Louisville, Louisville, KY, USA
Wound healing macrophages: The intervention of ATP to stimulate pro-wound healing phenotypes

Harshini Sarojini, Sarah Eichenberger, Sufan Chien | University of Louisville, Louisville, KY

Background: Macrophage phenotype changes occur in accordance with intracellular signaling during wound healing. We found that intracellular ATP delivery (ATP-vesicles) enhances wound healing by transitioning macrophages from pro-inflammatory (M1) to anti-inflammatory (M2), resulting in extremely rapid tissue regeneration. Methods: Twenty-seven rabbits were used with four wounds created on each ear. Two were treated with ATP-vesicles (10 mM Mg-ATP) and two with controls (normal saline or Regranex). They were sacrificed at 5h, 12h, and days 1, 2, 3, 4, 6, 9, and 15 post-surgery. Results: ATP-vesicle-treated wounds showed increased platelets, neutrophils, and cytokines as early as 5-12h by immunohistochemical analysis. Massive macrophage accumulation was detected by CD68, CD16, and Anti-Mac by 24h. Activation of BRG1/Brm, subunits of the SWI/SNF ATP-dependent chromatin remodeling complex, may be responsible. PPARalpha and PPARgamma are also involved in macrophage polarization. As early as day two, CD36, arginase, and collagen type 1 immunoreactivity were detected along with early neovascularization. Double immunostaining showed massive early M2 macrophage polarization and double staining of macrophages with pre-collagen markers showed active collagen synthesis as early as day 3. Conclusions: Intracellular ATP delivery causes rapid tissue regeneration by initiating SWI/SNF ATP-dependent chromatin remodeling complex subunits in macrophages, providing a new strategy for wound healing.

H1.04

PRECLINICAL PORCINE MODEL TO STUDY FACIAL SCAR CONTRACTURES AND THEIR MECHANISMS
Mithun Sinha1, Mohamed El-Masry1, Amitava Das1, Nandini Ghosh1, Shomita Steiner1, Kai Leung2, Sashwati Roy1, Chandan K. Sen1
1Indiana University, Indianapolis, IN, USA, 2US Army Institute of Surgical Research, Fort Sam Houston, TX, USA

H1.05

EPIGALLOCATECHIN-3-GALLATE PRIMED HUMAN WHARTON’S JELLY DERIVED MESENCHYMAL STEM CELLS SEEDED HYDROGEL CONSTRUCT IMPROVES HEALING OF THERMAL BURNS
Hira Butt, Azra Mehmood, Ansa Andleeb, Ramla Ashfaq, Hafiz Ghufran, Maryam Azam, Amna Ramzan, Sheikh Riazuddin
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan

H1.06

THE POLARIZATION OF TH2 RESPONSE IMPROVES THE HEALING OF DEEP PARTIAL-THICKNESS BURNS
Edna A. Mandujano, YI1, Francisco González1, Rosa M. Salgado1, Gerardo Arrellín2, Edgar Krötzsch1
1Instituto Nacional de Rehabilitación, Mexico, Mexico, 2Universidad Autónoma de Morelos, Mexico, Mexico
The polarization of Th2 response improves the healing of deep partial-thickness burns

Edna A. Mandujano, Francisco Gonzalez, Rosa M. Salgado, Gerardo Arrellin, Edgar Krotzsch | Instituto Nacional de Rehabilitacion, Mexico; Universidad Autonoma de Morelos, Mexico

Background: An imbalance in the local and systemic inflammatory response guides an impaired healing process. Severe burn patients show an enhanced and chronic type 2 T-helper cell (Th2) response while Th1 response is early suppressed. This study was designed to elucidate the effect of this Th2 inflammatory response during burn healing. Methods: 8-week Balb/c mice were irritated and challenged with toluene diisocyanate (TDI) to polarize the Th2 response. A deep partial-thickness burn was generated on TDI mice. We histologically characterized all stages of the healing process and evaluated Th1/Th2 cytokine levels by qRT-PCR and immunohistochemistry. Results: After irritation and challenge, mice exhibited increased levels of Th2-type cytokines (IL-13, IL-10, IL-4) in both skin tissue and inguinal nodes. Th2 polarized mice exhibited improvement in the reepithelization stage compared to the control group. Treated mice showed increased levels of Th2-type cytokines from 1 day post-burn (dpb) to 28 dpb, while this anti-inflammatory response decreased over time in control mice. IL-13 in the stroma was clearly elevated in TDI mice at early stages of wound healing. Conclusions: An exacerbated anti-inflammatory response, above normal condition, promotes the healing of burns.

H2.01

LOW ABUNDANCE OF MICRORNA-21 IN DIABETIC WOUND MACROPHAGES COMPROMISES RESOLUTION OF INFLAMMATION
Amitava Das, Mithun Sinha, Atul Rawat, Amit Madeshiya, Savita Khanna, Chandan K. Sen, Sashwati Roy
Department of Surgery, IU Health Comprehensive Wound Center, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, USA

H2.02

FAVORABLE MODULATION OF INFLAMMATION AND IMMUNITY IS ACHIEVED WITH BOTH SINGLE AND TRI-LAYER AMNIOTIC MEMBRANE ALLOGRAFTS
Victoria Stefanelli, Paul Bonvallet, Sita Damaraju, Qiaoling Lin, Heli Modi, Sunil Saini, Ankur Gandhi
Integra LifeSciences, Plainsboro, NJ, USA
Favorable modulation of inflammation and immunity is achieved with both single and tri-layer amniotic membrane allografts

Victoria Stefanelli, Paul Bonvallet, Sita Damaraju, Qiaoling Lin, Heli Modi, Sunil Saini, Ankur Gandhi | Integra LifeSciences, Plainsboro, NJ

Background: Amniotic-based products provide benefits including cytokines, growth factors, and ECM components that accelerate healing. The purpose of this study was to elucidate anti-inflammatory and immunomodulatory capabilities of a single-layer dehydrated amniotic membrane allograft (DAMA) and a tri-layer placental allograft membrane (TPAM). Methods: Anti-inflammatory function was evaluated via cytokine analysis following PBMC activation with lipopolysaccharide in the presence of DAMA or TPAM extracts. M1/M2 macrophage polarization was assessed via CCL22, CCL18, PDGF, IL-13, TNF-alpha, IFN-gamma, IL-6, and RANTES. T cell stimulation was also explored. Results: Amniotic extracts possess significant ability to reduce pro-inflammatory molecule secretion including TNF-alpha, IL-1beta, IL-10, MIP-1a, and IL-6 by over 90% in PBMC compared to controls (n=4 per group; p<0.05). Both DAMA and TPAM (n=3 per group) modulated an M2-preferred macrophage response via both soluble factors and physical contact. T cell responses were also favorably altered through exposure to amniotic extracts. Conclusions: The ability of DAMA and TPAM to constructively influence inflammatory and immunomodulatory processes likely constitutes a substantial part of their overall effectiveness in wound remediation.

H2.03

MACROPHAGE FUNCTIONAL PHENOTYPING BY METABOLIC IMMUNOMODULATION
Mary Cloud B. Ammons, PhD, Catherine B. Anders, PhD, Tyler Lawton, Hannah Smith, Jamie Garrett, RN, Sydney Nelson, Joanna Beck
IVREF-Boise VA Medical Center, Boise, ID, USA
Macrophage functional phenotyping by metabolic immunomodulation

Mary Cloud B. Ammons, PhD, Catherine B. Anders, PhD, Tyler Lawton, Hannah Smith, Jamie Garrett, RN, Sydney Nelson, Joanna Beck | IVREF-Boise VA Medical Center, Boise, ID

Background: In non-healing wounds, the healing process stalls at the transition from inflammation to tissue repair. Our central hypothesis is that macrophage plasticity is key to this transition. Recent advances in metabolomics have revealed a pivotal role for metabolites in immunomodulation. Methods: An ex vivo macrophage culture model produces six macrophage phenotypes. CD14+ peripheral blood monocytes are isolated, differentiated into M0 macrophages, and polarized into M1 (IFN-gamma/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) phenotypes. Each phenotype is characterized by ~50 secreted immunomodulating proteins, ~800 myeloid genes, and ~450 metabolites. Results: Protein profiles and pathway enrichment analysis generally groups phenotypes into pro-inflammatory macrophages (M1 and M2b) and tissue repair/regeneration macrophages (M2a, M2c, M2d). M1 macrophages activate processes controlling nitric oxide and ROS for acute inflammatory responses. M2b macrophages regulate chronic inflammation and chemotaxis of lymphocytes, endothelial cells, and epithelial cells. Both M2c and M2d promote angiogenesis, but M2c promotes ECM assembly while M2d activates ECM degradation. Metabolomics further validate that shifts between aerobic glycolysis, pentose phosphate pathway, and oxidative phosphorylation distinguish phenotypes. Conclusions: Metabolomics is fundamentally changing our understanding of immunomodulation. Integrating metabolomics into macrophage characterization provides insight into cellular functions fundamental to macrophage plasticity.

H2.04

THE ROLE OF LNCRNA LETHE IN THE REGULATION OF INFLAMMATION AND MACROPHAGE POLARIZATION
Liping Zhang, Junyi Hu, Kenneth W. Liechty, Junwang Xu
University of Colorado Anschutz Medical Campus, Aurora, CO, USA

H2.05

EPIGENETIC REGULATION OF TLR4 IN DIABETIC MACROPHAGES MODULATES IMMUNOMETABOLISM AND WOUND REPAIR
Frank M. Davis1, Aaron DenDekker1, Amrita Joshi1, Mahmoud El Azzouny2, Sonya J. Wolf1, Johann E. Gudjonsson1, Xianying Xing1, Kanakadurga Singer1, Charles Burant1, Steve Kunkel1, Katherine A. Gallagher1
1University of Michigan, Ann Arbor, MI, USA, 2Agilent Technologies, Santa Clara, CA, USA
Epigenetic regulation of TLR4 in diabetic macrophages modulates immunometabolism and wound repair

Frank M. Davis, Aaron DenDekker, Amrita Joshi, Mahmoud El Azzouny, Sonya J. Wolf, et al. | University of Michigan, Ann Arbor, MI; Agilent Technologies, Santa Clara, CA

Background: Transition of macrophages from an inflammatory to reparative state is critical for wound healing. In type 2 diabetes (T2D), chronic inflammation prevents wound repair. TLR4 is an important driver of myeloid-mediated inflammation in wounds. This study investigated whether epigenetic modifications to the TLR4 pathway lead to inflammatory derangements in T2D macrophages. Methods: Control and diet-induced obese (DIO) mice underwent cutaneous wounding with wound macrophage isolation. Human monocyte-derived macrophages were analyzed from T2D patients and age-matched controls. LC-MS was used to determine metabolic pathway alterations; gene expression and histone methylation were analyzed by qPCR and chromatin immunoprecipitation. Results: T2D patients and DIO mice display decreased wound healing rates with increased Tlr4 expression (p<0.05) and hyperinflammatory macrophage phenotype. LC-MS demonstrated altered metabolic pathways in diabetic macrophages, specifically increased palmitate (p<0.05) which stimulates TLR4. Mechanistically, macrophages from T2D patients and DIO mice had increased expression of MLL1, which upregulated Tlr4 via MLL1-mediated H3K4 trimethylation on the Tlr4 promoter. Myeloid-specific depletion of MLL1 reduced TLR4 expression in diabetic macrophages and improved inflammation. Pharmacological inhibition of TLR4 (TAK-242) and genetic depletion of Tlr4 improved diabetic wound healing. Conclusions: These results define an important role for MLL1-mediated epigenetic regulation of TLR4 in pathologic diabetic wound repair and suggest a target for therapeutic manipulation.

H2.06

THE INTERACTION OF MICROBIAL VIRULENCE WITH AGE ALTERS THE KERATINOCYTE WOUND HEALING AND INFLAMMATORY RESPONSE
Laurice J. Flowers, Aayushi Uberoi, Elizabeth A. Grice
University of Pennslyvania, Philadelphia, PA, USA
The interaction of microbial virulence with age alters the keratinocyte wound healing and inflammatory response

Laurice J. Flowers, Aayushi Uberoi, Elizabeth A. Grice | University of Pennsylvania, Philadelphia, PA

Background: Wound repair markedly declines with age, but how the microbiota interacts with age to promote or delay healing is unknown. We hypothesized that keratinocytes from aged donors co-cultured with commensal or pathogenic microbes would exhibit impaired innate immune and wound healing responses. Methods: In vitro migration assays were performed on primary keratinocytes isolated from aged and young human skin. Keratinocytes were co-cultured with commensal microbes from healthy human skin (Staphylococcus epidermidis, Staphylococcus warneri, Corynebacterium aurimucosum) or S. aureus isolated from skin infections and non-healing diabetic foot ulcers (DFU). Results: Aged and young keratinocytes showed differential wound healing responses. Young donors had increased IL-6 and CSF2 expression compared to aged donors. With commensal microbes, strain- and age-specific changes in innate immune and wound healing cytokines were observed. Young keratinocytes colonized with pathogenic S. aureus strains exhibited increased IL-10, CSF2, and TNF expression compared to aged keratinocytes. DFU S. aureus dramatically increased IL-10 (fold change = 48) in young keratinocytes relative to aged. Conclusions: Aged keratinocytes mount an innate inflammatory response when wounded in the presence of healthy skin microbiota but fail to produce a robust response when colonized by pathogens, suggesting that age and microbial virulence interact to modulate wound repair.

H3.01

A NON-CONTACT DEVICE FOR QUICK SCREENING OF WOUND INFECTIONS IN CHRONIC WOUND PATIENTS
Jon Senkowsky1, Hong Vu2, Ashwin Nair2, Lan Tran2, Suvra Pal3, Liping Tang2, Wenjing Hu2
1Texas Health Physician’s group, Arlington, TX, USA, 2Progenitec Inc., Arlington, TX, USA, 3University of Texas at Arlington, Arlington, TX, USA
A non-contact device for quick screening of wound infections in chronic wound patients

Jon Senkowsky, Hong Vu, Ashwin Nair, Lan Tran, Suvra Pal, Liping Tang, Wenjing Hu | Texas Health Physician’s group, Arlington, TX; Progenitec Inc., Arlington, TX; University of Texas at Arlington

Background: Wound infections are detected based on clinician visual assessment. Wound culture results take 2-3 days, by which time infection may worsen. No test strip devices exist for detecting neutrophils (a hallmark of infection) based on esterase levels in wounds. This study developed and investigated such a screening tool. Methods: A disposable device, DETEC esterase, was developed to detect wound infection by pressing a freshly recovered wound dressing against its test surface. Its ability to detect esterase was assessed in simulated wound fluids in vitro. A clinical study was conducted to correlate device results with microbiological culture results. Results: The device had high esterase detection sensitivity (>90%) using simulated wound fluids soaked onto wound dressings and upon testing discarded wound dressings from human patients. Results were not affected by wound dressing type. Statistical analyses confirmed a strong association between device output and microbiological culture results for neutrophil counts and infection. Conclusions: Without directly contacting the patient, this device quickly assesses (within 3 minutes) esterase activity in chronic wounds to detect neutrophil presence and confirm wound infection. DETEC esterase has been developed to screen for infections during routine wound assessment and inform the decision-making process at the point-of-care.

H3.02

COMPOSITE SCAFFOLD WITH INHERENT ANTIMICROBIAL ACTIVITY FOR TREATMENT OF COMPLEX WOUNDS
Navid Karimi, Reza Jalili, Demian Felix, Ruhangiz Kilani, Dirk Lange, Aziz Ghahary
University of British Columbia, Vancouver, BC, Canada
Composite scaffold with inherent antimicrobial activity for treatment of complex wounds

Navid Karimi, Reza Jalili, Demian Felix, Ruhangiz Kilani, Dirk Lange, Aziz Ghahary | University of British Columbia, Vancouver, BC

Background: Complex wounds including pressure ulcers are commonly affected by biofilm and infection. We developed and tested a flowable scaffold that not only enhances wound healing but also resists microbial infection via silver nanoparticle (AgNP) technology. Methods: Monodisperse spherical silver nanoparticles were synthesized via reduction of silver nitrate. A composite scaffold was prepared by loading AgNPs into a liquid bioengineered collagen-based scaffold. An infected wound model was generated by inserting a titanium implant into a subcutaneous pocket in rat dorsal skin with bacteria (Pseudomonas aeruginosa or Staphylococcus aureus). The pockets were filled with scaffold alone or scaffold+AgNP before closure. Results: Scaffold with 600 PPM AgNP was effective to clear wound infections induced by 10^6 P. aeruginosa. AgNPs were also effective on S. aureus. Mouse open wound healing experiments showed that scaffold+AgNP is safe to use and AgNPs would not interfere with the healing process. Conclusions: These findings provide support for the application of an AgNP-loaded flowable scaffold as a promising method to promote wound healing as well as preventing/treating complex wound infections.

H3.03

THE USE OF INDOCYANINE GREEN FLUORESCENCE IN A PRECLINICAL MODEL OF HUMAN WOUND HEALING
Aos Karim1, Aiping Liu1, Christie Lin2, Adam Uselmann2, Kevin Eliceiri1, Matthew Brown1, Angela Gibson1
1University of Wisconsin, Madison, WI, USA, 2OnLume, Madison, WI, USA
The use of indocyanine green fluorescence in a preclinical model of human wound healing

Aos Karim, Aiping Liu, Christie Lin, Adam Uselmann, Kevin Eliceiri, Matthew Brown, Angela Gibson | University of Wisconsin, Madison, WI; OnLume, Madison, WI

Background: Indocyanine Green (ICG) fluorescence evaluates microperfusion in murine models and humans. We characterized graft take in a mouse model of human skin xenograft utilizing ICG and immunohistochemistry to evaluate the utility of a murine-human xenograft as a model for human skin wound healing. Methods: Normal human skin from reconstructive operations was grafted onto immunocompromised mice as full thickness (FT) or partial thickness (PT) pieces. ICG (1 mg/kg) was injected retro-orbitally. ICG fluorescence and white light images were captured using a novel ambient-light compatible fluorescence imaging system at 8 and 12 weeks post-grafting. Results: PT grafts appeared well-engrafted at 8 weeks while FT grafts exhibited a central necrotic region with well-engrafted surrounding skin. The extent of ICG fluorescence indicated perfusion was greater in PT compared to FT (95% vs 10% of graft area) at 8 weeks. By 12 weeks, FT grafts showed complete perfusion. Cell viability and revascularization (evaluated histologically with LDH and CD31) were greater in PT at 8 weeks; by 12 weeks, both PT and FT grafts showed complete viability and revascularization. Conclusions: Delayed revascularization during engraftment leads to ischemia-induced cellular death. We demonstrated the feasibility of ICG fluorescence as a marker for monitoring human skin xenograft engraftment for future wound healing mechanistic studies.

H3.04

GLUTATHIONE CONJUGATED HYDROGELS: SIMPLE, FLEXIBLE VEHICLES FOR LOCAL THERAPY
Karol Sokolowski, Hai Pham, Eric R. Wenzler, Richard A. Gemeinhart
University of Illinois at Chicago, Chicago, IL, USA
Glutathione conjugated hydrogels: Simple, flexible vehicles for local therapy

Karol Sokolowski, Hai Pham, Eric R. Wenzler, Richard A. Gemeinhart | University of Illinois at Chicago, Chicago, IL

Background: Chronic wounds present a complex disease environment for which optimal treatment remains elusive. Development of a versatile delivery vehicle for small molecule and protein therapeutics could provide a personalized option over conventional systemic therapies and rigid topical dressings. Methods: Polyethylene glycol (PEG) hydrogels were prepared through UV-polymerization with PEGDA, 0.05% Irgacure 2959, and in the absence or presence of 60 mM glutathione (GSH-PEG). Loading of vancomycin and meropenem occurred through incubation post-polymerization. Antimicrobial activity was tested against Pseudomonas aeruginosa. Toxicity was examined in vitro using primary human dermal fibroblasts. Results: Incorporation of GSH improved deliverable drug quantity compared to PEG-only hydrogels while permitting simultaneous delivery of GST-fusion proteins. Vancomycin and meropenem loading were time- and concentration-dependent. After one-hour incubation in meropenem (0.16 mg/mL), significant differentiation between GSH-PEG and PEG loading was observed (51.9+/-8.6 vs. 4.35+/-11.6 mcg; p=0.037). GSH-PEG meropenem achieved bactericidal activity against meropenem-resistant carbapenemase-producing P. aeruginosa (MIC 128 mcg/mL). Deliverable concentrations of meropenem and vancomycin did not negatively influence fibroblast proliferation over 24 hours (p>0.05 all). Conclusions: GSH-PEG hydrogels demonstrate capacity to passively load and deliver small molecule agents alone and in combination with GST-fusion proteins, with favorable drug delivery properties to address the variable needs of chronic wound environments.

H3.05

DETECTION OF NECROSIS AVIDITY USING INDOCYANINE GREEN FLUORESCENCE IN HUMAN WOUND HEALING
Aos Karim1, Kishan Thadikonda1, Christie Lin2, Adam Uselmann2, Kevin Elicieiri1, Samuel Poore1, Angela Gibson1
1University of Wisconsin, Madison, WI, USA, 2OnLume, Madison, WI, USA
Detection of necrosis avidity using indocyanine green fluorescence in human wound healing

Aos Karim, Kishan Thadikonda, Christie Lin, Adam Uselmann, Kevin Elicieiri, Samuel Poore, Angela Gibson | University of Wisconsin, Madison, WI; OnLume, Madison, WI

Background: ICG fluorescence was recently shown to be an imaging biomarker for necrosis in a murine model of burn injury. No studies have shown ICG necrosis avidity in human wound healing. We hypothesized that necrosis avidity of ICG can be used to evaluate burn-induced necrosis in a human skin xenograft and flap necrosis in DIEP breast reconstruction patients. Methods: Human skin was grafted onto immunocompromised mice. Twelve weeks after grafting, the graft was burned at 150 degrees C for 5 seconds. ICG was injected at 1 mg/kg dose. ICG fluorescence images were captured immediately and 24 hours later. In a clinical study, breast reconstruction patients (n=4) were imaged intraoperatively after vascular anastomosis as well as 2, 24, 48, and 72 hours later using the SPY imaging system. Results: ICG fluorescence decreased immediately after burning. Twenty-four hours after injury, ICG signal was detected in the burn area (~5-6 mm diameter); a smaller area than perfusion analysis immediately post-burn suggested. In all breast reconstruction patients, ICG signal intensity was highest 2-24 hours after surgery, centered on areas of bruising at the edge of the flap. Follow-up revealed well-healed flaps with minimal epidermolysis around edges, consistent with ICG findings. Conclusions: Delayed ICG imaging validated its use as a marker for necrotic tissue in murine human xenograft burn wounds and in a human post-surgical model, highlighting potential for monitoring post-mastectomy patients.

H3.06

NRF2 ACTIVATING TOPICAL THERAPY DELIVERED BY A NOVEL BIOINSPIRED SYNTHETIC PROTEIN HYDROGEL PROMOTES CUTANEOUS HEALING OF DIABETIC WOUNDS
Joseph Kuhn1, Priya Katyal2, Michael Meleties2, Absara Hassan1, Jennifer Kwong1, Bonnie Lin2, Alvaro Villarreal-Ponce1, Jasmine Lee1, Jin Montclare1, Piul S Rabbani1
1NYU Langone Health, New York, NY, USA, 2NYU Tandon School of Engineering, New York, NY, USA

H4.01

DISRUPTION OF FOCAL ADHESION KINASE MECHANOTRANSDUCTION SIGNALING REGENERATES INTRADERMAL ADIPOSE TISSUE
Sun Hyung Kwon, Britta A. Kuehlmann, Chikage Noishiki, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA

H4.02

INHIBITING MECHANOTRANSDUCTION SIGNALING CHANGES FIBROBLAST HETEROGENEITY AND PROMOTES TISSUE REGENERATION IN HEALING WOUNDS
Kellen Chen, Sun Hyung Kwon, Dominic Henn, Britta A. Kuehlmann, Clark A. Bonham, Jagannath Padmanabhan, Chikage Noishiki, Janos Barrera, Michael T. Longaker, Michael Januszyk, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA
Inhibiting mechanotransduction signaling changes fibroblast heterogeneity and promotes tissue regeneration in healing wounds

Kellen Chen, Sun Hyung Kwon, Dominic Henn, Britta A. Kuehlmann, Clark A. Bonham, Jagannath Padmanabhan, Chikage Noishiki, Janos Barrera, Michael T. Longaker, Michael Januszyk, Geoffrey C. Gurtner | Stanford University

Background: Mechanical stress is a critical component of hypertrophic scar formation, acting via mechanisms that promote fibrotic cellular activities. Small molecule disruption of focal adhesion kinase (FAK)-mediated mechanotransduction promotes wound healing and improves scar quality. Methods: Large deep partial-thickness wounds on red duroc pigs were treated with FAK inhibitor (FAKI) or placebo-releasing hydrogels or standard bandages. Wound healing and scar quality were assessed by serial quantification of scar area and cutometer measurements. Dermal fibroblasts from porcine and human skin were incorporated into 3D collagen lattices and stretched with or without FAKI. Droplet-based microfluidic single cell RNA sequencing (scRNAseq) using 10X Genomics was performed. Results: FAKI resulted in significantly accelerated wound closure, lower Visual Analog Scale scar score, decreased firmness, increased elasticity, and regrowth of hair follicles and dermal appendages. FAKI-treated wounds showed significantly lower alpha-SMA expression. ScRNAseq indicated that mechanical stretch in 3D collagen shifted fibroblast gene expression to distinctly different subpopulations with higher ACTA2, PDGFR, and COL1A1 expression. FAKI shifted fibroblast heterogeneity into a subpopulation with decreased pro-fibrotic markers. These findings were consistent across porcine and human fibroblasts. Conclusions: FAKI-mediated disruption of mechanotransduction promotes wound healing and improves scar quality, likely through enhanced regeneration of dermal appendages and shifting fibroblast heterogeneity from pro-fibrotic to a more regenerative subpopulation.

H4.04

EMERGING BIOMARKERS IN IMPLANT-INDUCED FIBROSIS
Britta Kuehlmann1, Clark A. Bonham1, Lukas Prantl2, Geoffrey C. Gurtner1
1Stanford University, Stanford, CA, USA, 2University Hospital of Regensburg, Regensburg, Germany
Emerging biomarkers in implant-induced fibrosis

Britta Kuehlmann, Clark A. Bonham, Lukas Prantl, Geoffrey C. Gurtner | Stanford University; University Hospital of Regensburg, Germany

Background: Revealing the relationship between genes in fibrotic tissues from patients with capsular fibrosis around implants will help garner a better understanding of the pathophysiology of this disease. Methods: Using the largest breast capsule tissue bank in the world, 20 human samples of physical capsule (Baker I) and 20 samples of pathological fibrotic capsule (Baker IV) were compiled. A large systematic analysis was conducted to identify differentially expressed genes (DEGs) in fibrotic human tissue samples. Results: 2,559 DEGs were identified, followed by knowledge-based network generation and pathway association study to identify novel biomarkers for capsular fibrosis. 455 genes were distinguished as potential clinical biomarkers and 111 as drug targets. MMP9 was among the highest upregulated biomarkers (p-value: 0.00625), making it a useful potential biomarker as it can easily be detected in blood, sputum, and urine. Conclusions: Discovering biomarkers for the diagnosis of fibrosis at the earliest stages is clinically required. These results bring new hope for biomarker-based diagnosis for capsular fibrosis.

H4.05

ADIPOCYTE STEM CELLS AMELIORATE RADIATION INDUCED FIBROSIS VIA HEPATOCYTE GROWTH FACTOR SECRETION
Asim Ejaz, Michael W. Epperly, Joel S. Greenberger, Peter J. Rubin
University of Pittsburgh, Pittsburgh, PA, USA
Adipocyte stem cells ameliorate radiation induced fibrosis via hepatocyte growth factor secretion

Asim Ejaz, Michael W. Epperly, Joel S. Greenberger, Peter J. Rubin | University of Pittsburgh, Pittsburgh, PA

Background: Radiation therapy to the head and neck, chest wall, or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. Methods: Female C57BL/6J mice were irradiated to the right flank at 35Gy in a single fraction. Subgroups had irradiated sites injected with ASCs from luciferase+ GFP+ mice. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. In vitro Transwell co-cultures contained irradiated human foreskin fibroblasts (HFFs) in the bottom layer and human ASCs in the upper layer. Results: RF was detected in vivo at day 14 and increased by day 28. At day 14, upregulation of TGF-beta, CTGF, Collagen 1, 3, and 4 was confirmed in irradiated tissue. A single 1×10^6 ASC injection at day 28 significantly restored limb excursion and promoted migration of regenerative bone marrow cells to site of injury. Transwell co-culture revealed significant downregulation of profibrotic genes (TGF-beta, Col 1-4) in irradiated cells. Among genes expressed by ASCs, hepatocyte growth factor (HGF) was prominent. Addition of recombinant HGF to irradiated HFFs significantly downregulated pro-fibrotic gene transcripts. Conclusions: HGF secreted by ASCs reduces RF in vitro and in vivo.

H4.06

REGULATION OF PULMONARY FIBROSIS BY CD109 IN A MURINE MODEL
Maha Alsharqi, Liqin Xu, Meryem Blati, Anie Philip Mcgill
University, Montreal, QC, Canada
Regulation of pulmonary fibrosis by CD109 in a murine model

Maha Alsharqi, Liqin Xu, Meryem Blati, Anie Philip | McGill University, Montreal, QC

Background: TGF-beta signaling is transduced by the type I and type II TGF-beta receptors. TGF-beta co-receptors such as CD109 are known to strongly regulate TGF-beta signaling in a variety of cell types. The significance of CD109 function in pulmonary fibrosis in scleroderma (SSc) is unknown. Methods: We examined the effect of CD109 deficiency on fibrotic responses in the lung and skin using CD109 knockout (KO) versus wild-type (WT) littermate mice. Collagen deposition and tissue architecture were examined histologically. ECM protein expression was analyzed by immunohistochemistry and Western blot. Fibroblasts were isolated and analyzed for TGF-beta signaling via phospho-Smad2/3 levels. Results: Both lung and skin tissue from KO mice show markedly increased cellularity, collagen disorganization, and expression of collagen type I, fibronectin, CTGF, and alpha-SMA compared to WT littermates (p<0.05 in all cases), with lung exhibiting more dramatic increases than skin. KO lung and skin fibroblasts display significantly elevated phospho-Smad2/3 signaling and enhanced migration in vitro. Conclusions: CD109 deficiency results in markedly enhanced TGF-beta signaling and fibrotic responses in the lung. CD109 may represent a molecular target for therapeutic intervention in pulmonary fibrosis in SSc.

J1.01

DEEP LEARNING FOR AUTOMATED SEGMENTATION OF SKIN WOUNDS
Jake Jones, Kyle Quinn
University of Arkansas, Fayetteville, AR, USA
Deep learning for automated segmentation of skin wounds

Jake Jones, Kyle Quinn | University of Arkansas, Fayetteville, AR

Background: Histopathological staining and imaging using H&E has served as a gold-standard technique to evaluate wound healing. Neural networks have emerged as powerful image classification tools sensitive to subtle changes in tissue features. The goal of this study was to create a deep learning convolutional network capable of classifying and segmenting wound features from wound histology. Methods: C57BL/6J mice (n=24) received 6mm diameter full-thickness excisional wounds excised at days 3, 5, or 10 and sliced into 30 mcm cross-sections. Wound sections were stained with H&E and imaged using bright-field microscopy. A custom neural network (UNet architecture) was trained using 150 512×512 pixel H&E images over 25 epochs, designed to identify and segment regions including: epidermis, dermis, granulation, stratum corneum, hair follicles, and connective tissue. Results: Six entire wound tissue sections from days 3, 5, and 10 were segmented by the trained network and compared to manual segmentation. Each wound tissue region showed high true positive rates above 86%. Automated measurements of dermal thickness, wound size, and percent re-epithelization were within 5% error. The final network had an overall accuracy of 88.4% and was able to segment an entire section in ~10 seconds. Conclusions: This deep learning neural network can segment and classify wound features automatically without user input, creating new opportunities for rapid, quantitative analysis to assist in wound care.

J1.02

MECHANICAL SIGNALING CRITICALLY DRIVES HUMAN FOREIGN BODY RESPONSE TO IMPLANTABLE BIOMATERIALS
Jagannath Padmanabhan, Teruyuki Dohi, Kellen Chen, Zachary A. Stern-Buchbinder, Clark A. Bonham, Peter A. Than, Dominic Henn, Hadi S. Hosseini, Artem A. Trotsyuk, Sun Hyung Kwon, Babak Hajhosseini, Michael Januszyk, Zeshaan N. Maan, Geoffrey C. Gurtner
Stanford University, Stanford, CA, USA

J1.03

TEMPORAL DELIVERY OF POLYPEPTIDE BIOMATERIALS FOR ACCELERATED WOUND HEALING AND TISSUE REPAIR
Deepanjan Ghosh1, Suneel Kumar2, Francois Berthiaume2, David J. DiCaudo3, Jacquelyn Kilbourne1, Kaushal Rege1
1Arizona State University, Tempe, AZ, USA, 2Rutgers University, Piscataway, NJ, USA, 3Mayo Clinic, Scottsdale, AZ, USA
Temporal delivery of polypeptide biomaterials for accelerated wound healing and tissue repair

Deepanjan Ghosh, Suneel Kumar, Francois Berthiaume, David J. DiCaudo, Jacquelyn Kilbourne, Kaushal Rege | Arizona State University; Rutgers University; Mayo Clinic

Background: Complexity of the repair process, monotherapies, and delivery challenges limit the effectiveness of current treatment strategies for diabetic wounds. We demonstrate that temporal delivery of polypeptide biomaterials augments individual stages of wound repair and accelerates closure of acute and slow-healing diabetic wounds. Methods: Single full-thickness 5 mm excisional splinted wounds were made on dorsal skin of Balb/c and diabetic db/db mice. Results: A combination of silk fibroin protein and soluble exogenous histamine was more effective at wound closure compared to individual treatments and Tegaderm dressing. Histological and immunohistochemistry analyses showed the treatment reduced dermal gap, promoted angiogenesis (CD31+), myofibroblast-mediated wound contraction (aSMA+), and higher TGF-beta1 expression. Sequential delivery of growth factor nanoparticles (GFNPs) one day before the transition from inflammation to proliferative phases further accelerated wound closure. Sequential delivery of SDF1 or bFGF nanoparticles after silk+histamine treatment enhanced wound closure compared to simultaneous treatment. In diabetic wounds, GFNPs delivery in a sequential manner resulted in reepithelialization 2 days prior to control groups and ~20% improvement in healed skin strength. Conclusions: Multifactor therapy (silk fibroin dressing, exogenous histamine, and GFNPs) is a promising treatment option that outperforms clinically approved polyurethane wound dressings.

J1.04

NANOENGINEERED DNA HYDROGELS: A VERSATILE PLATFORM FOR DRUG DELIVERY AND TISSUE REPAIR
Arghya Paul1, Sayantani Basu2, Settimio Pacelli2
1The University of Western Ontario, London, ON, Canada, 2The University of Kansas, Lawrence, KS, USA
Nanoengineered DNA hydrogels: A versatile platform for drug delivery and tissue repair

Arghya Paul, Sayantani Basu, Settimio Pacelli | University of Western Ontario; University of Kansas

Background: The objective was to deliver a unique malleable DNA-based hydrogel scaffold that can be injected, will set and integrate to the injured site after placement, and will be resorbed over time as it induces rapid in situ tissue repair via sustained drug delivery. Methods: A DNA hydrogel was formulated by combining a network of interconnections between neighboring DNA strands with a second network of electrostatic interactions between charged silicate nanodisks and the DNA backbone. Rheological studies were conducted to investigate shear-thinning properties. Hydrogels were loaded with dexamethasone, a corticosteroid with osteoinductive properties. In vivo bone repair capacity was assessed using cranial bone defects in a rat model. Results: Inclusion of silicate nanodisks increased viscosity and shear-thinning properties of the hydrogels. Hydrogels maintained bioactivities of encapsulated drugs and demonstrated sustained release for more than a week. The hydrogels can be 3D bioprinted in layers and surface coated on allografts for drug delivery. Drug-carrying gels promoted bone formation at the periphery of the defect after one month post-treatment. Conclusions: A novel shear-thinning DNA-based hydrogel was developed and tested as a potential drug delivery vehicle for accelerated wound healing. Future preparation from patient’s own tissues may help fast-track this platform to the clinic.

J1.05

FIBROBLASTS OF DISTINCT SCARRING PHENOTYPES DISPLAY CHARACTERISTIC BIOENERGETIC METABOLISM PROFILES WHICH CAN BE REGULATED USING ENGINEERED BIOMATERIALS
Hima V. Vangapandu1, Harrison Strang1, Aditya Kaul1, Hui Li1, Sundeep Keswani1, Philip Jung2, Swathi Balaji3
1Baylor College of Medicine, Houston, TX, USA, 2Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, LA, USA, 3Surgery, Baylor College of Medicine, Houston, TX, USA
Fibroblasts of distinct scarring phenotypes display characteristic bioenergetic metabolism profiles which can be regulated using engineered biomaterials

Hima V. Vangapandu, Harrison Strang, Aditya Kaul, Hui Li, Sundeep Keswani, Philip Jung, Swathi Balaji | Baylor College of Medicine; Louisiana State University

Background: It is unknown why different people scar differently from identical injuries. Since energy metabolism governs wound healing and fibrosis, and fibroblasts are the main arbiters of fibrosis, we hypothesize differences in intrinsic fibroblast bioenergetic metabolism underlie differential scarring. Methods: Fibroblasts were isolated from uninjured normal-skin and c-section scars from abdominoplasty samples of “low-scarrers” (VSS <3) or "high-scarrers" (VSS >6). Oxidative phosphorylation (OCR), glycolysis (ECAR), ATP-production, mitochondrial membrane potential, and ROS were measured. Fibroblasts were cultured on gelatin methacrylate crosslinked with thiolated lignosulfonate (GelMA-TLS). Results: Both normal-skin and scar fibroblasts from high-scarrers had higher basal OCR and ECAR than low-scarrers (p<0.01). High-scarrer fibroblasts responded to stress with significant increase in spare respiratory and glycolytic reserve capacity than low-scarrers (p<0.01). Under hypoxia, high-scarrers showed significant increase in glycolysis. High-scarrer fibroblasts had more depolarized mitochondria and mitochondrial ROS, with significantly lower p-HSP27Ser82 (p<0.001). GelMA-TLS composites scavenged ROS and attenuated upregulated pHSP27 and fibrotic markers alpha-SMA and Col1-a1 in high-scarrer fibroblasts (p<0.05). Conclusions: Fibroblasts of distinct scarring phenotypes display characteristic bioenergetic metabolism profiles that may govern fibrotic differences, which can be regulated by engineered biomaterials.

J1.06

A NOVEL MURINE DISTRACTION DEVICE INVESTIGATING LONG BONE REGENERATION
Harsh N. Shah, Ankit Salhotra, Michael T. Lopez, Derrick C. Wan, Michael T. Longaker
Stanford University, Stanford, CA, USA
A novel murine distraction device investigating long bone regeneration

Harsh N. Shah, Ankit Salhotra, Michael T. Lopez, Derrick C. Wan, Michael T. Longaker | Stanford University

Background: The application of distraction osteogenesis (DO) has revolutionized treatment of many congenital and acquired defects. Here we describe the development of a novel mouse distraction model for the tibia. Methods: Tibial distraction devices were designed using computer-aided design software and 3D-printing. A 0.6mm hole was drilled on either side of the tibial crest, followed by osteotomy at the tibial crest using a diamond disc saw. Animals were divided into four groups: sham, fracture (osteotomy without distraction), acutely lengthened, and gradually distracted. The gradual distraction protocol consisted of a five-day latency period, 10 days of distraction at 0.15mm every 12 hours, and 28 days of consolidation. Results: Micro-CT images of the sham group presented native, unperturbed bone, while the acute lengthening group showed absence of bone regeneration. Bone regeneration occurred in the fracture and gradual distraction groups. Quantitative analysis showed bone volume per tissue volume (BV/TV) and callus volume were significantly higher in the distraction group compared to the sham group (***P<0.001 and ****P<0.0001 respectively). Conclusions: A new model for long bone distraction osteogenesis in the mouse has been developed to determine the underlying bone regeneration mechanisms.

J2.01

HOST SKIN LIPIDS ENABLE BIOFILM PATHOGENICITY COMPROMISING FUNCTIONAL CUTANEOUS WOUND CLOSURE
Nandini Ghosh1, Mithun Sinha1, Dayanjan S. Wijesinghe2, Kanhaiya Singh1, Amitava Das1, Shomita Steiner1, Savita Khanna1, Manabu Kawada3, Gayle M. Gordillo1, Sashwati Roy1, Chandan K. Sen1
1Indiana University, Indianapolis, IN, USA, 2Virginia Commonwealth University, Richmond, VA, USA, 3Institute of Microbial Chemistry Research Foundation, Tokyo, Japan

J2.02

HOST BIOFILM INTERACTION IN BREAST IMPLANT ASSOCIATED ANAPLASTIC LARGE CELL LYMPHOMA
Imran Khan1, Colby Neumann1, Mary Lester1, Bruce Van Natta2, Christine Kelley-Patteson2, Gayle M. Gordillo1, Chandan K. Sen1, Aladdin Hassanein1, Mithun Sinha1
1Indiana University, Indianapolis, IN, USA, 2Meridian Plastic Surgeons, Indianapolis, IN, USA

J2.03

INVESTIGATING BACTERIAL-FUNGAL INTERACTIONS WITHIN CHRONIC WOUND MICROBIOMES
Alex Cheong, Chad Johnson, PhD, Hanxiao Wan, Jeniel Nett, MD, PhD, Lindsay Kalan, PhD
University of Wisconsin-Madison, Madison, WI, USA
Investigating bacterial-fungal interactions within chronic wound microbiomes

Alex Cheong, Chad Johnson, PhD, Hanxiao Wan, Jeniel Nett, MD, PhD, Lindsay Kalan, PhD | University of Wisconsin-Madison

Background: Chronic wounds are host to a diverse microbiome. It is unclear how microbial communities, consisting of both bacteria and fungi, interact within the wound environment. Methods: An in vitro mixed-species biofilm model was used to investigate species-interactions of wound isolates during biofilm formation. High-resolution microscopy was applied to evaluate physical interactions in three-dimensional space. Results: Interaction between Candida albicans (CA) and Citrobacter freundii (CF) isolated from a diabetic foot ulcer was found to cooperatively form biofilms in vitro. CF competes with CA for substrate binding, resulting in significant reduction in CA colony forming units (2.8 log CFU, 95% CI [2.6, 3.0]) when seeded onto established CF biofilms. CF also competes with Staphylococcus aureus (SA) for binding to CA, reducing SA growth (1.3 log CFU, 95% CI [0.6, 2.0]; p<0.01) without affecting CF or CA proliferation. CF adhesion to CA was found to be mannose-sensitive, suggesting binding may be mediated by type 1 fimbriae. CF increases CA hyphae formation, and neutrophil extracellular trap release was observed in response to the co-culture, suggesting fungal-bacterial interactions may result in pro-inflammatory phenotypes. Conclusions: Fungal-bacterial interactions in chronic wounds appear crucial to understanding pathogenesis and have not been widely studied.

J2.04

HIGH LEVELS OF OS ARE CRITICAL FOR BACTERIAL BIOFILM-DEVELOPMENT IN DIABETIC CHRONIC WOUNDS
Jane H. Kim, Paul R. Ruegger, Elyson G. Lebig, Ansel Hsiao, James Borneman, Manuela Martins-Green
University of California, Riverside, Riverside, CA, USA
High levels of OS are critical for bacterial biofilm-development in diabetic chronic wounds

Jane H. Kim, Paul R. Ruegger, Elyson G. Lebig, Ansel Hsiao, James Borneman, Manuela Martins-Green | University of California, Riverside

Background: Diabetic foot ulcers (DFU) have global prevalence of 6.3%. Understanding the factors that contribute to healing and complication is essential for developing novel therapeutics and diagnostic tools. We hypothesized that high oxidative stress (OS) leads to wound chronicity by promoting colonization by biofilm-forming over commensal/beneficial bacteria. Methods: A db/db-/- mouse model for chronic wounds that develops pathogenic biofilms naturally after we induce high OS immediately after wounding was used. We sequenced the bacterial rRNA internal transcribed spacer (ITS) of the wound microbiome from chronic wound initiation to fully developed chronic wounds. Indicator species analysis was used to determine which bacterial species are strongly associated with healing or chronic wounds. Results: Healing wounds are colonized by a diverse and dynamic bacterial microbiome that never develops biofilms even though strong biofilm-forming bacteria are present. Species such as Cutibacterium acnes, Achromobacter sp., Delftia sp., and Escherichia coli were highly associated with healing wounds. In contrast, chronic wounds with high OS have low bacterial diversity and are colonized by clinically-relevant biofilm-forming bacteria such as Pseudomonas aeruginosa, Enterobacter cloacae, Corynebacterium frankenforstense, and Acinetobacter sp. Conclusions: Bacteria associated with non-chronic or chronic wounds can function as bioindicators of healing or non-healing respectively. Understanding bacterial interactions between pathogenic and beneficial bacteria will help find better solutions for biofilm management.

J2.05

BACTERIAL BIOFILM IMPACT ON MACROPHAGE PHENOTYPE IN DIABETICS
Sydney Nelson, Jamie Garrett, Hannah Smith, Tyler Lawton, Catherine Anders, PhD, Mary Cloud Ammons, PhD
IVREF-Boise VAMC, Boise, ID, USA
Bacterial biofilm impact on macrophage phenotype in diabetics

Sydney Nelson, Jamie Garrett, Hannah Smith, Tyler Lawton, Catherine Anders, PhD, Mary Cloud Ammons, PhD | IVREF-Boise VAMC, Boise, ID

Background: Diabetic ulcers cost the US economy an estimated $58 billion annually. Our central hypothesis is that macrophage phenotypic plasticity is essential to the transition from inflammation to tissue repair and that a disrupted metabolic environment combined with colonizing bacterial biofilms causes stalled healing in diabetic wounds. Methods: Monocytes were isolated from healthy/pre-diabetic/diabetic donor blood, differentiated into resting macrophages, and polarized into five functionally-defined phenotypes. During polarization, macrophages were co-cultured with biofilms composed of Pseudomonas aeruginosa, Staphylococcus aureus, or Clostridium perfunges established on 0.2 mcm pore tissue culture inserts. Macrophages were characterized by cell surface markers, immunomodulating proteins, expressed myeloid genes, and metabolites. Results: Similar patterns of early inflammatory markers were observed in healthy and diabetic macrophages; however, late inflammatory markers were prolonged and exaggerated in diabetic macrophages. Bacterial biofilm co-culture significantly inhibited macrophage polarization, both pro-inflammatory and anti-inflammatory, in healthy and diabetic macrophages. Conclusions: Prolonged inflammation in non-healing wounds may result from inherent macrophage dysfunction in diabetics, and the burden of colonizing bacterial biofilms may dramatically inhibit macrophage functional shift to tissue repair.

J2.06

HIGH-THROUGH PHENOTYPING OF STAPHYLOCOCCUS AUREUS ISOLATES FROM DIABETIC FOOT ULCERS REVEALS VIRULENCE BEHAVIORS ASSOCIATED WITH CLINICAL OUTCOMES
Amelia R. McCready-Vangi1, Amy E. Campbell1, Sue E. Gardner2, Elizabeth A. Grice1
1University of Pennsylvania, Philadelphia, PA, USA, 2University of Iowa, Iowa City, IA, USA
High-throughput phenotyping of Staphylococcus aureus isolates from diabetic foot ulcers reveals virulence behaviors associated with clinical outcomes

Amelia R. McCready-Vangi, Amy E. Campbell, Sue E. Gardner, Elizabeth A. Grice | University of Pennsylvania; University of Iowa

Background: Previous metagenomic analysis of a prospective cohort of neuropathic, uninfected DFU (n=46) demonstrated that strain-level variation of S. aureus was associated with clinical outcome. We hypothesized S. aureus strains isolated from DFUs would display differential virulence behaviors that correlate with clinical outcomes. Methods: A library of 224 S. aureus isolates cultured from a longitudinal, prospective cohort of n=100 DFU patients was screened. High-throughput, quantitative phenotyping assays examined: 1) biofilm formation; 2) production of the pigmented virulence factor staphyloxanthin; 3) siderophore production; and 4) phenol-soluble modulin-mediated colony spreading. Results: S. aureus strains producing high staphyloxanthin are significantly associated with a non-healing wound phenotype (T-test p-value=0.032). In vitro biofilm production was not correlated with healing phenotype (T-test p-value=0.544). Conclusions: Whole genomic sequencing of these isolates will enable genetic association analysis to identify genetic loci associated with virulence phenotypes and clinical outcomes. This work has the potential to inform DFU patient care by providing insight into what constitutes problematic S. aureus colonization, providing novel diagnostic and therapeutic targets.

J3.01

SINGLE-CELL RNA-SEQUENCING IDENTIFIES NOVEL MOLECULAR TARGETS OF ENDOTHELIAL MICRORNA-200B IN ISCHEMIC WOUND
Kanhaiya Singh, Edward Hernandez, Manishekar Kumar, Yashika Rustagi, Ahmed Abouhashem, Sujit Mohanty, Savita Khanna, Sashwati Roy, Chandan K. Sen
Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, USA
Single-cell RNA-sequencing identifies novel molecular targets of endothelial microRNA-200b in ischemic wound

Kanhaiya Singh, Edward Hernandez, Manishekar Kumar, Yashika Rustagi, Ahmed Abouhashem, Sujit Mohanty, Savita Khanna, Sashwati Roy, Chandan K. Sen | Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine

Background: Injury-induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis but the underlying mechanisms remain unclear. Methods: Single cell RNA sequencing (scRNA-seq) was performed on ~15000 human dermal microvascular endothelial cells at day 3 following treatment with miR-200b inhibitor or its mimic. Unsupervised clustering using CellRanger v3.0.2 identified 13 cell clusters, few of which were miR-200b responsive. SILAC-based proteomic analysis using mass spectrometry was used to verify novel potential targets. miR-200b-429fl/fl-Tie2 Cre mice (endothelial miR-200b depleted) were generated and studied using ischemic hind limb model. Results: 3818 proteins were detected; filtering identified 319 proteins targeted by miR-200b. Inhibition of endothelial miR-200b rescued hindlimb ischemia with improved perfusion (n=5). This rescue was associated with increased abundance of CD31+/vWF+ vasculogenic cells at the site of injury. Conclusions: This work identified miR-200b regulated endothelial cell clusters, the functionality of which is explained by SILAC proteomics data. These findings provide insight into novel mechanisms explaining how transient inhibition of endothelial miR-200b helps switch vascular homeostasis into an angiogenic fate in response to injury.

J3.02

DERMAL FIBROBLASTS UPTAKE EXOSOMES FROM ENDOTHELIAL CELLS AND PROMOTE MIGRATION
Anna Salapatas, Trevor Leonardo, Lin Chen, Sriram Ravindran, Luisa A. DiPietro
University of Illinois at Chicago College of Dentistry, Chicago, IL, USA
Dermal fibroblasts uptake exosomes from endothelial cells and promote migration

Anna Salapatas, Trevor Leonardo, Lin Chen, Sriram Ravindran, Luisa A. DiPietro | University of Illinois at Chicago College of Dentistry

Background: This study examines if exosomes from microvascular endothelial cells influence wound resolution by targeting fibroblasts. Our objectives were to isolate and characterize exosomes from human dermal microvascular endothelial cells, examine uptake by human dermal fibroblasts (FBs), and observe the effect on fibroblast migration. Methods: Exosomes were collected and purified from culture supernatant of endothelial cells (EC-exos). Nanoparticle tracking determined quantity and size distribution. Green fluorescently labeled EC-exos were placed on plated FBs for 24 hours to assess uptake using fluorescent microscopy. A transwell migration assay and scratch assay were used to assess fibroblast migration. Results: Nanoparticle analysis showed a mean particle size of 148.1 nm, characteristic of exosomes. Fluorescent and confocal microscopy showed uptake of exosomes from HDMVECs by HDFs at 15% concentration. FBs treated with EC-exos exhibited enhanced migration in the transwell migration assay and accelerated scratch closure. Conclusions: ECs produce functional exosomes that can be taken up by FBs causing changes in migration patterns, suggesting that endothelial cells can influence fibroblast function in wounds via exosomes.

J3.03

NRF2 MEDIATES ANGIOGENIC FUNCTION OF ENDOTHELIAL CELLS THROUGH REGULATION OF CXCR4/SDF-1 AXIS
Jasmine Lee1, Alvaro Villareal Ponce1, Joshua A. David2, Joseph F. Kuhn1, Daniel J. Ceradini1
1NYU Langone Medical Center, New York, NY, USA, 2University of Pittsburgh, Pittsburgh, PA, USA
Nrf2 mediates angiogenic function of endothelial cells through regulation of CXCR4/SDF-1 axis

Jasmine Lee, Alvaro Villareal Ponce, Joshua A. David, Joseph F. Kuhn, Daniel J. Ceradini | NYU Langone Medical Center; University of Pittsburgh

Background: The purpose of this study is to propose a mechanism by which Nrf2 directly regulates endothelial cell angiogenic potential in tissue regeneration via the CXCR4/SDF-1 axis. Methods: Cadherin5 (Cdh5)-CreER mice crossed with Nrf2flox/flox mice generated endothelial-specific Nrf2 knockout (KO) mice. Ten mm diameter full-thickness stented excisional wounds were created on 6-8-week-old WT, diabetic, and KO mice. C166 mouse endothelial cells were transfected with Nrf2 siRNA and placed in 5% oxygen hypoxia. Results: Nrf2 KO mice showed delayed wound closure compared to WT mice (KO 33.0+/-1.73 days vs. WT 14.0+/-0 days), closely approximating diabetic mice (31.0+/-1.41 days). Primary endothelial cells from KO mice demonstrated significantly decreased angiogenic functionality: fewer branches (KO 64.75+/-14.81 vs. control 282.8+/-46.7 branches/cm2, p=0.014) and fewer branching points (p=0.0031). Nrf2 KD reduced Nrf2 transcription by 76.5% (p=0.001), reduced NQO1 by 66.4% (p=0.02), and reduced CXCR4 and SDF-1 by 61.7% (p=0.001) and 47.6% (p=0.02) respectively. Conclusions: Nrf2 expression in endothelial cells is necessary for physiologic wound healing. Nrf2 dysregulation associated with diabetes may contribute to impaired angiogenesis via the CXCR4/SDF-1 axis.

J3.04

THE EFFECTS OF M1 MACROPHAGE ACTIVATION ON M1-TO-M2 PHENOTYPIC SWITCHING
Erin M. O’Brien, Kara L. Spiller, PhD
Drexel University, Philadelphia, PA, USA
The effects of M1 macrophage activation on M1-to-M2 phenotypic switching

Erin M. O’Brien, Kara L. Spiller, PhD | Drexel University, Philadelphia, PA

Background: There is growing evidence that early M1 macrophages initiate sprouting, and subsequent M2 activity stabilizes vascular structures. In pathologies where angiogenesis is inhibited, M1 macrophages are insufficiently activated and also fail to switch to the M2 phenotype. This study investigated how M1 activation affects the switch to the M2 phenotype. Methods: Primary human monocyte-derived macrophages were used to test whether M1 macrophages are primed to undergo phenotypic switching. IL-4 receptor-alpha (IL-4Ralpha) expression was measured via flow cytometry. We compared M0- and M1-derived M2 macrophage gene expression of M2 and angiogenesis markers, secretion of M2-associated angiogenic proteins, and migration of endothelial cells in response to macrophage-conditioned media. Results: M1 macrophages upregulated surface IL-4Ralpha expression compared to M0, suggesting they are primed to switch to M2. M2 macrophages derived from M0 (M0-M2) and M1 (M1-M2) differentially upregulated M2 markers and genes associated with angiogenesis. M1-M2 macrophages especially upregulated CCL17 and CXCR4, and secreted more CCL17 and PDGF-BB than M0-M2. M1-M2 macrophage-conditioned media induced significantly more migration of endothelial cells compared to M0-M2. Conclusions: M2 macrophages that have previously been M1-activated are phenotypically unique and exhibit enhanced M2 function, particularly in angiogenesis. Promoting the M1 macrophage phenotype may rescue dysfunctional angiogenesis.

J3.05

BIO-FUNCTIONAL COLLAGEN MATRIX SCAFFOLD COMPOSITION DIFFERENTIALLY PROMOTES PARACRINE ACTIVITY IN HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED VASCULAR SMOOTH MUSCLE CELLS
Kaiti Duan1, Biraja Dash2, Henry Hsia2
1Frank H. Netter MD School of Medicine, Hamden, CT, USA, 2Yale School of Medicine, New Haven, CT, USA
Bio-functional collagen matrix scaffold composition differentially promotes paracrine activity in human induced pluripotent stem cell derived vascular smooth muscle cells

Kaiti Duan, Biraja Dash, Henry Hsia | Frank H. Netter MD School of Medicine; Yale School of Medicine

Background: Human iPSC-derived vascular smooth muscle cells (iPSC-VSMC) have the potential to treat chronic wounds by secreting proangiogenic VEGF. Little is known about how ECM composition impacts the cells’ paracrine secretion profile. Methods: Type-I collagen scaffolds were incorporated with hyaluronic acid (HA), fibronectin, and laminin functional biomolecules at three different collagen densities (1.25, 2.5, and 5 mg/mL). iPSC-VSMC viability and paracrine secretion profile (VEGF, SDF, PDGF, bFGF, ANG-1, IL-8, TGF, IL-10) were investigated. Results: Functional biomolecule-collagen scaffolds showed increased cell viability across all collagen densities compared to non-functional scaffolds. 1.25 mg/mL scaffolds displayed the greatest cell proliferation (P=0.0001). Enhanced VEGF was observed in all three functionalized scaffolds in 1.25 mg/mL (P=0.0086). Fibronectin-functionalized 5 mg/mL collagen scaffold exhibited substantially elevated bFGF secretion (P=0.0049), with a positive correlation of increasing fibronectin embedment with increasing bFGF paracrine secretion (P=0.0001). IL-8 and IL-10 secretion were upregulated in fibronectin-functionalized collagen scaffolds at 1.25 and 2.5 mg/mL respectively. Conclusions: Functionalization along with density differentially regulate paracrine function of Human iPSC-VSMCs, optimizing them as a viable therapeutic agent for chronic wound healing.

J3.06

ANALYSIS OF SOX2-DEPENDENT ANGIOGENESIS EFFECTORS IN A MOUSE MODEL OF IMPROVED WOUND HEALING
Daniela Grassini1, Akihiko Uchiyama2, Stephen Brooks3, Maria Morasso1
1Skin Biology Lab, NIAMS/NIH, Bethesda, MD, USA, 2Gunma University, Gunma, Japan, 3Biodata Mining Section, NIAMS/NIH, Bethesda, MD, USA

J4.01

MEVASTATIN FOR TOPICAL USE IN DIABETIC FOOT ULCERS TO ACCELERATE WOUND CLOSURE BY INHIBITION OF CAVEOLIN-1 AND RESTORATION OF EGF SIGNALING
Andjela N. Egger, Andrew P. Sawaya, Ivan Jozic, Rivka C. Stone, Irena Pastar, Marjana Tomic-Canic
Wound Healing and Regenerative Medicine Research Program, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
Mevastatin for topical use in diabetic foot ulcers to accelerate wound closure by inhibition of caveolin-1 and restoration of EGF signaling

Andjela N. Egger, Andrew P. Sawaya, Ivan Jozic, Rivka C. Stone, Irena Pastar, Marjana Tomic-Canic | University of Miami Miller School of Medicine

Background: Epidermal growth factor receptor (EGFR) is mislocalized to the cytoplasm and downregulated in the epidermis from DFU patients, whereas Caveolin-1 (Cav1) is overexpressed in DFUs. Cav1 may act to inhibit and sequester EGFR to inhibit wound healing. This study evaluated the ability of topical statins to promote healing through Cav1 suppression and EGF restoration. Methods: Tissue samples from DFU patients were collected. DFUs were treated ex vivo and porcine in vivo wounds were treated with topical Mevastatin (MEV). Histology validations and protein assessments focused on Cav1 localization and expression and EGFR activation. Effects of MEV on proliferation and migration were evaluated using primary human keratinocytes. Results: DFU histology confirmed hyperproliferative epidermis and thickened cornified layer. Compared to vehicle-treated controls, MEV inhibited Cav1 and activated p-EGFR in MEV-treated DFUs. Immunoperoxidase staining of in vivo porcine wounds showed MEV decreased Cav1 expression and enhanced epithelization compared to controls. Conclusions: Topical application of MEV in DFUs can promote wound closure by inhibiting Cav1 and restoring EGF signaling. Our findings also highlight why recombinant EGF therapy failed to reach FDA efficacy in clinical trials: due to sequestration of EGFR and inhibition of its signaling by Cav1.

J4.02

DOWNREGULATED LONG INTERGENIC NONCODING RNAS (LINCRNA) EPS DURING DIABETIC WOUND HEALING
Junyi Hu, Liping Zhang, Kenneth W. Liechty, Junwang Xu
University of Colorado Anschutz Medical Campus, Aurora, CO, USA

J4.03

GENE EXPRESSION ANALYSIS OF DIABETIC FOOT ULCER DEBRIDED TISSUE TO STANDARD AND ADVANCED TREATMENTS
Jessica M. Eager1, Michael S. Weingarten, MD2, Kara L. Spiller, PhD1
1Drexel University, Philadelphia, PA, USA, 2Drexel University College of Medicine, Philadelphia, PA, USA
Gene expression analysis of diabetic foot ulcer debrided tissue to standard and advanced treatments

Jessica M. Eager, Michael S. Weingarten, MD, Kara L. Spiller, PhD | Drexel University; Drexel University College of Medicine

Background: Chronic DFUs are notoriously difficult to treat, with a major challenge stemming from the mystery of why some wounds respond to treatment while others do not. We determined differences in gene expression within wound tissue of healing vs. non-healing DFUs treated with standard of care (SOC) or amniotic membrane-derived advanced biomaterials. Methods: Debrided tissue was collected from 29 SOC-treated and 7 advanced biomaterial-treated patients for gene expression analysis via Nanostring. Samples were classified into responders and non-responders based on wound status at 12 weeks. A custom panel of 227 genes related to inflammation, macrophage phenotype, and bacterial communication was used. Results: Pro-inflammatory genes EBI3 and CCL1 were upregulated in responders to SOC compared to non-responders at all time points (adjusted p<0.05). Genes related to M1 macrophage phenotype and bacterial communication (CXCL9, CCL1, CXCL11, DEFB4) were significantly upregulated at week 1 in responders. Non-responders to amniotic membrane-derived biomaterials expressed lower levels of PRSS8 compared to SOC non-responders at week 4. Conclusions: Responders to SOC expressed higher levels of M1 macrophage-related genes at early time points, possibly capturing the conversion of a chronic to acute wound environment. Advanced biomaterials may have a slight anti-inflammatory effect, but more samples are needed for validation.

J4.04

COMPARATIVE TRANSCRIPTOMICS OF HUMAN DIABETIC FOOT ULCERS AND DIABETIC MOUSE WOUNDS REVEALS LIMITED OVERLAP
Sydney Resnik1, Rivka Stone1, Irena Pastar1, Elizabeth Grice2, Marjana Tomic-Canic1
1University of Miami, Miami, FL, USA, 2University of Pennsylvania, Philadelphia, PA, USA
Comparative transcriptomics of human diabetic foot ulcers and diabetic mouse wounds reveals limited overlap

Sydney Resnik, Rivka Stone, Irena Pastar, Elizabeth Grice, Marjana Tomic-Canic | University of Miami; University of Pennsylvania

Background: Diabetic foot ulcers (DFUs) have a global prevalence of 6.3% and are a leading cause of non-traumatic limb amputation. The diabetic murine model (db-/db-) is widely used in pre-clinical DFU studies, but wound closure eventually occurs in these mice, unlike in humans. Methods: Utilizing comparative genomics, we identified shared and/or unique genes and pathways between gene expression data from db-/db- mouse wounds (days 3, 7, and 14 post-wounding) and human DFUs. Normalized datasets were subjected to Ingenuity Pathway Analysis (IPA) for functional enrichment and comparative analysis. Results: Greater overlap exists between db-/db- and control db+/db- mice (~50%) than between db-/db- mice and DFUs (~15%). Genes involved in immune cell activation appear conserved to some extent, including the IL-36 subfamily of the IL-1 family, found activated in both db-/db- mice and DFUs. However, pathways involving functioning of immune cells and inflammation, cell movement, and cytoskeleton organization were active in db-/db- mice but absent in DFUs. Impairment of DNA repair, a unique hallmark of DFUs, was absent in db-/db- mice. Conclusions: Diabetic mouse wounds retain the transcriptional signature of acute wounds with minimal overlap with human DFUs, indicating caution should be taken when translating observations from pre-clinical diabetic mouse models to human DFU.

J4.05

TOPICAL DELIVERY OF CNP-MIR146A VIA NANOSILK ACCELERATES DIABETIC WOUND HEALING
Carlos Zgheib1, Sarah A. Hilton1, Lindel C. Dewberry1, Junwang Xu1, Sushant Singh2, Tamil S. Sakthivel2, Sudipta Seal2, Kenneth W. Liechty1
1Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver School of Medicine and Children’s Hospital Colorado, aurora, CO, USA, 2Department of Materials Science and Engineering, Advance Materials Processing Analysis Center, Nanoscience Technology Center, College of Medicine, University of Central Florida, Orlando, FL, USA

J4.06

DEFEROXAMINE IMPROVES WOUND HEALING IN A MOUSE MODEL OF DELAYED HEALING FROM PRESSURE INJURY
Lisa Tucker-Kellogg1, N. Jannah M. Nasir1, Johannes A. Heemskerk1, Ralph Bunte1, Paul Matsudaira2, Peter T.C. So3
1Duke-NUS Medical School, Singapore, Singapore, 2National University of Singapore, Singapore, Singapore, 3MIT, Cambridge, MA, USA
Deferoxamine improves wound healing in a mouse model of delayed healing from pressure injury

Lisa Tucker-Kellogg, N. Jannah M. Nasir, Johannes A. Heemskerk, Ralph Bunte, Paul Matsudaira, Peter T.C. So | Duke-NUS Medical School; National University of Singapore; MIT

Background: Pressure ulcer research requires animal models of impaired regeneration without diabetes to seek treatments to restore regeneration. We develop a mouse model of delayed muscle regeneration after gradual pressure-induced injury and investigate the therapeutic effect of deferoxamine, an FDA-approved drug that has been found to accelerate regeneration of skin. Methods: Pressure injuries were created in C57BL6 mice by applying a pair of 12mm magnets to the dorsal skinfold and panniculus carnosus muscle in two 12-hour intervals. Deferoxamine (or saline control) was injected subcutaneously for 16 days (n=7). Acute-control mice had the same muscles injured via cardiotoxin. Results: In acute-control mice, muscle completed regeneration by 16 days. In pressure injury mice, dead muscle showed minimal immune infiltrate or phagocytosis at 3 days, with lasting tissue compaction. At 40 days, muscle tissue was still actively regenerating. Deferoxamine treatment caused increased infiltration of macrophages with M2 (pro-regenerative) markers at 3 and 10 days, followed by greater muscle regeneration at 40 days, compared to saline control. Conclusions: A successful animal model of delayed regeneration after pressure injury was achieved, associated with delayed immune cell infiltration. Deferoxamine treatment improves muscle regeneration, associated with increased macrophage infiltration.

M1.01

HYPOPIGMENTED HYPERTROPHIC SCAR CAN BE TREATED WITH SYNTHETIC ALPHA MELANOCYTE STIMULATING HORMONE
Bonnie C. Carney1, Lauren T. Moffatt2, Cynthia M. Simbulan-Rosenthal1, Dean S. Rosenthal1, Jeffrey W. Shupp2
1Department of Biochemistry, Georgetown University School of Medicine, Washington, DC, USA, 2Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington, DC, USA
Hypopigmented hypertrophic scar can be treated with synthetic alpha melanocyte stimulating hormone

Bonnie C. Carney, Lauren T. Moffatt, Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal, Jeffrey W. Shupp | Georgetown University School of Medicine; MedStar Health Research Institute, Washington, DC

Background: Burn injuries often result in hypertrophic scar (HTS) with dyschromia. We previously showed that alpha-MSH expression is absent in hypo-pigmented scar. A nude mouse model of xenografted porcine dyschromic HTS was developed to test if supplying hypo-pigmented cells with alpha-MSH can re-pigment the scar. Methods: Dyschromic HTSs were created in Duroc pigs. Epidermal cells were derived from regions of hyper-, hypo-, or normally-pigmented scar or skin. Dermal fibroblasts (DFs) were isolated separately. Wounds were created on nude mice and a grafting dome was placed. DFs were seeded on day 0, epidermal cells on day 3, dome removed on day 7. Hypo-pigmented xenografts were treated with synthetic alpha-MSH delivered with microneedling on day 7. Xenografts were excised on day 10. Sections were stained using H&E and RNA was isolated for qRT-PCR of TYR, TYRP1, and DCT. Results: HTSD fibroblasts formed a dermis similar in structure to porcine HTS dermis. A fully stratified epithelium was formed. Treated hypo-pigmented xenografts showed increased pigmentation and increased transcription of TYR, TYRP1, and DCT compared to controls (TYR: 2.7 vs 0.3; TYRP1: 2.6 vs 0.3; DCT: 0.7 vs 0.3 fold change; n=3). Conclusions: Hypo-pigmented regions of burn scar can be stimulated to make melanin by synthetic alpha-MSH.

M1.02

IS HEDGEHOG SIGNALING CRITICAL FOR WOUND CHRONICITY? A WGCNA ANALYSIS OF CHRONIC WOUNDS OVER TIME
Proma Basu, Jane Kim, Manuela Martins-Green
University of California Riverside, Riverside, CA, USA

M1.03

FLUID FLOW IN TUMESCED SUBCUTANEOUS PIG TISSUE
John P. Koulakis, Joshua Rouch, Nhan Huynh, Holden H. Wu, James C. Y. Dunn, Seth Putterman
UCLA, Los Angeles, CA, USA
Fluid flow in tumesced subcutaneous pig tissue

John P. Koulakis, Joshua Rouch, Nhan Huynh, Holden H. Wu, James C. Y. Dunn, Seth Putterman | UCLA, Los Angeles, CA

Background: Administration of pharmaceuticals such as monoclonal antibodies via subcutaneous injection requires large volume doses that tumesce the extracellular matrix near the injection site. The properties of tissue in the tumesced state are not well known. Methods: We investigate the flow properties of externally tumesced subcutaneous pig tissue with MRI, CT, and 3D scanning. The evolution of 24 bolus injections of saline (2.5-40 mL volumes) was monitored on timescales from minutes to several hours. Results: Bolus injection into subcutaneous tissue results in accumulation of fluid at the injection site, not as a pool but as a hydrogel-bound state within the expanded interstitial matrix. Subcutaneous tissue can expand to a few times its initial volume. Stress around the tumesced region acts to spread the fluid to peripheral unexpanded regions over a few minutes, after which it remains in place for several hours. Eventually systemic circulation absorbs the excess fluid and tissue returns to its original state. Conclusions: The expansion of subcutaneous tissue and corresponding decrease in hydraulic resistance while tumesced may allow for dispersal of large pharmaceuticals. A procedure is proposed whereby tumescent antibiotic injections are used to treat drug-resistant skin infections and chronic wounds that extend into the subcutaneous tissue.

M1.04

MICE LACKING FUNCTIONAL CONCENTRATION-DEPENDENT SODIUM CHANNEL NAX (SCN7A) RESIST BLEOMYCIN-INDUCED DEVELOPMENT OF SKIN FIBROSIS
David Dolivo, Adrian Rodrigues, Chun Hou, Seok Hong, Thomas Mustoe, Robert Galiano
Northwestern University, Chicago, IL, USA

M1.05

A SYSTEMS BIOLOGY TO OVERCOME BIOLOGICAL VARIANCE IN THE STUDY OF FIBROSIS
Daniel J. Gibson
University of Florida, Gainesville, FL, USA
A systems biology approach to overcome biological variance in the study of fibrosis

Daniel J. Gibson | University of Florida, Gainesville, FL

Background: Biological variance has been observed in injury models in terms of actual fibrotic outcomes, leading to masking of key regulators or attenuation of statistical power. This variance can also be used as a powerful tool to reduce complex datasets and reveal core regulatory networks active during fibrosis. Methods: A rabbit corneal lamellar keratectomy model was used to injure 20 corneas on 10 rabbits. The scar formation process was quantified via automated image analysis by measuring light-reflecting cellular haze between days 5-14 after injury. Corneas with no haze growth were segregated from those with high haze growth prior to mRNA analysis via RNASeq. Results: Approximately 15,000 genes were mapped to the rabbit genome. Only 399 were differentially expressed between corneas with active fibrosis vs. those without. Two growth factors – placental growth factor (PGF, p=0.0002) and VEGF-A (p=5×10-5) – were the only so-named growth factors differentially regulated, along with a cluster of genes highly associated with cells derived from the myeloid lineage. None of the corneas presented with angiogenesis from the limbus. Conclusions: Common pro-fibrotic factors such as TGF-beta and CTGF were not found to be differentially expressed during the later, active fibrotic phase, whereas two growth factors typically associated with angiogenesis and vasculogenesis were highly differentially expressed but without angiogenic activity, suggesting a potential pleiotropic role.

M1.06

KNOCKDOWN OF A SODIUM CHANNEL NAX IN THE EPIDERMIS RELIEVES DERMATITIS SYMPTOMS
Jingling Zhao1, David Dolivo2, Chun Hou2, Robert Galiano2, Thomas Mustoe2, Seok Hong2
1The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China, 2Northwestern University, Chicago, IL, USA
Knockdown of a sodium channel Nax in the epidermis relieves dermatitis symptoms

Jingling Zhao, David Dolivo, Chun Hou, Robert Galiano, Thomas Mustoe, Seok Hong | Sun Yat-Sen University, Guangzhou, China; Northwestern University, Chicago, IL

Background: Nax (SCN7A) is an atypical voltage-gated sodium channel that acts as a sodium concentration-sensitive channel in the epidermis. Both atopic dermatitis (AD) and psoriasis are chronic inflammatory skin diseases with defective skin barrier function as a major contributor to their pathogenesis. Methods: AD-like and psoriasis-like models were established in rabbit ear skin by application of house dust mite extract and imiquimod, respectively. The effects of RNAi-mediated Nax knockdown on inflammatory phenotypes were evaluated. Epidermal hyperplasia, keratinocyte hyperproliferation, and inflammatory cell infiltration were determined by histology. Expression of S100A9 was quantified by immunohistological analysis. Results: In vivo knockdown of Nax using RNAi reduced epidermal hyperplasia and keratinocyte hyperproliferation in AD-like rabbit ear skin. Nax knockdown decreased skin erythema, scaling, and papule formation in psoriasis-like rabbit ear skin. Infiltration of inflammatory cells induced by house dust mite extract was reduced upon knockdown of Nax. Expression of S100A9, a downstream pro-inflammatory gene target of Nax, was also decreased by Nax-RNAi. Conclusions: Inhibition of Nax relieved dermatitis symptoms in vivo, suggesting Nax is a novel therapeutic target for AD and psoriasis.

M1.07

FLEXIBLE SMART BANDAGE FOR WIRELESS WOUND HEALING
Artem A. Trotsyuk, Yuanwen Jiang, Simiao Niu, Maddy Larson, Ethan Beard, Aref Saberi, Dominic Henn, Sun Hyung Kwon, Clark Andrew Bonham, Kellen Chen, Michael Januszyk, Zeshaan Maan, Janos Barrera, Jagannath Padmanabhan, Katharina S. Fischer, Zhenan Bao, Geoffrey C. Gurtner
Stanford University, Stanford University, CA, USA
Flexible smart bandage for wireless wound healing

Artem A. Trotsyuk, Yuanwen Jiang, Simiao Niu, Maddy Larson, Ethan Beard, Aref Saberi, et al. | Stanford University

Background: Current standard of care wound dressings are passive and cannot actively respond to variations in the wound environment. Smart bandages are well positioned to address these challenges. We report a multidisciplinary approach combining electrical and chemical engineering with the fundamentals of cellular and biomolecular processes in wound healing. Methods: A flexible printed wireless stimulator was designed and fabricated to deliver directional energy across a wound gradient. A low impedance PEDOT:PSS electrode was designed to optimize the skin-stimulator interface with a robust gel of tunable adhesion. The smart bandage was evaluated in excisional diabetic and C57BL6/J murine wound healing models. A parabiosis model and single cell analyses were used to evaluate changes in cell populations as a result of electrical stimulation. Results: Wireless electrical stimulation resulted in significantly accelerated wound closure in both diabetic and C57BL6/J murine excisional wound healing models. Complete epidermal recovery was observed, with thicker collagen network and increased dermal thickness. Greater neovascularization and appendage formation were observed in treatment groups. Single cell analyses revealed higher proliferation and remodeling regulatory markers in treated groups. In vitro co-culture validation demonstrated accelerated proliferation, mitotic rate, and tube formation compared to controls. Conclusions: This novel treatment modality will integrate AI processing components for a closed-loop functional stimulator, paving the way for the next generation of palliative wound care.

M1.08

BETA-CATENIN ACTIVATION IN CARDIAC FIBROBLASTS MODULATES THE IMMUNE MICRO-ENVIRONMENT TO PROMOTE FIBROSIS AND HEART FAILURE FOLLOWING CHRONIC INJURY
Meiling Melzer, David Beier, Pampee Young, Sarika Saraswati
Vanderbilt University Medical Center, Nashville, TN, USA

N1.01

REDUCTION OF SENESCENT CELLS IMPROVES HEALING IN AN IN VITRO MODEL OF AGED HEALING
Upasana Niyogi, Mark A. Carlson
University of Nebraska Medical Center, Omaha, NE, USA
Reduction of senescent cells improves healing in an in vitro model of aged healing

Upasana Niyogi, Mark A. Carlson | University of Nebraska Medical Center, Omaha, NE

Background: One of the likely factors associated with poor healing in the aged is the increase of senescent cells in the wound. We utilized the fibroblast-populated collagen matrix (FPCM) with near-senescent human dermal fibroblasts to study the role of fibroblast senescence on wound healing. We hypothesized that BCL-2 inhibition along with a mitogen stimulation would improve wound healing endpoints in the senescent FPCM model. Methods: Senescence was induced by replicative stress or oxidative stress. Characterization was performed with SA-beta-Gal activity, proliferation assays, migration assays, and expression of senescence and oxidative stress markers. The senescent FPCM was treated with BCL-2 inhibitor (ABT-737) or a combination of ABT-737 and FGF-2, followed by scratch healing assay. Results: A delay in wound healing was observed in the senescent FPCM compared to young FPCM (p<0.0001). Treatment of the senescent FPCM with ABT-737 reduced the fraction of senescent cells and accelerated the scratch wound healing rate (p<0.005). Combined treatment with ABT-737 and FGF-2 further improved the healing rate (p<0.003), reduced alpha-SMA-positive cells, and decreased collagen secretion, consistent with an anti-fibrotic effect. Conclusions: Accumulation of senescent cells with age was associated with decreased healing efficacy. Reduction of these cells with soluble factors improved healing outcomes in the aged in vitro model.

N1.02

EFFECT OF DEHYDRATED AMNION CHORION MEMBRANES ON MESENCHYMAL STEM CELLS IN VITRO
Katrina Harmon, John P. McQuilling, Katie Mowry
Organogenesis Inc, Birmingham, AL, USA
Effect of dehydrated amnion chorion membranes on mesenchymal stem cells in vitro

Katrina Harmon, John P. McQuilling, Katie Mowry | Organogenesis Inc., Birmingham, AL

Background: Mesenchymal stem cells (MSCs) have been shown to promote resolution of wound inflammation, increased angiogenesis, and regulation of ECM remodeling in vivo. Hypoxia has been shown to affect MSC responses including proliferation, differentiation, and secretory profile, relevant given that diabetic wounds are known to be hypoxic. We evaluated the response of bone marrow MSCs to dehydrated amnion chorion membranes (dACM) in vitro under normal and hypoxic conditions. Methods: bm-MSCs were treated with either assay media or media conditioned with dACM (CM). CM was obtained by incubating dACM grafts in basal media for 3-5 days at 4 degrees C (1 cm2 to 1 mL media). To characterize the role of hypoxia, bm-MSCs treated with CM were cultured under normoxic (18.6% O2) and hypoxic (2.5% O2) conditions for 3 days, then supernatants were analyzed using Quantibody arrays. Results: dACM CM promoted bm-MSC proliferation and migration (1.3 and 3.5-fold change compared to assay media respectively) in normoxic conditions. dACM CM treatment resulted in significant increased secretion of CCL3, CCL5, IL-6, and IL-8 from MSCs under normoxic and hypoxic conditions. Increased trends were also seen in IGFBP-3, IGFBP-4, and IGFBP-5, though not statistically significant. Conclusions: dACM promotes bm-MSC proliferation, migration, and stimulates secretion of factors related to inflammation and angiogenesis in vitro.

N1.03

HARNESSING NOVEL GENE EXPRESSION ANALYSES TO IDENTIFY DRIVERS OF REGENERATIVE EAR WOUND HEALING IN MRL MICE
Heather E. desJardins-Park, Katya L. Mack, Michael F. Davitt, Michelle Griffin, Hunter B. Fraser, Michael T. Longaker
Stanford University, Stanford, CA, USA
Harnessing novel gene expression analyses to identify drivers of regenerative ear wound healing in MRL mice

Heather E. desJardins-Park, Katya L. Mack, Michael F. Davitt, Michelle Griffin, Hunter B. Fraser, Michael T. Longaker | Stanford University

Background: The “super-healing” MRL/MpJ (MRL) mouse is uniquely able to regenerate ear punch wounds. We hypothesized that differential allele-specific expression in MRL/CAST hybrid offspring could represent a novel approach to identify genes driving MRL ear regeneration. Methods: MRL and CAST/EiJ (CAST) mouse genome sequences were obtained and variant calling performed. MRL and CAST mice were cross-bred and F1 offspring underwent dorsal excisional and ear punch wounding; wounds were harvested on POD7. Immune cells (CD45+) were isolated by FACS and underwent bulk RNA-sequencing. Reads were mapped to the MRL or CAST genome based on strain-specific variants. Results: Ear wounds in MRLxCAST hybrid mice grossly regenerated over time, while dorsal wounds formed scars. The number of genes with significantly different expression from MRL vs. CAST allele was substantially (>10-fold) greater in the ear than the dorsum (3096 vs. 194 unique genes), and the majority of allele-specific expression in the dorsum was also observed in the ear (1040 shared genes vs. 194 unique to dorsum). Conclusions: MRL hybrid mice exhibit markedly greater allele-specific gene expression in ear (regenerative) than dorsal (scarring) wound inflammatory cells, consistent with MRL-specific gene cis-regulation leading to improved ear healing. Probing differential allele-specific gene expression may represent a powerful approach for identifying molecular drivers of wound regeneration.

N1.04

ACTIVATION OF SKELETAL STEMS CELLS IN RESPONSE TO LONG BONE DISTRACTION OSTEOGENESIS
Ankit Salhotra, BS, Harsh N. Shah, BS, MPH, Michael T. Lopez, BS, Derrick C. Wan, MD, Michael T. Longaker, MD, MBA
Stanford University, Stanford, CA, USA
Activation of skeletal stem cells in response to long bone distraction osteogenesis

Ankit Salhotra, BS, Harsh N. Shah, BS, MPH, Michael T. Lopez, BS, Derrick C. Wan, MD, Michael T. Longaker, MD, MBA | Stanford University

Background: The underlying cellular response during Distraction Osteogenesis (DO) is not well understood. We hypothesize skeletal stem cells are responsible for regeneration during DO. Methods: 10-week old male Actin;R26VT2/GK3 (rainbow) mice underwent distraction surgery with a five-day latency period, twice-daily distraction of 0.15 mm for 10 days, and consolidation period of 28 days. At post-operative day 43, distracted tibias were imaged. Proliferation and osteogenic assays were performed. At POD 10, fracture and distracted tibias were harvested for FACS of skeletal stem cells (SSCs). Results: Rainbow labeling of distraction calluses reveals a distinct clonal expansion in response to DO. SSCs sorted from distraction calluses at POD 10 form robust colonies and larger bone nodules in comparison to fracture calluses in vitro. Distraction SSCs form more colonies (***p<0.001) and have increased osteogenic potential (****p<0.0001). Conclusions: Long Bone DO promotes bone regeneration through the activation of skeletal stem cells.

N1.05

NEUROGENIC TISSUE NANOTRANSFECTION IN THE MANAGEMENT OF CUTANEOUS DIABETIC POLYNEUROPATHY
Savita Khanna
Indiana University, Indianapolis, IN, USA
Neurogenic tissue nanotransfection in the management of cutaneous diabetic polyneuropathy

Savita Khanna | Indiana University, Indianapolis, IN

Background: Distal forms of diabetic peripheral neuropathy (DPN) are characterized by loss of axons through degeneration. Prior approaches using pharmacologic therapies like exogenous nerve growth factor (NGF) have failed past phase II/III clinical trials mainly for lack of efficacy and adverse side-effects. Tissue Nanotransfection (TNT) is a non-viral nanotechnology platform that may deliver ABM (Ascl1, Brn2, and Myt1l) and induce neurogenic reprograming of murine skin in vivo. Methods: Non-viral cell transfection was conducted via nanochannel electroporation (NEP). Mouse Embryonic Fibroblasts were grown on transwell membrane, mounted on a custom gold electrode in contact with plasmid solution, followed by pulsed electric field to deliver ABM plasmids. For in vivo reprogramming, topical cutaneous TNTABM was performed on C57Bl/6 mice and db/db mice using TNT chip device. Results: Nanoelectroporation-mediated transfection of MEF with ABM caused reprogramming into induced neurons and increased NGF expression in vitro. In vivo, TNTABM caused successful neurogenic conversion of cells, neurotrophic enrichment of skin stroma, elevation of endogenous NGF and other neurotrophic factors including Nt3. TNTABM spared loss of cutaneous PGP9.5+ mature nerve fibers in db/db diabetic mice. Conclusions: This is the first study demonstrating that under conditions of neurogenic in vivo reprogramming, changes in the skin tissue microenvironment can leverage rescue of pre-existing nerve fibers from predictable loss under diabetic conditions.

N1.06

CLINICAL EFFICACY OF NEW GENERATION VASCULAR AND TISSUE REGENERATIVE CELL (MNC-QQ) THERAPY FOR NON-HEALING ISCHEMIC ULCER
Rica Tanaka, Taro Fukuta, Satoshi Fujimura, Kayo Arita, Rie Hirano, Hiroshi Mizuno
Juntendo University School of Medicine, Tokyo, Japan
Clinical efficacy of new generation vascular and tissue regenerative cell (MNC-QQ) therapy for non-healing ischemic ulcer

Rica Tanaka, Taro Fukuta, Satoshi Fujimura, Kayo Arita, Rie Hirano, Hiroshi Mizuno | Juntendo University School of Medicine, Tokyo, Japan

Background: We reported the novelty of a serum-free ex vivo expansion system called Quantity and Quality Culture System (QQc) using peripheral blood mononuclear cells (PbMNC) as a non-invasive and effective vascular and tissue regenerative cell therapy. This study investigates the safety and efficacy of QQ cultured PbMNC (MNC-QQ therapy) on non-healing ischemic extremity wounds. Methods: 200ml of peripheral blood was drawn from patients with chronic (>3 months) ischemic extremity ulcers. Mononuclear cells were isolated and cultured in QQc for one week. Under local anesthesia, 2×10^7 cells were injected within 20 cm2 of the chronic wound. Wound closure, VAS scale, skin perfusion pressure (SPP), TcPo2, laser doppler, thermography, and angiography were performed at 2, 4, 8, and 12 weeks post-therapy. Results: Nine patients (ten limbs) were enrolled. Seven patients had diabetes with renal failure. All wounds extended into bone or tendon. All patients remained ambulant without major amputation. There was no death, serious adverse events, or major amputation within 12 weeks. Increased vascular perfusion with decrease in VAS scale were seen in all patients. SPP significantly increased post-therapy. Conclusions: The outcomes indicate safety and feasibility of MNC-QQc cell therapy in patients with ischemic nonhealing wounds, allowing transplantation of highly vasculogenic peripheral blood stem cells from small amount of blood draw.

N2.01

CLINICAL TRIAL EVALUATING CHANGES IN THE WOUND MICROENVIRONMENT IN PATIENTS WITH VLUS TREATED WITH A HYPOTHERMICALLY STORED AMNIOTIC MEMBRANE
John P. McQuilling1, Judy Fulton2, Marissa J. Carter3, Keyur Patel4, Bryan Doner5, Laura Serena, LPN5, Thomas E. Serena5, Katie Mowry1
1Organogenesis Inc, Birmingham, AL, USA, 2SerenaGroup, Hingham, MA, USA, 3Strategic Solutions, Cody, WY, USA, 4Armstrong CM Hospital, The Snyder Institute, Kittaning, PA, USA, 5SerenaGroup Inc, Cambridge, MA, USA
Clinical trial evaluating changes in the wound microenvironment in patients with VLUs treated with a hypothermically stored amniotic membrane

John P. McQuilling, Judy Fulton, Marissa J. Carter, Keyur Patel, Bryan Doner, Laura Serena, Thomas E. Serena, Katie Mowry | Organogenesis Inc.; SerenaGroup

Background: There have been few studies evaluating the in situ effect of amniotic tissues on the wound microenvironment. Hypothermically stored amniotic membrane (HSAM) has been shown to contain growth factors, cytokines, ECM, and cells; in vitro it promotes proliferation and migration of fibroblasts and keratinocytes. This study evaluated VLUs treated with HSAM. Methods: In this prospective, single arm study, 15 female patients with venous leg ulcers present for 8 months to 35 years were treated with HSAM from male donors along with standard of care multilayer compression therapy for 12 weeks. Wound exudate was collected and evaluated using a proteomic microarray. Biopsies were collected from the wound bed one week after the first, second, and fourth HSAM applications. Results: At 4 weeks, 60% (9/15) of subjects achieved >/=50% wound size reduction; by 8 weeks this increased to 73% (11/15). At 12 weeks, 53% (8/15) achieved 100% re-epithelialization. 28% of biopsies were positive for male DNA (presumably from HSAM graft present in wound). Proteomic analysis revealed that wounds on a healing trajectory had significantly higher MMP-10 and significantly lower IL-1ra, IL-1a, IL-9, IL-2, MCP-1, and TNF-b compared to stalled wounds. Conclusions: This study provides a novel approach to investigating VLUs treated with advanced therapies, demonstrating significant correlations between wound exudate biomarkers and healing trajectory.

N2.02

A UNIQUE, DEFINITIVE AND DURABLE SOLUTION TO THE CHRONIC DIABETIC FOOT ULCER
Lewis Freed, Todd Haddon
OrthoArizona, Mesa, AZ, USA

N2.03

A SYSTEMS BIOLOGY APPROACH TO IMPROVED GROUPING, TIMING, AND STATISTICAL POWER IN THE WISH STUDY PATIENT POPULATION OF CHRONIC WOUNDS
Daniel J. Gibson, Debra Lynch Kelly, Susan Millan, Debra Lyon, Joyce K. Stechmiller
University of Florida, Gainesville, FL, USA
A systems biology approach to improved grouping, timing, and statistical power in the WISH study patient population of chronic wounds

Daniel J. Gibson, Debra Lynch Kelly, Susan Millan, Debra Lyon, Joyce K. Stechmiller | University of Florida, Gainesville, FL

Background: The most common basis for comparison in wound biomarker studies is healing versus non-healing. However, many wounds oscillate or stagnate, reflecting at least 3 true groups. Timing of sampling relative to wound healing outcome presents another potential pitfall. These issues have severe consequences for biomarker discovery and clinical translation. Methods: Underlying assumptions: 1) A wound’s trajectory is based on its tissue environment at the time of measurement. 2) All information has an expiration date. 3) Each measurement should be predictive for each patient, at each visit. All patient data were compiled on a per-visit basis. The change in clinical measurements from both the week before and week after the progress visit were calculated. Each visit was categorized as consistently healing, consistently worsening, or mixed. Results: 36 patients yielded 150 visits; half (n=78) were visits with before and after measurements. Changes in wound area: 53% healing (n=41), 31% worsening (n=24), 17% mixed (n=13). Changes in wound volume differed: 28% healing (n=22), 45% worsening (n=35), 27% mixed (n=21). Visits with consistent healing of both area and volume: 28% (n=22); consistent worsening: 27% (n=21); remaining 45% had improvement in only area or volume. Conclusions: A novel basis with 9 groups was identified. Nearly equal numbers of patients with both consistent healing/worsening before and after any given visit (n=22/21) were found. Biological samples taken at these visits can be compared without the contaminating effects of patients who are currently “turning the corner” for better or worse.

N2.04

INCIDENCE AND PREDICTOR VARIABLES OF PRESSURE INJURIES IN PATIENTS UNDERGOING VENTRICULAR ASSIST DEVICE AND TOTAL ARTIFICIAL HEART SURGERIES
Tod Brindle
Virginia Commonwealth University, Richmond, VA, USA
Incidence and predictor variables of pressure injuries in patients undergoing ventricular assist device and total artificial heart surgeries

Tod Brindle | Virginia Commonwealth University, Richmond, VA

Background: A growing subset of cardiac surgery includes patients with end-stage heart failure who undergo VAD or TAH surgery. The specific risk factors for pressure injury (PI) development remain unexplored in this population. Methods: A retrospective study of all VAD-TAH surgeries between 2010-2018 was performed at a large academic health system. PI incidence was evaluated by case incidence, patient incidence, and incidence density per 1000 patient days. Variables significant in bivariate analysis were entered into a stepwise logistic regression model. Results: The final sample included 292 independent VAD-TAH cases in 265 patients. 32 patients developed 45 PIs. PI incidence per all surgical cases was 11% (32/292) and per patient was 12% (32/265). Incidence density was 1% for 2010-2012, 1.2% for 2013-2015, and 1.1% for 2016-2018. Logistic regression identified significant predictors: age, mechanical ventilation time, and preoperative Braden Risk Assessment score. Despite long OR and total immobility times, mean time to PI was 23 days after admission and over 14 days after surgery, indicating a low rate of intraoperative and ICU-associated PI. Conclusions: PI incidence was much lower than anticipated given historical incidence in non-device cardiac surgery patients. A prospective study to further investigate significant risk factors and identify potential preventive mechanisms is warranted.

N2.05

EFFECTIVENESS OF AMNIOTIC MEMBRANE AND UMBILICAL CORD PARTICULATE FOR PRESSURE ULCERS: A RETROSPECTIVE STUDY
Anne T. Mancino, Alison Acott, Kathryn P. Brinegar
CAVHS, Little Rock, AR, USA
Effectiveness of amniotic membrane and umbilical cord particulate for pressure ulcers: A retrospective study

Anne T. Mancino, Alison Acott, Kathryn P. Brinegar | CAVHS, Little Rock, AR

Background: Pressure ulcers (PUs) are a prevalent clinical problem and only ~43% of PUs heal, the lowest wound healing rate among uncomplicated wounds. Amniotic membrane and umbilical cord (AMUC) contains anti-inflammatory, anti-scarring, and pro-regenerative properties. This study evaluated effectiveness of AMUC particulate for recalcitrant PUs. Methods: Medical records were retrospectively reviewed to identify patients that received injection of AMUC particulate for recalcitrant PUs between 2017 and 2018. Data collected included patient age, comorbidities, wound location, PU stage, previous therapy, and wound dimensions. Time to wound healing and incidence of wound healing were assessed at 12, 24, 36, and 52 weeks. Results: 26 PU wounds (85% stage IV, average volume 47.1 +/- 81.9 cm3) received AMUC. After treatment, 14 (54%) PUs achieved complete wound closure in a median time of 12.4 weeks (range 5-52). Complete wound closure was observed in 7 (27%) at 12 weeks, 10 (38%) at 24 weeks, 13 (50%) at 36 weeks, and 14 (54%) at 52 weeks. One patient required amputation due to vascular issues; no adverse events or complications related to treatment were observed. Conclusions: These preliminary results suggest that injection of AMUC particulate may be a safe and promising treatment for promoting wound closure of difficult-to-treat PUs.

N2.06

RISK FACTORS FOR MAJOR AMPUTATION IN HOSPITALIZED DIABETIC PATIENTS WITH FOREFOOT ULCERS
Seung-Kyu Han, Kyung-Chul Moon
Korea University Guro Hospital, Seoul, Korea, Republic of
Risk factors for major amputation in hospitalized diabetic patients with forefoot ulcers

Seung-Kyu Han, Kyung-Chul Moon | Korea University Guro Hospital, Seoul, Korea

Background: The forefoot is the most frequently involved region in diabetic foot ulcers. Large-scale cohort studies specifically discussing outcomes and characteristics of diabetic forefoot ulcers are not widely available. This study investigated the risk factors for major amputation in patients hospitalized with diabetic forefoot ulcers. Methods: Between January 2003 and December 2018, 1,032 diabetic patients with forefoot ulcers were included from a total of 1,792 diabetic patients admitted to the diabetic wound center. 983 patients (95%) healed without major amputations while 49 patients (5%) healed after major amputations. Data related to 88 potential risk factors including demographics, ulcer condition, vascularity, bioburden, neurology, and serology were collected. Results: Among 88 potential risk factors, 34 showed statistically significant differences between groups. In univariate analysis, 33 showed statistically significant differences. In stepwise multiple logistic regression analysis, four risk factors remained significant. Multivariate-adjusted odds ratios for gender, magnesium levels, platelet levels, and HbA1c levels were 8.216, 2.480, 1.009, and 0.570 respectively. Conclusions: Risk factors for major amputation in hospitalized patients with diabetic forefoot ulcers include male gender, increased magnesium levels, increased platelet levels, and low HbA1c levels.

N3.01

INHIBITION OF CYSTEINE PROTEASE CATHEPSIN K ACCELERATES WOUND HEALING IN A DIABETIC PORCINE MODEL
Anne Chenchar1, Mary Freeman1, Brenda M. Alexander1, Amanda E. Amanda E. Louiselle2, Stephen M. Niemiec2, Mark W. Azeltine Bannerman2, Zgheib Zgheib3, Kenneth Liechty2, Sreejayan Nair1
1University of Wyoming, Laramie, WY, USA, 2University of Colorado Denver School of Medicine and Children’s Hospital Colorado, Aurora, CO, USA, 3University of Colorado Denver School of Medicine and Children’s Hospital Colorado, Laramie, WY, USA
Inhibition of cysteine protease cathepsin K accelerates wound healing in a diabetic porcine model

Anne Chenchar, Mary Freeman, Brenda M. Alexander, Amanda E. Louiselle, et al. | University of Wyoming; University of Colorado Denver/Children’s Hospital Colorado

Background: Elevated protease activity has been implicated in impaired wound healing. Cathepsin K is upregulated in diabetic wounds compared to non-diabetic wounds. We hypothesize that inhibition of cathepsin K could enhance healing of diabetic wounds. Methods: Ten female Yorkshire pigs were rendered diabetic by single intravenous injection of streptozocin (75 mg/kg). Four weeks later, 10 full-thickness wounds (2.54 x 2.54 cm) were created on the back of diabetic and non-diabetic pigs. Wounds were injected intradermally with cathepsin K inhibitor-II, odanacatib (a small molecule cathepsin K inhibitor), or vehicle at two doses (high-dose 30 ng/mm2/wound and low-dose 10 ng/mm2/wound) on days 0, 3, 7, and 14. Animals were euthanized on day 21. Results: Percent of original wound area in vehicle-treated wounds at day 21 was 30.5 +/- 2.7. Treatment with cathepsin K inhibitor-II low-dose, odanacatib low-dose, and odanacatib high-dose accelerated wound healing dramatically: percent of original wound area was 6.5+/-0.6 (p=0.05), 7.1+/-1.3 (p=0.004), and 8.4+/-2.3 (p=0.01) respectively. Conclusions: Pharmacological inhibition of cathepsin K resulted in significant improvement in diabetic wound healing, suggesting that targeting cathepsin K may offer a therapeutic option for treatment of diabetic wounds.

N3.02

STRUCTURAL AND FUNCTIONAL EQUIVALENCY BETWEEN LYOPRESERVED AND CRYOPRESERVED CHORIONS WITH VIABLE CELLS
Vimal Jacob1, Nicholas Johnson1, Anne Lerch1, Brielle Jones1, Sandeep Dhall2, Malathi Sathyamoorthy1, Alla Danilkovitch1
1Smith & Nephew, Columbia, MD, USA, 2Smith & Nephew, Elkridge, MD, USA
Structural and functional equivalency between lyopreserved and cryopreserved chorions with viable cells

Vimal Jacob, Nicholas Johnson, Anne Lerch, Brielle Jones, Sandeep Dhall, Malathi Sathyamoorthy, Alla Danilkovitch | Smith and Nephew, Columbia, MD

Background: Clinical studies demonstrate that cryopreserved trophoblast-free chorion with viable cells results in high wound closure rates. A new lyopreservation method has been developed for chorion that also retains endogenous viable cells. This study compared lyopreserved chorionic membrane (VLCM) to cryopreserved chorionic membrane (VCCM) and investigated the immunogenicity of chorion. Methods: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and LPS challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by live/dead fluorescent staining. Anti-inflammatory and pro-angiogenic properties were evaluated. An in vivo rabbit abdominal adhesion model was used to evaluate anti-fibrotic properties. Results: Chorion was found to be non-immunogenic. Tissue architecture, growth factors, and cell viability of fresh chorion were maintained in both VLCM and VCCM. Anti-inflammatory and angiogenic properties were retained in VLCM. VLCM prevents formation of post-surgical adhesions in the rabbit abdominal adhesion model. Conclusions: Similar to VCCM, VLCM retains native components of fresh chorion including collagen-rich ECM, growth factors, and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, pro-angiogenic, and anti-fibrotic, supporting structural and functional equivalency between VLCM and VCCM.

N3.03

TOPICAL (R)-ND-336 ACCELERATES WOUND HEALING IN INFECTED DIABETIC MICE
Trung Nguyen, Zhihong Peng, William R. Wolter, Bowen Anderson, Valerie A. Schroeder, Mayland Chang
University of Notre Dame, Notre Dame, IN, USA

N3.04

ELUCIDATING THE EFFECT OF ETHYL PYRUVATE HYDROGEL AGAINST OXIDATIVE STRESS IN RADIATION DERMATITIS
Deepti Sharma, Lajpreet Kaur, Sanhita Saxena, Sukhvir Singh, Himanshu Ojha, Vinod Kaushik
Institute of Nuclear Medicine and Allied Sciences, Delhi, India
Elucidating the effect of ethyl pyruvate hydrogel against oxidative stress in radiation dermatitis

Deepti Sharma, Lajpreet Kaur, Sanhita Saxena, Sukhvir Singh, Himanshu Ojha, Vinod Kaushik | Institute of Nuclear Medicine and Allied Sciences, Delhi, India

Background: Radiation exposure leads to formation of reactive oxygen species (ROS) and macromolecular damage. Excess ROS production prolongs inflammation leading to chronic wound formation. Ethyl pyruvate (EP) is an antioxidant with anti-inflammatory properties. This study tested topical hydrogel of EP on radiation-exposed dorsal skin of Sprague Dawley rats. Methods: Topical hydrogels of various concentrations of EP (0.5%, 1%, and 2%) were prepared and their efficacy was tested on radiation-exposed dorsal skin of Sprague Dawley (SD) rats. 2% EP was found to have highest healing efficacy, healing the wound in 40 days. Further studies were done to determine the healing potential of 2% EP hydrogel. Results: EP reduces oxidative stress, speeding up the wound healing process. Histopathological analysis showed 2% EP hydrogel has better and faster recovery. Ellman’s reagent assay suggests thiol content was retained in the EP hydrogel-treated groups. Protein carbonylation assay showed similar results. MDA concentration was lower in the EP hydrogel-treated groups. SOD and catalase were higher in EP hydrogel-treated groups. Conclusions: EP hydrogel plays a major role in curbing oxidative stress in radiation induced wounds and accelerates healing mechanisms.

N3.05

COMPARISION OF LOCAL RECURRENCE BETWEEN NON-NPWT(NEGATIVE PRESSURE WOUND THERAPY) AND NPWT AFTER LOCALIZED MELANOMA SURGERY
KyoungAe Nam1, YeongJoo Oh1, ByungHo Oh2, MiRyung Roh3, KeeYang Chung2
1Yonsei University Health System Severance Hospital, Seoul, Korea, Republic Of, 2severance Hospital Yonsei University College Of Medicine, Seoul, Korea, Republic Of, 3gangnam Severance Hospital Yonsei University College Of Medicine, Seoul, Korea, Republic Of

N3.06

FATTY ACID POTASSIUM HAD BENEFICIAL BACTERICIDAL AND BIOFILMS REMOVAL EFFECTS WITH REDUCED CYTOTOXICITY OF FIBROBLASTS AND KERATINOCYTES
Sadanori Akita1, Takayoshi Kawahara2, Akihiro Masunaga3, Miki Takita3, Hayato Morita3, Tadayuki Tsukatani4, Kouji Nakazawa5, Daisuke Go5
1Fukuoka University, Fukuoka, Japan, 2Shabondama, KItakyushu, Japan, 3Shabondama, Kitakyushu, Japan, 4Fukuoka Industrial technology Center, Fukuoka, Japan, 5Kitakyushu Municipal University, Fukuoka, Japan
Fatty acid potassium had beneficial bactericidal and biofilms removal effects with reduced cytotoxicity of fibroblasts and keratinocytes

Sadanori Akita, Takayoshi Kawahara, Akihiro Masunaga, et al. | Fukuoka University; Shabondama, Kitakyushu; Fukuoka Industrial Technology Center; Kitakyushu Municipal University

Background: Wounds frequently become infected with pathogens. Highly effective but less adversely reactive agents are expected in wound cleansing. Potassium salts of fatty acids were tested for bactericidal and biofilm removal properties. Methods: Several potassium salts of fatty acids were tested for bactericidal activity against Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Clostridium difficile. Biofilm removal from S. aureus was compared to sodium lauryl ether sulfate (SLES) and sodium lauryl sulfate (SLS). Cytotoxicity was evaluated using WST assay in mouse fibroblasts (BALB/3T3 clone A31) and LDH leakage assay. Results: Potassium oleate (C18:1K) caused >4 log CFU/mL reductions in S. aureus and E. coli within 10 min and >2 log CFU/mL reduction in C. difficile within 1 min. C18:1K (90.3% removed) was significantly more effective at removing S. aureus biofilms than SLES (74.8%, p<0.01) and SLS (78.0%, p<0.05). Mouse fibroblasts in C18:1K showed higher viability than those in SLES or SLS (relative viability 102.8% vs. 30.1% and 18.1%; p<0.05). C18:1K was associated with significantly lower LDH leakage than SLES or SLS (108.9% vs. 720.6% and 523.4%; p<0.05). Conclusions: Potassium oleate demonstrated bactericidal effects, removed significantly greater amounts of S. aureus biofilm than SLES and SLS, and maintained fibroblast viability, potentially useful for wound cleaning and peri-wound skin treatment.

N4.01

QUANTITATIVE ASSESSMENT OF ANATOMICAL, PHYSIOLOGICAL AND MECHANICAL PARAMETERS OF CUTANEOUS WOUND HEALING AND SCARRING IN HUMAN SKIN
Sara Ud-Din1, Emma Barrett2, John Belcher2, Douglas McGeorge3, Ardeshir Bayat1
1University of Manchester, Manchester, United Kingdom, 2Manchester University NHS Foundation Trust, Manchester, United Kingdom, 3Grosvenor Nuffield Hospital, Chester, United Kingdom
Quantitative assessment of anatomical, physiological and mechanical parameters of cutaneous wound healing and scarring in human skin

Sara Ud-Din, Emma Barrett, John Belcher, Douglas McGeorge, Ardeshir Bayat | University of Manchester; Manchester University NHS Foundation Trust; Grosvenor Nuffield Hospital, Chester

Background: Objective quantitative assessment of cutaneous skin scarring in humans is essential for accurate diagnosis, standardization, prognosis, and validation of methodology. This study quantitatively investigated objective technologies for evaluation of skin scars created in 40 healthy human volunteers using a skin-biopsy model. Methods: Scar anatomical, mechanical, and physiological parameters were assessed over a weekly time period up to eight weeks (8W) using objective devices, quantifying haemoglobin, collagen, melanin (SIAscopy), erythema (colorimeter), blood-flow (FLPI and D-OCT), and skin thickness (HFUS). Results: SIAscopy demonstrated 108% increase in melanin from uninjured intact skin (UIS) to week 3 scar and a 32% decrease from week 3 to week 8. HFUS showed skin thickness increased 20% from UIS (1.4mm) to W8 (1.7mm). Collagen increased 129% from baseline to W8. Erythema showed 250% increase at W1 then 53% reduction to W8. FLPI showed blood flow peaked 286% at W1 and decreased 59% at W8. Viscoelasticity decreased from UIS (25Au) to W8 scar (8Au) by 68%. Conclusions: These findings show the utility of non-invasive objective devices in the quantitative evaluation of skin scarring parameters in human skin, enhancing understanding and potentially leading to objective classification of response to scar therapy.

N4.02

LIPOTRANSFER EFFECTIVELY REVERSES SKIN FIBROSIS
Michelle Griffin, Faith Jeon, Aurora Almadori, Christopher Denton, Peter Butler
University College London, London, United Kingdom
Lipotransfer effectively reverses skin fibrosis

Michelle Griffin, Faith Jeon, Aurora Almadori, Christopher Denton, Peter Butler | University College London

Background: Oro-facial fibrosis in systemic sclerosis (SSc) causes significant impairment in mouth function and quality of life. This study aimed to prospectively assess the use of lipotransfer to reverse orofacial fibrosis and restore facial volume in SSc patients. We also investigated the mechanism by which adipose derived stem cells (ADSCs) within lipotransfer may mediate anti-fibrotic effects. Methods: Prospective analysis of 88 female SSc patients undergoing lipotransfer was performed at minimum 12 months following surgery. Mouth function was assessed using the validated MHISS Scale and 3D-surface imaging. Following isolation of ADSCs and dermal fibroblasts from 6 female patients, in vitro co-culture assays were performed for 14 days. SSc-HDF proliferation, invasion, and migration were analyzed. Fibrosis pathway-specific qPCR array and ELISA for TGF-beta1 and CTGF were performed. Results: All patients reported aesthetic and functional improvement in orofacial fibrosis without complications. Significant improvement in post-operative MHISS scores demonstrated enhanced mouth function (p<0.05). Proliferation, migration, and invasive capacity of SSc-HDFs was significantly enhanced in co-culture with SSc-ADSCs than monocultures (p<0.05). Downregulation of fibrotic genes (PDGF, MEK, Erk) and secretion of CTGF and TGF-beta1 by SSc-HDFs was observed over 14 days in co-culture (p<0.05). Conclusions: Lipotransfer effectively reverses orofacial fibrosis in SSc and may mediate this through MEK-Erk pathways.

N4.03

RADIATION-INDUCED FIBROSIS IN PORCINE SKIN IMPROVES WITH TRANSDERMAL DEFEROXAMINE TREATMENT
Sandeep Adem, Mimi R. Borrelli, Nestor M. Diaz Deleon, Abra H. Shen, Kellen Chen, Chikage Noishiki, Geoffrey C. Gurtner, Michael T. Longaker, Derrick C. Wan
Department of Surgery, Division of Plastic Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
Radiation-induced fibrosis in porcine skin improves with transdermal deferoxamine treatment

Sandeep Adem, Mimi R. Borrelli, Nestor M. Diaz Deleon, Abra H. Shen, Kellen Chen, Chikage Noishiki, Geoffrey C. Gurtner, Michael T. Longaker, Derrick C. Wan | Stanford University School of Medicine

Background: Radiation therapy can cause adverse collateral effects to surrounding soft tissue. We tested a novel transdermal drug delivery system (TDDS) to provide cutaneous delivery of deferoxamine (DFO), an FDA-approved drug that stabilizes HIF-1-alpha, to irradiated porcine skin. Methods: Three red Duroc pigs were irradiated with 30 Gray delivered to five equal localized regions. After eight weeks of recovery, pigs were divided into three treatment conditions: 1% DFO TDDS patch, control TDDS patch with no medication, and no treatment. Biopsies were acquired at 1, 2, 3, 4, and 8 week time points. Histology evaluated dermal thickness, collagen deposition, and vascularity. Mechanical testing and cutometer readings assessed skin stiffness and deformation. Results: DFO TDDS-treated irradiated skin demonstrated significant reduction in dermal thickness, lower collagen content, and higher vascularity compared to no patch (***p<0.001, **p<0.01, *p<0.05) and control patch (*p<0.05, ****p<0.0001, **p<0.01). DFO-treated skin also exhibited decreased stiffness and higher deformation from cutometer data. Conclusions: DFO TDDS patch treatment provides a method to mitigate radiation-induced skin damage. Histological and mechanical characteristics of irradiated skin after DFO treatment indicate similarities to normal skin, establishing potential to treat patients with radiation-induced soft tissue fibrosis.

N4.04

EXOSOMES MEDIATE ANTI-FIBROTIC EFFECTS OF PLACENTAL MEMBRANES
Yong Mao1, Vimal Jacob2, Amit Singal1, Shunyao Lei1, Min Sung Park2, Mariana R.N. Lima1, Chaoyang Li2, Sandeep Dhall2, Malathi Sathyamoorthy2, Alla Danilkovitch2, Joachim Kohn1
1Rutgers University, Piscataway, NJ, USA, 2Smith & Nephew, Columbia, MD, USA
Exosomes mediate anti-fibrotic effects of placental membranes

Yong Mao, Vimal Jacob, Amit Singal, Shunyao Lei, Min Sung Park, et al. | Rutgers University; Smith and Nephew

Background: Placental membranes have anti-fibrotic activity. Recent studies have shown that exosomes function as conduits for intercellular transfer, containing all necessary components to induce resolution of fibrosis. We tested the hypothesis that anti-fibrotic activity of placental membranes is mediated by exosomes. Methods: Amnion and chorion-derived exosomes from conditioned media were isolated by sequential ultracentrifugation. Particle size and morphology were analyzed using Dynamic Light Scattering and TEM. Exosomal anti-fibrotic activity was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Proliferation, migration, and expression of fibrotic markers were analyzed. Results: Exosomes from amnion or chorion-conditioned media had an average size of 75nm with spherical shape. Exosomes significantly inhibited proliferation of TGF-beta1 activated LX-2, but had no effect on resting LX-2 cells. In scratch wound assay, exosomes reduced migration of LX-2. Exosomes reduced gene expression of pro-fibrotic COL1A1, ACTA, and TGF-beta1, and increased expression of anti-fibrotic HGF and IL-1beta in LX-2. Conclusions: Exosome effects on LX-2 suggest potential use in cell-free treatment of fibrotic diseases including keloid and hypertrophic scars. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic and chorionic tissues.

N4.05

STRETCH MARKS ARE ABUNDANT IN CD26-POSITIVE HUMAN DERMAL FIBROBLASTS AND EXHIBIT INCREASEDPROFIBROTIC MECHANOSENSITIVE SIGNALING
Mimi R. Borrelli, Michelle Griffin, Ledibabari M. Ngaage, Shamik Mascharak, Nicolette Lewis, Michael Januszyk, Derrick C. Wan, Michael T. Longaker, Hermann P. Lorenz
Stanford, Menlo Park, CA, USA
Stretch marks are abundant in CD26-positive human dermal fibroblasts and exhibit increased profibrotic mechanosensitive signaling

Mimi R. Borrelli, Michelle Griffin, Ledibabari M. Ngaage, Shamik Mascharak, Nicolette Lewis, Michael Januszyk, Derrick C. Wan, Michael T. Longaker, Hermann P. Lorenz | Stanford University

Background: Striae distensae (stretch marks) are common disfiguring cutaneous lesions. Despite their prevalence, the etiology remains elusive, hindering development of effective treatment strategies. We investigated key cellular-molecular characteristics distinguishing human dermal fibroblasts (HDF) in striae distensae versus normal skin. Methods: Striae distensae and normal skin samples were isolated from surgical specimens (n=15). Tensile strength, H&E, Trichrome, and Picrosirius Red staining were performed. HDF were isolated by flow cytometry for mRNA microarray analysis. Immunofluorescence staining and flow cytometry were used for confirmation of gene expression data. Results: Skin of striae distensae had reduced tensile strength, fewer rete ridges, epidermal atrophy, and more disorganized collagen fiber bundles in the reticular dermis. Microarray analysis revealed 296 upregulated genes in HDF from striae distensae compared to normal skin, including CD26. Gene ontology indicated upregulation of key profibrotic signaling pathways including focal adhesion, TGF-beta, and FAK-PI3-AKT signaling. 174 genes were downregulated, mainly involved in immune function pathways including CD27 and AMPK signaling. Conclusions: Fibroblasts from striae distensae exhibit increased pro-fibrotic and decreased anti-fibrotic signaling. Significant upregulation of CD26 and downregulation of CD74 highlight these surface markers as potential therapeutic targets.

N4.06

REACTIVE OXYGEN SPECIES AND APOPTOTIC PROTEINS IN IRRADIATED MURINE SKIN DECREASE WITH DEFEROXAMINE TREATMENT
Abra H. Shen, Nestor M. Diaz Deleon, Sandeep Adem, Mimi R. Borrelli, Ankit Salhotra, Harsh Shah, Geoffrey C. Gurtner, Michael T. Longaker, Derrick C. Wan
Department of Surgery, Division of Plastic Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
Reactive oxygen species and apoptotic proteins in irradiated murine skin decrease with deferoxamine treatment

Abra H. Shen, Nestor M. Diaz Deleon, Sandeep Adem, Mimi R. Borrelli, Ankit Salhotra, Harsh Shah, Geoffrey C. Gurtner, Michael T. Longaker, Derrick C. Wan | Stanford University School of Medicine

Background: Radiation can lead to acute and chronic wounds with pain, ulceration, and fibrosis. Deferoxamine (DFO) is an FDA-approved iron chelator that has been shown to increase skin vascularization by stabilizing HIF-1-alpha levels and improve radiation-induced fibrosis. However, it is unclear whether there are other mechanisms underlying DFO’s therapeutic effects. This study evaluates effects of transdermal DFO on reactive oxygen species (ROS) and cellular apoptosis in the context of irradiation. Methods: The study period consisted of a 14-day pre-IR period, followed by a 12-day course of fractionated IR delivering a total of 30 Gray. CD-1 nude immunodeficient mice were randomized to receive daily control patch (n=4) or DFO patch (n=4). Mouse scalp skin was harvested one day after completion of IR. Tissues were stained for iron and the ROS detector dihydroethidium (DHE). ELISA measured levels of Bax and Cleaved Caspase-3, markers of apoptosis. Results: DFO treatment was associated with decreased iron in the skin, confirming its role as an iron chelator. DHE fluorescence was more intense in the control group than in the DFO-treated group (***p<0.0001), indicating decreased ROS generation with DFO treatment. Apoptosis was higher in the control group as shown by higher Bax protein (*p=0.028) and Cleaved Caspase-3 (**p=0.0038) levels. Conclusions: Transdermal delivery of DFO decreases levels of ROS and apoptotic markers, providing further insight into mechanisms underlying its restorative effects on radiation-induced skin fibrosis.