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2023 Abstracts

I.01

Wound Macrophage-Derived Exosomes Enable Keratinocyte Migration For Functional Wound Closure
Anu Sharma1, Adam Anthony2, Sashwati Roy1, David Clemmer2, Chandan Sen1, Subhadip Ghatak1
1Surgery, Indiana University, Indianapolis, IN, United States. 2Chemistry, Indiana University, Bloomington, IN, United States
Wound Macrophage-Derived Exosomes Enable Keratinocyte Migration For Functional Wound Closure

Anu Sharma1, Adam Anthony2, Sashwati Roy1, David Clemmer2, Chandan K. Sen1, Subhadip Ghatak1 1Surgery, Indiana University, Indianapolis, IN; 2Chemistry, Indiana University, Bloomington, IN

Background: In the wound microenvironment, cell-specific exosomes represent an important component of paracrine regulation of tissue repair. We tested the hypothesis that functional wound closure requires successful crosstalk between keratinocytes and wound macrophages (wmΦ) in vivo. Methods: WmΦ derived exosomes in mice were genetically labeled with RFP reporter (Exowmφ) using tissue nanotransfection. Wound-edge (WE) Exowmφ were characterized per MISEV 2018 guidelines and reported in EV-track that received an EV-metric score of 100%. Protein cargo in Exowmφ was detected, validated, and quantified using LC-MS/MS, dSTORM nanoimaging, and flow cytometry. Functional wound closure was evaluated using analytical histology and transepidermal water loss.

Results: Following injury, the Exowmφ was localized at the leading-edge keratinocytes. LC-MS/MS and dSTORM imaging identified the presence of outer mitochondrial membrane (OMM) protein TOMM70 in Exowmφ. At d5 post-wounding, 92.65±1.34% Exowmφ were TOMM70+ (p<0.001; n=6). Such TOMM70-enriched Exowmφ increased keratinocyte migration by 72.05±8.50% (p<0.001; n=5). Wound hypoxia significantly degraded TOMM70 in WE keratinocytes. Such hypoxic loss of keratinocyte TOMM70 inhibited cytosolic PTEN-induced kinase 1 (PINK1) translocation to inner mitochondrial membrane causing PINK1 oligomerization on OMM followed by mitophagy. Hypoxic loss of TOMM70 in keratinocytes was compensated by uptake of TOMM70+ Exowmφ. Downstream PINK1 oligomerization and mitophagy were thus spared in keratinocytes resulting in improved keratinocyte ATP pool necessary to support the metabolic cost of cell migration. Tetraspanins (CD9/CD63/CD81) are specific exosome markers. To block Exowmφ uptake by WE keratinocytes, "eat me not" Exowmφ were generated using LysM promoter-driven tetraspanins plasmid connected via IRES element with "eat me not" CD47 sequence and in-frame GFP reporter. Selective interruption of exosomal crosstalk between wmΦ and WE keratinocytes significantly delayed re-epithelialization and impaired functional wound closure. Interestingly, the same hypoxic signal that depleted WE keratinocyte TOMM70, simultaneously enhanced the endosomal packaging of TOMM70 in wmΦ and induced TOMM70+ Exowmφ release. Under diabetic conditions, Exowmφ uptake by WE keratinocytes is significantly blunted explaining impaired closure that is commonly reported under such conditions. Conclusion: This work lays the foundation for a novel paradigm that addresses the molecular bases of cell-cell crosstalk in wound microenvironment with direct reference to functional diabetic wound outcomes. Of extraordinary significance is the maiden observation that such crosstalk has a direct bearing on wound tissue bioenergetics, a critical factor necessary to pay for the metabolic cost of wound closure.

I.02

Macrophage Phenotype And Function In Response To Staphylococcus Aureus Biofilm Infection In Chronic Wounds
Piya DasGhatak, Fabio Oliveira, Pradipta Banerjee, Subhadip Ghatak, Chandan Sen, Sashwati Roy
Indiana University, Indianapolis, IN, USA
Macrophage Phenotype and Function in Response to Staphylococcus Aureus Biofilm Infection in Chronic Wounds

Piya DasGhatak, Fabio M. Oliveira, Pradipta Banerjee, Subhadip Ghatak, Chandan K. Sen, Sashwati Roy Surgery, Indiana University, Indianapolis, IN

Background: Staphylococcus aureus (SA) is one of highly abundant bacterial species found in infected human chronic wounds. Current studies claim that SA biofilm transforms host macrophages towards an alternatively activated (aka M2 or pro-resolution) phenotype that prevents phagocytosis and suppresses the inflammatory response. However, biofilm infection of wounds is also known to exhibit sustained inflammatory response resulting in non-healing chronic wounds. These two lines of development on wound biofilm infection are in conflict. This led us to investigate the phenotype and function of wound macrophages in wounds infected with SA biofilm. Methods: To specifically address the biofilm component of SA pathogenicity we comparatively used two isogenic mutant strains of SA which are known to possess varying degrees of biofilm-forming ability. Two isogenic mutant strains of the WT SA strain USA300LAC (USA300): the USA300::sarA (ΔsarA) and USA300::rexB (ΔrexB) were used for the studies. The ΔsarA and ΔrexB have hypo- and hyperbiofilm-forming ability, respectively. To study response of macrophages biofilm infected wounds a porcine model of chronic biofilm infection established in our laboratory was used. Mechanistic studies were performed on peripheral blood monocytes (PBMC) cells subjected to conditioned media derived from ΔsarA and ΔrexB biofilm cultures.
Results & Conclusion: scRNAseq and bulk RNAseq studies in our laboratory revealed the presence of a unique macrophage subtype that displays a distinct gene expression signature when exposed to CM derived from a hyperbiofim forming ΔrexB mutant as compared to CM from a hypobiofilm forming ΔsarA mutant. We identified MARCO, a macrophage receptor with collagenous structure, as one of the highly abundant uniquely expressed biofilm-responsive genes in wound macrophages. Significant (P<0.05; n=6) upregulation of MARCO was observed in the ΔrexB treated macrophages in a dose and time-dependent manner as compared to ΔsarA. MARCO is a member of the scavenger receptor that belong to larger group of pattern recognition receptors (PRR) primarily involved in immunosurveillance. MARCO plays important roles in host defense and apoptotic cell clearance activities. Knockdown of biofilm-induced MARCO in macrophages resulted in dysregulated inflammatory responses, suggesting a key role of MARCO in macrophage response to biofilm infection in wounds. Imaging mass cytometry studies in biofilm infected porcine wounds validated the in vitro findings. In summary, this study identifies a SA biofilm inducible wound macrophage receptor with significant role in wound healing.

I.03

Altered N-Glycoproteome of Murine Keratinocyte-Originated Exosomes Under Diabetic Conditions Compromised Resolution Of Wound Inflammation
Xuyao Zeng1, Adam Anthony1, Anu Sharma2, Subhadip Ghatak2, Jonathan Trinidad1, David Clemmer1
1Chemistry, Indiana University, Bloomington, IN, United States. 2School of Medicine, Indiana University, Indianapolis, IN, United States
Altered N-Glycoproteome Of Murine Keratinocyte-Originated Exosomes Under Diabetic Conditions Compromised Resolution Of Wound Inflammation

Xuyao Zeng1, Adam Anthony1, Anu Sharma2, Subhadip Ghatak2, Jonathan Trinidad1, David Clemmer1 1Chemistry, Indiana University, Bloomington, IN; 2School of Medicine, Indiana University, Indianapolis, IN

Background: Unresolved inflammation is a common complication of diabetic wounds. Successful resolution of inflammation relies on the 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 diabetes altered the exosome surface thereby impairing keratinocyte-macrophage crosstalk. Methods: Keratinocyte-derived exosomes (Exoκ) were genetically labeled with a fluorescent reporter (GFP) using tissue nanotransfection. Purified labeled Exoκ were isolated from dorsal murine skin and the wound-edge tissue of diabetic db/db mice and its littermate heterozygous control m+/db mice by differential ultracentrifugation followed by immunomagnetic isolation. Exoκ surface characterization was performed using Charge Detection Mass Spectrometry (CDMS) and glycoproteomic analysis was conducted using LC-MS/MS. Comparative analysis between diabetic keratinocytes and Exoκ with their non-diabetic counterparts was performed. Uptake of diabetic and non-diabetic Exoκ by wound macrophages was monitored using confocal microscopy.
Results: CDMS result from 54974 single Exoκ showed the absence of high mass particles among db/db mice compared to m+/db mice. Using a 2D-Gaussian model, Exoκ was partitioned into 16 subpopulations based on mass and charge. Diabetic wound-edge Exoκ demonstrated a significant decrease in high mass (≥44 MDa) and charge (≥650 e) sub-populations. These features were associated with compromised cargo packaging of Exoκ in diabetic conditions. Comparative glycoproteomics analysis between keratinocytes and Exoκ identified 1083 and 584 glycosylation events mapped to 234 and 66 glycoproteins, respectively. Gene ontology analysis identified glycoproteins functionally related to immune response, inflammation, and wound healing. Exoκ glycosylation patterns between db/db and m+db mice were correlated by converting the degrees of differences into correlation coefficient values using a multi-dimensional correlation method. Such metric allows us to empirically quantify which glycosylation sites most strongly regulated between m+/db and db/db mice. We observed a 19.8% higher fucosylation level in diabetic cells and exosomes, a trend shared among over 80% of fucosylated glycosylation sites. High fucosylation resulted in compromised uptake of Exoκ by macrophages resulting in the release of chemokines for recruitment of inflammatory cells resulting in persistent inflammation.
Conclusion: Findings of this work lay the framework of an emerging paradigm wherein the N-glycoproteome of the wound edge Exoκ determines the fate of wound healing outcomes. Such advancement in our understanding of wound healing may be exploited to design productive wound-care strategies.

I.04

Type III Collagen Regulates Matrix Architecture During Wound Healing
Daniel Stewart1, Becky Brisson1, William Yen1, Malcolm Maden2, Susan Volk1
1University of Pennsylvania, Philadelphia, PA, United States. 2University of Florida, Gainesville, FL, United States
Type III Collagen Regulates Matrix Architecture During Wound Healing

Daniel C. Stewart1, Becky K. Brisson1, William Yen1, Malcolm Maden2, Susan W. Volk1 1University of Pennsylvania, Philadelphia, PA; 2University of Florida, Gainesville, FL

Purpose: Post-natal healing of cutaneous wounds is characterized by development of scar tissue lacking normal architecture and functional integrity of unwounded tissue. Our laboratory has shown that type-III collagen (Col3) deficiency is associated with myofibroblast activation and increased scar formation suggesting Col3 plays a critical role in promoting regeneration and attenuating scar formation. Here we investigate the role of Col3 on collagen architecture in regenerative (Acomys spp.) and scar-permissive (Col3-/-) environments during healing and potential mechanisms by which it governs regenerative responses. Methods: Wound architecture was assessed in 6 mm wounds (paramedian on the dorsum) from Acomys mice and common lab mice, Mus, as well as a tamoxifen-induced Col3 knockdown (Col3F/F) mouse model (Col3B6/B6, wild-type control) at 7, 14, and 35-days post-wounding (DPW). Col3 expression was determined by immunofluorescence. Collagen was imaged with second harmonic generation (SHG) imaging and analyzed with CT-FIRE (fiber characteristics) and the aspect ratio of the FFT (alignment). Myofibroblast activation and mechanotransduction was assessed in Col3+/+ and Col3-/- murine embryonic (E18.5) fibroblasts (MEFs) by αSMA/F-actin co-localization and YAP nuclear translocation, respectively.
Results: Acomys and Mus had similar Col3 expression in unwounded skin with an initial increase in Col3 in wounds at 7 DPW. Acomys showed sustained increased Col3 expression in 14 and 35 DPW compared to Acomys unwounded dermis (p<0.01) and to Mus wounds (p<0.0001 for both timepoints). Acomys wounds had shorter and fewer collagen fibers at 7 and 14 DPW compared to Mus (p<0.01 for both). Acomys had significantly fewer fibers aligned in one direction and more basketweave-like compared to Mus (p<0.005) at 35 DPW. Col3F/F mice had significantly longer fibers by 35 DPW (p<0.01) and more aligned fibers (p<0.005) at 14 and 35 DPW compared to Col3B6/B6 mice. Col3-/- MEFs had significantly more co-localized αSMA+/F-actin stress fibers (p<0.01) and higher nuclear YAP expression (p<0.01) compared to Col3+/+ MEFs.
Conclusions: Our results show that Col3 plays a critical role in promoting tissue regeneration. Collagen architecture exhibited in Acomys suggest a pro-regenerative matrix consists of a Col3-rich matrix with architecture akin to unwounded skin rather than a Col3-attenuated and more-aligned architecture exhibited in most post-natal mammalian scars (illustrated in our scar-permissive Col3F/F model). Loss of Col3 also promotes myofibroblast activation by increasing mechanotransduction and directs cell-matrix dynamic reciprocity to further promote scar formation. Further understanding of Col3’s role in matrix architecture may inform better clinical strategies that harness pro-regenerative attributes and optimize tissue healing.

I.05

Engineered Antioxidant and Oxygen-Releasing Lignin Composites Improve Diabetic Wound Healing
Olutoye Oluyinka1, Benjamin W. Padon1, Walker D. Short1, Aditya A. Kaul1, Lane D. Yutzy2, Kenny L. Nguyen2, Fayiz Faruk1, Nabila N. Anika1, Olivia S. Jung2, Phillip Kogan1, Ling Yu1, Hui Li1, Jangwook P. Jung2, Swathi Balaji1
1Department of Pediatric Surgery, Texas Children’s Hospital & Baylor College of Medicine, Houston, TX. 2Department of Biological Engineering, Louisiana State University, Baton Rouge, LA
Engineered Antioxidant and Oxygen-Releasing Lignin Composites Improve Diabetic Wound Healing

Olutoye Oluyinka1, Benjamin W. Padon1, Walker D. Short1, Aditya A. Kaul1, Lane D. Yutzy2, Kenny L. Nguyen2, Fayiz Faruk1, Nabila N. Anika1, Olivia S. Jung2, Phillip Kogan1, Ling Yu1, Hui Li1, Jangwook P. Jung2, Swathi Balaji1 1Department of Pediatric Surgery, Texas Children’s Hospital & Baylor College of Medicine, Houston, TX. 2Department of Biological Engineering, Louisiana State University, Baton Rouge, LA

Introduction: Diabetic wounds are a major clinical problem with few targeted treatments available. Impaired neovascularization, increased levels of reactive oxygen species (ROS), chronic low-grade inflammation and hypoxia associated with diabetes disrupt mechanisms of wound healing. We engineered novel injectable lignin (a natural antioxidant from lignocellulose)-based composites with ROS-scavenging and oxygen-releasing properties and hypothesized that they enhance neovascularization and attenuate inflammation to promote diabetic wound healing.
Methods: We developed 1) Thiolated LignoSulfonate (TLS) nanoparticles to confer thiol-ene chemoselective crosslinking to methacrylated gelatin (GelMA) while maintaining ROS scavenging ability of lignosulfonate and 2) oxygen-producing LignoSulfonate nanoparticles (LS-np) via encapsulation of CaO2. Composites tested includ: Untreated (UNTX), GelMA, GelMA+TLS, GelMA+TLS+LS-np without CaO2 (CPOc), and GelMA+TLS+LS-np with CaO2 (CPO). Dermal fibroblast fibrosis phenotype (PCR array) and microvascular endothelial cell branching on the lignin composites under high glucose conditions was tested in vitro. 6mm stented skin wounds in WT C57BL/6N or diabetic (db/db) mice (8-10 wk;F/M;n=4) were treated on d0 and were harvested at d7,14,28 post wounding and examined for epithelial gap, granulation tissue (HandE), endothelial cells and vessels (CD31), macrophages (F4/80, CD206), VEGF and HIF1a expression. p values by ANOVA.
Results: Elemental analysis by microPXIE confirmed incorporation of 17 times more Calcium in CPO than in CPOc. Oxygen release from CPO lignin composites was maintained for 7 days (700 ppm/day). Weighted gene co-expression network analysis identified 3 hub genes (Mmp3, Stat6 and Col3a1) in fibroblasts on lignin composites. The dual function of CPO composites promoted endothelial cell branching and capillary-like network formation (p<0.05) and corrected high glucose-induced changes in their VEGF and HIF1a expression in vitro. In WT and db/db skin wounds, CPO composites promoted granulation tissue deposition and capillary lumen density at d7(p<0.05). In db/db mice, staining showed reduced VEGF expression in the hyperproliferative leading epidermis of CPO composite-treated wounds at d7, but quantification in the homogenized wound bed showed an increase in VEGF(p<0.01), suggesting dermal angiogenesis is promoted by CPO composites. CPO composite treatment decreased HIF1a expression and macrophage infiltration(p<0.05) in db/db wounds. Negligible scarring was grossly seen in CPO composite wounds, which was supported by robust wound bed and basket weave collagen staining, along with an increase in CD31+ lumens at d14 and 28.
Conclusion: Engineered lignin biomaterials with multiple wound healing-promotive functions, including pro-angiogenic, sustained oxygenation and ROS-scavenging properties can synergistically correct diabetes-associated cell and wound microenvironmental impairments to promote wound healing.

I.06

Cerium Oxide Nanoparticles Conjugated to miR146a and miR23a Decrease Inflammation/Oxidative Stress And Promote Angiogenesis
Elayaraja Kolanthai1, Aadith Menon1, Yifei Fu1, Craig Neal1, Ashwin Babu1, Udit Kumar1, Carlos Zgheib2, Kenneth Liechty2, Sudipta Seal1
1Material Science and Engineering, University of Central Florida, Orlando, FL, United States. 2Department of Surgery College of Medicine, University of Arizona, Tucson, AZ, United States
Cerium Oxide Nanoparticles Conjugated to miR146a and miR23a Decrease Inflammation/Oxidative Stress and Promote Angiogenesis

Elayaraja Kolanthai1, Aadith Menon1, Yifei Fu1, Craig J. Neal1, Ashwin Babu1, Udit Kumar1, Carlos Zgheib2, Kenneth W. Liechty2, Sudipta Seal1 1Material Science and Engineering, University of Central Florida, Orlando, FL; 2Department of Surgery College of Medicine, University of Arizona, Tucson, AZ

Background: Impairments in wound healing are a significant clinical problem in diabetic wounds that affect approximately 30% of diabetic patients. Diabetic wounds do not heal properly due to multiple deficiencies including chronic inflammation and decreased angiogenesis. We have previously shown that miR-146a, an anti-inflammatory microRNA, is downregulated in diabetic wounds, and that the delivery of miR-146a conjugated to cerium oxide nanoparticles (CNP-miR146a) improves diabetic wound healing by decreasing inflammation and oxidative stress. In this study, we hypothesize that CNPs can be conjugated to and efficiently deliver both miR-146a and miR23a (a pro-angiogenic miR) simultaneously.
Methods: CNPs were synthesized by the water-based chemical hydrolysis method. Using carbonyl diimidazole chemistry, miR-146a and miR23a mimics were conjugated to CNPS. After the conjugation process, the conjugated samples were dialyzed against RNAase-free water to remove the unbound miRNAs and nanoparticles. Further, the conjugated samples were cultured with HUVEC cells for 48 h and then analyzed the cytotoxicity using MTT assay. In addition, the commercial angiogenesis assay kit was used to analyze the angiogenesis on miRs transfected HUVECs. Further, the RT-PCR technique quantified pro-cytokine expression on macrophage cells cultured with miRs conjugated samples.
Results: UV-vis and fluorescence spectroscope/microscope was used to determine the angiogenesis and anti-inflammatories regulating miRNAs conjugated with the antioxidant nature of CNPs. In CNPs’ structure, Ce3+/ Ce4+ ratio plays a vital role in acting as antioxidant property. X-ray photoelectron spectroscopy determined that CNPs maintained their ROS scavenging capability following conjugation with miR146a and miR23a mimics. This was further confirmed using a superoxide dismutase activity assay. The cell studies show no cytotoxicity for miRs/CNPs treated samples. miRs transfected HUVECs were cultured according to the angiogenesis assay procedure, and we observed the angiogenic network formation increased as compared to untreated cells. Further, inflammation (IL-6) and ROS reduced in cells treated with miRs/CNPs. According to these results, the dual miRNA decorated CNPs have the potential to reduce inflammation and oxidative stress and increase angiogenesis, thus accelerating the healing of diabetic wounds.
Conclusion: The results demonstrate that CNPs are able to carry and deliver multiple miRs to target different dysregulated processes in diabetic wounds. Furthermore, our study shows that CNPS conjugated to miR146a and miR23a reduce inflammation and enhance angiogenesis, as well as scavenging the ROS in the cellular environment.

I.07

Single-Cell Analysis of p16-Positive Wound Cells During Acute Wound Healing
Maria Shvedova, David Lennon, Daniel Roh
Surgery, Boston University School of Medicine, Boston, MA, United States
Single-cell Analysis of p16-positive Wound Cells During Acute Wound Healing

Maria Shvedova, David D. Lennon, Daniel S. Roh Surgery, Boston University School of Medicine, Boston, MA

Background: Cellular senescence, a biologically active state of permanent growth arrest caused by various stressors like tissue injury, has been identified as an important mechanism of physiological wound healing. Senescent cells (SnC) that arise acutely in cutaneous wounds have proposed beneficial effects in wound healing when they are transiently present, however, specific SnC identities and their associated functions during wound healing are not thoroughly characterized. The purpose of this study was to characterize SnCs (based on high p16Ink4a expression, a common senescence marker) that transiently arise during acute cutaneous wound healing. Methods: Transgenic p16tdTomato mice were used for to visualize p16-positive cells in wound and skin during wound healing and to isolate p16-positive wound cells. A single 1cm full-thickness dorsal wound was created on a 2-month old p16tdTomato mice. In vivo IVIS imaging was used to detect fluorescent p16-positive cells during wound healing. On post-wound Day 6, wound tissue (with short rim of surrounding skin) was digested with collagenase and underwent FACS sorting for p16-high expressing and p16-negative cells followed by single cell RNA sequencing (scRNAseq). Cell clustering and pathway analysis were performed with Celda (cellular latent Dirichlet allocation) and GSVA (gene set variation analysis), respectively.
Results: p16-positive cells were detected during acute wound healing with in vivo IVIS fluorescent imaging of young p16tdTomato mice. There was a peak of p16-positive wound cells on post-wound Day 6 which resolved by Day 12. FACS analysis revealed that 2.1-3.8% of total wound cells were p16-positive. scRNAseq analysis of p16-postiive and p16-negative wound cells demonstrated multiple cell types with p16 expression with more extracellular matrix (ECM) interactions, angiogenesis, and anti-inflammatory signaling pathways in p16-positive cells than p16-negative wound cells. Sub-cluster analysis of macrophages demonstrated p16-positive macrophages were more phagocytic, promoted angiogenesis, cell migration, and fibroblast proliferation compared to p16-negative macrophages. p16-positive fibroblasts demonstrated increased signaling pathways associated with ECM organization and ECM interaction.
Conclusions: SnC cells, as indicated by high p16-expression, during wound healing are transiently present during the inflammatory/proliferative stages of wound healing. There are multiple types of wound SnCs with a majority being macrophages and fibroblasts, both which appear to display beneficial pro-healing signaling pathways.

I.08

Endothelial Dysfunction in Areas of Unburned Skin in a Burn Injury Model: Implications for Autologous Skin Grafting
Edward Kelly1, Bonnie Carney1, Eriks Ziedins1, Lauren Moffatt1, Jeffrey Shupp2
1Firefighter’s Burn and Surgical Research Laboratory, Medstar Washington Hospital Center, Washington, DC, United States. 2The Burn Center, Medstar Washington Hospital Center, Washington, DC, United States
Endothelial Dysfunction in Areas of Unburned Skin in a Burn Injury Model: Implications for Autologous Skin Grafting

Edward J. Kelly1, Bonnie Carney1, Eriks Ziedins1, Lauren Moffatt1, Jeffrey W. Shupp2 1Firefighter’s Burn and Surgical Research Laboratory, Medstar Washington Hospital Center, Washington; 2The Burn Center, Medstar Washington Hospital Center, Washington

Background: Burn shock is mediated by an inflammatory response leading to endothelial cell dysfunction (EnD) and vascular permeability in large total body surface area (TBSA) injuries. Smaller TBSA burns do not induce this systemic EnD. Previous studies have examined markers of EnD following thermal injury and have aimed to characterize this dysfunction in various end-organs. However, there is limited literature investigating EnD in the unburned skin of burn patients. Intact skin with compromised microvasculature could lead to unintended consequences during donor site harvesting for skin grafting to achieve definitive wound closure. For this reason, we sought to examine the presence of EnD in the unburned skin of burn injured animals and the magnitude of dysfunction in relation to other vital organs.
Methods: Sprague-Dawley rats underwent thermal injury creation or sham procedures. Rats were subjected to either 10% or 40% TBSA scald burn and were resuscitated with crystalloid and monitored for 24 hours. Blood was sampled at hour 2,4,8,12 and 24. At necropsy, Evans Blue Dye (EBD) was administered to assess vascular permeability and samples of lung, spleen, liver, kidney and healthy skin were assessed using spectrophotometry. One-way ANOVA was used to compare injured and control groups and two-way ANOVA with multiple comparisons was used to compare organ types.
Results: EnD was not evident in any organ, including intact, unburned skin in 10% TBSA injured animals. EBD extraction was significantly higher in the intact skin of 40% TBSA injured animals vs. controls (86.72 ± 22.53μg/g tissue vs. 31.57 ± 10.80μg/g tissue, p=0.015). EBD extraction was also found to be significantly higher in the lung and spleen of injured animals when compared to controls (901.2 ± 253.8μg/g tissue vs. 278.2 ± 83.14 μg/g tissue, p=0.001 and 566.8 ± 166.7μg/g tissue vs. 233.8 ± 28.66μg/g tissue, p=0.021, respectively). EBD extraction was not significantly different in the liver or kidney of injured vs control animals (p=0.137 and p=0.510, respectively). When comparing magnitudes of EBD extraction, lung and spleen showed significantly higher levels than intact skin while levels in the liver and kidney were not significantly different when compared to skin.
Conclusion: EnD is evident in the unburned skin of thermally injured rats based on a vascular permeability assay. The magnitude of dysfunction is comparable to the endotheliopathy measured in other major organs. Further characterizing this endotheliopathy may have implications for the optimization of skin graft placement and donor site harvesting, as well as healing, scar formation, and pain control associated with donor sites. Amelioration of EnD in the unburned skin prior to harvest may be an important step in improving outcomes.

I.09

ETRS Microfibril-Associated Protein 5 Regulates Skin Fibroblast Function And Scar Formation
Chen Han1, Trevor R. Leonardo1, Bruna Romana-Souza2, Junhe Shi3, Mohamad Altakriti1, Matthew J. Ranzer4, Sammy Ferri-Borgogno5, Samuel C. Mok5, Lin Chen1, Luisa A. DiPietro1
1Center for Wound Healing and Tissue Regeneration, University of Illinois Chicago, Chicago, IL, USA, 2Department of Histology and Embryology, Rio de Janeiro State University, Rio de Janeiro, RJ, Brazil, 3Institute of Clinical Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China 4Department of Surgery, University of Illinois Chicago, Chicago, IL, USA,5Department of Gynecologic Oncology and Reproductive Medicine, University of Texas MD Anderson Cancer Center, TX, USA
Microfibril-Associated Protein 5 Regulates Skin Fibroblast Function And Scar Formation

Chen Han1, Trevor R. Leonardo1, Bruna Romana-Souza2, Junhe Shi3, Mohamad Altakriti1, Matthew J. Ranzer4, Sammy Ferri-Borgogno5, Samuel C. Mok5, Lin Chen1, Luisa A. DiPietro1 1Center for Wound Healing and Tissue Regeneration, University of Illinois Chicago, Chicago, IL, USA, 2Department of Histology and Embryology, Rio de Janeiro State University, Rio de Janeiro, RJ, Brazil, 3Institute of Clinical Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China 4Department of Surgery, University of Illinois Chicago, Chicago, IL, USA, 5Department of Gynecologic Oncology and Reproductive Medicine, University of Texas MD Anderson Cancer Center, TX, USA

Background: Recent studies by our lab show that fibroblasts can act as non-professional phagocytes and engulf apoptotic cells in healing skin wounds. Following apoptotic cell engulfment, fibroblasts develop a fibrotic phenotype with enhanced migration and increased α-SMA expression, along with increased TGFβ1 and collagen production. RNA sequencing of these phagocytic fibrotic fibroblasts showed that microfibril-associated protein 5 (MFAP5, or microfibril-associated glycoprotein 2/MAGP2) is significantly upregulated. MFAP5, a 25 kD extracellular matrix ECM glycoprotein, is linked to fibrosis and angiogenesis in certain cancers and is increased in fibrotic diseases. However, it’s role in wound healing and scar formation is unknown. Therefore, the aim of this study was to investigate MFAP5’s role in wound healing in vivo and its effect on fibroblast phenotype in vitro.
Materials & Methods: Microarray, real-time PCR analysis, and immunofluorescence staining of MFAP5 in full-thickness excisional mouse skin wounds was performed. MFAP5 neutralization was performed in vivo by treating full-thickness excisional mouse skin wounds with anti-MFAP5 antibodies. Angiogenesis in treated wounds was quantified using immunofluorescent staining of CD31, while collagen deposition was evaluated by Masson’s trichrome staining. Wound closure was also measured. In vitro gain of function experiments was done by culturing fibroblasts with recombinant MFAP5. Cellular migration, contractile activity and genomics were then assessed. Results: Mfap5 expression was significantly upregulated during the later proliferative and remodelling phases of healing in vivo. Immunohistochemical localization showed that MFAP5 was predominantly in the cytoplasm of dermal cells and in scars of dermal ECM in vivo. Treatment of mouse wounds with anti-MFAP5 antibodies had no effect on wound closure. However, compared to control, antibody treated wounds had significantly decreased angiogenesis at 7 days post-wounding and decreased collagen deposition at 21 days post-wounding. In vitro, treatment with recombinant MFAP5 significantly enhanced fibroblast migration and contractility. Recombinant MFAP5 also significantly induced fibroblast expression of ACTA2, COL1A1, COL6A3, COL11A1, MMP1, MMP9 and TGFβ1 in vitro.
Conclusions: Together, our results support a novel role for MFAP5 in the regulation of fibroblasts, scar formation, and ECM reconstruction during skin wound healing.

K1.01

Flowable Dermal Hydrogel for Enhanced Biointegration of Acellular Dermal Matrix
Lillian DeCostanza1, Graham M. Grogan2, Anthony C. Bruce1, Kristen A. Atkins3, Evan A. Clark1, Jennifer C. Smith2, Kara L. Spiller4, Chris A. Campbell2, Patrick S. Cottler2
1Biomedical Engineering, University of Virginia, Charlottesville, VA, United States 2Plastic Surgery, University of Virginia, Charlottesville, VA, United States 3Pathology, University of Virginia, Charlottesville, VA, United States 4Biomedical Engineering, Drexel University, Philadelphia, PA, United States
Flowable Dermal Hydrogel for Enhanced Biointegration of Acellular Dermal Matrix

Lillian DeCostanza1, Graham M. Grogan2, Anthony C. Bruce1, Kristen A. Atkins3, Evan A. Clark1, Jennifer C. Smith2, Kara L. Spiller4, Chris A. Campbell2, Patrick S. Cottler2 1Biomedical Engineering, University of Virginia, Charlottesville, VA; 2Plastic Surgery, University of Virginia, Charlottesville, VA; 3Pathology, University of Virginia, Charlottesville, VA; 4Biomedical Engineering, Drexel University, Philadelphia, PA

Background: Functional wound healing requires the coordination of complex cellular processes, including immune response and extracellular matrix remodeling. Acellular dermal matrices (ADMs) have revolutionized implant-based breast reconstruction by providing mechanical and soft tissue support. Tissue integration of the ADM determines surgical outcomes; insufficient wound healing can lead to clinical complications. However, cellular infiltration required for matrix remodeling can be limited by insufficient contact of the ADM and host tissue. We introduce a gap-filling flowable hydrogel derived from decellularized dermis that shows promise in maintaining persistent contact and improving cellular infiltration with minimal immune response. Decellularized porcine dermal (DPD) hydrogel was created by detergent-based decellularization prior to enzymatic solubilization and gelation. C57BL/6J mice received bilateral dorsal subcutaneous ADM implants (5mm diameter). One pocket received 0.5mL of DPD hydrogel and the other served as an ADM-only control. Implants were harvested at 1, 3, and 6 weeks for analysis. Methods: The hydrogel flows easily into the surgical pocket and polymerizes at 37C. H&E staining reveals colocalization of DPD hydrogel with the ADM implant. Pathological analysis of the capsule formation, cellularity, foreign body response, and smooth muscle actin reveals no difference between control and DPD hydrogel (n=13). Decreasing trends as a function of time are seen in both groups (ns, 3-week n=6 and 6-week n=7).
Results: Flow cytometry at 1, 3, and 6 weeks evaluated inflammatory response via a panel of pan-, M1, M2, and transitory macrophage markers. There is a lower percentage of F4/80+ macrophages expressing Arginase 1, an M2 marker, in DPD hydrogel compared to ADM control at 1 week (p<0.05, n=5), but not at 3 weeks (n=6) or 6 weeks (n=5). There are no differences in other M2 markers.
Conclusion: Immunohistochemistry of vimentin, a mesenchymal cell marker, assessed cellular infiltration at 3 and 6 weeks. Percent area trends upward over time and the increase in DPD hydrogel outpaces that of ADM alone (ns, n=4 except 3-week DPD hydrogel n=3). DPD hydrogel flows into the surgical pocket, polymerizes at physiologic temperature, and remains in place for at least 6 weeks, maintaining persistent contact between the ADM and the skin. It does not elicit an increased fibrotic response. While there are decreased Arginase 1 macrophages at 1 week, DPD hydrogel does not cause additional inflammation at later timepoints. Trends of increased cellular infiltration in the presence of DPD hydrogel demonstrate strong wound healing potential.

K1.02

Effects Of A Bioengineered Allogeneic Cellular Construct On Immunosuppressive Macrophages
Lindsay Steele, Beatriz Hernaez-Estrada, Kara L. Spiller
School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, United States
Effects Of A Bioengineered Allogeneic Cellular Construct On Immunosuppressive Macrophages

Lindsay Steele, Beatriz Hernaez-Estrada, Kara L. Spiller School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA

Background: Severe burns are complex injuries that lead to a prolonged suppression of the inflammatory response. Consequently, patients are vulnerable to infection and other complications that impede wound healing. Macrophages are innate immune cells that regulate wound healing by changing their phenotype from M1, a pro-inflammatory phenotype, to M2, a class of phenotypes that can range from pro-reparative to immunosuppressive. In the context of burns, macrophages contribute to the dysregulated inflammatory response by polarizing to an immunosuppressive M2 phenotype. In clinical trials, a bioengineered allogeneic cellular construct (BACC) promotes healing in patients with deep partial thickness burns. We hypothesize that the pro-healing effects of the BACC are mediated, at least in part, by re-directing the macrophage response from an immunosuppressive M2 phenotype to a pro-reparative M2 phenotype. The purpose of this study is to investigate how the BACC modulates macrophage function in the context of burn wounds.
Methods: Primary human macrophages (N=4 healthy donors) were co-cultured with the BACC or collagen controls for up to one week. Macrophages were polarized to the M2b phenotype type, which displays immunosuppressive and immunoregulatory characteristics, by pre-treating with lipopolysaccharide (LPS) and immunoglobulin G (IgG), or left undifferentiated, known as M0, as a control. Macrophage-BACC co-culture was performed with or without LPS in the media to mimic infections that are relevant to burn wounds. Macrophage RNA was isolated and gene expression analysis was performed using a custom panel of 208 genes related to macrophage phenotype, including multiple M2 sub-phenotypes, and wound healing. To assess phenotype changes, we performed gene set analysis using single sample gene set enrichment (ssGSEA). Data was analyzed using paired t-test or one way ANOVA where appropriate and statistical significance was determined by p<0.05.
Results: Gene expression analysis revealed a shift in the undifferentiated macrophage phenotype towards a pro-reparative M2 phenotype, known as M2a, as demonstrated by an increase in the expression of multiple M2a genes, including MRC1, CCL18, and CCL24. Furthermore, the M2a gene set was increased in undifferentiated macrophages by day 6. Secretory analysis revealed a decrease in the secretion of inflammatory proteins, like interleukin 6. On day 5, MRC1 expression was increased in M2b macrophages and CCL18 was increased in M2b macrophages when LPS was present in the media.
Conclusion: These results demonstrate that the BACC promotes the transition of macrophage phenotype towards a pro-reparative M2a phenotype in both undifferentiated and immunosuppressive, M2b, macrophages. This result provides insight into the mechanism by which BACCs promote healing in the burn wound environment.

K1.03

Light-Activated Sealants for Skin Sealing in Diabetic and Immunodeficient Mice Models
Shubham Pallod3, Deepanjan Ghosh3, Russell Urie1, Jordan R. Yaron1, Jacquelyn Kilbourne2, Kaushal Rege1
1School of Engineering, Matter, Transport, and Energy, Arizona State University, Tempe, AZ, United States 2Department of Animal Care and Technology, Arizona State University, Tempe, AZ, United States 3Biological Design, Arizona State University, Tempe, AZ, United States
Light-Activated Sealants for Skin Sealing in Diabetic and Immunodeficient Mice Models

Shubham Pallod3, Deepanjan Ghosh3, Russell Urie1, Jordan R. Yaron1, Jacquelyn Kilbourne2, Kaushal Rege1 1School of Engineering, Matter, Transport, and Energy, Arizona State University, Tempe, AZ; 2Department of Animal Care and Technology, Arizona State University, Tempe, AZ; 3Biological Design, Arizona State University, Tempe, AZ

Background: Traditionally, skin incisions are sealed with non-absorbable nylon sutures. They can suffer from wound dehiscence, cause inflammation, and lead to extensive scarring – especially in immunocompromised or diabetic patients. To address this issue, we developed light-activated sealants (LASE) that are composed of a silk matrix and a chromophore that converts the light into heat upon near-infrared laser irradiation. This causes interdigitation of tissue and sealant proteins – forming a strong bond and sealing the incision. Previously, we developed LASE biomaterials consisting of NIR-absorbing chromophores such as gold nanorods (GNRs) and indocyanine green (ICG) dye and tested them in the conventional murine model – Balb/c. After optimizing the material composition and sealing process in Balb/c, we investigated the efficacy of the laser sealing approach in immunocompromised and diabetic animal models – Balb/c SCID (Severe Combined Immunodeficiency) and db/db respectively.
Methods: We incorporated ICG, copper chloride (CuCl2), or silver nanoprisms (AgNPr) within silk films. ICG-LASE was also loaded with vancomycin to combat bacterial infections and its efficacy in preventing MRSA growth was evaluated using a modified microdilution broth method in vitro and skin incision inoculation method in vivo in BALB/c. The efficiency of wound healing was determined by studying the ultimate tensile strength (UTS) of the healed skin, transepidermal water loss (TEWL), and immunohistochemistry (IHC) on day 2 and day 7 post-wounding and comparing them with the controls.
Results: The incisions were successfully sealed using LASE containing different chromophores in three species of mice – Balb/c, Balb/c SCID, and db/db. The mechanical strength restoration – measured by UTS – was significantly higher in Balb/c and db/db mice, while Balb/c incisions treated with ICG-LASE and db/db incisions treated with AgNPr-LASE also exhibited enhanced barrier function restoration in the early phase of wound healing (day 2). Vancomycin-loaded ICG-LASE exhibited anti-MRSA activity and reduced Methicillin-Resistant Staphylococcus aureus (MRSA) load significantly better than commercial antibacterial sutures in the Balb/c model. The immunohistochemical analysis illustrated increased migration of neutrophils in incisions treated with Cu-LASE towards the wound bed in Cu-LASE treated incisions compared to sutures and AgNPr-LASE-treated incisions.
Conclusion: Our results indicate that this novel approach can be used as an alternative to conventional wound approximation devices such as sutures and staples. These LASE films can also be loaded with bioactive to elicit desired immune phenotype.

K1.04

Synergistic Effect of Scar Reduction Using Topical Application of Captopril-Containing Silicone Gel in a Rabbit Model of Hypertrophic Scar
Eun Young Rha1
1Eunpyeong St. Mary’s hospital, Seoul, Korea (the Republic of)
Synergistic Effect of Scar Reduction Using Topical Application of Captopril-containing Silicone Gel in a Rabbit Model of Hypertrophic Scar

Eun Young Rha Eunpyeong St. Mary’s hospital, Seoul, Korea (the Republic of)

Background: Antihypertensive medications such as angiotensin-converting enzyme (ACE) inhibitors and calcium channel blockers have been used to treat pathological scars. We aimed to compare the difference in the scar-reducing effect of topical silicone and captopril (ACE inhibitor) gel and demonstrate the synergistic effect of scar reduction using a rabbit ear hypertrophic scar model.
Methods: Eight New Zealand white rabbits with 80 wounds in both ears were used. We established control (n = 16), vehicle (n = 16), silicone (n = 16), captopril 50 mg/g (CP50) (n=16), and captopril 50 mg/g-added silicone (silicone+CP50) (n = 16) groups, and started treatment on day 28. The scar elevation index (SEI) was measured by histopathological examination, and scarring was expressed as fibroblast counts, capillary counts, and epithelial thickness. The expression of Ki 67, type I and III collagens, and vascular endothelial growth factor (VEGF) was investigated using immunohistochemistry.
Results: There were significant differences in the median SEI between the five groups (P < 0.0001). However, there were no significant differences between the control and the vehicle, silicone, and CP50 groups. Among the five groups, it was significantly lower in the silicone+CP50 group. There was a significant difference in the median ETI and fibroblast count between the five groups (P < 0.0001). Among the five groups, ETI and fibroblast count were significantly lower in the silicone+CP50 group. The Ki67 proliferation index differed significantly among the five groups (P < 0.0001) and was significantly lower in the silicone, CP50, and silicone+CP50 groups than in the control and vehicle groups. However, there were no significant differences between the silicone, CP50, and silicone+CP50 groups. The median capillary count differed significantly in the five groups (P < 0.0001). Among the five groups, the median capillary count was significantly lower in the silicone+CP50 group. The VEGF expression score also differed significantly among the five groups (P < 0.0001). It was significantly lower in the silicone, CP50, and silicone+CP50 groups than in the control and vehicle groups. Collagen type I expression differed significantly among the five groups (P < 0.0001). It was significantly lower in the silicone and CP50 groups than in the control and vehicle groups. Among the five groups, it was significantly lower in the silicone+CP50 group. Collagen type III expression differed significantly among the five groups (P < 0.0001). It was significantly lower in the CP50 and silicone+CP50 groups than in the silicone group.
Conclusion: Topical application of captopril 50 mg/g alone and captopril 50 mg/g-added silicone effectively reduced scar formation. Captopril and silicone had a synergistic effect on the reduction of pathologic scars. Captopril is an option for scar management in various situations compared to conventional silicone.

K1.05

Incorporation Of Growth Factors Into Cyclodextrin-Based Hydrogels Accelerates Cutaneous Wound Closure
Beatriz Abdo Abujamra1, Jelena Marjanovic1, Fotios Andreopoulos2, Yarixa Diaz Cintron3, Marjana Tomic-Canic1, Stephen Davis1, Francisco Fernandez Lima3, Ivan Jozic1
1Dermatology, University of Miami School of Medicine, Miami, FL, United States 2Biomedical Engineering, University of Miami, Miami, FL, United States 3Chemistry and Biochemistry, Florida International University, Miami, FL, United States
Incorporation of Growth Factors into Cyclodextrin-Based Hydrogels Accelerates Cutaneous Wound Closure

Beatriz Abdo Abujamra1, Jelena Marjanovic1, Fotios Andreopoulos2, Yarixa Diaz Cintron3, Marjana Tomic-Canic1, Stephen Davis1, Francisco Fernandez Lima3, Ivan Jozic1 1Dermatology, University of Miami School of Medicine, Miami, FL; 2Biomedical Engineering, University of Miami, Miami, FL; 3Chemistry and Biochemistry, Florida International University, Miami, FL

Background: Chronic wounds represent a major healthcare burden, affecting more than 6 million people annually and hindering the health care system with $60 billion in costs. Although growth factor-based therapies exhibited tremendous promise, all but one (rhPDGF-BB, aka Regranex) failed in clinical trials. We have recently demonstrated that at in at least two types of chronic wounds (DFUs and VLUs), there is an exuberant upregulation of caveolin-1, which acts to sequester growth factor receptors and thus provides an explanation why growth factor therapy has been largely futile in treatment of chronic wounds. Thus, the purpose of this project was to develop topical hydrogel formulations that target caveolin-1 and release growth factor receptors from sequestration, and then assess whether incorporating growth factors into our cyclodextrin-based hydrogels can accelerate cutaneous wound closure in comparison to growth factors alone.
Methods: We first synthesized collagen and hyaluronic acid hydrogel formulation containing either methyl-beta-cyclodextrin, or (2-hydroxypropyl)-beta-cyclodextrin. Their efficacy to extract cholesterol from cells (primary human keratinocytes) and tissues (3D skin equivalents and ex vivo human skin) was tested using total cholesterol quantification kits and filipin staining assays. Next, we tested effectiveness of our hydrogel formulations to stimulate wound closure in multiple models of wound healing including in vitro organotypic skin equivalents, ex vivo human skin and splinted db/db mouse models in presence or absence of different growth factors (EGF, KGF, PDGF-BB, TGFa and VEGF. Our measures included wound re-epithelialization and epidermal thickness by H&E staining, angiogenesis macrophage polarization by immunohistochemistry, as well as penetration of our cyclodextrin formulations by time of flight secondary ion mass spectrometry (TOF-SIMS).
Results: First, we validated that formulations disrupted cholesterol synthesis using in vitro 3D human skin equivalent cultures and ex vivo human skin, followed by promotion of cell proliferation and migration. Using TOF-SIMS, we were also able to delineate penetration and localization of released from the topical hydrogels. Lastly, we observed that incorporation of each growth factor into our cholesterol depleting hydrogel formulations accelerated wound closure in each wound model tested in comparison to growth factors alone.
Conclusions: Together, these data demonstrate successful pre-clinical testing and provide a new therapeutic strategy for treatment of hard to heal chronic wounds.

K1.06

Twice Applied Nanosilk Cream Protects Against Pressure Ulcer Progression
Anisha Apte1, Tanner Lehmann2, James R. Bardill2, Alyssa E. Vaughn2, Bailey D. Lyttle2, Sudipta Seal3, Kenneth W. Liechty1, Carlos Zgheib1
1Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Arizona Tucson College of Medicine, Tucson, AZ, United States 2Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Colorado School of Medicine, Aurora, CO, United States 3Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL, United States
Twice Applied Nanosilk Cream Protects Against Pressure Ulcer Progression

Anisha Apte1, Tanner Lehmann2, James R. Bardill2, Alyssa E. Vaughn2, Bailey D. Lyttle2, Sudipta Seal3, Kenneth W. Liechty1, Carlos Zgheib1 1Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Arizona Tucson College of Medicine, Tucson, AZ; 2Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Colorado School of Medicine, Aurora, CO; 3Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL

Background: Pressure ulcers (PU) are caused by ischemic-reperfusion injuries from repeated or unrelieved pressure to the skin. PU are associated with significant morbidity and incur a considerable expense to our health care system. We have developed a novel therapeutic from nanofibers of silk fibroin called nanosilk. Silk fibroin is a naturally occurring polymer that is prized for its tensile strength. Nanosilk has been shown to strengthen biomechanical properties of human skin. In this study, we hypothesize that applying 4% nanosilk cream twice, once before and after injury, will prevent PU formation and reduce PU progression in a murine model.
Methods: 12–13-week-old C57BL/6 mice under anesthesia were surgically implanted with a steel disc under the external oblique. Following 2 weeks of recovery, mice were subjected to cyclic application of a disc magnet on skin overlying the implanted disc for a total of 10 cycles. Cycles consisted of 2 hours of the magnet on and 1 hour of the magnet off. Photos were taken at the conclusion of each cycle and PU were graded from 0 to 4 using the National Pressure Ulcer Staging System. Harvested tissue samples were stained for CD45 and trichrome to analyze leukocyte and collagen density, respectively, and quantified using 10 random high-powered field 20x images using NIS Elements – Advanced Research imaging software. Four study groups were included: a non-treated control group, two applications (2x) of control pluronic gel (PLG), 2x Cavilon spray, and 2x 4% nanosilk cream. Each treatment was applied once prior to cycle 1 and a second time prior to cycle 5. Statistical significance was determined by one-way ANOVA (p<0.05).
Results: Application of 4% nanosilk cream prior to injury did not prevent PU formation however it reduced PU severity starting at cycle 5 compared to the untreated control, 2x Cavilon spray, and 2x PLG control. When compared to 2x PLG alone, 2x 4% nanosilk significantly lowered inflammatory cell infiltrate (p=0.017) and increased collagen deposition (p=0.004) in PU at the end of 10 cycles, and there was a trend to significance when compared to 2x Cavilon.
Conclusion: 4% nanosilk cream applied prior to and after PU injury was found to be more effective at preventing high stage PU than both a control gel and Cavilon spray. Tissues treated with 4% nanosilk showed reduced inflammatory cellular infiltration and increased collagen deposition than controls. This is a potential mechanism to explain how nanosilk works.

K2.01

Application Of Supervised Machine Learning In Predicting Major Limb Amputation In Diabetic Foot Patients With Acute Infection
Ren-Wen Huang2, Szu-Han Wang2, Yu-Yao Huang1, Jiun-Ting Yeh2
1Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung Memorial Hospital, Linkou, Taiwan. 2Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan.
Application of Supervised Machine Learning in Predicting Major Limb Amputation in Diabetic Foot Patients With Acute Infection

Ren-Wen Huang2, Szu-Han Wang2, Yu-Yao Huang1, Jiun-Ting Yeh2 1Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung Memorial Hospital, Linkou, Taiwan; 2Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan

Background: Patients with diabetic foot ulcers (DFU) have poor prognoses in the overall 5-year survival rate. How to prevent and cure DFUs is a critical issue. If a patient doesn’t take good care of his ulcer, this patient may encounter acute systemic infection and need multiple debridements, even major limb amputations. In our cohort, we found that some patients received major limb amputation despite undergoing multiple debridements. To avoid unnecessary debridement, we used supervised machine learning to build a prediction model to predict major limb amputation in DFU patients with acute infection.
Methods: We retrospectively collected patients’ data, including age, gender, BMI ratio, initial wound condition, diabetes treatment, diabetes mellitus-related complications, outcome, discharge condition, and laboratory data from January 2018 to December 2020. The data were preprocessed by variable calculation. The low incidence (5-10%) of major limb amputation causes significant data imbalance among the study groups and is unfavorable for the prediction models. Synthetic Minority Oversampling Technique plus Tomek links (SMOTE-Tomek), was performed to balance the dataset. All variables were adjusted with Z-score standardization. The dataset was then divided into training and validation sets at a ratio of 80% to 20%. The prediction models were developed from three machine learning algorithms, K-Nearest Neighbors, Random Forest, and AdaBoost. The prediction models with the best test score were applied to the validation set for the performance evaluations. Model performance evaluation was performed by accuracy value, ROC curve, Precision-Recall curve, and Cohen’s Kappa coefficient. Shapley Additive exPlanations (SHAP) was then used to interpret the model’s prediction.
Results: Among 1241 patients, 92 patients received major limb amputation. Four algorithms proceeded, and Random Forest showed an accuracy of 97.93%, with the best prediction accuracy of all algorithms. It also presented good precision and recall in the model evaluation (Precision: 0.986, Recall: 0.972). Further analysis by SHAP multiclass summary plot showed that the C-reactive protein value was the most influential factor in predicting major limb amputation.
Conclusion: We have provided an applicable and precise model for predicting major limb amputation in diabetes mellitus foot patients with acute infection. This model can prevent patients from receiving non-beneficial debridement surgeries, and it also can reduce the operative risks and hospital stay and improve the prognosis at the same time.

K2.02

Novel Method For Spatial Lipidomic And Proteomic Profiling Of Acute And Chronic Wounds By Mass Spectrometry
Jamie L. Burgess1, Yarixa Cintron2, Beatriz Abdo Abujamra1, Ahmed Hawash1, Francisco Fernandez Lima2, Hadar Lev-Tov1, Marjana Tomic-Canic1, Ivan Jozic1
1Dermatology, University of Miami School of Medicine, Miami, FL, United States 2Chemistry and Biochemistry, Florida International University, Miami, FL, United States
Novel Method for Spatial Lipidomic and Proteomic Profiling of Acute and Chronic Wounds by Mass Spectrometry

Jamie L. Burgess1, Yarixa Cintron2, Beatriz Abdo Abujamra1, Ahmed Hawash1, Francisco Fernandez Lima2, Hadar Lev-Tov1, Marjana Tomic-Canic1, Ivan Jozic1 1Dermatology, University of Miami School of Medicine, Miami, FL; 2Chemistry and Biochemistry, Florida International University, Miami, FL

Background: Millions of Americans suffer from non-healing wounds annually due to aging population and the increasing incidence of obesity and diabetes. Wound healing is a multifaceted process that involves a sequence of biological events characterized by an inflammatory response, followed by proliferation and subsequent remodeling of the wound. Using TOF-SIMS, we demonstrated the localization of specific lipid molecules during acute and chronic wound healing, where healing wounds exhibit a spatiotemporal downregulation of cholesterol. We evaluated the molecular distribution of specific lipids in the epidermis of healed acute wounds and two types of chronic wounds, venous leg ulcers (VLUs) and a diabetic foot ulcers (DFUs). We also combined MS imaging with LC-timsTOF-MS to obtain both lipidomic and proteomic profiles for these wounds.
Methods: De-identified human skin samples were collected from individuals undergoing routine reduction surgeries at UM Wound Clinic. Samples were snap frozen upon collection, cryo-sectioned at 10um thickness and freeze-dried. A TOF-SIMS instrument with a high-spatial resolution LMIG analytical beam (25 keV, Bi3+) was used for chemical mapping of lipids. Samples were then fixed immunostained against K14, K10, K6 and filaggrin to confirm spatial localization. For proteomic analysis, discarded wound dressing exudate was isolated from wound edge and wound bed, and digested samples were analyzed by LC-timsTOF-MS/MS, characterized with PEAKS Studio and validated using commercially available proteome profiler kits.
Results: TOF-SIMS analysis in both positive and negative polarities provided high mass resolution and detection of endogenous signals like adenine (134.1m/z), fatty acids 24-28 (367.4, 381.4, 395.4, 409.4, and 426.4m/z), ceramides (264.3 and 282.3m/z) and cholesterol sulfate (465.3m/z) in both acute and chronic wounds. VLUs and DFUs exhibited upregulation and mislocalization of cholesterol sulfate to all epidermal layers. Likewise, both chronic wounds exhibited downregulation of ceramides and long chain fatty acids, thus suggesting a conserved mechanism of barrier disfunction resulting from disruption of these lipid species. To determine if wound exudate from wound dressings is reflective of this biology, we performed location specific analyses from the center and periphery of the wound dressing. We identified spatially distinct localization of molecules at the center of the wound dressing in comparison to the perimeter, resulting in specific profiles that reflect the biology of each. Upon hierarchical cluster analysis, we observed that both center or perimeter could identify subgroups of specimens that segregate into distinct groups based on their inflammatory, angiogenic, growth factor and MMP/TIMP signature.
Conclusions: Together, these data demonstrate a novel method to spatially map lipidome and proteome of acute and chronic wounds and identify biomarkers of chronic wounds.

K2.03

Comprehensive Transcriptional Characterization Of Genetic And Pathophysiologic Diabetic Wound Healing
Katharina S. Fischer1, Ben Litmanovich1, Sultana M. Mojadidi1, Javier Gonzalez1, Filiberto Quintero1, Mansi Singh1, Dharshan Sivaraj1, Hudson Kussie1, William Hahn1, Andrew Hostler1, Maia Granoski1, kellen Chen1, Geoffrey C. Gurtner1
Surgery, University of Arizona, Tucson, AZ, United States
Comprehensive Transcriptional Characterization of Genetic and Pathophysiologic Diabetic Wound Healing

Katharina S. Fischer, Ben Litmanovich, Sultana M. Mojadidi, Javier Gonzalez, Filiberto Quintero, Mansi Singh, Dharshan Sivaraj, Hudson Kussie, William Hahn, Andrew Hostler, Maia Granoski, kellen Chen, Geoffrey C. Gurtner Surgery, University of Arizona, Tucson, AZ

Background: Diabetes, especially diabetic wound healing, is a major public health concern. Approximately 30 million people in the United States suffer from the diagnosis of type 1 or type 2 diabetes, and these numbers are expected to double by 2050. The underlying mechanisms leading to impaired wound healing are defective signaling in the wound microenvironment and aberrant function of the recruited cells. Most groups use the genetically modified db/db mice to study diabetes, but the pathophysiology of a high-fat induced pre-diabetic and diabetic state more similarly matches the physiology of human diabetes. Here we analyze and compare the wound healing of the different diabetic models at an unprecedented depth. We show the changes and similarities in cell types within the wound bed and show that the HF model is comparable to the db/db diabetes model.
Methods: Full-thickness excisional wounds were created on the dorsum of C57/BL6 wild type (WT) mice, WT mice fed with a high-fat (HF) diet over multiple months to induce pathophysiologic diabetes, and leptin-receptor deficient B6.BKS(D)-Lepr/J (db/db) mice with genetically induced diabetes. Using gross photography, we analyzed wound closure kinetics. Tissue was explanted at days 0, 2, and 7 and processed into single cell suspensions for high-throughput single cell RNA sequencing. Bioinformatic analyses was used to characterize the cell populations within the WT, HF, and db/db mice.
Results: Genetic and pathophysiologic diabetes significantly impaired wound healing compared to healthy WT mice. By day 12, all WT mice had healed, whereas the wounds from the diabetes models stayed unhealed significantly longer, until days 18-20. Looking at the cellular ecology, we were able to identify the cell populations involved in diabetic wound healing. Cells collected from all the mice primarily consisted of fibroblast, macrophage, lymphatic, neutrophil, endothelial, epithelial, smooth muscle, and adipose-derived stromal cells. Specifically, both sets of diabetic mice exhibited an increase in inflammatory myeloid cells (Ptprc, Lyz2) at early time points (d2) and fibroblasts (Col1a1, Pdgfra) at later timepoints (d7), demonstrating a delay in the wound healing kinetics and a shift in gene expression compared to WT mice.
Conclusion: Many different diabetic murine models are being utilized in current research. Type 2 diabetes in humans occurs mainly through long-term consumption of highly fatty nutrition. The murine high-fat induced obesity model represents the most similarities in pathogenesis of diabetes compared to humans. Thoroughly identifying the perturbations in cellular ecology as well as defining if cell dysfunction, rather than cell depletion, drives diabetic wound healing will lead to novel therapeutic approaches to promote proper wound healing.

K2.04

Monocyte/macrophage Heterogeneity During Skin Wound Healing In Mice
Jingbo Pang2, Mark Maienschein-Cline1, Timothy J. Koh2
1Research Informatics Core, University of Illinois at Chicago, Chicago, IL, United States 2Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL, United States
Monocyte/macrophage heterogeneity during skin wound healing in mice

Jingbo Pang2, Mark Maienschein-Cline1, Timothy J. Koh2 1Research Informatics Core, University of Illinois at Chicago, Chicago, IL; 2Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL

Monocytes/macrophages (Mo/MΦ) help to orchestrate the inflammatory, proliferative and remodeling phases of skin wound healing, in part through their ability to adopt a spectrum of phenotypes. However, current understanding of skin wound Mo/MΦ heterogeneity is limited by traditional experimental approaches and the limited number of markers typically used to describe this heterogeneity. Therefore, we sought to more fully explore Mo/MΦ heterogeneity and associated state transitions over the course of both normal and impaired skin wound healing in mice using single cell RNAseq. Live CD45+CD11b+Ly6G- cells were isolated from excisional skin wounds of non-diabetic C57Bl/6 and diabetic db/db mice on day 3, 6, and 10 post-injury and captured using the 10x Genomics Chromium platform. Cells passing quality control filters were embedded into Uniform Manifold Approximation and Projection space and clusters/subsets of cells were identified. For non-diabetic mice, cluster dissimilarity and differentially expressed gene analysis categorized these clusters into three groups: early stage/pro-inflammatory, late-stage/pro-healing, and antigen-presenting phenotypes. There were striking differences between non-diabetic and diabetic mice in all cell clusters identified throughout the healing process. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/MΦ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, cell death, and antigen processing and presentation. qPCR assays validated characteristics of early- vs late-stage Mo/MΦ inferred from our scRNAseq dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity, as well as adoptive transfer experiments, indicated state transitions between early- and late-state Mo/MΦ as healing progressed in non-diabetic mice. Importantly, diabetic mice did not exhibit these state transitions. In summary, our data demonstrate the complexity of Mo/MΦ phenotypes during normal skin wound healing, as well as potential state transitions that likely help to orchestrate efficient healing.

K2.05

Aberrant Wound Healing Response in Hidradenitis Suppurativa
Nathan Balukoff, Jelena Marjanovic, Watcharee Amornpairoj, Nicole Vecin, Andrew Sawaya, Jamie L. Burgess, Paola Catanuto, Ivan Jozic, Hadar Lev-Tov, Marjana Tomic-Canic, Irena Pastar
Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States
Aberrant Wound Healing Response in Hidradenitis Suppurativa

Nathan Balukoff, Jelena Marjanovic, Watcharee Amornpairoj, Nicole Vecin, Andrew Sawaya, Jamie L. Burgess, Paola Catanuto, Ivan Jozic, Hadar Lev-Tov, Marjana Tomic-Canic, Irena Pastar Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL

Background: Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease manifesting with painful nodules and chronically draining tunnels at advanced disease stage. Despite its high prevalence and morbidity, HS remains understudied, with currently no consistently effective treatment strategies. While it is known that HS lesions can progress into large open wounds characterized by hypergranulation tissue, the wound healing process in HS has not been characterized yet. We aimed to evaluate wound closure and keratinocyte motility in HS using newly established ex vivo and in vitro models.
Methods: We have used comparative transcriptomic analysis of HS, human acute wound, and chronic wound profiles to decipher the wound healing signature in moderate to severe HS. Ingenuity Pathway Analysis (IPA) software confirmed robust inflammatory response in HS similar to levels found in acute healing wounds. However, transcriptomic analysis showed that crucial cell motility pathways are either suppressed or not regulated in HS tissue when compared to acute wounds. Some of the most important cell motility signaling we found suppressed in HS and activated in acute wounds were actin cytoskeleton pathway, signaling by Rho family GTPases, and integrin-mediated signal transduction. RhoGDI signaling, an inhibitor of Rho GTPases, was activated in HS and suppressed in acute wounds. Interestingly, absence of epidermal growth factor (EGF) signaling was also associated with HS. In line with these, we found that cell migration of primary keratinocytes isolated from HS is impaired when compared to healthy keratinocytes. Moreover, HS keratinocytes do not even respond to EGF, a well-known wound healing stimulus. Next, we established an ex vivo HS wound model and compared wound re-epithelialization between lesional and location-matched skin.
Results: Histomorphometric analyses demonstrated that HS ex vivo wounds lack re-epithelialization. Moreover, the marker of activated keratinocytes Keratin 17, induced in acute and chronic wound epidermis, was found suppressed in HS ex vivo wounds. Conclusion: Together, we identified unique wound signature in HS tissue characterized by non-resolved inflammation and impaired wound closure. HS keratinocyte migration and re-epithelialization were found impaired due to disorganization of actin cytoskeleton network and diminished expression of Keratin 17. Newly established ex vivo HS model can also enable pre-clinical testing of novel treatments.

K2.06

Recurrent Pressure Injury Risk What Can Transcriptomics Tell Us?
Kath M. Bogie1, Letitia Graves1, Katelyn Schwartz1, M K. Henzel1, MaryAnn Richmond1, Nannette Alvarado1, E R. Chan3, Josie Shiff1, Marinella Galea4, Anna Toth2, Christine Olney2
1Louis Stokes Cleveland VA Medical Center, Cleveland, OH, United States 2Minneapolis VA Health Care System, Minneapolis, MN, United States 3Case Western Reserve University, Cleveland, OH, United States 4James J Peter VA Medical Center, Bronx, NY, United States
RECURRENT PRESSURE INJURY RISK – what can transcriptomics tell us?

Kath M. Bogie1, Letitia Graves1, Katelyn Schwartz1, M K. Henzel1, MaryAnn Richmond1, Nannette Alvarado1, E R. Chan3, Josie Shiff1, Marinella Galea4, Anna Toth2, Christine Olney2 1Louis Stokes Cleveland VA Medical Center, Cleveland, OH; 2Minneapolis VA Health Care System, Minneapolis, MN; 3Case Western Reserve University, Cleveland, OH; 4James J Peter VA Medical Center, Bronx, NY

Background: Chronic wounds have a devastating impact on quality of life for many people with reduced mobility, in particular the elderly and those with spinal cord injury. Our team is investigating the conundrum of why many suffer from a continuous cycle of recurring PrI and long periods of hospitalization while others stay PrI free. We found that it is not just the quantity but the quality of the muscle which impacts tissue health. Increased intramuscular fat infiltration and decreased lean muscle are indicative of a history of severe or recurrent pressure injury. Moreover circulatory biomarkers provide clinically significant risk factors for recurrent pressure injuries. Current studies are identifying multivariate biomarkers, including genetic and epigenetic indicators, that may facilitate earlier identification of individuals at the highest risk for recurrent tissue breakdown.
Methods: RNA sequencing (RNA-Seq) of processed whole blood samples has been carried to examine repeated measures of the transcriptome activity of 60 persons with complete or incomplete SCI (AIS A-D) and known PrI history. Samples were collected at 6-12 month intervals for all participants. RNA was isolated and mixed with Deoxyribonuclease (DNase) to reduce the amount of genomic DNA. Quality control was carried out using the Qubit™ RNA broad range kit and normalized to 50ng/uL prior to sequencing using the NovaSeq 6000 (Illumina) system. Sequence data was analyzed using iPathwayGuide (Advaita Bioinformatics).
Results: Current results indicate 216 active genes that are differentially up- or down-regulated between persons with or without pressure.
Conclusions: Transcriptomic biomarkers appear to differentiate increased risk for recurrent pressure injuries. The huge impact of chronic wounds on quality of life remains challenging. The overall goal of our team is to develop user-centered tools and technologies to enable people to optimize their quality of life. An ongoing multisite repeated measures study is revealing further insights into multi-omic risk for recurrent pressure injuries.

K3.01

GelATA Wound Care Dressing To Combat Hostile Wound Biofilm And Promote Functional Wound Closure
Mohamed El Masry, Subhadip Ghatak, Jessica Jessica Smith, Sashwati Roy, Chandan K. Sen
Indiana Center for Regenerative Medicine & Engineering-Department of Surgery, School of Medicine-Indiana University, Indianapolis, IN, United States
GelATA Wound Care Dressing To Combat Hostile Wound Biofilm And Promote Functional Wound Closure

Mohamed El Masry, Subhadip Ghatak, Jessica Jessica Smith, Sashwati Roy, Chandan K. Sen Indiana Center for Regenerative Medicine & Engineering-Department of Surgery, School of Medicine-Indiana University, Indianapolis, IN

Background: Biofilms represent a major threat to health care and a clinical challenge to manage infected wounds resistant to antibiotic. Persister bacterial phenotypes are a subpopulation of antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested and can resume growth after a lethal stress. The key to managing biofilms of persister bacteria is complete eradication and one approach is to dismantle the structural framework of these biofilms. Extracellular DNA (eDNA) is a major component of the biofilm matrix that maintains structural integrity of the biofilm. DNase treatments eradicate standard biofilms but are ineffective against persister biofilms due to the presence of fragmented eDNA that enhances resistance to disruption by DNase. We reported that a DNase resistant SCV biofilm of Pseudomonas aeruginosa (PAO1DwspF strain) can be disrupted by the FDA repurposed drug aurine tricarboxylic acid (ATA), a chemical inhibitor of covalent binding between eDNA and protein. In the current study, we incorporated ATA into a polymer-based gel (ElastogelTM; SouthWest technologies) to create a dressing (GelATA) that was tested against polymicrobial persister biofilm infection in a preclinical porcine burn wound model.
Methods: Eight 2″x2″ full thickness burn wounds were created on the dorsum of female domestic white pigs (70-80lbs) using a standardized method published by us. The wounds were inoculated at day 3 post-burn with persister biofilm strains of clinical P.aeruginosa (PAO1DwspF) and Staphylococcus aureus (SArexB) at 108 colony forming units (CFU)/ml. Wounds were treated with either a standard-of-care dressing (Acticoat) or GelATA once weekly. At D28 postburn, GelATA treated wounds was switched to ElastogelTM alone until D56. Progression of burn wound healing was followed using non-invasive imaging; Digital images, Harmonic Ultrasound Doppler Imaging (HUSD) and Trans Epidermal Water Loss (TEWL). Histopathological examination and Scanning Electron Microscopy (SEM) of the burn wounds were performed at D56.
Results: SEM imaging of GelATA treated wounds showed disrupted biofilm formation with less bacterial colonization compared to Acticoat treatment. Furthermore, GelATA significantly (p<0.05; n =3) enhanced wound closure and re-epithelialization of persister biofilm-infected wounds (p<0.05; n = 3). Interestingly, improved wound closure with inhibition of biofilm formation resulted in functional healing which was evident by significant decrease in TEWL (p<0.05; n = 3) and improved skin barrier function in GelATA treated wounds.
Conclusion: This work presents first in vivo evidence for the efficacy of the GelATA in disrupting persister biofilm and promoting functional wound closure in a pre-clinical porcine burn wound model.

K3.02

Chronic Wound Microenvironment Mediates Selection Of Antibiotic Resistant And Biofilm Forming Staphylococcus Epidermidis
Miroslav Dinic1, Rebecca Verpile1, Jingjing Meng1, Jelena Marjanovic1, Jamie L. Burgess1, Seth Thaller2, Hadar Lev-Tov1, Marjana Tomic-Canic1, Irena Pastar1
1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States 2Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, United States
Chronic Wound Microenvironment Mediates Selection of Antibiotic Resistant and Biofilm Forming Staphylococcus Epidermidis

Miroslav Dinic1, Rebecca Verpile1, Jingjing Meng1, Jelena Marjanovic1, Jamie L. Burgess1, Seth Thaller2, Hadar Lev-Tov1, Marjana Tomic-Canic1, Irena Pastar1 1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2Department of Surgery, University of Miami Miller School of Medicine, Miami, FL

Background: As one of the most abundant skin-resident bacteria, Staphylococcus epidermidis is primarily known as beneficial as it modulates cutaneous immunity and improves barrier function. However S. epidermidis can also carry a reservoir of antimicrobial resistance genes (ARGs), adding this microbe to the list of “accidental” pathogens. In this study, we aimed to characterize S. epidermidis from chronic venous leg ulcers (VLUs) and healthy skin and evaluate their virulence potential and impact on wound healing.
Methods: Shotgun metagenomic sequencing was performed to analyze presence of ARG and virulence genes in isolates from both environments. Furthermore, antimicrobial susceptibility was tested using the microdilution method. To assess virulence traits biofilm formation and adhesion to extracellular matrix (ECM) were performed. Human ex vivo wound model was used to assess the effect of S. epidermidis isolates on healing.
Results: By employing shotgun sequencing we showed that signatures of ARG, biofilm formation and adhesion capability of VLU isolates strongly corelate with in vitro bacterial traits assessed in antimicrobial susceptibility tests and ECM binding assays. S. epidermidis isolates from VLU associated with methicilin, gentamicin, ampicillin, erythromycin, norfloxacin, tetracycline, and trimethoprim resistance. This was functionally confirmed, VLU isolates showed higher minimal inhibitory concentration values for these antibiotics and for benzalkonium chloride, a widely used wound disinfectant. All VLU strains exhibited stronger ability to bind to ECM and form biofim in vitro, compared to healthy skin strains. Moreover, by using human ex vivo wound model, we demonstrated that isolates from VLUs impaired re-epithelialization through biofilm-dependent induction of pro-inflammatory cytokines IL-1β and IL-8. In contrast, isolates from healthy skin were not capable of forming wound biofilm. Observed ability of VLU isolates to form the biofilm in ex vivo human wounds correlated with the healing outcomes in patients with VLU. We also show that VLU strains form a potent wound biofilm even in the absence of ica operon, aap and sdrF genes, previously thought to be indispensable for biofilm formation.
Conclusion: Our data suggests that chronic wound microenvironment influenced selection of S. epidermidis strains with the prevalence of antimicrobial resistance and biofilm formation. Our data also reflect the dangers of antibiotic and antimicorbial overuse due to the frequency of antibiotic resistance and virulence potential of S. epidermidis strains found in chronic VLU.

K3.03

Upregulation of TNFA via Nfkb Signaling in Human and Mouse Gene Expression Profiles after Exposure to Combusted and E-Cigarettes during Acute Wound Healing
Olutayo M. Alese1, Anna Wilson1, Marissa Macchietto2, Dorothy Hatsukami3, Amy Anne D. Lassig1
1Hennepin Healthcare Research Institute/ University of MN, Minneapolis, MN, United States 2Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN, United States 3Masonic Cancer Center and Department of Psychiatry, University of Minnesota, Minneapolis, MN, United States
Upregulation of TNFA via Nfkb Signaling in Human and Mouse Gene Expression Profiles after Exposure to Combusted and E-Cigarettes during Acute Wound Healing

Olutayo M. Alese1, Anna Wilson1, Marissa Macchietto2, Dorothy Hatsukami3, Amy Anne D. Lassig1 1Hennepin Healthcare Research Institute/ University of MN, Minneapolis, MN; 2Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN; 3Masonic Cancer Center and Department of Psychiatry, University of Minnesota, Minneapolis, MN

Background: Combusted and electronic cigarettes impair cutaneous physiology. We investigated the common gene expression pathways in human and mouse models of acute wound healing in the setting of such exposure.
Methods: A human cohort study of adult combusted cigarette, e-cigarette and non-smoking controls was recruited and enrolled. A punch biopsy was performed on days 0 and 7. For the mouse study, adult C57BL/6J male mice were exposed to research combusted cigarette, JUUL e-cigarette, or control (room air) via whole body inhalation using the Buxco EVT smoke generator for 5 days per week, for 4 weeks prior to a McFarlane dorsal surgical flap elevation. Exposure continued throughout the postoperative period. Punch biopsies were obtained from the flap suture line on day 7. Human and mouse samples were snap-frozen in liquid nitrogen and stored at -80C until downstream processing. RNA was extracted and libraries prepared by standard protocol. RNA was sequenced using the TakaraBio SMARTer Stranded RNA-Seq v2 methods. Differential expression of genes (FDR < 0.05) was performed in edgeR. All tested genes were ranked by their log10(FDR) x direction of their fold change to run through Gene set enrichment analysis which was used for comparisons across the groups. To determine changes in gene expression over time and between treatment groups, we implemented Next maSigPro in R for RNA-seq time-course analysis. A step-wise backward regression ANOVA was applied.
Results and Conclusion: On day 7 of the experiments, twenty common pathways involved in the cell cycle were downregulated in both cigarette and e-cigarette groups compared to non-smokers in humans and mice; supporting impairment in this critical process of acute wound healing in exposure groups. Similarly, TNFA signaling via NFKB, Adipogenesis, Hypoxia, PPAR signaling, and Xenobiotic metabolism pathways were upregulated in cigarette-exposed study groups relative to controls. Out of all pathways, the highest number of shared genes was found in the TNFA signaling via NFKB pathway. Such upregulation of TNFA is associated with a strong pro-inflammatory response as well as effects of cell proliferation, differentiation, and apoptosis. In human and mouse models of acute wound healing, exposure to combusted and e-cigarettes results in similar alterations of gene expression. In particular, overexpression of the pro-inflammatory pathway via TNFA and NFKB at 7 days post-wounding, suggests dysregulated inflammation as key means of delayed acute wound healing and a possible therapeutic target.

K3.04

Mechanical Signaling Induced Changes in PKM2 and Hsp27 Phosphorylation in Dermal Fibroblasts Regulate Energy Metabolism and Fibrotic Responses
Nabila Anika, Pranav Gowda, James Chen, Mallika Singh, Mahika Dawar, Fayiz Faruk, Hui Li, Ling Yu, Sundeep Keswani, Swathi Balaji
Surgery, Baylor College of Medicine, Houston, TX, United States
Mechanical Signaling Induced Changes in PKM2 and Hsp27 Phosphorylation in Dermal Fibroblasts Regulate Energy Metabolism and Fibrotic Responses

Swathi Balaji, Nabila N. Anika Surgery, Baylor College of Medicine, Houston, TX

Background: Fibroblasts (FB) respond to changes in the mechanical forces in the wound milieu by reorganizing the actin cytoskeleton to promote healing. Heat shock protein (Hsp27) phosphorylation is known to regulate actin rearrangement under mechanical tension. This is an energy intensive process that is fueled by oxidative phosphorylation and glycolysis to regulate ATP. Emerging evidence shows aerobic glycolysis drives pathologic fibrosis, but its role in physiologic FB wound healing responses is unknown. Pyruvate Kinase M2 (PKM2) is a rate limiting enzyme of glycolysis, whose phosphorylation (p) and dimerization increases aerobic glycolysis, but its interaction with Hsp27 in FB is unknown. We hypothesize PKM2/Hsp27-mediated alterations in FB energy metabolism are influenced by wound biomechanical forces to drive fibrotic responses.
Methods: FB were isolated from 8wk C57BL/6J mice, then cultured on either flexible silicone membranes (flex – 930 kPa Young’s modulus) that provide a softer substrate, or standard plastic culture dishes (TCP – 1×107 KPa). Cells were stimulated with TGFb (10ng/ml) for 24hr +/- TEPP (100uM) which is a PKM2 tetramer activator that inhibits aerobic glycolysis. Total and phosphorylated PKM2 and Hsp27 were analyzed using immunoblotting. Hsp27 intracellular expression and alignment with actin was assessed by staining. Lactate, ADP/ATP balance, and a-SMA (qRT-pcr; immunoblotting) were analyzed. n=3 biologic replicates/group, p-values by ANOVA.
Results: The expression of both total PKM2 and p-PKM2 were lower in cells on flex membrane than TCP at baseline. TGFb significantly increased p-PKM2/PKM2 ratio in cells on TCP (2 fold) compared to 1 fold in flex, and TEPP treatment reduced the effect of TGFb only in cells on flex membrane. In contrast, total Hsp27 was higher (by 1.5 fold) and p-Hsp27 was lower (by 2 fold) in cells on flex membrane at baseline compared to cells on TCP. p-Hsp27 was more induced by TGFb in cells on flex membrane than on TCP (6- vs. 3-fold). There was no effect of TEPP on p-Hsp27 in TCP, but it lowered p-Hsp27 in cells on flex. Lactate was similar at baseline in both conditions, but TGFb had a more pronounced induction of lactate in cells on TCP compared to flex, and TEPP treatment was effective in reducing the effect of TGFb on lactate similarly in both conditions. ADP/ATP ratio in cells on TCP was reduced by TGFb, but not on flex and TEPP treatment was ineffective on TCP, but it increased the ratio in cells on flex membrane. Lastly, the cells on flex membrane had reduced a-SMA than those on TCP, and TGFb induced more a-SMA in cells on flex than on TCP (2- vs. 1.5-fold). TEPP treatment reduced a-SMA in cells on flex but not TCP.
Conclusion: These data show that fibroblasts display distinct PKM2 and Hsp27 activation and a-SMA and bioenergetic metabolism profiles in different microenvironmental stiffness conditions, and indicate that cells are more amenable to treatment to regulate aerobic glycolysis and fibrosis markers in less stiffer environments.

K3.05

Threapeutic Effects of Postbiotics Against Intracellular Staphylococcus Aureus Wound Infections
Jamie L. Burgess1, Miroslav Dinic2, Rebecca Verpile1, Jovanka Lukic2, Nicole Vecin1, Jelena Marjanovic1, Paola Catanuto1, Natasa Golic2, Ivana Strahinic2, Marjana Tomic-Canic1, Irena Pastar1
1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States 2Group for Probiotics and Microbiota-Host Interaction, Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia
Therapeutic effects of postbiotics against intracellular Staphylococcus aureus wound infections

Jamie L. Burgess1, Miroslav Dinic2, Rebecca Verpile1, Jovanka Lukic2, Nicole Vecin1, Jelena Marjanovic1, Paola Catanuto1, Natasa Golic2, Ivana Strahinic2, Marjana Tomic-Canic1, Irena Pastar1 1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2Group for Probiotics and Microbiota-Host Interaction, Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia

The intracellular bacterial niche has been recently discovered and is associated with the non-healing phenotype of chronic wounds. However, therapeutic approaches targeting intracellular cutaneous infections remain unexplored. Probiotic lactobacilli are known for their ability to modulate the immune system and exhibit antimicrobial effects against various pathogens, but their role in cutaneous immunity remains largely unknown. Postbiotics – bioactive lysates of probiotics – also show beneficial immune modulating effects while minimizing the risks associated with the application of live bacteria. This study focused on understanding the mechanisms of epidermal response to postbiotics from probiotic Lactobacillus strains during wound healing and intracellular infection. We evaluated the potential of 13 Lactobacillus strains to inhibit intracellular growth of methicillin resistant Staphylococcus aureus (MRSA) post infection of human keratinocytes. By using a lysostaphin protection infection assay, we identified 10 postbiotics with the capability to significantly decrease MRSA counts. Lactobacillus curvatus BGMK2-41 was selected as the optimal candidate for further evaluation on wound healing, based on the highest potential to eliminate intracellular MRSA. Further, we confirmed a reduction in intracellular bacteria by immunostaining keratinocytes infected with GFP-labeled MRSA, showing that application of BGMK2-41 resulted in a decreased percentage of infected cells in comparison to untreated controls. In addition to its antimicrobial potential, we investigated BGMK2-41 postbiotic effect on wound healing by performing an in vitro scratch assay. The postbiotic treatment significantly enhanced wound closure in vitro. Finally, using a human ex vivo wound model, we showed that topical application of the postbiotic BGMK2-41 stimulated re-epithelialization and increased expression of antimicrobial peptide LL-37 and anti-inflammatory cytokine IL-10, with no effects on pro-inflammatory cytokines. Altogether, our results showed that BGMK2-41 postbiotic, and the postbiotic concept in general, could be further explored to develop novel therapeutic approaches for wound healing disorders and persistent cutaneous MRSA infections.

K3.06

An Initial Mechanistic Study of Lipid Ether Amine Antimicrobials
Daniel Gibson1, Artem S. Svetlov2
1University of Alabama, Tuscaloosa, AL, United States 2Integumed, LLC, Gainesville, FL, United States
An Initial Mechanistic Study of Lipid Ether Amine Antimicrobials

Daniel Gibson1, Archibald Svetlov2 1University of Alabama, Tuscaloosa, AL; 2Integumed, LLC, Gainesville, FL

The purpose of this study was to determine if the initial efficacy of lipid ether amine-based (LEA) against in vitro bacterial biofilms was due to membrane disruption by the lipid moiety. To test this, organic fatty acids (OFA) with the equivalent lipid chains were tested for each of 3 LEAs: 1) octanoic acid vs. LEA80, 2) oleic acid vs LEA181, and 3) arachidonic acid vs LEA204 in a standardized minimum inhibitory concentration (MIC)-based assay. Saline was used as a non-inhibitory control while vancomycin (0.2 μg/ml and 2.0 μg/ml) was used as an pro-inhibitory control. The study was performed twice, once with tryptic soy broth (TSB) and once with Muller-Hinton broth (MH). Two bacterial strains were tested, P. aeruginosa (PAO1) and a methicillin resistant S. aureus (BAA-44). A 2-fold dilution series for each agent was utilize from 1.25mM – 0.02mM (n = 3) for the TSB experiment and 256 – 0.125 μg/ml for the MH experiment (n = 5). The plates were sealed and incubated at 35 C overnight, and the wells’ turbidity was measured both visually and with an absorbance-mode plate reader at 625nm. Neither the LAE or the OFA fully inhibited PAO1 in either experiment; though LEA80 (1.25 mM/700 μg/ml) and LEA181 (1.25 mM/522.5 μg/ml) had some evidence of reduced optical density at higher concentrations when tested in TSB (p = 0.0012 and 0.003, respectively via t-Test). From both experiments, the LEA80 had an MIC greater than 256 μg/ml (350 μg/ml in the TSB), LEA181 and LEA204 both had an MIC of 8.0 μg/ml. Only arachidonic acid from the OFAs had an MIC within the testable range of 47.5 μg/ml. On an experiment-by-experiment basis, equivalent amounts of OFAs did not have the antibacterial activity that the LEAs did. Structurally, the physically largest moiety in both classes of molecules are the lipid chains. These lipid chains are hypothesized to be membrane disruptors. However, our findings do not support this hypothesis. Additional studies with a focus on cell surface and intracellular targets for LEAs are needed to explain the divergent activities of LEAs and OFAs.

K4.01

From Prediction to Pre-clinical Animal mMdel: Proof-of-concept for Retinoic Acid Receptor Agonist Ch55 as an Anti-fibrotic Agent in the Dermis
David Dolivo, Adrian Rodrigues, Robert Galiano, Thomas Mustoe, Seok Jong Hong
Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL, United States
From Prediction to Pre-clinical Animal Model: Proof-of-concept for Retinoic Acid Receptor Agonist Ch55 as an Anti-fibrotic Agent in the Dermis

David Dolivo, Adrian Rodrigues, Robert Galiano, Thomas Mustoe, Seok Jong Hong Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL

Introduction: The prevalence of fibrotic diseases and the lack of pharmacologic modalities to effectively treat them impart particular importance to the discovery of novel anti-fibrotic therapies. One paradigm that pervades all fibrotic disease is the pathological myofibroblast, a collagen-secreting, contractile cell that is responsible for deposition of fibrotic tissue. Though myofibroblasts are generally classified by their function, recent technical advances have enabled transcriptomic analysis at single cell-resolution in order to better characterize expression profiles characteristic of cells with known functions. Here we predicted known drugs that antagonize expression of a universal myofibroblast marker gene set in order to identify putative anti-fibrotic compounds.
Methods: To predict drugs, we utilized the Connectivity Map to query a conserved, previously identified myofibroblast marker gene set, and identified compounds predicted to antagonize myofibroblast marker gene expression. We screened a shortlist of these compounds using primary human fibroblasts and assessed their ability to antagonize fibroblast activation by measuring expression of fibrotic genes using qRT-PCR. We followed up on the most promising hit compound by assessing dose-dependency, antagonism of myofibroblast-associated gene and protein expression, and ability to antagonize hypertrophic scar formation via intradermal injection in a rabbit ear model.
Results: Of initially screened compounds, we determined that the retinoic acid receptor agonist Ch55 potently antagonized fibroblast activation and significantly decreased expression of myofibroblast-associated genes. The anti-fibrotic effects of Ch55 on fibroblasts were demonstrated over a range of concentrations and in multiple donors, including in rabbit dermal fibroblasts, and were phenocopied by retinoic acid receptor agonism by other compounds. Intradermal injection of Ch55 over the course of hypertrophic scar formation lessened alleviated scar hypertrophy, erythema, and collagen deposition in a rabbit ear model.
Conclusion/Discussion: Our data demonstrate plausibility of using antagonism of pathological gene expression paradigms as a predictive screening metric to identify novel anti-fibrotics from pools of existing compounds. In particular, we demonstrated that Ch55 antagonized myofibroblast gene expression in fibroblasts in vitro and ameliorated hypertrophic scar formation in vivo in a rabbit ear model. Future research of this compound and other lead compounds generated using this method will be performed to further investigate their anti-fibrotic potential.

K4.02

Fractional Ablative CO2 Laser Revision Results in Measurable Changes to Collagen Fibers in Hypertrophic Scars
Kabir Q. Al-Tariq1, Lesle Jimenez2, Eriks Ziedins2, Cameron D’Orio2, Brooke Pierson2, Lauren Moffatt2, Jeffrey W. Shupp2, Taryn Travis2, Bonnie Carney2
1Georgetown Universtiy School of Medicine, Washington, DC, United States 2Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington, DC, United States
Fractional Ablative CO2 Laser Revision Results in Measurable Changes to Collagen Fibers in Hypertrophic Scars

Kabir Q. Al-Tariq1, Lesle Jimenez2, Eriks Ziedins2, Cameron D’Orio2, Brooke Pierson2, Lauren Moffatt2, Jeffrey W. Shupp2, Taryn Travis2, Bonnie Carney2 1Georgetown Universtiy School of Medicine, Washington; 2Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington

Background: The sequelae of burn injuries and resultant hypertrophic scars (HTS) are well known to negatively impact a patient’s quality of life and skin function. HTS is characterized by the production of excess and differentially organized collagen. Recently, the use of fractional ablative CO2 laser revision (FLSR) has been shown to be effective in treating burn scar symptoms; however, a specific mechanism by which FLSR alleviates symptoms is unknown. This is in part due to the lack of standardized, quantitative metrics to evaluate treatment. It was hypothesized that using FLSR for HTS in a reproducible porcine model will alter collagen to become more similar to normal skin, and that these changes will be detectable quantitatively.
Methods: Red Duroc pigs were used as their wounds heal similarly to humans via re-epithelization over granulation tissue and form HTS. Each pig had 4 HTS sites and 2 normal skin (NS) sites. Punch biopsies were taken from each site and were paraffin-embedded, sectioned, and stained using Picrosirius Red at Post-operative days 49 and 70, which also serve as the time points for pre- and post-FLSR treatment. FLSR was delivered for 3 consecutive weeks on days 49, 56, and 63. All stained sections were then imaged at 40x (n=3 images/tissue) at the papillary dermis and were analyzed using CT-Fire and CurveAlign softwares. Using CT-Fire, values were extracted for individual collagen fiber width, length, straightness, angle, and number. Using CurveAlign, values were obtained for overall collagen fiber orientation and alignment. Subsequent analysis was performed on GraphPad Prism to compare the above parameters in the HTS and NS sites before and after laser treatment using a 2-way ANOVA with multiple comparisons.
Results: Before FLSR treatment, there was a significant difference in collagen alignment in NS vs. HTS sites (p=0.0119). After FLSR, this difference was no longer significant (p=0.1299). There was a significant difference in collagen width between NS and HTS both before (p=0.0019) and after FLSR (p=0.0021). There was also a significant difference in collagen length only after FLSR treatment (p=0.0489), but not before FLSR (p=0.3958). Differences in all other parameters were non-significant at each timepoint.
Conclusion: Before FLSR, NS and HTS were significantly different from each other in alignment and width. After FLSR, the alignment of collagen fibers within HTS became more similar to NS, but width remained different. Additionally, FLSR lengthened collagen fibers after treatment. While the mechanism of FLSR has yet to be fully understood, this study illustrates via objective and quantitative evaluation, that FLSR works by altering collagen fiber alignment and length in HTS.

K4.03

Evaluating Skin Color Diversity in the Validation of Scar Assessment Tools
Stuti P. Garg, Tokoya Williams, Iulianna C. Taritsa, Rou Wan, Chirag Goel, Raiven Harris, Robert Galiano
Northwestern University, Chicago, IL, United States
Evaluating Skin Color Diversity in the Validation of Scar Assessment Tools

Stuti P. Garg, Tokoya Williams, Iulianna C. Taritsa, Rou Wan, Chirag Goel, Raiven Harris, Robert Galiano Northwestern University, Chicago, IL

Background: Across scar studies, there is a lack of dark-skinned individuals. Darker-skinned patients have a predisposition for keloid formation, altered pigmentation, and poorer quality-of-life. There is a need for patients-of-color to be included in scar scale development and validation. In this study, we evaluate the racial diversity of patients included in the validation of scar assessment scales.
Methods: A systematic review was conducted for articles reporting on the validation of a scar assessment tool. Racial, ethnic, and Fitzpatrick skin type (FST) data was extracted.
Results: Thirteen scar scale validation studies were included. Eight of the studies did not mention FST, race, or ethnicity of the patients. Two of the studies that did report FST or race information only included White patients or included no FST V or VI patients: MAPS and VAS. Only three studies included non-white patients or dark-skinned patients in the validation of their scar scale: the modified Vancouver Scar Scale, Modified POSAS, and SCAR-Q scales. The patients included in the modified VSS validation were 7 and 13% FST V or VI, 14% African in the modified POSAS, and 4.5% FST V or VI in the SCAR-Q.
Conclusion: This study highlights the severe lack of diversity in scar scale validation, with only 3 out of 13 studies including dark-skinned patients. Given the susceptibility of darker-skinned individuals to have poorer scarring outcomes, it is critical to include patients-of-color in the very assessment tools that determine their scar prognosis. Inclusion of patients-of-color in scar scale development will improve assessment of scar pathology and better inform scar care decision-making.

K4.04

Mechanisms Of Small Intestine Submucosa Matrix In Attenuation Of Profibrotic Pathways In Normal Human And Keloid Fibroblasts
Pradipta Banerjee1, Grace Lander1, Amitava Das1, Rae Ritchie2, Michael Hiles2, Chandan K. Sen1, Sashwati Roy1
1Dept. of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States 2Cook Biotech Inc, West Lafayette, IN, United States
Mechanisms Of Small Intestine Submucosa Matrix In Attenuation Of Profibrotic Pathways In Normal Human And Keloid Fibroblasts

Pradipta Banerjee1, Grace Lander1, Amitava Das1, Rae Ritchie2, Michael Hiles2, Chandan K. Sen1, Sashwati Roy1 1Dept. of Surgery, Indiana University School of Medicine, Indianapolis, IN; 2Cook Biotech Inc, West Lafayette, IN

Background: Uncontrolled fibrosis caused by excess deposition of extracellular matrix (ECM), is a hallmark of hypertrophic scars and keloids. A clear understanding of underlying mechanism and therapeutic approaches to address this problem are areas of unmet need. Small intestine submucosa (SIS, Cook Biotech Inc), a decellularized ECM scaffold derived from porcine SIS, is widely used in clinical applications as a scaffold for tissue repair and reduce scarring.
Objective: Current study was performed to determine effects of SIS scaffolds on normal and keloid fibroblast profibrotic responses and determine underlying mechanisms of action of such activity.
Methods: Immortalized human fibroblast (hFB) and human keloid fibroblasts (hKF) were used. Cells were cultured on SIS discs, 2D collagen gel coated plates, 3D collagen gels or Integra® Matrix wound dressing were used as controls for the experiments. The cells grown on SIS for the period of experiments retained normal morphology and viability as compared to corresponding controls suggesting no adverse effects of SIS on these cells. To determine a role of SIS on profibrotic gene responses in fibroblasts, a human ECM focused RT2 Profiler PCR Array (QIAGEN) assay was performed.
Results: Two pro-fibrotic genes that significantly (p<0.05) attenuated following SIS treatment were Thrombospondin-1 (TSP1) and Fibronectin-1 (FN1). RT-PCR validation confirmed the RT2 profiler findings. ELISA analysis of TSP1 and FN1 proteins showed significant (n=5-6; p<0.05) downregulation of both proteins in hFB and hKF as compared to culturing with collagen (2D or 3D) or Integra controls. TSP1, a matricellular protein, is a major activator of latent transforming growth factor (TGFb). A central role of TGFb1 in fibrotic responses is well known. Culturing both hFB and hKF with SIS significantly attenuated (n=5; p<0.05) activated TGFb1 level in culture media. SIS has been reported to contain minor quantities of endogenous FN1 and TSP1. Treatment of cells with the equivalent quantities of FN1 and TSP1 present in SIS did not significantly change the gene expression or protein levels of TSP1 or FN1 suggesting that the endogenous levels of these proteins in SIS has no direct effect on the expression. A knock down of FN1 by siRNA significantly attenuated (n=5, p<0.05) profibrotic responses including expression of Col1A1 and TGFb1 in hKF suggesting downregulation of FN1 by SIS is one of the primary underlying mechanisms of attenuated profibrotic responses in keloid fibroblasts.
Conclusion: The current study reports that a decellularized ECM scaffold, SIS, may significantly attenuate profibrotic responses in both normal and keloid fibroblasts via TSP1 and FN1 dependent mechanisms.

K4.05

Scar Patterning After Addition of Autologous Skin Cell Suspensions to Meshed Autografting in a Porcine Burn Model
Monica Collins1, Dillon Williams1, Mary Oliver2, Lauren Moffatt2, Jeffrey W. Shupp3, Taryn Travis3, Bonnie Carney2
1Georgetown University School of Medicine, Washington, DC, United States 2Firefighters’ Burn and Surgical Research Laboratory, Medstar Research Institute, Washington, DC, United States 3Department of Surgery, Georgetown University, Washington, DC, United States
Scar Patterning After Addition of Autologous Skin Cell Suspensions to Meshed Autografting in a Porcine Burn Model

Monica Collins1, Dillon Williams1, Mary Oliver2, Lauren Moffatt2, Jeffrey W. Shupp3, Taryn Travis3, Bonnie Carney2 1Georgetown University School of Medicine, Washington, ; 2Firefighters’ Burn and Surgical Research Laboratory, Medstar Research Institute, Washington, ; 3Department of Surgery, Georgetown University, Washington,

Background: Wound patterning is a common aesthetic complication for patients who receive a meshed split-thickness skin graft after burn injury. This patterning, caused by the differential healing of the mesh and interstices of the graft, can result in dyschromia from either erythema or melanin differences at these sites. Autologous skin cell suspensions (ASCS) can be created through enzymatic digestion of a small piece of split-thickness donor skin and can be used to “over-spray” a meshed autograft. It was hypothesized that the use of ASCS with autologous meshed skin graft would decrease patterning in healed burn wounds.
Methods: Full thickness burn or excisional wounds (n=4 each) were created in pigs and received 4:1 meshed autologous skin grafts after wound bed preparation. Half of the wounds received ASCS and half did not at the time of grafting. ASCS viability was assessed prior to spraying with trypan blue. Photographs were taken of the wounds on days 14, 21, and 35 to observe patterning. The photographs underwent a blinded scoring process whereby the degree of patterning of the healed wounds was scored on a scale of 0-100. In this scale, 0 indicated no patterned area while 100 indicated complete scar coverage with a pattern. Three images of burn scars that did not receive meshed skin grafting were used as controls. Interrater reliability (IRR) was assessed between the two scorers.
Results: ASCS count and viability was 5.78e5±5.72e4 cells/cm2 and 72.3±3.3% viable and 8.03e5±1.97e5 cells/cm2 and 78.05±10.92% viable in excisional and burn wounds respectively at the time of application. The results between the two scorers are consistent with an IRR score of 71% agreement. For both raters, the wounds that did not receive meshed skin grafting were given scores of 0. Overall, average patterning scores showed that meshed pattern was observable to the level of 40-60% in all conditions. No significant difference was found in patterning scores between the wounds that received ASCS and the wounds that did not when averaged across time points (p=0.69). Additionally, at all time points, no significant differences in patterning scores were found between groups. Wound etiology likewise did not produce differences in patterning score.
Conclusion: While ASCS may benefit wound healing in other capacities, the addition of ASCS to autologous 4:1 meshed skin grafts for burn and excisional wounds does not impact the degree of patterning, suggesting that melanocytes may not robustly transfer in ASCS application. Future work should aim to optimize melanocyte viability during enzymatic digestion and trace ASCS cells from application to ultimate scar location.

K4.06

Application of a Topical Mineralocorticoid Inhibitor to Full Thickness Wounds Improves Epithelialization
Pooja Humar, Yadira Villalvazo, Fuat Baris Bengur, shawn Loder, Wayne V. Nerone, Thomas Mitts, Aleksander Hinek, Lauren Kokai, J. Peter Rubin
University of Pittsburgh, Gibsonia, PA, United States
Application of a Topical Mineralocorticoid Inhibitor to Full Thickness Wounds Improves Epithelialization

Pooja Humar, Yadira Villalvazo, Fuat Baris Bengur, shawn Loder, Wayne V. Nerone, Thomas Mitts, Aleksander Hinek, Lauren Kokai, J. Peter Rubin University of Pittsburgh, Gibsonia, PA

Background: There is a delicate balance between physiologic healing and formation of unfavorable scars. We previously demonstrated that systemic mineralocorticoid receptor (MR) inhibition improves epithelialization and diminishes collagen deposition without eroding scar strength. MR-inhibition, however, has many off-target effects when applied systemically. Consequently, herein we evaluated the role of local MR-inhibition in wound healing and hypothesize that signaling through the MR on macrophages contributes to a pro-fibrotic phenotype in wound healing.
Methods: Female C57Bl/6 mice sustained bilateral 6 mm full-thickness biopsies with stenting and were stratified into either a) vehicle control, b) 5% topical spironolactone, or c) systemic spironolactone. Systemic spironolactone was delivered intraperitoneally every three days, and topical cream formulations of spironolactone and vehicle control were reapplied every three days. Tegaderm was placed over the mice to allow topical cream to remain over wound. Mice were followed photographically for 6 weeks for time to re-epithelialization and terminal scar area. Wound biopsies were collected for gross architectural analysis and assess collagen, fibrin, and elastic fibers with Movat’s pentachrome.
Results: By day 5, both spironolactone groups demonstrated epithelization with minimal contracture. Scar area was noted to be diminished in both the spironolactone groups versus the control group. In mice receiving systemic spironolactone, all wounds had epithelialized by day 14, while wounds with topical application continued to remain open. All wounds had epithelialized by day 17 in mice receiving topical spironolactone and by day 21 receiving the topical control agent. Skin elasticity was improved with topical spironolactone application as compared to topical control. As opposed to human wounds, murine injures begin to contract immediately post-epithelialization. This was not impaired by MR-inhibition and consequently we noted a significant reduction in scar area in both spironolactone treatment groups. Histologic evaluation demonstrated persistence of inflammation, wound edema, and immature ECM.
Conclusion: These results corroborate our prior findings of the efficacy of MR-inhibition in improving scar resolution with a systemic delivery. Mice receiving systemic and topical spironolactone healed their wounds quicker than mice receiving a topical control agent. Topical application further highlighted the promising role of ECM-modifying mechanism involved with MR manipulation, with the benefit to minimize side effects and maximize the treatment efficacy. Given that wounds in the systemic spironolactone group healed the earliest, next steps would be to test different concentrations of topical Spironolactone to determine how this enhances wound healing.

L1.01

Wound Healing Over Exposed Critical Structures: A Novel 3D-Printed Wound Frame Model to Evaluate Different Dermal Matrices
Fuat Baris Bengur, Chiaki Komatsu, Yadira Villalvazo, Pooja Humar, shawn Loder, Benjamin Schilling, Mario Solari
Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, United States
Wound Healing Over Exposed Critical Structures: A Novel 3D-printed Wound Frame Model to Evaluate Different Dermal Matrices

Fuat Baris Bengur, Chiaki Komatsu, Yadira Villalvazo, Pooja Humar, Shawn Loder, Benjamin Schilling, Mario Solari Plastic Surgery, University of Pittsburgh, Pittsburgh, PA

Background: Defects with exposed critical structures commonly require reconstruction with vascularized composite tissues. Skin grafts and biological wound matrices are traditionally dependent on nutrients absorbed from the wound bed and often inadequate to provide durable coverage because of the lack of blood supply from the wound bed in complex wounds with avascular structures. Current models to evaluate these materials in a clinically relevant avascular wound bed are inadequate or not easily reproducible. We aimed to develop an affordable rodent model to demonstrate the efficacy of non-vascularized materials over a poorly vascularized wound bed.
Methods: We created 20×20 mm full thickness wounds on the dorsal skin of Lewis rats and secured 1-mm thick silicone sheets to the wound bed. A similarly sized custom-made 3D-printed wound contraction frame made of polylactic acid (PLA) filaments was placed around the wound bed and the wound edges remained within the exterior notch of the frame to isolate the inner wound environment. Upon establishing the model, following groups were assessed for viability to cover the silicone sheet inside the wound frame: 1) split thickness skin graft, 2) single layer bovine tendon collagen and glycosaminoglycan dermal matrix, 3) porcine urinary bladder matrix, 4) free flap anastomosed to the vessels in the neck. The rats were followed for 4 weeks with weekly dressing changes and photography. Samples were retrieved at the endpoint for histologic analysis with H&E and Trichrome.
Results: The total wound surface area was constant throughout the duration of the experiment. Necrosis of the portion of the skin graft and dermal matrix that corresponds to the silicone sheet was observed with exposure of the silicone sheet at the 4-week endpoint. When the size of the silicone sheet was reduced from 50% of the wound surface area, the portion surviving over the silicone sheet increased. For the silicone surface area of 25% of the wound surface area, bovine collagen and porcine bladder groups performed worse with a higher percent of exposed silicone surface area than skin grafting and free flap (p<0.05). The free flap provided complete coverage over the silicone sheet.
Conclusion: We developed a novel model of rodent wound healing that prevents contracture and isolates the wound environment in a clinically relevant complex wound environment with an avascular structure in the wound bed. The model was able to maintain the same wound size up to 4 weeks. Skin graft and dermal matrices failed to cover the exposed structure, whereas the free flap was able to provide viable coverage. This cost-effective model will establish an easily reproducible platform to evaluate more complex bioengineered wound coverage solutions.

L1.02

Robust Critical Limb Ischemia Porcine Model Involving Skeletal Muscle Necrosis
Mohamed El Masry, Surya Gnyawali, Chandan K. Sen
1Indiana Center for Regenerative Medicine & Engineering-Department of Surgery, School of Medicine-Indiana University, Indianapolis, IN, United States
Robust Critical Limb Ischemia Porcine Model Involving Skeletal Muscle Necrosis

Mohamed El Masry, Surya Gnyawali, Chandan K. Sen Indiana Center for Regenerative Medicine & Engineering-Department of Surgery, School of Medicine-Indiana University, Indianapolis, IN

Background: Critical limb Ischemia (CLI) is the advanced stage of peripheral arterial disease (PAD), defined by impaired circulation to the lower extremities and is characterized by ischemic rest pain, non-healing ischemic ulcers, and gangrene with both life and limb threatening complications. CLI carries about 25% to 40% risk of major amputation with 20% annual mortality. Currently, there is no specific treatment that target ischemic myopathy characteristic of CLI. Current swine models of CLI, with tolerable side-effects, fail to achieve sustained ischemia followed by a necrotic myopathic endpoint. Such limitation in experimental model hinders development of effective interventions. Thus, this work sought to develop a robust and clinically relevant swine model of critical limb ischemia (CLI) involving ischemic muscle necrosis.
Methods: CLI was induced unilaterally by ligation-excision of one inch of the common femoral artery (CFA) via infra-inguinal minimal incision in female Yorkshire pigs (n=5). X-ray arteriography was done pre- and post-CFA transection to validate successful induction of severe ischemia. Weekly assessment of the sequalae of ischemia on limb perfusion, and degree of ischemic myopathy was conducted for 1 month using X-ray arteriography, laser speckle imaging (LSI), CTA angiography, femoral artery duplex, high resolution ultrasound and histopathological analysis.
Results: Successful induction of CLI was achieved in all pigs (n=5) and was confirmed using invasive and non-invasive imaging modalities. Gait disturbance and limping were recorded in all pigs. The arteriography and CT angiography showed complete cessation of blood flow downstream the CFA without jeopardizing the pelvic vessels and organs. Arterial duplex indicis (Pulsatility index, Resistive index and Systolic to diastolic ratio were significantly lower in ischemic limb compared to contra lateral limb demonstrating the successful vessel occlusion and development of Ischemia at all timepoints (p<0.05; n = 5). The non-invasive tissue analysis of the elastography images showed specific and characteristic pattern of increased muscle stiffness indicative of the fibrotic and necrotic outcome expected with associated total muscle ischemia. The prominent presence of skeletal muscle necrosis was evident upon direct inspection of the affected tissues. Ischemic myopathic changes associated with inflammatory infiltrates and deficient blood vessels were objectively validated by histopathological analysis.
Conclusions: A translational model of severe hindlimb ischemia causing ischemic myopathy was successfully established adopting an approach that enable long-term survival studies in compliance with regulatory requirements pertaining to animal welfare.

L1.03

The Effect of Anatomic Location On Pig Models Of Burn Injury And Wound Healing
Aiping Liu1, Alex Cheong2, Sameeha Hassan1, Jen Meudt3, Elizabeth Townsend2, Dhanu Shanmuganayagam3, Lindsay Kalan2, Angela Gibson1
1Surgery, University of Wisconsin-Madison, Madison, WI, United States 2Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, United States 3Animal & Dairy Sciences, University of Wisconsin-Madison, Madison, WI, United States
The Effect Of Anatomic Location On Pig Models Of Burn Injury And Wound Healing

Aiping Liu1, Alex Cheong2, Sameeha Hassan1, Jen Meudt3, Elizabeth Townsend2, Dhanu Shanmuganayagam3, Lindsay Kalan2, Angela Gibson1 1Surgery, University of Wisconsin-Madison, Madison, WI; 2Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI; 3Animal & Dairy Sciences, University of Wisconsin-Madison, Madison, WI

Background: Pigs are frequently used for burn wound studies due to the structural and functional similarities of pig and human skin. However, factors including anatomic location, lack of standardized wound method, and pig breed can impact the interpretation of wound data which may affect translation to patients. The objectives of this study are to examine the influence of anatomical locations on uniformity of burn creation and evaluate the location-dependent healing in pig burn wound models.
Methods: Burns were created on ventral and dorsal surfaces from ex vivo excised pig skin or in situ in a freshly euthanized pig, with a customized burn device set at 100 C in contact with the skin for 5, 15, 30, or 60 seconds. To determine the location-dependent healing in vivo, burn wounds as well as excisional wounds of similar size and depth were created on the ventral and dorsal surfaces and at the cranial and caudal ends of two living pigs. Full thickness biopsies of wounds were obtained, processed and stained for LDH and H&E for burn depth assessment (immediately after injury) and wound re-epithelization (14 days after injury). Burn depth and re-epithelization were quantified by cell non-viability in LDH slides and the distance between the neo-epithelial tongues in H&E slides, respectively.
Results: Burn depth increased with longer contact time for both ventral and dorsal pig skin. However, at identical thermal conditions, burn depth on the ventral skin was significantly deeper than that of the dorsal skin. Compared to burns created ex vivo, burns created in situ immediately post-mortem were significantly deeper in the ventral location only. After 14 days, 2 out of 12 burn wounds were fully re-epithelialized in contrast to complete re-epithelization of all excisional wounds. Among the 12 burn wounds, no significant differences in re-epithelization was found between dorsal and ventral skin. However, burn wounds at the cranial-dorsal site exhibited faster healing than those at the caudal-dorsal site. The ventral skin exhibited large variations in healing rate regardless of site.
Conclusions: Dorsal and ventral pig skin have different contact time and burn depth relationships, indicating that anatomical location of porcine skin is an important consideration for the consistency of burn depth creation and healing. These data support location randomization of wound conditions in the pig model taking into consideration dorsal, ventral, cranial and caudal sites to prevent misinterpretation of results, and increase the translatability of pig model findings to humans.

L1.04

A Novel Ex Vivo Human Fascio cutaneous Flap Perfusion Model to Investigate Skin Injuries
Yusuf Surucu, Jeffery Gusenoff, Katherine Yang, Fuat Baris Bengur, shawn Loder, Alexey Altman, Antonio Arellano, Asim Ejaz
Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, United States
A Novel Ex Vivo Human Fascio cutaneous Flap Perfusion Model to Investigate Skin Injuries

Yusuf Surucu, Jeffery Gusenoff, Katherine Yang, Fuat Baris Bengur, shawn Loder, Alexey Altman, Antonio Arellano, Asim Ejaz Plastic Surgery, University of Pittsburgh, Pittsburgh, PA

Skin is the first line of defense against burns, chemicals, radiation, and trauma injuries. Recent research discovered a wide range of pathways and agents to treat skin injuries but still, there is a wide gap in the knowledge due to the complex nature of the injuries. Often, animal models are used for testing new agents, yet they lack anatomical feature resemblance to human tissue. Human tissue-based models are ideal; however, maintaining complex tissue ex vivo is challenging. Here we describe a novel, optimized, and well-characterized model of a full-thickness human skin perfusion system that utilizes surgical waste skin to cultivate flaps ex vivo. Abdominal panniculectomy samples were collected as surgical waste. Under sterile conditions, we isolated and cannulated perforators of the superficial and deep inferior epigastric systems. We perfused the cannulated tissue using a bioreactor system capable of real-time monitoring of pressure, flow rate, fluidic temperature, and tissue temperature. Albumin-supplemented culture media at 60mmHg pressure with 6ml/min inflow was perfused throughout the run time of approximately three weeks. Angiosome distribution was confirmed by fluorescein angiography and infrared imaging. Flow rate measurements, vascular reactivity, daily tissue biopsy samples for histology and electron microscopy, cell viability, lactate production, and gene expression levels were measured to assess the viability of the flap. Utilization of the skin perfusion model for chemical and burn injuries was assessed by induction of chemical (Nitrogen Mustard) and burn wounds. Angiography verified that the SIEA to SIEV flap system successfully fed 90% surface area of a large flap. Flow rate, temperature, and pressure remained steady throughout ex vivo cultivation. The vascular reactivity test showed a physiological response of the vasculature upon application of a vasoconstrictor (epinephrine) and vasodilator (papaverine). H&E staining and TUNEL immunofluorescence staining revealed healthy and viable cells during the perfusion run. Isolated adipose stem cells and dermal fibroblasts at different time points during perfusion showed viability and proliferation dynamics compared to fresh tissue isolates. We observed a decrease in circulatory glucose levels and increased lactate levels upon insulin challenge. Nitrogen mustard wounds showed a gradual increase in the dead TUNEL-positive cells. We observed the epithelium layer disruption and damage upon burn injuries. Our results suggest that this novel model system can keep the tissue viable for an extended period (app. 3 weeks) ex vivo. This system can be used to perfuse the tissue during and after pathology, allowing immediate and longitudinal data collection and be used as a subclinical drug testing model.

L1.05

A One-Stage Approach: Trilaminar Skin Reconstruction After a Full Thickness Burn Injury
Yadira Villalvazo1, Fuat Baris Bengur1, Shawn Loder1, Pooja Humar1, Rachel Ricketts1, Wayne V. Nerone1, Phoebe Lee1, Lauren Kokai1, Kacey G. Marra1, Elof Eriksson2, J. Peter Rubin1
1Department of Plastic and Reconstructive Surgery, University of Pittsburgh, Pittsburgh, PA, United States 2Harvard Medical School, Boston, MA, United States
A One-Stage Approach: Trilaminar Skin Reconstruction After a Full Thickness Burn Injury

Yadira Villalvazo1, Fuat Baris Bengur1, Shawn Loder1, Pooja Humar1, Rachel Ricketts1, Wayne V. Nerone1, Phoebe Lee1, Lauren Kokai1, Kacey G. Marra1, Elof Eriksson2, J. Peter Rubin1 1Department of Plastic and Reconstructive Surgery, University of Pittsburgh, Pittsburgh, PA, United States; 2Harvard Medical School, Boston, MA, United States

Background: The complex pathophysiology that occurs to the skin after a burn presents a difficult challenge. The insult disrupts the skin’s contour, mobility and appearance. The standard-of-care split thickness skin grafts are time consuming, fragile to motion, and have increased donor site morbidity. This leads to a patient requiring multiple debridements and prolonged reconstruction. Currently, there are no simple, single-stage procedures available for extensive full-thickness burns. Our team has previously demonstrated the viability of an adipose-first reconstruction to address hypodermal defects and provide a well vascularized reconstruction for complex burns. In this study, we demonstrate the efficacy of a combined fat plus pixel skin graft to achieve a single-stage trilaminar skin reconstruction.
Methods: Female Yorkshire swine sustained 16 full-thickness burns with a custom burn device. Delayed escharectomies were performed to the level of the fascia. Upon removal of the eschars, adipose from female Yorkshire swine was used on the wound as the initial layer of the reconstruction. One group had standard-of-care split thickness skin grafts. The other group had autologous pixel skin grafts (0.3×0.3 mm) that were applied on top of the adipose grafts. Pigs were maintained for 4-weeks with weekly photography, ultrasound, cutometer and biopsies, followed by histology and tension measurements upon sacrifice.
Results: At the end of the 4-week period, adipose combined with pixel graft demonstrated improved epithelialization and less contracture (p<0.01) by photography and surface area measurements. Thickness and mobility measurements were consistent in both groups and were similar to our previous approaches using adipose following debridement. Tissue pliability in the pixel grafting group was maintained to a high degree. On histologic analyses the presence of distinct, viable epidermal, dermal, and hypodermal elements was noted on cross-sections, suggesting the reconstitution of full-thickness trilaminar cutaneous architecture. Immunofluorescence staining further captured the reconstruction, and demonstrated the viability of adipose.
Conclusion: We found that a basal layer of particulate fat provided enough nutrient exchange to support immediate pixel-grafted skin. This particulate skin-plus-fat approach allowed us to generate a single-stage trilaminar reconstruction in complex burn defects in a highly translatable swine model. Immediate, single-stage trilaminar reconstruction of full-thickness complex burns reduces contracture, mitigates adhesion, and restores normal soft-tissue thickness, therefore, presenting a paradigm changing approach in the current practice of burn injuries to the mobile surfaces.

L1.06

Local Ischemia Induced by Arterial Division Impairs Healing in the Presence of S. aureus Infection in a Rabbit Ear Wound Model
Adrian Rodrigues, David Dolivo, Yingxing Li, Chun Hou, Thomas Mustoe, Robert Galiano, Seok Jong Hong
Northwestern University-Feinberg School of Medicine, USA
Local Ischemia Induced by Arterial Division Impairs Healing in the Presence of S. aureus Infection in a Rabbit Ear Wound Model

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

Introduction: The presence of chronic wounds is ever-increasing, due to high rates of obesity and diabetes, yielding enormous economic and clinical burden. Wound chronicity results in susceptibility to infection, further increasing patient morbidity. This heightened susceptibility to infection is exacerbated by tissue ischemia characteristic of diabetic wounds, such as diabetic foot ulcers, creating a distinctive need to better understand the relationship between infected wound chronicity and the ischemic tissue environment. Here we demonstrate that an early ischemic environment exacerbates subsequent wound healing in S. aureus-infected wounds in a rabbit ear wound healing model.
Methods: Female New Zealand white rabbits were used for a wound healing model on ventral ear skin. In each rabbit, one ear underwent central artery division in order to reduce blood flow to the ear and decrease tissue oxygen, while the contralateral ear underwent a sham operation absent arterial manipulation. Subsequently, six full-thickness wounds were created on the ventral surface of each ear down to the perichondrium using a sterile 7-mm biopsy punch. Wounds were infected by inoculation with S. aureus (UAMS-1 or USA300 strains) on POD3, and animals were maintained until harvest on POD10, at which time wound tissue was fixed for histological analysis to quantify de novo granulation tissue deposition and epithelial migration. Tissue oxygenation was determined at the center of each ear using the OxyLite fiberoptic probe on PODs 0,1,2 and 10.
Results: Arterial ligation resulted in a significant reduction in tissue oxygenation early in the wound healing process, which resulted in deceleration of wound healing, as assessed by reduced granulation tissue deposition and decreased epithelial migration at the time of harvest.
Conclusion/Discussion: These data suggest that early tissue ischemia in the widely used rabbit ear wound healing model induced by arterial division is sufficient to delay healing in wounds infected with clinically relevant strains of S. aureus. These findings suggest that this model may be used to better elucidate the mechanisms by which lack of tissue oxygen drives pathological chronicity in wounds that develop infection.

L2.01

Single Application of Nanosilk Cream Promotes Wound Healing in Murine Pressure Ulcer Model
Bailey Lyttle1, Alyssa Vaughn1, James Bardill1, Tanner Lehmann1, Anisha Apte2, Sudipta Seal3, Kenneth Liechty2, Carlos Zgheib2
1University of Colorado, USA; 2University of Arizona, USA; 3University of Central Florida, USA
Single Application of Nanosilk Cream Promotes Wound Healing in Murine Pressure Ulcer Model

Bailey Lyttle1, Alyssa Vaughn1, James Bardill1, Tanner Lehmann1, Anisha Apte2, Sudipta Seal3, Kenneth Liechty2, Carlos Zgheib2 1University of Colorado, USA; 2University of Arizona, USA; 3University of Central Florida, USA

Background: Pressure ulcers (PU) are chronic wounds that are common in both inpatient and rehabilitation settings but carry significant morbidity. Multiple biochemical mechanisms contribute to the formation of PU, including increased inflammation and impaired angiogenesis. The NFkB pathway largely regulates inflammation in wound healing, with elevated inflammatory markers including NFkB and TRAF6 noted in PU. VEGF promotes endothelial angiogenesis and is decreased in PU. There are few treatments available for prevention of PU formation; Cavilon® spray is currently the only marketed skin barrier protectant and has mixed efficacy. Nanosilk is a nanostructure formation of the biocompatible polymer silk fibroin and is associated with a high strength-to-density ratio that improves the biomechanical properties of skin. We have previously demonstrated that application of an 8% spray formulation of nanosilk reduces the incidence of PU development. Here, we hypothesize that a single application of 4% nanosilk cream will prevent PU development through reduced inflammation and improved angiogenesis.
Methods: 12-13 week C57BL/6 mice were implanted with a 6.35mm diameter steel disc beneath the external oblique muscle latero-caudally (n=41). After the mice were allowed to fully heal for approximately two weeks, a disc magnet (5 mm diameter, 2 mm thick) was applied to the skin superficial to the implanted disk for 10 cycles, during which the magnet was applied for two hours to mimic pressure injury, then followed by magnet removal for one hour to allow the tissue to rest. Treatments were applied prior to Cycle 1 and included three treatment groups: one application (1x) of control pluronic gel, 1x Cavilon® spray, and 1x 4% nanosilk cream (pluronic gel base). At the conclusion of 10 cycles, wound tissue was collected and processed for RT-qPCR. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (p<0.05 significant).
Results: Mice treated with 4% nanosilk had significantly lower gene expression levels of NFkB than mice treated with pluronic gel (p=0.0295), as well as significantly lower gene expression levels of TRAF6 than mice treated with Cavilon® spray (p=0.0022). Mice treated with 4% nanosilk also had significantly increased gene expression levels of VEGF-A compared to mice treated with Cavilon® spray (p=0.0256).
Conclusion: Treatment with 4% nanosilk reduces PU formation in a murine model compared to treatment with pluronic gel or Cavilon® spray, as evidenced by reduced inflammation through the NFkB pathway as well as improved angiogenesis demonstrated by increased levels of VEGF-A. Topical nanosilk demonstrates high potential as an effective preventative measure against PU formation.

L2.02

CNP-miR146a Improves Diabetic Wound Healing and Angiogenesis in a Porcine Model by Synergistically Targeting Inflammation and Oxidative Stress
Bailey D. Lyttle1, James R. Bardill1, Anne Chenchar4, Sreejayan Nair4, Brenda Alexander4, Tanner Lehmann1, Anisha Apte2, Alyssa E. Vaughn1, Sudipta Seal3, Carlos Zgheib2, Kenneth W. Liechty2
1University of Colorado, Denver, CO, United States 2University of Arizona, Tucson, AZ, United States 3University of Central Florida, Orlando, FL, United States 4University of Wyoming, Laramie, WY, United States
CNP-miR146a Improves Diabetic Wound Healing and Angiogenesis in a Porcine Model by Synergistically Targeting Inflammation and Oxidative Stress

Bailey D. Lyttle1, James R. Bardill1, Anne Chenchar4, Sreejayan Nair4, Brenda Alexander4, Tanner Lehmann1, Anisha Apte2, Alyssa E. Vaughn1, Sudipta Seal3, Carlos Zgheib2, Kenneth W. Liechty2 1University of Colorado, Denver, CO; 2University of Arizona, Tucson, AZ; 3University of Central Florida, Orlando, FL; 4University of Wyoming, Laramie, WY

Background: Diabetes is a common but highly morbid disease, with 1 in 3 Americans currently projected to develop diabetes by 2050. Chronic wounds affect nearly one third of diabetic patients in their lifetime, but limited treatments are available. Impaired diabetic wound healing occurs through multiple mechanisms including increased inflammation and impaired angiogenesis, and we have previously shown that diabetic wounds are deficient in the immune-regulating microRNA(miR)-146a. We subsequently developed CNP-miR146a, a nanoparticle in which miR146a is conjugated to a reactive oxygen species (ROS)-scavenging cerium oxide nanoparticle (CNP). Our prior studies demonstrated that CNP-miR146a improves rate of wound closure in murine and porcine models compared to phosphate buffered saline (PBS). We hypothesize that the efficacy of CNP-miR146a depends on both the ROS scavenging properties of CNP and the anti-inflammatory properties of miR146a, and that the synergistic effect outweighs the individual effects. To test our hypothesis, we compared the rate of wound closure with CNP-miR146a in a porcine diabetic model to CNP and miR146a alone.
Methods: 10-12 week pigs (n=6) were injected with streptozotocin to induce a diabetic phenotype and were wounded once expressing persistently elevated blood glucose levels at 4 months of age. 10 full-thickness wounds measuring 1 square inch each were created on the dorsal skin of each pig for a total of 60 wounds. Each wound was injected with either PBS (n=20), CNP-miR146a (n=20), CNP alone (n=10), or miR146a alone (n=10). The pigs were allowed to heal until Day 7 or Day 14, at which point the pigs were sacrificed for tissue collection. Photographs of the wounds were obtained on Days 0, 7, and 14. Wounds were measured using ImageJ software (NIH) and calculated as a percentage of the original wound area (Day 0). Wounds were also examined for the gene expression of the proangiogenic growth factor VEGF using q-PCR.
Results: Wounds treated with CNP-miR146a were significantly smaller on Day 14 than those treated with PBS (p=0.0457), CNP alone (p=0.0136), and miR146a alone (p=0.002). In addition, this significantly improved rate of wound healing with CNP-miR146a was associated with much higher levels of VEGF than those treated with PBS (p=0.0126).
Conclusion: CNP-miR146a corrects diabetic wound healing by targeting both ROS scavenging and inflammation, and the correction is synergistic compared to that of CNP or miR146a alone. This correction is additionally associated with increased proangiogenic gene expression. These results help elucidate the mechanism of action of CNP-miR146a in diabetic wound healing. CNP-miR146a holds promise as a potential therapeutic for the treatment of chronic diabetic wounds.

L2.03

Diabetic Wound Inflammation Rescue By Correction Of Mir-21 Promoter Hypermethylation Using Macrophage-Targeted Lipid Nanoparticles
Pradipta Banerjee, Amitava Das, Kanhaiya Singh, Subhadip Ghatak, Nandini Ghosh, Chandan K. Sen, Sashwati Roy
Dept. of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States
Diabetic Wound Inflammation Rescue By Correction Of Mir-21 Promoter Hypermethylation Using Macrophage-Targeted Lipid Nanoparticles

Pradipta Banerjee, Amitava Das, Kanhaiya Singh, Subhadip Ghatak, Nandini Ghosh, Chandan K. Sen, Sashwati Roy Dept. of Surgery, Indiana University School of Medicine, Indianapolis, IN

Background: Epigenetic modifications silence miRNA expression via promoter methylation. Previous work from our laboratory has highlighted the critical significance of wound macrophage miR-21 in enabling resolution of diabetic wound inflammation. In this work, tested the hypothesis that wound macrophages miR-21 is lowered because of promoter hypermethylation thus complicating resolution of inflammation.
Methods & Results: Macrophages were isolated from wounds of diabetic (Leprdb) or control (littermate heterozygous (non-diabetic, Leprdb/+) mice. RNA sequencing studies reveled that significantly lower miR-21 (p<0.05; n=6) levels in diabetic wound macrophages derived from both human and mice were associated with persistent inflammatory phenotype. To determine the role underlying epigenetic mechanisms, promoter methylation studies of miR-21 under hyperglycemic conditions was performed. Bisulfite sequencing showed hypermethylation of miR-21 promoter region (p<0.05; n=6) under conditions of hyperglycemia. Such finding was associated with higher abundance of 5-methyl cytosine in macrophages from diabetic wounds revealing an overall increase in DNA hypermethylation. DNMT and TET enzymes play crucial roles in DNA hypermethylation. Loss and gain of function studies of DNMT and TET revealed a central role of DNMT3b in hypermethylation of miR-21 promoter in diabetic wound macrophages. Hyperglycemia resulted in increased expression DNMT3b and higher miR-21 promoter hypermethylation concomitant with low abundance of miR-21 in wound macrophages. Knockdown of DNMT3b in macrophages resulted in rescue (p<0.05; n=6) of hyperglycemia-mediated attenuation in miR-21 abundance. Macrophage-targeted lipid nanoparticles (LNPmp) carrying specified cargo including miR-21 mimic or siDNMT (inhibits hypermethylation) were prepared and characterized for efficacy and specificity. Treatment of diabetic wounds with LNPmp carrying either miR-21 mimic or siDNMT3b significantly (p< 0.05; n=6) improved miR-21 level in diabetic wound macrophages. Such correction was associated with improved resolution of inflammation and wound healing.
Conclusion: This work recognizes hyperglycemia-associated epigenetic processes as a key barrier to timely resolution of diabetic wound inflammation. Specific therapeutic targets have been identified and found to be productive in restoring resolution of diabetic wound inflammation and improving healing outcomes.

L2.04

Application of Avena Sativa Derived Therapeutics B-Glucan and Avenanthramide Accelerate Wound Healing in Mice Via Angiogenic and Anti-inflammatory Mechanisms
Hudson C. Kussie2, William Hahn2, Filiberto Quintero2, Dharshan Sivaraj1, Katharina S. Fischer2, Andrew Hostler2, Maia Granoski2, Dominic Henn4, Abigail Miller3, Delaney Schurr3, Vincent Li3, William Li3, Geoffrey C. Gurtner2, kellen Chen2
1Stanford University, New York City, NY, United States 2Surgery, University of Arizona, Tucson, AZ, United States 3The Angiogenesis Foundation, Cambridge, MA, United States 4Surgery, University of Texas Southwestern Medical Center, Dallas, TX, United States
Application of Avena Sativa Derived Therapeutics β-Glucan and Avenanthramide Accelerate Wound Healing in Mice Via Angiogenic and Anti-inflammatory Mechanisms

Hudson C. Kussie2, William Hahn2, Filiberto Quintero2, Dharshan Sivaraj1, Katharina S. Fischer2, Andrew Hostler2, Maia Granoski2, Dominic Henn4, Abigail Miller3, Delaney Schurr3, Vincent Li3, William Li3, Geoffrey C. Gurtner2, kellen Chen2 1Stanford University, New York City, NY; 2Surgery, University of Arizona, Tucson, AZ; 3The Angiogenesis Foundation, Cambridge, MA; 4Surgery, University of Texas Southwestern Medical Center, Dallas, TX

Background: Improving wound healing after cutaneous injury decreases complications, prevents chronic wound formation, and improves patient wellbeing. β-Glucan and Avenanthramide (AVE) from oat extracts have previously shown potential to promote wound healing; however, both the mechanism and efficacy of how β-Glucan and AVE affect wound healing remain incompletely understood. In this study, we investigated the dose- and mechanistic effects of subcutaneous injections of β-Glucan and AVE on excisional wound healing in a murine model.
Methods: We utilized 15-week-old C57BL/6 mice in a splinted excisional wounding model to mimic human wound healing. Two dorsal excisional wounds per mouse and three mice per treatment group were used. Each respective group received one of five (0%, 0.1%, 0.5%, 1.0%, 2.0%) differing dosages of AVE dissolved in phosphate buffered saline (PBS) and administered via subcutaneous injection to the wound periphery. In a second set of mice, excisional wounds of each mouse received one of four (0%, 0.1%, 0.5%, 1.0%) differing dosage treatments of β-Glucan dissolved in PBS. Each wound was treated every other day until full wound closure. The wound areas were quantified and expressed as a percentage of the original area. Wound tissue sections were stained for collagen (Masson’s Trichrome, Picrosirius Red), for microvessels (CD31), endothelial progenitor cells (EPC) (CD133, CD34), and inflammatory cells (CD45).
Results: 1% β-Glucan and 1% AVE treatment significantly accelerated the rate of wound healing as compared with control mice. Other doses did not significantly improve healing. The mean time for complete wound healing was 10.67 days in the 1% AVE-treated group compared to 12.44 days in the control group (p= 0.032). At day fourteen, 1% AVE also promoted a significant decrease in length and alignment of collagen fibers compared to control (p= 0.033, p= 0.048), and significantly decreased CD45-stained inflammatory cells (p= 0.002). 1% β-Glucan significantly decreased wound size relative to controls at days 8 and 10 (p= 0.009, p= 0.016). At day fourteen, 1% β-Glucan promoted a significant increase in width and length of collagen fibers compared to control (p=0.031, p=0.036). 1% β-Glucan treatment upregulated CD133, CD34, and CD31 expression compared to the control (p=0.001, p=0.036, p=0.040).
Conclusions: 1% β-Glucan and 1% AVE treatment resulted in faster wound closure compared to controls (0%). 1% AVE treatment downregulated inflammatory cell infiltration, which may lead to a more regenerative extracellular matrix (ECM) architecture and accelerate wound healing. 1% β-Glucan treated tissue exhibited more blood vessels than in the control, potentially due to an increase in EPCs, and the resulting ECM with increased collagen fiber size accelerated wound healing. A better understanding of the differing mechanisms driving these naturally derived therapeutics may lead to improved patient wound outcomes.

L2.05

Assessment of the Stability of miR-146a Conjugated Cerium Oxide Nanoparticles in Different Conditions
Anisha Apte1, James R. Bardill2, Alyssa E. Vaughn2, Bailey D. Lyttle2, Tanner Lehmann2, Kenneth W. Liechty1, Carlos Zgheib1
1Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Arizona College of Medicine, Tucson, AZ, United States 2Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Colorado Denver School of Medicine, Aurora, CO, United States
Assessment of the Stability of miR-146a Conjugated Cerium Oxide Nanoparticles in Different Conditions

Anisha Apte1, James R. Bardill2, Alyssa E. Vaughn2, Bailey D. Lyttle2, Tanner Lehmann2, Kenneth W. Liechty1, Carlos Zgheib1 1Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Arizona College of Medicine, Tucson, AZ; 2Pediatric Surgery, Laboratory for Fetal and Regenerative Biology, University of Colorado Denver School of Medicine, Aurora, CO

Background: The use of microRNA to alter gene expression has become a novel method of drug development for a wide range of disease processes. In inflammatory disease states, microRNA(miR)-146a is critical to downregulating pro-inflammatory signaling. Correcting dysregulated levels of miR146a can mitigate inflammation, improve healing, and reduce disease severity. Therapeutic miR delivery remains challenging due to its instability and susceptibility to degradation. We conjugated miR146a to cerium oxide nanoparticles (CNP-miR146a) to both target reactive oxygen species and facilitate stable delivery of miR146a. We have previously shown that CNP-miR146a is a promising therapeutic with multiple applications, including diabetic wounds and acute lung injury. The observed therapeutic activity exceeded the efficacies of individual conjugate components (CNPs and miR146a) alone. We hypothesized that this novel conjugation functions to stabilize and improve delivery of this potential therapeutic, allowing for broad use in various clinical and field settings.
Methods: To investigate long-term stability, stock samples of CNP-miR146a were placed at room temperature (RT), 4C, -20C, and -80C storage conditions. 1μL aliquots were removed at 2 weeks, 1 month, 3 months, and 6 months and real time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate miR146a expression levels. Freeze-thaw stability was assessed over 10 cycles for stock samples stored at -20C and -80C. For each cycle, the sample was removed from the freezer and allowed to thaw, a 1uL aliquot was removed, and the sample was returned to the freezer. RT-qPCR was performed to evaluate miR146a expression levels of each aliquot after each cycle.
Results: CNP-miR146a remained stable for up to 6 months at both -20C and -80C storage conditions when compared to miR146a expression levels of a stock control sample. There was minimal loss in stability at RT and 4C storage. Additionally, CNP-miR146a remained stable after 10 freeze-thaw cycles, in both 20C and -80C storage conditions.
Conclusions: The ability of CNP-miR146a to remain stable at -20C with minimal loss in gene expression at RT and 4C supports easy and practical storage of CNP-miR146a therapeutics in a clinical setting. Additionally, the freeze-thaw stability of CNP-miR146a will allow for storage in larger quantities without concern for reduced potency throughout multiple administrations. Longer stability studies are underway.

L2.06

A Mammalian Target Of Rapamycin (mTOR) Inhibitor, Sirolimus, SRL, is Effective In Lymphatic Malformation-Related Wounding And Skin Abnormality
Sadanori Akita
Fukushima Medical University/Jonaikai, Shimabara, Nagasaki, Japan
A Mammalian Target of Rapamycin (mTOR) Inhibitor, Sirolimus, SRL, is Effective in Lymphatic Malformation-Related Wounding and Skin Abnormality

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

Background: A lymphatic malformation, LM, commonly infiltrates soft tissues, and it can be found anywhere on the body, from the extremities to the abdominal or thoracic cavities. The lesion contours are ill-defined, with invasive involvement of the adjacent tissues and structures, resulting in severe complications, including organ dysfunction and impairment of breathing, swallowing, or wounding. A mammalian target of rapamycin (mTOR) inhibitor, sirolimus, SRL is received pharmaceutical approval and is fully reimbursed by a universe health insurance by the Japanese government dated September 27, 2021. Eight lymphatic malformation patients, 10 to 37 years, 5 males and 3 females, with wounds and skin lesions, are tested with sirolimus pills, 1 mg to 3 mg per day up to 6 months.
Methods: 3 cases resulted in complete wound healing, 2 cases over 50% wound healing and 3 demonstrated unchanged wounding. Even in the five remaining wound cases, subjective and objective skin condition scales improved in the amount of exudate, irritation, and texture at 45% and 60% respectively compared to pre-treatment.
Results: There were no severe adverse reactions or complications such as interstitial pneumonitis or systemic infection but there were 3 cases of stomatitis and 2 cases of local infection such as atheroma as minor complications. In such minor complication cases, local antimicrobial cleansing and ointment subsided the symptoms in a few days.
Conclusions: The blood trough concentrations were investigated in all cases at 2 weeks of starting medication or when a negative symptom occurred, and all are set to 5 to 15 n/mL by managing the dose from 1 to 4 mg per day. An mTOR inhibitor, sirolimus is easily manageable and leads to healing of lymphatic malformation-related wounding.

L3.01

Modulation of Innate Immunity Through Exosomes Restores Delayed Diabetic Wound Healing Via Macrophage Plasticity
Kody P. Mansfield, Dianny Almanzar, Bibi S. Subhan, Juan Troncoso, Lesly Honore, Piul S. Rabbani
Hansjörg Wyss Department of Plastic Surgery, NYU Langone, Brooklyn, NY, United States
Modulation Of Innate Immunity Through Exosomes Restores Delayed Diabetic Wound Healing Via Macrophage Plasticity

Kody P. Mansfield, Dianny Almanzar, Bibi S. Subhan, Juan Troncoso, Lesly Honore, Piul S. Rabbani Hansjörg Wyss Department of Plastic Surgery, NYU Langone, Brooklyn, NY

One of the most remarkable qualities of skin is its regenerative capacity following injury. This, however, is lost in diabetic foot ulcers. These chronic wounds are untreatable and in turn are the number one cause of lower-limb amputation. Using cultured human bone marrow multipotent stromal cells and their secreted nanoscale exosomes, we have been able to accelerate wound closure in a genetic animal model of diabetic delayed healing. However, the large paucity in knowledge surrounding the cellular events engaged during exosome-induced wound healing represents a major hurdle for translating this tissue engineering approach clinically. Cutaneous administration of 3×109 exosomes at post-operative day 1 (POD1) significantly decreased time to closure of excisional LepRdb/db diabetic wounds to 17±1.4 days vs 28±1.5 days observed in vehicle controls (VC) (p<0.01, n=9). This treatment also corresponded with reduced wound burden in a dose dependent manner. In addition to accelerated healing, exosome treated wounds displayed extensive CD31+ neovascularization in expanded areas of granulation tissue in the diabetic wound bed by POD10, phenocopying typical healing wounds of wildtype mice. Gene expression analysis of the wound beds demonstrated significant upregulation of TNFα, IL6, NOS2, VEGF and PDGF which accompanied both dynamic fluctuations and phenotypic shifts in innate immunity. By POD5, exosome administration increased the presence of monocytes and macrophages in the diabetic wound tissue by 1.6-fold and 6.2-fold of that in VC-treated wounds, respectively. The ratio of F4/80+iNOS+ pro-inflammatory to F4/80+Arg1+ pro-repair macrophages in diabetic wounds decreases from POD5 to POD10 with exosome treatment, unlike in VC-treated wounds. This ratio progression and monocyte/macrophage cell density in exosome-treated diabetic wounds is similar to that in non-diabetic mice of similar genetic background. The shift of active wound macrophages is not observed in diabetic mice treated with vehicle control. Further confirming our results, exosome treatment failed to rescue the delayed healing in clodronate liposome treated, macrophage depleted mice, indicating that the phenotypic shift is paramount for exosome-induced wound healing. Furthermore, the endogenous control of macrophage plasticity throughout the sequential phases of skin healing remains elusive even in the context of typical healing wounds. Our results demonstrate that exosomes mediate therapeutic potency, at least through macrophages. This critical point positions exogenous exosome treatment as a novel and effective tool to uncover temporally regulated mechanisms of macrophage functions throughout wound healing.

L3.02

CDMS of Murine Wound-Edge Tissue Exosomes: Observation Of 13 MDa Particles At Five Days Post-Wounding
Adam Anthony
Analytical Chemistry, Indiana University, Bloomington, IN, United States
CDMS of 1.10 – 1.14 gmL-1 Murine Wound-Edge Tissue Exosomes: Observation of 13 Mda Particles at Five Days Post-Wounding

Adam Anthony Analytical Chemistry, Indiana University, Bloomington, IN

The study of extracellular vesicles (EVs) and the subclass of particles known as exosomes has been rapidly expanding, in part due to their roles in communication between various cell types. Wound edge tissue from mice was harvested immediately and five days after wounding from the same mice then processed using differential centrifugation followed by density gradient ultracentrifugation to produce four EV populations with defined densities. Proteomic experiments indicated that all fractions were found to contain the exosome marker proteins CD9 and CD81. From these fractions the density range 1.10 – 1.14 g/mL was chosen for further analyses due to previous reports that this density range is depleted for high- and low-density lipoproteins, microvesicles and protein aggregates. The selected fractions displayed intact vesicles when subjected to transmission electron microscopy (TEM) that yielded similarly broad populations at day 0 and day 5. Charge Detection Mass Spectrometry (CDMS) provides mass and charge information for particles greater than 10 megadaltons. CDMS data reveals the day 5 EV fraction contains a similar population to the day 0 EV fraction centered at 26 MDa and 370 charges; however, CDMS was able to resolve a new, smaller EV population centered at 18 MDa and 250 charges not present in the day 0 sample. Combining a density-based isolation with CDMS allows for the particle masses to be converted to diameters and directly compared to TEM. The population found across day 0 and day 5 was centered around 41 nm in diameter, and the five days post-wounding population was centered around 35 nm in diameter. Proteomic measurements found 197 proteins enriched more than threefold across all day 5 densities including CD177 and CD209d, as well as 194 proteins enriched more than twofold across all day 0 densities including Aldoa. The data suggests that the population found across day 0 and day 5 is enriched in energy transport and metabolic proteins, and the new population seen at day 5 is enriched in mRNA processing proteins and ribosomal proteins for RNA translation. CDMS shows a major population of wound edge exosomes present in both day 0 and day 5 exosomes. Additionally, CDMS shows a lower mass population present in day 5 wound edge exosomes that is not present at day 0. Correlating CDMS and proteomics data suggests that proteins enriched at day 0 will be representative of the shared, higher mass population and proteins enriched at day 5 will correlate to the day 5 lower mass population. Thus, at both timepoints, exosomes expressing metabolic and energy related proteins are present; however, at day five, the lower mass exosome population containing proteins to ensure proliferative healing is present. While the cellular origin of these exosome subpopulations is still unknown, they are likely related to one or more cells involved in the immune response.

L3.03

Topical Treatment With Exosomes In Bioinspired Protein Hydrogel Delivers Wound Closure Efficacy In Diabetic Wound Model
Ramazan Yildiz1, Bibi S. Subhan1, Priya Katyal2, Juan Cortes Troncoso1, Andrew Wang2, Michael Meleties2, Jin K Montclare2, Piul S. Rabbani1
1Grossman School of Medicine, New York University, New york, NY, United States 2Tandon School of Engineering, . New York University, New York, NY, United States
Topical Treatment with Exosomes in Bioinspired Protein Hydrogel Delivers Wound Closure Efficacy In Diabetic Wound Model

ramazan yildiz1, Bibi S. Subhan1, Priya Katyal2, Juan Cortes Troncoso1, Andrew Wang2, Michael Meleties2, Jin K Montclare2, Piul S. Rabbani1 1Grossman School of Medicine, New York University, New york, NY; 2Tandon School of Engineering, New York University, New York, NY

We accelerated excisional wound closure in diabetic animal models of delayed healing by using cultivated human bone marrow multipotent stromal cells and their released nanoscale small extracellular vesicles (exosomes). To apply this tissue engineering technique practically, we require feasible delivery platforms for targeted dosing and physical retention at the wound site without reducing exosome potency. Our team bioengineered Exo-Q hydrogel, a thermoresponsive soft protein matrix packed with exosomes. An engineered version of the cartilage oligomeric matrix protein’s coiled-coil domain, Q, self-assembles into a fibrous matrix at low temperatures like 4-10 Celsius and displays an upper critical solution temperature phase behavior. Exo-Q hydrogels are a unique substance produced by incorporating exosomes during the gelation of Q. In transmission electron micrographs of Exo-Q, protein fibers are consistently entangled and interspersed with exosomes, increasing the storage modulus of Q for a spreadable gel. Rigorous characterization using cell surface phenotype, nanoparticle tracking analysis and transmission electron micrography confirmed that our culture and isolation methods generate consistent exosomes (median diameter 137nm) across different human bone marrow donors (n≥3) and ones that encapsulate in Q with reproducible efficiency. Following topical administration onto excisional wounds at post-op day 1, we detected sustained presence of fluorescently-labeled exosomes in Exo-Q at the wound site, but not in non-target organs, indicating sustained and targeted delivery. In wound closure analysis, a single Exo-Q topical application significantly decreased time to closure of excisional LepRdb/db diabetic wounds to 17±1.4 days, relative to that with Q alone at 28±1.5 days, showing a 78.5% reduction in pathological time to closure (n=6, p<0.05). Genes associated with driving wound healing, such as TNFα, IL6, NOS2, VEGF and PDGF, were significantly upregulated at least 2-fold in the Exo-Q-treated diabetic wound beds as compared to their Q-only counterparts. Additionally, Exo-Q therapy resulted in extensive neovascularized granulation tissue. Our results demonstrate that exosomes retain therapeutic potency when delivered through Exo-Q hydrogels. Exo-Q hydrogels enable biodistribution at the targeted wound site following topical application, but do not interfere with wound healing progression. The ease of application of Exo-Q hydrogel and efficacy with limited administration show promise for clinical translation.

L3.04

Obesity Dysregulates Chromatin Response To Hypoxia That Governs Metabolic Programming And Phagocytosis In Macrophages
Kentaro Takahashi, Jasmine R. Jackson, Jinghua Liu, Norifumi Urao
Pharmacology, SUNY Upstate Medical University, Syracuse, NY, United States
Obesity Dysregulates Chromatin Response to Hypoxia that Governs Metabolic Programming and Phagocytosis in Macrophages

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

Obesity is associated with poor wound healing. Macrophages play integral roles in wound healing and obesity-associated conditions, however, how obesity dysregulates machinery that ensures proper macrophage functions in response to their environment remains to be understood. Oxygen-sensing chromatin remodeling regulated by histone posttranslational modification is an emerging mechanism of cellular response to hypoxia. In mouse bone marrow-derived macrophages (BMDMs), we found that 1% hypoxia rapidly (in 1h) increases the global levels of histone 3 methylations. Comparing BMDMs from mice fed a high-fat diet (HFD) with ones from mice fed a normal-fat diet (ND), immunoblotting of isolated histone and high-content imaging revealed that hypoxia-induction of histone 3-lysine 4 tri-methylation (H3K4me3) is specifically inhibited in HFD-BMDMs, while the induction of hypoxia-inducible factor-1α remained intact. ChIP-seq identified that hypoxia-induced H3K4me3 marks the promoter lesions of the genes associated with multiple metabolism-related pathways including glycolysis in ND-BMDMs. Interestingly, no pathway is associated with hypoxia-induced H3K4me3 marks in HFD in gene ontology analysis, suggesting a broad impairment of oxygen-sensing H3K4me3 in obesity. We further found glycolytic genes, Aldoa, ldha, and ldhb are dysregulated in HFD-BMDMs, along with the reduced lactate levels and histone lactylation in hypoxia. Furthermore, HFD-BMDMs exhibit lower phagocytosis of dying cells under hypoxia due to the reduced capacity of anaerobic glycolysis. In summary, oxygen-sensing H3K4me3 enrichment promotes glycolytic capability and phagocytosis in hypoxia and that obesity impairs this mechanism of chromatin response. Dysregulated chromatin response to hypoxia may be a key disrupted mechanism in obesity.

L3.05

Depletion of Natural Killer Cells Enhances Skin Wound Healing in Mice
Jacqueline Cavalcante Cavalcante Silva, Timothy J. Koh
Center for Wound Healing and Tissue Regeneration, University of Illinois at Chicago, Chicago, IL, United States
Depletion of Natural Killer Cells Enhances Skin Wound Healing in Mice

Jacqueline Cavalcante Cavalcante Silva, Timothy J. Koh Center for Wound Healing and Tissue Regeneration, University of Illinois at Chicago, Chicago, IL

NK cells are defined by their “natural killing” function, especially against viruses and tumor cells, but also can also produce cytokines and growth factors, shaping immune responses through crosstalk with other immune and non-immune cells. In this study, we aimed to investigate the role of NK cells in wound healing in C57BL/6J mice. Full-thickness 8-mm excisional skin wounds were made on the dorsum of each mouse, and wounds were collected on days 3, 5, or 7 after wounding for immunohistochemical, flow cytometry, and qPCR analysis. Immunohistochemical and flow cytometry assays showed that NK cells accumulate in excisional skin wounds, peaking on day 5 post-injury. We also found that NK cells proliferate locally in wounds and that IL-15 promotes NK cell proliferation and accumulation in wounds. NK cells in wounds present a primarily mature CD11b+CD27- and NKG2A+NKG2D- phenotype and express pro-inflammatory cytokines such as IFN-y, Tnf-a, and Il-1b. Importantly, depletion of NK cells resulted in enhanced re-epithelization and collagen deposition, suggesting a negative role for these cells in skin wound healing. NK cell depletion did not influence accumulation of other immune cells such as neutrophils or monocytes/macrophages in wounds but reduced expression of pro-inflammatory markers such as IFN-y, Tnf-a, and Il-1b, indicating that NK cells contribute to pro-inflammatory cytokine expression in wounds. In short, NK cells may negatively regulate physiological wound healing via production of pro-inflammatory cytokines.

L3.06

Investigating Exosome-Induced Macrophage Polarization Relevant To Chronic Wound Healing
Jasmina Abdalla, Bibi S. Subhan, Lesly Honore, Dianny Almanzar, Kody P. Mansfield, Piul S. Rabbani
New York University School of Medicine, New York, NY, United States
Investigating Exosome-Induced Macrophage Polarization Relevant to Chronic Wound Healing

Jasmina Abdalla, Bibi S. Subhan, Lesly Honore, Dianny Almanzar, Kody P. Mansfield, Piul S. Rabbani New York University School of Medicine, New York, NY

Almost 75 million patients with diabetes worldwide are at risk of developing a diabetic foot ulcer (DFU) in their lifetime. DFUs increase the health and economic burden these patients live with. Therefore, there is a need for novel treatments for diabetic patients suffering from chronic wounds. In the diabetic wound environment, the prolonged elevation of inflammatory cytokines such as IL-1β and TNFα leads to an unfavorable environment, partially characterized by presence of macrophages (Mφ), which are a key cell population involved in modulating wound healing. Current research indicates that Mφ infiltrate the wound area and that the pro-inflammatory type give way to the anti-inflammatory type through the course of healing. In type 2 diabetic mouse wounds, anti-inflammatory Mφ are rare. However, the anti-inflammatory Mφ numbers rise following local treatment with multipotent stromal cell-derived small extracellular vesicles, also called exosomes. Our goal was to isolate the interaction between monocyte-macrophages and exogenously administered multipotent stromal cell exosomes into an in vitro study. Exosomes are nanoscale extracellular vesicles secreted by all cells and ones from multipotent stromal cells promote angiogenesis and re-epithelialization when applied in tissue engineering approaches in diabetic wound healing models. We cultured bone-marrow derived monocyte-macrophages (BMDM) from adult wild type (WT) and LepRdb/db mice. We administered varying doses of exosomes to either cultured monocytes or in vitro-differentiated pro-inflammatory Mφ, and assessed for polarization into anti-inflammatory Mφ using cell surface phenotyping via flow cytometry. We found that 1e7 to 1e8 exosomes when applied to pro-inflammatory Mφ, drove higher expression of typical anti-inflammatory or pre-repair Mφ markers such as CD206, CD80. Simultaneously, we found reduction in typical pro-inflammatory markers, such as CD38. Our results suggest that a direct interaction exists between macrophages and exosomes. The interactions potentially model the cellular and molecular events occurring in vivo in preclinical diabetic wound models following application of multipotent stromal cell exosomes.

L4.01

Prediction Of Non-Alkaline Wound Healing Outcomes Based On One-Time Wound Alkalinity Measurement
Jon Senkowsky1, Liping Tang2, Maha Khan3
1Texas Health Physician’s group; 2Progenitec Inc; 3TCU School of Medicine
Prediction Of Non-Alkaline Wound Healing Outcomes Based On One-Time Wound Alkalinity Measurement

Jon Senkowsky1, Liping Tang2, Maha Khan3 1Texas Health Physician’s group; 2Progenitec Inc; 3TCU School of Medicine Purpose: The objective of this study was to predict the healing outcomes by assessing the change of wound alkalinity following the initial wound treatment.

Methods: The planar alkalinity of heterogeneous wounds was monitored by assessing discarded wound fluid-soaked wound dressings from 24 various types of wounds using a disposable and non-contact device – DETEC® pH. 12 wounds were further classified as venous stasis ulcers. The wound alkalinity and size at the 1st, 2nd, and 3rd visits were correlated with healing wound determination over 8 weeks of follow-up from the device output.
Results: 3 venous stasis ulcers’ (VSUs) dressings were randomly chosen to showcase the heterogenous wound alkalinity and healing outcome in various wound regions. In Fig 1A and 1B, the lateral edges of the wound are non-alkaline, whereas it becomes more alkaline towards the center. This wound healing outcome aligns with second intension, where the granulation tissue fills in the edges (Fig. 1C) and works inwards. The wound was then filled with novo tissue and vasculature and replacing necrotic tissue until replacement by scar tissue (Fig 1D).
Conclusion: Our results show that wound alkalinity measurements of venous ulcer wounds at a routine follow-up visit between 5 to 14 days after the first visit could predict the healing outcome and wound size reduction for heterogenous wounds. This innovative outcome and knowledge could help the clinician design the most optimum wound management process for the patient.
Disclosure: Data collection for this study was undertaken while Jon Senkowsky and Liping Tang were affiliated to Texas Health and Progenitec Inc. All opinions presented in this manuscript belong to the authors alone, and not to any institution to which they are or were affiliated. The authors declare that they have no competing interests.

L4.02

Role of Oxygen Sensing Histone Demethylase KDM5A in Wound Healing
Jasmine R. Jackson1, Kentaro Takahashi1, Jinghua Liu1, Yoshikiyo Akasaka2, Norifumi Urao1
1Pharmacology, SUNY Upstate Medical University, Syracuse, NY, United States 2Pathology, Toho University, Tokyo, Japan
Role of Oxygen Sensing Histone Demethylase KDM5A in Wound Healing

Jasmine R. Jackson1, Kentaro Takahashi1, Jinghua Liu1, Yoshikiyo Akasaka2, Norifumi Urao1 1Pharmacology, SUNY Upstate Medical University, Syracuse, NY; 2Pathology, Toho University, Tokyo, Japan

Oxygen sensing within a wound is widely acknowledged to be important in the overall healing process, however, the underlying mechanisms involved in the cellular response to hypoxia remain elusive. Studies suggest that hypoxia-induced transcription is calibrated by chromatin remodeling through histone modifications, particularly tri-methylated histone 3-lysine 4 (H3K4me3), which is associated with open chromatin and regulated by histone demethylases KDM5A. Here, we show that hypoxia rapidly induces H3K4me3 in bone marrow-derived cultured macrophages (n=3, p<0.05). Using inducible KDM5A knockout (KO) mice, we found that hypoxia-induced H3K4me3 response is enhanced in KDM5A-KO macrophages (n=3, p<0.05). Chromatin immunoprecipitation followed by sequencing for H3K4me3 revealed that H3K4me3 is marked at the genome loci of genes related to multiple metabolic pathways, such as glycolysis, oxidative phosphorylation, and fatty acid metabolism, suggesting that hypoxia-induced H3K4me3 may have a specific influence on the macrophage functions regulated by metabolism such as immunophenotype and phagocytosis. In vivo, KDM5A-KO mice exhibited promoted fibrotic response in the early phase of dorsal skin wound injury, whereas both the rate of wound closure and scar formation appears unchanged (n=4, p<0.05). Together these results suggest that H3K4me3 induction by hypoxia promotes metabolic and pro-healing responses in macrophages during wound healing and that inhibition of KDM5A may be a strategy to harness the hypoxia-promoted healing response without incurring scar formation.

L4.03

Monitoring Ischemic Skin Necrosis Through Autofluorescence Lifetime Imaging
Hallie Ramser1, Alan Woessner1, S. Morteza Seyed Jafari2, Suneel Kumar3, Jake Jones1, Francois Berthiaume3, Robert Hunger2, Amiq Gazdhar2, Kyle Quinn1
1University of Arkansas, Fayetteville, AR, United States 2Bern University Hospital, Bern, Switzerland. 3Rutgers University, New Brunswick, NJ, United States
Monitoring Ischemic Skin Necrosis Through Autofluorescence Lifetime Imaging

Hallie Ramser1, Alan Woessner1, S. Morteza Seyed Jafari2, Suneel Kumar3, Jake Jones1, Francois Berthiaume3, Robert Hunger2, Amiq Gazdhar2, Kyle Quinn1 1University of Arkansas, Fayetteville, AR; 2Bern University Hospital, Bern, Switzerland; 3Rutgers University, New Brunswick, NJ

Skin necrosis can be difficult to predict due to the lack of quantitative biomarkers. Laser doppler imaging, ultrasound, and fluorescence angiography can be used to monitor blood flow in tissue, but they do not directly measure the cellular response to ischemia, trauma or infection. Label-free multiphoton microscopy is well suited for non-invasively monitoring skin metabolism through NAD(P)H and other intrinsic fluorophores, and offers immediate future directions for assessing necrosis in the clinic. By measuring the time elapsed between excitation and emission, fluorescence lifetime imaging (FLIM) has emerged as a powerful method for discriminating fluorophores and detecting changes in the molecular microenvironment. The objective of this study was to assess whether FLIM could be used to evaluate skin necrosis and treatment efficacy in ex vivo skin sections from an ischemic skin flap model. We employed a simplified fit-free approach to analyzing FLIM data through phasor analysis of the fluorescence lifetime decay. Phasor maps of fluorescence lifetime revealed differences among skin flap regions and growth factor treatments. These changes in skin flap autofluorescence lifetime were not associated with NADH autofluorescence. Rather, a very short lifetime component accumulated in the ischemic regions of skin, and we partitioned phasor space to classify necrotic tissue based on this short lifetime signature. This analysis technique demonstrated high accuracy (96%) in detecting distal necrotic regions from fresh frozen tissue section and revealed a sensitivity to IL-10/VEGF and IL-10/HGF gene transfer therapy in ischemic skin flaps (p≤0.0072). Future work will explore the temporal dynamics of ischemia-induced necrosis through longitudinal in vivo fluorescence lifetime imaging.

L4.04

Continuous Measurement of Sensory Responses in a 3-Dimensional Dermal Substitute for Neuropathic Research.
Justin Klug1, Alex Bunk2, Jennifer Hahn3, Jennifer Westphal1, Maksym Krutko1, Anh Vu1, Ilya Whitaker1, Leyla Esfandiari1, Greg Harris4, Dorothy Supp3, Steve Davidson2, Stacey Schutte1
1Biomedical Engineering, University of Cincinnati, Cincinnati, OH, United States 2Department of Anesthesiology, University of Cincinnati, Cincinnati, OH, United States 3Department of Surgery, University of Cincinnati, Cincinnati, OH, United States 4Department of Chemical Engineering, University of Cincinnati, Cincinnati, OH, United States
Continuous Measurement of Sensory Responses in a 3-Dimensional Dermal Substitute for Neuropathic Research.

Justin Klug1, Alex Bunk2, Jennifer Hahn3, Jennifer Westphal1, Maksym Krutko1, Anh Vu1, Ilya Whitaker1, Leyla Esfandiari1, Greg Harris4, Dorothy Supp3, Steve Davidson2, Stacey Schutte1 1Biomedical Engineering, University of Cincinnati, Cincinnati, OH; 2Department of Anesthesiology, University of Cincinnati, Cincinnati, OH; 3Department of Surgery, University of Cincinnati, Cincinnati, OH; 4Department of Chemical Engineering, University of Cincinnati, Cincinnati, OH

Creating an innervated dermal substitute with a guided neural sensory network capable of measuring sensation continuously. The model consists of 3-layers formed inside a Transwell insert. The bottom layer consists of a 1.5 mm thick collagen 1 hydrogel seeded with GCaMP6s modified sensory neurons (SNs) harvested from mouse dorsal root ganglia. The middle layer consists of a 1.5mm thick collagen 1 hydrogel, seeded with human fibroblasts (hDFs). The top layer consists of a thin layer of piezoelectric PVDF-Trfe, which is adhered to the middle layer with hDFs. After 14 days of culture, the engineered tissue was transferred to a calcium imaging suite where the bottom layer is scanned for neuronal cell bodies where the neuronal cell bodies are treated with Capsaicin. With the GCaMP6s modification, SNs that respond to the stimuli fluoresce as they depolarize and stop fluorescing as repolarization begins. Responses to stimuli were measured in AU. Collagen 3 and Laminin were added to observe the effect on tissue innervation. A one-way ANOVA with a 95% confidence interval was performed to identify statistical significance. Innervation, measured in percent neurite extensions per neuronal cell, was measured to be 52.9%. 30% addition of collagen 3 (w/w) to the ECM inhibited neurite growth, N = 20. After the initial stimulus is removed from the neuronal cell bodies, fluorescent response returned to base line within 450s where it could then be successfully stimulated again for a measurable response. We have developed a 3D construct capable of providing continuous numerical measurements of sensory responses to painful stimuli. By combining 2D neuronal electrophysiology techniques with 3D tissue engineering strategies we have documented a measurable tactile sensory response in an 3D engineered tissue. This model has proven to be effective despite no optimization of the 2D neurons or their 2D media for a 3D environment, making it a cost-effective method to study sensory neuronal populations in 3D. Our model is created in three distinct layers where the neuronal cell body is accessible for calcium imaging while still embedded in collagen hydrogel. SN populations from DRG are heterogeneous, with many different phenotypes specific to detecting different stimuli. Since we used DRG isolated SNs, we cannot yet say if this technique is effective for achieving measurable responses from all SN subtypes. In addition, without further testing of different stimuli aside from capsaicin, we have yet to determine if the SNs in the engineered tissue respond to other stimuli.

L4.05

Non-Invasive Measurements Of A Novel Skin Graft Substitute Using The Mexameter And The Dermascan
Charles Arcand, Dominique Mayrand, Danielle Larouche, Lucie Germain, Auger A. François, Véronique Moulin
Université Laval, Quebec, QC, Canada
Non-invasive Measurements of a Novel Skin Graft Substitute Using the Mexameter and the Dermascan.

Charles Arcand, Dominique Mayrand, Danielle Larouche, Lucie Germain, Auger A. François, Véronique Moulin Université Laval, Quebec, QC, Canada

Background: Severely burned patients requiring large skin coverage face a paucity of donor sites. Self-Assembled Skin Substitute (SASS), a bilayered autologous skin substitute, has been developed at our laboratory to remediate this situation. Its mechanical and physical properties following graft must be evaluated.
Methods: We were following the patients enrolled in the ongoing Canadian-wide trial SASS 2 (Self Assembled Skin Substitute for the Autologous Treatment of Severe Burn Wounds in Acute Stage of Burn Trauma). These severely burned patients were grafted using SASS and the conventional split-thickness skin graft (STSG). SASS and STSG measuring 50-100 cm2 were selected, grafted side-by-side on the same day and randomized as site A and site B. Measurements using two instruments: the Dermascan©(Cortex Technology) and the Mexameter MX 18© (Courage+Khazaka electronic GmbH) were performed on both sites at 3, 6-, 12-, 24- and 36-months post graft. Images generated with the Dermascan©, a 20 MHz ultrasound, allowed the measurement of dermal thickness. The thickness of SASS was compared with the adjacent STSG. The Mexameter MX 18©, a spectrophotometry device emitting three specific wavelengths, measures the skin melanin and hemoglobin (erythema) content using reflectance. As with the Dermascan, measurements were done on the two sites aforementioned.
Results: Dermal thickness as measured with the Dermascan© was comparable between SASS (3.01 mm +/- 0.24 mm) and the STSG (2.71 mm +/- 0.14 mm). These results persisted at 36 months. Heamoglobin and melanin being very patient specific, comparison between patients was omitted. For all patients, hemoglobin values were non statistically different between STSG and SASS regardless of time post-graft. Melanin values were significantly lower in the SASS than the STSG. This result was expected as skin culture medium used for the production of SASS hinder melanocyte replication.
Conclusion: This preliminary data supports the hypothesis that SASS is comparable to STSG with respect to dermal thickness and erythema. These biophysical properties strengthen our belief that SASS is a potential alternative to STSG. Conversely, melanin shows a lower value. As a result, the cosmetic appearance is significantly different, and UV sensitivity may be altered. With proper sun protection, this effect can be minimized. We believe that SASS has the potential to become an alternative to STSG in the treatment of severely burned patients.

L4.06

Decoding and Modulation Of Spiking Activity Of The Sciatic Nerve In An Awake And Moving Rodent
Katharina S. Fischer1, Eric T. Zhao3, Zeshaan Maan4, Janos Barrera4, Chikage Noishiki4, Ben Litmanovich2, Dominic Henn4, Sultana M. Mojadidi1, Javier Gonzalez1, Dharshan Sivaraj1, Andrew Hostler1, William Hahn1, Kellen Chen2, Nicholas Melosh3, Geoffrey C. Gurtner4
1Surgery, University of Arizona, Tucson, AZ, United States 2Surgery, University of Arizona, Tucson, AZ, United States 3Department: Materials Science and Engineering, Stanford University, Stanford, CA, United States 4Surgery, Stanford University, Stanford, CA, United States
Decoding and Modulation of Spiking Activity of the Sciatic Nerve in an Awake and Moving Rodent

Katharina S. Fischer1, Eric T. Zhao3, Zeshaan Maan4, Janos Barrera4, Chikage Noishiki4, Ben Litmanovich2, Dominic Henn4, Sultana M. Mojadidi1, Javier Gonzalez1, Dharshan Sivaraj1, Andrew Hostler1, William Hahn1, Kellen Chen2, Nicholas Melosh3, Geoffrey C. Gurtner1 1Surgery, University of Arizona, Tucson, AZ; 2Surgery, University of Arizona, Tucson, AZ; 3Department: Materials Science and Engineering, Stanford University, Stanford, CA; 4Surgery, Stanford University, Stanford, CA

Background: Conventional prostheses do not interface directly with neural signals but use either body motion or muscle activity as a proxy. Consequently, these prostheses are difficult to use, tiring, have limited function, and provide no sensory feedback. For the development of an advanced anthropomorphic prosthetic arm that can recapitulate the degrees of freedom and sensory feedback of the human hand, it is critical to design a low damage, high fidelity, and stable peripheral nerve interface (PNI). The key innovation of our microfabricated device, is that our electrodes are orders of magnitudes more compliant than existing PNIs, approximately 1500 and 40,000,000 times more compliant than the Transverse Intrafascicular Multichannel Electrode (TIME) and Utah Slanted Electrode Array (USEA) respectively.
Methods: The device consists arrays of individual 1 μm thick, and 4 μm wide electrodes, each with a 15 μm diameter recording/stimulation pad, mimicking the dimensions, compliance, and spatial distribution of axon bundles in the peripheral nerve. An electrochemically etched, 80 μm tungsten microwire was threaded through a hole on the device and drawn through the sciatic nerve of a C57BL/6J mouse. A custom designed circuit board was mounted on a 3D-printed backpack to facilitate chronic recording and stimulation.
Results: Mice ran voluntarily on a cylindrical treadmill in a head strained condition, while we recorded the neural activity with our device. Spiking activity was readily observed in our electrodes, where we isolated 6 single units across our 16 electrodes. Using markerless pose estimation, we extracted the joint angles innervated by the sciatic nerve and found a robust correlation between spiking activity and gait. The ultra-small size of the electrodes and their proximity to individual axons permitted extremely local stimulation – down to eliciting movement in a single toe.
Conclusions: To our knowledge, we are the first to record single neuron spiking activity in the peripheral nervous system of an awake and moving rodent. This represents a substantial advance, in that we overcame a series of challenges involving: (1) Microfabrication of ultra-thin electrodes to mirror the surrounding biomechanical environment to minimize the foreign body response, (2) Device robustness under substantial movement and strain, (3) Microsurgical device implantation to minimize acute insertion damage. Future work will involve electrical modulation to augment movement in peripheral nerve neuropathies and translation into larger animal models with increased channel counts for movement decoding.

O1.01

Distinct Oral Fibroblast Population Expedites Wound Healing By Modulating Innate Immunity
Kang Ko, Zhaoxu Chen, Rahul Debnath, Dana Graves
University of Pennsylvania, USA
Distinct Oral Fibroblast Population Expedites Wound Healing By Modulating Innate Immunity

Kang Ko, Zhaoxu Chen, Rahul Debnath, Dana Graves Periodontics, University of Pennsylvania, Philadelphia, PA

Background: Most injuries in humans heal by scarring, which impairs organ function. Oral wounds contrast this by healing with rapid resolution of inflammation and minimal scar formation. Despite interdisciplinary interest in harnessing this property, the cellular mechanism remains unclear. Here, we identify oral fibroblasts with an active 2.4kb Prx1 promoter element (Prx1+) as a progenitor population and tested the hypothesis that Prx1+ cells accelerate wound healing by modulating innate immune response.
Methods: For lineage tracing analysis, Prx1CreERT-eGFP.R26RtdTomato mice were used to label Prx1+ cells and tdTomato+ lineage cells in healing wounds. For wound induction, 1mm full-thickness wounds were created in hard palate, and healing parameters were assessed by histology and by collagen 3 staining. Single cell RNA sequencing (scRNA-seq) of unperturbed hard palate was used to characterize Prx1high fibroblasts and infer their lineage trajectory. Flow cytometry was used to immunophenotype macrophages (MΦ) and neutrophils in healing wounds that were enriched or deficient in Prx1+ cells. In series of animal studies, wound healing and inflammation were assessed in mice that had Prx1+ cells ablated by diphtheria toxin or in oral and skin wounds that had Prx1+ cells augmented via transplantation for rescue effect. Animal studies were carried out with n = 6-11 each, with statistical significance set to p<0.05 by t-test.
Results: Prx1+ cells were spindle-shaped and co-expressed pan-fibroblast marker PDGFRA. These fibroblasts exhibited differential spatial density and were found mostly in anterior palate but not in posterior region. Lineage tracing and scRNA-seq analyses revealed that Prx1+ cells differentiate towards stem cell antigen-1 (SCA1)+ fibroblasts which highly expressed chemokines such as CCL2. Prx1+ enriched oral wounds exhibited rapid influx of MΦ and subdued neutrophil response, whereas wounds lacking Prx1+ cells had persistent inflammation and healed slowly. In mice that had Prx1+ cells ablated, wounds had a high proportion of pro-inflammatory MΦ and decreased Col3+ stromal area. Autografting Prx1+ enriched oral tissue to Prx1-deficient area prior to wound induction improved stromal healing response, demonstrating rescue effect. Importantly, adoptive transfer of Prx1+ cells to skin accelerated wound closure, resolution of inflammation, and facilitated more complete healing as shown by new hair follicle formation and reduced α-smooth muscle actin expression. Conclusion: Our study provides a mechanism by which distinct Prx1+ cells mediate rapid wound healing by differentiating into chemotactic fibroblasts that regulate resolution of inflammation. This also explains how oral wounds display primed immune response and rapidly switch to a pro-healing environment, which may be exploited for treating wounds that heal by scar formation.

O1.02

Sensor-Enabled Multilayer Artificial Intelligence Analysis For Predictive Wound Healing And Real-Time Patient Monitoring
Artem Trotsyuk 2, Serena Jing 1, Kellen Chen 2, Dominic Henn 1, Yuanwen Jiang 1, Simiao Niu 1, Dharshan Sivaraj 2, Ronjon Nag 1, Michael Snyder 1, zhenan bao 1, Geoffrey Gurtner 2
1 Surgery, Stanford University, Stanford, CA, USA; 2 Surgery, University of Arizona, Tucson, AZ, USA
Sensor-enabled Multilayer Artificial Intelligence Analysis for Predictive Wound Healing and Real-Time Patient Monitoring

Artem A. Trotsyuk2, Serena Jing1, Kellen Chen2, Dominic Henn1, Yuanwen Jiang1, Simiao Niu1, Dharshan Sivaraj2, Ronjon Nag1, Michael Snyder1, zhenan bao1, Geoffrey C. Gurtner2 1Surgery, Stanford University, Stanford, CA; 2Surgery, University of Arizona, Tucson, AZ

Introduction: Approximately thirty million people in the United States suffer from chronic wounds. Patients who currently develop infection are at risk for sepsis, multisystem organ failure and death. Wound infections are also a leading cause of hospital re-admissions, increasing healthcare costs. 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 that by using patient wound etiology as well as predictive machine learning analytics, we can identify and incorporate sensors into a flexible smart bandage that will pave the way for high resolution, in situ tissue regeneration. Materials and Methods: Continuous patient wound data was collected at the Stanford Advanced Wound Care Center for a 9-month period. The dataset included basic patient demographics for 135 patients, as well as both quantitative and categorical information on patient wounds. Further, commercially available sensors were used to obtain additional wound characterizations that are outside the scope of standard-of-care data collection. This included temperature, pH, impedance and trans epidermal water loss. Wounds were split into training and test sets by random assignment. Data was used to determine which sensors were most correlated with high predictive accuracy for wound healing outcomes. Informed by the results, we prototyped and integrated sensors into a wireless flexile printed circuit board (PCB) and validated the device on patients.
Results and Discussion: By combining impedance and temperature sensor data with patient co-morbidly data and wound etiology, we were able to achieve an area under the curve of 0.939 (95% confidence interval) for predictive wound healing outcomes. Informed by these data, we then used a wireless flexible PCB to continuously record impedance and temperature data in real time. We observed that with normal healing, temperature gradually decreased, and impedance slowly increased. However, with an impeding infection, both temperature and impedance fluctuated sharply.
Conclusion: Our data demonstrates that by incorporating sensors into wound dressings, early and accurate prediction of an onset of infection and overall wound healing is possible. Further, our patient sensing platform can integrate treatment modalities to deliver closed-loop wound therapies, decreasing overall healthcare costs, improving patient quality of life, and allowing for clinicians to more accurately diagnose and prevent wound infections.

O1.03

Macrophage/T Cell Crosstalk to Promote Resolution of Inflammation
Erin O’Brien, Kara Spiller
School of Biomedical Engineering, and Health Systems, Drexel University, Philadephia, PA, USA
Macrophage/T Cell Crosstalk to Promote Resolution of Inflammation

Erin M. O’Brien, Kara L. Spiller School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA

Macrophages play an active role in multiple phases of wound healing, transitioning from a predominantly pro-inflammatory (M1) population to less inflammatory, pro-regenerative phenotypes (M2). As high-level regulators of healing, macrophages pose a strong influence on other cell types through the secretion of cytokines and the expression of immunomodulatory ligands. In some pathologies where wound healing is inhibited, M1 macrophages are insufficiently activated and also fail to switch to the M2 phenotype. Therefore, macrophages are an attractive target for cell therapies aiming to remedy dysfunctional wound healing characterized by chronic inflammation, such as that seen in aging patients. Previous work has shown that M2 macrophages that have previously been M1-activated, compared to those that have not, are phenotypically unique and exhibit enhanced M2 function. These “M1àM2” macrophages are characterized by expression of the M2 marker CCL17, a chemokine known to attract CCR4+ skin-homing T cells, particularly IL-4-secreting Th2 cells and anti-inflammatory regulatory T cells (Tregs). This finding, along with the upregulation of immunosuppressive ligands PD-L1 and PD-L2 on M1 and M1àM2 macrophages, respectively, begs the question of how macrophages may influence T cell differentiation (and vice versa) in the context of wound healing. However, interactions between macrophages and T cells during wound healing are poorly understood. In this study, we cultured human primary CD4+ T cells directly with autologous M0, M1, or M2 macrophages, or with conditioned media generated by those phenotypes. After three days of co-culture, both macrophages and T cells were characterized via flow cytometry. Compared to T cells cultured alone, T cells co-cultured with macrophages exhibited increased differentiation into the Th2 and Treg phenotypes, and decreased differentiation into the inflammatory Th1 phenotype. These findings suggest that macrophages may direct the resolution of inflammation via crosstalk with T cells. Interestingly, these results were not replicated in T cells cultured in macrophage-conditioned media, suggesting this crosstalk is dependent on cell-cell interactions. Finally, macrophages co-cultured with T cells upregulated the expression of PD-L1, indicating that T cells may also promote immunosuppressive activity in macrophages. Together, these findings point to a potential mechanism for the resolution of inflammation directed by macrophages, which may be leveraged to promote healing in chronic wounds.

O1.04

Wound Healing Impairments in Ischemic Limbs Reversed by a Novel Gene-Modified Mesenchymal Stem Cell Therapy
Carlos Huerta, Yulexi Ortiz, Yan Li, Antoine Ribieras, Francesca Voza, Nga Le, Zhao-Jun Liu, Omaida Velazquez
University of Miami, Miami, FL, USA
Wound Healing Impairments in Ischemic Limbs Reversed by a Novel Gene-Modified Mesenchymal Stem Cell Therapy

Carlos T. Huerta, Yulexi Y. Ortiz, Yan Li, Antoine J. Ribieras, Francesca A. Voza, Nga Le, Zhao-Jun Liu, Omaida C. Velazquez Surgery, University of Miami, Miami, FL

Background: Tissue loss resulting from chronic wounds significantly worsens the risk of extremity amputation among patients with chronic limb-threatening ischemia (CLTI). Cell-based therapies utilizing mesenchymal stem/stromal cells (MSCs) hold major promise for promoting wound healing and therapeutic angiogenesis. Here we report biologic effects of E-selectin modified MSCs, a cell-adhesion molecule capable of inducing post-natal neovascularization, on a translational murine model that creates critical hindlimb ischemia with cutaneous tissue loss.
Methods: Bone marrow cells harvested from 8-10 week old FVB/Rosa26SormTmG donor mice were cultured in MesenCult™ medium to enrich MSCs. MSCs were verified as CD44+/CD73+/CD105+/CD29+/Sca-1+ by flow cytometry. MSCs were transduced with E-selectin-GFP/AAV or GFP/AAV, and tri-lineage differentiation assay was conducted in vitro. Femoral artery ligation was performed in 12-14 week old recipient FVB mice immediately followed by a 4 mm cutaneous wound in the ipsilateral limb and local injection of PBS or 1×10^6 donor GFP+/MSCs or E-selectin+-GFP+/MSCs (E-sel+/MSC). Wound closure was monitored daily for 7 days, and tissue was harvested. Confocal microscopy and whole-body Dil perfusion were conducted to evaluate wound tissue vascular density.
Results: Untransduced MSCs do not express E-selectin, and E-sel+/MSCs retained MSC phenotype and trilineage differentiation capability. Mice receiving E-sel+/MSC treatment (n=10) demonstrated accelerated rates of wound closure compared to both GFP+/MSCs (n=10) and PBS (n=10) treated mice at each post-injury day (PID) with the highest degree of difference observed at PID five (94 ± 3% vs. 79 ± 10% GFP vs. 72 ± 9% PBS, p<0.001) and seven (98 ± 2% vs. 86 ± 8% GFP vs. 87 ± 6% PBS; p<0.010). Collagen deposition was more abundant in wounds treated with E-sel+/MSCs (21 ± 4% vs. 5 ± 1% GFP vs. 9 ± 1% PBS). Wound healing focused PCR-Array identified upregulation of target genes (>2-fold Cxcl3, Cxcl5, Mmp9, Wnt5a) in wound tissues responding to E-sel+/MSCs. DiI perfusion demonstrated increased vascular density in wound beds treated with E-sel+/MSCs (76 ± 15% vs. 29 ± 7% GFP vs. 9 ± 5% PBS relative vascular density) at PID 10. Immunofluorescence revealed that E-sel+/MSCs exhibit stronger viability given the presence of significantly more mTmG+-E-sel+/MSCs (13.2 ± 2.6 cells/high powered field (HPF)) in the wound bed compared to GFP+/MSCs (3.4 ± 1.6 cells/HPF) and PBS (0 ± 0 cells/HPF) controls at PID 7 (both p<0.010).
Conclusion: E-selectin modified MSC therapy augments wound healing in a murine hindlimb ischemia model. These results demonstrate a potential role for E-selectin supercharged MSCs as a novel cell-based therapy in future clinical applications for delayed and non-healing wounds associated with CLTI.

O1.05

CNP-miR146a Decreases Inflammation in a Two-Hit Model of Lung Injury
Alyssa Vaughn 1, Alison Wallbank 2, Stephen Niemiec 1, Tanner Lehmann 1, Sudipta Seal 3, Bradford Smith 2, Carlos Zgheib 4, Kenneth Liechty 4
1 Surgery, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2 Bioengineering, University of Colorado Denver, Denver, CO, USA; 3 Advanced Materials Processing and Analysis Center, University of Central Florida, Orlando, FL, USA; 4 Surgery, University of Arizona Tucson College of Medicine, Tucson, AZ, USA
CNP-miR146a Decreases Inflammation in a Two-Hit Model of Lung Injury

Alyssa E. Vaughn1, Alison Wallbank2, Stephen M. Niemiec1, Tanner Lehmann1, Sudipta Seal3, Bradford Smith2, Carlos Zgheib4, Kenneth W. Liechty4 1Surgery, University of Colorado Anschutz Medical Campus, Aurora, CO; 2Bioengineering, University of Colorado Denver, Aurora, CO; 3Advanced Materials Processing and Analysis Center, University of Central Florida, Orlando, FL; 4Surgery, University of Arizona Tucson College of Medicine, Tucson, AZ

Introduction: Acute respiratory distress syndrome (ARDS) exerts a substantial disease burden with significant morbidity and mortality. Acute inflammation is a central feature in the pathophysiology of acute lung injury (ALI) and subsequently ARDS. The need for mechanical ventilation can further exacerbate lung damage by ventilator-induced lung injury (VILI). Current treatment for ARDS is mainly limited to supportive care. We have previously shown that intratracheal (IT) delivery of CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA(miR)-146a, reduces bleomycin-induced acute lung injury by decreasing inflammatory infiltrates and oxidative stress. We hypothesized that CNP-miR146a treatment would similarly decrease inflammation in a lipopolysaccharide (LPS) bacterial endotoxin model of ALI and further protect against secondary VILI by decreasing pro-inflammatory signaling in this model.
Methods: 8-10 week old C57BL/6 mice were given IT LPS (50μg) to induce lung injury or 50μL saline. Four hours following injury, mice received IT administration of 1ng/50μL CNP-miR146a or an equivalent volume of saline. At 24 hours post-injury, a subset of mice were anesthetized, tracheostomized, and mechanically ventilated for 3 hours at tidal volume 20cc/kg, respiratory rate 75 breaths/minute, and positive end expiratory pressure 3 cmH20. Whole lungs were then harvested and processed for total RNA extraction. Real-time quantitative polymerase chain reaction analysis was performed to evaluate expression levels of pro-inflammatory genes. Statistical significance was determined by one-way ANOVA (p<0.05 significant).
Results: In this two-hit model, we found that LPS-injured lungs were prone to a second-hit injury from mechanical ventilation. Compared to ventilated controls, ventilated LPS-injured mice had significantly higher relative gene expression of pro-inflammatory IL-6 (p<0.0001), IL-8 (p<0.0001), and IL-1b (p<0.0001). Treatment with CNP-miR146a significantly lowered expression of IL-6 (p=0.04, 0.0003) and IL-8 (p=0.02, 0.15) compared to LPS-injured lungs in both the one- and two-hit models, respectively.
Conclusion: Treatment of LPS-induced lung injury with CNP-miR146a significantly decreases pro-inflammatory gene expression and provides additional protection against inflammation due to secondary VILI. CNP-miR146a has significant promise as a potential therapeutic for the treatment of ARDS and mitigation of secondary VILI.

O1.06

Interrogation of Wound Healing with Single Cell Analysis using a Wireless Smart Bandage
Artem Trotsyuk2, Yuanwen Jiang1, Simiao Niu1, Kellen Chen2, Dominic Henn1, Dharshan Sivaraj2, Zhenan Bao1, Geoffrey Gurtner2
1Surgery, Stanford University, Stanford, CA, United States. 2Surgery, University of Arizona, Tucson, AZ, United States
Interrogation of Wound Healing with Single Cell Analysis using a Wireless Smart Bandage

Artem A. Trotsyuk2, Yuanwen Jiang1, Simiao Niu1, Kellen Chen2, Dominic Henn1, Dharshan Sivaraj2, zhenan bao1, Geoffrey C. Gurtner2 1Surgery, Stanford University, Stanford, CA; 2Surgery, University of Arizona, Tucson, AZ

Introduction: 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 and evaluate the biology of wound healing in real time. We believe this can be achieved using a multidisciplinary approach combining electrical, biological and chemical engineering with the fundamentals of cellular and biomolecular processes in wound healing directed towards high resolution, in situ tissue regeneration.
Materials and 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 and Discussion: 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, namely activation of pro-regenerative macrophages. 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 that was used to evaluate wound healing in real time, measuring the biologic cellular response. 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.

O1.07

Targeting Anaerobic Metabolism in Pseudomonas aeruginosa Resolves Chronic Wound Infections and Results in Wound Healing
Melanie Spero1, Jane Kim2, Zachery Lonergan3, Ines Trindade3, Dianne Newman3, Manuela Martins-Green2
1Biology, University of Oregon, Eugene, OR, USA; 2Molecular, Cell and Systems Biology, University of California Riverside, Riverside, CA, USA; 3California Institute of Technology, Pasadena, CA, USA
Targeting Anaerobic Metabolism in Pseudomonas aeruginosa Resolves Chronic Wound Infections and Results in Wound Healing

Melanie Spero1, Jane Kim2, Zachery Lonergan3, Ines Trindade3, Dianne Newman3, Manuela Martins-Green2 1Biology, University of Oregon, Eugene, OR; 2Molecular, Cell and Systems Biology, University of California Riverside, Riverside, CA; 3California Institute of Technology, Pasadena, CA

Chronic wound infections contribute to impaired wound healing. Unfortunately, antibiotic treatment often fails to resolve chronic wound infections, in part because many antibiotics are ineffective at killing bacteria in the absence of oxygen. The wound environment is largely anoxic, and pathogens grow as biofilms within wounds, which harbor anoxic populations that display antibiotic tolerance. One novel approach for combatting antibiotic failure is to identify drugs that target pathogen-specific anaerobic processes. Anaerobic nitrate respiration is a widespread energy metabolism used by pathogens like P. aeruginosa. We found that the small molecule, chlorate, acts as a pro-drug to selectively kill pathogens that employ nitrate respiration. Specifically, chlorate is reduced by the nitrate reductase enzyme, Nar, thereby generating toxic chlorite inside pathogen cells. We showed that chlorate kills anoxic, antibiotic-tolerant P. aeruginosa biofilm populations in vitro. To test whether chlorate treatment is effective against P. aeruginosa in vivo, we used a diabetic chronic wound mouse model. Briefly, db/db-/- mice received full thickness excision wounds, which were inoculated with P. aeruginosa. Wounds were untreated for 10 days, allowing them to become chronic. Using tissue-clearing and confocal microscopy, we visualized P. aeruginosa biofilm formation deep within wounds. Starting on day 10, we treated wounds daily with chlorate or a vehicle (control). All wounds increased in size during the first 15 days, but control wounds showed no decrease in size by day 40, whereas chlorate treated wounds were significantly smaller at day 40. Histology and immunolabeling studies of day 40 wounds showed that chlorate-treated wounds had markers of proper healing, such as re-epithelization, granulation tissue formation, basement membrane formation, and blood vessel development, whereas control wounds did not display these markers. Finally, we found that mouse wounds healed naturally (i.e. without treatment) when they were infected with a P. aeruginosa nar mutant strain, demonstrating for the first time that P. aeruginosa requires this anaerobic metabolism to establish a chronic infection. Importantly, in prior work we used an in vitro genetic screen to show that Nar inactivation is the primary mechanism of chlorate resistance in P. aeruginosa. Taken together, these results are particularly promising because they suggest that acquiring chlorate resistance (i.e. Nar inactivation) will severely hinder this pathogen’s ability to perpetuate a chronic infection. Our work demonstrates that targeting anaerobic metabolism in P. aeruginosa infected wounds is an effective approach for resolving chronic wound infections.

O1.08

Cutaneous Wounds Occurring After Exposure To Spaceflight Do Not Exhibit Impacts To Re-Epithelialization: Analyses Of Mice Returned From The International Space Station
Bonnie Carney 1, Eriks Ziedins 1, Allison Hoke 2, Stephen Butler 2, Lauren Moffatt 1, Abdulnaser Alkhalil 1, Aarti Gautam 2, Nabarun Chakraborty 2, Carolynn Conley 3, James Mcleroy 3, Rasha Hammamieh 2, Jeffrey Shupp 1
1 MedStar Health Research Institute, Washington, DC, USA; 2 Walter Reed Army Institute of Research, Washington, DC, USA; 3 Space Test Program, NASA, Houston, TX, USA
Cutaneous Wounds Occurring After Exposure to Spaceflight Do Not Exhibit Impacts to Re-Epithelialization: Analyses of Mice Returned from the International Space Station

Bonnie Carney1, Eriks Ziedins1, Allison Hoke2, Stephen Butler2, Lauren Moffatt1, Abdulnaser Alkhalil1, Aarti Gautam2, Nabarun Chakraborty2, Carolynn Conley3, James Mcleroy3, Rasha Hammamieh2, Jeffrey W. Shupp1 1MedStar Health Research Institute, Washington; 2Walter Reed Army Institute of Research, Washington; 3Space Test Program, NASA, Houston, TX

Background: As commercial spaceflight(SFLT) expands and astronauts spend extended time in space, a need exists to better understand the impacts of SFLT conditions on health, including cutaneous healing. More broadly, alterations in gravitational force are known to modulate mechanical forces in tissues; hence SFLT offers an opportunity to modulate cell-cell and cell-extracellular matrix dynamics in the skin, and thereby modulate skin wound healing and regeneration. It was hypothesized that exposure of mice to SFLT on-board the International Space Station (ISS) would lead to slower wound healing. Methods: Mice were exposed to SFLT for 2 months on the ISS and were then returned to gravity and exposed for 48 hours before wounding. These animals were termed the flight(FLT) group(n=4). There were two control groups. The 1st was housed in identical caging, feed, and environmental conditions as the FLT group and were termed the habitat ground controls(HGC)(n=4). The 2nd was housed per vivarium standards in standard caging and feed and were termed vivarium ground controls(VGC)(n=4). All animals were shaved and depilated. Bilateral 6 mm wounds were created and were stented open to promote re-epithelialization. Wounds were collected at hour 108 post-wounding and were processed and stained with H&E or immuno-stained with cytokeratin-14 and Ki-67. Epithelial tongue length, Ki67 positive cells, and % cellularity in the wounds was measured. One-way ANOVA with multiple comparisons and Tukey’s was used and p<0.05 was significant.
Results: There were no differences in epithelial tongue length between the FLT, HGC, and VGC groups (227.6±97.9 vs. 180.0±33.7 vs. 114.8±9.4 mm, respectively). There were no differences in Ki67 positive cells between the groups (1.31±0.06 vs. 1.40±0.20 vs. 1.23±0.04 CY3:DAPI ratio, P<0.05) and each group showed robust proliferation within the epithelial tongues. The FLT group had significantly increased dermal % cell infiltrate under the region of epithelialization compared to the VGC group (29.31±16.21% vs. 18.90±1.63%, respectively, p=0.0105). The HGC group was also decreased compared to the FLT group.
Conclusion: Exposure to SFLT and subsequent return to terrestrial gravity conditions does not inhibit a rodent’s ability to undergo re-epithelialization after wounding. Infiltration of proliferative inflammatory cells is increased in FLT groups indicating a robust wound healing potential. These findings may have implications for astronauts injured shortly after return to earth. Given the conditions created by variable gravitational forces on cell-cell and cell-ECM interactions, the model may also offer broader insight to therapeutic targets for non-astronauts.

P1.01

Neutrophilic 5-LO Promotes Burn Wound Healing By Supporting The Early Inflammatory Phase
Shannon M. Clayton1, Miguel Gijon2, Alan Nguyen1, Glendalyn Smith1, Marilyn Wang1, Paul Kennedy1, Rivkah Isseroff1, Athena Soulika1
1Dermatology, UC Davis, Sacramento, CA, United States 2Cayman Chemical Company, Ann Arbor, MI, United States
Neutrophilic 5-LO Promotes Burn Wound Healing by Supporting The Early Inflammatory Phase

Shannon M. Clayton1, Miguel Gijon2, Alan Nguyen1, Glendalyn Smith1, Marilyn Wang1, Paul Kennedy1, Rivkah Isseroff1, Athena Soulika1 1Dermatology, UC Davis, Sacramento, CA; 2Cayman Chemical Company, Ann Arbor, MI

Neutrophils are among the first responders in burn wound injury. However, their migration to the affected site is not well understood. One of the most potent neutrophil chemoattractant is LTB4, which can be produced in large levels by neutrophils themselves. LTB4 is synthesized by 5-lipoxygenase (5-LO) mediated metabolism of arachidonic acid. Using a murine scald burn wound model, we show that neutrophils are persistently increased in burn wounds compared to healthy skin. We also show that both the transcripts of the 5-LO pathway associated genes (p<0.05) and LTB4 levels (p<0.01) are increased at the wound site both during the inflammatory and the proliferative phases, following burn injury. In other models, studies have shown that neutrophilic 5-LO promotes neutrophil swarming to the affected site, likely via LTB4. Thus, we formed the hypothesis that in burn injury, neutrophilic 5-LO also increases and sustains neutrophil numbers at the wound site, likely delaying healing. To test our hypothesis, we generated a mouse in which 5-LO is specifically deleted in neutrophils (MRP8-Cre+Alox5fl/fl) and its control (MRP8-Cre+Alox5wt/wt). MRP8-Cre mice employ the human MRP8 promoter, which has been previously shown to restrict Cre expression to neutrophils. After inflicting scald burn injury, immunohistochemical analysis of wound tissues of MRP8-Cre+Alox5wt/wt mice showed 5-LO immunoreactivity colocalized both with neutrophils (Ly6G+) and non-neutrophil (Ly6G-) cells. In burn tissues of MRP8-Cre+Alox5fl/fl mice, Ly6G+ cells did not show 5-LO immunoreactivity, while Ly6G- cells did, suggesting that 5-LO deletion is specific to neutrophils. Unexpectedly, flow cytometric analysis of burn wounds during the inflammatory and proliferative phases showed that neutrophilic 5-LO deletion did not affect either the relative frequencies or the total numbers of neutrophils. Interestingly, the relative frequencies of inflammatory monocytes were decreased in MRP8-Cre+Alox5fl/fl wounds compared to their controls during the early stages post burn injury (p<0.0498, n= 4). Furthermore, and contrary to our original hypothesis, MRP8-Cre+Alox5fl/fl mice showed delayed healing compared to control mice as shown by decreased re-epithelialization rates on day 3 (3.02 ± 3.62 versus 4.52 ± 0.967, respectively; p<0.0214, n=4) and day 14 (38.9 ± 10.2 versus 68.5 ± 28.8, respectively; p<0.0495, n=3) post burn injury. In conclusion, our data show that neutrophilic 5-LO does not affect neutrophil recruitment, which is likely mediated through other mechanisms or by LTB4 synthesized by other 5-LO expressing cells, in burn injury. Instead, neutrophilic 5-LO promotes the presence of inflammatory monocytes at the wound site during the early stages following burn injury, which may promote the timely transition to the proliferative phase, thus promoting healing.

P1.02

Topical treatment with Non-Euphoric Phytocannabinoid Elixir 14 (NEPE14) Reduces Inflammation And Mitigates Burn Wound Progression
Sean Christy2, Anders Carlson2, David Larson2, Robert Christy2, Kristo Nuutila1
1USAISR, San Antonio, TX, United States 2Metis Foundation, San Antonio, TX, United States
Topical Treatment with Non-Euphoric Phytocannabinoid Elixir 14 (NEPE14) Reduces Inflammation and Mitigates Burn Wound Progression

Sean Christy2, Anders Carlson2, David Larson2, Robert Christy2, Kristo Nuutila1 1USAISR, San Antonio, TX; 2Metis Foundation, San Antonio, TX

Background: Combat-related burn wounds are caused by exposure to a wide variety of agents including heat, electricity, radiation, chemicals, and friction. Early intervention can decrease injury severity by preventing excess inflammation and improve long term healing outcomes. In recent years, numerous studies have demonstrated that cannabinoids can trigger anti-inflammatory responses and promote wound closure. Therefore, the purpose of this study was to investigate whether a topical application of NEPE14 containing a full complement of phytocannabinoids (<0.3% delta-9-THC or CBD) and other phytochemicals in a cosmetic lotion base would mitigate burn wound progression in the treatment of deep partial-thickness (DPT) wounds.
Methods: DPTs were created on the dorsum of four anesthetized swine using a thermocoupled burn device. One hour following burn, biopsies from each site were collected and either NEPE14, Silverlon® or gauze was placed on the wound. Wounds were assessed on post-burn days 3, 7 and 14. Assessments consisted of digital photographs, Laser-Speckle imagery (blood perfusion), MolecuLight® imagery and biopsies for histology and immunohistochemistry (IL6 and TNFa).
Results: Topical treatment with NEPE14 significantly (p < 0.001) decreased inflammation (IL6 and TNFa) in comparison to control groups. It was also demonstrated that the reduction in inflammation led to mitigate burn wound progression. The results showed that burn depth on day 3 was 528±35 μm, 839±111 μm and 991±99 μm in the NEPE14, Silverlon® and gauze treated burns respectively. The difference between the NEPE14 and the gauze treated burns was statistically significant (p < 0.05). In terms of wound healing and presence of bacteria no statistically significant differences were observed. However, there was trend showing that NEPE14 treated wounds had re-epithelialized better by day 14 than the control groups.
Conclusion: Topical treatment of deep partial-thickness wound with NEPE14 decreased wound inflammation and mitigated burn wound progression in comparison to control treatments.

P1.03

AutoMated Burn diagnostic System for Healthcare (AMBUSH)
Mohamed El Masry1, Surya Gnyawali1, Maxwell Jacobson2, Maria Tricas2, Yexiang Xue2, Juan Wachs2, Chandan K. Sen1, Gayle Gordillo1
1Surgery, Indiana University, Indianapolis, IN, United States 2Purdue University, West Lafayette, IN, United States
AutoMated Burn diagnostic System for Healthcare (AMBUSH)

Mohamed El Masry1, Surya Gnyawali1, Maxwell Jacobson2, Maria Tricas2, Yexiang Xue2, Juan Wachs2, Chandan K. Sen1, Gayle Gordillo1 1Surgery, Indiana University, Indianapolis, IN; 2Purdue University, West Lafayette, IN

Background: In the United States, about 1.25 million people are treated each year for burns, and 40,000 are hospitalized for the treatment of these injuries resulting in high medical costs, approximately $7.9 billion per year. Early assessment of burn depth, considered a predictor of pathological scarring that occurs in 30%-91% of burn injuries, and prioritizing burns that require surgical intervention is a critical task. However, it continues to be an open clinical challenge. We sought to develop a high accuracy automated system, that relies on multimodal Harmonic B-mode ultrasound (HUSD B-mode) and Tissue Elastography imaging (TEI), to classify burn pathology using novel techniques based on machine learning and artificial intelligence (AI).
Methods: Burn wounds of different degrees (superficial, partial, and full thickness; n=2; size 2″x2″) were created on the dorsum of female domestic pigs (70-80lbs) (n=6 pigs) using a standardized burner. Progression of burn wounds was followed by non-invasive imaging using digital photographs, HUSD B-mode, and TEI videos at day 0 – post-burn, and on days 3, 7, 14, 21, 28, 35 and 42 postburn. Burn depth was validated by histopathological analysis and results were compared with ultrasound-acquired data at different time points. State-of-the-art deep learning methods to analyze images and videos such as convolutional neural networks (CNNs) were employed. These features were used to train task-specific networks. In the case of depth classification, the classifier was further enhanced using traditional computer vision features.
Results: Burns of different degrees were successfully created on all the pigs. HUSD B-mode and TEI showed characteristic biomechanical and biological response patterns unique to the different degrees of burn which was validated by H&E staining. Histological pattern graded the burn injury from superficial involving only epidermal layer to the full thickness burn involving all skin and subcutaneous layers. Data labelling, segmentation and augmentation was done and fed into the AI system. Our system was able to classify burn wounds with a mean accuracy greater than 90% which is significantly exceeds that of baseline algorithms. In burn segmentation, our system achieved a mean global accuracy greater than 0.87. Further, we calculated a mean intersection over union (IoU) score of 0.8. These scores represent a statistically significant improvement over our baseline segmentation model. Critically, this part of our system presented a clear and human-readable masks to understand the surface of burn wounds, allowing a high degree of explainability often required to interpret AI-produced results.
Conclusion: This work presented elements of an autonomous AI system to analyze and predict burn depth via texture-based image processing algorithms using multiple common medical modalities.

P1.04

Full-Thickness Skin Columns: A Novel Method for Skin Replacement Therapy
Corey Keenan1, Laura Cooper2, Rodney Chan3, Anders Carlson2, Kristo Nuutila2
1General Surgery, US Army, El Paso, TX, United States 2USAISR, San Antonio, TX, United States 3Plastic Surgery, San Antonio Military Medical Center, Ft. Sam Houston, TX, United States
Full-Thickness Skin Columns: A Novel Method for Skin Replacement Therapy

Corey Keenan1, Laura Cooper2, Rodney Chan3, Anders Carlson2, Kristo Nuutila2 1General Surgery, US Army, El Paso, TX; 2USAISR, San Antonio, TX; 3Plastic Surgery, San Antonio Military Medical Center, Ft. Sam Houston, TX

Background: A novel method of skin replacement therapy utilizing full-thickness skin columns (FTSCs) takes advantage of the functional benefits of full thickness skin grafts with little downside. This study aimed to determine the optimal implantation density of FTSCs as well as the maximal harvest density of FTSCs from donor sites in a porcine model.
Methods: Fifty deep-partial thickness burns were created on five anesthetized swine. FTSCs were harvested and implanted evenly across varying density ranges (1:1, 1:2, 1:4, 1:9 columns/cm2). Burned, untreated skin was utilized as a negative control. For the maximal harvest density, ten donor sites were created on one anesthetized swine with the highest possible harvest ratio of sixteen 1.5mm-diameter skin columns/cm2 (28%). Split thickness skin graft (STSG) donor sites were harvested with a dermatome as comparison. In both studies, healing was assessed via re-epithelialization, contraction, pigmentation, and scar thickness on post-burn day (PBD) 7, 14, 21, 28, 60, & 90.
Results: Regarding implantation density, by post burn day (PBD) 28 the wounds treated with the 1:1 and 1:2 densities had a mean re-epithelialization of 99% and 98%, respectively. Both were significantly more re-epithelialized as compared to the burned, untreated control, with mean re-epithelialization of 84%, at PBD 28 (p=0.02 and 0.03, respectively). At PBD 90, wounds treated with 1:1 and 1:2 implantation densities visually appeared to contract less than the burned, untreated control; however, these results were not significant. Implantation densities of 1:4 and 1:9 showed no significant difference compared to burned, untreated control. When studying donor site morbidity, harvest densities of 7% was found to have faster re-epithelialization (100% at 7d vs. 28d) and less dyspigmentation as compared to STSG donor sites. At a maximal FTSC harvest density of 28%, FTSC and STSGs were found to have similar patterns of being initially hypopigmented until around day 28 where they became hyperpigmented until day 90 when no significant difference was noted, compared to uninjured skin. In terms of scar thickness, FTSC’s donor sites were found to be significantly thicker compared to STSG and control on PBDs 28 and 60, until day 90 when no significant differences in thickness were observed (FTSC v. control: p=0.01 and 0.01, respectively; FTSC v. STSG: p=0.001 & 0.02, respectively). FTSC and STSG were significantly more vascular as compared to intact skin control; however, there was no significant difference in vascularity between FTSC and STSG. There were no significant differences in re-epithelialization at PBD 14 or contraction at PBD 90 between FTSC and STSG donor sites. All donor sites were completely re-epithelialized by PBD 21.
Conclusion: Implantation ratios of 1:1 and 1:2 improve re-epithelialization and appear less contracted. At 28% harvest density, donor site morbidity was not superior to STSGs, exceeding the optimal harvest density.

P1.05

The Advent of a Complete Approach to Complex Burns: The Combination of Pixel and Fat Grafting with a Moist Environment
Yadira Villalvazo1, Pooja Humar1, Fuat Baris Bengur1, Shawn Loder1, Wayne V. Nerone1, Alexandra Vagonis1, Bahaa Shaaban1, Rachel Ricketts1, Lauren Kokai1, Kacey G. Marra1, Elof Eriksson2, J. Peter Rubin1
1Department of Plastic and Reconstructive Surgery, University of Pittsburgh, Pittsburgh, PA, United States 2Harvard Medical School, Boston, MA, United States
The Advent of a Complete Approach to Complex Burns: The Combination of Pixel and Fat Grafting with a Moist Environment

Yadira Villalvazo1, Pooja Humar1, Fuat Baris Bengur1, Shawn Loder1, Wayne V. Nerone1, Alexandra Vagonis1, Bahaa Shaaban1, Rachel Ricketts1, Lauren Kokai1, Kacey G. Marra1, Elof Eriksson2, J. Peter Rubin1 1Department of Plastic and Reconstructive Surgery, University of Pittsburgh, Pittsburgh, PA; 2Harvard Medical School, Boston, MA

Background: Thermal burns are highly morbid injuries risking severe disfigurement, and functional and psychosocial impairment. Management requires consideration not only to the insult, but a patient’s quality of life. Interventions often require stage-reconstruction, revision for increasingly unsatisfactory results and a burdensome dressing during healing. This standard is costly, limited by donor-site availability, and often morbid. Currently there are no single-stage interventions with simple dressings to address complex burns. Here we aim to provide a complete approach by demonstrating the efficacy of single-staged minced skin grafting plus autologous adipose grafting in an incubator-like microenvironment with a platform wound device (PWD) to achieve rapid reconstruction after burns.
Methods: A customized burn device was used to induce 16 standardized full-thickness burns on Female Yorkshire swine. Delayed escharectomies were performed to the level of fascia. In one group, autologous split-thickness skin was cut into pixel size grafts, and combined with or without autologous adipose. The groups were then divided by bolster or PWD dressing. Wounds were followed for 5 weeks with photography, cutometer, ultrasound, histology and tension measurements.
Results: The PWDs provided an optimized environment and protected the wounds from graft displacement despite no limitations in mobility. There was accelerated wound closure in the PWD group. As early as one week, epithelialization started in the PWD group with visible epithelioid islands on the wound bed granulation tissue. This progressed with a similar trend throughout the 5-week period leading to complete epithelization and keratinization. Gross appearance of the scars showed distinct differences in topography between the groups. Cutometer illustrated an enhanced trend to return to baseline gross elasticity in the PWD group. Overall, distinct differences in contour were noted between the bolster and PWD groups.
Conclusion: The management of complex burns has stalled since the introduction of excision and grafting. Little focus has been placed on the overall patient burden during recovery. This study demonstrates the efficacy of a single-staged approach and the use of a sustainable dressing that both provides enhanced wound healing and decreased burden. In both the bolstered and PWD group, pixel and fat grafts survived to form a viable basal layer. The use of a negative-pressure PWD versus a positive-pressure bolster demonstrated critical implications for accelerated and aesthetic reconstruction. This study introduces a wholistic approach that would challenge the need for the status quo staged reconstruction often performed in complex burns.

P1.06

A Healing Assessment of Full Thickness Burns Compared to Excisional Wounds After Meshed Split-Thickness Skin Grafting
Brooke Pierson, Mary Oliver, Dillon Williams, Monica Collins, Lauren Moffatt, Taryn Travis, Jeffrey W. Shupp, Bonnie Carney
MedStar Research Institute, Washington DC, DC, United States
A Healing Assessment of Full Thickness Burns Compared to Excisional Wounds After Meshed Split-Thickness Skin Grafting

Brooke Pierson, Mary Oliver, Dillon Williams, Monica Collins, Lauren Moffatt, Taryn Travis, Jeffrey W. Shupp, Bonnie Carney MedStar Research Institute, Washington DC

Background: Excisional wounds have been used in animal models to study full-thickness injury to the skin. Burn wounds are excised after a certain time point, thus this model provides a more efficient, repeatable, and controlled experimental process. However, it is not well understood what impact the lack of a burn has on a gross, histologic, and cellular level. We seek to understand whether there is differential healing, potentially due to the differential recruitment of inflammatory cells to the wound or the local burn signaling cascade causing damage to peri-burn cells necessary for migration and wound healing. It was hypothesized that excisional wounds would heal faster than burns followed by excising and that histologic metrics of healing would reflect this increased rate. Methods: Red Duroc pigs had full thickness excisional wounds(n=4) or full thickness burn wounds(n=6) created on their flanks(n=5 total animals). Two days prior to excision, full thickness burn wounds were created with a branding iron. On day 0 all burns and skin were excised to the level of subcutaneous tissue and grafted 4:1 using an autologous meshed split-thickness skin graft (mSTSG). Healing was measured at days 3, 5, 9, 28, and 49 by punch biopsies and digital imaging. Biopsies were taken specifically over the area of mSTSG to increase homogeneity. They were processed and stained. Digital images were analyzed in ImageJ to quantify % re-epithelialization. All were analyzed using two-way ANOVA with multiple comparisons. P<0.05 was considered significant.
Results: At days 3 and 5, the excisional wounds had a higher percentage of re-epithelialization as compared to the burn plus excision group(Day 3: 53.77±1.46 vs. 34.72±2.70 %, p<0.01, Day 5: 60.22±1.66 vs. 40.81±2.61%, p<0.01). At days 7 and 9 % re-epithelialization was similar between the groups. In both groups, dermal thickness increased from day 5(557.3±198.1 μm) to day 49(4076.0±597.7 μm). There were no significant differences between the groups. In both groups, epidermal thickness peaked at day 9(208.9±39.35 μm) and then resolved at days 14, 28, and 49(102.5±12.28, 86.50±17.50, 148.2±59.70μm). There were no significant differences between the groups. In both groups, rete ridge ratio peaked at day 9(1.86±0.12 RRR) and then resolved at days 14, 28, and 49(1.68±0.16, 1.52±0.12, 1.40±0.04 RRR). There were no significant differences between the groups.
Conclusion: This data suggests that the incorporation of a burn wound may be important for studies seeking to examine burn wound healing in the acute time frame. However, other histologic metrics of healing were not different among the groups. Ongoing studies are aimed at examining other molecular markers of injury and healing to help elucidate local and regional cutaneous signaling that may influence ability to heal after the specific injury type.

P2.01

Comparative Profiling of mRNA Expression in Infected Wounds Following Electrotherapy in the Porcine Model
Nava Rijal, Taylor Wallen, Michael Goodman, Andrei Kogan, Melanie Cushion, Daria Narmoneva
Biomedical Engineering, University of Cincinnati, USA
Comparative Profiling of mRNA Expression in Infected Wounds Following Electrotherapy in the Porcine Model

Nava Rijal, Taylor Wallen, Michael Goodman, Andrei Kogan, Melanie Cushion, Daria Narmoneva Biomedical Engineering, University of Cincinnati, USA

Background: Poor vascular supply and infection are significant factors in impaired healing of chronic wounds. We have recently developed a non-contact wireless electric field (EF) therapy that can stimulate cells within the entire wound and demonstrated its positive effects on wound vascularization and healing. The objective of this study is to quantify the effects of the electrotherapy on gene profile in the infected wounds in the porcine model. Hypothesis: The wireless electrotherapy augments infection-induced changes in gene expression profile 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. Experimental groups included electrotherapy vs. untreated control, each with or without infection, and additional non-injured skin control group (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 TegadermTM dressing. Electrotherapy was applied 1 hr/day, 3-5 days/week, for 2 weeks, at which time 2-mm punch biopsies were collected; tissue was used to quantify bacterial load or to extract RNA for gene expression (RNAseq) analyses.
Results: The results demonstrate significantly reduced levels of both types of bacteria in electrotherapy-treated wounds at 2 weeks post inoculation. RNA-Seq–based differential expression analysis 369 DEG differentially expressed genes (DEGs) satisfied the comparison filtering criteria (P < 0.05; log2 fold change > ±1, electrotherapy vs control). Hierarchical clustering analyses identified specific sets of upregulated DEGs in the infected wounds following electrotherapy, with the most significant differences in signaling pathways associated with antimicrobial humoral immune response, IL-6, MMPs, pro-angiogenic VEGF and MAPK pathways, with additional analyses currently ongoing. The findings are consistent with our previous results for improved wound healing following electrotherapy and provide mechanistic insights. Interestingly, electrotherapy resulted in a highly-significant (log10(p)=40) reversal of the >100-fold downregulation of keratin-associated genes (e.g. KRT2) in the infected wounds, which is associated with compromised wound healing.
Conclusion: The results demonstrate that wireless electrotherapy is effective in reducing infection-induced impairments in cellular responses in the wound, suggesting the promise of this strategy to improve healing outcomes in patients with infected non-healing ulcers.

P2.02

Predictive Factors for Limb Salvage Versus Amputation in Diabetic Patients with Lower Extremity Wounds
Pooja Humar, Elizabeth Moroni, Joseph Mocharnuk, Maryanna Owoc, Yadira Villalvazo, J. Peter Rubin, Brodie Parent
University of Pittsburgh, Gibsonia, PA, United States
Predictive Factors for Limb Salvage Versus Amputation in Diabetic Patients with Lower Extremity Wounds

Pooja Humar, Elizabeth Moroni, Joseph Mocharnuk, Maryanna Owoc, Yadira Villalvazo, J. Peter Rubin, Brodie Parent University of Pittsburgh, Gibsonia, PA

Background: Lower extremity (LE) wounds are common sequela of diabetes and vascular disease. The American Diabetes Association estimates 11% of the United States (US) population had diabetes in 2018. Approximately 15% of patients with diabetes develop a foot ulcer during their lifetime. Of those patients, 6% are hospitalized due to infection or other ulcer-related complication, which may progress to amputation. This study analyzes wound healing in patients with non-traumatic LE wounds to determine predictive factors for delayed wound healing, limb loss, and limb salvage.
Methods: A retrospective review of patients seen at an outpatient wound care clinic network from 2015-2022 was completed. Patients with diagnosed diabetes and at least 1 non-traumatic LE wound were included. Multinomial logistic regression was performed on a combination of numerical and categorical variables. Backwards selection was used, optimizing for the Akaike Information Criterion (AIC) estimator of prediction error, and the likelihood ratio test was used to assess the significance of each variable.
Results: 3,632 patients had non-traumatic LE ulcers with an average of 3 wounds per patient. Of these patients, 542 (14.9%) underwent amputation. Average age at the time of first wound care clinic visit was 66.7yrs ± 12.8yrs. 60.4% of the cohort was male and the rest female. In this cohort, only 41.2% of patients healed all their wounds at the time of their last clinic visit. Patient with unhealed LE ulcers had an average of 2 open wounds present at their last clinic visit. Area deprivation index (ADI) (p<0.01), number of wound clinic encounters (p<0.01), and active smoking (p=0.046) were all associated with limb salvage. Patients with unhealed LE wounds had significantly higher ADI when compared to those with healed wounds (78.2 vs 69.4, p<0.01). Analysis of odds ratio showed that a one point increase in ADI and active smoking were associated with a 5% and 27% decrease in probability of wound healing, respectively, whereas an increase of one wound care clinic visit was associated with a 12% increased probability of wound healing. The predictive model for amputation included the same significant covariates. An increase of one point in ADI (p=0.022) and active smoking (p<0.01) was associated with a 4% and 34% increase in the likelihood of amputation. An increase of one wound care clinic encounter (p<0.05) was associated with a 3% decrease in the likelihood of amputation
Conclusions: In patients with diabetic LE wounds, high ADI and active smoking are associated with wound progression while wound clinic encounters are protective. Limb loss is a critical population health issue. Based on these data, future limb salvage efforts should focus on increasing patient access to wound centers, optimizing social support, and complete cessation of smoking.

P2.03

Atypical and Regionally-Heterogenous Keratinocyte Differentiation In Volar Skin
Christiane Fuchs, Joshua Tam
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, United States
Atypical and Regionally-Heterogenous Keratinocyte Differentiation in Volar Skin

Christiane Fuchs, Joshua Tam Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA

Background: Volar skin (skin covering the palms and soles) harbors many specializations, including lack of hair, poor barrier function, and unique keratin structure/composition. However, these specializations are generally ignored in wound research. We previously reported broad functional changes in volar keratinocytes, and found inherently delayed healing in porcine plantar wounds. In this study we investigated whether keratinocyte differentiation is also altered in volar skin.
Methods: Post-amputation, deidentified human pedal tissue was obtained under IRB approval. Biopsies were taken from the heel (weightbearing volar), plantar arch (nonweightbearing volar), and dorsal foot (non-volar) skin. Immunohistochemistry was used to characterize established markers corresponding to different stages of keratinocyte differentiation: keratins K5/K14 for basal keratinocytes, K1/K10 for post-mitotic, suprabasal keratinocytes, and involucrin and loricrin for terminal differentiation. The volar skin marker K9, wound-associated keratins K6, K16 and K17, and proliferative marker Ki-67 were also investigated.
Results: K1, K5, and lorcrin conformed to their expected presentations in all skin types examined. In heel skin K14 (typically restricted to basal keratinocytes) surprisingly persists in all layers of the suprabasal epidermis, but only along furrows between friction ridges (the small foldings in volar skin that give rise to dermatoglyphs). The wound-associated keratins K6, K16 and K17 follow a similar pattern, while K9 and K10 are distributed in the opposite pattern – i.e. present throughout the suprabasal epidermis except along friction ridge furrows. The “ectopic” expression of K14, K6, K16, and K17 in friction ridge furrows was not accompanied by increased cell proliferation. These patterns are absent in non-weightbearing plantar arch skin, indicating that the unique expression and distribution of keratins in heel skin are not solely due to the presence of friction ridges (which are also present in non-weightbearing volar skin), and that mechanical forces related to weightbearing are likely involved. Involucrin is primarily confined to granular keratinocytes in non-volar skin, but in both heel and arch skin there is extensive pericellular expression in spinous keratinocytes, which is typically not seen in otherwise healthy skin, but observed in various skin pathologies including psoriasis, seborrheic keratosis, and actinic keratosis.
Conclusion: The volar epidermis shows substantial alterations in keratinocyte differentiation endpoints. Our results call into question the prevailing (if often unstated) assumption that volar and non-volar keratinocytes are essentially identical for wound healing purposes, with implications both for our understanding of the pathophysiology of plantar ulcers, and how we approach therapy development for these devastating lesions.

P2.04

Factors Affecting the Variability of Compression Therapy
Harrison J. Shawa1, Pallas Lim2, Sara Dahle3, Rivkah Isseroff2
1Kirk Kerkorian School of Medicine at UNLV, Las Vegas, NV, United States 2Dermatology, VA Northern California Health Care System, Mather, CA, United States 3Podiatry, VA Northern California Health Care System, Mather, CA, United States
Factors Affecting the Variability of Compression Therapy

Harrison J. Shawa1, Pallas Lim2, Sara Dahle3, Rivkah Isseroff2 1Kirk Kerkorian School of Medicine at UNLV, Las Vegas, NV; 2Dermatology, VA Northern California Health Care System, Mather, CA; 3Podiatry, VA Northern California Health Care System, Mather, CA

Background: Venous leg ulcers, the most common leg ulcer, occur in patients with chronic venous insufficiency due to venous hypertension. Evidence supports the conservative treatment with lower extremity compression, ideally between 30-40 mm Hg. Pressures in this range provide enough force to partially collapse lower extremity veins without restricting arterial flow in patients without peripheral arterial disease. There are many options for applying such compression, and those who apply these devices have varying levels of training and backgrounds. Thus, this project sought to determine the inter-applicator and inter-compression device variability of compression.
Methods: In this quality improvement project, a single observer utilized a reusable pressure monitor to compare pressures applied using different devices by individuals in wound clinics with diverse training from specialties of dermatology, podiatry, and general surgery. Results: Average compression was higher in the dermatology wound clinic (n=153) compared to the general surgery clinic (n=53) (35.7 ± 13.3 mm Hg and 27.2 ± 8.0 mm Hg, respectively, p<.0001), and wraps applied by clinic staff (n=194) were nearly twice as likely as a self-applied wrap (n=71) to have pressures greater than 40 mm Hg (relative risk 2.2, 95% confidence interval 1.136-4.423, p = .02). Pressures were also dependent upon the specific compression device used, with CircAid®s (35.5 mm Hg, SD 12.0 mm Hg, n=159) providing higher average pressures than Sigvaris Compreflex (29.5 mm Hg, SD 7.7 mm Hg, n=53, p=.009) and Sigvaris Coolflex (25.2 mm Hg, SD 8.0 mm Hg, n=32, p<.0001).
Conclusion: These results indicate that the device-provided pressure may be dependent on both the compression device and the background and training of the applicator. We propose that standardization in the training of compression application and increased use of a point-of-care pressure monitor may improve the consistency of applied compression, thus improving adherence to treatment and outcomes in patients with chronic venous insufficiency.

P2.05

Skin Strain Modeling To Assess The Strains Imparted To Tissue During Negative Pressure Wound Therapy With A Felted Foam With 10mm Holes
Amy K. McNulty, Robert Wilkes, James Sieracki
MSD, 3M Company, St. Paul, MN, United States
Skin Strain Modeling to Assess the Strains Imparted to Tissue During Negative Pressure Wound Therapy with a Felted Foam With 10mm Holes

Amy K. McNulty, Robert Wilkes, James Sieracki MSD, 3M Company, St. Paul, MN

Background: Not all patients with wounds are good surgical candidates and therefore it is not always possible to conduct good surgical debridement. Recently a felted, reticulated open cell foam with an array of 10 mm holes was introduced (VCCC). This foam was designed to be used in conjunction with instillation therapy to initiate wound cleansing and facilitate removal of wound exudate and infectious materials. In clinic this dressing in combination with instillation therapy has been shown to be able to eliminate non-viable material from the wound bed. The mechanisms by which this dressing facilitates the cleaning of the wound bed have not been fully elucidated. The current study attempts to elaborate the biomechanical stresses, strains and work imparted to tissue with VCCC versus the most often used negative pressure wound therapy reticulated open cell foam (ROCF) dressing. Finite element analysis was used to measure strain and deformation that occurs at the tissue interface with VCCC or ROCF.
Methods: For this in silico model, skin was modeled as Neo-Hookean with a Young’s Modulus of 0.05 MPa and Poisson’s ratio of 0.15. Foam samples were compressed between steel platens to 70% compression to generate stress strain curves.
Results: Foam testing showed that VCCC densifies at 50% compression while the ROCF densifies at 80%. The peak maximum principal strain imparted to tissue at -125mmHg with VCCC was over 20% while with ROCF the maximum peak principal strain was less than 5%. The frictional work around the holes in the VCCC was approximately 0.015 mJ while the frictional work around the pores in ROCF was less than 0.005 mJ. The strain energy density imparted to tissue with VCCC was approximately 0.53 mJ and with ROCF less than 0.1 mJ.
Conclusions: VCCC leads to higher forces imparted to the tissue. Specifically, there is friction work around the 10mm holes which may pin the tissue in place under the holes and therefore allow for higher strain energy density as the tissue is pulled up into the holes. This work done by the dressing may allow for fracturing of the devitalized tissue which then may be removed during instillation therapy.

P2.06

Fluoxetine delivery through iontophoresis bandage promotes wound healing in mice
Hsin-ya Yang 1, Houpu Li2, Andrea Medina Lopez 1, Anthony Gallegos1, Kan Zhu3, Cynthia Recendez3, Moyasar AlHamo1, Narges Asefifeyzabadi2, Prabhat Baniya2, Mircea Teodorescu2, Min Zhao3, Marco Rolandi2, Rivkah Isseroff1
1Dermatology, University of California, Davis, USA; 2Electrical and Computer Engineering, UC Santa Cruz, USA; 3Ophthalmology, UC Davis, USA
Fluoxetine Delivery Through Iontophoresis Bandage Promotes Wound Healing in Mice

Hsin-ya Yang1, Houpu Li2, Andrea Medina Lopez1, Anthony Gallegos1, Kan Zhu3, Cynthia Recendez3, Moyasar AlHamo1, Narges Asefifeyzabadi2, Prabhat Baniya2, Mircea Teodorescu2, Min Zhao3, Marco Rolandi2, Rivkah Isseroff1 1Dermatology, University of California, Davis, USA; 2Electrical and Computer Engineering, UC Santa Cruz, USA; 3Ophthalmology, UC Davis, USA

Epidermal wound healing is a highly coordinated process including hemostasis, inflammation, cell proliferation, cell migration, and tissue remodeling. To optimally promote wound closure, delivery of wound-modifying drugs within the micro wound environment should be temporally and spatially controlled. In this study, we demonstrated a proof-of-concept approach that uses a light-weight, iontophoresis bandage with programmable bioelectronic ion pumps to deliver fluoxetine, a drug that we have previously demonstrated to improve healing when topically applied, for wound treatment. By using a polyelectrolyte-hydrogel-based electrophoresis ion pump constructed in polydimethylsiloxane (PDMS) base that abuts the wound bed, the protonated fluoxetine molecules in the reservoir can be released to the wound under electrical modulation, while minimal amount of solvent is delivered to avoid drug diffusion. The efficacy of this fluoxetine delivery bandage device was examined in a mouse model for wound closure. Full-thickness, 6mm circular wounds were created on the back of C57B6 male mice and the bioelectronic ion pump bandage loaded with fluoxetine or a mock control device were applied to the wounds. Fluoxetine was targeted to delivered at 100nM or 0.04mg/wound/day during the daily 6-hour delivery program. On the post-operative day 3, wound tissue was harvested to examine healing. When compared to the control, the wounds treated with daily fluoxetine demonstrated a 39.9% increase in re-epithelization (21.6 +/- 11.6% in control vs 30.2 +/-13.5% with fluoxetine, n=15-22 mice, p<0.05), showing a significant improvement of early stage healing. The anti-inflammatory macrophage M2 subtype was also increased in the wound bed relative to the M1 subtype in the fluoxetine-treated wounds, resulting in a decrease of the M1/M2 cell ratio by 27.2% (1.80 +/- 0.62 in control vs 1.31 +/- 0.46, n=13 mice, p<0.05) on day 3. Serum levels of fluoxetine were below the detection limit (fluoxetine < 20 ng/mL and norfluoxetine< 40 ng/mL) in the treated animals. The design of this bioelectronic ion pump bandage not only provides a new approach for topical, programmed drug delivery, but also demonstrates that the fluoxetine released from the bandage retains its reparative biological activity. We hope to further optimize the design of the bandage for potential clinical use in the future.

P3.01

Enrichment of NK cells by Pro-Regenerative Biomaterials In Muscle Wounds Correlates with XCL-1 secretion and cDC1 Recruitment To Injury
Daphna Fertil, Ravi Lokwani, Tran Ngo, Sabrina DeStefano, Kenneth Adusei, Minhaj Bhuiyan, Aditya Josyula, Mondreakest Faust, Aaron Lin, Maria Karkanitsa, Parinaz Fathi, Kaitlyn Sadtler
Section on Immunoengineering, Biomedical Engineering and Technology Acceleration Center, National Institutes of Health, Bethesda, MD, United States
Enrichment of NK cells by Pro-Regenerative Biomaterials in Muscle Wounds Correlates with XCL-1 Secretion and cDC1 Recruitment to Injury

Daphna Fertil, Ravi Lokwani, Tran Ngo, Sabrina DeStefano, Kenneth Adusei, Minhaj Bhuiyan, Aditya Josyula, Mondreakest Faust, Aaron Lin, Maria Karkanitsa, Parinaz Fathi, Kaitlyn Sadtler Section on Immunoengineering, Biomedical Engineering and Technology Acceleration Center, National Institutes of Health, Bethesda, MD

Understanding the immune imbalances associated with pathogenesis in wound healing and surgical implantation could advance regenerative therapeutics to assist in more favorable treatment outcomes. To evaluate immune response to biomaterials in injury, we employed a murine volumetric muscle loss (VML) model consisting of a 3mm defect in the quadriceps muscle of mice. Three conditions were investigated. Polyethylene (PE) was implanted into the injury site inducing a profibrotic response, extracellular matrix (ECM) was used to promote a pro-regenerative environment, as well as an injury (untreated) control. Using flow cytometry, ELISA, and RT-PCR, we evaluated immune responses in each trauma site. At 7 days post injury, NK cells (CD49b+TCRβ-) were increased in ECM environment compared to PE and control (2.498% versus 0.216% and 0.0734% ± 0.017, P < 0.0001). As seen by researchers evaluating cancer models, these NK cells were accompanied by an enrichment of XCR1+CD103+ conventional dendritic cells (cDC1s) that are capable of antigen cross-presentation. Furthermore, ECM treatment induced heightened levels of XCL-1 in the injury microenvironment and peripheral blood. There was no significant difference in the concentration of XCL-1 between wild type and Rag1-/- mice suggesting XCL-1 secretion is mediated by a RAG1-independent cell type such as NK cells. This correlated with an increase in XCR1+CD103+ dendritic cells that promote regenerative behavior. In the absence of these cells in Batgf3-/- mice, there were physical manifestations such as necrotic muscle fibers and giant cells more distal from the injury site showing a spread of trauma beyond the initial injury. Evaluating these data together, within the first week after injury there was in upregulation of XCL-1, in addition XCR1+ CD103+ dendritic cells and CD49b+ NK Cells. The presence of XCL-1 provides a possible mechanism of XCR1+ CD103+ cell recruitment. In Rag1-/- mice, the concentration of XCL-1 was not significantly different compared to the wild type, indicating that a RAG-independent recruitment was the source of XCL-1. NK cells were found to be present at ECM injury site at a greater proportion when compared to the control and PE environment. In previous studies of melanoma, it was shown that NK cells have the capability to recruit XCR1+ cDC1s to tumors through an XCL-1 pathway. In future research, NK cells will be investigated more closely to identify their role in supporting a pro-regenerative environment.

P3.02

Controlled Release of Inflammasome Modulators to Promote Tissue Repair
Jordan R. Yaron1, Selin Bakkaloglu1, Sophia Macko2, Samantha Rhodes2, Kaushal Rege1
1School for Engineering of Matter, Transport, & Energy, Arizona State University, Tempe, AZ, United States 2School of Life Sciences, Arizona State University, Tempe, AZ, United States
Controlled Release of Inflammasome Modulators to Promote Tissue Repair

Jordan R. Yaron1, Selin Bakkaloglu1, Sophia Macko2, Samantha Rhodes2, Kaushal Rege1 1School for Engineering of Matter, Transport, & Energy, Arizona State University, Tempe, AZ; 2School of Life Sciences, Arizona State University, Tempe, AZ

Purpose/Objectives: The inflammasome is a multiprotein complex critical for the innate immune response to injury. Inflammasome activation is important for healthy wound healing, but comorbidities with poor healing (e.g., diabetes) exhibit pathologic, sustained activation with delayed resolution that prevents healing progression. The inflammasome is readily druggable and thus an ideal target to promote diabetic wound repair with drug-releasing dressings. Extracellular ATP (eATP) is detected by P2X7 purinergic receptors, resulting in potassium (K+) and calcium (Ca2+) flux across cellular membranes and NLRP3 inflammasome activation, primarily in macrophages in wounds but in other cells as well. We have shown the competitive P2X7 antagonist A438079 inhibits NLRP3 signaling by preventing ion flux, mROS generation, NLRP3 assembly, mature IL-1b release, and pyroptosis. Here, we develop a controlled release scaffold to deliver A438079 as an inflammasome-modulating wound dressing.
Methodology: Silk fibroin was isolated from Bombyx mori silkworm cocoons. Films (6-mm diameter) of tunable thickness were prepared by solvent evaporation and made insoluble by autoclaving. Thickness was measured by micrometer and rheological properties were measured by dynamic mechanical analysis (DMA). Degradation was determined in PBS over 40 days by film thickness and BCA assay. Spectroscopy of A438079 identified analytical wavelengths at 226 nm and 258 nm. A438079, a water-insoluble drug solubilized in 50% DMSO, was passively loaded into films, dried, and stored before use. Drug release into PBS was quantified by spectroscopy over 5 days. Released drug was tested in J774-DUAL mouse macrophages induced for inflammasome activation with LPS and eATP and IL-1beta release was measured by ELISA.
Results: Insoluble silks films of tunable thickness demonstrated no evidence of swelling or degradation over 40 days. Drug release followed an exponential plateau (R2=0.82 to 0.98) and achieved a maximum cumulative release of 200 μM A438079 from 55 μm-thick films or 360 μM A438079 from 100 μm-thick films at 3 days. Released drug was tested in J774-DUAL macrophages primed with LPS and stimulated with eATP. Conditioned media from 4 hours of drug release exhibited near-complete inhibition of IL-1b release and evidence of pyroptosis, indicating robust inflammasome inhibition in vitro.
Conclusion/Significance: These studies demonstrate the controlled, tunable release of small molecule inflammasome modulators from a biocompatible drug delivery scaffold. Studies in vivo will evaluate the potential for accelerating wound healing in a full-thickness splinted model to explore this novel approach to treat diabetic wounds by controlled release of small molecule inflammasome modulators.

P3.03

Integrative Pathway Analysis to Computationally Model Dysregulation of Functional Plasticity and Metabolic Immunomodulation in Macrophages from Veterans with Diabetes
Catherine B. Anders1, Tyler M. Lawton2, Hannah L. Smith3, Bridget Brackney4, Mary Cloud B. Ammons1
1Idaho Veteran Research & Education Foundation – Boise VA, Boise, ID, United States 2Boise State University, Boise, ID, United States 3Texas A & M University, College Station, TX, United States 4Brigham Young University, Rexburg, ID, United States
Integrative Pathway Analysis to Computationally Model Dysregulation of Functional Plasticity and Metabolic Immunomodulation in Macrophages from Veterans with Diabetes

Catherine B. Anders1, Tyler M. Lawton2, Hannah L. Smith3, Bridget Brackney4, Mary Cloud B. Ammons1 1Idaho Veteran Research & Education Foundation – Boise VA, Boise, ID; 2Boise State University, Boise, ID; 3Texas A & M University, College Station, TX; 4Brigham Young University, Rexburg, ID

Non-healing in diabetics is a complex medical problem that requires integration of metabolism with immunity; therefore, development of Precision Medicine tools based on Systems Biology are essential to any therapeutic advancement. Within the Veterans Affairs healthcare system, around 25% of military veterans have diabetes and the economic burden of lower limb amputations exceeded $200 million for fiscal year 2010. Despite innovations in both wound care and diabetes management, diabetic ulcers remain the leading cause of amputation for VA patients. Normal wound healing in healthy individuals initiates quickly and proceeds through well-characterized, iterative steps; however, in diabetic ulcers, the healing process stalls at the transition between resolution of inflammation and initiation of tissue reorganization. This transition is characterized by a population shift from neutrophils to macrophages (Mθ). Our research indicates that Mθ have unique phenotypic plasticity that enables diverse functional capacity driven by metabolic reprogramming; however, in diabetic donors Mθ phenotypic plasticity appears disabled and has not been well characterized in the context of wound healing. To address the critical need for Precision Medicine tools to treat diabetic ulcers, we are developing an algorithm of clinically actionable biomarkers based on a systems biology integration of high-dimensional metabolomics data and Mθ phenotypic plasticity modeling. Using our ex vivo Mθ model, biomarkers correlating Mθ metabotype to functional phenotype have been probed for critical deviations using specimens from diabetic donors. Specifically, we have identified biomarkers associated with inhibited functional plasticity in diabetic Mθs and characterized the relationship between metabolism and Mθ functionality. Utilizing our ex vivo Mθ modeling of global metabolomics, proteomic markers, and myeloid gene expression profiles of phenotypic plasticity, deviations have been quantified using Mθs derived from diabetic patients to establish key biomarkers. In addition, comparative data sets from diabetic and non-diabetic donors have been computationally modeled using Integrative Pathway Analysis, a machine-learning computational approach to identifying novel relationships across functional pathways. These insights provide both the backbone for predictive modeling that will eventually translate to diagnostic and therapeutic innovations in Precision Wound Care, as well as provide fundamental discovery on the dysregulation of Mθ functional plasticity and metabolic immunomodulation that contribute to non-healing in patients with diabetes. Approaching the issue of non-healing wounds in patients with diabetes through integration of metabolism and innate immunity at a systems level is a high-dimensional approach intended to address the high complexity of a problem recalcitrant to conventional therapeutic interventions.

P3.04

Cassia Angustifolia Primed ASCs Carrier Amniotic Membrane Accelerate Burn Healing by Modulation of Inflammatory Response
Saba Tasneem1, Azra Mehmood1, Hafiz Ghufran1, Maryam Azam1, Amna Arif1, Musab Bin Umair1, Shehla Javed Akram2, Kausar Malik1, Sheikh Riazuddin1
1Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore, Pakistan. 2Akram Medical Complex, Lahore, Pakistan
Cassia Angustifolia Primed ASCs Carrier Amniotic Membrane Accelerate Burn Healing by Modulation of Inflammatory Response

Saba Tasneem1, Azra Mehmood1, Hafiz Ghufran1, Maryam Azam1, Amna Arif1, Musab Bin Umair1, Shehla Javed Akram2, Kausar Malik1, Sheikh Riazuddin1 1Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore, Pakistan; 2Akram Medical Complex, Lahore, Pakistan

The purpose of this study was to evaluate the effectiveness of Cassia angustifolia extract primed adipose derived stem cells (ASCs) scaffolded with denuded amniotic membrane in ameliorating the thermal burn injury in rats. For ex-vivo studies, ASCs were pretreated with optimized dose (30μg/ml) of C. angustifolia for 24hrs and subsequently exposed to in-vitro thermal injury at 51 o C for 10minutes in water bath. For in-vivo studies rats were randomly divided into four groups (n=8 rats per group); 1) burn wounds without treatment, 2) burn wounds transplanted with non-primed ASCs with amniotic membrane, 3) burn wounds transplanted with C. angustifolia primed ASCs with amniotic membrane, 4) excisional wounds without burns. Burn wound was induced by placing round copper disc of 2 cm diameter on dorsum of the rats for 10 seconds. ASCs (2.0 x 10 5 ) pretreated with C. angustifolia, then cultured on stromal side of amniotic membrane (a natural 3D scaffold) and transplanted in rat heat injury model for in-vivo studies. Wound-healing potential of transplantation groups was assessed morphologically and histologically in-vivo by wound closure analyses, histology, and gene expression analyses. The results revealed significantly upregulated gene expression of IGF1, SDF1A, TGFβ1, VEGF, GSS, GSR, PIK3CA, IL4, BCL2 to BAX ratio and downregulation of IL6 and NFkB1 in SM primed ASCs (P<0.001) confirming its potential in promoting cell proliferation, migration, wound healing, angiogenesis, antioxidant, cell survival and anti-inflammatory activity respectively. In-vivo data depicted that SM primed ASCs induce early wound healing, better architecture, normal thickness of epidermis, orderly arranged collagen fibers (cri-cross arrangement), and well-developed increased number of skin appendages in healed skin of rats transplanted with C. angustifolia primed ASCs. Gene expression studies of the same showed increased expression of cell survival (Bcl2), angiogenic (Vegf and PeCam), proliferation (Pcna, Tgfβ1), growth factors (Igf1, bFgf), antioxidant (Gss, Gsr) and structural (Krt1, Krt8, Krt19, Desmin, Vimentin, α-Sma) genes while reduced expression of inflammatory (Il1β, Il6, Tnfα & Nfkb1 (P65) and apoptotic genes (Bax) in comparison to untreated ASCs laden amniotic membrane transplanted rats and heat injured rats. In conclusion, the data in this study demonstrated that C. angustifolia is effective in enabling ASCs to counter in-vitro and in-vivo thermal injury by significantly modulating gene expression and enhancing cell survival upon transplantation. The present study provides bases for development of effective natural and regenerative medicine combinational therapies with clinical prospects for treating burn wounds.

P3.05

Radiation Alters Macrophage Response to Acellular Dermal Matrix Implants
Lillian DeCostanza1, Anthony C. Bruce1, Graham M. Grogan2, Kara L. Spiller3, Chris A. Campbell2, Patrick Cottler2
1Biomedical Engineering, University of Virginia, Charlottesville, VA, United States 2Plastic Surgery, University of Virginia, Charlottesville, VA, United States 3Biomedical Engineering, Drexel University, Philadelphia, PA, United States
Radiation Alters Macrophage Response to Acellular Dermal Matrix Implants

Lillian DeCostanza1, Anthony C. Bruce1, Graham M. Grogan2, Kara L. Spiller3, Chris A. Campbell2, Patrick Cottler2 1Biomedical Engineering, University of Virginia, Charlottesville, VA; 2Plastic Surgery, University of Virginia, Charlottesville, VA; 3Biomedical Engineering, Drexel University, Philadelphia, PA

Immune response to implantable biomaterials is a critical component of wound healing. Acellular Dermal Matrices (ADMs) have revolutionized implant-based breast reconstructions by providing mechanical and soft tissue support. The use of ADM triggers inflammation and a poorly understood macrophage response. ADM also positively modulates inflammation, directing the immune response. The microenvironment is further shaped by radiation, a treatment many breast reconstruction patients have recieved. Here we characterize and compare the post-ADM implantation macrophage response of normal and irradiated environments in vivo. C57BL/6J mice received 0Gy or 35Gy of dorsal skin radiation 12 weeks prior to subcutaneous, 5mm ADM implantation. Implants were harvested for flow cytometry at 1 and 3 weeks (n=6 except 1-week 0Gy n=5), and H&E staining at at 3 and 6 weeks (3-week 0Gy n=6, 6-week 0Gy n=7, all 35Gy n=4). Flow cytometry was performed using a panel of pan-, M1, M2, and transitory macrophage markers and quantified as percent of F4/80+ macrophages expressing each marker of interest. H&E slides were evaluated for fibrotic encapsulation. At 1 week, radiation caused an increase in macrophages expressing the classical M1 marker CD86 (p<0.01) and a decrease in those expressing classical M2 markers CD163 (p<0.0001), Arg1 (p<0.05), and CD301b (p<0.0001) compared to normal mice. Thus, radiation induces a greater propensity for an early inflammatory response with fewer pro-regenerative macrophages. At 3 weeks, only the difference in CD163+ M2 macrophages (p<0.001) remains; all other M2 macrophage populations are comparable regardless of radiation dose. There is an increased percentage of CD184+ transitory macrophages (p<0.01) and CD192+ M1 macrophages (p<0.0001). CD192 has been implicated in additional macrophage recruitment. The increase of the latter two markers suggests a more rapid shift towards a pro-regenerative environment in irradiated mice, a conclusion confounded by the decreased presence of CD163+ M2 macrophages. Temporal expression profiles were also altered. Pathological analysis of capsule formation, cellularity, foreign body response, and smooth muscle actin shows no variation by timepoint or radiation level, indicating that radiation does not alter 6-week fibrotic response. We demonstrate that the effect of radiation is more complex than depletion of the immune microenvironment. An irradiated environment provokes a greater early inflammatory response upon ADM implantation, with complex differences observed at 3 weeks. A comprehensive review of macrophage and fibrotic response to ADM will inform immunomodulatory strategies for use in the next generation of regenerative biomaterials.

P3.06

Impact of Cigarette Smoke and Nicotine on Secretome Profile and Matrix Degradation of Human Dermal Fibroblasts in 3D Culture
Angelin T. Mathew, Jackson Parker, Henry Hsia
Department of Plastic Surgery, Yale University, New Haven, CT, United States
Impact of Cigarette Smoke and Nicotine on Secretome Profile and Matrix Degradation of Human Dermal Fibroblasts in 3D Culture

Angelin T. Mathew, Jackson Parker, Henry Hsia Department of Plastic Surgery, Yale University, New Haven, CT

Wound healing is a complex biological process that requires the coordination of several molecular events. Patients who smoke have significantly increased risk of complications after surgery (e.g. infection, tissue flap necrosis, and anastomotic leakage). Our research aimed to elucidate the mechanisms of cigarette smoke impaired wound healing and compare that to nicotine impaired wound healing by studying the secretome profile of dermal fibroblasts in vitro. Cigarette smoke extract was collected by cigarette combustion smoke through unsupplemented media prior to 0.2 μm filtration. Human dermal fibroblast cells were cultured in Type 1 collagen scaffolds and incubated with cigarette smoke extract, nicotine (30 ng/mL), or untreated control all at 10% FBS supplementation. Conditioned media was collected over 7 days and used to conduct cell proliferation and cytoxicity assays. Relative expression amounts of TGFB, PDGF, MMP2, and IL8 secretion were measured. Most notably, we noticed a time dependent pattern in MMP2 secretion profile. Initially on Day 1 and Day 2, the control untreated fibroblasts had a higher secretion of MMP2 compared to cells treated with nicotine or smoke cigarette extract solution. MMP2 secretion by cells treated with nicotine or smoke cigarette extract were relatively the same and statistically insignificant. However, on Day 5 there was a significant change in this trend with a pronounced difference between the relative levels of MMP2 secretion of the fibroblast cells treated with nicotine or smoke cigarette extract. The control cells had relative secretion levels of MMP2 that were 3 times greater than those of cells treated with smoke cigarette extract. The control cells’ MMP2 secretion was also 2x greater than that of cells treated with nicotine. This trend was maintained on Day 7 and statistically significant for both Day 5 and Day 7. These findings are significant because MMP2 plays a central role in coordinating matrix remodeling and degradation which is an essential process in regulating scar formation. Irregular scar formation is a complication of wound healing that may lead to tissue fibrosis, keloid formation, and chronically unclosed wounds. Additionally, for patients who smoke and are undergoing elective or non immediate surgeries, physicians often advise using nicotine patches. Our results indicate that the fibroblasts treated with a nicotine solution still had drastic reductions in MMP2 secretion when compared to fibroblast cells not treated with anything.

P4.01

Improved Wound Healing Outcomes for Large Surface Area Burn Wounds with a Novel Spray on Antimicrobial Wound Dressing
Kerriann Greenhalgh1, Marc Thompson2, Robert Christy2
1KeriCure Medical, Wesley Chapel, FL, United States 2Burn Surgical Research Team, USAISR, Ft. Sam Houston, TX, United States
Improved Wound Healing Outcomes for Large Surface Area Burn Wounds with a Novel Spray on Antimicrobial Wound Dressing

Kerriann Greenhalgh1, Marc Thompson2, Robert Christy2 1KeriCure Medical, Wesley Chapel, FL; 2Burn Surgical Research Team, USAISR, Ft. Sam Houston, TX

Background: Large total body surface area (TBSA) burn injuries are uniquely difficult to treat in acute care. The debilitating effects of large TBSA burns and their associated complications, such as infection, inflammation and hypermetabolism, may be further heightened when surgical intervention is delayed. This study evaluated a novel wound dressing with capabilities in acute and long term care to promote wound healing for large surface area, deep partial thickness burn wounds in a porcine model. KeriCure Medical’s Field Shield Wound Dressing (FSWD) products are spray on dressings that establish semiocclusive antimicrobial barrier over any size/shape burn with silver hydrosol (antimicrobial) and lidocaine (anesthetic) in a moisture donating hydrogel. FSWD was compared to sterile gauze and Silverlon dressings. Objectives were to evaluate FSWD products to: 1. Improve wound closure when applied in acute care through continuum of care and 2. Mitigate infection development. We hypothesized that FSWD would promote wound healing with a greater wound closure rate and lower severity of early wound expansion, and support healing through aided autolytic debridement and better moisture maintenance equally or better than SOC treatments. Methods: Yorkshire swine (55kg) were employed to create twelve burns of 9x15cm surface area for a TBSA of 17% or greater. A brass block with thermocouple was applied to dorsum for 15 sec at 100C with constant pressure. Burn injury treatments were block randomized to avoid cross application from spray dressings. Treatments were applied within 2 hours of burn creation and covered with secondary dressings. Wounds were sharp debrided Day 3 then managed following the recommended treatment regimen of Silverlon, with dressing changes every 3 to 4 days until Day 14. Only secondary dressing changes occured from Day 15 to Day 35 (termination). Imaging quantified wound size, re-epithelialization and fluid flow. Histological assessments with H&E staining were obtained from biopsies taken throughout the study.
Results: Use of FSWD resulted in a statistically greater propensity for wound closure (50-65% closure) compared to gauze and Silverlon® (32.2% and 23.3% respectively) by Day 35. Data indicated that FSWD managed burns had a significantly higher rate of blood flow post-debridemnt that may have contributed to the limited formation of necrotic tissue and faster wound closure rates. Visually, FSWD products produced moister wounds and aided autolytic debridement, whereas wounds treated with saline soaked gauze and Silverlon were drier to the touch, with notable regions of necrosis begining Day 14. No infection development was observed.
Conclusion: Field Shield Wound Dressing provided clinically significant re-epithelialization by Day 35 with visible improvements to wound healing compared to SOC treatments.

P4.02

Novel Peel and Place Negative Pressure Wound Therapy (NPWT) Dressing: Histologic, Proteomic, and Dressing Peel Force Evaluation in a Porcine Wound Model
Diwi Allen, Samantha A. Mann, Marisa Schmidt, Kris Kieswetter
Medical Solutions Division, 3M Company, San Antonio, TX, United States
Novel Peel and Place Negative Pressure Wound Therapy (NPWT) Dressing: Histologic, Proteomic, and Dressing Peel Force Evaluation in a Porcine Wound Model

Diwi Allen, Samantha A. Mann, Marisa Schmidt, Kris Kieswetter Medical Solutions Division, 3M Company, San Antonio, TX

Though reticulated open cell foam (ROCF) is well-established for use with negative pressure wound therapy (NPWT), it necessitates frequent dressing changes by a wound care professional every 2–3 days to mitigate the potential of tissue ingrowth. Therefore, new approaches to extending the length of time between ROCF dressing changes have been considered. A novel, easy-to-use peel and place dressing has been designed that utilizes the advantages of ROCF while addressing this challenge. All animal work was approved by the relevant Institutional Animal Care and Use Committee (IACUC) and complied with all applicable national and local regulations. Full-thickness excisional wounds were created along the spine of 11 swine. Dressings were applied with continuous -125mmHg negative pressure for 7 days with a dressing change at day 4. Dressing removal/peel force measurements were performed at dressing change and term. At term, day 7, wounds were collected en bloc for histomorphometry measurements of granulation tissue thickness. A board-certified histopathologist assessed granulation tissue quality. Biopsies were collected to analyze wound healing biomarkers via multiplex immunoassays. Peel force analysis confirmed improved ease of dressing removal using the peel and place dressing. The force required to remove the dressing was 6.7 times lower for the peel and place dressing at dressing change (p<0.0001) and 26 times lower at day 7 (p<0.0001) versus ROCF. In correlation, the amount of tissue ingrowth into the dressing was limited to only ROCF. The peel and place dressing promoted more granulation tissue than ROCF (p<0.001). Although the collagen quality and maturity were comparable between dressings, the level of vasculature was greater with the peel and place dressing (p<0.001). Wound healing-associated cytokines/chemokines and growth factors also suggested differences between the treatments. Greater levels of interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (ra), IL-8, and IL-18 were evident with the peel and place dressing relative to ROCF (p<0.05). The following growth factors were also elevated with the peel and place dressing versus ROCF (p<0.05): heparin binding-endothelial like growth factor (HB-EGF), platelet derived growth factor (PDGF)-AA, PDGF-AB/BB, and transforming growth factor (TGF)α. The results of this study illustrate that the peel and place dressing performed well as a longer-wear NPWT dressing with substantially decreased tissue ingrowth and notably lower peel forces compared to ROCF. In addition, increased levels of pro-wound healing biomarkers support greater granulation tissue formation and comparable, if not slightly improved, tissue quality, in the peel and place dressing treatment compared to ROCF.

P4.03

Priming with Caffeic Acid Enhances the Potential and Survival Ability of Human Adipose derived Stem Cells to Counteract Hypoxia
Muhammad Shifa ul Haq, Ramla Ashfaq, Azra Mehmood, Warda Shahid, Hafiz Ghufran, Saba Tasneem, Maryam Azam, Sheikh Riazuddin
Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore, Pakistan
Priming with Caffeic Acid Enhances the Potential and Survival Ability of Human Adipose derived Stem Cells to Counteract Hypoxia

Muhammad Shifa ul Haq, Ramla Ashfaq, Azra Mehmood, Warda Shahid, Hafiz Ghufran, Saba Tasneem, Maryam Azam, Sheikh Riazuddin Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore, Pakistan

The therapeutic effectiveness of stem cells after transplantation is hampered by the hypoxic milieu of chronic wounds. Prior research has established antioxidant priming as a thorough plan to improve stem cell performance. The purpose of this study was to ascertain how Caffeic Acid (CA) priming affected the ability of human adipose-derived stem cells (hASCs) to function under hypoxic stress. In order to study the cytoprotective properties of CA, hASCs were primed with CA in CoCl2 hypoxic conditions. Microscopy was used to assess cell morphology, XTT, Trypan Blue, X-gal, LDH, Live Dead, and scratch wound healing assays were used to analyze viability, senescence, cell death, and proliferation. According to our findings, the cultured ASCs showed high expression of positive cell surface markers of hASCS including CD71+, CD73+, CD90+, and CD105+ while negligible expression of negative markers including CD14- and CD45- . Results suggested that cellular death is directly proportional to the increasing concentration of hypoxia stress and thus that dose (500 μM) of CoCl2 was selected where almost 50% of cells were found to be dead. While for keeping the cells in the natural morphological shape, 75 μM CA priming dose was selected for further experiments. The percentage of live cells in the Pre.hASCs+Stress group was found to be 97 ± 12%, showing a marked cell survival increase with more stained live cells as compared to the Hypoxia Stress group (72 ± 8%). The percentage of senescent cells in the Hypoxia Stress group was found to be 6.2 ± 1.6% while in that of the Pre.hASCs+Stress group was 2.2 ± 1.1%, suggesting the protective role of CA against senescence. Results of the wound healing assay displayed that CA Pre.hASCs+Stress group of hASCs exhibited enhanced migration of cells towards the empty streak area as compared to the Hypoxia Stress group. Overall results of these experiments indicated that antioxidant treatment while using CA has significantly enhanced the survival and regenerative potential of hASCs to survive in a hypoxic environment. Semi-quantitative real-time PCR analysis portrayed significantly enhanced expression of pro-apoptotic markers including BAX, FADD, and Casp-3 in the Hypoxia Stress group as compared to the Pre.hASCs+Stress group. Significantly enhanced expression of cell survival (IGF-1, FGF-7, AKT, PCNA, PI3-K, BCL-XL), angiogenic (VEGF, SDF-1) and inflammation associated markers (IL-6 and TGFβ1) was found in CA primed hASCs as compared to the untreated cells. Our in-vitro research suggested that pre-treatment of hASCs with CA could be a unique way to enhance their therapeutic efficacy and ability to survive in hypoxic microenvironments.

P4.04

Preclinical and Proteomic Evaluation of a Novel Peel and Place Negative Pressure Wound Therapy (NPWT) Dressing After 7 Days of Continuous Therapy
Diwi Allen, Samantha A. Mann, Marisa Schmidt, Kris Kieswetter
Medical Solutions Division, 3M Company, San Antonio, TX, United States
Preclinical and Proteomic Evaluation of a Novel Peel and Place Negative Pressure Wound Therapy (NPWT) Dressing After 7 Days of Continuous Therapy

Diwi Allen, Samantha A. Mann, Marisa Schmidt, Kris Kieswetter Medical Solutions Division, 3M Company, San Antonio, TX

Negative pressure wound therapy (NPWT) with reticulated open cell foam (ROCF) has evolved the practice of wound care. To date, ROCF has not been utilized as an extended wear dressing due to the potential for tissue ingrowth that may occur if left in place for >72 hours. This study evaluated a novel, easy-to-use, peel and place dressing designed to utilize the advantages of ROCF while addressing tissue ingrowth as an extended-wear NPWT dressing. All animal work was approved by the relevant Institutional Animal Care and Use Committee (IACUC). Animal care complied with all applicable national and local regulations. Full-thickness excisional wounds were created along the spine of 11 swine and dressings applied using continuous negative pressure at -125mmHg for 7 days. No dressing changes were performed. At day 7, the peel forces required to remove the dressings from the wound beds were measured and tissues collected for histology. Granulation tissue thickness and percent re-epithelialization were assessed via morphometric analysis and tissue quality was evaluated by a board-certified pathologist. Biopsies were collected and multiplex immunoassays performed on extracted total protein for wound healing biomarkers. The peel and place dressing promoted more granulation than ROCF, 5.7mm versus 2.8mm, respectively (p<0.01). Granulation tissue ingrowth into the dressing material was limited to only ROCF-treated wounds. The level of re-epithelialization was 1.6 times greater with the peel and place dressing versus ROCF (p<0.01). Elevated levels of collagen deposition (p<0.05) and vascularization (p<0.001) were also observed with the peel and place dressing compared to ROCF. Analysis of wound healing-associated cytokines/chemokines and heparin-binding endothelial like growth factor (HB-EGF) also indicated differences between the two dressings. Greater levels of interleukins (IL)-1α, IL-1β, IL-1 receptor antagonist (ra), IL-8, IL-12, and HB-EGF were evident with the peel and place dressing versus ROCF (p<0.05). As expected, there was a correlation between peel force and tissue ingrowth. The force required to remove the peel and place dressing was considerably lower (36 times) compared to ROCF, 0.3N versus 10.7N, respectively (p < 0.0001). Elevated wound healing biomarkers supported enhanced granulation tissue formation and improved tissue quality with the peel and place dressing compared to ROCF. Increased promotion of re-epithelialization was also observed. The substantially reduced tissue ingrowth and notably lower dressing peel forces compared to ROCF may facilitate easier dressing changes. This lends further support for the peel and place dressing as an extended-wear (7-day duration) NPWT dressing.

P4.05

Hyaluronic Acid Binding Peptide Instruct Dermal Fibroblasts Towards A Pro-Regenerative Phenotype
Tugba Ozdemir
Nanoscience and Biomedical Engineering, South Dakota School of Mines and Technology, Rapid City, SD, United States
Hyaluronic Acid Binding Peptide Instruct Dermal Fibroblasts Towards A Pro-Regenerative Phenotype

Tugba Ozdemir Nanoscience and Biomedical Engineering, South Dakota School of Mines and Technology, Rapid City, SD

Fibroblasts are central to variety of homeostatic events such as wound healing. However, their pathologic activation thought to play roles in variety of diseases not only limited to fibrosis, foreign body reaction, scleroderma but also cancer metastasis. Biophysical properties of the extracellular matrix (ECM) deposited through an activated fibroblast determines whether there is a pro-regenerative or scarring response. Compared to aged fibroblasts, embryonic fibroblasts were shown to deposit a pro-regenerative ECM characterized with early hyaluronic acid (HA) deposition, increased levels of pro-regenerative collagens such as type III collagen. Since HA is also a regulator of collagen organization, we propose that early accumulation of HA by fibroblasts can facilitate a pro-regenerative matrix formation. Since molecular weights of HA present in pro-regenerative matrix is higher than synthetic HA, we strategize to attracting HA synthesized by fibroblasts. In this study, we used a synthetic peptide sequence known to have affinity to HA and used as a strategy to instruct fibroblasts to retain HA on the surface. We hypothesized that hyaluronic acid binding peptide (HABP) may instruct fibroblasts to express more HA. Increased HA expression may lead to fibroblasts to adopt a regenerative phenotype thus further regulate collagen assembly. We functionalized silica glass surfaces with HABP using aminoorganosilane mediated chemisorption and screened primary human dermal fibroblasts (hDF) for cell morphology, cytoskeletal arrangement, and alpha-smooth muscle actin (α-SMA) expression. Further, we assessed hDF derived decellularized matrices for ECM components, more specifically hyaluronan deposition, collagen organization using fiber tracing methods. Finally, we analyzed hyaluronan synthase gene expression using quantitative PCR. Our results show HABP treated surfaces retain higher levels of HA on silica glass compared to control surfaces on fibroblast derived matrices. Analysis of α-SMA shows increased α-SMA expression on hDFs and increased stress fiber formation. The physical features of collagen fibers deposited by fibroblasts were also organized differently in the presence of HABP. We propose that HABPs are potentially viable strategy to instruct pro-regenerative fibroblasts and can be used as a therapeutic strategy to treat fibrotic diseases.

P4.06

In Vitro Testing of the Protease Modulating Activity of a Nitric Oxide Generating Foam
Daniel Gibson
University of Alabama, Tuscaloosa, AL, United States
In Vitro Testing of the Protease Modulating Activity of a Nitric Oxide Generating Foam

Daniel Gibson University of Alabama, Tuscaloosa, AL

The purpose of this study was to test a two-component foaming liquid’s effects on a variety of proteases. A mix-and-read fluorescent substrate-based assay was used to measure the protease activity of matrix metalloprotease (MMP) -1, -2, -8, -9, human neutrophil elastase (hNE), and a type-I collagenase mixture from Clostridium histolyticum. The assay was run in a 96-well plate format using a phosphate buffered saline buffer augmented with 1mM CaCl2 and 5μM ZnCl2 + 0.05% polysorbate 20. The enzymes were tested at levels associated with extremely high proteolytic activity in human chronic wounds (5-10 μg/ml). A standard curve was generated for each enzyme to ensure a proportional response. The test product comes as a two part unmixed pair of solutions (Part A and Part B); when applied the two solutions as foams are mixed 1:1 prior to application to the wound and periphery. Three test groups were utilized consisting of Part A, Part B, and a 1:1 mixture of Part A:B. Dilutions of each agent with diH2O resulting in 100%, 50%, 25%, and 12.5% active agent were tested. The reaction was monitored for fluorescence generation with 485nm excitation and 528nm emission, 1 reading per minute for 10 minutes. The relative fluorescence unit (RFU) per minute slope was calculated for each sample while the reaction was still in the linear phase. The residual activity was reported as the RFU/min observed divided by the RFU/min of the non-treated control. When exposed to the undiluted test agents, an effect was found among the control, Part A, Part B, and 1:1 mixture (multiple p-values, all p ≤ 1.69 x 10-8). All components and the final mixture reduced or eliminated detectable protease activity. Dilution had no apparent effect on activity reduction for Part A, and there was a dose-dependent loss of inhibition with Part B. The dilution of the 1:1 mixture had a slight reduction of residual protease activity; with the most pronounced effect being with the C. histolyticum collagenase (0.7% residual activity in the 100%, down to 0.1% in the 12.5% diluted sample). These initial results indicate a definite broad-spectrum anti-protease activity when tested in vitro. The anti-protease activity appears to be stable up to at least a 12.5% dilution. Additional testing in wound fluids or wound fluid surrogates is necessary to determine if the effects will be diminished in the presence of additional biomolecules.