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2022 Abstracts

I.01

Comparing Transcriptomes Of Paired Acute And Chronic Wounds
Harrison J. Shawa1, Alexander Merleev1, Alina Marusina1, Nicholas R. Hum2, Aimy Sebastian2, Gabriela G. Loots2, Sara Dahle3, Emanuel Maverakis1, Rivkah Isseroff1
1Dermatology, University of California, Davis, Las Vegas, NV, United States. 2Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, United States. 3Podiatry Section, VANCHCS, Mather, CA, United States.
Comparing Transcriptomes Of Paired Acute And Chronic Wounds

Harrison J. Shawa1, Alexander Merleev1, Alina Marusina1, Nicholas R. Hum2, Aimy Sebastian2, Gabriela G. Loots2, Sara Dahle3, Emanuel Maverakis1, Rivkah Isseroff1 1Dermatology, University of California, Davis, Las Vegas, NV, United States. 2Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, United States. 3Podiatry Section, VANCHCS, Mather, CA, United States

Chronic wounds remain a challenge for the United States healthcare system, yet the molecular pathogenesis of chronic, nonhealing wounds remains to be fully elucidated.

Here, we analyzed the transcriptomic signatures of paired acute and chronic wounds collected from the same patients, same site, and same time to accurately delineate the molecular differences between chronic, nonhealing wounds and acute wounds. Patients included 7 male individuals aged 55 to 69 years with chronic, nonhealing, full-thickness diabetic foot ulcers. Tissue was collected from the chronic wound edge at the time of amputation. 10 minutes thereafter, tissue was collected from the amputation wound edge (acute wound). RNA was extracted and next generation RNA-seq was performed on an Illumina NextSeq 500. In total, 1,698 differentially expressed genes were identified. Gene set enrichment analysis revealed significant differences cytokine-cytokine interaction (68/294 genes; p<10-13), arachidonic acid metabolism (16/63 genes; p<0.001), linoleic acid metabolism (10/29 genes; p<0.001), IL-17 signaling (20/93 genes; p<0.001), NF-kappa B signaling (20/95 genes; p<0.001), alpha-linolenic acid metabolism (9/25 genes; p<0.001), ether lipid metabolism (11/47 genes; p<0.001), TNF signaling (19/110 genes; p<0.005), and histidine metabolism (7/23 genes; p<0.005). Analysis of the most up- or down-regulated genes indicated differences in keratinocyte proliferation and differentiation, neutrophil and lymphocyte attraction, and extracellular remodeling in chronic wounds. Additionally, differences were detected in dopamine, epinephrine, norepinephrine, and serotonin metabolism and signaling. Analysis of the immune-cell landscape using CIBERSORT algorithm inferred increased numbers of M1 macrophages (9.4-fold; p=0.02), plasma cells (2.2-fold; p=0.02), resting mast cells (86-fold; p=0.003) and activated NK cells (259-fold; p=0.005), while increased numbers of M0 macrophages (2.5-fold; p=0.01), activated dendritic cells (9-fold; p=0.04) and mast cells (7.2-fold; p=0.006), and resting NK cells (9.7-fold; p=0.008) were detected in chronic wounds. Additionally, resting dendritic cells were present in 5 of 7 acute wounds and were absent in each chronic wound. This comprehensive, same-site, paired acute and chronic wound analysis indicates the presence of dysregulation of immune response, catecholamine signaling, epithelialization, and tissue remodeling in chronic wounds, providing specific targets to heal chronic wounds or prevent acute wounds from becoming chronic. NRH, AS and GGL were supported by the USDOE, LLNL (DE-AC52-07NA27344).

I.02

Healing Phase-Specific Signatures Of Wound Fibroblasts And Matrix Patterns Define Cancer-Associated Fibroblast Subtypes And Predict Cancer Outcome
Mateusz S. Wietecha1, David Lauenstein1, Sybille Seiler1, Juyoung Jin1, Andreas Goppelt2, Manfred Claassen3, Mitchell Levesque4, Reinhard Dummer4, Sabine Werner1
1Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland. 2Ottobock SE & Co. KGaA, Duderstadt, Germany. 3Department of Computer Science, Eberhard-Karls University, Tübingen, Germany. 4Department of Dermatology, University of Zurich, Zurich, Switzerland.
Healing Phase-Specific Signatures Of Wound Fibroblasts And Matrix Patterns Define Cancer-Associated Fibroblast Subtypes And Predict Cancer Outcome

Mateusz S. Wietecha1, David Lauenstein1, Sybille Seiler1, Juyoung Jin1, Andreas Goppelt2, Manfred Claassen3, Mitchell Levesque4, Reinhard Dummer4, Sabine Werner1 1Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland. 2Ottobock SE & Co. KGaA, Duderstadt, Germany. 3Department of Computer Science, Eberhard-Karls University, Tübingen, Germany. 4Department of Dermatology, University of Zurich, Zurich, Switzerland

Background: Cancer encapsulates the wound healing process becoming chronically dysregulated and has been described by Dr. Harold Dvorak as a “wound that fails to heal”. Despite the long-standing academic interest in the cellular and molecular parallels between healing and carcinogenesis, a comprehensive molecular comparison between wounds and tumors in vivo is still missing, and the specific roles of the wound healing phases have largely been overlooked. Therefore, we performed a comprehensive multi-omics comparison of wounds and tumors at the whole tissue and single cell levels. Methods: We first deconstructed the complex wound healing spectrum via a new peak detection bioinformatics package, yielding healing phase-specific gene expression signatures in mice and humans that we then used to enrich healing phases within thousands of samples across twelve cancer types based on The Cancer Genome Atlas (TCGA). We used machine learning to identify prognostic healing phase-predictive signatures followed by pathway enrichment analyses. We performed comprehensive meta-analyses of bulk and single cell transcriptomes of wound- and cancer-associated fibroblasts (CAFs) that we further integrated with published spatial transcriptomics datasets. To determine the translational relevance of our findings, we validated our bioinformatics data by high-throughput extracellular matrix (ECM) imaging of a primary melanoma tissue microarray (TMA). Results: The most phase-predictive and prognostic genes in the TCGA enriched in ECM-related pathways and pointed toward fibroblasts as key drivers of these signatures. Meta-analyses of wound fibroblasts and CAFs revealed healing phase-related CAF subtypes across several cancer types that expressed divergent ECM repertoires within spatially-defined niches. An “early-wound” CAF subtype localized to the inner tumor stroma, correlated with poor prognosis, and enriched for collagen-related ECM genes. In contrast, a “late-wound” CAF subtype localized to the outer tumor stroma, correlated with better prognosis, and enriched for elastin-related ECM genes. ECM imaging and analysis of the primary melanoma TMA linked healing-associated CAF subtypes and their divergent collagen- vs elastin-rich matrix signatures and architectures with patient survival and cancer recurrence. Conclusion: The results identify healing phase-specific genes and matrix patterns with prognostic potential in cancer, and pave the way for the development of innovative wound healing- and matrix-based diagnostic tools to predict and potentially influence cancer outcome.

I.03

Adipocytes Transition To Pro-Fibrotic Fibroblasts And Contribute To Muscle Fibrosis Following Nerve Injury
Amanda F. Spielman, Michelle Griffin, Nicholas Guardino, Heather E. desJardins-Park, Kristian E. Bauer-Rowe, Jason L. Guo, Jennifer Parker, Darren B. Abbas, Derrick Wan, Michael T. Longaker
Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University, Stanford, CA, United States.
Adipocytes Transition To Pro-Fibrotic Fibroblasts And Contribute To Muscle Fibrosis Following Nerve Injury

Amanda F. Spielman, Michelle Griffin, Nicholas Guardino, Heather E. desJardins-Park, Kristian E. Bauer-Rowe, Jason L. Guo, Jennifer Parker, Darren B. Abbas, Derrick Wan, Michael T. Longaker Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University, Stanford, CA, United States

Background: Recent studies have suggested that interconversion between adipocytes and fibroblasts (the cellular mediators of skin scarring) occurs in the wound environment. Muscle fibrosis occurs due to aging, muscle injury, and muscular dystrophies causing weakness and pain. The role of adipocytes in muscle fibrosis has not been fully explored. Our study examines whether mature adipocytes participate in muscle fibrosis following nerve injury via conversion into dermal fibroblasts. Methods: AdipoqCre transgenic driver mice were crossed to R26mTmG reporter mice to generate AdipoqCre;ROSA26mTmG mice, in which mature adipocytes (Adipoq/adiponectin-expressing cells) express green fluorescent protein (GFP), and all other cells express a red fluorescent protein (RFP). AdipoqCre;ROSA26mTmG mice underwent sciatic nerve injury per established protocol. Injured tibialis anterior and gluteus maximus muscle were harvested at postoperative day-14 (POD-14) and underwent immunofluorescent staining for fibroblast (collagen type IV/col-IV, alpha-smooth muscle actin/a-SMA) and adipocyte (perilipin) cell markers. To achieve local adipocyte ablation, AdipoqCre;ROSA26mTmG;R26tm1(HBEGF)Awai mice were generated and wounded, and diphtheria toxin (DT;400ng/mouse) was injected into the muscle at the nerve injury site. DT- and vehicle control-treated muscle underwent histologic analysis. Results: We identified a significantly greater number of adiponectin-lineage-positive cells (GFP+) within injured muscle at POD-14 compared to uninjured muscle (*P<0.05,n=5). The GFP+ cells that infiltrated the muscle injury exhibited upregulation of fibrotic markers including col-IV and α-SMA, and downregulation of adipocyte markers including perilipin, indicating conversion of adipocytes into pro-fibrotic fibroblasts. DT-induced ablation of Adipoq-lineage-positive cells resulted in reduced muscle scarring, with decreased collagen deposition compared to vehicle-control injured muscle (*P<0.05,n=5). Conclusion: Our study demonstrates that adipocytes undergo conversion to pro-fibrotic fibroblasts in response to nerve injury and contribute to muscle fibrosis. Targeted adipocyte ablation resulted in reduced muscle scarring, suggesting that adipocyte-derived fibroblasts are important contributors to muscle fibrosis.

I.04

Deferoxamine Improves The Wound Microenvironment And Improves Muscle Regeneration In A Mouse Model Of Pressure Ulcers
Nurul Jannah M. Nasir1, Johannes Heemskerk1, Peter T. So2, Lisa Tucker-Kellogg1
1Duke-NUS Medical School, Singapore, Singapore. 2Massachusetts Institute of Technology, Cambridge, MA, United States.
Deferoxamine Improves The Wound Microenvironment And Improves Muscle Regeneration In A Mouse Model Of Pressure Ulcers

Nurul Jannah M. Nasir1, Johannes Heemskerk1, Peter T. So2, Lisa Tucker-Kellogg1 1Duke-NUS Medical School, Singapore, Singapore. 2Massachusetts Institute of Technology, Cambridge, MA, United States

Introduction: Deferoxamine (DFO) is an FDA-approved iron-chelating drug that has been found to accelerate skin wound closure, but its effect on muscle and its mode of action are unclear. This study aims to investigate the therapeutic effect of DFO on the wound microenvironment and subsequent muscle regeneration following pressure ulcers in a transgenic mouse model. Methods: Multicolour (confetti) fluorescent protein expression was induced in muscle satellite stem cells and their progeny by crossing inducible Pax7cre and Brainbow2 transgenic C57BL6 mice. This construct causes newly regenerated muscle to express confetti fluorescent proteins while the pre-existing tissue remains dark. Pressure ulcers were created in the mice by applying a pair of 12 mm magnets to the dorsal skinfold and panniculus carnosus muscle, in two intervals of 12 hours. Mice with PU were subcutaneously injected with DFO in the treatment group and 0.9% saline in the control group (n=7 each). Ex vivo wound tissues were analysed at different time-points: 3, 10, 40 and 90 days after injury. Results: DFO treatment caused an eight-fold decrease in free iron (p < 0.01) compared to saline control, according to Perl's Prussian blue staining. At 3 days following injury, DFO treatment caused a two-fold decrease in lipid peroxidation (p < 0.001) and DNA oxidation (p < 0.05), and a five-fold decrease in extracellular traps (p < 0.001). At 10 days post-pressure injury, DFO-treated wound tissues had two-fold higher angiogenesis (p < 0.05) and two-fold greater infiltration of alternately activated macrophages (p < 0.01), than saline-treated tissues. At 40 days and even 90 days following PU, saline-treated muscle tissues failed to regenerate or were partially regenerated, with deformed and split fibers, while DFO-treated tissues exhibited larger volumes of regenerated muscle (p < 0.01), and fewer malformations in myofiber morphology (p < 0.0001). Conclusions: In summary, we show that DFO causes a decrease in oxidative stress, and promotes granulation and infiltration of pro-regenerative macrophages. Furthermore, DFO improved the extent and quality of muscle regeneration following pressure ulcers. Given that pressure ulcers are a huge healthcare burden with an unmet need for clinical interventions, our study advances DFO towards clinical trials for PU treatment.

I.05

Diabetic Dysfunctional Exosomes (Diaexosomes) Impair Resolution Of Human Wound Inflammation
Poornachander Guda1, Adam Anthony2, Anu Sharma1, Andrew Course2, Sashwati Roy1, David Clemmer2, Chandan K. Sen1, Subhadip Ghatak1
1Surgery, Indiana University, Indianapolis, IN, United States. 2Indiana University, Bloomington, IN, United States.
Diabetic Dysfunctional Exosomes (Diaexosomes) Impair Resolution Of Human Wound Inflammation

Poornachander Guda1, Adam Anthony2, Anu Sharma1, Andrew Course2, Sashwati Roy1, David Clemmer2, Chandan K. Sen1, Subhadip Ghatak1 1Surgery, Indiana University, Indianapolis, IN, United States. 2Indiana University, Bloomington, IN, United States

Background: Paracrine effects via exosome emerged as the primary mechanisms of cell-cell crosstalk at the wound-site. Resolution of wound inflammation relies on successful crosstalk between keratinocytes that must re-epithelialize, and the blood borne wound-site macrophage, which must mount and timely resolve inflammation. We tested the hypothesis that wound fluid diaexosomes impair resolution of wound inflammation. Methods: Of the different cell types that are present in wound-edge, keratinocytes originate from ectoderm and are uniquely characterized by presence of intermediate filament keratins. Two independent databases (Vesiclepedia and Exocarta) identified type I keratin 14 (Krt14) as unique to exosomes originating from keratinocytes. Using magnetic Krt14 immunoprecipitation method, we successfully separated exosomes of keratinocyte origin from a heterogenous pool of extracellular vesicles (EV) in human wound fluid of 17 non-diabetic healing and 32 diabetic patients. Diaexosomes were characterized and reported in EV-track. The multiplexing direct stochastic optical reconstruction microscopy (dSTORM) technology (resolution 20nm) were used to quantitatively analyze the abundance of miRNA cargo in hExoκ and surface modification at single exosome resolution. Results: Human keratinocyte-derived exosomes (hExoκ) were isolated from wound fluid and characterized as per EV-track guidelines. The size of hExoκ ranged from 75-105 nm. Super resolution dSTORM microscopy demonstrated abundance of Krt14 partner Krt5 in hExoκ. The hExoκ compromises of 10% of total exosome pool and 1% of the total EV present in wound fluid normalized with albumin. Compared to non-diabetic subjects, there was a significant reduction of hExoκ in wound fluid of diabetic subjects. Co-immunofluorescence staining of heterogeneous ribonucleoprotein (hnRNP) A2B1 and Q, which are critical for miRNA packaging in hExoκ showed significant low abundance in wound-edge keratinocytes of diabetic subjects. dSTORM microscopy using molecular beacons with Cy5 reporter and BHQ3 quencher demonstrated abundance of pre-miR-21 in hExoκ that were positively correlated with wound closure. Unlike hExoκ diaexosomes of diabetic subjects, addition of hExoκ isolated from non-diabetic subjects to macrophages resulted in conversion to a pro-resolution phenotype. Conclusion: This work provides a novel insight into the pathophysiological mechanism of diaexosomes of chronic wound patients. Hyperglycemia dysregulates exosome packaging, release, and surface modification of keratinocyte originating diaexosomes. These perturbations compromise crosstalk between keratinocyte and wound macrophages complicating resolution of wound inflammation.

I.06

Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing
Kellen Chen, Michelle Griffin, Dominic Henn, Clark A Bonham, Katharina Fischer, Jagannath Padmanabhan, Artem A. Trotsyuk, Dharshan Sivaraj, Melissa C. Leeolou, Hudson C. Kussie, Savana L. Huskins, Sydney Steele, David Perrault, Michael T. Longaker, Geoffrey C. Gurtner
Stanford University, Stanford, CA, United States
Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing

Kellen Chen, Michelle Griffin, Dominic Henn, Clark A Bonham, Katharina Fischer, Jagannath Padmanabhan, Artem A. Trotsyuk, Dharshan Sivaraj, Melissa C. Leeolou, Hudson C. Kussie, Savana L. Huskins, Sydney Steele, David Perrault, Michael T. Longaker, Geoffrey C. Gurtner Stanford University, Stanford, CA, United States

Background: Repair after tissue injury involves a dynamic interplay among not just tissue resident cells (e.g., fibroblasts), but also cells recruited from the circulation. Myeloid cells, such as monocytes and macrophages, are derived from hematopoietic precursors and migrate to sites of injury where they play a role in modulating all stages of wound healing and scar formation. There is mounting evidence that mechanical stimuli are also able to modulate monocyte and macrophage response during tissue healing, but the exact mechanisms behind this “mechano-immunomodulation” remain incompletely understood. Methods: We attached a mechanical strain device to the mouse dorsum to initiate a uniform and consistent strain profile across an incisional wound to create hypertrophic scar (HTS) formation in mice. To investigate mechano-responsive immune cells, we performed parabiosis of wildtype (WT) and GFP+ mice, allowed the mice to develop a shared blood circulation, initiated HTS formation in the WT mouse, and analyzed the cells using single cell RNA sequencing (scRNA-seq), fluorescent-activated cell sorting (FACS), and immunofluorescent staining. Results: Mechanical modulation significantly upregulated the presence of inflammatory subtypes within the healing tissue, characterized by an increase in infiltrating GFP+ cells from 5.4% to 12.2%. In the GFP+ circulating cells, mechanical strain directly increased the proportion of fibrotic myeloid cells, primarily defined by the monocyte marker Ly6c2 as well as the TGFB responsive and macrophage activating gene Thbs1. Mechanical strain also increased the proportion of inflammatory myeloid cell populations, defined by Ccl and Il6 chemoattractants, and Cd74+ migratory myeloid cells. Utilizing both a pharmacological blocker of focal adhesion kinase (FAK) as well as a myeloid specific FAK knockout (KO), we demonstrated that modulating mechanical signaling abrogated those responses and instead promoted homeostatic myeloid transcriptional fates. Conclusions: Tissue injury activates a cascade of signaling pathways to recruit and orchestrate various cell types during healing. Our study indicates that modulating mechanical stress directly affects myeloid cell phenotypes and interactions with other cell types in the complicated, multicellular milieu of wound healing. This principle has been previously unexplored in the context of fibrosis and regeneration, with most previous studies focused on fibroblast heterogeneity and transcriptional profiles. To our knowledge, this is the first study to directly investigate the effects of modulating mechanotransduction on immune cell response at the single cell level utilizing parabiosis and wound healing. Collectively, we demonstrate that mechano-immunomodulation of the “early responders” of healing can trigger a cascade of downstream regenerative healing.

I.07

Overcoming Radiation Induced Oral Fibrosis Through The Down Regulation Of WNT Signaling Using BMP-7 Inhibitors
Nicholas Guardino, Michelle Griffin, Amanda F. Spielman, Darren B. Abbas, Megan King, Jennifer Parker, Derrick Wan, Michael T. Longaker
Surgery, Stanford, Menlo Park, CA, United States.
Overcoming Radiation Induced Oral Fibrosis Through The Down Regulation Of WNT Signaling Using BMP-7 Inhibitors

Nicholas Guardino, Michelle Griffin, Amanda F. Spielman, Darren B. Abbas, Megan King, Jennifer Parker, Derrick Wan, Michael T. Longaker Surgery, Stanford, Menlo Park, CA, United States

Background: Oral scarring is a common debilitating side effect of head and neck radiation therapy. With no effective treatment patients suffer long-term pain, taste dysfunction, and infection from radiation induced oral fibrosis. Previous work has suggested that Wnt signaling may contribute to radiation scaring in the oral mucosa. We have developed a preclinical in vivo murine model to study the effects of radiation damage on the oral mucosa. Our study evaluates the effects of Wnt pathway inhibitors to reduce scarring in the oral mucosa following irradiation. Methods: Adult C57Bl/6 mice (6–8-week-old) (n = 6) were randomized to one of three conditions: 1. Treated with 30 Gy external beam irradiation to the oral mucosa delivered as six fractionated doses of 5Gy over a period of 12 days (irradiation group), 2. Irradiation to the oral mucosa with simultaneous treatment with a Bone Morphogenetic Protein 7 (BMP-7) inhibitor (BMP-7) (treatment group), and 3. No irradiation and no treatment (control group). Mice were euthanized at 4 weeks post irradiation for histological and flow cytometry analysis. Oral sections underwent immunofluorescent staining for fibrosis (e.g., collagen type I/col-I, alpha smooth muscle actin/a-SMA), vascularization (CD31 staining), inflammation (F4/80 antigen), and Wnt signaling (e.g., axin2, Wnt1) markers and were imaged on a confocal microscope. Results: Histological analysis by H&E staining confirmed dermal thickening and increased collagen content in the irradiated group compared to the control group (p < 0.05), confirming the reproducibility of the murine model to induce oral fibrosis. BMP-7 inhibitor treatment decreased the dermal and epidermal thickening observed in the irradiated group (p < 0.05). The treatment group also demonstrated reduced dermal fibrosis with decreased col-I and a-SMA staining compared to the irradiation group (p < 0.05). Flow cytometry analysis further showed reduced numbers of profibrotic fibroblasts (CD26+) in the treatment compared to the irradiation group (p < 0.05). Immunofluorescence staining confirmed significantly high levels of Wnt pathway protein signaling (Axin2, Wnt1) in the irradiation group compared to the treatment and control group (p < 0.05). Mice receiving the BMP-7 inhibitor also demonstrated improved vascularization as shown by CD31 staining and a decreased inflammatory infiltrate evidenced by F4/80 macrophage staining (p < 0.05). Conclusions: Our novel murine model suggests that canonical Wnt signaling may be responsible for oral scarring post irradiation. Targeting Wnt signaling through BMP-7 inhibitors may be an effective method to reduce the burden of radiation induced oral fibrosis.

I.08

A Double-Blind Randomised Placebo-Controlled Clinical Trial Demonstrates That Pre-Emptive Priming Of Human Skin With Topical Epigallocatechin-3-Gallate Improves Cutaneous Skin Scarring
Sara Ud-Din1, Traci A. Wilgus2, Douglas McGeorge3, Ardeshir Bayat1
1The University of Manchester, Manchester, United Kingdom. 2The University of Ohio, Columbus, OH, United States. 3Grosvenor Nuffield Hospital, Chester, United Kingdom
A Double-Blind Randomised Placebo-Controlled Clinical Trial Demonstrates That Pre-Emptive Priming Of Human Skin With Topical Epigallocatechin-3-Gallate Improves Cutaneous Skin Scarring

Sara Ud-Din1, Traci A. Wilgus2, Douglas McGeorge3, Ardeshir Bayat1 1The University of Manchester, Manchester, United Kingdom. 2The University of Ohio, Columbus, OH, United States. 3Grosvenor Nuffield Hospital, Chester, United Kingdom

Epigallocatechin-3-gallate (EGCG) enhances cutaneous wound healing due to its antiangiogenic, anti-inflammatory, and antioxidant properties. We previously demonstrated the role of EGCG in scarring in ex vivo human scar models and in a double-blind randomised placebo-controlled clinical trial which showed that immediate and delayed application of this anti-scarring topical improved skin scarring by reducing scar thickness and mast cell count and increasing hydration and elasticity. The concept of pre-emptive priming of skin pre-injury offers a novel approach in optimizing cutaneous scarring outcome. The objective was to deliver an active compound at the optimal time in order to maximize its impact and improve cutaneous scarring. Therefore, pre-emptive application of anti-scarring topical, EGCG, pre-injury compared with post-injury can potentially be superior on scarring outcome. Thus, we conducted another double-blind randomised placebo-controlled trial comparing the effects of pre-emptive priming of skin with EGCG topical pre-injury versus post-injury. Healthy volunteers (n = 40) were split into 4-groups; each undergoing different modes of application versus placebo: Group-1 = priming (7Days) pre-injury, Group-2 = priming (3D) pre-injury, Group-3 = immediate (0D) day-of-injury, Group-4 = delayed application (14D) post-injury. Excisional skin-biopsies in upper-arms were evaluated weekly with multiple non-invasive objective quantitative devices over 8-weeks. Histological, immunohistochemical, mRNA sequencing and QRT-PCR studies were performed on tissue-biopsies. EGCG reduced mast cells at weeks-4 and 8 by gene and protein analyses (p < 0.01). Group 1 was superior to other groups (p < 0.01) in both clinical (blood flow) and laboratory parameters (elastin and immune marker expression). Additionally, there was down-regulation of angiogenic-markers by mRNA-sequencing and of CD31 and VEGF-A at weeks-4 and 8 (p < 0.01) by immunohistochemistry and at week-4 (p < 0.05) by QRT-PCR. EGCG increased antioxidant levels (HO-1) at week-4 (p < 0.01). Immunohistochemical analysis of elastin was up-regulated at week-8 in EGCG-treated samples compared to placebo samples in all groups (p < 0.01) The greatest differences between groups were in group-1 (p < 0.01). This was also supported by clinical elastin measurements in group-1 at week-8 (p = 0.029). In conclusion, pre-emptive priming of skin pre-injury has significant beneficial effects on skin scarring shown by reducing mast cells, blood flow and angiogenesis plus increasing elastin content.

I.09

Macrophage Acute Contact Induces Fibroblast Intracellular Stress In A Calcium- And Gap-Junction-Dependent Process
Maya Ezzo1,2, Jun Bo Wang1, Gilbert Pecoraro1, Joao Firmino1, Pardis Pakshir1, Boris Hinz1,2
1Laboratory of Tissue Repair and Regeneration, Faculty of Dentistry, University of Toronto, Toronto, Canada, 2Laborartoy of Tissue Repair and Regeneration, Keenan Research Centre for Biomedical Science of the St. Michael’s Hospital, Toronto, Ontario, Canada
Macrophage Acute Contact Induces Fibroblast Intracellular Stress In A Calcium- And Gap-Junction-Dependent Process

Maya Ezzo1,2, Jun Bo Wang1, Gilbert Pecoraro1, Joao Firmino1, Pardis Pakshir1, Boris Hinz1,2 1Laboratory of Tissue Repair and Regeneration, Faculty of Dentistry, University of Toronto, Toronto, Canada. 2Laborartoy of Tissue Repair and Regeneration, Keenan Research Centre for Biomedical Science of the St. Michael’s Hospital, Toronto, Ontario, Canada

Rationale: Fibrosis (organ scarring) is responsible for >40% disease-related deaths worldwide. Key effector and target cells in fibrosis are fibroblasts activated to myofibroblasts to produce and contract collagen into detrimental scar tissue. We published fibroblasts make contact with mechanically attracted macrophages (Mϕ) and found that first contact with Mϕ results in fibroblast contractile events. However, the mechanisms of contact-induced acute fibroblast contraction are unknown. Hypothesis: Touching Mϕ initiate fibroblast contraction and acute intracellular stress by inducing an increase in intracellular calcium levels. Objective: Quantify fibroblast contractions and calcium transients before, during, and after contact with Mϕ, with and without connexin-43 (Cx43) formation. Methods: The change in fibroblast stress was quantified before, during, and after contact formation with Mϕ by measuring nuclear translocation of mechanosensitive transcription factor YAP. In addition, changes in fibroblast contractions were computed from the displacement of embedded fluorescent beads on a collagen matrix. Fluorescent reporters were used to detect contact-induced cytosolic calcium changes as fast indicator of contractile response. As one possible source of calcium, we inhibited Cx43 gap junctions formed between Mϕ and fibroblasts through siRNA knockdown of Cx43. Gap junctions allow intercellular passage of small molecules such as tracer molecule calcein-AM. Both, calcium dyes and gap-junction permeable tracers were assessed by immunofluorescence and flow cytometry. Results: Fibroblasts in short contact with Mϕ exhibited 1.5-fold higher nuclear levels of YAP and 1.7-fold increased contraction speed compared to control fibroblasts. Increased fibroblast stress upon Mϕ contact correlated with a change in frequency and 1.6-fold enhanced intensity of cytosolic calcium transients. Fibroblasts and Mϕ both expressed Cx43 and exchanged calcein-AM indicating formation of hetero-cellular gap junctions that in principle – allow passage of calcium. Consequently, knockdown of Cx43 in co-cultures of fibroblasts and Mϕ affected the regularity and frequency of fibroblasts calcium increases. Conclusion: Our results show Mϕ contact formation enhances fibroblast cytoskeletal stress and contractile activities in a calcium- and Cx43 channel-dependent process. We propose the molecular components that mediate Mϕ-fibroblast interaction as potential targets for anti-fibrosis strategies

K1.01

Allometric Tissue-Scale Forces Activate Mechanoresponsive Immune Cells To Drive Pathological Foreign Body Response To Biomedical Implants
Jagannath Padmanabhan1, Kellen Chen1, Dharshan Sivaraj1, Britta Kuehlmann1, Clark Bonham1, Teruyuki Dohi1, Dominic Henn1, Zachary Stern-Buchbinder1, Peter Than1, Hadi Hosseini2, Janos Barrera1, Hudson Kussie1, Noah Magbual1, Mimi Borrelli1, Artem A. Trotsyuk1, Sun Hyung Kwon1, James Dunn2, Zeshaan Maan1, Michael Januszyk1, Lukas Prantl3, Geoffrey C. Gurtner1
1Division of Plastic Surgery, Department of Surgery, Stanford University, Stanford, CA, United States 2Division of Pediatric Surgery, Department of Surgery, Stanford University, Stanford, CA, United States 3Department of Plastic and Reconstructive Surgery, University Hospital Regensburg, Regensburg, CA, United States
Allometric Tissue-Scale Forces Activate Mechanoresponsive Immune Cells To Drive Pathological Foreign Body Response To Biomedical Implants

Jagannath Padmanabhan1, Kellen Chen1, Dharshan Sivaraj1, Britta Kuehlmann1, Clark Bonham1, Teruyuki Dohi1, Dominic Henn1, Zachary Stern-Buchbinder1, Peter Than1, Hadi Hosseini2, Janos Barrera1, Hudson Kussie1, Noah Magbual1, Mimi Borrelli1, Artem A. Trotsyuk1, Sun Hyung Kwon1, James Dunn2, Zeshaan Maan1, Michael Januszyk1, Lukas Prantl3, Geoffrey C. Gurtner1 1Division of Plastic Surgery, Department of Surgery, Stanford University, Stanford, CA, United States. 2Division of Pediatric Surgery, Department of Surgery, Stanford University, Stanford, CA, United States. 3Department of Plastic and Reconstructive Surgery, University Hospital Regensburg, Regensburg, CA, United States

Background: The longevity of biomedical implants is limited by pathological foreign body response (FBR), leading to implant failure. Nearly 90% of all implant failures are associated with FBR and up to 30% of all implantable devices will undergo failure during their lifetime. Despite the prevalence of FBR-mediated implant failure, the underlying mechanisms are not well understood. Our incomplete understanding of the FBR is exacerbated by the inability of standard laboratory models to recapitulate the highly fibrotic response associated with implant failure in humans. Allometric scaling principles govern inter-species differences in biological properties resulting from changes in the size of the organism and predict that tissue-scale forces increase exponentially as the body size increases. The impact of these allometric tissue-scale forces on the activation of mechanical signaling and its consequences for overall FBR has not been investigated. Methods: We analyzed a unique, large volume human tissue library consisting of breast implant FBR capsules with different degrees of fibrosis. We analyzed the human tissue specimens using a next generation sequencing-based quantitative assay against 2500+ known biomarkers for inflammation and fibrosis. Further, we modeled the local mechanical stress patterns resulting from tissue-scale forces at the implant-tissue interface in mice and humans using finite element modeling. Next, we developed a mechanically stimulated implant (MSI) model to recapitulate human-like tissue-scale forces in mice. We compared the fibrous capsules formed around standard implants and MSIs in mice using histopathology and single cell sequencing, and compared the results with human implant capsules. Finally, we blocked mechanical signaling in the human-like MSI mouse model and analyzed the resultant FBR using histopathology and immunostaining. Results: We first demonstrated that pathological (highly fibrotic) FBR in humans is mediated by immune cell-specific Rac2 mechanotransduction signaling, independent of implant chemistry or mechanical properties. We then showed that mice, which are typically poor models of human FBR, can be made to induce a strikingly human-like pathological FBR by altering tissue forces. Altering tissue forces alone activates Rac2 signaling in a unique subpopulation of immune cells and results in a human-like pathological FBR at the molecular, cellular, and local tissue levels. Finally, we demonstrated that blocking Rac2 signaling negates the effect of increased tissue forces, dramatically reducing FBR. Conclusions: For decades, it has been assumed that the foreign body response to biomedical implants is primarily a reaction to the chemical and mechanical properties of the implant. Here, we show for the first time that a third independent variable, allometric tissue-scale forces (which increase exponentially with body size), can activate mechanoresponsive immune cells and drive the biology of FBR.

K1.02

Mechanical Regulation Of Provisional Matrix Assembly In 3D Fibrous Microtissues
Jeroen Eyckmans1, Shoshana Das1, Kazunori Shimizu2, Christopher Chen1
1Biomedical Engineering, Boston University, Boston, MA, United States 2Nagoya University, Nagoya, Japan
Mechanical Regulation Of Provisional Matrix Assembly In 3D Fibrous Microtissues

Jeroen Eyckmans1, Shoshana Das1, Kazunori Shimizu2, Christopher Chen1 1Biomedical Engineering, Boston University, Boston, MA, United States. 2Nagoya University, Nagoya, Japan

Background: During tissue repair, fibroblasts lay down a provisional matrix which serves as a migration base for other cells to close the wound and restore tissue architecture. Although the field has come to appreciate that mechanical forces modulate tissue repair, how tissue mechanics regulate provisional matrix assembly by fibroblasts is poorly understood. To address this problem, we’ve developed a biomimetic wound-on-chip model wherein fibroblasts ensconced in collagenous matrices close open wounds by assembling a fibronectin-rich provisional matrix and demonstrate that this process is dependent on cellular contractility and extracellular matrix (ECM) alignment of the surrounding tissue. Methods: A cell suspension of NIH3T3 fibroblasts in collagen type I matrix was seeded in microfabricated polydimethylsiloxane (PDMS) arrays of microwells containing 4 or 8 standing micropillar structures (diameter 100μm, height is 250μm). After tissue compaction around the pillars, microtissues were wounded with a microdissection knife and the tissue’s reaction was monitored with time lapse microscopy. To investigate the contribution of fibronectin to the closure process, fluorescently labeled fibronectin was added to the culture medium, or experiments were conducted with fibronectin null cells or under serum free conditions. Actomyosin inhibitors were used to study the role of cell contractility on provisional matrix assembly. To investigate the effect of ECM alignment on provisional matrix assembly, wound closure and provisional matrix assembly was measured in injured rectangular (aligned ECM) versus octagonal (isotropic ECM) microtissues. Results: Within 24 to 48 hours, 3T3 fibroblasts closed full-thickness wounds in microtissues by assembling a provisional matrix that bridged the gap. Pulse-chase experiments suggested that the provisional template was made of both cellular fibronectin and plasma fibronectin. Tissue closure was abrogated in fibronectin null fibroblasts, but not in microtissues grown under serum free conditions, suggesting that newly synthesized cellular fibronectin was critical for provisional matrix assembly. In presence of contractility inhibitors, the rate of provisional matrix assembly to close the wound was reduced. Intriguingly, the rate of provisional matrix assembly correlated with local ECM alignment of the tissue adjacent to the wound. As such, wounds in octagonal tissues closed faster compared to wounds in rectangular microtissues. Conclusion: Together, these data suggest that provisional matrix assembly is controlled by tissue contractility and alignment of the ECM, which constitutes the mechanical boundary conditions of our model system. A better understanding of how mechanical boundary conditions regulate de novo tissue formation may inform new strategies to promote tissue repair and regeneration.

K1.03

Mechanical Signaling Mediated by IQGAP1 Promotes Pathologic Foreign Body Response
Dharshan Sivaraj, Jagannath Padmanabhan, Kellen Chen, Dominic Henn, Hudson C. Kussie, Melissa C. Leeolou, Artem A. Trotsyuk, Katharina Fischer, David Perrault, Geoffrey C. Gurtner
Surgery, Stanford University, Stanford, CA, United States
Mechanical Signaling Mediated by IQGAP1 Promotes Pathologic Foreign Body Response

Dharshan Sivaraj, Jagannath Padmanabhan, Kellen Chen, Dominic Henn, Hudson C. Kussie, Melissa C. Leeolou, Artem A. Trotsyuk, Katharina Fischer, David Perrault, Geoffrey C. Gurtner Surgery, Stanford University, Stanford, CA, United States

Background: Foreign body response (FBR) begins as a wound healing reaction to tissue trauma created from the insertion of a foreign material, such as a biomedical implant. The continued presence of these implants within the body exacerbates this response, promoting the formation of a fibrous capsule around the implant. Over time, this process often leads to implant failure and patient morbidity. The identification of rational therapeutics that inhibit the effects of mechanical stimulation on a cellular level, and therefore reduce the pathologic inflammation present around biomedical implants, represents a promising area of investigation. In this study, we utilized a genomics and proteomics based approach to analyze both murine and human FBR tissue. Our goal was to identify a molecular mediator of mechanical signaling pathways that may ultimately lead to the design of novel precision therapeutics for mitigating pathologic FBR. Methods: FBR capsules from explanted human biomedical devices, murine FBR capsules, and control human and murine subcutaneous tissue were analyzed using mass spectrometry. Pathway analysis of the top upregulated proteins in human and murine FBR tissue was generated via STRING Functional Enrichment Analysis. To investigate FBR in mice, we employed a murine model of mechanically stimulated implants (MSIs). Finally, we performed single cell RNA sequencing (scRNA seq) of cells isolated from murine FBR capsules and further assessed the FBR capsules using histological and immunohistochemical staining. Results: Our STRING pathway analysis revealed IQ motif containing GTPase Activating Protein (IQGAP1) as being a central meditator common to several of the top upregulated pathways in both human and murine FBR. In our mouse model of FBR, we found that IQGAP1 deficient mice displayed a significantly reduced FBR as evidenced by thinner capsules, lower levels of collagen deposition, collagen maturity, and myofibroblast activation. Our scRNA seq analysis revealed that IQGAP1 deficiency resulted in diminished transcription of mechanotransduction (Rac1), inflammation (Ccl3, Ccl4, Il1b), and fibrosis related (Acta2, Cxcl2, Cxcl5, Ptx3) genes, which was confirmed on the protein level with immunofluorescent staining. Conclusion: Collectively, our results demonstrate that targeting mechanical signaling at the implant tissue interface may improve the biocompatibility of biomedical implants, with broad implications for clinical application. We investigated a potential molecular target involved in pathologic FBR around biomedical implants and showed that genetic deficiency of IQGAP1 mitigates mechanically stimulated FBR by diminishing downstream mechanical, inflammatory, and fibrotic pathways. Therefore, IQGAP1 may be a promising molecular target for rational therapeutic design to mitigate pathologic FBR.

K1.04

Wound Stratification Elucidates Novel Genomic Persepctive Of Negative Pressure Therapy In A Porcine Model
Jacob Hodge1, David Zamierowski2, Jennifer Robinson3, Adam Mellott2
1Bioengineering Graduate Program, University of Kansas, Lawrence, KS, United States 2Plastic Surgery, University of Kansas Medical Center, Kansas City, KS, United States 3Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS, United States
Wound Stratification Elucidates Novel Genomic Persepctive Of Negative Pressure Therapy In A Porcine Model

Jacob Hodge1, David Zamierowski2, Jennifer Robinson3, Adam Mellott2 1Bioengineering Graduate Program, University of Kansas, Lawrence, KS, United States. 2Plastic Surgery, University of Kansas Medical Center, Kansas City, KS, United States. 3Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS, United States

Statement of Purpose: Negative Pressure Wound Therapy (NPWT) has been shown to modulate the behavior of wound tissue. Yet, the dynamic mechanism(s) of how NPWT exerts its effects at a molecular and cellular level remains unresolved; specifically, the key early signaling pathways initiated and the roles of different cellular populations upon exposure to negative pressure and different dressing materials. To date, wound analyses have only evaluated gene expressional profiles of whole wound isolates. In this study we provide a novel perspective that compares the expressional profile of “Full” wounds relative to each independent layer of wounded skin (epidermis vs dermis vs subcutaneous). Additionally, we investigate how different dressing materials augment the early wound environment in the setting of NPWT. Methodology: An acute incisional porcine wound model was developed by inflicting 2cm (long) x 2cm (deep) incisions on the flanks of two female Yucatan pigs (n=2). Four different wound dressings were applied within the incisional wounds, in addition to one control group. Dressing materials included Owen’s Rayon™, Granufoam™, Polycaprolactone (PCL) Mesh, and Fibrin Glue. Wounds were then treated with or without NPWT and evaluated over 8 hours. Tissue samples were collected at 0-, 2-, 4-, and 8-hours post-wounding and processed for RNA sequencing. Specimens, transverse to the incision, were bisected along the incision, with one side representing “Full” wound samples and the opposing side further dissected into the epidermis, dermis, and subcutaneous layers. Results: RNA sequencing demonstrated substantial differences in expressional profiles between individual layers relative to each other and between each independent layer relative to the “Full” wounds. Localization of expressional changes in key wound signaling pathways to specific layers was also observed, including angiogenesis, inflammation, proliferation, and remodeling. Lastly, dressing material and/or NPWT demonstrated the capacity to modulate early wound signaling. Conclusion: This study provides valuable information on medical material interactions with wound tissue and/or NPWT and provides insight into early genomic changes of wound tissue. Together this experimental data offers a new perspective and methodology for characterizing wound tissue responses and provides new insight into localizing expressional patterns and genomic profiles within different wound regions to ultimately help improve clinical therapies.

K1.05

Determining How Early Disruption Of Mechanotransduction Affects Acute Wound Healing
Hudson C. Kussie, Dharshan Sivaraj, Melissa C. Leeolou, Savana L. Huskins, Sydney Steele, Dominic Henn, Artem A. Trotsyuk, Geoffrey C. Gurtner, Kellen Chen
Stanford University, New York City, NY, United States
Determining How Early Disruption Of Mechanotransduction Affects Acute Wound Healing

Hudson C. Kussie, Dharshan Sivaraj, Melissa C. Leeolou, Savana L. Huskins, Sydney Steele, Dominic Henn, Artem A. Trotsyuk, Geoffrey C. Gurtner, Kellen Chen Stanford University, New York City, NY, United States

Background: Previous work has determined that focal adhesion kinase inhibitor (FAKI) can mitigate mechanotransduction-mediated fibrosis and inflammation after 14 days of treatment. We sought to determine how targeted inhibition of FAK affected incisional wound healing during acute time periods. Methods: A 3 cm incision was made on the mouse dorsum and sutured closed. After 4 days, we attached a mechanical strain device to the dorsum to apply mechanical strain across the wound to create hypertrophic scar (HTS) formation. Of a total of 18 mice, (n=2) received an incision but no device (control); (n=10) received an incision with mechanical strain (HTS); and (n=6) were treated with FAKI hydrogels in conjunction with mechanical strain (S+FAKI). After 3 days of treatment, the mice were sacrificed. These groups and unwounded skin were compared by measuring scar width, analyzing collagen ultrastructure using picrosirius staining and collagen quantification algorithms, and immunofluorescent staining of a TGFB1 dependent fibrotic marker Thrombospondin1 (THBS1). Results: The average width of the HTS was significantly greater than the width of both the FAKI treated and control scars. The HTS collagen fibers were also significantly longer and more highly aligned than FAKI treated and control scars, as well as unwounded skin. The scars in the HTS mice also had significantly elevated levels of a pro-fibrotic extracellular matrix marker Thrombospondin1 compared to FAKI treated and control scars. Conclusion: Acute FAKI therapy effectively narrowed scar widths and promoted regenerative collagen architecture formation. Scar formation has been previously found to be TGFB1 dependent, and we measured extracellular matrix marker Thrombospondin1 as a downstream surrogate for TGFB1 expression. Thus, early FAKI could promote healing through the FAK–>TGFB1–>Thrombospondin1 signal pathway. Further understanding of targeting and timing of FAKI therapy can prove important for future therapies.

K1.06

Comparison Of Fibroblast Versus Macrophage Specific Mechanotransduction Knockout In A Wound Healing Model
Sydney Steele, Michelle Griffin, Dharshan Sivaraj, Savana L. Huskins, Hudson C. Kussie, Michael T. Longaker, Geoffrey C. Gurtner, Kellen Chen
Surgery, Hagey Laboratory for Pediatric and Regenerative Medicine, Palo Alto, CA, United States
Comparison Of Fibroblast Versus Macrophage Specific Mechanotransduction Knockout In A Wound Healing Model

Sydney Steele, Michelle Griffin, Dharshan Sivaraj, Savana L. Huskins, Hudson C. Kussie, Michael T. Longaker, Geoffrey C. Gurtner, Kellen Chen Surgery, Hagey Laboratory for Pediatric and Regenerative Medicine, Palo Alto, CA, United States

Background: For an injury to heal, a variety of cell types are recruited to begin the healing process. To better understand the impact of certain cell types and their influence on healing, we investigated how fibroblasts and macrophages responded to mechanical signals to influence healing, fibrosis, and regeneration. Methods: In order to simulate human-like hypertrophic scarring (HTS), we utilized an HTS device that applies mechanical strain on the incisional wounds of mice. These devices are steel palatal expanders with rod extender arms that are attached to the backs of the mice using skin adhesive and staples to allow for a uniform strain profile. We waited 4 days after the incision to mount the device, allowing the wound to close. Next, we extended the device every other day for a 14 day time period. To analyze the scar tissue, we took photos of the scars before we explanted. Then, once we explanted the scar tissue, we stained it using picro-sirius red, a type of staining that magnifies collagen formation and structure. With the stained tissue, we were able to use CurveAlign and CT-Fire collagen architecture algorithms. CurveAlign quantifies all fiber angles within the given image, while CT-Fire analyzes individual fiber measurements. We specifically bred mice to knockout FAK signaling in either fibroblasts (Col1a2 Cre, Ptk2-flox) or myeloid cells (Lyz2-Cre, Ptk2-flox) and ran both breeds of mice through the HTS experiment explained above. Results: Utilizing these devices created scars in wildtype mice with increased levels of fibrotic tissue and highly aligned collagen as compared to the control wildtype mice without extension. With the photos, we took measurements of both length and width of the scar to compare the fibroblast and myeloid FAK KO scars with wildtype scars. We observed that both knockout strains generated visually less scarring than in wildtype mice. Utilizing the software programs, we analyzed collagen fiber alignment and length. Both of the knockout strains had scar tissue with significantly less aligned, smaller fibers contrasting with the highly aligned, long fibers of wild type HTS mice scar tissue. With these measurements, we are able to demonstrate that the blocking fibroblast or macrophage mechanotransduction almost completely eliminated scar formation, decreased collagen alignment, and promoted skin regeneration. Conclusions: Our findings point toward the importance of myeloid cell mechanotransduction signaling during healing, possibly opening new doors for therapeutic treatment of fibrosis.

K2.01

A Medium Throughput 3D Wound-On-A-Chip Screening Platform To Assess Provisional Matrix Assembly During Stromal Wound Closure
Anish Srinivas Vasan1, Megan Griebel1, Nico Pederoncelli2, Christos Michas1, Christopher Chen1, Jeroen Eyckmans1
1Biomedical Engineering, Boston University, Boston, MA, United States 2Bioengineering, University of California, Los Angeles, Los Angeles, CA, United States
A Medium Throughput 3D Wound-On-A-Chip Screening Platform To Assess Provisional Matrix Assembly During Stromal Wound Closure

Anish Srinivas Vasan1, Megan Griebel1, Nico Pederoncelli2, Christos Michas1, Christopher Chen1, Jeroen Eyckmans1 1Biomedical Engineering, Boston University, Boston, MA, United States. 2Bioengineering, University of California, Los Angeles, Los Angeles, CA, United States

Background: Provisional matrix assembly is a crucial event during healing of full-thickness wounds and involves dynamic interactions between the fibroblast populations in the wound bed and the extracellular matrix. In previous work, we have demonstrated that this process can be recapitulated in engineered collagenous microtissues in vitro. In this work, we describe the development of a 3D wound-on-a-chip screening platform to assess provisional matrix assembly during wound closure in a 96-well format. Methods: 2-photon polymerization (2PP) 3D printing was used to create a master mold of a micropillar device consisting of 4 micropillars of 150 μm diameter with spherical caps of diameter 200 μm at a height of 350 μm. A polydimethylsiloxane (PDMS) replicate of the master mold was inserted in a stereolithography (SLA) 3D printed holder for casting PDMS micropillar devices in a 96-well plate. After curing and sterilization of the plate, cells seeded in a collagen type I matrix and were allowed to self-assemble into microtissues (1.0 mm X 0.6 mm) anchored to these micropillars. A pulsed nanosecond laser was used to ablate full-thickness wounds with a diameter of 100 μm to 200 μm. To demonstrate the utility of the medium-throughput platform, we optimized seeding density of three different types of fibroblasts (neonatal and adult human dermal fibroblasts (NHDF-neo and NHDF-Ad), and cardiac fibroblasts (CF)) to make microtissues and assessed gap closure area and rate using time-lapse microscopy. To characterize the ECM of the provisional matrix, injured microtissues were fixed and stained for collagen I, collagen III, collagen IV, and fibronectin 24h after injury. Results: The optimal cell seeding density for forming stable microtissues in a collagen hydrogel of 2.2 mg/mL was 7500 cells/well. Upon injury, CFs (N=2, n=12) closed the wounds in 11.6 ± 0.48 hours, NHDFneo (N=2, n=7) in 26.2 ± 3.53 hours and NHDF-Ad (N=2, n=15) in 59.8 ± 22.05 hours. All three cell types closed the gap by depositing a provisional matrix that contained fibronectin, collagen I, III and IV. Interestingly, based on fluorescent intensity, the levels of fibronectin in the provisional matrix produced by CF were higher than NHDF-neo, which were higher than NHDF-Ad. Conclusion: We successfully developed a medium throughput screening platform for assessing provisional matrix assembly during stromal wound closure and show that fibroblasts of different tissue origin have different closure rates, which correlates with the expression of fibronectin in the provisional matrix. References: Sakar, M. S. et al. Nat. Commun. 7, 1–8 (2016).

K2.02

A Non-Contact Device For Fast Screening Of Infections In Diabetic Foot Ulcer And Venous Leg Ulcer
Jon Senkowsky2, Shuxin Li1, Suvra Pal3, Wenjing Hu1, Liping Tang1
1Progenitec, Arlington, TX, United States 2Wound Care Limb Salvage Clinic, Texas health Physician Group, Arlington, TX, United States 3Mathematics, University of Texas at Arlington, Arlington, TX, United States
A Non-Contact Device For Fast Screening Of Infections In Diabetic Foot Ulcer And Venous Leg Ulcer

Jon Senkowsky2, Shuxin Li1, Suvra Pal3, Wenjing Hu1, Liping Tang1 1Progenitec, Arlington, TX, United States. 2Wound Care Limb Salvage Clinic, Texas health Physician Group, Arlington, TX, United States. 3Mathematics, University of Texas at Arlington, Arlington, TX, United States

Purpose: Infection is the most likely single cause of delayed healing in chronic wounds. Recent study shows that infected wounds have elevated leukocyte esterase activities as compared with non-infected wounds as determined by a portable device – Detec® Esterase. Here we investigated whether the same device, Detec® Esterase, can be used to screen and diagnose the infection of diabetic foot ulcer (DFU) and venous leg ulcer (VLU) at point-of-care. Method: Wound dressings from 71 DFU patients and 31 VLU patients at the Texas Health Arlington Memorial Wound Care and Limb Salvage Clinic were tested with DETEC® esterase and the device output was compared with subsequent clinical determination of infection on the same wounds. We evaluated the efficiency of the device through sensitivity, specificity, accuracy, and the increase in post-test risk of infection with positive test result. Results: For DFU patients, the results show that DETEC® Esterase device had good sensitivity (89.8%), fair specificity (45.5%), moderate accuracy (76.1%), and the increase in post-test risk of infection with positive test result of 9.56%. As for VLU patients, the results show that DETEC® Esterase device had comparably good sensitivity (82.1%), moderate specificity (66.7%), good accuracy (80.6%), and a smaller increase in post-test risk of infection with a positive test result of 5.51% because of high prevalence of the condition. Through fisher’s exact test to compare the diagnosis efficiency between DFU and VLU, two tailed p-values for sensitivity, specificity, and accuracy are 0.4825, 0.593, and 0.7975, respectively. Therefore, there are no significant differences in the device diagnosis efficiency between two different wounds. Using a logistic regression model, we determined whether other factors (such as age, gender, race, initial wound size, wound location, and sign of infection) would affect the accuracy of DETEC® Esterase device on diagnosis of infection and found none of these factors has significant effect on diagnosis accuracy of DETEC® Esterase device (age, P=0.4597; gender, P=0.1886; race, P=02401; initial wound size, P=0.9020; wound location, P=0.8549; signs of infection, P=0.456). Conclusion: Our results support that DETEC® esterase can effectively and accurately diagnose and/or screen for infections in DFUs and VLUs. The diagnosis accuracy of the device is not influenced by the patients’ age, gender, race, initial wound size, wound location, and sign of infection.

K2.03

Pullulan-Collagen Hydrogel Wound Dressing Promotes Dermal Remodeling and Healing in an Excisional Wound Model
Melissa C. Leeolou, Dharshan Sivaraj, Michael Davitt, Dominic Henn, Sydney Steele, Savana L. Huskins, Artem A. Trotsyuk, Hudson C. Kussie, Autumn Greco, David Perrault, Jagannath Padmanabhan, Michael T. Longaker, Kellen Chen, Geoffrey C. Gurtner
Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University, Stanford, CA, United States
Pullulan-Collagen Hydrogel Wound Dressing Promotes Dermal Remodeling and Healing in an Excisional Wound Model

Melissa C. Leeolou, Dharshan Sivaraj, Michael Davitt, Dominic Henn, Sydney Steele, Savana L. Huskins, Artem A. Trotsyuk, Hudson C. Kussie, Autumn Greco, David Perrault, Jagannath Padmanabhan, Michael T. Longaker, Kellen Chen, Geoffrey C. Gurtner Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University, Stanford, CA, United States

Background: The fundamental objectives of wound healing therapies are to provide protection against external factors and to sustain optimal moisture levels within the wound bed. Biological scaffolds such as hydrogels provide an ideal, physio-mimetic of native ECM that can improve wound healing outcomes after cutaneous injury. While most studies have focused on the benefits of hydrogels in accelerating wound healing, there is minimal data directly comparing different hydrogel material compositions. Methods: In this study, we utilized a splinted excisional wound model that recapitulates human-like wound healing in mice and treated wounds with three different collagen hydrogel dressings. The first dressing was composed of 90% collagen and 10% alginate. The second dressing was composed of 55% collagen and 44% cellulose. Finally, the third dressing was composed of 5% collagen and 95% pullulan. We assessed the feasibility of applying each dressing during standard dressing changes and took photographs of the wounds over time to determine the rate of wound closure. We then performed histologic and histopathologic analysis on the explanted scar tissues to assess the effects each hydrogel dressing on collagen architecture and alignment, as well as tissue response. Results: Wound area changes were measured by analyzing digital photographs. At PODs 10 and 12, the wound area was significantly smaller in wounds treated with the collagen-pullulan hydrogel compared to control wounds. Quantitative analysis of collagen architecture demonstrated that collagen-pullulan treated wounds had a lower amount of mature collagen within the healed scar. Untreated murine wounds showed a trend towards higher collagen fiber alignment, measured by the angle skewness of the fibers. Collagen-pullulan treated wounds showed significantly more random tissue alignment compared to collagen-cellulose treated wounds. Further, collagen-pullulan treated wounds demonstrated significantly shorter tissue fiber length compared to the other wound groups. Collectively, collagen-pullulan hydrogel treatment promoted restoration of a more normal skin phenotype with a basket-weave collagen architecture. Conclusion: Our data indicate that the material compositions of hydrogel dressings can significantly influence healing time, cellular response, and resulting architecture of healed scars. Collagen-pullulan hydrogel therapy accelerated wound closure and promoted healed tissue with shorter, less dense, and more randomly aligned collagen fibers, compared to other dressings. Further understanding of how specific hydrogel properties affect the healing and resulting tissue architecture of wounds may lead to novel insights and further optimization of the material properties of wound dressings.

K2.04

Toward Immunocompetent, Vascularized, Autologous 3D Skin Model Reconstructed By Tissue Engineering For Wound Healing Studies
Emilie Attiogbe1, Sébastien Larochelle1, Carine Mainzer2, Adèle Mauroux2, Sylvie Bordes2, Brigitte Closs2, Caroline Gilbert3, Véronique Moulin1
1LOEX institute, Université Laval, Québec, QC, Canada. 2R&D department, Silab, Brive la gaillarde, France. 3Université Laval, 2Centre de Recherche du CHU, Québec, QC, Canada
Toward Immunocompetent, Vascularized, Autologous 3D Skin Model Reconstructed By Tissue Engineering For Wound Healing Studies

Emilie Attiogbe1, Sébastien Larochelle1, Carine Mainzer2, Adèle Mauroux2, Sylvie Bordes2, Brigitte Closs2, Caroline Gilbert3, Véronique Moulin1 1LOEX institute, Université Laval, Québec, QC, Canada. 2R&D department, Silab, Brive la gaillarde, France. 3Université Laval, Centre de Recherche du CHU, Québec, QC, Canada

Skin wound healing is a process occurring after injury and is categorized into four phases: hemostasis, inflammation, proliferation, and remodeling. The early phases require the recruitment of several immune cells from the blood that regulate the fate of wound healing. However, the role of skin’s resident immune cells is not well defined. They could perpetuate a distinct role in the process compared to circulating immune cells. To investigate their contribution to wound healing, we first developed a cell extraction technique that isolates skin resident cells from the same donor (keratinocytes, fibroblasts, immune and endothelial cells). Analysis by flow cytometry reveals the presence of resident skin macrophages CD45+ CD14+; lymphocytes, CD45+CD3+; dendritic cells CD45+, CD14-, CD1a+ and endothelial cells CD45-CD31+ in freshly isolated skin cells. From this isolation, an autologous three-dimensional (3D) human skin model was developed in vitro by self-assembled method. Their presence was then confirmed in the 3D skin model by immunofluorescence staining. Endothelial cells self-assemble to form capillary like networks. The number of immune cells is constant after 14 days in culture and cells were able to produce TNF-α cytokine. The rate of proliferation at basal layer of the epidermis characterized by Ki67 staining, reveal an important contribution of immune and endothelial cells in reconstructed skin (N=4). This complete and autologous skin model provides a great tool to study the interaction of skin’s dermal resident immune cells in the context of wound healing.

K2.05

Real-Time Measurement And Tracking Of Wound Healing
Connor Povoledo1, Jacob Damant1, Daniel Brick1, Savannah Beavers2, Robert Burrell1
1Biomedical Engineering, University of Alberta, Edmonton, AB, Canada. 2Emergency Care, Delaney Veterinary Services, Sherwood Park, AB, Canada
Real-Time Measurement And Tracking Of Wound Healing

Connor Povoledo1, Jacob Damant1, Daniel Brick1, Savannah Beavers2, Robert Burrell1 1Biomedical Engineering, University of Alberta, Edmonton, AB, Canada. 2Emergency Care, Delaney Veterinary Services, Sherwood Park, AB, Canada

The goal of this study was to evaluate the use of a new smart phone imaging technique to assess healing of skin wounds at the point-of-care in real time. Initially photos of a wound were collected after each dressing change (every 3 to 4 days). Photoshop was used to isolate and standardize the wound. For the purposes of this analysis, ‘wounded’ areas were defined as light-red granulation tissue while ‘unwounded areas’ were defined as everything else. The wounded areas were masked in black and the intact areas were electronically removed leaving a white background. This produced a black and white image of the wound. A Python script was written to calculate the proportion of black pixels and white pixels of the image to determine the relative proportion of damaged to undamaged/healed areas. The time to time rate of change in the wound size, or healing rate, is analogous to the rate at which the black pixels vanish. The data generated was very useful but was not point-of-care. To convert this to a point-of-care wound healing tool the Python script was converted to Swift and C++ language platforms for processing on iPhones. The new program allowed the smart phone to use visual analysis algorithms and the RGB (Red Green Blue) coordinates of the wound to map out the wound surface and mask it. The software then calculated the change in wound size since the last reading. The system was used to follow the healing of a deep wound with a missing flap and exposed 10cm of canon bone in a horse. The results were plotted as rate of healing over time and as percentage change in wound size between dressing changes. The initial work with Photoshop showed that the wound area could be mapped and followed using software. The second stage showed that the wound surface area calculated by the new algorithms was within 1% of the surface area calculated by Photoshop. Asymmetry of the wound or its healing process did not cause any problems in the assessment. Using a visual analyses algorithm and a smart phone, it has been demonstrated that mapping the entire wound healing process could be done at the point-of-care. This will greatly facilitate clinical care.

K2.06

Endothelial PLCG2: The missing link that makes VEGF Therapy Robust
Khanna, Sashwati Roy, Chandan K. Sen
ICRME, Department of Surgery, Indiana University, Indianapolis, IN, United States
Endothelial PLCG2: The missing link that makes VEGF Therapy Robust

Kanhaiya Singh, Yashika Rustagi, Priyanka Verma, Edward Hernandez, Manishekhar Kumar, Poornachander Guda, Rajneesh Srivastava, Sujit Mohanty, Sumit Verma, Savita Khanna, Sashwati Roy, Chandan K. Sen ICRME, Department of Surgery, Indiana University, Indianapolis, IN, United States

Critical limb threating ischemia (CLTI) is a severe form of peripheral arterial disease with markedly reduced blood flow in lower extremities. Type 2 diabetes mellitus increases the incidence and severity of CLTI by 2-4-fold. Hyperglycemia induced endothelial cell dysfunction is recognized as a contributor to such limitation. For CLTI, VEGF therapy has met with limited success. To address such limitation, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using Chromium Next GEM Single Cell 3′ Gel Bead Kit v3.1 (10x Genomics, Inc.) and sequenced on NovaSeq 6000 (Illumina, Inc). Using our published quality control measures, 36,487 cells (from 3 diabetic skin and 5 non-diabetic skin) were chosen for downstream analysis. Global communications analyses of scRNA-seq data using CellChat identified that although VEGF signaling was not significantly altered in diabetic vs non-diabetic skin, phospholipase C Gamma 2 (PLCG2) was significantly down-regulated. The significance of PLCG2 in VEGF mediated increase in endothelial mitochondria function was assessed in cultured human microvascular endothelial (HMEC) cells using extracellular flux (Seahorse) assay. In HMEC, VEGF enhanced mitochondrial function as indicated by elevation in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). PLCG2 inhibition impaired the action of VEGF on mitochondrial metabolism (n = 5, *P < 0.05, one-way ANOVA). Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCG2 in murine diabetic ischemic tissue. Non-viral tissue nanotransfection technology (TNT2.0) mediated delivery of PLCG2 to endothelial cells was achieved through cadherin-5 promoter driven PLCG2-ORF. Targeted delivery of endothelial PLCG2 promoted the rescue of hind-limb ischemia in diabetic mice (n = 6, *P < 0.05, one-way ANOVA). Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining (n = 6, *P < 0.05, one-way ANOVA). TNT-based gene delivery was tolerated well by diabetic ischemic tissue. Taken together, this work identifies endothelial PLCG2 as an important contributor to the success of VEGF therapy in a diabetic ischemic limb setting.

K3.01

Activation Of Lasr And Specific Enzyme Defense Genes Results In Pseudomonas Aeruginosa Changing From Plancktonic To Biofilm Forming
Jane Kim, Proma Basu, Manuela Martins-Green
University of California, Riverside, Fullerton, CA, United States
Activation Of Lasr And Specific Enzyme Defense Genes Results In Pseudomonas Aeruginosa Changing From Plancktonic To Biofilm Forming

Jane Kim, Proma Basu, Manuela Martins-Green University of California, Riverside, Fullerton, CA, United States

Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. Certain PA strains are highly virulent, establish strong biofilm and are antibiotic resistant. PA growth is regulated by the quorum sensing system lasR to manage virulence and response to high oxidative stress (OS) generated by phagocytes during inflammation. How PA forms biofilm in chronic wounds in response to OS is still unknown. We hypothesize that PA in the microbiome of the skin becomes biofilm-forming in chronic wounds when high levels of OS activate lasR, and catalase (katA and katB) and superoxide dismutase (sodA and sodB), enzymes that break down H2O2 released by neutrophils in the wound into water and oxygen. To test this possibility, we used a biofilm-forming PA strain isolated from our murine chronic wounds and measured the transcriptional response of PA in the presence and absence of H2O2-induced OS. RNA was extracted, treated with RNAlater overnight and reversed transcribed into cDNA for quantitative polymerase chain reaction. We show that PA in mature biofilm, when compared to planktonic cells in log phase, has significantly increased basal expressions of lasR, sodA, sodB and katB (log2FC=13.22, 7.43, 10.67, and 7.36, respectively, p<0.05). After 4hr of H2O2 treatment, expressions of lasR, sodA, katA and katB increases (log2FC=16.03, 13.65, 12.58 and 13.50, respectively), showing that PA in biofilm respond to OS readily compared to planktonic cells. This is clearly shown when planktonic cells, treated with H2O2 are compared to untreated cells, respond poorly to OS as shown by significantly decreased expressions of lasR, sodA, katA and katB (log2FC=-0.04, -3.97, -5.54 and -4.03, respectively). Taken together, these results indicate that PA in biofilm has the advantage of withstanding OS through the proper activation of the lasR quorum sensing system, and catalase and superoxide dismutase genes, two major antioxidant systems. In conclusion, PA survives the harsh, high OS microenvironment present in chronic wound and colonizes these wounds with biofilm by turning on specific survival genes. Therefore, effective biofilm removal may be accomplished by disrupting lasR sodA, katA and katB expression. Because it is well known that biofilm in human chronic wounds readily returns after debridement, these finding could have major implications for treatment of human chronic wounds immediately after debridement.

K3.02

Wound Macrophage-Derived Oncostatin M Induces Antimicrobial S100a9 In Cutaneous Wound Epithelium
Amitava Das, Karthik Gourishetti, Pradipta Banerjee, Nandini Ghosh, Tanner Guith, Chandan K. Sen, Sashwati Roy
Surgery, Indiana University, Indianapolis, IN, United States
Wound Macrophage-Derived Oncostatin M Induces Antimicrobial S100a9 In Cutaneous Wound Epithelium

Amitava Das, Karthik Gourishetti, Pradipta Banerjee, Nandini Ghosh, Tanner Guith, Chandan K. Sen, Sashwati Roy Surgery, Indiana University, Indianapolis, IN, United States

Background. Wound macrophages play multifaceted role in cutaneous wound healing. Emergent line of evidence argues in favor of a strong macrophage-keratinocyte cross-talk. This work builds on previous observation that human wound-site macrophages and wound fluid is rich in Oncostatin M (OSM). OSM, unlike other macrophage-derived cytokines exclusively involved in inflammation, primarily functions by its effect on keratinocytes. A diverse and networked antimicrobial defense system in keratinocytes defends against skin infections. Antimicrobial peptides/proteins (AMPs) like β-defensins and S100 proteins (S100A7, S100A8, and S100A9) represent a key innate defense mechanism of the human skin. We sought to test the significance of OSM on antimicrobial defense system of the cutaneous wound epithelium (WE). Methods and Results. Laser capture microdissection and proteomics studies identified S100A9 as being highly expressed in the human chronic cutaneous WE. Importantly, OSM present in human chronic wound fluids potently induced (p<0.001; n = 4) the expression of S100A9 in human keratinocytes. In addition, OSM induced β-defensins, establishing a cause and effect link between wound-site OSM and keratinocyte AMPs. OSM repressed keratinocyte SOCS3, a negative regulator of JAK-STAT signaling. In turn, in keratinocytes with repressed SOCS3, OSM-induced S100A9 was potentiated. OSM receptor knockdown abrogated (p<0.05; n = 4) OSM-induced S100A9 expression suggesting that OSM mediated suppression of SOCS3 drives JAK-STAT signaling leading to S100A9 induction. S100A9 over-expression in human keratinocytes induced (p<0.05; n = 4) the expression of specific β-defensins and increased the killing of S. aureus. The ability to fight S. aureus infection was compromised (p<0.05; n = 4) in S100A9-null mice. Finally, wound fluid from human chronic wounds with poor infection outcomes was associated with low (p<0.05; n = 7) OSM and S100A9 in wound fluid. Conclusion. Taken together, this work establishes the critical significance of wound macrophage-keratinocyte cross-talk in endowing the WE with antimicrobial defenses. Wound macrophage derived OSM enables such cross-talk adding a new dimension to the significance of wound inflammation.

K3.03

RNA-Seq Analysis Displays Divergent Early Gene Expression Profiles In Aureus Infecting The Wounds Of Wild Type Compared To DB/DB Mice
David Dolivo1, Steven Lanier1, Kai P. Leung2, Thomas Mustoe1, Seok Jong Hong1, Robert Galiano1
1Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States 2Combat Wound Care Group, United States Army Institute of Surgical Research, San Antonio, TX, United States
RNA-Seq Analysis Displays Divergent Early Gene Expression Profiles In Aureus Infecting The Wounds Of Wild Type Compared To DB/DB Mice

David Dolivo1, Steven Lanier1, Kai P. Leung2, Thomas Mustoe1, Seok Jong Hong1, Robert Galiano1 1Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States. 2Combat Wound Care Group, United States Army Institute of Surgical Research, San Antonio, TX, United States

Purpose: Since skin wounds compromise the epidermal barrier, development of infections is always a possibility. For delayed healing wounds in particular, such as those that tend to occur in diabetic or otherwise healing-impaired patients, the threat of wound colonization by infectious microorganisms, such as Staphylococcus aureus, is of particular concern. The ways in which bacteria adapt to the wound environment, and how these adaptation processes differ in healing-impaired wounds, are not fully understood. Methods: Here we used a murine full-thickness splinted excisional wound model infected with S. aureus (UAMS-1 strain) in both wild type (WT) C57BL/6 mice and diabetic model db/db mice. At post-infection day 3 and day 7 we harvested bacterial RNA from the wound bed and performed bulk RNA-seq to compare gene expression profiles. We also compared these gene expression profiles to those of planktonic S. aureus cultured in vitro. Results: Mapping to the S. aureus and Mus musculus genome revealed that, in addition to S. aureus RNA, samples also contained murine RNA, which could be quantified and excluded based on mapping to the constructed genomes. Differential analysis of S. aureus genes revealed dramatically different gene expression profiles at post-infection day 3 between bacteria from WT and db/db mice. The majority of this difference resolved by post-infection day 7, though a number of differences remained. All samples harvested from wound-infecting S. aureus revealed dramatic differences in gene expression from in vitro cultured S. aureus, irrespective of time point or host mouse genotype. Conclusions: These data suggest that the early wound environment in WT and db/db mice drives dramatically different gene expression paradigms, and that some aspects of this differential expression are retained until later in the wound healing process. Future experiments will seek to characterize the host organism responses that accompany these variable expression paradigms in the hopes of better understanding the characteristics of the wound environment that yield differential.

K3.04

Cerium Nitrate Protects Skin Models From Bacterial Penetration
Ping Chen, Shankar J. Evani, Sai Lakshmi Rajasekhar Karna, Kai P. Leung
Combat Wound Care Group, US Army Institute of Surgical Research, Fort Sam Houston, TX, United States
Cerium Nitrate Protects Skin Models From Bacterial Penetration

Ping Chen, Shankar J. Evani, Sai Lakshmi Rajasekhar Karna, Kai P. Leung Combat Wound Care Group, US Army Institute of Surgical Research, Fort Sam Houston, TX, United States

Background: Infection is the leading cause of mortality among burn patients and responsible for up to 50% of burn-related deaths. Multiple topical products have been developed to control burn infections to improve the healing. Among wound care products, Silver Sulfadiazine cream (SSD) is the standard of care in the US for the management of deep burn wounds. In contrast, in Europe and South America, for this purpose SSD cream combined with cerium nitrate (CeN), Flammacerium, has been widely used. Compared to SSD cream, CeN+SSD cream has been recognized by burn surgeons to have the following benefits: hardening of the eschar resulting in less bacterial colonization, infection, and invasion. To investigate these recognized benefits of CeN, we studied Pseudomonas penetration into skin models (in vitro 3-D collagen model and an ex-vivo porcine burn skin model), and the potential mechanism by which CeN can protect against penetration. Methods: 3-D collagen matrix (rat tail collagen) or porcine skin was treated with CeN (40 mM) or water (control) for 30 min. In the collagen matrix, mechanical changes due to these treatments were measured by rheometry, and the penetration of P. aeruginosa was visualized by confocal microscopy. In the burned porcine skin model, the penetration of P. aeruginosa was assessed by recovering the bacteria on a filter membrane (0.2 μm) underlying the skin. Lastly, Pseudomonas-inoculated burned porcine skin was treated with silver dressing and viable counts were determined. Results: CeN treatment caused the 3-D collagen matrix to become more brittle, characterized by increased stiffness and reduced yield strength. In the deeper layers of the inoculated CeN-treated 3-D collagen gel, compared to water-treated collagen gel, fewer P. aeruginosa were present. Similarly, CeN-treatment resulted in fewer P. aeruginosa invading the burned porcine skin. Lastly, fewer Pseudomonas counts were found in the burned porcine skin following treatment with silver dressing Conclusions: CeN treatment resulted in less Pseudomonas penetration and fewer viable counts in the burned porcine skin after treatment with silver dressing.

K3.05

Filamentous Bacteriophage Delay Healing Of Pseudomonas-Infected Wounds
Arya Khosravi1, Michelle S. Bach1, Christiaan R. de Vries1, Johanna M. Sweere1, Medeea Popescu1, Qingquan Chen1, Aviv Hargil2, Jonas D. Van Belleghem2, Gernot Kaber2, Elizabeth B. Burgener3, dan Liu2, Quynh-Lam Tran2, Tejas Dharmaraj1, Vivekananda Sunkari1, Swathi Balaji4, Nandini Ghosh5, Shomita S. Mathew-Steiner5, Sundeep G. Keswani4, Niaz Banaei6, Laurence Nedelec1, Chandan K. Sen5, Venita Chandra7, Patrick R. Secor8, Gina Suh9, Paul Bollyky1
1Infectious Diseases and Geographic Medicine, Stanford Universtiy, Stanford, CA, United States 2Immunology, Stanford University, Stanford, CA, United States 3Pediatrics, Stanford University, Stanford, CA, United States 4Surgery, Baylor College of Medicine, Houston, TX, United States 5Surgery, Indiana University, Indianapolis, IN, United States 6Pathology, Stanford University, Stanford, CA, United States 7Vascular Surgery, Stanford University, Stanford, CA, United States 8University of Montana, Missoula, MT, United States 9Infectious diseases, Mayo Clinic, Rochester, MN, United States
Filamentous Bacteriophage Delay Healing Of Pseudomonas-Infected Wounds

Arya Khosravi1, Michelle S. Bach1, Christiaan R. de Vries1, Johanna M. Sweere1, Medeea Popescu1, Qingquan Chen1, Aviv Hargil2, Jonas D. Van Belleghem2, Gernot Kaber2, Elizabeth B. Burgener3, dan Liu2, Quynh-Lam Tran2, Tejas Dharmaraj1, Vivekananda Sunkari1, Swathi Balaji4, Nandini Ghosh5, Shomita S. Mathew-Steiner5, Sundeep G. Keswani4, Niaz Banaei6, Laurence Nedelec1, Chandan K. Sen5, Venita Chandra7, Patrick R. Secor8, Gina Suh9, Paul Bollyky1 1Infectious Diseases and Geographic Medicine, Stanford Universtiy, Stanford, CA, United States. 2Immunology, Stanford University, Stanford, CA, United States. 3Pediatrics, Stanford University, Stanford, CA, United States. 4Surgery, Baylor College of Medicine, Houston, TX, United States. 5Surgery, Indiana University, Indianapolis, IN, United States. 6Pathology, Stanford University, Stanford, CA, United States. 7Vascular Surgery, Stanford University, Stanford, CA, United States. 8University of Montana, Missoula, MT, United States. 9Infectious diseases, Mayo Clinic, Rochester, MN, United States

We have identified a novel role for Pf, a filamentous bacteriophage (phage), in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. Pf is prevalent in chronic Pa wound infections and may promote disease by contributing to biofilm formation and antibiotic resistance as well as directly impairing host immune defenses. We now demonstrate that Pf directly delays wound re-epithelialization by impeding epithelial migration. Utilizing murine and porcine models of wound infection, we reveal Pf impairs healing and epithelial barrier integrity of wounds infected with Pa, as determined by histology, morphometric analysis, and transepidermal water loss studies (TWELS). Further, Pf is able to inhibit healing even in the absence of live Pa, indicative that Pf promotes chronic wounds independent of Pa pathogenesis. Mechanistically, Pf phage directly inhibits autocrine signaling of CXCL1 (KC) resulting in to keratinocyte migration and wound closure. In agreement with these studies, a prospective cohort study of 36 human patients with chronic Pa wound infections revealed that wounds infected with Pf positive strains of Pa took longer to heal and were more likely to increase in size compared to wounds infected with Pf negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian target cells. We propose that Pf phage may have potential as a biomarker and therapeutic target in delayed wound healing.

K3.06

Low Abundance Of Microrna-21 In Diabetic Wound Macrophages Compromises Resolution Of Inflammation
Amitava Das, Pradipta Banerjee, Karthik Gourishetti, Nandini Ghosh, Tanner Guith, Kanhaiya Singh, Mithun Sinha, Chandan K. Sen, Sashwati Roy
Surgery, Indiana University, Indianapolis, IN, United States
Low Abundance Of Microrna-21 In Diabetic Wound Macrophages Compromises Resolution Of Inflammation

Amitava Das, Pradipta Banerjee, Karthik Gourishetti, Nandini Ghosh, Tanner Guith, Kanhaiya Singh, Mithun Sinha, Chandan K. Sen, Sashwati Roy Surgery, Indiana University, Indianapolis, IN, United States

Background. Unresolved inflammation impairs diabetic wound healing. Previous work by our laboratory have demonstrated decreased clearance of apoptotic cells (efferocytosis) by diabetic wound macrophages that compromises timely resolution of wound inflammation. Interestingly, we also observed that delivery of miR-21 to human monocyte-derived macrophages (MDM) bolsters efferocytosis resulting in a net anti-inflammatory phenotype. Thus we hypothesized that diabetic wound macrophages have lower abundance of miR-21 that leads to persistent inflammation. Method. To address our hypothesis, wound macrophages were isolated from well-developed negative-pressure wound therapy (NPWT) dressings (for human) and subcutaneously implanted PVA sponges (for mice). Results. Both human and murine diabetic wound macrophages showed significantly lower miR-21 levels compared to non-diabetics (p<0.05; n=6). To determine the significance of lower miR-21 in wound macrophages fate and resolution of inflammation, a myeloid cell-specific miR-21 knockdown mice (miR-21/LysM) was developed in our laboratory by crossbreeding mice carrying floxed miR-21 allele (miR-21fl/fl) with LysM-Cre mice. Expression of pro-inflammatory markers in miR-21/LysM wound macrophages were significantly higher while those of reparative phenotype was attenuated (p<0.05; n=6). Wounds of miR-21/LysM animals suffered from increased neutrophil abundance and persistent inflammation and impairment in wound closure (p<0.05; n=6). Bioinformatics analysis predicted the neutrophil chemoattractant GRO-α to be a direct target of miR-21. Using 3'-UTR firefly luciferase reporter, GRO-α was discovered to be a direct target of miR-21 (p<0.05; n=6). Lower miR-21 in diabetic and miR-21/LysM wound macrophages desilenced GRO-α (p<0.05; n=6) resulting in increased abundance of neutrophils at the wound-edge tissue. Delivery of nanoparticle-based delivery of miR-21 to diabetic wound-site macrophages resolved inflammation and accelerated wound closure (p<0.05; n=6). Conclusion. This study directly implicates a miR-21-GRO-α pathway responsible for compromised resolution of inflammation in diabetic wounds.

K4.01

Myo-Inositol In Fermented Sugar Matrix Improves Human Macrophage Function
Nandini Ghosh1, Amitava Das1, Yuki Hayashi2, Savita Khanna1, Chandan K. Sen1, Sashwati Roy1
1Surgery, Indiana University, Indianapolis, IN, United States 2OSATO Research Institute, Gifu, Japan
Myo-Inositol In Fermented Sugar Matrix Improves Human Macrophage Function

Nandini Ghosh1, Amitava Das1, Yuki Hayashi2, Savita Khanna1, Chandan K. Sen1, Sashwati Roy1 1Surgery, Indiana University, Indianapolis, IN, United States. 2OSATO Research Institute, Gifu, Japan

Background: Reactive oxygen species production by innate immune cells plays a central role in host defense against invading pathogens at wound-site. A weakened host-defense may result in persistent infection leading to wound chronicity. Fermented Papaya Preparation (FPP), a complex sugar matrix, bolstered respiratory burst activity and improved wound healing outcomes in chronic wound patients. The objective of the current study was to identify underlying molecular factor/s responsible for augmenting macrophage host defense mechanisms following FPP supplementation. Methods & Results: In depth LC-MS/MS analysis of cells supplemented with FPP led to identification of myo-inositol as a key determinant of FPP activity towards improving macrophage function. Myo-inositol, in quantities that is present in FPP, significantly improved macrophage respiratory burst and phagocytosis via de novo synthesis pathway of ISYNA1. Additionally, myo-inositol transporters, HMIT and SMIT1, played a significant role in such activity. Blocking these pathways using siRNA attenuated FPP-induced improved macrophage host defense activities. FPP supplementation emerges as a novel approach to increase intracellular myo-inositol levels. Such supplementation also modified wound microenvironment in chronic wound patients to augment myo-inositol levels in wound fluid (clinicaltrials.gov; NCT02332993). Conclusion: These observations indicate that myo-inositol in FPP influences multiple aspects of macrophage function critical for host defense against invading pathogens.

K4.02

Thermal Burn Injury To A Rabbit Ear Shows Decreased Scar Hypertrophy Following Topical Application Of Amiloride
Adrian Rodrigues, David Dolivo, Yingxing Li, Lauren Sun, Chun Hou, Thomas Mustoe, Seok Jong Hong, Robert Galiano
Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States
Thermal Burn Injury To A Rabbit Ear Shows Decreased Scar Hypertrophy Following Topical Application Of Amiloride

Adrian Rodrigues, David Dolivo, Yingxing Li, Lauren Sun, Chun Hou, Thomas Mustoe, Seok Jong Hong, Robert Galiano Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States

Purpose: Thermal injury wounds produce skin disfigurement in the form of hypertrophic scars. Victims of severe contact thermal injuries, like deep dermal burns, require an array of treatments to prevent infection, dehydration, and metabolic instability. Although treatments that promote adequate wound healing are critical, limited pharmacological interventions exist that target the resulting scars. Methods: In this study, we induced 1-cm-diameter contact burn wounds onto the ears of rabbits and analyzed scars following topical application of either amiloride, celecoxib or a control vehicle. We harvested the scars at post-burn day 29 and analyzed tissues for scar elevation index and gene expression. Results: The results showed that topical amiloride, when applied after wound reepithelization, can significantly reduce the scar elevation index (SEI) of burn-induced scars. Although Celecoxib also showed a reduction in SEI, this decrease was not statistically significant. Tissue from amiloride-treated scars were also analyzed for expression of PTGS2, ACTA2 and COL1A1, but there were no significant differences between the scars of vehicle control-treated and drug-treated wounds. Conclusions: Our results demonstrate that topical administration of amiloride was sufficient to reduce scar elevation, an important clinical indicator of scar severity. Future experiments are required to better understand the mechanisms underlying amiloride-induced antagonism of scar hypertrophy.

K4.03

A Novel Hyaluronic Acid Matrix Quells The Inflammatory Phase Through The Activation Of NFKB, TNF-A And Arg1 To Accelerate Wound Healing In A Diabetic Mouse Preclinical Model
Mitchell Sanders1, Da Wei1, Lindsay Polan1, Mia Hanna1, Nicole Siegmund1, ted bundrick2, Thomas E. Serena3
1Alira Health Boston LLC, Framingham, MA, United States 2Lacerta Life Sciences, Broussard, LA, United States 3Serena Group, Cambridge, MA, United States
A Novel Hyaluronic Acid Matrix Quells The Inflammatory Phase Through The Activation Of NFKB, TNF-A And Arg1 To Accelerate Wound Healing In A Diabetic Mouse Preclinical Model

Mitchell Sanders1, Da Wei1, Lindsay Polan1, Mia Hanna1, Nicole Siegmund1, ted bundrick2, Thomas E. Serena3 1Alira Health Boston LLC, Framingham, MA, United States. 2Lacerta Life Sciences, Broussard, LA, United States. 3Serena Group, Cambridge, MA, United States

In normal wound healing, fibroblasts produce hyaluronic acid (HA) which is an important extracellular matrix component required for tissue repair and regeneration by driving cell proliferation, protecting growth factors and stem cells, and preventing infection through bacteriostasis. However, in a diabetic wound, fibroblasts are senescent and as a consequence are often stalled in the inflammatory and proliferative phases of wound healing. We utilized a diabetic mouse wound healing model to demonstrate that a novel hyaluronic acid matrix derived from American Alligator (aHA) can accelerate wound healing through the gene activation of important biomarkers that quell the inflammatory phase and stimulate wound healing. Immunohistochemistry and qPCR indicate that the novel aHA matrix activates the important transcription factor NF-kB which plays a pivotal role in mediating the inflammatory response with a 2.6 fold increase in NF-kB on day 3, concomitant with the upregulation of TNF-a (1.7 fold) and an activation of M2A macrophages based on Arg 1 gene expression and immunofluorescence staining. Our recent findings indicate that replenishing a diabetic wound with aHA will accelerate wound healing by quelling the inflammatory phase and activating M2A macrophages to jump start the cell proliferation. These findings have profound clinical implications for restarting chronic wound healing prior to using more advanced and expensive therapies such as NPWT or human allografts.

K4.04

Transdermal Deferoxamine Enhances Wound Healing In Chronically Irradiated Skin In Mice
Hendrik Lintel, Darren B. Abbas, Christopher V. Lavin, Michelle Griffin, Nicholas Guardino, Jason L. Guo, Amanda F. Spielman, Geoffrey C. Gurtner, Michael T. Longaker, Derrick Wan
Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, United States
Transdermal Deferoxamine Enhances Wound Healing In Chronically Irradiated Skin In Mice

Hendrik Lintel, Darren B. Abbas, Christopher V. Lavin, Michelle Griffin, Nicholas Guardino, Jason L. Guo, Amanda F. Spielman, Geoffrey C. Gurtner, Michael T. Longaker, Derrick Wan Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, United States

Background: Radiation-induced fibrosis is a common chronic sequelae of radiation therapy. Chronic radiation dermatitis is characterized by a hypovascular dermis and a fibrotic state which presents many clinical challenges to healthcare providers, including impaired wound healing. Transdermal deferoxamine (DFO), an iron chelator, has been shown to mitigate radiation-induced skin fibrosis in mice through improved angiogenesis and a reduction in reactive oxygen species production. In this study, we aim to investigate the effects of DFO on excisional wound healing in chronically irradiated skin in a mouse model. Methods: Thirty C57BL/6 mice had their dorsal skin irradiated with 30 Gy fractionated over 6 doses of 5 Gy every other day. After waiting 4 weeks post-radiation to allow for the development of chronic fibrosis, two 5mm excisional wounds were created and stented open on the irradiated dorsum of each mouse. The mice were equally divided into 3 groups: an irradiated (IR) control, a vehicle-only patch, and a 1 mg/cm2 DFO-loaded patch. Every two days, patches and dressings were changed, wounds were photographed, and wound perfusion was assessed via Laser doppler until wound closure. Data analysis was done via one-way ANOVA to compare means between the treatment groups with statistical significance determined at p<0.05. Results: DFO-treated mice grossly demonstrated faster wound closure rates than the vehicle and IR control groups (A). Laser doppler analysis also revealed increased perfusion in the DFO-treated wounds throughout the wound healing process (B-C). In particular, DFO-treated wounds were found to reach a statistically significant higher perfusion index than vehicle-only patch and IR control wounds at post-operative day 14 (POD14) (D). Conclusion: Transdermal DFO demonstrates potential as a treatment modality to improve wound healing outcomes in chronically irradiated skin due to the advanced wound closure rate and elevated perfusion index noted in this study.

K4.05

Simvastatin Cream Alleviates Dermal Fibrosis In A Rabbit Ear Hypertrophic Scar Model
David Dolivo1, Adrian Rodrigues1, Lauren Sun1, Chun Hou1, Yingxing Li1, Eugene Chung2, Kai P. Leung2, Robert Galiano1, Thomas Mustoe1, Seok Jong Hong1
1Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States 2Combat Wound Care Group, United States Army Institute of Surgical Research, San Antonio, TX, United States
Simvastatin Cream Alleviates Dermal Fibrosis In A Rabbit Ear Hypertrophic Scar Model

David Dolivo1, Adrian Rodrigues1, Lauren Sun1, Chun Hou1, Yingxing Li1, Eugene Chung2, Kai P. Leung2, Robert Galiano1, Thomas Mustoe1, Seok Jong Hong1 1Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States. 2Combat Wound Care Group, United States Army Institute of Surgical Research, San Antonio, TX, United States

Purpose: Hypertrophic scars result from traumatic injury to the skin and represent a clinical burden with limited treatment options. Our group and others demonstrated that statin drugs (HMG-CoA reductase inhibitors) could attenuate hypertrophic scar formation in preclinical models, but convenient and topical delivery and retention of these drugs at the wound site remains a challenge. Methods: Here we describe a cream that can deliver statin drugs and enables simple, convenient topical application, and we tested this cream in our well-characterized excisional wound model performed in rabbit ear ventral skin. Results: We demonstrated that application of simvastatin in a cream formulation significantly attenuated hypertrophy of the resultant scars, compared to application of the identical vehicle cream without the drug. Despite causing symptoms of transient dryness and heightened trans-epidermal water loss, simvastatin cream application resulted in a decrease in macrophage and myofibroblast density at post-operative day 28 harvest. Withdrawal of statin cream treatment resulted in alleviation of dryness and substantial decrease in TEWL back to normal levels. Conclusions: Together, these data demonstrate that cream formulation is a viable vehicle for delivery of simvastatin, which maintains its anti-fibrotic capacity, and enables rapid recovery of epidermal barrier function after its withdrawal.

K4.06

Cerium Nitrate Treatment Diminishes Pseudomonas Aeruginosa Pathogenic Potential
Sai Lakshmi Rajasekhar Karna, Shankar J. Evani, Ping Chen, Kai P. Leung
Combat Wound Care Group, United States Army Institute of Surgical Research, Converse, TX, United States
Cerium Nitrate Treatment Diminishes Pseudomonas Aeruginosa Pathogenic Potential

Sai Lakshmi Rajasekhar Karna, Shankar J. Evani, Ping Chen, Kai P. Leung Combat Wound Care Group, United States Army Institute of Surgical Research, Converse, TX, United States

Background: Burn wound infections are significant causes of military and civilian casualties. Cerium Nitrate (CeN), a nitrate salt of cerium, a rare earth element, has been used in combination with Silver Sulfadiazine (SSD) in a cream for the management of deep burn wounds. This combination has shown added benefits over SSD alone in lessening bacterial colonization, infection, and invasion of deep burn wounds. However, details of the CeN activity on burn wound pathogens have yet to be defined. Pseudomonas aeruginosa is one of the most damaging pathogens encountered in burn wound infections. Therefore, we chose to study the impact of CeN on P. aeruginosa growth, motility, invasiveness, inflammation, and phagocytosis in vitro. Methods: Pseudomonas growth after incubation in different concentrations of CeN at 37oC for 24 h was measured by colony-forming-units. Motility of CeN-treated Pseudomonas was measured by a swarming assay. The penetration of Pseudomonas into burned porcine skin was assessed by recovering bacteria that passed through the skin onto an underlying filter membrane (0.25 μm). In vitro inflammation and phagocytosis were studied using THP-1 monocytic cells. Results: CeN treatment, between 0.1 to 10 mM, reduced P. aeruginosa viability dose-dependently. In the swarming assay, P. aeruginosa treated with CeN were less motile. Additionally, the penetration of Pseudomonas into the burned porcine skin was reduced by CeN treatment. In the inflammation assay, Pseudomonas treated with CeN induced lower TNF-α and IL-8 responses from THP-1 cells. Additionally, P. aeruginosa lipopolysaccharide treated with CeN was less inflammatory to ThP1 cells. Furthermore, CeN-treated P. aeruginosa (live or heat-killed) were more susceptible to phagocytosis by ThP-1 cells. Conclusions: These results suggest that CeN treatment lessened the virulence of P. aeruginosa, making it less invasive, less pro-inflammatory, and more susceptible to phagocytosis by immune cells.

N1.01

Role Of Type 1 Regulatory T-Lymphocytes In The Regulation Of Neovascularization In Dermal Wounds
Walker D. Short, Oluyinka Olutoye, Benjamin Padon, Hui Li, Ling Yu, Lily Cheng, Swathi Balaji, Sundeep G. Keswani
Department of Surgery, Baylor College of Medicine, Houston, TX, United States
Role Of Type 1 Regulatory T-Lymphocytes In The Regulation Of Neovascularization In Dermal Wounds

Walker D. Short, Oluyinka Olutoye, Benjamin Padon, Hui Li, Ling Yu, Lily Cheng, Swathi Balaji, Sundeep G. Keswani Department of Surgery, Baylor College of Medicine, Houston, TX, United States

Objective(s): CD4+ T-lymphocytes are essential in regulating dermal fibrosis. SCID mice, which lack lymphocytes, heal with worse angiogenesis and fibrosis compared to wild type mice. We have demonstrated that IL-10-producing type 1 regulatory T lymphocytes (Tr1) improve vascularity in the healing wound, accelerate wound closure, and reduce dermal fibrosis in SCID mice. How the immune-modulatory Tr1 regulate angiogenesis is unknown. We therefore hypothesize that Tr1 modulate the pro-angiogenic cytokine balance (VEGF; CXCL12; MIF) to improve fibroblast and endothelial cell angiogenic functions, thereby enhancing neovascularization and wound healing outcome. Methods: 10BiT murine (M/F; 8-12wk) splenocytes were sorted into Tr1 precursors and Naive T-lymphocytes (Tnaive). In vitro, dermal fibroblasts were co-cultured with Tnaive or Tr1 for 24h and gene expression of VEGF and CXCL12 was measured using qRT-PCR. Dermal endothelial cells were cultured with conditioned medium from Tr1 or Tnaive and branching assay was performed. In vivo, we made 6mm bilateral dorsal full-thickness splinted wounds on SCID or C57BL/6J (WT) mice (female, 8-10wk), followed by adoptive transfer of 10^6 Tr1 cells. Wounds were harvested at days 3, 7, 30. D3 wound protein was measured for VEGF, CXCL12, and MIF. Angiogenic and inflammatory cellular interactions were visualized with imaging mass cytometry (IMC) at D7. D30 wounds were examined for dermal architecture and neovascularization (H&E; CD31). Results: Fibroblasts co-cultured with Tr1 had increased expression of VEGF (1.5 fold increase; p<0.001) and CXCL12 (1.5 fold increase; p<0.05) compared to untreated fibroblasts. Endothelial cells treated with Tr1 conditioned medium demonstrated significantly greater branching and network formation (6.05+/-0.15 vs 4.37+/-0.56mm, p<0.05). Compared to WT, SCID mice deficient in T-lymphocytes showed abnormally elevated wound VEGF levels at D3 (325.3+/-24.6 vs 187.4+/-42.1 pg/mg total protein, p<0.05), and treatment with Tr1 normalized VEGF in SCID mice to levels similar to WT (177.8+/-55.5 pg/mg total protein). Similarly, the positive effect of Tr1-treatment was seen in normalizing the CXCL12 expression in D3 SCID wounds (SCID: 1.8+/-0.5 vs WT: 0.6+/-0.4, SCID+Tr1: 0.8+/-0.5 ng/mg total protein, p<0.05). MIF levels were also reduced by Tr1-treatment in D3 SCID wounds (SCID: 800.6+/-86.2 vs SCID+Tr1 ng/mg total protein, p<0.01). IMC performed on D7 wounds revealed more mature vessels (alpha-SMA positive) with close cellular approximation of transferred Tr1 cells, indicating their paracrine effects on angiogenesis. WT mice wounds enriched with Tr1 application to wounds at the time of wounding resulted in improved wound maturation with a basket weave collagen structure, more dermal appendages, and increased neovascularization at D30. Conclusions: Tr1 improve the balance of angiogenic modulators VEGF, CXCL12, and MIF to promote improved neovascularization and scarring. Enrichment of Tr1 to wounds may be a therapeutic target to enhance wound healing.

N1.02

Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing
Kellen Chen, Michelle Griffin, Dominic Henn, Clark A Bonham, Katharina Fischer, Jagannath Padmanabhan, Artem A. Trotsyuk, Dharshan Sivaraj, Melissa C. Leeolou, Hudson C. Kussie, Savana L. Huskins, Sydney Steele, David Perrault, Michael T. Longaker, Geoffrey C. Gurtner
Stanford University, Stanford, CA, United States
Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing

Kellen Chen, Michelle Griffin, Dominic Henn, Clark A Bonham, Katharina Fischer, Jagannath Padmanabhan, Artem A. Trotsyuk, Dharshan Sivaraj, Melissa C. Leeolou, Hudson C. Kussie, Savana L. Huskins, Sydney Steele, David Perrault, Michael T. Longaker, Geoffrey C. Gurtner Stanford University, Stanford, CA, United States

Background: Repair after tissue injury involves a dynamic interplay among not just tissue resident cells (e.g., fibroblasts), but also cells recruited from the circulation. Myeloid cells, such as monocytes and macrophages, are derived from hematopoietic precursors and migrate to sites of injury where they play a role in modulating all stages of wound healing and scar formation. There is mounting evidence that mechanical stimuli are also able to modulate monocyte and macrophage response during tissue healing, but the exact mechanisms behind this “mechano-immunomodulation” remain incompletely understood. Methods: We attached a mechanical strain device to the mouse dorsum to initiate a uniform and consistent strain profile across an incisional wound to create hypertrophic scar (HTS) formation in mice. To investigate mechano-responsive immune cells, we performed parabiosis of wildtype (WT) and GFP+ mice, allowed the mice to develop a shared blood circulation, initiated HTS formation in the WT mouse, and analyzed the cells using single cell RNA sequencing (scRNA-seq), fluorescent-activated cell sorting (FACS), and immunofluorescent staining. Results: Mechanical modulation significantly upregulated the presence of inflammatory subtypes within the healing tissue, characterized by an increase in infiltrating GFP+ cells from 5.4% to 12.2%. In the GFP+ circulating cells, mechanical strain directly increased the proportion of fibrotic myeloid cells, primarily defined by the monocyte marker Ly6c2 as well as the TGFB responsive and macrophage activating gene Thbs1. Mechanical strain also increased the proportion of inflammatory myeloid cell populations, defined by Ccl and Il6 chemoattractants, and Cd74+ migratory myeloid cells. Utilizing both a pharmacological blocker of focal adhesion kinase (FAK) as well as a myeloid specific FAK knockout (KO), we demonstrated that modulating mechanical signaling abrogated those responses and instead promoted homeostatic myeloid transcriptional fates. Conclusions: Tissue injury activates a cascade of signaling pathways to recruit and orchestrate various cell types during healing. Our study indicates that modulating mechanical stress directly affects myeloid cell phenotypes and interactions with other cell types in the complicated, multicellular milieu of wound healing. This principle has been previously unexplored in the context of fibrosis and regeneration, with most previous studies focused on fibroblast heterogeneity and transcriptional profiles. To our knowledge, this is the first study to directly investigate the effects of modulating mechanotransduction on immune cell response at the single cell level utilizing parabiosis and wound healing. Collectively, we demonstrate that mechano-immunomodulation of the “early responders” of healing can trigger a cascade of downstream regenerative healing.

N1.03

Topical Treatment With Antagomir-193B-3P Accelerates Diabetic Wound Healing
Jelena Marjanovic, Ivan Jozic, Rivka C. Stone, Cheyanne Head, Beatriz Abdo Abujamra, Robert S. Kirsner, Hadar Lev-Tov, Irena Pastar, Marjana Tomic-Canic
Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States
Topical Treatment With Antagomir-193B-3P Accelerates Diabetic Wound Healing

Jelena Marjanovic, Ivan Jozic, Rivka C. Stone, Cheyanne Head, Beatriz Abdo Abujamra, Robert S. Kirsner, Hadar Lev-Tov, Irena Pastar, Marjana Tomic-Canic Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States

Background: One of the major contributing factors of healing impairment of diabetic foot ulcer (DFU) is impaired re-epithelialization marked by diminished migration and hyperproliferation of keratinocytes. We used laser capture microdissection (LCM) and genomic approaches to identify specific miRNAs that potentially can contribute to this keratinocyte non-healing phenotype and found microRNA-193b-3p (miR-193b-3p) as one of the top upregulated. Thus, we focused on deciphering the role of miR-193b-3p in epithelialization and therapeutic potential of miR-193b-3p inhibition. Methods: We collected tissue samples from DFUs (n=15), control skin samples (n=15), and human acute wounds (n=3) and performed LCM and transcriptomic analyses using Ingenuity Pathway Analysis (IPA). qPCR was used to analyze miR-193b-3p and expression of its target genes. We performed gain- and loss-of function experiments. Cell migration was analyzed using scratch assay, and re-epithelialization was evaluated using human ex vivo and diabetic murine in vivo wound models. Formation of stress fibers involved in cell migration was studied using immunocytochemistry and RhoA pull-down assay. Results: miR-193b-3p was found significantly induced in the epidermis of DFUs, but not in the human acute wounds. Knockdown of miR-193b-3p promoted migration of human keratinocytes, without affecting cell proliferation or cell viability. Moreover, inhibition of miR-193b-3p expression in ex vivo human wounds and diabetic murine in vivo wounds accelerated re-epithelialization. Conversely, overexpression of this miR impaired keratinocyte migration and re-epithelialization in a 3D organotypic wounds. We identified the underlying mechanism of miR-193b-3p anti-migratory activity through a disruption of stress fiber formation and a reduction of RhoA activity, with no effect on lamellipodia or filopodia formation. IPA analysis revealed miR-193b-3p target network that contributes to poor healing in DFUs. We confirmed suppression of downstream targets of miR-193b-3p in DFUs (ARHGEF6, CCND1, IGFBP5, JAK2, KIT, KRAS, MAP3K1, NFAT3, SOS2, TCF4, TNS1) and further showed that this transcriptomic landscape found in non-healing DFUs can be reversed upon miR-193b-3p suppression by topical treatment with antagomirs. Conclusion: We identified miR-193b-3p as an important suppressor of keratinocyte migration in DFUs. Topical treatment with miR-193b-3p antagomiR represents a novel therapeutic approach for promotion of epithelialization in DFUs.

N1.04

Aerobic Glycolysis Drives Physiological Responses To Injury Resulting In Heterogeneous Wound Repair
Oluyinka Olutoye, Fayiz Faruk, Walker D. Short, Benajmin Padon, Tanuj Prajaparti, Aditya Kaul, Hui Li, Ling Yu, Sundeep G. Keswani, Christian Coarfa, Swathi Balaji
Surgery, Baylor College of Medicine, Houston, TX, United States
Aerobic Glycolysis Drives Physiological Responses To Injury Resulting In Heterogeneous Wound Repair

Oluyinka Olutoye, Fayiz Faruk, Walker D. Short, Benajmin Padon, Tanuj Prajaparti, Aditya Kaul, Hui Li, Ling Yu, Sundeep G. Keswani, Christian Coarfa, Swathi Balaji Surgery, Baylor College of Medicine, Houston, TX, United States

Background: Identical dermal injuries heal with varying degrees of fibrosis in different patients. Wound repair is an energy-intensive process, fueled by oxidative phosphorylation (OXPHOS) and glycolysis to generate ATP. Emerging evidence shows aerobic glycolysis is a preferred mechanism underlying pathologic fibrosis, but its role in physiologic wound healing is unknown. As proliferation and ECM production of fibroblasts to rebuild the dermis relies on lactate production, a prelude to shift to aerobic glycolysis, we hypothesize that differences in intrinsic fibroblast bioenergetic metabolism/aerobic glycolysis underlie differential scarring in patients. Methods: We created a biorepository of paired normal-skin, c-section scar tissue, and fibroblasts from abdominoplasty samples from patients with clinically stratified low scar (LS) and high scar (HS) phenotypes by the Vancouver Scar Scale, <3 vs.>6 respectively. Expression of Pyruvate Kinase M2 (PKM2), a modulator of aerobic glycolysis, and lactate dehydrogenase (LDHA) were measured in patient skin (IHC). OXPHOS (OCR), glycolysis (ECAR), and ATP production at resting, under stress, and with TGF-beta treatment were evaluated in fibroblasts (seahorse assays), along with expression of PKM2 and LDHA (Protein simple WES). LS/HS human fibroblasts (10^6 cells/wound) were transferred to 6mm full-thickness stented wounds on SCID mice (female, 8-10weeks). Wounds were harvested on days 7 and 28. Wound closure (H&E), alpha-SMA, PKM2, LDHA (staining), inflammatory profile (Luminex; IHC), and collagen expression (trichrome) were analyzed. Data are presented as n=3 biologic replicates/group; p-values by ANOVA. Results: Expression of PKM2 and LDHA was higher in both normal skin and scar tissue from HS. In vitro, RNA sequencing and GO analysis revealed several altered metabolic pathways in HS vs. LS normal skin fibroblasts. Normal skin and scar fibroblasts from HS had higher basal OCR and ECAR than LS (p<0.01). HS fibroblasts responded to stress (FCCP/Oligomycin treatments) with significantly higher increases in spare respiratory and glycolytic reserve capacity than LS (p<0.01), along with an increase in phospoPKM2 (p<0.05). TGF-beta treatment resulted in increased phospoPKM2 and LDHA in HS vs. LS (p<0.05). In vivo, murine wounds with HS fibroblasts showed expedited wound closure at day 7 with reduced IL-10, IL-17, MIP-1a/b, and G-CSF expression via immunoplexing and displayed increases in alpha-SMA (25-fold) gene expression and staining. On day 28, there were increased PKM2+ and LDHA+ spindle-shaped cells in wounds with HS, along with pronounced collagen staining and thick epidermis. Conclusion: Fibroblasts of different scarring phenotypes display characteristic bioenergetic metabolism profiles and produce distinct scarring in murine wounds. Our data suggest that a shift to aerobic glycolysis (Warburg effect) is associated with increased fibrosis (high scar).

N1.05

Mechanical Stimulation Reverses Pro-Fibrotic Transcriptional States in Senescent Fibroblasts
Jason L. Guo, Michelle Griffin, Nicholas Guardino, Kellen Chen, Geoffrey C. Gurtner, Michael T. Longaker
Surgery, Stanford University, Stanford, CA, United States
Mechanical Stimulation Reverses Pro-Fibrotic Transcriptional States in Senescent Fibroblasts

Jason L. Guo, Michelle Griffin, Nicholas Guardino, Kellen Chen, Geoffrey C. Gurtner, Michael T. Longaker Surgery, Stanford University, Stanford, CA, United States

Background: Senescent fibroblasts accumulate with age, contributing to fibrosis and impaired healing. It is of longstanding clinical interest to reverse this pro-fibrotic transcriptional state. Interestingly, senescence downregulates mechanotransduction in fibroblasts, but the effects of this phenomenon on skin fibrosis are unclear. We utilized a stretchable collagen I hydrogel (Fig. 1A) to investigate if exogenous mechanical stimulation could reverse the pro-fibrotic transcriptome of senescent fibroblasts. Methods: Dermal fibroblasts were harvested from C57BL/6 mice by enzymatic digestion and flow cytometry, cultured in growth media, and exposed to 0-250 nM doxorubicin (72h) to induce senescence, quantified by beta-galactosidase. Fibroblasts were encapsulated (1,000,000 cells/mL) in 1.92 mg/mL collagen I, 0.8x MEM, and 16mM HEPES. Gels were formed in 20x20x5mm cruciforms and cultured 7 days (media replaced each 2-3 days) with 10% equibiaxial strain for mechanical stimulation. RNA was retrieved by collagenase digestion and TRIzol extraction. RNAseq was performed (40M paired-end reads), aligned to mm10 (HISAT2), and quantified (StringTie). Differential expression analysis and gene ontology (GO) analysis of the top 500 differential genes were performed (edgeR; alpha=0.05). All experiments performed at n=3. Results: Beta-galactosidase showed optimal 76.7% senescence induction with 125nM doxorubicin (Fig. 1B). RNAseq showed increased Glb1 (senescence) and decreased Mki67 (non-senescence/proliferation) expression for senescent fibroblasts, which were reversed by mechanical stimulation (Fig. 1C). Yap1 (mechanosensing) expression increased with mechanical stimulation of non-senescent and senescent fibroblasts. Pdgfra and Acta2 (pro-fibrotic, canonical) were highly enriched in senescent fibroblasts. Critically, these pro-fibrotic markers were reversed by mechanical stimulation in senescent fibroblasts only. Col1a1 was downregulated by senescence and mechanical stimulation, while Col1a2 was downregulated by mechanical stimulation of senescent fibroblasts only. GO analysis revealed that mechanical stimulation differentially modified transcriptomes of non-senescent and senescent fibroblasts. The top 5 enriched terms for senescent fibroblasts were characterized by systemic modifications to biological quality/development (Fig. 1D), while the top 5 enriched terms for non-senescent fibroblasts were largely superficial responses to stretched matrix/gel (Fig. 1E). Conclusions: Mechanical stimulation differentially modified normal vs. senescent fibroblasts, increasing pro-fibrotic transcription for non-senescent fibroblasts but reversing pro-fibrotic transcription and systemically modifying biological qualities for senescent fibroblasts. Furthermore, mechanical stimulation mitigates inherent features of the senescent phenotype (Glb1+/Mki67-). Ultimately, our model suggests that exogenous mechanical stimulation reverses the pro-fibrotic phenotype of senescent dermal fibroblasts.

N1.06

Endothelial PLCG2: The missing link that makes VEGF Therapy Robust
Kanhaiya Singh, Yashika Rustagi, Priyanka Verma, Edward Hernandez, Manishekhar Kumar, Poornachander Guda, Rajneesh Srivastava, Sujit Mohanty, Sumit Verma, Savita Khanna, Sashwati Roy, Chandan K. Sen
ICRME, Department of Surgery, Indiana University, Indianapolis, IN, United States
Endothelial PLCG2: The missing link that makes VEGF Therapy Robust

Kanhaiya Singh, Yashika Rustagi, Priyanka Verma, Edward Hernandez, Manishekhar Kumar, Poornachander Guda, Rajneesh Srivastava, Sujit Mohanty, Sumit Verma, Savita Khanna, Sashwati Roy, Chandan K. Sen ICRME, Department of Surgery, Indiana University, Indianapolis, IN, United States

Critical limb threating ischemia (CLTI) is a severe form of peripheral arterial disease with markedly reduced blood flow in lower extremities. Type 2 diabetes mellitus increases the incidence and severity of CLTI by 2-4-fold. Hyperglycemia induced endothelial cell dysfunction is recognized as a contributor to such limitation. For CLTI, VEGF therapy has met with limited success. To address such limitation, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using Chromium Next GEM Single Cell 3′ Gel Bead Kit v3.1 (10x Genomics, Inc.) and sequenced on NovaSeq 6000 (Illumina, Inc). Using our published quality control measures, 36,487 cells (from 3 diabetic skin and 5 non-diabetic skin) were chosen for downstream analysis. Global communications analyses of scRNA-seq data using CellChat identified that although VEGF signaling was not significantly altered in diabetic vs non-diabetic skin, phospholipase C Gamma 2 (PLCG2) was significantly down-regulated. The significance of PLCG2 in VEGF mediated increase in endothelial mitochondria function was assessed in cultured human microvascular endothelial (HMEC) cells using extracellular flux (Seahorse) assay. In HMEC, VEGF enhanced mitochondrial function as indicated by elevation in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). PLCG2 inhibition impaired the action of VEGF on mitochondrial metabolism (n = 5, *P < 0.05, one-way ANOVA). Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCG2 in murine diabetic ischemic tissue. Non-viral tissue nanotransfection technology (TNT2.0) mediated delivery of PLCG2 to endothelial cells was achieved through cadherin-5 promoter driven PLCG2-ORF. Targeted delivery of endothelial PLCG2 promoted the rescue of hind-limb ischemia in diabetic mice (n = 6, *P < 0.05, one-way ANOVA). Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining (n = 6, *P < 0.05, one-way ANOVA). TNT-based gene delivery was tolerated well by diabetic ischemic tissue. Taken together, this work identifies endothelial PLCG2 as an important contributor to the success of VEGF therapy in a diabetic ischemic limb setting.

N1.07

Microfibril Associated Protein 5 in Skin Wound Healing
Lin Chen1, Bruna Romana-Souza2, Trevor R. Leonardo1, Junhe Shi3, Chen Han1, Luisa A. DiPietro1
1Center for Wound Healing and Tissue Regeneration, University of Illinois Chicago, Chicago, IL, United States 2Department of Histology and Embryology, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro State University, Brazil 3Institute of Clinical Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
Microfibril Associated Protein 5 in Skin Wound Healing

Lin Chen1, Bruna Romana-Souza2, Trevor R. Leonardo1, Junhe Shi3, Chen Han1, Luisa A. DiPietro1 1Center for Wound Healing and Tissue Regeneration, University of Illinois Chicago, Chicago, IL, United States. 2Department of Histology and Embryology, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro State University, Brazil. 3Institute of Clinical Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China

Recent studies from our lab demonstrated that fibroblasts are phagocytic in healing skin wounds. RNA sequencing of phagocytic fibroblasts led to the identification of Microfibril associated protein 5 (MFAP5, or microfibril-associated glycoprotein 2/MAGP2) as a molecule that is significantly unregulated in phagocytic fibroblasts. MFAP5, a 25 kD serine and threonine rich small protein, is a component of microfibrils in the extracellular matrix (ECM). MFAP5 is known to be closely associated with fibrosis and angiogenesis in certain cancers. Given the role of MFAP5 in cancer, the aim of this study was to investigate the production of MFAP5 in fibroblasts and in a murine model of skin wound repair. Flow cytometric analysis demonstrated that skin fibroblasts constitutively express MFAP5 and that MFAP5 levels increase markedly in actively phagocytic fibroblasts. Microarray and real time PCR analysis of full thickness excisional wounds showed that MFAP5 mRNA expression is significantly upregulated during the proliferative and remodeling phases of healing (day 3 to 21). Immunohistochemical staining demonstrated that MFAP5 was strongly present in the cytoplasm of dermal cells and in the dermal ECM of scars. In addition, MFAP5 was found to be strongly expressed in human keloids. The data shows that fibroblasts can be a source of MFAP5 in wounds, and that the expression of MFAP5 changes significantly over the course of skin wound healing. The findings support a role for MFAP5 in the regulation of fibroblasts, scar formation, and ECM function in skin wound healing. Supported by NIH R01 GM50875, R35 GM139603-01, and F31 DE028747-01

N1.08

Cellular vs. Acellular Tissue-Based Products For The Treatment Of Diabetic Foot Ulcers: Final Report Of A Longitudinal Comparative Effectiveness Study
Harrison J. Shawa1, Ramajot Kaur2, Catherine Tchanque-Fossuo2, Hadar Lev-Tov2, Kaitlyn West2, Nuen Yang3, David Rocke3, Sara Dahle4, Rivkah Isseroff1
1Dermatology, University of California, Davis, Las Vegas, NV, United States 2Dermatology Section, VA Northern California Healthcare System, Mather, CA, United States 3Public Health Sciences, University of California, Davis Medical Center, Sacramento, CA, United States 4Podiatry Section, VA Northern California Healthcare System, Mather, CA, United States
Cellular vs. Acellular Tissue-Based Products For The Treatment Of Diabetic Foot Ulcers: Final Report Of A Longitudinal Comparative Effectiveness Study

Harrison J. Shawa1, Ramajot Kaur2, Catherine Tchanque-Fossuo2, Hadar Lev-Tov2, Kaitlyn West2, Nuen Yang3, David Rocke3, Sara Dahle4, Rivkah Isseroff1 1Dermatology, University of California, Davis, Las Vegas, NV, United States. 2Dermatology Section, VA Northern California Healthcare System, Mather, CA, United States. 3Public Health Sciences, University of California, Davis Medical Center, Sacramento, CA, United States. 4Podiatry Section, VA Northern California Healthcare System, Mather, CA, United States

Diabetic foot ulcers (DFU) present a significant challenge to the United States healthcare system. With the current of standard of care (SOC), less than 30% of wounds heal after 20 weeks. This dismal cure rate prompted development of advanced therapies, albeit with great cost and only modest improvement, with limited data showing even 50% of wounds healing within 12 weeks. While the cost of these newly developed approaches varies widely, studies comparing them directly against each other have not been done. We addressed this knowledge-gap via comparative analysis of effectiveness between a cellular matrix (Dermagraft) and an acellular matrix (Oasis), hypothesizing no difference in healing effected by either matrix. This randomized, single-blinded, controlled clinical trial compared DFU-healing effectiveness of two intervention arms: cellular matrix and acellular matrix, and SOC. Over 600 patients were screened, yielding 120 randomized patients from October 2011 – February 2017. Subjects in either intervention arm received up to 8 weekly matrix applications. 103 patients reached the primary endpoint of 12 weeks. 96 patients reached the secondary endpoint of 31 weeks. No significant inter-group demographic or wound characteristic differences were noted (p-values>0.05). 57.14% and 73.53% in the cellular matrix group, and 78.57% and 69.23% in the acellular matrix group healed by 12 and 31 weeks, respectively (p-values >0.05). In the SOC group, 61.2% and 78.3% of wounds healed by 12 and 31 weeks, respectively. The average rate of ulcer healing among three treatment arms was similar (p>0.05). The average wound size in each treatment arm at week 1, week 15, and week 19 were similar (p>0.05). No association was found among offloading methods among groups (p=0.87). The cost of the cellular and acellular matrices was $1081.50 and $107.57 per application, respectively. This comparative analysis reveals that treatment effected by cellular and acellular matrices is comparable at both 12 and 31 weeks, suggesting that the acellular matrix is a more cost-effective treatment than the cellular matrix.

O1.01

Wireless Electrotherapy Improves Healing Of Infected Wounds In Porcine Model
Maksym Krutko1, Nava Rijal1, Rebecca Ruppel1, Taylor Wallen3, Michael Goodman3, Andrei Kogan2, Melanie Cushion4, Daria Narmoneva1
1Biomedical Engineering, University of Cincinnati, Cincinnati, OH, United States 2Physics, University of Cincinnati, Cincinnati, OH, United States 3Surgery, University of Cincinnati, Cincinnati, OH, United States 4Internal Medicine, University of Cincinnati, Cincinnati, OH, United States
Wireless Electrotherapy Improves Healing Of Infected Wounds In Porcine Model

Maksym Krutko1, Nava Rijal1, Rebecca Ruppel1, Taylor Wallen3, Michael Goodman3, Andrei Kogan2, Melanie Cushion4, Daria Narmoneva1 1Biomedical Engineering, University of Cincinnati, Cincinnati, OH, United States. 2Physics, University of Cincinnati, Cincinnati, OH, United States. 3Surgery, University of Cincinnati, Cincinnati, OH, United States. 4Internal Medicine, University of Cincinnati, Cincinnati, OH, United States

Background: Poor vascular supply and infection are significant factors in impaired healing of chronic wounds. We have developed a non-contact wireless electric field (EF) therapy that can stimulate cells within the entire wound. An optimal therapeutic signal for the in vivo experiments is determined using numerical in-situ simulation (ANSOFT/HFSS) for the interactions between the applied EF and underlying wound tissues. The objective of this study is to determine efficacy of this wireless electrotherapy on healing of infected wounds in the porcine model. Hypothesis: the wireless electrotherapy enhances vascularization, reduces bacterial load and promotes wound microenvironment that is conducive to healing. Methods: N=14 wounds (2×2 cm) were created on dorsum of two female white Yorkshire pigs. The wounds were divided into four groups: electrotherapy vs. untreated control, each with or without infection (n=3-4 wounds per group). Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) were delivered post-wounding (1x10e9 of each bacteria) in 0.3mL total volume per wound. Control wounds were inoculated with sterile PBS only. Wounds were covered with Tegaderm dressing. Electrotherapy was applied 1 hr/day, 3-5 days/week, for 4 weeks. The effects of the electrotherapy on wound closure, bacterial load (scab or swab analyses), and gene expression (RNAseq analyses) were quantified. Results: Electrotherapy resulted in significant reduction in the wound area at 2 and 3 weeks post-surgery for both infected and control (non-infected) wounds (p<0.001) and significantly faster wound closure (p<0.01). The results demonstrate significantly reduced levels of both types of bacteria in electrotherapy-treated wounds at 2 weeks post inoculation. Genomic analysis revealed significant upregulation in key pathways and individual genes responsible for wound healing such as angiogenesis, antibacterial humoral response, ECM remodeling, and activating immune responses (p<10-4-10-6, ssGSEA and DEseq), with analyses currently ongoing. Successful application of the combined experimental approach demonstrates that wireless electrotherapy enhances infected wound healing via deep stimulation of wound tissues, increasing rate of wound closure, reducing bacterial colony count, and upregulating genes wound healing related genes. Conclusions: The results demonstrate that wireless electrotherapy is effective in reducing wound infection and improving healing, suggesting the promise of this strategy to improve healing outcomes and decrease amputations in patients with infected non-healing ulcers.

O1.02

Galvanotactic Smart Bandage for Chronic Wound Management and Tissue Regeneration
Artem A. Trotsyuk, Yuanwen Jiang, Simiao Niu, Dominic Henn, Kellen Chen, Maddy Larson, Ethan Beard, Aref Saberi, Dharshan Sivaraj, Alana Malaika Mermin-Bunnell, Smiti Mittal, Serena Jing, Sun Hyung Kwon, Clark Bonham, Jagannath Padmanabhan, David Perrault, Melissa C. Leeolou, Michael Januszyk, Zhenan Bao, Geoffrey C. Gurtner
Surgery, Stanford University, Stanford, CA, United States
Galvanotactic Smart Bandage for Chronic Wound Management and Tissue Regeneration

Artem A. Trotsyuk, Yuanwen Jiang, Simiao Niu, Dominic Henn, Kellen Chen, Maddy Larson, Ethan Beard, Aref Saberi, Dharshan Sivaraj, Alana Malaika Mermin-Bunnell, Smiti Mittal, Serena Jing, Sun Hyung Kwon, Clark Bonham, Jagannath Padmanabhan, David Perrault, Melissa C. Leeolou, Michael Januszyk, Zhenan Bao, Geoffrey C. Gurtner Surgery, Stanford University, Stanford, CA, United States

Background: Approximately thirty million people in the United States suffer from diabetes. The prevalence of diabetic foot ulcers (DFUs) in this population is 13%. 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. To our knowledge there have not been significant advancements in incorporating sensing technologies to deliver active wound care. We believe this can be achieved using a multidisciplinary approach combining electrical and chemical engineering with the fundamentals of cellular and biomolecular processes in wound healing directed towards high resolution, in situ tissue regeneration. Methods: A flexible printed wireless stimulator was designed and fabricated to deliver directional energy across a wound gradient. Subsequently a low impedance PEDOT:PSS electrode was designed to optimize the skin and stimulator interface, producing a robust gel with tunable adhesion properties. The smart bandage was evaluated in an excisional diabetic and C57BL6/J murine wound healing model. A parabiosis model was used to evaluate circulating cell migration into the wound bed. Single cell analyses were performed to evaluate changes in cell populations as a direct result of induced electrical stimulation. In vitro validation was performed to elucidate in vivo results. Results: Wireless electrical stimulation resulted in significantly accelerated wound closure, when compared to controls, in both a diabetic and C57BL6/J murine excisional wound healing model. Complete epidermal recovery was observed, with a thicker collagen network and increased dermal thickness. Greater neovascularization and appendage formation were observed in the treatment groups. Single cell analyses revealed higher proliferation and remodeling regulatory markers expressed across treated groups. In vitro co-culture validation experiments demonstrated accelerated proliferation, mitotic rate and tube formation when compared to controls. Conclusion: Our data demonstrates the functionality of a robust wireless interface for wound healing. This novel treatment modality will integrate AI processing components for the development of a closed-loop functional stimulator. By combining the domain expertise of nanofabrication, mechanotransduction, fibrosis and molecular/cellular analyses, we are developing a novel chronically stable and robust smart bandage that will pave the way for the next generation of palliative wound care.

O1.03

Using Membrane Acting Agents And Ultrasound To Overcome Biological Barriers And Eradicate A Methicillin Resistant Staphylococcus Aureus Wound Infection In Diabetic Mice
Sarah E. Rowe-Conlon1, Ashelyn Sidders1, Phillip Durham2, Paul Dayton2, Brian Conlon1, Virginie Papadopoulou2
1Microbiology and Immunolgy, UNC Chapel Hill, Chapel Hill, NC, United States 2Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, Chapel Hill, NC, United States
Using Membrane Acting Agents And Ultrasound To Overcome Biological Barriers And Eradicate A Methicillin Resistant Staphylococcus Aureus Wound Infection In Diabetic Mice

Sarah E. Rowe-Conlon1, Ashelyn Sidders1, Phillip Durham2, Paul Dayton2, Brian Conlon1, Virginie Papadopoulou2 1Microbiology and Immunolgy, UNC Chapel Hill, Chapel Hill, NC, United States. 2Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, Chapel Hill, NC, United States

Bacterial biofilms are the leading cause of delayed healing in chronic wounds. Biofilms display remarkable tolerance to antibiotics as they impede penetration of drugs and even when drugs do penetrate, cells within a biofilm are in an antibiotic tolerant state. Aminoglycosides, such as gentamicin (Gent), are ineffective against biofilm cells as they maintain proton motive force below the threshold for drug uptake. We employ a novel aminoglycoside adjuvant, palmitoleic acid (PA), to facilitate drug uptake. Here we propose a dual strategy to eradicate a chronic wound infection; utilizing ultrasound-stimulated phase change contrast agents (US+PCCA) to improve the penetration of the novel drug combination Gent/PA. A chronic wound infection model was established in SKH-1 hairless mice; 6-8 week old mice were treated with streptozocin to induce diabetes. A circular wound was created on the back of the mice and infected with methicillin resistant Staphylococcus aureus (MRSA). Wounds were treated topically with Gent and/or PA, twice daily for 4 days. One daily antibiotic treatment was combined with US+PCCA. On day 5, mice were euthanized, the wound area was excised and plated to enumerate bacterial survivors. Neither Gent nor Gent/PA reduced bacterial burden but both treatments were significantly improved by applying US+PCCA to improve drug penetration (Kruskal Wallis One-Way ANOVA with Dunn’s multiple comparison test). Importantly, bacteria were eradiated from 3 out of the 8 wounds in the Gent/PA group that were treated with US+PCCA. This data shows that improving penetration of a novel anti-biofilm drug combination is a viable strategy to eradicate biofilms in chronic wounds. Palmitoleic acid is an attractive antibiotic adjuvant as it has previously shown to reduce inflammation and accelerate wound healing. This therapeutic strategy of utilizing ultrasound to improve penetration of anti-biofilm drugs has the potential for clinical translation as ultrasound is already used in wound debridement and the PCCA formulation is a variant of FDA approved contrast agents currently used in diagnostic imaging.

O1.04

Fibroblast Clearance Of Damaged Tissue Following Laser Ablation Of Engineered Microtissues In Vitro
Megan Griebel, Anish Srinivas Vasan, Christopher Chen, Jeroen Eyckmans
Boston University, Brookline, MA, United States
Fibroblast Clearance Of Damaged Tissue Following Laser Ablation Of Engineered Microtissues In Vitro

Megan Griebel, Anish Srinivas Vasan, Christopher Chen, Jeroen Eyckmans Boston University, Brookline, MA, United States

Background: Clearance of damaged and necrotic tissue is a critical step for wounds to heal. Although the role of immune cells, such as neutrophils and macrophages, in the autolytic debridement of wounds is appreciated, it is unclear if fibroblasts are able to contribute to this process. Herein, we demonstrate fibroblast-mediated wound clearance following laser ablation of engineered microtissues prior to wound closure. Methods: Collagen type I laden with 3T3 fibroblasts is added to polydimethylsiloxane (PDMS) micro-wells, each containing four PDMS pillars. Following collagen compaction, a rectangular (1 mm X 0.6 mm) microtissue is suspended between the pillars. Full-thickness wounds between 100 mm and 200 mm in diameter are generated either mechanically, using a microdissection knife, or by ablation, using a nanosecond-pulsed Nd:YAG laser. Following injury, live microscopy is used to track and quantify the change in wound area. To study the role of cytoskeletal dynamics in clearance of the wound extracellular matrix (ECM), inhibitors of cellular contractility (Y-27632, blebbistatin, MLCK inhibitor peptide-18), microtubule polymerization (Nocodazole), and clathrin-mediated endocytosis (dynasore) were added to the microtissue prior to injury. Results: Ablated tissues displayed significantly more cell death (up to 10% of tissue area) at the wound perimeter than microtissues injured using a microdissection knife (<1% by area). Microtissues injured by knife displayed smooth wound edges and began closure (marked by a decreasing wound area) within 2 hours following injury. In contrast, ablated microtissues first displayed wound opening for an average of 6 hours before closure commenced. The rate of tissue opening was decreased by 28% (p<0.001) and 26% (p<0.001) when microtissues were treated with Y-27632 and dynasore, respectively. Transmission electron microscopy imaging of injured microtissues showed elevated number of vesicles containing collagen fibrils, thus suggesting a role for endocytic processes during wound clearance by fibroblasts. Conclusions: By using laser ablation, we are able to generate wounds in vitro that contain damaged tissue, as might be seen in burn wounds or chronic wounds where debridement is impeded. Our data suggest that fibroblasts are capable of clearing damaged tissue prior to wound closure, a process that is mediated by ROCK activity and endocytosis.

O1.06

Electroceutical Technology Against Wound Microbial Biofilm Infection
Nandini Ghosh, Mohamed El Masry, Chandan K. Sen, Sashwati Roy
Surgery, Indiana University, Indianapolis, IN, United States
Electroceutical Technology Against Wound Microbial Biofilm Infection

Nandini Ghosh, Mohamed El Masry, Chandan K. Sen, Sashwati Roy Surgery, Indiana University, Indianapolis, IN, United States

Background: Wound biofilms are a challenging and perennial problem encountered by physicians. The CDC estimates that 65% of all human infectious disease is caused by bacteria with a biofilm phenotype and NIH estimates that this number is closer to 80%. Surgical approaches are temporarily productive, however, remain ineffective in eradication of this problem. Electrical principles influence fundamental processes in bacterial biofilm formation biology. In past few years, we have reported the efficacy of electroceutical-based wound dressing (EDTlo and EDThi) treatment in vitro biofilm infection and in vivo wound infection models. Objective: The objective of this work is to develop and optimize an electroceutical dressing based protocol for management of biofilm infected clinical chronic wounds. Methods: An established pre-clinical porcine burn wound biofilm model infected with Pseudomonas aeruginosa (PA01) and Staphylococcus aureus USA 300 (MRSA) was used to establish chronic biofilm infection in wounds. Full-thickness burn wounds (2″x2″) infected with biofilm were treated with EDThi for 28 days post-infection followed by EDTlo until the end of the study (d56). The EDT dressings were changed twice a week throughout the duration of the study (d56). Placebo group included treatment with EDhi (power off) and EDlo (silver only) dressings. Analyses of wound closure (digital planimetry), skin barrier function (TEWL), wound biofilm infection assay (by scanning electron microscopy, immunohistochemistry) were performed. Results: Scanning electron microscope (SEM) images of d14 post-infected wound biopsies showed thick aggregates of bacteria encased in extrapolymeric matrix (EPS) characteristic of bacterial biofilm. The biofilm structures significantly reduced in the EDT treated wounds. Confocal fluorescence microscopy analysis of biofilm infection in wounds exhibited diminished bacterial aggregates in the EDT treated wounds indicative of reduction in biofilm infection. Next, the effect of EDT treatment on host responses was investigated. A significant (p<0.05; n=6) improvement in wound closure accompanied by a robust (p<0.05; n=6) reepithelialization (keratin 14 stating) response was noted in EDT treatment group as compared to sham treated group suggesting a clear improvement in host wound repair response. Conclusion: The data indicated that electroceutical treatment protocol effectively eradicated wound biofilm infection and improved wound repair response in host suggesting therapeutic use of EDT dressings in recalcitrant biofilm infection.

O2.01

Unique Physiology And Intrinsically Delayed Wound Healing In Plantar Skin
Christiane Fuchs1, Katherine J. Stalnaker1, Linh Pham1, Ying Wang1, Clifton L. Dalgard2, Sunghun Cho3, Jon H. Meyerle3, Joshua Tam1
1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, United States 2The American Genome Center, Uniformed Services University of the Health Sciences, Bethesda, MD, United States 3Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States
Unique Physiology And Intrinsically Delayed Wound Healing In Plantar Skin

Christiane Fuchs1, Katherine J. Stalnaker1, Linh Pham1, Ying Wang1, Clifton L. Dalgard2, Sunghun Cho3, Jon H. Meyerle3, Joshua Tam1 1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, United States. 2The American Genome Center, Uniformed Services University of the Health Sciences, Bethesda, MD, United States. 3Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States

Background: Diabetic foot ulcers (DFUs) have devastating health and socioeconomic impacts. Previous research in DFU has focused primarily on diabetes-related pathologies, while the “foot” aspect has been mostly neglected, despite the many well-characterized specializations of plantar skin, including lack of hair, unique epidermal structural, accelerated epidermal turnover, poor barrier function, and keratin 9 expression. In this study we investigated how plantar-specific properties could impact wound healing. Methods: This study utilized the porcine model because of anatomic and physiologic similarities between porcine and human skin. Skin samples were collected from the trunk, snout, plantar and dorsal foot. Structural features were characterized by standard histology and transmission electron microscopy. Tissue calcium distribution and calcium signaling in primary keratinocytes were evaluated using calcium indicator dyes. Gene expression profiles were compared by RNAseq. Immunohistochemistry confirmed protein expression and distribution in the porcine skin samples, with key findings validated in human plantar and abdominal skin samples. A new porcine plantar wound model was developed to compare the wound healing process between plantar, trunk, and dorsal foot skin. Results: Plantar skin showed many unique characteristics, including greatly thickened epidermis composed of hypertrophic keratinocytes, a compact stratum corneum that is structurally distinct from non-plantar skin, absence of epidermal calcium gradient (thought to be critical for keratinocyte differentiation), and a gene expression profile that clusters away from other skin types, but shares certain similarities with activated wound keratinocytes. Of particular interest was stromal interaction molecule 1 (STIM1), which showed diminished protein expression in both porcine and human plantar skin. STIM1 is a key regulator of intracellular calcium storage, and a critical component of the store-operated calcium entry (SOCE) response which, due to its ubiquitous utility in calcium second messenger signaling, is involved in many wound healing processes. Reduced STIM1 in plantar epidermis is consistent with its diminished calcium gradient, and predicts the blunting of SOCE and associated downstream pathways. Using primary keratinocytes, we verified that plantar keratinocytes have a substantially diminished SOCE response. Finally, wound contraction and re-epithelialization are both delayed in porcine plantar wounds, causing wound closure time to more than double that of non-plantar control wounds. Conclusions: We have found that plantar skin is more distinctive from “normal” skin than previously realized, with intrinsically delayed wound healing, and that these properties are likely related to altered calcium signaling. The atypical physiology and wound healing process in plantar skin are likely to contribute significantly to the pathology of plantar ulcers.

O2.02

Genomic And Phenotypic Fibrotic Cutaneous Response To Radiation Injury Extends Beyond The Treatment Field In A Murine Model Of Radiation-Induced Skin Fibrosis
Nathan Balukoff1, Jamie L. Burgess1, Tongyu Wikramanayake1, Gregory Azzam2, Stuart Samuels2, Sharon Elliot3, Marjana Tomic-Canic1, Rivka C. Stone1
1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States 2Department of Radiation Oncology, University of Miami Miller School of Medicine, Miami, FL, United States 3Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States
Genomic And Phenotypic Fibrotic Cutaneous Response To Radiation Injury Extends Beyond The Treatment Field In A Murine Model Of Radiation-Induced Skin Fibrosis

Nathan Balukoff1, Jamie L. Burgess1, Tongyu Wikramanayake1, Gregory Azzam2, Stuart Samuels2, Sharon Elliot3, Marjana Tomic-Canic1, Rivka C. Stone1 1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States. 2Department of Radiation Oncology, University of Miami Miller School of Medicine, Miami, FL, United States. 3Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States

Progressive skin fibrosis develops in a subset of patients following high-dose radiation exposure in the oncology and interventional radiology settings. Radiation-induced skin fibrosis is a debilitating disease with devastating progression that results in long-term disability, discomfort, and negative impact of quality of life. For this study we sought to establish a murine model in which fibrosis is induced by high-dose irradiation of skin. C57BL/6 wild-type mice (n=6) were irradiated with a single dose of 18 Gy for 8 minutes, maintained under standard conditions for 24 weeks, and sacrificed for collection of skin from irradiated and adjacent non-irradiated skin and compared with non-irradiated murine controls (n=3). To quantify fibrosis, dermal thickness was measured in hematoxylin and eosin (H&E)- and Masson Trichrome-stained tissue sections. Hydroxyproline content was measured via colorimetric assay (Sigma) as an assessment of total collagen levels. RNA-Sequencing of murine skin from irradiated and non-irradiated sites was performed and compared with untreated controls, and transcriptomic profiles were analyzed using Partek Flow (Partek Inc.) and Ingenuity Pathway Analysis (Qiagen) for pathway enrichment analyses. We observed that in addition to developing alopecia and hypopigmentation at the irradiated site, mice developed histologic evidence of fibrosis as demonstrated by increased dermal thickness on H&E and Masson Trichrome-stained sections (p<0.005). There was no significant difference in hydroxyproline (collagen) content in irradiated versus non-irradiated areas of each mouse; however, collagen was increased in irradiated mice compared with untreated (non-irradiated) controls (p<0.05). Expression of 665 fibrosis-associated genes were similar in RNA-seq profiles from irradiated and non-irradiated areas, but distinct from mice that had no exposure to radiation. In conclusion, we successfully induced fibrosis in murine skin via delivery of high-dose radiation as the source of injury. Fibrosis in irradiated mice was evidenced by increased dermal thickness, increased hydroxyproline content, and evidence of fibrosis-associated gene and pathway induction at the genomic level in RNA-seq profiles. Notably, the irradiated and non-irradiated areas of each mouse appeared phenotypically distinct, but the pro-fibrotic effects of radiation extended beyond the treatment field. Evidence of fibrosis in areas outside of the radiation treatment field highlight the importance of prophylactic and therapeutic approaches to minimize off-target effects and to prevent severe progressive skin fibrosis following radiotherapy.

O2.03

Cellular Mechanism of Highly-Active or Antiretroviral Therapy-Induced Lipodystrophy: Atazanavir, A Protease Inhibitor, Compromises Adipogenic Conversion of Adipose-Derived Stem/Progenitor Cells through Accelerating ER Stress-Mediated Cell Death in Differentiating Adipocytes
Sadanori Akita
Fukushima Medical University/Jonaikai, Shimabara, Nagasaki, Japan
Cellular Mechanism of Highly-Active or Antiretroviral Therapy-Induced Lipodystrophy: Atazanavir, A Protease Inhibitor, Compromises Adipogenic Conversion of Adipose-Derived Stem/Progenitor Cells through Accelerating ER Stress-Mediated Cell Death in Differentiating Adipocytes

Sadanori Akita Fukushima Medical University/Jonaikai, Shimabara, Nagasaki, Japan

Background: Lipodystrophy is a common complication in human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART) or antiretroviral therapy (ART). Previous studies demonstrated that endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) is involved in lipodystrophy; however, the detailed mechanism has not been fully described in human adipogenic cell lineage. Methods: Adipose tissue-derived stem cells (ADSCs) obtained from human subcutaneous adipose tissue was used, and atazanavir (ATV), a protease inhibitor (PI), was administered to ADSCs and ADSCs undergoing adipogenic conversion. Results: Greater repression of adipogenic differentiation was observed when ATV was administered during 10 days of ADSC culture in adipogenic differentiation medium. Although ATV had no effect on ADSCs, it significantly induced apoptosis in differentiating adipocytes. ATV treatment also caused the punctate appearance of CCAAT-enhancer-binding (C/EBP) protein homologous protein (CHOP), and altered expression of CHOP and GRP78/Bip, which are the representation of ER stress, only in differentiating adipocytes. Administration of UPR inhibitors restored adipogenic differentiation, indicating that ER stress mediated UPR was induced in differentiating adipocytes in the presence of ATV. It is also observed autophagy, which was potentiated in differentiating adipocytes by ATV treatment. Thus, adipogenic cell atrophy leads to ATV-induced lipodystrophy, which is mediated by ER stress-mediated UPR and accelerated autophagy, both of which would cause adipogenic apoptosis. Conclusion: As our study demonstrated for the first time that ADSCs are unsusceptible to ATV and its deleterious effects are limited to the differentiating adipocytes, responsible target(s) for ATV-induced lipodystrophy may be protease(s) processing adipogenesis-specific protein(s).

O2.04

The Role Of Sp Transcription Factors In Appendage Regeneration, From Fins To Feet
David A. Brown1, Grant Darner2, Timothy Curtis2, Jianhong Ou2, Thomas Dvergsten2, Veronica Han2, Sydney Jeffs3, Kenneth Poss2
1Plastic Surgery, Duke University, Durham, NC, United States 2Cell Biology, Duke University, Durham, NC, United States 3School of Medicine, Duke University, Durham, NC, United States
The Role Of Sp Transcription Factors In Appendage Regeneration, From Fins To Feet

David A. Brown1, Grant Darner2, Timothy Curtis2, Jianhong Ou2, Thomas Dvergsten2, Veronica Han2, Sydney Jeffs3, Kenneth Poss2 1Plastic Surgery, Duke University, Durham, NC, United States. 2Cell Biology, Duke University, Durham, NC, United States. 3School of Medicine, Duke University, Durham, NC, United States

Background: A first critical step in limb or appendage regeneration is re-epithelialization of the amputation stump to form a specialized regeneration epidermis, which functions as a signaling center to initiate reformation of the structure from underlying mesenchyme. We hypothesized that common factors exist in the regeneration epidermis among species with innate abilities in appendage regeneration. Methods: The regeneration epidermis of zebrafish caudal fin was profiled with single-cell RNA sequencing. Immunofluorescence and in situ hybridization were used to characterize gene expression. Zebrafish null mutants were generated with the CRISPR/Cas-9 system. Epidermal-specific, inducible deletion mutants in mice were produced by crossing mice expressing CreER under the Krt14 promoter to mice harboring floxed alleles of the target genes. Results: Single-cell RNA sequencing in regenerating zebrafish fin revealed an enriched cell cluster marked by the transcription factor Sp6. Immunofluorescence and in situ hybridization confirmed the expression of Sp6 and other Sp family members in the basal epidermis of regenerating caudal fin as well as regenerating mouse digits. CRISPR/Cas-9 was used to generate null mutants in zebrafish for several Sp genes, which demonstrate a variety of skeletal defects and lethality. Mice deficient for Sp6 and Sp8 exhibit distal limb defects, while induced deletion of Sp8 in the basal epidermis of adult mice before digit amputation display impaired bone outgrowth, suggesting a critical role in this process. Intriguingly, we also detected Sp6 and Sp8 in the wound epidermis of human leg amputations. Upcoming work will identify transcriptional targets of Sp8 in regenerating mouse digits and human leg amputation wounds with CUT&RUN sequencing. Conclusions: Sp transcription factors may represent conserved genes that participate in appendage regeneration across various species. Future work will define the role of these factors in regeneration-linked gene expression, as well as explore the idea that their expression may be remnants of an incomplete limb regeneration program in humans.

O2.05

Topical Treatment With Antagomir-193B-3P Accelerates Diabetic Wound Healing
Jelena Marjanovic, Ivan Jozic, Rivka C. Stone, Cheyanne Head, Beatriz Abdo Abujamra, Robert S. Kirsner, Hadar Lev-Tov, Irena Pastar, Marjana Tomic-Canic
Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States
Topical Treatment With Antagomir-193B-3P Accelerates Diabetic Wound Healing

Jelena Marjanovic, Ivan Jozic, Rivka C. Stone, Cheyanne Head, Beatriz Abdo Abujamra, Robert S. Kirsner, Hadar Lev-Tov, Irena Pastar, Marjana Tomic-Canic Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States

Background: One of the major contributing factors of healing impairment of diabetic foot ulcer (DFU) is impaired re-epithelialization marked by diminished migration and hyperproliferation of keratinocytes. We used laser capture microdissection (LCM) and genomic approaches to identify specific miRNAs that potentially can contribute to this keratinocyte non-healing phenotype and found microRNA-193b-3p (miR-193b-3p) as one of the top upregulated. Thus, we focused on deciphering the role of miR-193b-3p in epithelialization and therapeutic potential of miR-193b-3p inhibition. Methods: We collected tissue samples from DFUs (n=15), control skin samples (n=15), and human acute wounds (n=3) and performed LCM and transcriptomic analyses using Ingenuity Pathway Analysis (IPA). qPCR was used to analyze miR-193b-3p and expression of its target genes. We performed gain- and loss-of function experiments. Cell migration was analyzed using scratch assay, and re-epithelialization was evaluated using human ex vivo and diabetic murine in vivo wound models. Formation of stress fibers involved in cell migration was studied using immunocytochemistry and RhoA pull-down assay. Results: miR-193b-3p was found significantly induced in the epidermis of DFUs, but not in the human acute wounds. Knockdown of miR-193b-3p promoted migration of human keratinocytes, without affecting cell proliferation or cell viability. Moreover, inhibition of miR-193b-3p expression in ex vivo human wounds and diabetic murine in vivo wounds accelerated re-epithelialization. Conversely, overexpression of this miR impaired keratinocyte migration and re-epithelialization in a 3D organotypic wounds. We identified the underlying mechanism of miR-193b-3p anti-migratory activity through a disruption of stress fiber formation and a reduction of RhoA activity, with no effect on lamellipodia or filopodia formation. IPA analysis revealed miR-193b-3p target network that contributes to poor healing in DFUs. We confirmed suppression of downstream targets of miR-193b-3p in DFUs (ARHGEF6, CCND1, IGFBP5, JAK2, KIT, KRAS, MAP3K1, NFAT3, SOS2, TCF4, TNS1) and further showed that this transcriptomic landscape found in non-healing DFUs can be reversed upon miR-193b-3p suppression by topical treatment with antagomirs. Conclusion: We identified miR-193b-3p as an important suppressor of keratinocyte migration in DFUs. Topical treatment with miR-193b-3p antagomiR represents a novel therapeutic approach for promotion of epithelialization in DFUs.

O2.06

Microfibril Associated Protein 5 in Skin Wound Healing
Lin Chen1, Bruna Romana-Souza2, Trevor R. Leonardo1, Junhe Shi3, Chen Han1, Luisa A. DiPietro1
1Center for Wound Healing and Tissue Regeneration, University of Illinois Chicago, Chicago, IL, United States 2Department of Histology and Embryology, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro State University, Brazil 3Institute of Clinical Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
Microfibril Associated Protein 5 in Skin Wound Healing

Lin Chen1, Bruna Romana-Souza2, Trevor R. Leonardo1, Junhe Shi3, Chen Han1, Luisa A. DiPietro1 1Center for Wound Healing and Tissue Regeneration, University of Illinois Chicago, Chicago, IL, United States. 2Department of Histology and Embryology, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro State University, Brazil. 3Institute of Clinical Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China

Recent studies from our lab demonstrated that fibroblasts are phagocytic in healing skin wounds. RNA sequencing of phagocytic fibroblasts led to the identification of Microfibril associated protein 5 (MFAP5, or microfibril-associated glycoprotein 2/MAGP2) as a molecule that is significantly unregulated in phagocytic fibroblasts. MFAP5, a 25 kD serine and threonine rich small protein, is a component of microfibrils in the extracellular matrix (ECM). MFAP5 is known to be closely associated with fibrosis and angiogenesis in certain cancers. Given the role of MFAP5 in cancer, the aim of this study was to investigate the production of MFAP5 in fibroblasts and in a murine model of skin wound repair. Flow cytometric analysis demonstrated that skin fibroblasts constitutively express MFAP5 and that MFAP5 levels increase markedly in actively phagocytic fibroblasts. Microarray and real time PCR analysis of full thickness excisional wounds showed that MFAP5 mRNA expression is significantly upregulated during the proliferative and remodeling phases of healing (day 3 to 21). Immunohistochemical staining demonstrated that MFAP5 was strongly present in the cytoplasm of dermal cells and in the dermal ECM of scars. In addition, MFAP5 was found to be strongly expressed in human keloids. The data shows that fibroblasts can be a source of MFAP5 in wounds, and that the expression of MFAP5 changes significantly over the course of skin wound healing. The findings support a role for MFAP5 in the regulation of fibroblasts, scar formation, and ECM function in skin wound healing. Supported by NIH R01 GM50875, R35 GM139603-01, and F31 DE028747-01

O3.01

A Novel Prehabilitation Protocol: Improving Outcomes And Reducing Morbidity In A Complex Surgical Wound Population
Kylie R. McMath, Subhas Gupta
Wound Care/Plastic Surgery, Loma Linda University Health, Loma Linda, CA, United States
A Novel Prehabilitation Protocol: Improving Outcomes And Reducing Morbidity In A Complex Surgical Wound Population

Kylie R. McMath, Subhas Gupta Wound Care/Plastic Surgery, Loma Linda University Health, Loma Linda, CA, United States

Purpose: Major surgery is associated with a significant decline in functional capacity. While most reconstructive procedures are not considered “major” surgery, some of our procedures do carry significant risks and have associated morbidity. Multiple surgical specialties have begun to implement preoperative patient optimization in an effort to reduce postoperative complications. This has been defined as Prehabilitation. Prehabilitation is defined as a series of activities designed to enhancing an individual’s functional capacity to enable him or her to withstand a forthcoming stressor, such as surgery. Most prehabilitation programs have moved towards a multimodal approach, including medical optimization, preoperative physical exercise, nutritional support, and stress/anxiety reduction. Methods: We developed detailed goals for a prehabilitation program including: – Improving cardiovascular, pulmonary, and/or musculoskeletal function. – Improve balance and reduce the risk of falls. – Reduce anxiety and improve coping with specific cognitive behavioral strategies – Improve quality of sleep with sleep hygiene education. – Optimize surgical outcomes with smoking cessation interventions. – Optimize diet with nutrition counseling. – Educate around postoperative routines to prepare a safe home environment. Following institutional protocol, we enrolled 65 patients undergoing combination procedures and assigned each of them our prehabilitation protocol (Figure 1). The program was designed to occur over an eight-week period. The patients then underwent their scheduled procedures. Outcome variables including hospital length of stay, re-operation within 30 days, major complications, and minor complications were measured. A historical cohort of similar preoperative morbidity-matched was used as a source for control data. Results: The results are summarized in the table attached. Significant differences were found in the complications per patient and the number of patients with any complication. Likert scale patient satisfaction also revealed significantly improved scores in patients in the intervention group frequently attributed to “feeling more connected” to their surgeon and office. Conclusion: Our prehabilitation protocol reduced complications and improved overall satisfaction with combination procedures. While there was almost certainly an increase in preoperative functional capacity in our study patients, this was not discretely measured. As has been well established with enhanced recovery after surgery protocols, a more rapid return to functional capacity after surgery is considered a major factor in complication reduction. With prehabilitation, the patient’s preoperative functional capacity is increased. This implies that the physiologic impact of surgery reducing functional capacity results in higher postoperative capacity, and a likely quicker return to baseline capacity.

O3.02

Racial Disparities In Hidradenitis Suppurativa Management At A Single Center
Nikhi P. Singh2, Kaitlin Burge2, Sean Drummond2, Ryan Zaniewski2, Spencer MacLeod2, Lauren Moradi3, Reagan Hattaway2, Timothy W. King1, Tiffany Mayo4, Jorge de la Torre5
1Division of Plastic Surgery, Department of Surgery, Loyola University Medical Center, Maywood, IL, United States 2Heersink School of Medicine University of Alabama at Birmingham, Birmingham, AL, United States 3School of Medicine, University of South Alabama, Mobile, AL, United States 4Department of Dermatology, University of Alabama at Birmingham,, Birmingham, AL, United States 5Division of Plastic Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, United States
Racial Disparities In Hidradenitis Suppurativa Management At A Single Center

Nikhi P. Singh2, Kaitlin Burge2, Sean Drummond2, Ryan Zaniewski2, Spencer MacLeod2, Lauren Moradi3, Reagan Hattaway2, Timothy W. King1, Tiffany Mayo4, Jorge de la Torre5 1Division of Plastic Surgery, Department of Surgery, Loyola University Medical Center, Maywood, IL, United States. 2Heersink School of Medicine University of Alabama at Birmingham, Birmingham, AL, United States. 3School of Medicine, University of South Alabama, Mobile, AL, United States. 4Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, United States. 5Division of Plastic Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, United States

Introduction: Hidradenitis Suppurative (H/S) disproportionately affects African Americans, requiring medical and/or surgical care. Within we describe those affected by this disease at our institution in order to evaluate potential racial disparities. Methods: IRB exemption (IRB-300006003) was obtained and a retrospective review of 1,187 patients with H/S cared for by plastic surgery and/or dermatology at our institution was performed. Patients were excluded if older than 65. Demographic information, medical history, dermatology treatment history, surgical history, and insurance information was collected. Independent sample t-tests and chi square tests determined statistical significance. Results: Table 1 illustrates demographics, medical comorbidities, and location of H/S across all cohorts. African Americans were more likely than non-African Americans to be afflicted by H/S of the axilla (p=0.006) and groin (p<0.001); and were more likely to undergo medical management only (p<0.001). White patients were more likely to undergo initial excision surgery (p=0.001) and revision surgery (p<0.001) and had higher rates of postoperative infection (p<0.001) compared to non-white cohorts. There was no difference in the rate of resolution of symptoms across cohorts. Time from initial presentation to surgery was shorter for African Americans (107 days) than for non-African Americans (162 days) (p=0.045). Non-African American patients had a shorter time to follow-up following surgery than African American patients, 4.7 to 6.2 days (p=0.032). Non-African American patients had more clinic visits than their African American counterparts, 11 to 6 (p<0.001). Conclusions: African Americans may face disparities in management of this complex and often chronic disease course. Thus, it is important those affected by H/S are managed by a multi-disciplinary team to ensure optimal patient care.

O3.03

GRANZYME K: An Important Mediator Of Cutaneous Inflammation And Psoriasis
Katlyn Richardson1, Christopher Turner1, Hongyan Zhao1, Angela Burleigh2, Richard Crawford1, David Granville1
1Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada. 2Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada
GRANZYME K: An Important Mediator Of Cutaneous Inflammation And Psoriasis

Katlyn Richardson1, Christopher Turner1, Hongyan Zhao1, Angela Burleigh2, Richard Crawford1, David Granville1 1Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada. 2Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada

Background: Granzyme K (GzmK) is a serine protease once believed to induce immune cell-mediated killing. However, recent evidence has challenged this paradigm, suggesting a role for this protease in inflammation. GzmK is abundant in human psoriasis, a cutaneous condition characterized by persistent inflammation and epidermal proliferation forming thick, scaly plaques. The purpose of the present study was to assess the role of GzmK in psoriasis. We hypothesized that GzmK contributes to the onset and progression of psoriasis through the augmentation of inflammation and epidermal proliferation. Methods: GzmK levels and cellular sources were assessed histologically in skin biopsies with and without psoriasis (n=4 each). The role of GzmK was investigated in a murine model of psoriasis, comparing GzmK knockout (K-KO) to wild-type (WT) mice (n=6 per genotype). Psoriasis severity (erythema, squamae) was assessed macroscopically for onset and severity using a modified Psoriasis Area and Severity Index (PASI). Skin tissue extracts were examined for relevant pro-inflammatory markers and epidermal thickness via histology, ELISA, qPCR, and validated in vitro. Results: GzmK-positive cell numbers were elevated 40-fold (p=0.045) in lesional psoriasis skin compared to healthy skin. Mast cells, macrophages, and neutrophils were identified as the predominant cell sources of GzmK. K-KO mice exhibited an average 60% decrease in psoriasis severity compared to WT mice (scores of 4.25 and 7.25 on 8-point modified PASI scale, p<=0.001 at end of trial). K-KO mice exhibited a reduction by approximately 50% (p=0.0053) in inflammatory cell infiltrate compared to WT mice. Pro-inflammatory cytokines, IL-17A and IL-23, commonly elevated in human psoriasis, were reduced an average of 46% (2147 vs 4657 pg IL-17A/mg total protein, p=0.044) and 48% (7448 vs 15491 pg IL-23/mg total protein, p<0.001) in K-KO mice, respectively. K-KO mice also exhibited an average 30% decrease (p=0.014) in epidermal thickness, and 98% decrease (p=0.03) in proliferation markers, compared to WT mice. Conclusion: In conclusion, GzmK is elevated in human psoriasis and may contribute to pathogenesis by provoking inflammation and epidermal proliferation. Our combined murine and in vitro human models of psoriasis suggest that GzmK depletion attenuates psoriasis-like skin severity through reduced inflammatory markers IL-17A and IL-23 as well as keratinocyte proliferation.

O3.04

Nanosilk Spray Prevents The Development Of High Stage Pressure Ulcers
Alyssa E. Vaughn1, Stephen M. Niemiec1, James R. Bardill1, Tanner Lehmann1, Amanda E. Louiselle1, Mark A. Bannerman1, Sudipta Seal2, Kenneth W. Liechty1, Carlos Zgheib1
1Laboratory for Fetal and Regenerative Biology, University of Colorado School of Medicine and Children’s Hospital Colorado, Aurora, CO, United States 2Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL, United States
Nanosilk Spray Prevents The Development Of High Stage Pressure Ulcers

Alyssa E. Vaughn1, Stephen M. Niemiec1, James R. Bardill1, Tanner Lehmann1, Amanda E. Louiselle1, Mark A. Bannerman1, Sudipta Seal2, Kenneth W. Liechty1, Carlos Zgheib1 1Laboratory for Fetal and Regenerative Biology, University of Colorado School of Medicine and Children’s Hospital Colorado, Aurora, CO, United States. 2Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL, United States

Background: Pressure ulcers (PU) are chronic wounds associated with significant morbidity and are a substantial burden to the healthcare system. There are limited therapeutics available for prevention and treatment of PU. Silk fibroin is a biocompatible polymer that can be fabricated into nanostructures, termed nanosilk. Nanosilk is characterized by a high strength-to-density ratio and is hypothesized to strengthen the biomechanical properties of skin. We have previously shown that one application of nanosilk reduces the incidence of PU development. Here, we propose that multiple applications of a nanosilk spray would have improved efficacy at preventing PU progression compared to both a control spray as well as Cavilon spray.

Methods: At 12-13 weeks of age, a 6.35mm diameter steel disk is implanted beneath the external oblique muscle latero-caudally in C57BL/6 mice (n = 59). Mice are given approximately two weeks for the wound to completely heal after implantation. A neodymium bar magnet (6.35 mm diameter, 12.7 mm thick) is applied to the skin superior to the implanted disk for ten, three-hour cycles consisting of two hours with the magnet applied and one hour of rest with the magnet off, with photos taken at the conclusion of each cycle for staging. Six treatment groups were included: one application (1x) of phosphate-buffered saline spray (PBS), two applications (2x) of PBS, 1x and 2x Cavilon spray, and 1x and 2x 8% nanosilk spray. The first treatment application occurred before cycle one, and the second treatment application before cycle five.

Results: PU development was evaluated using the established National Pressure Ulcer Staging System with grades from 0 to 4. Statistical significance was determined by one-way ANOVA (p < 0.05 significant). Two applications of 8% nanosilk significantly decreased the PU stage compared to control, PBS treatment both 1x (p = 0.03) and 2x (p = 0.008), a difference that achieved statistical significance at cycle seven. Additionally, 2x 8% nanosilk was superior to 1x 8% nanosilk at improving ulcer stage (p = 0.02) by the end of 10 cycles. 2x 8% nanosilk also improved ulcer stage compared to 2x Cavilon, though this did not reach statistical significance.

Conclusion: Two applications of an 8% nanosilk spray was more effective at preventing high stage PU than both control and Cavilon solutions. Nanosilk shows significant promise as a potential therapeutic for prevention and treatment of PU.

O3.05

Skin Grafting With Morselized Full Thickness Skin In Recalcitrant Wounds And A Clinical Correlation With A High Viability Full Thickness Morselation Device
Thomas A. Davenport
Plastic Surgery, LIPSG, Syosset, NY, United States
Skin Grafting With Morselized Full Thickness Skin In Recalcitrant Wounds And A Clinical Correlation With A High Viability Full Thickness Morselation Device

Thomas A. Davenport Plastic Surgery, LIPSG, Syosset, NY, United States

Purpose: The purpose of this study is to assess the efficacy of morselized full thickness skin grafting in difficult to heal wounds

Introduction: Full thickness skin grafts are convenient grafts because the closed donor site makes post-op care easier and less painful. Full thickness skin grafts, by the nature of their thickness and limited donor areas, are not optimal grafts for compromised graft beds or very large wounds. Full thickness grafts that could be morselized into thinner segments and spread out over larger areas would have the benefits of the both the full thickness skin graft donor site and the expandability and thin profile of a split graft. We present a series of patients where full thickness skin grafts are morselized manually with surgical scissors and grafted into full thickness wounds. We also describe a device for morselizing fulling thickness skin to deliver high viability skin morsels for grafting.

Methods: Twenty-Seven patients 13 male, 14 females with non-healing wounds were retrospectively studied. All patients initially received conservative management consisting of dressings and regular debridement. Wounds that failed to progress despite standard of care dressings and were stable for at least 6 weeks and who were candidates for split thickness skin grafting were grafted with manually morselized full thickness grafts. Morselized grafts were treated in the same post procedure protocol as the treatment of a split thickness graft with a non-stick xeroform dressing followed by dry sterile dressing. Pictures were taken at each post visit to document wound healing and wounds were declared healed with complete epithelialization.

Results: Twenty-two patients were followed for the 4 months study period with 20/27 (74%) of patients healed at the end of the follow-up period. Of the 7 non-healed patients 4/6 (67%) of patients had partial graft take and 3 patients had complete graft loss. All patients with some degree of failed grafts had multiple co-morbidities with poor graft bed wound etiologies including radiation, vasculitis, pressure wound, diabetic foot wound, lymphedema, and chronic infection.

Conclusion: These results are consistent with split thickness skin graft results for patients with poor skin graft beds and multiple co-morbidities. Prospective studies with wound type stratification will be needed to further access this approach.

O3.06

Early Prognosis Of Chronic Wound Healing By Monitoring Wound Alkalinity: A Survivial Analysis
Pei Wang2, Suvra Pal2, Jon Senkowsky3, Shuxin Li1, Wenjing Hu1, Liping Tang1
1Progenitec, Arlington, TX, United States 2Mathematics, University of Texas at Arlington, Arlington, TX, United States 3Wound Care Limb Salvage Clinic, Texas health Physician Group, Arlington, TX, United States
Early Prognosis Of Chronic Wound Healing By Monitoring Wound Alkalinity: A Survivial Analysis

Pei Wang2, Suvra Pal2, Jon Senkowsky3, Shuxin Li1, Wenjing Hu1, Liping Tang1 1Progenitec, Arlington, TX, United States. 2Mathematics, University of Texas at Arlington, Arlington, TX, United States. 3Wound Care Limb Salvage Clinic, Texas health Physician Group, Arlington, TX, United States

Purpose: The objective of this study was to determine whether wound alkalinity can be used as a monitoring biomarker to prognosticate the healing outcomes of chronic wounds at an early stage.
Methods: Wound alkalinity was monitored by assessing discarded wound fluid-soaked wound dressings, via a disposable and non-contact device – DETEC pH, from 59 patients at their 1st, 2nd, 3rd, 4th, and 5th visits during routine standard of care.

Results: To identify the relationship between wound alkalinity and wound healing progress, survival analysis was used to investigate the time to complete wound healing. For this purpose, the Cox proportional hazards model with time-dependent variables was used that can accommodate both binary (alkaline or non-alkaline) and continuous (wound area) variables. The outcome is the time to complete wound healing in days, with the event of interest being complete wound healing in 90 days. The overall estimated effect of alkalinity was such that the instantaneous healing rate of a patient with non-alkaline (pH<7) wound was 6.87 times that of a patient with alkaline (pH >=7) wound. This effect was significant, with a p-value of = 0.008. Furthermore, the overall estimated effect of percentage wound area was such that an increase in wound area by one percentage would reduce the healing rate by a factor of 0.06 (=0.06%). This effect was also significant, with a p-value = 0.0001. Predicted non-healing probability curves were generated to show how the non-healing (or survival) probabilities would change over time for different alkalinity measurements and different values of wound area. Furthermore, just from the first three measurements of alkalinity condition (corresponding to the first three visits), we can predict the non-healing probability pattern for the entire 12-week duration.

Conclusions: The results support that the survival model can used to prognosticate the complete wound healing outcome based on the alkalinity measurements during their initial and follow-up visits, and the probability of non-healing will decrease when the wound alkalinity is changed from alkaline to non-alkaline following the initial treatments.

O4.01

Diet-induced Obesity Dysregulates Rapid Epigenetic Reprogramming To Hypoxia In Macrophages
Kentaro Takahashi, Jinghua Liu, Jasmine R. Jackson, Norifumi Urao
Pharmacology, SUNY Upstate Medical University, Syracuse, NY, United States
Diet-induced Obesity Dysregulates Rapid Epigenetic Reprogramming To Hypoxia In Macrophages

Kentaro Takahashi, Jinghua Liu, Jasmine R. Jackson, Norifumi Urao Pharmacology, SUNY Upstate Medical University, Syracuse, NY, United States

Background: Macrophages play key roles in tissue homeostasis and wound healing response. Their functions are dysregulated in the patients with metabolic syndrome such as obesity and diabetes, resulting in poor prognosis of wound healing. Epigenetic reprogramming including histone modification causes macrophage dysregulation in response to external stimuli. The response to hypoxic environment has implications in macrophage functions in inflammation. Whether and how obesity influences histone modification under hypoxic environment is unknown. Methods and Results: B6 wild-type mice were fed normal diet (ND) or high-fat diet (HFD) for 4-5 months to induce diet-induced obesity. Bone marrow-derived macrophages (BMDMs) were obtained from both groups. BMDMs were treated in hypoxic condition (1% O2) for 1 hour, and the enrichment of histone 3 lysine 4 tri-methylation (H3K4me3) in BMDM was assessed with high-content imaging analysis. We found that hypoxia increases total H3K4me3 levels in BMDMs from ND, whereas this hypoxia-induced H3K4me3 upregulation was abolished in BMDMs from HFD. ChIP-seq was performed to investigate H3K4me3 peaks downregulated in HFD after hypoxia. Interestingly, 79 genes were identified with downregulated H3K4me3 enrichment at the promoter regions in BMDM from HFD compared to ND after hypoxia. As H3K4me3 in the promoter is associated with active transcription, RNA-seq also revealed that hypoxia-induced upregulation was dampened in 42 genes in BMDM from HFD. Gene ontology analysis further revealed ‘carbohydrate metabolic process’ is associated with the genes identified by multi-omics approach including glycolysis-related genes such as Pgk1, Nupr, and Pfkfb3. Seahorse assay revealed the shift of glycolysis pattern after hypoxia treatment was impaired in HFD BMDM. These results highlight the role of hypoxia-inducible H3K4me3 enrichment in expression of glycolysis-related genes in response to hypoxia and suggest that impairment of this mechanism may cause dysregulation of macrophage metabolism in HFD. Conclusion: Diet-induced obesity impairs hypoxia-induced shift of glycolysis pattern in macrophages via dysregulated H3K4me3 enrichment followed by transcriptional change of glycolysis-related genes.

O4.02

Ossabaw Pigs As A Natural Preclinical Model Of Metabolic Syndrome Mediated Impaired Wound Healing
Nandini Ghosh, Shomita S. Mathew-Steiner, Amitava Das, Mithun Sinha, Savita Khanna, Sashwati Roy, Chandan K. Sen
Surgery, Indiana University, Indianapolis, IN, United States
Ossabaw Pigs As A Natural Preclinical Model Of Metabolic Syndrome Mediated Impaired Wound Healing

Nandini Ghosh, Shomita S. Mathew-Steiner, Amitava Das, Mithun Sinha, Savita Khanna, Sashwati Roy, Chandan K. Sen Surgery, Indiana University, Indianapolis, IN, United States

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

O4.03

Systems Biology Approaches To Understanding Mechanisms Of Chronic Wound Progression
Proma Basu, Manuela Martins-Green
Molecular Cell and Systems Biology, University of California, Riverside, CA, United States
Systems Biology Approaches To Understanding Mechanisms Of Chronic Wound Progression

Proma Basu, Manuela Martins-Green Molecular Cell and Systems Biology, University of California, Riverside, CA, United States

Our previous studies showed that gene expression in cutaneous chronic wounds differs significantly from non-chronic wounds in the first 48 hours post-wounding. Here, we performed an RNASeq analysis using tissue from both non-chronic and chronic wounds to study progression of chronicity. We collected tissues from early stages of chronic wound development (mild chronicity), and when the wound was chronic (full chronicity). A Weighted Gene Correlation Network Analysis (WGCNA) was performed in R on 21919 genes identified in the RNAseq using Kallisto. WGCNA segregated the 21919 genes into 14 modules and also found modules significantly correlated (p<0.05) to mild and full chronicity. Genes belonging to these modules were used for a pathway analysis using pathfindR. Modules significantly correlated to mild chronicity showed enrichment of several pathways. The 'Ephrin mediated growth cone collapse' pathway, leads to poor healing of motor and sensory neurons which often accompany chronic wounds. The 'ErbB signaling' pathway showed that various ligands that bind to EGFR were dysregulated in chronic wounds. For example, Hbegf is highly upregulated in healing wounds but in chronic wounds Hbegf is not upregulated. Application of rHB-EGF restores re-epithelialization in mice. The 'Focal adhesion' pathway showed dysregulated integrins and laminins, molecules that are important in proliferation and migration of fibroblasts and endothelial cells. Finally, in mild chronicity, several pathways were related to fibroblast/myofibroblast dysfunction. The major gene involved in this dysfunction is alpha-smooth muscle actin (Acta1) which was upregulated in chronic wounds. Acta1 overexpression hampers proliferation and fibroblast-mediated repair. In full chronicity, the 'JAK-STAT signaling' pathway was suppressed, a finding that indicates similarity to melanoma development, and undermines the immune system impairing defenses against microbes. Furthermore, the pathway 'Syndecan interactions' was enriched with the gene Sdc1 overexpressed in chronic wounds. In melanoma cells Sdc1 overexpression modulates MMP9 to enhance metastasis. Moreover, the 'Neuroactive ligand receptor interaction' pathway was enriched and Adra2b was overexpressed which downregulates cAMP. Applying cAMP analog to open wounds improved re-epithelialization and granulation tissue formation. In conclusion, our studies show that during development of chronicity, numerous cell and molecular signaling pathways involved in arresting healing are present, and that there are parallels between the processes involved in chronic wounds and melanoma. Testing these pathways in vivo, could lead to the discovery of candidates for treatment of chronic wounds.

O4.04

Fibronectin-Derived Peptide P12 Promotes In Vitro And In Vivo Angiogenesis
Richard A. Clark, Marcia G. Tonnesen, Monica McTigue
Dermatology, Stony Brook University, East Setauket, NY, United States
Fibronectin-Derived Peptide P12 Promotes In Vitro And In Vivo Angiogenesis

Richard A. Clark, Marcia G. Tonnesen, Monica McTigue Dermatology, Stony Brook University, East Setauket, NY, United States

Background: Cyclized P12 (cP12), administered intravenously (iv) at 0.01mg/kg, 1-4h post-burn, limits burn injury progression, speeds healing, and reduces scarring in our porcine vertical injury progression burn model (WRR 24:501-513, 2016). Study Goal: 1. To determine whether P12 promotes cultured human endothelial cells (HEC) growth and in vitro angiogenesis. 2. To determine whether P12 in an angio-reactor mouse model stimulates in vivo angiogenesis. Method: For studies on angiogenesis we used three established methods: 1. XTT assay to determine HEC growth on collagen-coated plates 2. An in vitro angiogenesis assay using HEC cultured on collagen-coated microbeads that were dispersed in a human fibrin gel (Feng, et al. Int J Cell Biol, 13:231-279, 2013). 3. An in vivo angiogenesis assay using nude mice and a commercially available assay kit containing angioreactors and matrix for implanting into the flanks of the mice. Although the kit contained Matrigel as a hydrogel delivery system for potential angiogenesis factors, we found that human fibrin gel for delivery of peptides was superior. Results: P12 increased angiogenesis both in vitro and in vivo. 1. In endothelial cell defined medium with or without vascular endothelial cell growth factor (VEGF), P12 stimulated HDMEC growth in a dose response manner from 3-30uM without VEGF or with 1 or 50ng/ml VEGF. P12 stimulated optimal HDMEC growth when 10uM P12 was added to cultures containing 50ng/ml VEGF. 2. The in vitro angiogenesis assay demonstrated that 3uM P12 with VEGF and Fibroblast Growth Factor-2 (FGF-2) at suboptimal doses (15 ug/ml and 12.5 ug/ml, respectively) doubled sprout angiogenesis from EC-covered beads compared to no P12 under the same conditions. 3. The in vivo angiogenesis assay demonstrated that the addition of 10 or 30uM cP12 to a low dose of FGF-2 (50ng/ml) in fibrin gel increased angiogenesis 7-fold and 15-fold compared to no cP12. Similarly, 10 or 30uM cP12 addition to a low dose of VEGF (125ng/ml) in fibrin gel increased angiogenesis 80% and 130% compared to control. Conclusions: P12, a fibronectin-derived peptide, promotes HEC growth and stimulates angiogenesis by enhancing the effect of angiogenic growth factors in vitro and in vivo.

O4.05

Accelerated Wound Closure In Diabetic Mice Mediated By Extracellular Vesicles With A Defined Protein Payload
Dong Jun Park1, Erika Duggan2, John Nolan2, Brian P. Eliceiri1
1Surgery, University of California in San Diego, San Diego, CA, United States 2Scintillon Institute, San Diego, CA, United States
Accelerated Wound Closure In Diabetic Mice Mediated By Extracellular Vesicles With A Defined Protein Payload

Dong Jun Park1, Erika Duggan2, John Nolan2, Brian P. Eliceiri1 1Surgery, University of California in San Diego, San Diego, CA, United States. 2Scintillon Institute, San Diego, CA, United States

Impaired wound healing in diabetic patients is a major clinical problem in the US, affecting 25% of the diabetic population in their lifetime. Dysregulation of endogenous intercellular communications between stromal and immune cells in the wound bed is hypothesized as the basis for impaired vs. normal wound healing. Here, we focus on the role of extracellular vesicles (EVs) as mediators of these intercellular communications and identify candidate EV protein payloads that distinguish EVs in the wound bed of a mouse model of diabetic (db/db) vs. wildtype (WT) mice. First, we establish an approach for the culture and collection of EVs from a donor site that can be used for adoptive transfer to a recipient wound site. These donor EVs are collected from subcutaneous polyvinyl alcohol sponges of db/db or WT mice, and used to demonstrate that the WT donor EVs promote accelerated wound closure in db/db recipients compared to EVs from db/db donors. These studies define conditions in which WT EVs are sufficient to increase closure in an impaired model of wound healing. Second, we subject EVs from db/db vs WT mice to an unbiased analysis of protein payload using mass spectrometry and identify a hypermetabolic protein profile and evidence to support an impaired remodeling of the extracellular matrix in db/db vs. WT mouse EVs. Finally, we report on a lentiviral-mediated gene delivery system of a reporter fusion construct whereby green fluorescent protein (GFP) is expressed inside an EV to follow trafficking and uptake. With the demonstration that EVs from the WT and db/db wound are distinct on the basis of the biological activity and protein payload, we have a candidate ‘hit’ list and a delivery strategy to test their efficacy and uptake in the wound healing response.

O4.06

Human And Mouse Chronic Wounds Share Common Signaling Pathways
Proma Basu, Manuela Martins-Green
Molecular Cell and Systems Biology, University of California, Riverside, CA, United States
Human And Mouse Chronic Wounds Share Common Signaling Pathways

Proma Basu, Manuela Martins-Green Molecular Cell and Systems Biology, University of California, Riverside, CA, United States

We have previously shown that our murine model of chronic wounds has many characteristics of human chronic wounds. In this study we have compared the data gathered from our mouse model with chronic wounds to that collected from human chronic wounds, to understand the similarities and discover important biomarkers for wound chronicity as well as identify candidates for treatment. We compared our RNAseq analysis to that obtained in human chronic wounds in the last 10 years. Three transcriptomic studies that compared human diabetic chronic wounds to either diabetic or non-diabetic skin were identified. One bulk RNASeq and two single cell transcriptomic studies were identified for comparison. Comparison to the bulk RNASeq study done by Ramirez et al. (2018), identified 318 genes which are significantly differentially expressed (FDR<0.05) and show a similar trend of log fold change in expression in humans and mice chronic wounds. A Reactome pathway analysis showed a prevalence of pathways related to 'EGFR signaling'. Our data indicate that SHC1 and GRB2 mediated stimulation of EGFR activates mitogenic pathways downstream via tgfa, epiregulin, proepiregulin and areg. This overstimulated EGFR signaling is a known biomarker of oncogenic processes. The single cell transcriptomic study published by Theocharidis et al. (2020) identified 206 similarly expressed genes. Overexpression of mmp3, mmp14 and ctsk explained the enrichment of the pathway 'Degradation of ECM'. Additionally, 'Syndecan interactions' was enriched and consisted of fn1, col1a1 and postn, all overexpressed. The upregulation of all three genes is a hallmark of tumor growth and metastasis in melanoma and breast cancer. Postn overexpression in particular reduces cell adhesion augmented by COL1 and FN1 and facilitates metastasis in humans. The study by Januszyk et al. (2020), identified 24 genes clustering significantly in fibroblasts, of those, 22 genes were common with mouse chronic wounds. The pathway with the highest fold enrichment was 'Activation of AP-1 family of transcription factors'. Both jun and fos, which form AP1, were overexpressed in chronic wounds. Again, the pathway 'Syndecan interactions' was enriched and consisted of the genes fgf2, col1a1 and col3a1 which were all overexpressed. Fgf2 overexpression is linked to tumor progression in melanoma. In conclusion, these studies substantiate the efficacy of our mouse model for chronicity studies and provide biomarkers to identify candidates for proof-of-concept studies in humans.