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2024 Abstracts

I.01

Single-Cell Transcriptomics Reveals Reduction Of A Population Of Monocyte-Derived Dendritic Cells Associated With Impaired Healing In Diabetic Wounds Of Mice
Jingbo Pang1, Mehrdad Zandigohar2, Yang Dai2, Timothy J. Koh1
1Kinesiology & Nutrition, University of Illinois at Chicago, Chicago, IL, United States 2Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, IL, United States
Single-Cell Transcriptomics Reveals Reduction Of A Population Of Monocyte-Derived Dendritic Cells Associated With Impaired Healing In Diabetic Wounds Of Mice

Jingbo Pang1, Mehrdad Zandigohar2, Yang Dai2, Timothy J. Koh1 1Kinesiology & Nutrition, University of Illinois at Chicago, Chicago, IL; 2Department of Biomedical Engineering, University of Illinois at Chicago, CHICAGO, IL

Diabetes induces dysregulation of a spectrum of inflammatory cells which contributes to persistent inflammation and defective tissue repair responses. To explore dysregulation of subsets of monocyte/macrophage lineage, we isolated and pooled Live CD45+CD11b+Ly6G- cells from excisional skin wounds of non-diabetic C57Bl/6 and diabetic db/db mice on day 3, 6, and 10 post-injury and performed scRNAseq analysis using the 10x Chromium platform. Cells from non-diabetic vs diabetic wounds exhibited strikingly different phenotypes throughout the healing process. Although wound cells in non-diabetic mice showed an expected transition from pro-inflammatory to pro-healing phenotypes, cells from diabetic mice did not show this transition. Interestingly, a cluster expressing high levels of Cd209a, Cd74 and other genes associated with antigen presenting cells (APC) was populated primarily by cells from non-diabetic mice with significantly lower contribution from diabetic mice. These data indicate decreased accumulation of APCs in wounds of diabetic mice and were confirmed by flow cytometry analysis using markers for this APC cluster. To determine the origin of these cells, we examined this APC population in skin wounds from CCR2 knock-out (KO) mice which lack circulating monocytes. Accumulation of these APCs was almost completely ablated in wounds of CCR2 KO mice compared to their wild-type counterparts, suggesting these cells originate from circulating monocytes. Importantly, adoptive transfer of bone marrow naïve Ly6C+ monocytes to skin wounds indicated that donor cells gained dendritic cell markers including MHC II, CD11c, CD74 and CD209a after 20-hour incubation in the wound environment, confirming the monocyte origin of these APCs. Finally, to begin to understand the regulation of this APC population, we used the BITFAM model to infer TF activity in these cells during skin wound healing, incorporating both scRNAseq and scATACseq data. Predicted TF activity associated with this APC population included PRDM1, IRF4 and STAT4 activity, which was supported by significantly higher protein levels in these cells compared to other wound monocyte and macrophage populations. Previous studies have demonstrated monocytes have the capacity to differentiate into APCs called monocyte-derived dendritic cells which can adopt multi-functional capabilities during inflammation. We are currently performing experiments to elucidate the function of these cells during wound healing. Together, our data demonstrated the significant reduction of monocyte-derived APC in skin wounds of diabetic mice, which may contribute to impaired healing in these mice.

I.02

Macrophage RNA Binding Protein Pcbp2 Interacts With Pre-Mir-21 In Keratinocyte-Derived Exosome For Resolution Of Inflammation
Anita Yadav1, Anu Sharma1, Chandan K. Sen1, Sashwati Roy1, Subhadip Ghatak1
1Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States
Macrophage RNA Binding Protein Pcbp2 Interacts With Pre-Mir-21 In Keratinocyte-Derived Exosome For Resolution Of Inflammation

Anita Yadav, Anu Sharma, Chandan K. Sen, Sashwati Roy, Subhadip Ghatak Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA

Background. Resolution of inflammation is contingent on successful crosstalk between keratinocytes and wound-edge (WE) macrophages (mΦ). We tested the hypothesis that in mΦ, RNA binding adaptor poly(C)-binding protein 2 (PCBP2) interacts with pre-miR21 in keratinocyte-derived exosome (Exok) for timely resolution of inflammation.
Methods. Murine WE Exok were genetically labeled with GFP reporter, isolated, and characterized per MISEV 2018 guidelines. This method, reported in EV track, received an EV-metric score of 100%. dSTORM imaging (res:20nm) was performed in conjunction with sequence-specific molecular beacons (MB) to quantify pre-and mature miR21 abundance in Exoκ at single exosome resolution. Resolution of inflammation was studied using laser captured microdissection of WE mΦ, RT-qPCR, and ELISA of inflammatory cytokines and immunostaining of iNOS, ARG1, MPO, and annexinV. Day 5 pro-inflammatory mΦ (mΦpro) from murine WE tissue was isolated from single cell suspension using immunomagnetic sorting of anti-F4/80 and CD11b+ microbeads. Results. Automated electrophoresis of RNA isolated from WE Exoκ showed predominance of small RNA (<200 bp). In silico study identified pre-miR21 with an exomotif sequence in its stem-loop structure. Using MB specific to pre-miR21 and dSTORM imaging, significant high abundance of pre-miR21 was observed in WE Exoκ compared to skin (n=6, p<0.01). PCBP2, reported to stabilize mRNA transcript of pro-inflammatory cytokines, was also found in significantly high abundance in mΦpro (n=7, p<0.001). Uptake of Exoκ by mΦpro resulted in significant downregulation of PCBP2 with significant low transcript levels of TNF-α, IL-2, INF-g, GM-CSF (n=4-6, p<0.01). The transcript abundance of pro-resolution Cl3 and Arg1 was also increased. Immunoproximity ligation assay demonstrated binding of pre-miR21 with PCBP2 in mΦ. Immunoprecipitation of PCBP2 from murine WE tissue indicated presence of miR21. PCBP2-pre-miR21 interactions in day 5 WE mΦpro resulted in degradation of the mRNA transcript of pro-inflammatory cytokines. In diabetic mice, WE mΦpro demonstrated significant low abundance of pre-miR21 (n=4, p<0.001), suggesting the notion that uptake of pre-miR21 loaded Exok was compromised. To test our hypothesis and block Exok uptake by mΦpro, K14 promoter-driven tetraspanins plasmid connected via IRES element with "eat me not"-CD47 sequence with "in-frame" GFP reporter was generated. Selective interruption of Exok uptake resulted in the persistence of mΦpro with high PCBP2 and iNOS expression (n=4,p<0.01), high annexinV+ dead cells burden, and compromised re-epithelialization.
Conclusion. This work addresses the molecular dynamics of keratinocyte-mΦ crosstalk in wound microenvironment. Notably, it identifies the pivotal role of WE keratinocytes in directing resolution of inflammation, a process often disrupted in non-healing diabetic wounds.

I.03

Using Microbial Transcriptional Profiles As A Biomarker For Diabetic Wound Healing
Alex Cheong1, Oluchi Aroh2, Caitlin Sande2, Jessica Irvine1, Anna Nora1, Meghan Brennan1, Lindsay Kalan2
1University of Wisconsin, Madison, WI, United States. 2McMaster University, Hamilton, ON, Canada
Using Microbial Transcriptional Profiles As A Biomarker For Diabetic Wound Healing

Alex Cheong1, Oluchi Aroh2, Caitlin Sande2, Jessica Irvine1, Anna Nora1, Meghan Brennan1, Lindsay Kalan2 1University of Wisconsin, Madison, WI; 2McMaster University, Hamilton, ON, Canada

Introduction: The diabetic foot ulcer (DFU) microbiome has been associated with healing outcomes and is hypothesized to be a source of biomarkers. Profiling the microbiome by 16S rRNA sequencing is limited by species level classification and functional analysis of these communities, especially over time and in response to treatments. Here we apply metatranscriptomics to profile microbial transcription to identify key metabolic activities of the microbiome and use these to predict clinical outcome.
Methods: 100 patients with Wagner grade 1-3 DFUs were enrolled and matched specimens of debrided tissue and deep ulcer swabs were processed for DNA-based 16S rRNA amplicon sequencing and RNA-based metatranscriptomics. Specimens were collected at clinical visits up to week 12 of enrollment. Comprehensive clinical metadata and wound outcomes were collected for each patient. Data was processed to remove human reads, taxonomically classified, and metabolic pathways reconstructed.
Results: 16S rRNA microbiome composition significantly overlapped between the two specimen types, indicating that both are viable sampling methods. However, the metatranscriptome showed swabs are more diverse for species richness and functional diversity as compared to tissue, suggesting an increased sensitivity of RNA-based methods. We used the ratio of RNA to DNA abundance for each genera as a marker for microbial transcriptional activity. Anaerobes were found to be more transcriptionally active in wounds resulting in an amputation. The summed relative abundance of anaerobic transcripts significantly predicted the likelihood of amputation at the end of study (12 weeks; odds ratio 14.17, 95% CI: 1.14 – 232.95, logistic regression, p<0.05). Notably, anaerobic 16S amplicon sequencing relative abundances were not significantly associated with amputation (logistic regression, p>0.05), supporting the increased sensitivity of RNA-based methods. No individual taxa were associated with this outcome (logistic regression, unadjusted p>0.05), but summed transcript relative abundances of Clostridium, Anaerococcus, Peptoniphilus, and Finegoldia species were sufficient to detect a significant association with amputation (logistic regression, p<0.05), highlighting the importance of Gram-positive anaerobic cocci (GPAC) in this dataset. Anaerobic taxa abundance was not associated with peripheral arterial disease and decreased vascular flow, suggesting that there may be local factors (e.g. biofilms) allowing for anaerobes to persist, and that they may function as a separate indicator for low oxygen microenvironments.
Conclusions: These data support the wound microbiome as a promising clinical biomarkers. Further analysis of these datasets will yield testable microbial biomarkers that are translatable into clinical tools to be validated in a prospective cohort in collaboration with the Diabetic Foot Consortium.

I.04

Perinatal Upregulation of CCL11 In Skin Fibroblasts Is Essential For Subcutaneous Adipogenesis And Wound Healing
Rahul Debnath, Zhaoxu Chen, Kang Ko
Dental Medicine, University of Pennsylvania, Philadelphia, PA, United States
Perinatal Upregulation Of Ccl11 In Skin Fibroblasts Is Essential For Subcutaneous Adipogenesis And Wound Healing

Rahul Debnath, Zhaoxu Chen, Kang Ko Dental Medicine, University of Pennsylvania, Philadelphia, PA

Background: Fibroblasts are the most abundant mesenchymal cells in the dermis. Though traditionally known for its role in extracellular matrix synthesis, recent advances have provided insight into unexpected immunomodulatory properties of skin fibroblasts during homeostasis and in diseased conditions. Here, we hypothesized that immuno-regulatory secretome by cutaneous fibroblasts is age-dependent and tested if CCL11, a chemokine expressed largely by fibroblasts, is necessary for skin tissue homeostasis that may affect the wound healing process.
Materials and methods: Publicly available scRNA-seq datasets (GSE189210, GSE183031, GSE172226) were pooled, and fibroblast subsets were categorized as neonatal (P0) or adolescent (P22 and P28) for downstream analysis using Seurat package. Among fibroblast transcriptome, CCL11 upregulation was most notable, which was validated using FACS followed by RT-qPCR and RNAScope on WT C57BL/6N mice. To determine biological significance, CCL11null mice were examined, and skin specimens were compared to P15 and P28 of age. In a series of animal studies, wound healing was examined in WT and CCL11null mice by histologic analysis and flow cytometry. Autogenous fat grafting in CCL11null mice and CCL11 blocking by neutralizing antibody were examined for healing parameters. Animal studies were carried out with N=4-6 mice each, and statistical significance was determined at p<0.05 by one-way ANOVA and T-test.
Results: Bioinformatics analysis demonstrated that CCL11 transcripts were highly upregulated in the fibroblasts of adolescent mice compared to that of perinatal mice. Further validation showed that CCL11 mRNA was minimally expressed in PDGFRa+ fibroblasts from P3 skin, compared to P15 skin. RNAscope analysis confirmed the age-dependent expression of CCL11, which was largely localized to the dermal layer. CCL11 knockout mice showed a significant reduction in subcutaneous adipose layer width compared to wildtype littermates at P28, suggesting a role for adipogenesis in developing skin. Full-thickness wounds in CCL11null mice had fewer aSMA+ myofibroblasts and delayed re-epithelization compared to WT mice. Fat autografting prior to wound induction in CCL11null mice rescued the healing parameters compared to sham control groups, whereas CCL11 neutralization in WT mice did not have an impact on the wound healing process, indicating that wound healing deficit is dependent on intact adipose tissues and not CCL11.
Conclusion: Our study demonstrates that early-life production of CCL11 by dermal fibroblasts is essential for normal adipogenesis, and in turn, cutaneous injury response. The study highlights an important role of fibroblast-derived cytokine production that may unveil pathologic mechanisms behind dysregulated adipogenesis and impaired wound healing.

I.05

Host-Biofilm Interaction derived Oxylipin Impairs Diabetic Foot Ulcer Healing by Targeting T-Cell Immune Checkpoint Pathway
Sunil Kumar1, Miguel Jorge1, Imran Khan1, Ethan Rinne1, Bryce Hockman1,2, Kaitlyn Depinet2, Beth Altenburger2, Jaimee Hann2, Gregory Westin2,3, Mithun Sinha1
1Department of Surgery, Division of Plastic Surgery, Indiana University School of Medicine, Indianapolis, IN 2Comprehensive Wound Center, Indiana University Health, Indianapolis, IN 3Department of Surgery, Division of Vascular Surgery, Indiana University School of Medicine, Indianapolis, IN
Host-Biofilm Interaction Derived Oxylipin Impairs Diabetic Foot Ulcer Healing by Targeting T-Cell Immune Checkpoint Pathway

Sunil Kumar1, Miguel Jorge1, Imran Khan1, Ethan Rinee1, Bryce Hockman1, Kaitlyn Depinet2, Beth Altenburger2, Jaimee Hann2, Gregory Westin1, Mithun Sinha1 1Surgery, Indiana University, Indianapolis, IN; 2Comprehensive Wound Center, Indiana University Health, Indianapolis, IN

Background: Diabetic foot ulcers (DFU) are a leading cause of lower limb amputations. Current DFU treatments rely on pharmacological drugs and standard of care which often results in poor prognosis. The role of bacterial biofilm and its interaction with the host immune system in the context of wound healing is poorly understood.
Purpose: This study delves into the connection between DFU and bacterial biofilms, exploring the role of biofilm-derived oxylipins, particularly 10-hydroxy-8-octadecenoic acid (10S-HOME) and 9-hydroxy-octadecadienoic acid (9S-HODE) in immune cell impairment. Oxylipins have been reported to be immunomodulatory and capable of polarizing CD4+T cells. Methods: To test our hypothesis, we collected wound samples (N=27) from consenting subjects under a protocol approved by IRB. The samples underwent biofilm analysis using scanning electron microscopy (SEM) and 16S rRNA next-generation sequencing (NGS). Metabolite profiling of 10(S)-HOME and 9(S)-HODE was conducted via liquid chromatography-mass spectrometry (LC-MS/MS) using deuterated analogs of 10(S)-HOME and 9(S)-HODE as internal standard. T-cells from a healthy donor were studied for their immunological role and correlated with human keratinocytes-derived cell line mimicking wound under oxylipin-challenged conditions. Quantitative gene expression and protein abundance were performed using RT-qPCR, ELISA, and dot-blot techniques.
Results: Our data from a cohort of 27 patient specimens (wound 17 and control 10) showed a discernible augmentation in bacterial biofilm abundance in the wound samples relative to the control sample. NGS unveiled the presence of microorganisms, specifically Finegoldia magna, Pseudomonas aeruginosa, Staphylococcus aureus, and Anaerococcus vaginalis within the wound tissue. Moreover, oxylipin profiling revealed the upregulation of 10(S)-HOME and 9(S)-HODE in the wound tissue compared to normal unwounded skin. Treatment with 10(S)-HOME induced a discernible retardation in human keratinocyte proliferation compared to sham control (n=10, p<0.0003). Additionally, dysregulation in the expression of the immune checkpoint gene CTLA4 was observed after treatment with the oxylipins 10(S)-HOME (n=7) and 9(S)-HODE (n=7) in CD4+T-cells derived from healthy donors.
Conclusion: These findings suggest that host-pathogen interaction mediated via biofilm-derived oxylipins may interfere with the healing outcomes of DFU. This alteration seems to be mediated through the regulation of intracellular immune checkpoint genes. Therefore, CTLA4 and other intracellular checkpoints may hold promising candidates in controlling DFUs by targeting specific immune cell subsets. In addition, other checkpoint genes like PD-1 and CISH are under investigation. Further research and verification are essential to establish the potential of this approach in DFU management.

I.06

Shell or the Cargo? Significance of Keratinocyte-Derived Exosomes Surface Molecules In Tissue Repair
Anu Sharma1, Anita Yadav1, David Clemmer2, Sashwati Roy1, Chandan K. Sen1, Subhadip Ghatak1
1Surgery, McGowan Institute for Regenerative Medicine, Pittsburgh, PA, United States. 2Chemistry, Indiana University, Bloomington, Indianapolis, IN, United States
Shell Or The Cargo? Significance of Keratinocyte-Derived Exosomes Surface Molecules In Tissue Repair

Anu Sharma1, Anita Yadav1, David Clemmer2, Sashwati Roy1, Chandan K. Sen1, Subhadip Ghatak1 1Surgery, McGowan Institute for Regenerative Medicine, Pittsburgh, PA; 2Chemistry, Indiana University, Bloomington, Indianapolis, IN

Background – Exosomes, endocytically originated extracellular vesicles, play a pivotal role in cellular communication, encapsulating a myriad of biomolecules like DNA, proteins, and metabolites. This study posits that an in-depth analysis of the unique surface composition of exosomal is critical to unravel the intricacies of wound healing.
Methods – Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT). Exoκ-GFP were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Wound-edge keratinocyte-derived exosome uptake was blocked in mice by generating “eat me not” Exoκ-GFP-RFP using a KRT-14 promoter-driven tetraspanins plasmid connected via IRES element with “eat me not”-CD47 sequence with in-frame GFP and RFP reporter (Exoκ-GFP-RFP). Keratinocyte-targeted nanocarriers (TLNκ) were designed using pH-responsive lipid components with keratinocyte-targeting peptide sequence ASKAIQVFLLAG and loaded with siRNA of hnRNP to selectively inhibit cargo packaging within Exoκ-GFP. The isolated exosomes was characterized as per MISEV 2018 guidelines and by flow cytometry. Additionally, the surface of these exosomes was studied by various spectroscopic techniques such as Raman and FTIR measurements. Functional wound closure was evaluated using analytical histology and Transepidermal Water Loss (TEWL).
Results – Our results demonstrate that both compromising cargo packaging within Exoκ-GFP without compromising uptake by blood borne wound macrophages and inhibiting Exoκ-GFP uptake results in the persistence of proinflammatory macrophages at the wound site at day 12 (n=6, n=6). The macrophages exhibited high expression of iNOS (n=4, p<0.001; n=5, p<0.001). However, although no significant difference in re-epithelialization was observed compromising cargo packaging, "eat me not" Exoκ-GFP-RFP significantly compromised wound reepithelialization (n=6, p<0.001). Surface N-glycan analysis demonstrated a high abundance of mannose on Exoκ-GFP-RFP. Raman and FTIR spectroscopic analyses demonstrated spectral differences across the protein, lipids, and nucleic acid domains.
Conclusion – The findings from this research highlight the critical role of exosomal surface molecules in various physiological and pathological conditions. Expanding research on exosomal surfaces is essential for a deeper understanding of the complex healing processes at sites of injury.

I.07

Structural Equation Model To Quantify Importance Of Patient Factors And Wound Microbiome On Healing
Jacob Ancira4, Rebecca Gabrilska1, Craig Tipton4, Clint Miller2, Zachary Stickley3, Khalid Omeir4, Joseph Wolcott2, Todd D. Little3, Caleb Phillips4
1Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, United States. 2Southwest Regional Wound Care Center, Lubbock, TX, United States. 3Department of Educational Psychology and Leadership, Texas Tech University, Lubbock, TX, United States. 4Department of Biological Sciences, Texas Tech University, Lubbock, TX, United States
Structural Equation Model To Quantify Importance Of Patient Factors And Wound Microbiome On Healing

Jacob Ancira4, Rebecca Gabrilska1, Craig Tipton4, Clint Miller2, Zachary Stickley3, Khalid Omeir4, Joseph Wolcott2, Todd D. Little3, Caleb Phillips4 1Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX; 2Southwest Regional Wound Care Center, Lubbock, TX; 3Department of Educational Psychology and Leadership, Texas Tech University, Lubbock, TX; 4Department of Biological Sciences, Texas Tech University, Lubbock, TX

Background: The contribution of microbiome and host factors to driving chronicity and rate of healing in wounds is widely appreciated. However, there is currently little ability to account for the many variables and dynamics influencing differences in healing. Here, with the goal of developing a predictive framework, a novel structural equation modelling (SEM) approach was employed to model the chronic wound environment in relation to healing. Methods: The dataset consisted of 565 chronic wound microbiomes detected at initial patient visit using 16S sequencing paired with patient medical information. A novel pre-modeling parcel optimization routine was developed to transform the microbiome species table into two latent variables that related to healing time either positively or negatively. These latent constructs in addition to specific species correlating with healing, and patient/wound data were evaluated for model fit in the SEM using backward selection and delta chi-squared testing.
Results: A microbiome latent construct significantly associated with improved healing was validated, and the final SEM included this latent construct plus three species associated with diminished healing (Anaerococcus vaginalis, Finegoldia magna, Pseudomonas aeruginosa), as well as smoking, wound volume, slough, exudate, edema, percent granulation, and wound type. This model explained 49% of variation in healing time with the microbiome contributing the largest proportion of variance explained. Percent granulation and wound volume accounted for the second and third most variance explained. Species that formed the latent construct tended to correlate with each other less than did the remaining species (p < 0.001), potentially reflecting that species associated with faster healing act individually rather than synergistically. Also, species that formed the latent construct that was associated with faster healing also included species that are routinely treated with biofilm-based wound care.
Conclusions: This study provides a novel pre-modeling approach allowing the integration of microbiome data into SEM. The final model validated the importance of many variables on differences in healing time, with wound microbiome species being the most important. The importance of microbes in this model advocates for the efficacy of guiding treatment based on results of DNA sequencing-based microbiota profiling.

I.08

The Histone Methyltransferase Whsc1 Regulates TGF?-driven Macrophage to Myofibroblast Transition During Wound Healing
Kevin Mangum1, Amrita Joshi1, Sonya Wolf1, Jadie Moon1, Andrea Obi1, Beth Moore2, Frank Davis1, Katherine Gallagher1
1Vascular Sugery, University of Michigan, Ann Arbor, MI, United States 2Department of Immunology and Microbiology, University of Michigan, Ann Arbor, MI, United States
The Histone Methyltransferase Whsc1 Regulates TGFβ-Driven Macrophage To Myofibroblast Transition During Wound Healing

Kevin Mangum1, Amrita Joshi1, Sonya Wolf1, Jadie Moon1, Andrea Obi1, Beth Moore2, Frank Davis1, Katherine Gallagher1 1Vascular Sugery, University of Michigan, Ann Arbor, MI; 2Department of Immunology and Microbiology, University of Michigan, Ann Arbor, MI

Introduction: Diabetes contributes to significant morbidity and mortality in the United States, and one of the major features includes poor wound healilng, which leads to high rates of amputation due to ineffective treatment options. In diabetes, macrophages exhibit a pro-inflammatory phenotype that prevents normal wound healing. During normal tissue repair, macrophages undergo transition to myofibroblasts, which aid in late wound repair by promoting wound contraction and closure. Our lab and others have shown that macrophages express key fibrotic genes (e.g., SMA, Col1a1, Col3a1), which increase throughout wound healing. However, this fibrotic gene program is disrupted in diabetic macrophages, and fibroblast numbers are reduced in diabetic wounds. Here, we identify Whsc1 as a TGFβ-dependent histone methyltransferase that is essential for macrophage to myofibroblast transition (MMT), and we show that Whsc1 is disrupted in diabetic macrophages. Methods: The goal of this study was to identify TGFβ-dependent transcription mechanisms that regulate macrophage phenotype during wound healing. Bone marrow derived macrophages (BMDMs) and wound macrophages (CD11b+CD3-CD19-NK1.1-Ly6G-) were isolated for experiments from a diet-induced obese (DIO) diabetic mouse model and compared with those from normal diet (ND) mice. We performed an epigenetic superarray to identify key chromatin modifying enzymes in macrophages from murine wounds treated ex vivo with TGFβ. siRNA knockdown of Whsc1 and NFkβ p65 (RelA) and downstream chromatin immunoprecipitation (ChIP) and qPCR were also used.
Results: Whsc1 was significantly decreased in DIO macrophages with TGFβ treatment compared to ND macrophages (p<0.05). siRNA knockdown of Whsc1 in BMDMs decreased TGFβ-dependent expression of fibrotic (SMA, Col1a1, Col3a1) genes (p<0.05). In ChIP experiments, Whsc1 and its mark H3K36me2 were enriched at fibrotic promoters in ND macrohages in response to TGFβ but decreased in DIO BMDMs. The Whsc1 inhibitor LEM-14 significantly increased wound size and reduced fibrotic gene expression in BMDMs. Interestingly, the pro-inflammatory transcription factor RelA bound more strongly to Whsc1 in DIO BMDMs. Finally, siRNA knockdown of RelA reversed the dysregulated effect of TGFβ on fibrotic gene expression in DIO BMDMs by increasing Whsc1 enrichment at fibrotic promoters.
Conclusion: The current study delineates the TGFβ-Whsc1 signaling axis, which is altered in the diabetic macrophage, thereby leading to defective MMT. Specifically, we show that in ND macrohages Whsc1 controls expression of the fibrotic gene program; however, in DIO macrophages RelA inhibits Whsc1 enrichment at fibrotic gene promoters. Future studies will test whether macrophage-specific targeting of the Whsc1-RelA interaction improves diabetic wound healing in vivo.

I.09

ETRS: Soluble CD83 Improves Wound Healing in a 3D Wound Healing Model and Promotes Resolution of the Inflammatory Milieu In Chronic Wounds
Christian Hollard1,2, K. Peckert-Maier1, C. Erfurt-Berge2, A. Steinkasserer1, D. Royzman1
1Departent of Immune Modulation, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany, 2Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany
Soluble Cd83 Improves Wound Healing And Promotes The Resolution Of Chronic Inflammation In A 3d Wound Healing Model

Christian Hollard1,2, K. Peckert-Maier1, C. Erfurt-Berge2, A. Steinkasserer1, D. Royzman1 1Departent of Immune Modulation, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany; 2Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany

Macrophages (Mϕ) play a pivotal role in orchestrating the wound healing response after skin injury. In the early post-injury phase, pro-inflammatory Mϕ accumulate and sterilize the wound site. Subsequently, these Mϕ undergo a conversion into a pro-regenerative phenotype, initiating the proliferative phase by releasing essential anti-inflammatory cytokines and growth factors. However, when the transition from pro- to anti-inflammatory Mϕ is impaired, the resolution of inflammatory processes is disturbed, thus contributing to the development of chronic wounds. Currently, clinical options for the treatment of chronic wounds are limited and characterized by time-consuming, labor-intensive, and costly procedures. Consequently, there is an urgent need for the identification and development of novel therapeutic agents. In a recently published study, we demonstrated that soluble CD83 (sCD83) enhances and accelerates skin wound healing in mice by inducing pro-resolving Mϕ. Therefore, the focus of this study is to explore the therapeutic potential of sCD83 in the human system using a translational 3D skin wound healing model.
To investigate the effects of sCD83, we generated a 3D wound model using skin equivalents consisting of human keratinocytes, fibroblasts, and Mϕ. Wound infliction was mimicked using a 4 mm biopsy punch. Skin equivalents were treated with either sCD83 (25 μg/ml) or an equivalent volume of PBS as a control. 3D skin equivalents were analyzed by histology and flow cytometry 2 and 4 days after wounding. In addition, the cytokine composition in the supernatants was determined using cytometric bead arrays. In further experiments, chronic wound exudate from patients with venous or arterial/ischemic ulcers was added to the culture medium in order to simulate an inflammatory environment. Under these conditions, the regulatory mechanisms induced by sCD83 under non-healing conditions were analyzed.
The presence of sCD83 resulted in a significantly accelerated wound healing, characterized by an increased influx of cells into the wound area. This effect is mediated by the induction of a pro-regenerative Mϕ phenotype in 3D skin equivalents, as indicated by increased CD163 and decreased CD204 expression levels. Moreover, sCD83 modulates the expression of specific growth factors (increased levels of VEGF, TGF-α, PDGF) and cytokines (increased levels of IL-4, IL-33, decreased levels of IL-1β, IL-23) in wounded 3D skin equivalents. In conclusion, we demonstrate that sCD83 improves wound healing conditions in a 3D wound model, which is a further step towards establishing sCD83 as a therapeutic agent for the treatment of hard-to-heal wounds.

K1.01

What is Slough? Defining the proteomic and microbial composition of wound slough and its implications for wound healing
Elizabeth Townsend1, Alex Cheong1, Michael Radzietza2, Blaine Fritz3, Matthew Malone2, Thomas Bjarnsholt3, Karen Ousey4, Terry Swanson4, Gregory S. Schultz4, Angela Gibson1, Lindsay Kalan1
1University of Wisconsin, Madison, WI, United States 2Western Sydney University, Penrith, NSW, Australia. 3University of Copenhagen, Copenhagen, Denmark. 4International Wound Infection Institute, London, United Kingdom
What is Slough? Defining the Proteomic And Microbial Composition Of Wound Slough And Its Implications For Wound Healing

Elizabeth Townsend1, Alex Cheong1, Michael Radzietza2, Blaine Fritz3, Matthew Malone2, Thomas Bjarnsholt3, Karen Ousey4, Terry Swanson4, Gregory S. Schultz4, Angela Gibson1, Lindsay Kalan1 1University of Wisconsin, Madison, WI; 2Western Sydney University, Penrith, New South Wales, Australia; 3University of Copenhagen, Copenhagen, Denmark; 4International Wound Infection Institute, London, United Kingdom

Intro: Slough is a well-known feature of chronic wounds. However, the tissue and microbial composition of wound slough are not well defined. Clinically, it’s difficult to define what is normal slough and identify wounds likely to heal versus deteriorate. Aims: To determine the proteomic and microbiologic components of slough and their associations with wound healing.
Methods: 23 subjects with chronic wounds and visible slough were enrolled. Etiologies included venous stasis ulcers, post-surgical site infections, and pressure ulcers. Patient co-morbidities and wound healing outcome 3-months post sample collection were recorded. Debrided slough was analyzed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-rRNA gene sequencing.
Results: Wound age ranged from 6 weeks to 15 years. Microscopic imaging revealed slough to be amorphous in structure. 16S-profiling found slough microbial communities associate with wound etiology and location on the body (both PERMANOVA p<0.01). Across all subjects, slough predominantly consisted of proteins involved in skin structure and formation, blood-clot formation, and immune processes. To predict variables associated with wound healing, protein, microbial, and clinical datasets were integrated into a supervised partial least squares-discriminant analysis. This analysis found wounds that healed 3-months after sample collection were enriched for proteins involved in skin barrier development (eg cornifin B) and negative regulation of immune responses (eg cystatin F). Differential enrichment of these proteins in healed wounds compared to those that deteriorated was confirmed via DEqMS (all absolute Log2(fold change)>1.5 and p<0.01). Wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment score and were enriched for anerobic bacteria (eg Fingoldia & Peptoniphilus) and chronic inflammatory proteins (eg activator protein-1, vasodilator-stimulated phosphoprotein, & complement factor H; all absolute Log2(fold change)>1.5 and p<0.01).
Discussion: Slough is an underutilized reservoir for potential biomarkers of wound healing. This is the first study to integrate clinical, microbiome, and proteomic data to systematically characterize wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing. Future studies aim to explore these predicted biomarkers in a larger cohort. Development of a comprehensive patient-centered assessment will lead to more effective identification of patients’ wounds that may benefit from triage into specialty care and ultimately reduce the healthcare, financial, and personal burden of living with a chronic wound.

K1.02

Analysis Of 9,241 Wound Specimens Reveals Six Major Microbiome Community Types And Meteorological Associations
Craig Tipton1, Rebecca Gabrilska2, Jacob Ancira1, Courtney Jarvis3, Lars Koenig3, Karin Ardon-Dryer4, Kendra Rumbaugh2, Caleb Phillips1
1Biological Sciences, Texas Tech University, Lubbock, TX, United States 2Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, United States 3MicroGen DX, Lubbock, TX, United States 4Atmospheric Science Group, Texas Tech University, Lubbock, TX, United States
Analysis Of 9,241 Wound Specimens Reveals Six Major Microbiome Community Types And Meteorological Associations

Craig Tipton1, Rebecca Gabrilska2, Jacob Ancira1, Courtney Jarvis3, Lars Koenig3, Karin Ardon-Dryer4, Kendra Rumbaugh2, Caleb Phillips1 1Biological Sciences, Texas Tech University, Lubbock, TX; 2Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX; 3MicroGen DX, Lubbock, TX; 4Atmospheric Science Group, Texas Tech University, Lubbock, TX

Background: Colonizing microbiota are known contributors to chronic wound persistence. Though differential microbes likely relate to wound management success, there is limited understanding of the major types of microbial communities that are encountered. Prior work in the skin microbiome has shown that geographic and seasonal meteorological variation relates to differences in external microbiota, however it is unknown whether that link translates to a chronic infected wound.
Methods: We set out to describe the microbial composition of the wound microbiome by retrospective analysis of 9,241 specimens submitted from clinics in 43 states for microbial profiling that included matched qPCR, 16S rRNA and ITS gene sequencing. Historical sequence and qPCR data were obtained from MicroGen DX, a CAP/CLIA diagnostic sequencing provider, as matched to patient samples submitted under relevant wound test service between 2018 and 2020. Community state types (CSTs) were identified using the Dirichlet multinomial method. The CSTs were modeled for association to meteorological variables by multinomial logistic regression.
Results: Six CSTs were identified based on bacterial profiles that significantly related to differences in qPCR-estimated bacterial load, alpha diversity, fungal positivity, and bacterial morphotype (e.g., anaerobes). CSTs were unequally distributed across demographic factors such as sex, age, and wound location (p<0.05). For example, men had a 3.6-14.5% increased prevalence of CSTs with high load and anaerobic content (CST3/4) versus a 5.5-18.2% decreased prevalence of CSTs characterized by low bacterial load (CST2/6). The probability of observing CSTs was linked to meteorological variance, particularly temperature and relative humidity. For example, the relative probability of encountering a given CST ranged from -243% to +61% depending on local weather patterns. The most climate sensitive CSTs were characterized by high anaerobic content and load, or low bacterial load not suggestive of infection.
Conclusions: The CSTs identified provide an intuitive description of the types of microbes commonly found and are suggestive of microbial profiles that may warrant different therapeutic approaches. These CSTs provide valuable insights into common assemblages of microbes that will appear in a wound infection, though study in a prospective cohort is required to determine whether the CSTs relate to patient outcomes. Lastly, a statistical association linked the likelihood of observing different CSTs with local meteorological variation, providing the first evidence that wound clinics may encounter different types of infectious profiles and that differences in observation are potentially explained by spatiotemporal weather gradients.

K1.03

A Disposable Device For Fast Screening Of Diabetic Foot Ulcer Infection
Jon Senkowsky2, Shuxin Li1, Wenjing Hu1, Liping Tang1
1Progenitec, Arlington, TX, United States 2Texas Health Physician’s group, Arlington, TX, United States
A disposable Device For Fast Screening Of Diabetic Foot Ulcer Infection

Jon Senkowsky2, Shuxin Li1, Wenjing Hu1, Liping Tang1 1Progenitec, Arlington, TX; 2Texas Health Physician’s group, Arlington, TX

PURPOSE: The initial identification of infection in DFU mostly depends on the clinical signs and symptoms of local inflammation. The wound specimens are then collected via wound swab or tissue biopsy for microbiological analysis. Unfortunately, many infected wounds present subtle or no signs and symptoms of infection. Thus, there is an urgent need for the development of a complimentary approach or device that can quickly screen and identify suspected infection in DFU. Recent study shows that infected wounds have elevated leukocyte esterase (LE) activities as compared with non-infected wounds by a portable device – Detec® Esterase. Here, we try to answer two questions. Are LE activities elevated in wound fluids isolated from infected DFU wounds compared to those from non-infected wounds? Can Detec® Esterase be used to screen infection of DFU at point-of-care?
METHOD: Wound dressings from 15 infected DFU wounds and 5 non-infected DFU wounds based on the culture results were isolated for LE activities measurement. Dressings from 20 DFU patients (14 infected and 6 non-infected) at the Texas Health Arlington Memorial Wound Care and Limb Salvage Clinic were tested with DETEC® esterase and the device output was compared with subsequent clinical determination of infection on the same wounds. We evaluated the efficiency of the device through sensitivity, specificity, accuracy, and the increase in post-test risk of infection with positive test result.
RESULTS: Our results show that LE activities are significantly higher in infected DFUs (>0.02794 U/mL) than in non-infected DFUs (0.003902-0.02275 U/mL). By comparing our device output with the clinical determination, our results show that pre-optimized DETEC® Esterase has a high sensitivity (85.7%) and accuracy (75.0%) for screening infected DFU wounds. Further analyses revealed that the diagnosis accuracy of the device is not influenced by the patients’ age, gender, race, initial wound size, wound location, and sign of infection.
CONCLUSION: Our results support that LE activities are elevated in infected DFU wounds, and LE may be used as a biomarker for DFU infection. DETEC® esterase can effectively and accurately diagnose and/or screen for infections in DFUs.

K1.04

Pseudomonas Aeruginosa Activates Lasi/R Quorum Sensing, Selective Antioxidant Enzymes, And Type Vi Secrection System During Biofilm Formation And Chronic Wound Initiation
Jane Kim, Brandon Le, Weifeng Gu, Manuela M. Martins-Green
University of California, Riverside, Fullerton, CA, United States
Pseudomonas Aeruginosa Activates Lasi/R Quorum Sensing, Selective Antioxidant Enzymes, And Type Vi Secrection System During Biofilm Formation And Chronic Wound Initiation

Jane Kim, Brandon Le, Weifeng Gu, Manuela M. Martins-Green University of California, Riverside, Fullerton, CA

Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. Some PA strains are highly virulent, establish strong biofilm and are antibiotic resistant. How PA colonizes chronic wounds with biofilm formation in response to oxidative stress (OS), is still unknown. The purpose of this study is to investigate the changes in gene expression when PA is challenged with high levels of OS, and how it increases virulence, decreases OS and becomes biofilm-forming in chronic wounds. We used a biofilm-forming PA strain isolated from the chronic wounds of our murine model and performed qPCR to obtain gene expression patterns as PA developed biofilm in vitro in the presence of high levels of OS and then verified the findings in vivo. We found that the planktonic bacteria while forming biofilm under OS conditions, overexpressed the Quorum Sensing genes lasI and lasR, rhlI, and rhlR, which are important for the bacteria to communicate with each other to form biofilm. We also found that the antioxidant stress genes sodA, sodB, katA, katB and oxyR important in reducing the OS in the microenvironment for survival, were also overexpressed. In addition, expression of biofilm formation genes such as pelA and pelB and pslA and pslB and virulent genes phzA and phzM for phenyzine production were increased. In vivo, we found that the levels of gene expression for quorum sensing, biofilm formation, and virulence were all upregulated during biofilm development. However, of the antioxidant genes, only oryR, katA and sodB were upregulated. To identify unique genes, we performed a broader transcriptomic analysis of PA using the RNAseq in vivo and identified the activation of novel genes/pathways of the Type VI Secretion Systems involved in PA pathogenicity. Several genes of the core secretion structure, including tssB and tssC, and phospholipase effector proteins, such as tli5, are significantly upregulated. PA is known to activate the Type VI Secretion System to compete with other bacteria in its microenvironment and target mammalian cells for infection. In conclusion, PA survives the harsh, high OS microenvironment present in chronic wounds and colonizes these wounds with biofilm by turning on virulent, biofilm-forming and survival genes. Therefore, effective biofilm removal and or return after debridement may be accomplished by disrupting the lasR system and/or sodA, katA and katB expression and potentially also the Type VI Secretion System. Because it is well known that biofilm in human chronic wounds readily returns after debridement, these findings could have implications for treatment of human chronic wounds to eliminate PA containing biofilm immediately after debridement.

K1.05

Detection And Photoablation Of Wound Biofilm Infections With Theranostic Gold-In-Gold Cage Nanoparticles
Maryam Hajfathalian1, Christiaan R. de Vries1, Yuxi C. Dong2, Ahmad Amirshaghaghi2, Pallavi Jonnalagadda2, Jessica HSU2, Aimen Zlitni1, David Cormode2, Paul Bollyky1
1Stanford University, Mountain View, CA, United States 2University of Pennsylvania, Philadelphia, PA, United States
Detection And Photoablation Of Wound Biofilm Infections With Theranostic Gold-In-Gold Cage Nanoparticles

Maryam Hajfathalian1, Christiaan R. de Vries1, Yuxi C. Dong2, Ahmad Amirshaghaghi2, Pallavi Jonnalagadda2, Jessica HSU2, Aimen Zlitni1, David Cormode2, Paul Bollyky1 1Stanford University, Mountain View, CA; 2University of Pennsylvania, Philadelphia, PA

Introduction: Bacterial biofilms colonize wounds and delay healing. These biofilms are difficult to treat with existing clinical therapies due to antimicrobial resistance. Therefore, a critical need exists to effectively diagnose and treat biofilm infections. Here, we present a theranostic agent to image and treat harmful biofilms. We developed gold-in-gold cage nanoparticles (PTNP) with enhanced photothermal (PTT) and photoacoustic imaging (PA) properties that are promising candidates for biofilm detection, treatment, and infectious disease control. We found PTNP can control virulent biofilms and treat infectious disease via activation with near infrared region (NIR) laser with precise spatial control and in a short timeframe. A strong biocidal effect against Staphylococcus aureus (S. aureus) within biofilms was observed, considerably more effective than currently clinically used skin antimicrobials. Therefore, here, we introduce a fast, precise, and unique topical therapeutic method to image and treat costly skin biofilm-associated infections.
Methods: PTNP were synthesized via galvanic replacement reaction. After purification, the collected nanoparticles were coated with dextran-10kDa (DEX) to provide stability in biological media. These structures were characterized using transition electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), and UV-visible spectroscopy. The anti-biofilm efficacy of the PTNP was examined in vitro using S. aureus biofilms. The untreated or treated biofilm with PTNP was analyzed using high-resolution confocal fluorescence imaging, while PA imaging of biofilms treated with PTNP was carried out to investigate the theranostic potential of these structures within the biofilm in vivo. Furthermore, PTNP was examined for its efficacy as an anti-biofilm agent to control, image, and treat wound infections.
Results: TEM of PTNP showed the Au seeds core and the shells that contain AuAg alloys with some porosities. The optical extinction spectra of PTNP showed the plasmonic peak at 808 nm. Uptake of PTNP by S. aureus biofilms was confirmed by SEM in backscattered electron (BSE) mode which showing the morphology of PTNP-treated biofilm. We found almost complete bacteria killing in biofilms incubated with PTNP and after 30 s laser irradiation, while no dead cells were observed in the area without laser irradiation. We found an exceptionally strong biocidal effect against S. aureus within biofilms when exposed to NIR laser irradiation and PTNP. Treatment of S. aureus infected mouse wounds was studied using bioluminescence imaging (BLI), which showed potent in vivo bacterial killing. Robust PA contrast can be seen in wound infection treated with PTNP.
Conclusion: PTNP is a promising approach as a theranostic anti-biofilm agent.

K1.06

Extracellular Granzyme B Contributes To Delayed Healing Of Cutaneous Leishmaniasis In A Murine Model
Layla Nabai1, Yasaman Kaviani1, Katlyn Richardson1, Alexandre Aubert1, Karen Jung1, Farhad Handjani2, Reza Yaghoobi3, Nicholas Carr4, Hongyan Zhao1, Robert McMaster5, David Granville1
1Pathology and laboratory Medicine, University of British Columbia, Vancouver, BC, Canada. 2Molecular Dermatology Research Centre, Shiraz university of Medical Sciences, Shiraz, Iran (the Islamic Republic of). 3Department of Dermatology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (the Islamic Republic of). 4Department of Surgery/Division of Plastic Surgery, University of British Columbia, Vancouver, BC, Canada. 5Faculty of Medicine, Medical Genetics, University of British Columbia, Vancouver, BC, Canada
Extracellular Granzyme B Contributes To Delayed Healing Of Cutaneous Leishmaniasis In A Murine Model

Layla Nabai1, Yasaman Kaviani1, Katlyn Richardson1, Alexandre Aubert1, Karen Jung1, Farhad Handjani2, Reza Yaghoobi3, Nicholas Carr4, Hongyan Zhao1, Robert McMaster5, David Granville1 1Pathology and laboratory Medicine, University of British Columbia, Vancouver, BC, Canada; 2Molecular Dermatology Research Centre, Shiraz university of Medical Sciences, Shiraz, Iran; 3Department of Dermatology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 4Department of Surgery/Division of Plastic Surgery, University of British Columbia, Vancouver, BC, Canada; 5Faculty of Medicine, Medical Genetics, University of British Columbia, Vancouver, BC, Canada

Background and Hypothesis: Cutaneous leishmaniasis (CL) is an infectious disease caused by different species of leishmania parasite, resulting in a variety of chronic skin lesions that leave severe scarring and disfigurement. Granzyme B (GzmB), a serine protease, has been implicated in pathogenesis of CL caused by Leishmania braziliensis. Although the cytotoxic role of intracellular GzmB in parasite killing and targeted apoptosis in CL is well known, the contribution of extracellular GzmB to chronic inflammation, tissue damage, and scarring seen in CL is poorly understood. Here, we hypothesized that GzmB is elevated in CL caused by Leishmania major (L. major) and contributes to impaired healing through the cleavage of cell-cell and cell-basement membrane adhesion proteins.
Methods: Paraffin-embedded human skin biopsy specimens with confirmed diagnosis of L. major induced CL were subjected to the histological analysis using hematoxylin& eosin, immunohistochemistry (IHC), and immunofluorescent staining for GzmB, cellular markers of different immune cells, and GzmB substrates. Cell-free in vitro cleavage assay was used to identify new GzmB substrates. Twelve wild type and 11 GzmB knockout C57BL/6 mice were inoculated with L. major and the clinical course of the disease was documented over 8 weeks. Skin samples were collected for histological analysis at study endpoint.
Results: GzmB was highly expressed in human CL lesions compared to normal skin. The expression of E-cadherin, a cell-cell junction protein was significantly reduced in areas with higher number of GzmB expressing cells (n=11, p<0.001). Collagen VII and collagen XVII, were also reduced in areas with accumulation of GzmB+ cells. Cell-free in vitro cleavage assay resulted in identification of two novel substrates of GzmB, desmoglein 4 and annexin A2. Further, IHC staining of human samples showed reduction of desmoglein 4 in samples demonstrating psoriasiform dermatitis, and annexin A2 on endothelial cells of thrombotic blood vessels. In vivo study revealed that while 54% of GzmB KO mice showed no visible lesion/scarring, all WT mice still had clinical lesion/scarring at the study end point.
Conclusion: Our results show that extracellular GzmB is associated with epidermal changes and vascular thrombosis in L. major induced CL and deletion of GzmB significantly reduces the healing time of the lesions in a murine model of CL. Collectively, our findings suggest a prominent role for extracellular GzmB in pathogenesis of skin lesions caused by L. major.

K2.01

Extracellular Granzyme B Contributes To Delayed Healing Of Cutaneous Leishmaniasis In A Murine Model
Kellen Chen2, Dominic Henn2, Dharshan Sivaraj2, Katharina S. Fischer2, Jagannath Padmanabhan1, Michael Januszyk1, Geoffrey C. Gurtner2
1Surgery, Stanford University, Irvine, CA, United States 2Surgery, University of Arizona, Tucson, AZ, United States
Extracellular Granzyme B Contributes To Delayed Healing Of Cutaneous Leishmaniasis In A Murine Model

Layla Nabai1, Yasaman Kaviani1, Katlyn Richardson1, Alexandre Aubert1, Karen Jung1, Farhad Handjani2, Reza Yaghoobi3, Nicholas Carr4, Hongyan Zhao1, Robert McMaster5, David Granville1 1Pathology and laboratory Medicine, University of British Columbia, Vancouver, BC, Canada; 2Molecular Dermatology Research Centre, Shiraz university of Medical Sciences, Shiraz, Iran; 3Department of Dermatology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 4Department of Surgery/Division of Plastic Surgery, University of British Columbia, Vancouver, BC, Canada; 5Faculty of Medicine, Medical Genetics, University of British Columbia, Vancouver, BC, Canada

Background and Hypothesis: Cutaneous leishmaniasis (CL) is an infectious disease caused by different species of leishmania parasite, resulting in a variety of chronic skin lesions that leave severe scarring and disfigurement. Granzyme B (GzmB), a serine protease, has been implicated in pathogenesis of CL caused by Leishmania braziliensis. Although the cytotoxic role of intracellular GzmB in parasite killing and targeted apoptosis in CL is well known, the contribution of extracellular GzmB to chronic inflammation, tissue damage, and scarring seen in CL is poorly understood. Here, we hypothesized that GzmB is elevated in CL caused by Leishmania major (L. major) and contributes to impaired healing through the cleavage of cell-cell and cell-basement membrane adhesion proteins.
Methods: Paraffin-embedded human skin biopsy specimens with confirmed diagnosis of L. major induced CL were subjected to the histological analysis using hematoxylin& eosin, immunohistochemistry (IHC), and immunofluorescent staining for GzmB, cellular markers of different immune cells, and GzmB substrates. Cell-free in vitro cleavage assay was used to identify new GzmB substrates. Twelve wild type and 11 GzmB knockout C57BL/6 mice were inoculated with L. major and the clinical course of the disease was documented over 8 weeks. Skin samples were collected for histological analysis at study endpoint.
Results: GzmB was highly expressed in human CL lesions compared to normal skin. The expression of E-cadherin, a cell-cell junction protein was significantly reduced in areas with higher number of GzmB expressing cells (n=11, p<0.001). Collagen VII and collagen XVII, were also reduced in areas with accumulation of GzmB+ cells. Cell-free in vitro cleavage assay resulted in identification of two novel substrates of GzmB, desmoglein 4 and annexin A2. Further, IHC staining of human samples showed reduction of desmoglein 4 in samples demonstrating psoriasiform dermatitis, and annexin A2 on endothelial cells of thrombotic blood vessels. In vivo study revealed that while 54% of GzmB KO mice showed no visible lesion/scarring, all WT mice still had clinical lesion/scarring at the study end point.
Conclusion: Our results show that extracellular GzmB is associated with epidermal changes and vascular thrombosis in L. major induced CL and deletion of GzmB significantly reduces the healing time of the lesions in a murine model of CL. Collectively, our findings suggest a prominent role for extracellular GzmB in pathogenesis of skin lesions caused by L. major.

K2.02

Direct Contact With Mechanically Activated Myofibroblasts Drives Macrophages Into Distinct Transcriptional And Functional States
Li Diao1, Ronen Schuster2, Fereshteh Younesi1, Boris Hinz1
1LTRR, Unity Health Toronto, Toronto, ON, Canada. 2CytoReason, Tel Aviv, Israel
Direct Contact With Mechanically Activated Myofibroblasts Drives Macrophages Into Distinct Transcriptional And Functional States

Li Diao1, Ronen Schuster2, Fereshteh Younesi1, Boris Hinz1 1LTRR, Unity Health Toronto, Toronto, ON, Canada; 2CytoReason, Tel Aviv, Israel

Background: Both fibroblasts and macrophages (MΦ) are key in promoting the formation and remodeling of extracellular matrix following organ injury, but aberrant crosstalk can contribute to the development of fibrosis. MΦ provide cytokines like TGF-β1 that stimulate fibroblast activation into contractile myofibroblasts (MF). We have published that MF activation by MΦ-derived TGF-β1 requires spatial proximity and a ‘scar-stiff’ tissue environment. However, little is known how, in turn, MFs control MΦ phenotypes in the context of tissue repair and fibrosis. We hypothesized that mechanically activated MFs control distinct MΦ states in contact–dependent signaling processes.
Methods: MΦ were obtained by treating mouse bone marrow-derived monocytes with M-CSF in vitro for 5 d. Subcutaneous fibroblasts were isolated from Col1a-GFP reporter mice. To mechanically establish fibroblast and MF populations, fibroblasts were cultured on skin-soft or scar-stiff gelatin-coated silicone substrates for 2 passages, respectively. MΦ were then co-cultured for 3 d with fibroblasts/MFs on the respective substrates in setups that allowed either direct contact or medium-sharing only. Cells obtained from all experimental combinations were separately analyzed using immunofluorescence (IF) confocal microscopy and flow cytometry. Fibroblastic cells and MΦ were flow-sorted for subsequent RNA sequencing, further analyzed for principal components, differentially expressed genes and enrichment of signaling pathways and transcription factors binding motifs.
Results: Fibroblasts cultured alone on stiff substrates exhibit MF protein and RNA profiles absent from soft-cultured fibroblasts. Substrate stiffness in the chosen range does not affect RNA profiles of MΦ in monoculture. Conversely, co-culture with fibroblastic cells results in significant changes in MΦ transcriptomes, with unique features depending on the activation state of the co-cultured fibroblasts and the ability to form direct contact. Specifically, (1) MΦ in direct but not medium-shared-only co-culture with fibroblastic cells acquire an activated MAPK signaling. (2) Direct contact with fibroblasts results in suppression of stress response to stimuli and inter- and intra-cellular signal transduction. (3) In direct contact with MFs, MΦ exhibit upregulated pro-fibrotic signaling pathways, mediated by the activation of sema-plexin-GTPase axis and IL-17, NF-kB, and C-type lectin signaling. IF and flow cytometry validate RNA sequencing data. For instance, MΦ exhibit up to 1.6-fold significantly increased expression of CD206 in MΦ co-cultured with MFs versus fibroblasts. Increased expression of MΦ CD206 is 68-fold more pronounced in co-cultures with direct contact.
Conclusion: Direct contact with MFs generate a unique MΦ polarization state. The recognition of such a state offers novel therapeutic targets and potential for the prevention and treatment of fibrosis.

K2.03

The Role of Periostin and Hyaluronan Crosstalk in the Regulation of Wound Fibrosis
Sonya Keswani, Tanuj J. Prajapati, Hui Li, Ling Yu, Swathi Balaji
Baylor College of Medicine, Houston, TX, United States
The Role of Periostin and Hyaluronan Crosstalk in the Regulation of Wound Fibrosis

Sonya Keswani, Tanuj J. Prajapati, Hui Li, Ling Yu, Swathi Balaji Baylor College of Medicine, Houston, TX

Postnatal dermal injury triggers inflammatory and fibrogenic reactions involving feedback signals between ECM mediators that contribute to either wound regeneration or fibrosis. We identified a role for high molecular weight hyaluronan (HMW-HA) in promoting wound regeneration. Upon injury, HA and periostin (POSTN), a matricellular protein, are upregulated. However, HMW-HA expression is not sustained and quickly tapers down in postnatal wounds, while POSTN is sustained. We hypothesize that HA and POSTN crosstalk regulate ECM balance and wound fibrosis.
In vitro, murine dermal fibroblasts (mdF) from C57BL/6J mice were stimulated with TGFβ for 24h to promote fibrogenic milieu. HA and POSTN crosstalk was analyzed using gain- and loss-of-function of HA and POSTN signaling. The effect of siRNA knockdown of POSTN on HA synthesis and HA degradation enzymes was assessed. In vivo, 6mm full-thickness stented wounds in C57BL/6J (8wk;n=3) mice were studied with +/- lentiviral overexpression of HMW-HA, and the effect on HA and POSTN expression at d7 and d28 were examined along with changes in collagen and ECM architecture. Human dermal fibroblasts (hdF) from patients with low(LS) and high scar(HS) phenotypes clinically stratified with VSS <3 vs. >6 were tested. p values by ANOVA.
TGFβ treatment upregulated both HA-synthase (HAS2) that synthesizes HMW-HA and POSTN gene expression in mdF. To determine if POSTN regulates HAS2 in this model, we knockdown POSTN using siRNA, which significantly reduced the expression of HAS2 (p<.05). Further, mdF treated with 50ng/ml POSTN for 48h showed significantly higher mRNA levels of HAS1 and 2 than controls. To further assess the impact of HAS2 on a reciprocal feedback mechanism that regulates POSTN, HAS2-specific siRNA knockdown was done, which caused a significant increase in POSTN mRNA and protein. In vivo, POSTN was upregulated upon wounding. HMW-HA induction further increased POSTN at d7 post-wounding, but reduced its expression compared to PBS wounds at d28 of the remodeling stage which coincided with reduced collagen and improved ECM architecture in HMW-HA induced wounds at d28. This suggests that sustained HMW-HA production could counter POSTN expression. It is cogent that when HAS2 is low, POSTN expression is upregulated, but when HAS2 expression increases, POSTN is inhibited. Treatment of patient skin fibroblasts LS-hdF vs. HS-hdF with POSTN resulted in differential regulation of HAS2 expression, indicating that each subject responded to POSTN differently, which may contribute to the heterogeneity observed in human wound healing.
Our data suggest a mutual HAS2/POSTN interdependence. While injury-induced POSTN is necessary for HAS2 signal induction, the HAS2/HMW-HA then suppresses POSTN to promote wound regeneration. Understanding these basic mechanisms will facilitate improved wound healing and lead to regenerative tissue repair.

K2.04

Microfibril Associated Protein 5 And The Regulation Of Skin Scar Formation
Chen Han1, Heidi Yuan1, Lin Chen1, Timothy J. Koh1, Robert P. Mecham2, Luisa A. DiPietro1
1Center for Wound Healing and Tissue Regeneration, University of Illinois College of Medicine at Chicago, Chicago, IL, United States 2Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, United States
Microfibril Associated Protein 5 And The Regulation Of Skin Scar Formation

Chen Han1, Heidi Yuan1, Lin Chen1, Timothy J. Koh1, Robert P. Mecham2, Luisa A. DiPietro1 1Center for Wound Healing and Tissue Regeneration, University of Illinois College of Medicine at Chicago, Chicago, IL; 2Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO

Background: Studies in our lab have shown that Microfibril Associated Protein 5 (MFAP5, or microfibril-associated glycoprotein2/MAGP2) is upregulated in skin wound healing and modulates fibroblast phenotype. MFAP5, a 25 kD extracellular matrix (ECM) glycoprotein, is linked to fibrosis and angiogenesis in cancers and fibrotic diseases. The aim of this study was to use MFAP5 deficient (Mfap5-/-) mice to investigate MFAP5’s role in wound healing and on fibroblast transcriptome and function.
Materials & Methods: Full-thickness excisional wounds were made on dorsal skin of Mfap5-/- and C57BL/6J control (Mfap5+/+) mice. External wound closure was assessed, and wound tissue was collected during healing to assess key features of wound repair (N=9-10). Wound angiogenesis and inflammatory cell content were assessed by immunofluorescent staining of CD31 or Ly6G and CD68, respectively. Collagen deposition and maturity were assessed by Masson’s Trichrome and Herovici staining, respectively. To examine how loss of MFAP5 affects fibroblast transcriptome and phenotype, skin fibroblasts were isolated from neonatal Mfap5+/+ and Mfap5-/- mice. RNA-sequencing was performed on Mfap5+/+ and Mfap5-/- fibroblasts (N=6). All significantly downregulated genes (padj<0.05) in Mfap5-/- versus Mfap5+/+ fibroblasts underwent gene ontology enrichment analysis and annotated to biological processes (BP). Cellular migration, contractility, and proliferation were compared between Mfap5+/+ and Mfap5-/- fibroblasts (N=14-20). To examine changes to ECM composition, Mfap5+/+ and Mfap5-/- fibroblasts (N=8) were grown to confluency, treated with ascorbic acid, and then immunocytochemistry stained for ECM proteins.
Results: Mfap5-/- mice had significantly reduced rates of skin wound closure and wound angiogenesis (p<0.05). Mfap5-/- mice also had significantly enhanced inflammatory cell content as compared to Mfap5+/+ mice (p<0.05). Collagen deposition in Mfap5-/- mice was significantly reduced in normal skin (NS) and at 21 days post-wounding, while only NS of Mfap5-/- mice had significantly altered collagen maturity versus Mfap5+/+ mice (p<0.05). RNA-sequencing revealed down-regulated BP related to ECM organization, cellular migration, and proliferation. Mfap5-/- fibroblasts had significantly reduced cellular migration, contractility, proliferation, and COL1A2 deposition (p<0.05).
Conclusions: Our results suggest that MFAP5 is important for wound closure and angiogenesis. MFAP5 is also likely involved in the maturation and organization of collagen and may influence skin wound inflammation. Lastly, our in vitro assays demonstrate that MFAP5 is a regulator of fibroblast characteristics important for scar formation.
Funding: R01 GM050875, R35 GM139603, F31 DE028747, F31 AR082287.

K2.05

Elucidating the Role of sFRP2 in Modulating Organ Fibrosis
Delany Bradford1, Pampee Young2, Sarika Saraswati1
1Tennessee State University, Nashville, TN, United States 2American Red Cross, Nashville, TN, United States
Elucidating the Role of sFRP2 in Modulating Organ Fibrosis

Delany Bradford1, Pampee Young2, Sarika Saraswati1 1Tennessee State University, Nashville, TN; 2American Red Cross, Nashville, TN

Background: Fibrosis is the typical response to injury, which leads to distorted architecture, pathologic signaling and ultimately organ dysfunction. In cardiac tissue specifically, a fibrotic response to injury can lead to a decrease in the heart’s ability to function, which plays a significant role in the pathogenesis of most heart diseases. Secreted frizzled-related protein (sFRP2) has been identified as a mesenchyme derived factor that augments post-myocardial infarction repair, in part by down-regulating fibrosis. Yet, the molecular mechanism that regulates sFRP2’s effect on fibroblasts in modulating tissue fibrosis is incompletely understood.
Methods: We have generated a transgenic mouse model in which we can temporally and spatially regulate the expression of sFRP2 in injury-induced activated FSP1+ fibroblasts. These transgenic mice received bone marrow transplantation (BMT) from C57Bl/6 mice to ensure specificity of sFRP2 protein in fibroblasts, since FSP1 is also expressed in hematopoietic cells. sFRP2 expression was induced post-infarct following tamoxifen treatment in mice expressing sFRP2 under FSP1 promoter. These mice were assessed in vivo and in vitro for injury-induced fibrotic responses in two distinct models, heart and skin.
Results: Post-injury induction of sFRP2 exerted an anti-fibrotic effect in comparison to control Cre mice in heart as well as skin. sFRP2 overexpression in heart following myocardial infarction resulted in reduced scar size, improved function, and reduced adverse cardiac remodeling. In addition, assessment of the collagen content following excisional wound (skin injury) demonstrated significant reduction in dermal collagen deposition in sFRP2 overexpressing transgenic mice. Significant reduction in Wnt signaling, TGF beta signaling and collagen production was identified in mice overexpressing sFRP2.
Conclusion: Post-injury sFRP2 over-expression in mouse FSP1-fibroblasts resulted in reduction in fibrosis in two different organ injury models. Elucidating the mechanism that modulate sFRP2’s antifibrotic role will provide valuable insights on how this protein targets fibrosis and promotes regenerative repair. Identification of sFRP2’s role will also provide a potential use for such regulators as a way to target specific post-injury fibrotic processes such as Wnt and TGF beta signaling and inhibit pathological fibrosis without interfering with normal wound healing.

K2.06

Divergent Contributions Of Systemic Immune Cells And Local Fibroblasts To Wound Closure And Fibrosis
Andrew Hostler, William Hahn, Jenne Stensland, Katharina Fischer, Maria Gracia Mora Pinos, Jared S. Holley, Abdelrahman Alsharif, Jonathan P. Yasmeh, Fidel Saenz, Autumn Lester, Hudson C. Kussie, Eamonn McKenna, Maia Granoski, Amelia B. Knoch
Surgery, University of Arizona College of Medicine -Tucson, Tucson, AZ, United States
Divergent Contributions Of Systemic Immune Cells And Local Fibroblasts To Wound Closure And Fibrosis

Andrew Hostler, William Hahn, Jenne Stensland, Katharina Fischer, Maria Gracia Mora Pinos, Jared S. Holley, Abdelrahman Alsharif, Jonathan P. Yasmeh, Fidel Saenz, Autumn Lester, Hudson C. Kussie, Eamonn McKenna, Maia Granoski, Amelia B. Knochel, maisam Jafri, Jose Vasquez, Geoffrey C. Gurtner, Kellen Chen Surgery, University of Arizona College of Medicine -Tucson, Tucson, AZ

Background: Tissue repair is a dynamic process requiring various cellular populations and non-cellular elements coalescing to restore functional tissue integrity. Chronological aging has been associated with impaired physiological wound healing via diminished inflammatory and fibroblast cells. While we know that impaired immune cells and dysfunctional fibroblasts impede wound healing within aged populations, limited evidence exists isolating the importance of one cell type over the other. To further characterize the age-related cellular mechanisms of wound healing, we developed a novel skin graft murine model to isolate the effects of impaired, aged systemic immune cells versus dysfunctional local fibroblasts.
Methods: We established a novel heterochronic full-thickness skin graft (FTSG) murine model utilizing both 8-week (young) and 80-week (old) C57BL/6J mice. We harvested FTSGs to transplant old skin (OS) onto the dorsum of young mice (Y+OS). Conversely, the harvested young skin (YS) was transplanted onto the backs of old mice (O+YS). Young mice receiving young mouse skin transplants were used as controls (CO). Additionally, unwounded skin also analyzed (US). Upon FTSG integration (40 days), we utilized our splinted excisional wounding model to create two cutaneous, dorsal wounds at the graft region. Gross wound size was measured to quantify wound closure rate, and healed wound tissue was explanted at postoperative day (POD) 20. Hematoxylin & Eosin (H&E), Masson’s Trichrome, and Picrosirius Red staining was performed to access epidermal and dermal thickness, dermal collagen density, and dermal integrity.
Results: Control and Y+OS mice healed at around POD 15.5 while O+YS took up to POD 20 (p=0.0001). The rate of wound closure was significantly decreased in O+YS mice compared to Y+OS mice on PODs 6 (p=0.007), 8 (p=0.005), 10 (p=0.003), 12 (p<0.0001), 14 (p=0.002), 16 (p=0.02), and 18 (p=0.03). Subsequently, the rate of wound closure was significantly decreased in O+YS mice compared to control mice on POD10 (p=0.005), 12 (p<0.0001), 14 (p=0.0002), 16 (p=0.006), and 18 (p=0.02). Softwares CT-FIRE and CurveAlign were used to assess collagen fiber structure. We observed that CO and O+YS significantly increased collagen density (p<0.001) compared to US while Y+OS did not demonstrate an increase.
Conclusions: Our results demonstrate an intriguing divergence of immune and fibroblast contributions during the process of wound healing and fibrosis. Regardless of the transplanted skin, young mice demonstrate accelerated wound closure, thus showing that the young circulating immune cells primarily contribute to the wound healing process as opposed to local cells. In contrast, regardless of the age of the mouse, mice receiving young skin demonstrated more fibrotic skin after injury, demonstrating that the old local fibroblasts dominate the fibrotic response over the systemic contributions.

K3.01

Single-Cell RNA-Sequencing Identifies Novel Molecular Targets of Endothelial MicroRNA-200b in the Diabetic Ischemic Wound
Kanhaiya Singh, Manishekhar Kumar, Sujit K. Mohanty, Savita Khanna, Sashwati Roy, Chandan K. Sen
McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, United States
Single-Cell RNA-Sequencing Identifies Novel Molecular Targets of Endothelial MicroRNA-200b in the Diabetic Ischemic Wound

Kanhaiya Singh, Manishekhar Kumar, Sujit K. Mohanty, Savita Khanna, Sashwati Roy, Chandan K. Sen McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA

Injury-induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis, but the underlying mechanisms remain unclear. Single cell RNA sequencing was performed on 15000 human endothelial cells under high (mimic treated) miR-200b conditions. Unsupervised clustering using CellRanger identified five cell clusters, three of which were responsive to changes in miR-200b abundance. Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based proteomic analysis was employed to look for novel potential targets of endothelial miR-200b. A total of 3818 proteins were detected which were then filtered using statistical cut-offs (p value < 0.05; % change > 10%) identifying 319 proteins targeted by miR-200b. To determine the potential role of these candidates in regulating vascular function, miR-200b-429fl/fl-Tie2 Cre mice were generated where endothelial miR-200b levels could be specifically depleted in vivo. The therapeutic significance of endothelial miR-200b inhibition was studied using ischemic hindlimb in these mice. The significance of miR-200b depletion was also studied in streptozotocin-induced diabetic miR-200b-429fl/fl-Tie2 Cre mice. Perfusion imaging was performed using Laser Speckle imaging (Perimed Inc.) at different time-points (d3, d7, d10, d14). Inhibition of endothelial miR-200b rescued hindlimb ischemia with improved perfusion (n=11). Such effect of miR-200b inhibition on rescuing HL-ischemia was markedly increased in diabetic animals (n =8). Such rescue was associated with increased abundance of CD31+/vWF+ vasculogenic cells (n=6). This work identified miR-200b regulated endothelial cell clusters, the functional significance of which have been experimentally validated. These findings provide insight into novel mechanisms explaining how transient inhibition of miR-200b in the endothelial compartment improves angiogenic outcomes in the diabetic ischemic limb.

K3.02

Tissue Nanotransfection Based Endothelial-Targeted Epigenetic Gene Editing In Vivo To Rescue Diabetic Ischemic Wounds
Sumit S. Verma, Surya Gnyawali, Chandan K. Sen, Kanhaiya Singh
Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States
Tissue Nanotransfection Based Endothelial-Targeted Epigenetic Gene Editing In Vivo To Rescue Diabetic Ischemic Wounds

Sumit S. Verma, Surya Gnyawali, Chandan K. Sen, Kanhaiya Singh Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA

Our previous work (PMID: 35192691) has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. Hyperglycemia caused hypermethylation of endothelial specific gene promoter. Given that epigenetic changes are reversible, this work tests the significance of gene-targeted therapeutic DNA demethylation in improving blood flow to diabetic ischemic wounds. Bipedicle ischemic wounds were placed in streptozotocin (STZ) induced acute diabetic C57BL6 mice. Diabetic VWF+ dermal endothelial cells were isolated using flow sorting. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. Next, to specifically demethylate PLCγ2 promoter in endothelial cells, a demethylation cocktail was designed: (i) (scFv)-TET1 catalytic domain (TET1CD) system capable of inducing targeted DNA methylation, and (ii) endothelial promoter driven gene specific guide RNAs. This demethylation cocktail was delivered at the diabetic ischemic wound-edge employing non-viral topical tissue nano-transfection (TNT) technology. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging (Perimed Inc.) and ultrasonography (Vevo 2100) at different time-points post-surgery (days 3, 7&10). Overall DNA hyper methylation was prominent in murine ischemic flaps as demonstrated by the increased ratio of 5-methylcytosine (5-mc, methylation mark) to 5-hydroxymethylcytosine (5-hmC, demethylation mark) (n = 5). Specifically, the PLCγ2 promoter was hypermethylated (n=5). TNT mediated endothelial-targeted demethylation of the PLCγ2 promoter increased the expression of this gene in endothelial cells (n=4). Such demethylation-based upregulation of PLCγ2 improved wound tissue blood flow with increased abundance of VWF+/PLCG2+ vascular elements (n=4). Taken together, topical TNT-based endothelial demethylation of the PLCγ2 gene promoter improved perfusion of cutaneous diabetic wounds resulting in improved closure.

K3.03

AMPK and Rac1 Activity Regulation Promotes Wound Healing via Induction of Actin Cable Formation
Kento Takaya1, Yuka Imbe2, Qi Wang2, Shigeki Sakai1, Keisuke Okabe1, Noriko Aramaki-Hattori1, Kazuo Kishi
1Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan. 2Faculty of Pharmacy, Keio University, Minato-ku, Tokyo, Japan
AMPK and Rac1 Activity Regulation Promotes Wound Healing via Induction of Actin Cable Formation

Kento Takaya1, Yuka Imbe2, Qi Wang2, Shigeki Sakai1, Keisuke Okabe1, Noriko Aramaki-Hattori1, Kazuo Kishi1 1Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan; 2Faculty of Pharmacy, Keio University, Minato-ku, Tokyo, Japan

Background: Unlike adults, early developing fetuses can completely regenerate tissue, and replicating this could lead to developing treatments to reduce scarring. Mice epidermal structures, including texture patterns, are regenerated until embryonic day (E) 13, leaving visible scars thereafter. Although the formation of actin cables at the wound margin and epidermal cell migration are known to be involved in this transition, the detailed mechanism remains unclear. We focused on AMP-activated protein kinase (AMPK) and Rac1, factors involved in regulating cell migration and actin dynamics, and investigated their effects on skin regeneration through regulation of AMPK and Rac1 activity using a unique mouse fetal wound healing model.
Methods: (1) Regulation of Rac1 activity: The mouse epidermal cell line PAM212 was treated with the Rac1 inhibitor NSC23766 and the effect on migration ability was evaluated by scratch assay. We generated genetically engineered mice (K14-CreERT2;Rac1flox/flox) that can suppress epidermis-specific Rac1 cell migration and observed the wound healing process and actin dynamics in fetuses and adults. (2) Regulation of AMPK activity: Keratinocytes were established from fetal mouse tissues and treated with AMPK activator salicylate, and the effect on migration ability was evaluated by scratch assay. Embryos of ICR mice E13, E14, and E15 were wounded and salicylate was administered into the amniotic fluid and collected at multiple time points. Wound morphology was analyzed by 3D reconstruction of the wound images, and the presence of actin cable formation and the behavior of related molecules were observed.
Results: Keratinocyte migration was suppressed by NSC23766 and salicylate administration. In a mouse model in which epidermis-specific Rac1 can be knocked out conditionally, actin cable-like stress fiber formation occurred in the epidermis of wounds after E14. In salicylate-treated fetal wounds, significant improvement in wound area and depth was observed at E14 and E15, and AMPK was activated to induce actin cable formation during wound healing, a process that normally disappears.
Conclusion: The actin cables are involved in the complete regeneration of skin, and the induction of actin cable formation through AMPK activation and Rac1 regulation was observed to induce skin regeneration or promote healing. This finding suggests that regulation of AMPK and Rac1 may be a candidate therapeutic approach to improve the wound healing process.

K3.04

A Novel Ex Vivo Human Fascio cutaneous Flap Perfusion Model to Investigate Skin Injuries
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

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. Samples were collected from the wounded and control tissue for histology, protein, and gene expression analysis.
Angiography verified that the SIEA to SIEV flap system successfully fed 90% surface area of a large flap for two-three weeks. 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 viable system can help us understand the pathways and be used as a subclinical drug testing model.

K3.05

Subcutaneous Injection Of Zein, A Dietary Protein, Has Positive Wound Healing Effects That Are Prevented By Fingolimod (Fty720) Treatment
Isabela Beatriz C. Nóbrega, Angélica Vitória S. Andrade, Geraldo Magela Azevedo, Claudia R. Carvalho
Morphology, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Subcutaneous Injection Of Zein, A Dietary Protein, Has Positive Wound Healing Effects That Are Prevented By Fingolimod (Fty720) Treatment

Isabela Beatriz C. Nobrega, Angélica Vitória S. Andrade, Geraldo Magela Azevedo, Claudia R. Carvalho Morphology, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

Pathological scars resulting from impairment or modification in wound healing are serious problems for patients’ health and strongly impact the financial cost of their treatment. Pathological scars, such as hypertrophic scars and keloids, are common even in cases of scheduled surgery. Exacerbated and prolonged inflammation is an important factor in the occurrence of wound healing problems. The search for more effective forms of intervention that prevent pathological scars requires a better understanding of the cellular interactions that occur during repair. Previous work has shown that parenteral injection of dietary proteins, concomitant with skin lesions in mice, reduces the inflammatory infiltrate in the wound bed and improves healing. Zein is a corn protein present in mouse food and one injection of zein in Al(OH)3 concomitantly with skin wounds reduces the inflammatory infiltrate, increases the number of T lymphocytes in the wound bed and improves healing (https://doi.org/10.1590/1414-431X2021e11735). To evaluate whether the positive effects of zein injection on the repair of skin lesions depend on the circulation of lymphocytes, we used FTY720 (Fingolimod), a drug that sequesters lymphocytes in lymph nodes. Male C57BL/6 mice, 8 weeks old, received intraperitoneal injections of 1mg/kg of FTY720 two days before injury, on the day of injury and two days after skin injury (CEUA/UFMG protocol 253/2021). Two lesions on the back skin were made with a 6mm dermatological punch and the animals in the experimental group also received one subcutaneous injection of 10 microgram of zein in 1.6 mg of Al(OH)3, at the base of the tail. Skin analyzes performed at 7 and 40 days after the injuries showed that, in the absence of FTY720, the injection of zein concomitantly with the injuries reduced the inflammatory infiltrate, increased the number of T lymphocytes in the wound bed and improved the organization of the extracellular matrix in the neodermis. At 7 days after the injuries, it was noted that the injection of FTY720 delayed the detachment of the wound crust and increased the number of leukocytes (CD45+), neutrophils (Ly6G+) and macrophages (F4/80+) in the wound bed. Furthermore, FTY720 treatment altered the morphological characteristics of fibroblasts seen after H&E staining, suggesting greater activation of these cells. At 40 days after the injuries, the neodermis of animals that received injection of zein showed an organization of the extracellular matrix similar to that of intact skin but, on the contrary, those that received FTY720 and zein showed a larger scar, with thinner fibers organized parallel to the epidermis, with characteristics of a loose matrix and larger scar. In conclusion, FTY720 prevented the increase of T lymphocytes in the wound bed and the positive effects of zein on wound healing, showing that the positive effects of zein depend on circulating lymphocytes.

K3.06

Changes In The Catecholamine Adrenergic Network Support Wound Healing In Healthy Pigs
Anthony Gallegos3, Ksenia Zlobina1, Hsin-ya Yang3, Moyasar A. Alhamo3, Athena Soulika3, Mo Siadat3, Marcella Gomez1, Marco Rolandi2, Rivkah Isseroff3
1Applied Mathematics, UC Santa Cruz, Santa Cruz, CA, United States 2Electrical and Computer Engineering, UC Santa Cruz, Santa Cruz, CA, United States 3Dermatology, UC Davis, Sacramento, CA, United States
Changes In The Catecholamine Adrenergic Network Support Wound Healing In Healthy Pigs

Anthony Gallegos3, Ksenia Zlobina1, Hsin-ya Yang3, Moyasar A. Alhamo3, Athena Soulika3, Mo Siadat3, Marcella Gomez1, Marco Rolandi2, Rivkah Isseroff3 1Applied Mathematics, UC Santa Cruz, Santa Cruz, CA; 2Electrical and Computer Engineering, UC Santa Cruz, Santa Cruz, CA; 3Dermatology, UC Davis, Sacramento, CA

Predictable series of chemical and biological changes in each wound stage propel the healing process forwards. The catecholamines norepinephrine (NOR), epinephrine (EPI), and dopamine (DOP) are small molecule neurotransmitters present in the wound that can affect healing. Previously, we showed that physiological levels of EPI activate the alpha 2b adrenergic receptors (A2AR) on keratinocytes and accelerate their migration in-vitro, while activation of beta 2 adrenergic receptors (B2AR) by supra-physiological levels decreased their migration (Yang et al., 2021). Thus, either changes in the wound concentration of epinephrine or in alpha/beta receptor expression may affect re-epithelialization. Here we examined catecholamines and related gene expression in the wound environment during normal wound healing in porcine skin wounds. Twelve full-thickness 20 mm diameter circular wounds were placed on 6 pigs and blood and wound tissue were sampled at intervals over 21 days. Wound outcomes were imaging, tissue RNASeq and histology, and reverse phase HPLC analysis of serum catecholamines. Wound image area and histologic re-epithelialization demonstrated that most wounds healed by days 16-21. Expression of the alpha 2a AR (ADRA2A) was immediately decreased following wounding and remained so through day 7 of healing; expression was increased on days 9 through day 21, peaking at day 16. Expression of the alpha 2b AR (ADRA2B) was bi-modal with peaks on days 2 and 13, and a gradual return to baseline on intervening days. Beta 2 AR (ADRB2) expression increased after wounding, peaking at day 2 and gradually returning to baseline by day 6 through day 21. Expression of dopa decarboxylase (DDC) only began to increase on day 7 of healing, peaking on day 11 and returning to baseline by day 21. DDC is essential to convert L-dopa to DOP, a known mediator of healing. Serum DOP remained at baseline through day 11 and then increased significantly through day 21, concurrent with elevated DDC expression in the wound. The concurrent peaking of DDC, ADRA2A, and ADRA2B suggest their role in enhancing keratinocyte migration in the latter half of the healing process. Serum EPI levels increased immediately after wounding and remained elevated through day 7, gradually returning to baseline by day 12, but effects could be mitigated by the concurrent increase of degrading enzyme monoamine oxidase B (MAOB) in the wound tissue. Likewise, the elevated expression of solute carrier family 6 a2 (SLC6A2, responsible for NOR reuptake) in the first half of the healing process could abrogate the anti-migratory effects of elevated ADRB2 expression in early healing. Taken together, these results show how the complex dynamics of the adrenergic network in the skin change during normal wound healing to support wound closure.

K4.01

Type III Collagen Biomaterials Improve Cutaneous Wound Healing In Diabetic Mice
Daniel C. Stewart1, Yasumasa Iimori1, Becky K. Brisson1, William Yen1, David Chenoweth2, Claudia Loebel3, Jason Burdick4, Susan W. Volk1
1School of Veterinary Medicine , University of Pennsylvania, Philadelphia, PA, United States 2College of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, United States 3College of Engineering, University of Michigan, Ann Arbor, MI, United States 4College of Engineering and Applied Science, University of Colorado Boulder, Boulder, CO, United States
Type III Collagen Biomaterials Improve Cutaneous Wound Healing In Diabetic Mice

Daniel C. Stewart1, Yasumasa Iimori1, Becky K. Brisson1, William Yen1, David Chenoweth2, Claudia Loebel3, Jason Burdick4, Susan W. Volk1 1School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; 2College of Arts and Sciences, University of Pennsylvania, Philadelphia, PA; 3College of Engineering, University of Michigan, Ann Arbor, MI; 4College of Engineering and Applied Science, University of Colorado Boulder, Boulder, CO

Background: Dysregulated collagen production and proteolytic degradation of collagen in diabetic wounds is well known to contribute to impaired healing in diabetic patients. Altered dynamic reciprocity between cells in the healing microenvironment in turn leads to altered epithelial, stromal, and immune cell responses after injury. Based on our previous work showing a vulnerary and pro-regenerative role for type III collagen (Col3) in acute wound repair, we hypothesized that delivery of Col3 to diabetic wounds could improve efficiency and quality of repair in a diabetic mouse model. Furthermore, we have developed a proteolytic-resistant Col3 (aza-Col3) which we hypothesize will further improve efficacy of Col3 biomaterials in diabetic wound healing.
Methods: Two 8 mm full-thickness excisional wounds were made paramedian on the dorsum of LepRdb/db (db/db) mice at 14-16 weeks of age. Shear-thinning, self-healing hydrogels were utilized to deliver rhCol3 (200 μg) or vehicle to the wound bed at the time of wounding. Wounds were collected and gross wound closure was assessed at 7-, 10-, and 14-days post-wounding (DPW). Histomorphometry was determined for total re-epithelialized distance (RE), epidermal gap (EG), and granulation tissue (GT) area on H&E sections. Immune responses were characterized by immunohistochemical staining for MPO and F4/80 (neutrophils and macrophages, respectively). rhCol3 degradation with or without aza-glycine peptides (aza-Col3) was assessed with circular dichroism (CD) spectroscopy in the presence of collagenases.
Results: We observed that Col3 biomaterial-containing wounds had improved wound healing as evidenced by increased RE at 7, 10, and 14 DPW compared to control biomaterial wounds (p<0.01 for each timepoint); decreased EG at 7 and 14 DPW (p<0.05); and increased GT formation at 10 DPW (p<0.05). At 10 DPW, Col3 biomaterial wounds had decreased neutrophil (MPO+ cells) and macrophage (F4/80+ cells) infiltration compared to controls (p<0.05 each). CD spectra revealed increased resistance to degradation in aza-Col3 samples dose-dependently with increasing aza-glycine concentration compared to rhCol3 alone.
Conclusions: Our results demonstrate that Col3 improves wound healing outcomes in a diabetic mouse model through its effects on re-epithelization, GT formation, and immune response. While the incorporation of Col3 alone improved wound healing outcomes, preliminary data also indicate that incorporation of aza-peptides with Col3 prevents degradation in the presence of collagenases and suggest that aza-Col3 biomaterials may further improve healing outcomes in diabetic wounds.

K4.02

Intravesicular Cytokine Profiling Of Stage Iv Pressure Ulcers Treated With NPWT vs. NPWT and Porcine Extracellular Matrix Dressing
Lauren Fang1, Richard Simman2
1University of Toledo College of Medicine, Monroe, OH, United States 2ProMedica-Jobst Vascular Institute, Toledo, OH, United States
Intravesicular Cytokine Profiling Of Stage Iv Pressure Ulcers Treated With Npwt vs. Npwt and Porcine Extracellular Matrix Dressing

Lauren Fang1, Richard Simman2 1University of Toledo College of Medicine, Monroe, OH; 2ProMedica-Jobst Vascular Institute, Toledo, OH

Background: Extracellular vesicles (EVs) are involved in all phases of wound healing. They carry cytokines, which possess a wide range of pro-inflammatory and pro-healing functions. While inflammation is critical in wound healing, it must be resolved so that wounds can progress to proliferation and remodeling. Otherwise, wounds become chronic. The purpose of this study is to analyze intravesicular cytokines in wound fluid to understand how healing and non-healing wounds behave at the molecular level when treated with negative pressure wound therapy (NPWT) versus a combination of NPWT and Porcine Extracellular Matrix dressing (Oasis Ultra).
Methods: Wound fluid samples were obtained from 16 patients with stage IV trunk pressure ulcers. The patients were divided into two groups (n= 8 in each): a control group on NPWT alone and a study group on NPWT plus Oasis Ultra. A canister of patient wound fluid was collected from the NPWT device (wound VAC) every four weeks over the course of the 12-week study. Microvesicles were isolated and analyzed for concentration and content. The following were assayed: growth factors, proinflammatory interleukins and molecules, anti-inflammatory interleukins, and enzymes.
Results: In previous experiments, we found that the overall wound healing rate was significantly higher in the Porcine Extracellular Matrix study group compared to the control group (p< 0.05), as determined by wound size at 12 weeks. Study group wounds also healed faster. In our molecular data analysis, wounds in the study group expressed higher intravesicular pro-healing growth factor concentrations earlier in the study compared to wounds treated with NPWT (control) alone. For example, the intravesicular fibroblast growth factor (FGF) concentration at 4 weeks in the study group was statistically significantly higher than the control group (112.93 pg/mL vs. 28.51 pg/mL, p< 0.01). As wounds progressed toward healing, the EV concentration of proinflammatory molecules such as IL-5, IL-6, IL-8, IL-12, IFN-γ, and TNF-α decreased in the study group over time and were lower than control group levels by 12 weeks. Anti-inflammatory molecules IL-1ra and IL-10 were produced at higher concentrations in the faster healing study group as compared to the NPWT only group.
Conclusion: Wound healing is a regulated process involving both proinflammatory and inflammation-resolving mediators. Given the correlations between our molecular data and prior clinical wound healing data, our results suggest critical interactions between intravesicular cytokines and cells in the actual wound site. To our knowledge, this is the first study to successfully isolate microvesicles containing wound healing specific cytokines at multiple time intervals for a prolonged period. Intravesicular molecular profiling can help characterize wound progression, serving as a wound healing biomarker.

K4.03

Laser Micropatterned Dermal Templates Improved Cultured Epithelial Autograft Handleability And Development In Vivo
Britani Blackstone1, Molly E. Baumann1, Summer Gallentine1, Dorothy Supp3, Kevin Bailey2, Heather Powell1
1Materials Science and Engineering, The Ohio State University, Columbus, OH, United States 2Surgery, Wake Forest University, Winston Salem, NC, United States 3Dermatology, University of Cincinnati, Cincinnati, OH, United States
Laser Micropatterned Dermal Templates Improved Cultured Epithelial Autograft Handleability And Development In Vivo

Britani Blackstone1, Molly E. Baumann1, Summer Gallentine1, Dorothy Supp3, Kevin Bailey2, Heather Powell1 1Materials Science and Engineering, The Ohio State University, Columbus, OH; 2Surgery, Wake Forest University, Winston Salem, NC; 3Dermatology, University of Cincinnati, Cincinnati, OH

The success of cultured epithelial autografts (CEAs) as a treatment for large surface area burns is limited by graft fragility and resulting issues with handleability and blistering. Rete ridges play a significant role in epidermal adhesion; however, they are commonly missing from cultured skin grafts and CEA-treated wounds for up to one year. The purpose of this study was to explore whether a fibroblast-seeded dermal template (DT), fabricated with a dermal papillae-like architecture, could facilitate faster rete ridge development and improve the outcomes of CEA application in vivo. A porcine burn-excise-autograft model was used to establish a viable, partial thickness wound bed for grafting of CEAs alone or in conjunction with a flat (CEA+Flat) or micropatterned (CEA+Ridged) DT. Porcine keratinocytes and fibroblasts were isolated from split-thickness skin grafts from four subjects. Keratinocytes for each pig were cultured for 20 days to form CEAs. After expansion, fibroblasts from all pigs were pooled and seeded onto disinfected, hydrated electrospun collagen scaffolds to form DTs. A fractional carbon dioxide laser was used to micropattern the surface of half of the DTs prior to combination with the CEAs. For 9 weeks, grafts were assessed for contraction, epidermal barrier function, pigmentation, erythema, biomechanics, basement membrane formation and vascularization. Combinatorial use with a DT improved handleability and graft integration at the margins, and speeded restoration of epidermal barrier function by 2 weeks. At 2 weeks post-grafting, increased epidermal proliferation and localization of collagens IV and VII near the dermal-epidermal junction were observed in CEA+Ridged grafts, over CEA alone and CEA+Flat groups. Additionally, rete ridges were observed in all CEA+Ridged grafts from week 2, while these features were less frequent and shallower in CEA alone and CEA+Flat grafts over the course of the study. No differences between the groups were found in graft contraction or post-grafting biomechanics, and a stiffer substrate may better mitigate graft contraction for future use with epidermal sheets or suspensions. Overall, inclusion of a laser micropatterned dermal template improved graft development and decreased variability overall.

K4.04

Sustained Oxygenation And Ros-Scavenging By Lignin Composites Promote Diabetic Wound Healing
Tanuj J. Prajapati1, Lane Yutzy2, Oluyinka Olutoye1, Benjamin Padon1, Walker D. Short1, Fayiz Faruk1, Sonya S. Keswani1, Nabila N. Anika1, Olivia Jung2, Phillip Kogan1, Ling Yu1, Hui Li1, Jangwook Jung2, Swathi Balaji1
1Pediactric Surgery, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX, United States 2Department Of Biological Engineering, Louisiana State University, Baton Rouge, LA, United States
Sustained Oxygenation And Ros-Scavenging By Lignin Composites Promote Diabetic Wound Healing

Tanuj J. Prajapati1, Lane Yutzy2, Oluyinka Olutoye1, Benjamin Padon1, Walker D. Short1, Fayiz Faruk1, Sonya S. Keswani1, Nabila N. Anika1, Olivia Jung2, Phillip Kogan1, Ling Yu1, Hui Li1, Jangwook Jung2, Swathi Balaji1 1Pediactric Surgery, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX; 2Department Of Biological Engineering, Louisiana State University, Baton Rouge, LA

Excessive reactive oxygen species (ROS) potentiate inflammation and impair neovascularization resulting in impaired diabetic wound healing. We engineered novel lignin (a natural antioxidant from lignocellulose)-based composites with ROS-scavenging and oxygen-releasing properties and hypothesized that they enhance neovascularization and attenuate inflammation and fibrosis to promote diabetic wound healing.
Injectable lignin composites were prepared in methacrylated gelatin with test groups including antioxidant nanoparticles (Thiolated Lignosulphonate-TLS), antioxidant with O2 generation via incorporation of CaO2 in the nanoparticles (CPO) and its control (CPOc), and untreated. Full-thickness 6mm stented wounds were made in db/db (8-10 wk, F/M) mice and treated immediately on d0. Wounds were examined for epithelial gap (K14), granulation tissue (H&E), endothelial cells and lumens (CD31), VEGF and HIF1α, leukocytes (Ly6g,CD45) and macrophage (F4/80,CD206, arginase1) at d7 and d14. Weighted Gene Co-expression Network Analysis (WGCNA) was used to perform correlation network analysis on gene sets from a fibrosis PCR array data from dermal fibroblasts cultured on lignin composites. p by ANOVA.
In db/db skin wounds, CPO composites promoted wound closure and granulation tissue deposition (p<.05) and CD31+ capillary lumen density at d7 (p<.01). VEGF expression in wound homogenates was also significantly higher at d7 (p<.05), suggesting CPO composites promote angiogenesis in diabetic wounds.
Interestingly, decreased HIF1α expression was noted in the leading wound epithelium, with reduced expression in wound beds in CPO wounds (p<.05). CPO wounds also had reduced IL-6 (p<.05) and macrophage infiltration while maintaining the highest proportion of CD206 and arginase1 dual stained macrophages that are pro-healing. We then determined the effect of the lignin composite treatment on wound remodeling at d14. Improved healing in CPO wounds was supported by the presence of a robust granulating wound bed (H&E), along with an increase in the CD31+ lumens. ClueGO functional enrichment analysis revealed thyroid stimulating hormone as a key hub gene from fibroblasts cultured in vitro on lignin composites. Since thyroid hormone receptors regulates TGF-b signaling, specifically, the binding of thyroid hormone triiodothyronine (T3) through nuclear receptors regulating the TGF-b/SMAD pathway, our findings indicate a role for lignin composites in governing TGF-b signaling to attenuate fibroblast fibrotic responses. Our data showed that the dual function of antioxidation and oxygen production capacity of the lignin composites improved wound healing associated with enhanced neovascularization and reduce inflammation, representing new frontiers in improving diabetic wound healing by engineered biomaterials.

K4.05

In-Situ Bioprinting With Pro-Reparative Bioinks Improves Impaired Diabetic Wound Healing
Eleftheria Angeliki Valsami1, Seol-Ha Jeong2, zhuqing li1, Enya Wang1, Jihyun Kim2, Lance Fiondella3, Su Ryon Shin2, Aristidis Veves1, Georgios Theocharidis1
1Beth Israel Deaconess Medical Center, Boston, MA, United States 2Brigham and Women’s Hospital, Boston, MA, United States 3Electrical and Computer Engineering, University of Massachusetts- Dartmouth, Boston, MA, United States
In-Situ Bioprinting With Pro-Reparative Bioinks Improves Impaired Diabetic Wound Healing

Eleftheria Angeliki Valsami1, Seol-Ha Jeong2, zhuqing li1, Enya Wang1, Jihyun Kim2, Lance Fiondella3, Su Ryon Shin2, Aristidis Veves1, Georgios Theocharidis1 1Beth Israel Deaconess Medical Center, Boston, MA; 2Brigham and Women’s Hospital, Boston, MA; 3Electrical and Computer Engineering, University of Massachusetts- Dartmouth, Boston, MA

Existing in-situ printing techniques exhibit critical challenges, such as time-consuming calibration and restricted adaptability, limiting their application in clinical settings. We present INSIGHT (INtelligent in_SItu printing Guided by Eye_in Hand robot Technology), an innovative Artificial Intelligence_driven system that identifies arbitrary areas at various angles without predesigned code and calibration procedures. Diabetic foot ulcers is a substantial problem for patients and clinicians. In this study we hypothesized that we could harness advanced bioinks containing oxygen releasing particles and human mesenchymal stem cells (MSCs) to improve wound repair by regulating wound microenvironment and inflammatory response. MSCs are key cells in all phases of wound healing. We performed in vivo and in vitro studies to evaluate INSIGHT’s application of diverse bioinks on diabetic wound healing. 2 wounds were created on the dorsal skin of db/db mice and real_time scanning allowed for automatic generation of printing paths specific to each wound. Three different groups of microgels were studied: Gel, gel with oxygenating microparticles (Gel_OMP), gel with OMP and encapsulated MSCs (Gel_OMP_MSCs). Tegaderm covered wounds served as additional control. 10 wounds per treatment were studied. Wound closure measurements demonstrated better wound healing in Gel_OMP_MSCs group. Masson’s Trichrome staining revealed well_developed granulation tissue and higher cell density within the wound area, indicating advanced healing in microgel_treated groups. Microgel_treated wounds exhibited a significantly increased area of migrating hyperproliferative neoepidermis. RTqPCR gene expression data showed upregulation of markers related to extracellular matrix remodeling in Gel_OMP_MSCs group. Immunofluorescence staining detected higher number of macrophages (MΦ) in Gel_OMP and Gel_OMP_MSCs treated wounds. Significantly lower number of M1 and significantly higher number of M2 MΦ were detected in Gel_OMP_MSCs treated wounds. These findings were also supported by RTqPCR, where Gel_OMP_MSCs showed significantly higher expression of genes for M2 MΦ. In in vitro studies we investigated the effect of these microgels on human monocyte THP_1 cells. RTqPCR results showed that Gel_OMP_MSCs group exhibited significantly higher expression of M2 MΦ genes, that are in accordance with in vivo results. Taken together, these findings suggest that Gel_OMP_MSCs bioink promotes the switch to M2 MΦ in the wound that is critical for the transition to proliferative phase and the accomplishment of wound healing. Our data demonstrated that MSCs microgels in combination with INSIGHT’s practicality enables customized care for treatment of patients with diabetic foot ulcers and could accelerate diabetic wound healing.

K4.06

Biodegradable Magnesium Wires Promote Neovascularization And Neuronal Growth For Wound Regeneration
Nabila N. Anika1, Ajeet Nagi1, Sonya S. Keswani1, Pranav Bommekal1, Ling Yu1, Sarah Pixley2, Swathi Balaji1
1Pediatric Surgery, Baylor College of Medicine, Houston, TX, United States 2Dept. of Pharmacology & Systems Physiology, University of Cincinnati Medical Center, Cincinnati, OH, United States
Biodegradable Magnesium Wires Promote Neovascularization And Neuronal Growth For Wound Regeneration

Nabila N. Anika1, Ajeet Nagi1, Sonya S. Keswani1, Pranav Bommekal1, Ling Yu1, Sarah Pixley2, Swathi Balaji1 1Pediatric Surgery, Baylor College of Medicine, Houston, TX; 2Dept. of Pharmacology & Systems Physiology, University of Cincinnati Medical Center, Cincinnati, OH

Magnesium (Mg) is co-factor for critical enzymes important in wound healing. Biodegradable Mg metal alloys can promote wound healing by releasing Mg ions. We optimized alloy chemistry and thermomechanical processing conditions to manufacture fine Mg wire with sufficient mechanical properties to withstand wound implantation and in-service loading with excellent tissue tolerance. We hypothesize that Mg metal devices will provide physical guidance during early phases of wound closure and promote neovascularization, neuronal growth and wound regeneration. Mg alloy wires (WE43B, 127 μm, 90% cold work, and 250C heat treated) were cut to 6mm length, and 5 wires were placed in 6mm stented full-thickness flank skin wounds in C57BL/6J mice (n=6, F, 8 weeks). Contralateral flank wound was treated with PBS as an internal control. Wound sections were stained with H&E to measure epithelial gap and granulation tissue at d7; neovascularization (CD31), leucocyte and macrophage infiltration (CD45; F4/80), and neurons (tuj1) were assessed at d7. Scar area, collagen density, lumen density, epithelial thickness and dermal appendages were analyzed at d28. MicroCT was performed at d7 and d28 to see Mg wires. p values by ANOVA. All mice tolerated Mg wire placement. Gross observation showed no difference in exudate compared to PBS. Mg wires were visible at d7 and microCT revealed very small fragments even at d28, suggesting Mg wire degradation was appropriate to support all phases of wound healing. At d7 there was no difference in epithelial gap closure, but Mg significantly improved granulation tissue (0.56±0.19 vs 0.29±0.09 mm2, p<0.001). Mg also reduced inflammatory cell infiltration of both leukocytes (21.4±4.3 vs 34.1±5.1 cells/HPF, p<0.01) and macrophages (27.1±4.1 vs 50.1±7.5 cells/HPF, p<0.01), and improved wound neovascularization (21.2±5.7 vs 12.7±4.3 lumens/HPF, p<0.01) and tuj1 fluorescence expression compared to PBS. At d28, a very small scar remained in Mg wounds. Scar area was reduced in Mg wounds, with improvement in ECM organization and subepithelial nuclear counts in papillary dermis, significantly increased lumen density (16±3.6 vs 11.5±1.7 lumens/HPF, p<0.05) and dermal appendages. Our data demonstrate that Mg metal wires reduce inflammation and promote granulation tissue formation, neovascularization and neuron growth early in wound healing to support regenerative dermal wound healing. This provides a strong rationale to harness Mg metal use in wound healing applications, specially to treat infected or chronic wounds without creating adverse responses such as antibody resistance or rejection of the treatments.

L1.01

What Makes A Wound Become Chronic: Cellular And Molecular Processes That Lead To Chronic Wound Initiation
Parnian Jabbari1, Jane Kim1, Wei Zhang2, Brandon Le2, Manuela M. Martins-Green1
1Department of Molecular, Cell and Systems Biology, University of California, Riverside, Riverside, CA, United States 2Institute for Integrative Genome Biology, Department of Botany and Plant Sciences. College of Natural and Agricultural Sciences, University of California, Riverside, Riverside, CA, United States
What Makes A Wound Become Chronic: Cellular And Molecular Processes That Lead To Chronic Wound Initiation

Parnian Jabbari1, Jane Kim1, Wei Zhang2, Brandon Le2, Manuela M. Martins-Green1 1Department of Molecular, Cell and Systems Biology, University of California, Riverside, Riverside, CA; 2Institute for Integrative Genome Biology, Department of Botany and Plant Sciences. College of Natural and Agricultural Sciences, University of California, Riverside, Riverside, CA

Chronic wounds (CW) develop because of defective regulation of one or more processes needed for successful tissue repair, but how these alterations affect CW initiation, is still not known. The purpose of this study is to understand cellular and molecular processes that contribute to initiation of wound chronicity. We used a diabetic mouse model of CWs, scRNAseq, and bioinformatic approaches. Skin tissue was collected from the wound during the first 3 days of wounding from non-chronic (NCWs) and CWs, with four samples/group. After tissue processing, single cell isolation, and RNA sequencing, a total of 102,737 cells were profiled. After quality control and filtering (e.g. doublet removal), 75,511 cells were subjected to principal component analysis and cell clustering. We identified 17 clusters that were annotated using marker gene expression for different skin cell types: fibroblasts, keratinocytes, vascular and lymphatic endothelial cells, and leukocytes. Comparison of the clusters between NCWs and CWs, showed that there were significant differences between the same type of cluster under the two conditions. We identified 2 types of fibroblasts: one more prevalent in NCWs, healing fibroblasts (H-Fib), and the other more prevalent in CWs, non-healing fibroblasts (NH-Fib). Comparison of these 2 types of fibroblasts revealed several altered pathways. In NH-Fib, the Wnt signaling (upregulated SFRPs), integrin signaling (downregulation of Lamc3 and Megf6) and fibroblast growth factor signaling (downregulated Ppp2r2c), were all impaired. These are all processes needed for proper healing. We identified 5 subtypes of keratinocytes and of these, 2 showed significant differences. The cluster identified as channel keratinocytes is more abundant in CWs. However, the gene expression profile showed that the antiproliferative and apoptosis-inducing genes, IGFBP3 and Ptpn14, which inhibit IGF and YAP signaling, were overexpressed in these keratinocytes. The other type of keratinocyte is less abundant in CWs and shows upregulation of apoptotic pathways (e.g. Fas) and downregulation of migratory pathways (e.g. Mylk) suggesting impaired re-epithelialization in CW. Noteworthy, all 5 subtypes of keratinocytes show downregulation of electron transport chain complex 1 in CWs (e.g. mt-Nd1, mt-Nd4). The vascular and lymphatic endothelial cells in CWs had downregulation of mitochondrial complex 1 and antioxidant response genes (e.g. mt-Rnr2). The downregulation of mitochondrial genes indicates that the cells in CWs are not producing sufficient levels of ATP for proper function, which can contribute to the lack of: granulation tissue formation, re-epithelialization, blood flow, and excess of exudate produced by CWs. In conclusion, our findings show for the first time that the path for chronicity is established very early after wounding providing opportunities for the development of treatments that can reverse the path from non-healing to healing after debridement.

L1.02

Quantifying The Interaction Between Age And Diabetes On Skin Wound Metabolism Using In Vivo Multiphoton Microscopy
Marcos R. Rodriguez1, Kyle Quinn2, Malavika Nidhi1, Divya M. Gollapalli1
1Biomedical Engineering, University of Arkansas, Fayetteville, AR, United States 2Arkansas Integrative Metabolic Research Center, University of Arkansas, Fayetteville, AR, United States
Quantifying The Interaction Between Age And Diabetes On Skin Wound Metabolism Using In Vivo Multiphoton Microscopy

Marcos R. Rodriguez1, Kyle Quinn2, Malavika Nidhi1, Divya M. Gollapalli1 1Biomedical Engineering, University of Arkansas, Fayetteville, AR; 2Arkansas Integrative Metabolic Research Center, University of Arkansas, Fayetteville, AR

Diabetic foot ulcers are a significant health care burden. Current diagnostic approaches are primarily limited to symptomatic assessment and wound size monitoring, and new diagnostic tools are lacking. Therefore, there is a need for non-destructive imaging methods capable of characterizing and predicting healing outcomes. We have previously demonstrated that label-free multiphoton microscopy (MPM) can serve as a non-invasive diagnostic tool for skin wounds. Through MPM, we can visualize the autofluorescence of metabolic cofactors NADH and FAD, as well as collagen organization in 3D at a high-resolution. We have shown an optical redox ratio of FAD/(NADH+FAD) fluorescence intensities of the wound epithelium is sensitive to both diabetes- and age-related delays in healing. However, the interaction of these comorbidities on the epithelial redox ratio has not been elucidated. The goal of this study was to quantify the interaction of diabetes and age effects on wound closure and metabolism using in vivo label-free MPM. Young (5 mo.) and aged (24 mo.) streptozotocin-induced diabetic (n=17; 50/50 sex split) and control (n=17; 50/50 sex split) C57BL/6J mice received full-thickness, excisional wounds on their dorsum. Wounds were imaged longitudinally over 10 days using a multiphoton microscope that collected NADH (755nm excitation/460nm emission) and FAD (855nm excitation/525nm emission) autofluorescence. Image z-stacks were acquired at three wound edge locations using a 20x, 1.0 NA objective. Wound boundaries were manually traced to evaluate the rate of closure. Changes in the optical redox ratio within the epithelium were assessed using a multi-factor ANOVA and posthoc Tukey’s HSD tests. Wound size measurements indicated that closure was most delayed in aged diabetic mice, while closure was fastest in young non-diabetic controls. The epithelial redox ratio at the wound edge changed over 10 days similar to previous studies. The epithelial redox ratio of young non-diabetic control mice significantly decreased (p<0.0001) by day 3 during the initial inflammatory phase, which then increased during re-epithelialization, allowing the redox ratio to return to baseline levels by day 10. Aged control mice, however, did not exhibit a significant change in wound metabolism until day 7 (p<0.0001), suggesting delays during the inflammatory phase. The epithelial redox ratio of young and aged diabetic mice significantly decreased by day 3 (p<0.0029) like young control mice, but did not increase at later time points. While not statistically different from other groups, aged diabetic mice had the lowest redox ratio at day 10. In conclusion, these findings suggest that in vivo label-free MPM is sensitive to diabetes-induced delays in healing, independent of the metabolic effects associated with advanced age.

L1.03

Pten Is A Master Regulator Of Non-Healing Phenotype In Venous Leg Ulcers
Jelena Marjanovic1, Jamie L. Burgess1, Ivan Jozic1, Beatriz Abdo Abujamra1, Robert S. Kirsner1, Hadar Lev-Tov1, Harold Brem2, Momoh Ojirese2, Irena Pastar1, Marjana Tomic-Canic1
1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States 2Rutgers New Jersey Medical School, Newark, NJ, United States
Pten Is A Master Regulator Of Non-Healing Phenotype In Venous Leg Ulcers

Jelena Marjanovic1, Jamie L. Burgess1, Ivan Jozic1, Beatriz Abdo Abujamra1, Robert S. Kirsner1, Hadar Lev-Tov1, Harold Brem2, Momoh Ojirese2, Irena Pastar1, Marjana Tomic-Canic1 1Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2Rutgers New Jersey Medical School, Newark, NJ

Venous leg ulcers (VLUs) represent one of the most prevalent types of chronic wounds yet only 44.1% of VLUs heal with standard of care, underscoring the critical need to better understand the molecular and cellular pathology of VLUs. This study focused on multi-omic approaches to identify novel therapeutic targets and key cellular and molecular mechanisms that are associated with clinical outcomes of VLU healing. Patients with chronic VLU were recruited from 2 clinical centers. To identify the unique non-healing gene signatures, we performed bulk and single cell RNA sequencing on healing (n=5) and non-healing VLUs (n=6) and utilized Ingenuity Pathway Analysis to perform comparative genomics to human acute wounds (AW) collected at day 3. Immune response of healing VLUs showed a high resemblance to acute wounds, while non-healing VLUs signature showed a strong suppression of the cellular inflammation and, much less studied, lymphangiogenesis. We confirmed these findings in prospectively collected tissue (n=4 per group) by immunostaining. We demonstrated a significant decrease in CD45, CD3, CD4, CD19, CD68 and podoplanin positive cell population in non-healers compared to healers. Further, the absence of a proper inflammatory response was a consequence of multiple impaired cellular processes: suppressed transmigration, improper chemotaxis, and immune cell recruitment, which was corroborated by significant suppression of CCL4, CCL13, CXCL9, CXCL12, CX3CL1, ICAM1, ITGB2, VCAM1, TLR4, and XCR1 in non-healing VLUs. To identify the molecular mechanisms contributing to a non-healing VLU signature, we performed proteome profiler arrays and found suppression of AKT, ERK, p38 MAPK, SRC, STAT and PDGFR signaling pathways in non-healers when compared to healers. Upstream regulator analyses pointed to PTEN as a master negative regulator of all these pathways. PTEN protein analyses showed significant induction in non-healers when compared to healers. Indeed, PTEN inhibitor accelerated wound closure in murine wounds in vivo. In addition to increasing immune cell response, PTEN inhibitor also induced IFNγ and CXCL13. Thus, we identified PTEN as a master regulator of non-healing phenotype of VLUs that orchestrates impaired immune response and lymphangiogenesis. Identifying PTEN signaling and cellular makeup that is critical in promoting VLU healing provides key targets for novel therapeutic approaches that will successfully shift a non-healing to a healing VLU and promote closure.

L1.04

Transcriptional Analysis of Fibroblasts in Diabetic Wound Healing
Abdelrahman Alsharif, Katharina S. Fischer, Filiberto Quintero, Mansi Singh, Amelia B. Knochel, Ben Litmanovich, Sultana M. Mojadidi, Javier Gonzalez, Dharshan Sivaraj, Hudson C. Kussie, William Hahn, Andrew Hostler, Maia Granoski, Geoffrey C Gurtner, Kellen Chen
Department of Surgery , University of Arizona, Tucson, AZ, United States
Transcriptional Analysis of Fibroblasts in Diabetic Wound Healing

Abdelrahman Alsharif, Katharina S. Fischer, Filiberto Quintero, Mansi Singh, Amelia B. Knochel, Ben Litmanovich, Sultana M. Mojadidi, Javier Gonzalez, Dharshan Sivaraj, Hudson C. Kussie, William Hahn, Andrew Hostler, Maia Granoski, Geoffrey C. Gurtner, Kellen Chen Department of Surgery, university of Arizona, Tucson, AZ

Background: Diabetic wound healing poses a significant public health concern, impacting approximately 30 million individuals in the United States alone, with projections indicating a doubling by 2050. The cellular mechanisms that prevent proper wound healing are still not fully understood. Fibroblasts are crucial contributors to all stages of wound healing. Given their significance, this study is dedicated to a comprehensive exploration of how heterogeneous populations of fibroblasts contribute to diabetic wound healing using transcriptomic analysis and various different mouse models over time.
Methods: We created full-thickness excisional wounds on the dorsum of C57/BL6 (WT) mice, WT mice subjected to a high-fat (HF) diet to induce pathophysiologic diabetes, and leptin-receptor deficient (DB) mice with genetically induced diabetes. Mice were sacrificed at post-operative days (POD) 0, 2, 7 and 30. Tissues were processed into single-cell suspensions for single-cell RNA sequencing. Bioinformatic analyses were used to identify and further analyze fibroblasts (Col1a1+, Col1a2+, Col5a1+, and Vim+).
Results: The focused analysis of fibroblasts revealed nine distinct subpopulations defined by a unique gene expression profile, indicative of its functional characteristics, including fibrotic (F), keloid-initiating (KI), altered epithelial-like (AEL), and angiogenic (A) clusters. Notably, on POD2, WT fibroblasts demonstrated considerable enrichment in A (12%) and F (14%) clusters, while HF exhibited a deficiency (<1%) in both. Contrarily, DB only displayed a slight growth in A (3%), and F (4%). Across the different conditions at POD2, KI cluster displayed zero growth. At POD7, WT increased expression in A (15%), and declined in F (10%). These cells also demonstrated a sharp growth in KI (20%). DB rose to 8% in A and skyrocketed in F and KI (26% and 35% respectively). On the other hand, HF displayed a marginal increase of 3% in A and 1% in F with a substantial increase in KI (10.5%). Interestingly, HF displayed a significant increase in the AEL subpopulation with 40% vs <0.1% in DB and WT. By POD30, A, KI and AEL subpopulations dropped back to <0.7%. F remained marginally expressed in DB and HF at both 2% and WT regressed to 0%.
Conclusion: This study discovered distinct temporal patterns of A, F, AEL, and KI subpopulations of fibroblasts within WT, HF, and DB mice. Interestingly, A and F subpopulations were significantly weakened in the diabetic groups at POD2. At POD7, unique HF AEL clusters may critically affect impaired diabetic healing. Proportionally, the number of fibroblasts decreased by up to 100-fold by POD 30 compared to POD 7, demonstrating a resolution of the wound healing process. Creating therapies to increase A and fibrotic F subpopulations could improve diabetic wound healing.

L1.05

Vlu Wound Bed Preperation Is Highly Correlated With Wound Closure - Results From The Chronex Multicenter Rct
Marissa Carter1, Robert Snyder3, Keren David2, Yael Katz-Levy2, Ety Klinger2, Ofra Barnett-Griness4, Chaviva Peretz-Rozenblum4, John Lantis5
1Strategic Solutions, Inc., Bozeman, MT, United States 2MediWound Ltd, Yavne, Israel. 3Barry University, Miami, FL, United States 4Bioforum Ltd, Ness-Ziona, Israel. 5Mount Sinai West, Icahn School of Medicine, New York, NY, United States
Vlu Wound Bed Preperation Is Highly Correlated With Wound Closure – Results From The Chronex Multicenter Rct

Marissa Carter1, Robert Snyder3, Keren David2, Yael Katz-Levy2, Ety Klinger2, Ofra Barnett-Griness4, Chaviva Peretz-Rozenblum4, John Lantis5 1Strategic Solutions, Inc., Bozeman, MT; 2MediWound Ltd, Yavne, Israel; 3Barry University, Miami, FL; 4Bioforum Ltd, Ness-Ziona, Israel; 5Mount Sinai West, Icahn School of Medicine, New York, NY

Background: Wound bed preparation (WBP) is a critical step in transition of a chronic wound from a disrupted to normal healing process. Debridement of non-viable tissue and promotion of granulation tissue are key components of WBP, that can support secondary healing or facilitate effectiveness of advanced measures. Previously published results from the ChronEx RCT, assessing a novel bromelain-based enzymatic debridement (BBD) in the treatment of chronic venous leg ulcers (VLUs), showed the efficacy of BBD in WBP. The purpose of this analysis was to assess the correlation between WBP and wound closure (WCL).
Methods: In the ChronEx study patients with chronic VLUs were randomized (3:2:2 ratio) to daily treatment with either BBD, placebo gel vehicle, or non-surgical standard of care (NSSOC), for up to 2 weeks or until reaching complete debridement. Patients were then followed-up weekly with standardized NSSOC for 12 weeks. WBP was defined as complete debridement of non-viable tissue and wound bed completely covered with granulation tissue, both assessed clinically. Complete WCL was defined as complete re-epithelialization of the wound surface without drainage or dressing, confirmed at two visits. The incidence of WCL during the study was compared between those who achieved or did not achieve WBP, in all randomized patients. Incidence of WCL during the follow-up was compared between patients who achieved or not WBP by 16 days (end of daily treatment). The correlation between time to WBP and time to WCL was assessed using a time-dependent proportional hazards Cox regression model.
Results: A total of 119 patients were randomized to the study, with an average wound size of 15.5 cm2 (SD19.4), average non-viable tissue of 73.2% (SD15.2) and wound present for an average of 31.2 weeks (SD23.9).
Overall, 80 patients (67%), reached WBP anytime throughout the study and 39 (33%) did not. Of those that reached WBP, 34 patients (42.5%) achieved WCL within the study period, while only 4 (10.3%) of the patients that did not achieve WBP achieved WCL, with relative risk (RR) of 4.1 (95%CI=1.58-10.85, p=0.0004).
Patients reaching WBP by 16 days were 2.2 more likely to achieve WCL within 12 weeks follow-up compared to patients who did not reach WBP by that time, (50.0% vs. 22.7%, 95%CI for RR 1.31-3.71, p=0.0028). Time to WBP correlated with time to WCL (hazard ratio-11.96, 95%CI= 4.24-33.79, p<0.0001).
Conclusions: The ChronEx study demonstrates that wound bed preparation of chronic VLUs significantly increased the likelihood of complete WCL. These findings support the critical importance of adequate WBP in the process of wound healing. A future phase 3 pivotal study with BBD will utilize these new data to assess the efficacy of this novel treatment in early facilitation of active WCL in VLU.

L1.06

Spinal Cord Injury And Postoperative Complications In Pressure Ulcer Closure
Namrata V. Chintalapati, Robert Galiano
Plastic Surgery, Northwestern University, Chicago, IL, United States
Spinal Cord Injury And Postoperative Complications In Pressure Ulcer Closure

Namrata V. Chintalapati, Robert Galiano Plastic Surgery, Northwestern University, Chicago, IL

Background: Prior literature has shown that patients with spinal cord injury have higher incidence of pressure ulcers (PUs). However, differences in the rate of complications following debridement and closure, as well as variations in wound size, have not been thoroughly investigated. The purpose of this study is to evaluate these specific criteria to determine whether PU wound healing outcomes following surgical closure are impacted by a past medical history of spinal cord injury. In doing so, the study aims to inform targeted interventions that can enhance the overall quality of care for individuals with a history of spinal cord injury.
Methods: In this prospective single-institution study, patients with stage III/IV PUs undergoing surgical debridement and closure were included. Follow-up assessments were conducted at 14 days, 1 month, 6 months, and 1 year postoperatively. Complications assessed at 2 weeks included maceration, major or minor dehiscence, epidermolysis, drainage, congestion, and skin necrosis.
Results: A total of 38 patients were included. Of them, 36.8% (n = 14) had a past medical history of spinal cord injury. Patients with a history of spinal cord injury had a significantly higher number of post-operative complications 2 weeks following surgical PU wound closure (1 vs. 0.36, p < 0.05). The complication with the highest prevalence (21%, n = 8) was major or minor wound dehiscence. Patients with a history of spinal cord injury also had significantly higher risk of open wound status at 14 days post-operatively (RR = 1.4, p < 0.05) and at 1 month post-operatively (RR = 2.18, p < 0.05) compared to those without. However, this increased risk of an open wound was lost at 6 months (RR = 0, p = 0.18) and 1 year (RR = 2.83, p = 0.13) post-operatively. There was no significant difference found in wound length (6.46 cm vs. 5.39 cm, p = 0.20), width (4.59 cm vs. 3.67 cm, p = 0.20), or depth (2.99 cm vs. 2.41 cm, p = 0.20) between patients with a history of spinal cord injury and those without.
Conclusion: In conclusion, this study sheds light on the impact of a history of spinal cord injury on pressure ulcer (PU) wound healing outcomes following surgical closure. The findings reveal that patients with a past medical history of spinal cord injury experienced a significantly higher incidence of post-operative complications, particularly major or minor wound dehiscence, at 2 weeks post-operatively. Additionally, these individuals faced an increased risk of an open wound status short-term (2 weeks and 1 month post-operatively). Interestingly, this heightened risk diminished long-term (6 months and 1 year post-operatively). While wound dimensions did not exhibit significant differences between patients with and without a history of spinal cord injury, the study underscores the need for tailored interventions and closer monitoring in this patient population to optimize PU wound healing outcomes.

L2.01

Deep Vein Thrombosis Affects Healing Outcomes In Patients With Pyoderma Gangrenosum: A Single-Center Prospective Case-Control Study
Hannah Zhao, Sidharth Sengupta, Jonathan Sisley, Olivia M. Haddadin, Alex Ortega-Loayza
OHSU, Lake Oswego, OR, United States
Deep Vein Thrombosis Affects Healing Outcomes In Patients With Pyoderma Gangrenosum: A Single-Center Prospective Case-Control Study

Hannah Zhao, Sidharth Sengupta, Jonathan Sisley, Olivia M. Haddadin, Alex Ortega-Loayza OHSU, Lake Oswego, OR

While inflammation and thrombosis are closely integrated processes, there has been little exploration between PG and thromboembolism. In this single-center case-control study, we assess the association between a history of DVT and healing outcomes in PG patients. This prospective case-control study was approved by the OHSU IRB and was conducted between December 2019 and July 2023. Inclusion criteria was defined as patients with classic, ulcerative PG of the lower extremity confirmed by a PARACELSUS score of ≥10 from the Pyoderma Gangrenosum Study Registry (PYGAS) at OHSU. Data regarding patient demographics, comorbidities, and healing outcomes were extracted for further analysis. Patients were then grouped based on the presence or absence of ultrasound-proven lower extremity DVT prior to PG diagnosis. Cox regression and Kaplan-Meier survival analysis were applied to detect the effect of DVT on healing status within a 1-year period from initial presentation. Chi-squared tests were used to compare the descriptive statistics of the DVT and non-DVT groups. Statistical significance was established as P ≤0.05. In total, 99 patients met inclusion criteria, with 13 (13.1%) having a history of DVT and 86 (86.9%) without. 121 total ulcers were included in the analysis—patients presenting with sequential or recurrent ulcers were recorded as separate encounters. The mean age of presentation for patients with and without DVT was 57 (SD +/-11) and 53 (SD +/-18), respectively (P=0.60). There were significantly more males presenting with DVT than females (P =0.04). There were no significant differences in race, ethnicity, or smoking history between the two groups. There were also no significant differences in confounding comorbidities such as body mass index, history of solid or hematological malignancy, or history of autoimmune or inflammatory disorder between the DVT and non-DVT groups. Evaluation of median healing time between DVT and non-DVT groups using Kaplan-Meier survival analysis confirmed a significant increase in median healing time among patients with a history of DVT. The median healing time for the DVT and non-DVT group was 224 and 145 days, respectively (p<0.04). Univariate and multivariate Cox proportional hazard modelling was also used to assess the influence of various predictor variables on wound healing. When adjusting for obesity and smoking, the two factors most prevalent in our patient population and factors known to influence wound healing, a history of DVT remained the most significant influence on the healing time (p=0.05). Single predictor modelling also demonstrated that variables such as diabetes mellitus, solid malignancy, and multi-ulcer presentation did not significantly alter healing time. In conclusion, these findings suggest a novel association between a history of DVT and poorer healing in patients with PG, indicating the importance of identifying a history of thromboembolism when evaluating PG patients.

L2.02

Selective Agonism Of Histamine Receptors Augments Tissue Repair
Jordan R. Yaron, Shubham Pallod, Nicole Grigaitis, Samantha Rhodes, Dirghau M. Patel, Deepanjan Ghosh, Kaushal Rege
Biodesign Center for Biomaterials Innovation and Translation, Arizona State University, Tempe, AZ, United States
Selective Agonism Of Histamine Receptors Augments Tissue Repair

Jordan R. Yaron, Shubham Pallod, Nicole Grigaitis, Samantha Rhodes, Dirghau M. Patel, Deepanjan Ghosh, Kaushal Rege Biodesign Center for Biomaterials Innovation and Translation, Arizona State University, Tempe, AZ

Purpose: Histamine is produced by mast cells and non-canonically by other cells during tissue injury. Four G-protein coupled receptors for histamine (HRH1-4) regulate diverse vascular, fibrotic, and immune signaling. Histamine signaling is critical to healthy wound healing and is dysregulated in diabetic wounds. To understand the role of specific histamine receptors during tissue repair, we performed a systematic investigation of HRH agonism during wound closure in vitro and biomaterial-assisted incisional wound repair in vivo.
Methodology: Incisions were made in Balb/c mice and sealed with silk fibroin-based laser-activated sealants (LASE) with codelivery of saline, histamine, or selective agonists for HRH1 (2-pyridylethylamine), HRH2 (dimaprit), HRH3 (immethridine), or HRH4 (4-methylhistamine), hereafter referred to as HRHn-ag (where n is 1-4). Trans-epidermal water loss (TEWL) was measured daily for 3 days. Ultimate tensile strength (UTS) was measured after histamine treatment. IHC was performed for HRH1-4 post-wounding, and for M1/M2 macrophage and neutrophil infiltration, angiogenesis, proliferation, and EMT. Expression of HRH1-4 in HaCaT keratinocytes was evaluated by immunofluorescence and western blot. Migration in response to histamine or agonists was performed by scratch assay.
Results: Histamine treatment of LASE-sealed incisional wounds results in a significant early increase in UTS without alteration of TEWL vs. saline-treated wounds. In contrast, selective agonism of HRHs results in diverse TEWL responses: HRH1-ag (N=3) increased TEWL (p<0.05), HRH2-ag (N=3) and HRH4-ag (N=3) decreased TEWL (p<0.05), and HRH3-ag (N=4) causing an initial increase in TEWL (p<0.001) with a return to physiologic levels after day 1. We found that HaCaT cells express all HRHs. Interestingly, while histamine (p<0.01) and HRH1-ag (p<0.0001) accelerate scratch closure, HRH2-ag almost completely prevents migration (p<0.0001). IHC for tissue responses during repair indicated a pro-resolution macrophage environment (high Arg1, low CD86) with HRH1-ag and HRH4-ag (p<0.01), and a milder response by HRH2-ag (p<0.05) and no response by HRH3-ag. HRH2-ag and HRH4-ag robustly suppressed neutrophil presence (p<0.0001), with a milder response by HRH1-ag (p<0.01) and no response by HRH3-ag. Angiogenesis (CD31+ vessels) was stimulated by HRH1-ag (p<0.01) and HRH4-ag (p<0.0001), with no response by HRH2-ag and HRH3-ag. Epidermal EMT (loss of E-cadherin integrity) was promoted by HRH1-ag (p<0.05) and suppressed by HRH2-ag (p<0.01), with no response by HRH3-ag and HRH4-ag. Keratinocyte proliferation was unaffected by all receptors.
Conclusion: HRH1 and HRH4 activation drive a pro-resolution immune response with enhanced angiogenesis, while HRH1 also stimulates increased epidermal EMT. HRH2 activation slows epidermal healing, and HRH3 activation has minimal effect on healing.

L2.03

Preclinical And Clinical Development Of Sli-F06, A Novel Dermal Fibroblast Modulating Drug, In Cutaneous Wound Healing
Zhong Zheng1, Pin Ha2, Elisabeth Leeflang1, Zhaohan Zeng1, Joshua Yang2, Alyssa Miao2, Robert Galiano3, Paul Glat4, Donald Buck6, John Felder6, Kang Ting5, Chia Soo2
1Scarless Laboratories Inc., Cherry Hill, NJ, United States 2University of California, Los Angeles, Los Angeles, CA, United States 3Northwestern University, Chicago, IL, United States 4Dr. Paul Glat Clinic, Bala Cynwyd, PA, United States 5American Dental Association Forsyth Institute, Cambridge, MA, United States 6Washington University, St. Louis, MO, United States
Preclinical And Clinical Development Of Sli-F06, A Novel Dermal Fibroblast Modulating Drug, In Cutaneous Wound Healing

ZHONG ZHENG1, Pin Ha2, Elisabeth Leeflang1, Zhaohan Zeng1, Joshua Yang2, Alyssa Miao2, Robert Galiano3, Paul Glat4, Donald Buck6, John Felder6, Kang Ting5, Chia Soo2 1Scarless Laboratories Inc., Cherry Hill, NJ; 2University of California, Los Angeles, Los Angeles, CA; 3Northwestern University, Chicago, IL; 4Dr. Paul Glat Clinic, Bala Cynwyd, PA; 5American Dental Association Forsyth Institute, Cambridge, MA; 6Washington University, St. Louis, MO

SLI-F06, a fibromodulin (FMOD)-based peptide, enhances wound healing. In comprehensive animal models, including mice, rats, and Yorkshire pigs (a gold standard for normal human wound healing), SLI-F06 exhibited FMOD’s pro-migration, pro-tensile strength, and anti-fibrotic properties. Here, red Duroc pigs, which more closely mimic human hypertrophic scarring, were wounded to further assess the translational potential of SLI-F06 to humans. Large ellipses were designed to create excessive-mechanical-loading (EML) to represent a high-tension wound relative. Primary closed wound edges were immediately injected with 10 mg/ml SLI-F06 or triamcinolone acetonide (TAC), a commonly used corticosteroid for repressing scar. At 8 weeks, TAC did not considerably improve gross scar appearance or reduce scar size, while it significantly reduced scar tensile strength. Meanwhile, SLI-F06 markedly improved visual scar appearance (P < 0.0001; N =12) and significantly reduced scar size, especially in the EML wounds (resulting in 56% Scar Index reduction; P = 0.0024; N = 5). More importantly, SLI-F06 resulted in a 29% and 160% tensile strength increase in the normal (P = 0.0066; N =6) and EML wounds (P < 0.0001; N = 6), respectively. Nonclinical safety studies include a 5-day and a 28-day intravenous bolus repeat-dosing in rats, a 5-day subcutaneous repeat-dosing in pigs, and a 3-day intradermal repeat-dosing in a wounded pigs. All toxicology studies demonstrated no observable adverse effects at maximum feasible doses. Ames and Episkin testing have shown no genotoxicity or local irritation, respectively. After FDA clearance, we performed a multicenter, double-blind, First-in-Man study to compare the safety of the cGMP-compliant SLI-F06 drug product to control formulation buffer (vehicle). Twenty-four (24) subjects were enrolled and 21 completed the study. Each subject served as his or her own control. The study was divided into 2 parts: Part A was a safety and proof-of-concept study of small scars pre-abdominoplasty, and Part B was a phase IIa study of post-abdominoplasty scars. There were no drug-related adverse events or clinically meaningful abnormal laboratory values, and all subjects were negative for antidrug antibodies in immunogenicity testing. Moreover, the POSAS assessment indicated that the non-optimized, low-dose, one-time injection of SLI-F06 led to a 24.6% Surface Area improvement at the high-tension abdominoplasty wound segments (P = 0.0405; N = 42). Therefore, SLI-F06 shows promising preliminary efficacy in minimizing cutaneous scarring and excellent tolerability in human subjects, while further refinement of dosage and treatment regimens are essential for optimal results.

L2.04

Prophylactic, One Time Dose Of Rac Inhibitor Mitigates Foreign Body Response Through Immunomodulation At Both Early And Late Time Points
Hudson C. Kussie, Jonathan P. Yasmeh, Dharshan Sivaraj, Katharina S. Fischer, Eamonn McKenna, Brodi Stevens, Gabriel Starling, Maia Granoski, Andrew Hostler, Maisam Jafri, Geoffrey C. Gurtner, Kellen Chen
University of Arizona, Tucson, AZ
Prophylactic, One Time Dose Of Rac Inhibitor Mitigates Foreign Body Response Through Immunomodulation At Both Early And Late Time Points

Hudson C. Kussie, Jonathan P. Yasmeh, Dharshan Sivaraj, Katharina S. Fischer, Eamonn McKenna, Brodi Stevens, Gabriel Starling, Maia Granoski, Andrew Hostler, Maisam Jafri, Geoffrey C. Gurtner, Kellen Chen University of Arizona, Tucson, AZ

Purpose: Our study explores how a single prophylactic dose of Rac inhibitor can significantly diminish the foreign body response around biomedical implants, potentially improving the efficacy and durability of such devices in patient care. Methods: We used our previously published mechanically stimulated implants (MSIs) (Padmanabhan et al, 2023, Nat Biomed Eng) to create severe, pathological human FBR in mice. These MSIs were made of polydimethylsiloxane (PDMS) and encapsulated coin motors connected to external 3V batteries to initiate mechanical stimulation through vibration to create significant FBR fibrosis. Mice either received saline only (vehicle) or a prophylactic 5mg/kg NSC Rac inhibitor dose. After 7 days of vibration (POD 11), samples from 5 no vibration (NV), 5 MSI with saline (MSI+S) and 5 MSI with Rac Inhibitor (MSI+RI) were collected. After a month (POD30), 5 NV, 10 MSI+S, and 10 MSI+RI samples were collected. For this explanted tissue, FBR tissue was analyzed using staining including picrosirius red, hematoxylin and eosin, trichrome, and immunohistochemistry (IHC) for aSMA, F4/80, and DAPI.
Results: At POD 7, MSI+S significantly increased FBR compared to NV (p= 0.0465), and MSI with Rac Inhibitor treatment significantly reduced FBR by 47% from 284.8 to 133.8 μm compared to MSI+S (p=0. 0331) to be close to NV levels. Similarly, at POD30, MSI+S significantly increased FBR compared to NV (p=0.005), and MSI with Rac Inhibitor treatment significantly reduced FBR by 51% from 370.2 to 183.2 μm compared to MSI+S (p=0. 0.008), and not significantly different than NV levels. At early time points, MSI+S significantly reduced the length (p=0.037) while increasing alignment (p=0.001) and density (p=0.004) compared to NV. MSI+RI restored these collagen metrics (p=0.016, p=0.104, p=0.001) back to NV levels. At late time points, these effects were blunted. Using trichrome imaging, distinct architecture layers were evident in the MSI+S capsule, with a unique inner layer that had less wide, dense, and aligned collagen fibers (p=0.0020, p=0.0078, p=0.0051, respectively) compared to mechanically inhibited FBR which possessed no layering. Finally, we observed that Rac inhibition significantly reduced the number of macrophages (F4/80, p=0.02) and myofibroblasts (ASMA, p=0.03) compared to MSI+S.
Conclusions: Our study demonstrates that disrupting mechanical signaling with a one time prophylactic dose of Rac inhibitor notably reduces FBR capsule size and fibrotic ECM architecture. We observed a previously unreported layering in severe, pathological FBR, which seemed to linked with distinct layers of macrophages and fibroblasts within the capsule. Disrupting mechanical signaling with a one time, prophylactic therapeutic could have clinically meaningful effects for patients, and future biomedical implants could even be coated in a one time dose of this drug.

L2.05

Comparison Of Bromelain-Based Enzymatic Debridement To Collagenase Satyl® Ointment - Analyses From The Chronex Multicenter Rct
Dove Cyaandi1, Robert Snyder2, Keren David3, Yael Katz-Levy3, Ety Klinger3, Felix Sigal4
1University Health, San Antonio, TX, United States 2Barry University, Miami, FL, United States 3MediWound Ltd, Yavne, Israel. 4Angel City Research, Los Angeles, CA, United States
Comparison Of Bromelain-Based Enzymatic Debridement To Collagenase Satyl® Ointment – Analyses From The Chronex Multicenter Rct

Dove Cyaandi1, Robert Snyder2, Keren David3, Yael Katz-Levy3, Ety Klinger3, Felix Sigal4 1University Health, San Antonio, TX; 2Barry University, Miami, FL; 3MediWound Ltd, Yavne, Israel; 4Angel City Research, Los Angeles, CA

Background: Results from the ChronEx RCT, assessing a novel bromelain-based enzymatic debridement (BBD) in the treatment of chronic venous leg ulcers (VLU) were published previously. BBD was superior to hydrogel placebo and non-surgical standard of care (NSSOC), in complete debridement (CD) and complete granulation (CG), key components of wound bed preparation (WBP). One of the NSSOC used in the study was collagenase Santyl® Ointment, approved in the US for debridement of chronic dermal ulcers.
Post-hoc analyses assess the efficacy of BBD vs. Santyl in VLU in ChronEx.
Methods: In ChronEx patients with chronic VLU were randomized (3:2:2 ratio) to daily treatment with BBD, placebo, or NSSOC, for up to 2 weeks or until reaching CD and then followed-up weekly with NSSOC for 12 weeks. NSSOC included Santyl, hydrogels, medical grade honey, and non-active dressings. Surgical or mechanical debridement were not allowed.
Post-hoc analyses assessed incidence and time to CD, CG and WBP in patients treated with BBD compared to Santyl, placebo, and NSSOC excluding Santyl (NSSOCES). WBP was defined as CD and CG, both assessed clinically. Log-rank test was used to compare survival distributions and Fisher Exact test to compare incidences.
Summary of results: Of the 119 patients randomized, 46 were treated with BBD, 43 with placebo and 30 with NSSOC. Of the NSSOC arm, 8 patients were treated with Santyl and 22 were treated with NSSOCES. Overall, the average wound size was 15.5 cm2 (SD19.4), average non-viable tissue 73.2% (SD15.2) and average wound age 31.2 weeks (SD23.9). Baseline characteristics were comparable across all groups.
Median time to complete debridement (95% CI) was 9 days (5-15 days) for BBD vs. not achieved for Santyl (22-NA, P=0.023), 63 (21-93) and 44 days (21-67) on placebo and NSSOCES respectively.
Incidence of CD (95% CI) during the two weeks daily treatment was 63.0% (47.5-76.8) for BBD vs. 0% for Santyl (p=0.001), 30.2% (17.2-46.1) and 18.2% (5.2-40.3) for placebo and NSSOCES respectively.
Median time to CG as well as to WBP (95% CI) was 11 days (7-50 days) for BBD vs. not achieved for Santyl (22-NA, P=0.014), 85 (24-99) and 61 days (30-85) on placebo and NSSOCES respectively.
Incidence of WBP (95% CI) during the study was 78.3% (63.6-89.1) on BBD vs. 37.5% on Santyl (8.5-75.5, P=0.03), 60.5% (44.4-75.0), and 68.2% (45.1-86.1) on placebo and NSSOCES respectively.
Conclusions: Debridement of non-viable tissue and promotion of a well vascularized granulation tissue are key components of WBP, that can support secondary healing or facilitate the effectiveness of other advanced measures. Analysis from the ChronEx RCT demonstrates a clinically meaningful and statistically significant reduction in time to CD, CG and WBP and increased incidence of WBP in VLU patients treated with BBD compared to those treated with Santyl.

L2.06

A Longitudinal Single-Cell Transcriptomic Atlas Of Diabetic Porcine Wounds Reveals Distinct Fibroblast Subtypes And Trajectories In Response To Tissue Mechanical Manipulation
Ming Guan1, Nikolaos Kalavros2, Zhuqing Li1, Enya Wang1, Mauricio Contreras1, Jingjing Wu3, Xuanhe Zhao3, Ioannis Vlachos2, Georgios Theocharidis1, Aristidis Veves1
1Joslin-Beth Israel Deaconess Foot Center and The Rongxiang Xu, MD, Center for Regenerative Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States 2Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States 3Department of Mechanical Engineering, Massachusetts Institute of Technology, Boston, MA, United States
A Longitudinal Single-Cell Transcriptomic Atlas Of Diabetic Porcine Wounds Reveals Distinct Fibroblast Subtypes And Trajectories In Response To Tissue Mechanical Manipulation

Ming Guan1, Nikolaos Kalavros2, zhuqing li1, Enya Wang1, Mauricio Contreras1, Jingjing Wu3, Xuanhe Zhao3, Ioannis Vlachos2, Georgios Theocharidis1, Aristidis Veves1 1Joslin-Beth Israel Deaconess Foot Center and The Rongxiang Xu, MD, Center for Regenerative Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 3Department of Mechanical Engineering, Massachusetts Institute of Technology, Boston, MA

Background: Diabetic foot ulceration (DFU) is a complex complication of diabetes, and its pathogenesis remains elusive. Drawing on the success of a strain-programmed patch in expediting wound closure, we conducted a groundbreaking single-cell analysis of diabetic Yucatan minipig skin injuries to unravel the intricate mechanisms of diabetic wound healing at distinct time points.
Methods: In a longitudinal study, we examined various time points (3, 7, 14, and 28 days post-wounding) in Alloxan-induced diabetic swine treated with either Tegaderm, a non-strain patch formulation, or our improved strain-programmable patch formulation. Three wounds per group per timepoint underwent mechanical and enzymatic digestion, generating highly viable single-cell suspensions. Subsequently, we performed detailed single-cell analyses to unveil the molecular landscape of diabetic porcine wound healing.
Results: Sequencing 280,000 cells enabled the identification of expected cell types, including keratinocytes, fibroblasts, endothelial cells, smooth muscle cells, myeloid, and lymphoid cells. We identified several separate subsets of fibroblasts, which are transcriptionally and functionally distinct and change in abundance along the time and treatment axes, with some changes reaching statistical significance. Through differential expression and pathway analyses, we deeply characterize the fibroblast subtypes uncover their functional significance, and reveal, through cell-cell communication analysis, extensive inflammatory pathway activation on Day 7, consistent with an enhanced inflammatory wound healing process. Finally, through trajectory analysis, we propose a model of fibroblast differentiation for each subtype. In addition, leveraging our diabetic foot ulcer dataset and comparing it against mouse wounding studies, we highlight the porcine model’s superior resemblance to human skin wounds.
Conclusion: Our study unveils distinct fibroblast subsets in diabetic porcine wounds undergoing transcriptional and functional changes over time and treatments. Porcine wound cells closely resemble human wounded tissue in both cellular proportions and activated pathways. This comprehensive understanding of diabetic wound healing mechanisms opens avenues for targeted interventions and emphasizes the translational relevance of the porcine model in advancing diabetic wound research.

L3.01

Resolution of Bioburden and Wound Closure in a Porcine Full Thickness Wound Model
Justin Avery1, Joel Gil2, Kelly Kimmerling1, Stephen Davis2, Katie Mowry1
1Research & Development, Organogenesis, Birmingham, AL, United States 2Department of Dermatology and Cutaneous Surgery, University of Miami, Miami, FL, United States
Resolution of Bioburden and Wound Closure in a Porcine Full Thickness Wound Model

Justin Avery1, Joel Gil2, Kelly Kimmerling1, Stephen Davis2, Katie Mowry1 1Research & Development, Organogenesis, Birmingham, AL; 2Department of Dermatology and Cutaneous Surgery, University of Miami, Miami, FL

Collagen matrices have been shown to provide a target for aberrant proteolytic activity in a chronic wound environment. Combining these matrices with antimicrobials such as silver or polyhexamethylene biguanide (PHMB) has demonstrated both in vitro and in vivo to prevent the reformation of biofilm. However, a broader understanding of wounds treated with these devices is limited. We designed a porcine full thickness wound model infected with methicillin-resistant Staphylococcus aureus (MRSA) to model the clinical environment of chronic wounds. Evaluation of the impact of a native cross-linked collagen matrix with PHMB (PCMP^) on biofilm reformation, wound closure, and gene expression with STRING and Gene Ontology (GO) term assessment over the course of treatment was performed.
4cm x 4cm x 3mm full thickness wounds were generated on the backs of pigs (n=3), infected with MRSA (USA300), and allowed to form a biofilm for 72 hours. After biofilm formation, wounds were sharply debrided to model standard of care and subsequently PCMP was applied. PCMP was changed every 5 days, at which point wounds were assessed at Days -3 (unwounded baseline, N=4), 0 (infected and debrided baseline, N=6), 5 (N=2), 10 (N=5), 15 (N=5), and 20 (N=3) post-treatment initiation. Gene expression within the wound bed was assessed using RT2 Profiler™ PCR Arrays (Pig Wound Healing, Qiagen). Genes that were statistically different from unwounded skin were used for STRING analysis with Markov Cluster Algorithm (MCL) inflation parameter of 3. GO terms from these clusters were then plugged into “reduce and visualize gene ontology” (REVIGO) to determine high level GO terms related to various phases of the wound healing process.
Compared to unwounded skin, two pathways were identified: macrophage recruitment/activation and ECM remodeling. We observed statistically increased expression of genes associated with monocyte/macrophages recruitment (CCL2), activation (CD40LG), and subsequent response (IL-1α, IL-1β, IL-10, and TNF), which trended back towards baseline as the wounds healed and bioburden was eliminated. We also observed statistically greater expression of collagenase (MMP1), gelatinase (MMP9), and stromelysin (MMP3) in the wound that resolved as the wound healed. REVIGO analysis found GO terms associated with normal wound repair, starting with inflammation that progressed towards cellular proliferation and migration and ECM remodeling as wounds closed.
Together, these findings highlight that sharp debridement followed by a native cross-linked collagen matrix with PHMB helped prevent biofilm reformation and supported normal wound healing, while the changes in gene expression over the course of closure were characterized.
^PuraPly® AM, Organogenesis, Canton, MA

L3.02

Efficacy and Safety Of Dressing Containing Novel Antimicrobial Peptide For Managing Wound Biofilm
Jennifer Neff1*, John C. Vitucci2,3, Ryan Cummings1, Danir Bayramov1, Ashley Miller2,3, Jonathan Benaye2,4, Abigail Livingston2,4, John A. Cleary2,4, Jesse Smith2,3, Rhonda Lizewski2, Noah Gold2,4, Katy Garcia2,4, Yoann Le Breton2, Frederic Poly2, Alexandria Kesterson2
1Allvivo Vascular, Inc., Lake Forest, CA. 2 Naval Medical Research Command (NMRC), Silver Spring, MD. 3The Henry M. Jackson Foundation for the Advancement of Military Medicine (HJF), Bethesda, MD. 4 General Dynamics Information Technology (GDIT), Reston, VA.
Efficacy and Safety Of Dressing Containing Novel Antimicrobial Peptide For Managing Wound Biofilm

Jennifer Neff, Ryan Cummings, Danir Bayramov Allvivo Vascular, Inc., Lake Forest, CA

Purpose: Biofilm colonization, delayed healing and drug resistance remain important challenges in wound care. The purpose of this work was to evaluate a wound treatment comprising a novel broad-spectrum antimicrobial peptide (ASP-2) formulated in a biodegradable chitosan sponge (Gatekeeper™) for activity against multi-drug resistant bacterial biofilm, to assess the propensity for ASP-2 resistance to evolve with exposure, and to evaluate product safety.
Methods: Efficacy was evaluated using an ex vivo porcine skin biofilm model. Disks of porcine skin were incubated in bacterial culture (106 CFU/mL of Methicillin resistant Staphylococcus aureus, USA 300 ATCC BAA-1717 (MRSA) or Pseudomonas aeruginosa ATCC 15692 (P. aeruginosa)) for 72h to develop biofilm. Skin disks were then treated once with Gatekeeper™ and bacteria CFU remaining on skin were measured after 24, 48, and 72 hours. To evaluate potential for resistance development, MRSA was serially passaged in 0.5 MIC ASP-2 for 30 days. MICs for ASP-2, vancomycin, and mupirocin, were measured daily for ASP-2 exposed cultures with the CLSI M07-A8 microdilution method. The tolerability and toxicokinetics (TK) of ASP-2 and Gatekeeper™ in minipigs were determined following daily administration over 7 days, respectively.
Results: In the ex vivo porcine skin biofilm model, Gatekeeper™ application resulted in a 5.7 log reduction in MRSA CFU/mL by 24 hours with full eradication by 48 hours. P. aeruginosa biofilms experienced 3-4 log reductions in CFU/mL by 24 and 48 hours and were eradicated by 72 hours. All reductions were statistically significant relative to saline controls (p≤0.05, n=3 for all groups). Exposure to sub-inhibitory concentrations of ASP-2 did not select for resistance in MRSA. There was a 2-fold increase in ASP-2 MICs after the first passage, with no significant increases measured over the remaining 29 days of passaging. Moreover, bacteria exposed to thirty passages of sub-MIC ASP-2 did not display cross-resistance to vancomycin or mupirocin. In toxicology studies, the no observable adverse effect level of ASP-2 in rats with subcutaneous administration (N=10) was 100 mg/kg (>20X the equivalent human clinical dose). In minipigs, no toxicologically meaningful effects were found with Gatekeeper™ treatment of full thickness wounds over 2.5% body surface area (BSA) (N=2) or abraded skin over 10% BSA (N=2). Systemic exposure to ASP-2 in minipig TK studies was also found to be minimal.
Conclusions: Gatekeeper™ exhibited strong activity against MRSA and P. aeruginosa biofilms and initial toxicology studies indicate a good therapeutic window for its active component, ASP-2. Serial passaging of MRSA in subinhibitory concentrations of ASP-2 did not result in emergence of ASP-2 resistance or cross resistance to front line antibiotics tested. Collectively, data presented indicate a promising efficacy and safety profile for Gatekeeper™ in managing wound biofilm.

L3.03

Novel Antibiotic-Free Biomimetic Wound Matrix Provides Antimicrobial Protection And Superior Healing
Brunno F. Caetano, Trudy-Ann Grant, Bishnu P. Joshi, Adam Finzen, Tarak Bhakda, Rebecca Salamone, Manav Mehta, Ana Tellechea
Gel4Med Inc., Lowell, MA, United States
Novel Antibiotic-Free Biomimetic Wound Matrix Provides Antimicrobial Protection And Superior Healing

Brunno F. Caetano, Trudy-Ann Grant, Bishnu P. Joshi, Adam Finzen, Tarak Bhakda, Rebecca Salamone, Manav Mehta, Ana Tellechea Gel4Med Inc., Lowell, MA

Background: Pathogenic colonization is a major risk factor for acute and chronic wound complications. Current approaches have serious limitations, and the rise of multidrug-resistant organisms (MDROs) and biofilms further complicates treatment. To address this growing issue, we developed and tested a polypeptide biomimetic wound matrix (BWM) that prevents infection while promoting healing. This self-assembling cationic nanofiber technology was engineered to offer a mechanism of action that evades microbial resistance, a scaffolding matrix with cell attachment sites that encourage tissue regrowth, and wound-conforming properties for tissue void filling.
Methods: Antimicrobial efficacy against Gram-positive [Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus, MRSA] and Gram-negative [Pseudomonas aeruginosa (PaO1), ESBL Escherichia coli, ESBL Klebsiella pneumoniae, Acinetobacter baumannii] bacteria, as well as fungi [Candida albicans, Aspergillus fumigatus] was tested by standard time-kill assays. Efficacy against 72 hour-aged biofilms (PaO1 ± MRSA) was assessed in vitro and ex vivo (porcine skin). MRSA-inoculated murine wounds were treated with BWM and assessed by microbiology at 24 h. BWM mechanical properties were confirmed by rheology and wound bed coverage. In a swine model of full-thickness excisional wounds, BWM healing efficacy was tested vs. silver and collagen gels using the Tissue Analytics platform and histopathology.
Results: In vitro, BWM demonstrated bactericidal efficacy against ≥6 log10 CFU of PaO1 and MRSA at 5, 10, 15, 30, 60 min, and 24 h (p<0.0001, n=3). Within 24 h, BWM eliminated ≥6 log10 CFU of Gram-positive and Gram-negative clinical isolates, as well as sporulating and non-sporulating fungal pathogens. Notably, BWM showed superior efficacy against PaO1 and MRSA biofilms when compared to marketed antimicrobial gels (p<0.0001, n=6), while a single application eradicated mature PaO1 biofilms in pig skin explants by 24 h (n=3). Rapid bioburden reduction was confirmed in MRSA-inoculated murine full-thickness wounds (p=0.02, n=10). In a trypsin proteolysis assay, matrix stability was demonstrated for up to 14 days. BWM-treated swine full-thickness excisional wounds showed superior closure rates (96%, n=5) vs. collagen gel and silver gel (p=0.01) and reduced inflammation. BWM singularly achieved complete re-epithelialization with healthy granulation tissue repletion by day 14.
Conclusions: BWM demonstrates potent broad-spectrum antimicrobial activity against MDROs and biofilms. With controlled biodegradation in a highly proteolytic environment, BWM shows a wound healing profile superior to commercial silver and collagen dressings, including greater closure, increased re-epithelialization, granulation tissue formation, and reduced inflammation. Together, the data supports BWM potential to overcome the current challenges in managing complex and infected wounds.

L3.04

Treating Diabetic Fibroblasts Through Tunable Hyalurononan-Binding Silk Fibroin Therapeutic Hydrogels
Amelia Huffer, Tugba Ozdemir
Nanoscience and Biomedical Engineering, South Dakota School of Mines and Technology, Rapid City, SD, United States
Treating Diabetic Fibroblasts Through Tunable Hyalurononan-Binding Silk Fibroin Therapeutic Hydrogels

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

Granulation tissue formation in diabetic wounds is predominantly controlled by proliferation and extracellular matrix (ECM) deposition of diabetic fibroblasts (DFs). Accumulating evidence shows the proliferation and ECM deposition of DFs are significantly different than their normal counterparts. More specifically DFs are known to deposit altered ECM such as lower levels of Hyaluronan (HA) and higher crosslinking of collagens. The altered ECM in diabetic wounds results in altered mechanical properties and accumulating evidence shows surrounding mechanical properties of the matrix have an impact on cell behavior. We hypothesized that wound dressings designed towards use in diabetic wounds would address matrix mechanics alongside with matrix composition. We designed a tunable stiffness Silk Fibroin (SF) hydrogel matrix with an ability to attract and retain endogenous HA to accommodate for altered stiffness and compromised HA levels in these pathologies. The SF hydrogels will be grafted with a novel HA binding peptide (HABP) to aid in the attraction of these HA. Our goal is to create a therapeutic biomaterial platform to treat diabetic fibroblasts into a healthy phenotype. Creation of silk hydrogels. Mechanical mixing and lyophilization were used to develop the 3D SF hydrogels of varying stiffnesses. Silk binding peptide (SBP) motifs were used to aid in the grafting of the HABP to the SF hydrogels. SEM imaging, swelling ratio, and DMA were used to physically characterize the hydrogels. A carbazole assay was used to measure the endogenous HA binding capacity. Diabetic fibroblast physiology Metabolic activity, proliferation, and cell morphology assays were used to measure the effected of the SF hydrogel HA binding capacity on dhDF. To evaluate the role of HABP directed HA deposition on dhDF physiology we performed a PCR array targeting wound healing associated genes. We found softer substrate stiffness caused the dhDF to behave more like the hDF in both metabolic activity and with the cell morphology. There was a 15.6% decrease in the aspect ratio and a 16.4% increase in circularity for the dhDF when placed on a softer surface. This data was indicative that the dhDF were beginning to exhibit more fibroblast like behaviors on a softer surface. In light of these results, we developed microporous SF hydrogels and current studies undergoing to incorporate HABPs through silk binding peptide sequences. The swelling ratio of these SF hydrogels are 26.04 1.77% and the pore size is 62.056 ± 31.985 mm. Based on our findings we anticipate that the SF hydrogels with HABP to will retain endogenous HA and cause therapeutic changes in the physiology of the dhDF similar to our stiffness studies.

L3.05

Finite Element Analysis, Preclinical, And Proteomic Assessment Of A Novel 7-Day Extended Wear Peel And Place Negative Pressure Wound Therapy Dressing
Diwi Allen1, Samantha A. Mann1, Balakrishna Haridas2, Brenda Marchand1, Marisa Schmidt1, Kris Kieswetter1
1Medical Solutions Division, 3M Company, San Antonio, TX, United States 2Device & Implant Innovations, College Station, TX, United States
Finite Element Analysis, Preclinical, And Proteomic Assessment Of A Novel 7-Day Extended Wear Peel And Place Negative Pressure Wound Therapy Dressing

Diwi Allen1, Samantha A. Mann1, Balakrishna Haridas2, Brenda Marchand1, Marisa Schmidt1, Kris Kieswetter1 1Medical Solutions Division, 3M Company, San Antonio, TX; 2Device & Implant Innovations, College Station, TX

Problem: Negative pressure wound therapy (NPWT) with reticulated open cell foam (ROCF)^ necessitates frequent dressing changes as tissue ingrowth may occur if left in place for greater than 72 hours.
Objective: These studies evaluated key wound healing-associated characteristics of a novel NPWT peel and place dressing† designed for extended wear use.
Methods: Finite element analysis (FEA) was conducted using computer simulation to evaluate the effects of NPWT on tissue deformations produced by the peel and place dressing. Wound models were developed, with clinical input, incorporating dimensional specifications and material properties for the relevant tissue layers (epidermis, dermis, subcutaneous fat, muscle, and bone). An additional preclinical assessment was conducted using a swine model with full-thickness, excisional paraspinal wounds in 11 animals, and continuous -125mmHg NPWT for 7 days. The study was designed to assess long term wear; therefore, no dressing changes were performed throughout the 7-day study. The wounds were dressed with either peel and place dressing or ROCF. Biopsies were collected for protein extraction and tissues excised for histology at study termination. The extracted protein was assessed using a multiplex immunoassay to quantify wound healing biomarkers, and histologic morphometry was used to measure granulation tissue thickness. The study was approved by an Institutional Animal Care and Use Committee (IACUC) and animal care complied with all applicable national and local regulations.
Results: FEA revealed homogenous tissue displacements, uniform tissue tensile strains, and notable volume of tissue engagement. Preclinical results demonstrated significantly more granulation tissue than ROCF (p<0.05) with tissue ingrowth being limited to only the ROCF treatment (p<0.0001). Proteomic analysis of wound healing-associated cytokines/chemokines and heparin-binding endothelial like growth factor (HB-EGF) demonstrated elevated levels of interleukins (IL)-1α, IL-1β, IL-1ra, IL-8, IL-12, and HB-EGF in the peel and place dressing treatment as compared to ROCF (p<0.05).
Conclusion: Greater mechanical stimulation generated by the peel and place dressing, as illustrated by FEA, is likely to have contributed to cell signal transduction, promoting elevated levels of wound healing biomarkers. The elevated presence of these critical biomarkers, in turn, supported greater granulation tissue formation promoted by the peel and place dressing. This outcome, along with mitigated tissue ingrowth, support the effectiveness of the peel and place NPWT dressing for 7-day extended-wear.
3M™ V.A.C.® Therapy; ^3M™ V.A.C.® Granufoam™ Dressing; † Peel and Place Dressing (3M, San Antonio, TX)

L3.06

Wound Histology Through Virtual Staining Using Generative Adversarial Networks
Malavika Nidhi, Jake Jones, Alan Woessner, Kyle Quinn
Biomedical Engineering, University of Arkansas, Fayetteville, AR, United States
Wound Histology Through Virtual Staining Using Generative Adversarial Networks

Malavika Nidhi, Jake Jones, Alan Woessner, Kyle Quinn Biomedical Engineering, University of Arkansas, Fayetteville, AR

Hematoxylin and Eosin (H&E) staining is the gold standard for visualizing wound microstructural features. However, traditional biopsy, sectioning, and staining is destructive, variable, and cannot capture dynamic events. Label-free multiphoton microscopy (MPM) allows for non-invasive in vivo 3D visualization of tissue microstructure using the endogenous fluorescence of cellular metabolic cofactors as well as second harmonic generation of collagen. However, MPM-generated images are unfamiliar to pathologists, clinicians, and biologists accustomed to interpreting H&E images. Bridging this familiarity gap is crucial for integrating advanced imaging into established diagnostic practices. Generative Adversarial Networks (GANs) are deep learning models used for style transfer and image-to-image translation tasks. Typical GANs consist of a Generator network that can create or modify images and a Discriminator network that tries to distinguish real and computer-generated images, guided by loss probabilities. A Cycle GAN is a specific GAN architecture useful for unpaired image translation and employs two Generators (along with two adversarial Discriminators) to translate images between two domains. The objective of this project was to develop a Cycle GAN capable of translating unstained MPM images of wounds to resemble H&E sections. A Cycle GAN architecture was written in Python utilizing PyTorch. The Generator networks learned to transform MPM images of unstained tissue to resemble H&E stained sections and vice versa, while the Discriminator networks learned to tell the difference between the generated images and real H&E or MPM images. All the networks learned from mistakes of the Discriminators during training, and the Generators also learned from how similar computer-generated images look when transferred from one image domain and back. Our Cycle GAN was trained over 80 epochs on diverse MPM and H&E image datasets of excisional skin wounds. Visual comparisons between virtually- and chemically-stained images revealed promising outcomes, with the Generator preserving tissue morphology at a high resolution, while accurately staining epidermis and follicles purple and collagen pink. Most measures of loss (i.e. network error) decreased throughout training, with the noteworthy exception of the Discriminator loss. This suggests a successful outcome, indicating the Generators created convincing virtual staining that the Discriminators ultimately could not discern from real chemically-stained sections. This study demonstrates the potential of virtual staining via Cycle GAN to bridge the familiarity gap between MPM and traditional H&E staining. It provides a framework for obtaining high-resolution H&E images of wounds from in vivo imaging without the need for a tissue biopsy, sectioning, or staining.

L4.01

Easing The Pressure In Wound Therapy: Evaluating Chatgpt’s Management And Treatment Of Wounds
Daniel Najafali1, Timothy W. King2
1Plastic and Reconstructive Surgery, Carle Illinois College of Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA, Urbana, IL, United States 2Plastic and Reconstructive Surgery, Loyola University Chicago Stritch School of Medicine, Maywood, IL, United States
Easing The Pressure In Wound Therapy: Evaluating ChatGPT’s Management And Treatment Of Wounds

Daniel Najafali1, Timothy W. King2 1Plastic and Reconstructive Surgery, Carle Illinois College of Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA, Urbana, IL; 2Plastic and Reconstructive Surgery, Loyola University Chicago Stritch School of Medicine, Maywood, IL

Background: ChatGPT, a large language model utilizing generative artificial intelligence (AI), is the fastest-growing consumer application. As an emerging tool that is convenient, it has been accessed for medical advice and applied to healthcare with potential in providing patient education and guidance to practitioners in the management of cases. Wound care is a critical and common medical concern. This study comprehensively evaluates ChatGPT’s capacity to answer users’ questions regarding wound care, efficacy in providing evidence-based recommendations, and effectiveness in offering treatment strategies extrapolated from images from performed cases.
Methods: ChatGPT (GPT-3.5) was prompted using open-ended frequently asked questions that were web scraped and adapted from institutional medical websites. Common Questions About Wound Care from the American Academy of Family Physicians (AAFP) were also used. Agreement metrics between modalities were calculated and overlapping keywords were compared. The chatbot’s references were scrutinized. Images from published open access case reports pertaining to wound care were provided to GPT-4 to determine its assessment and recommendations for treatment support.
Results: Across 6 institutions and the prompts (22 questions) provided, 22 (100%) answers were in agreement with the chatbot’s response and 55/56 (98%) keywords were overlapping. The request for references retrieved 22 publications with 21 (95%) being legitimate (9 were open access). From the AAFP prompts, 8 (100%) agreed and 23 (74%) keywords were overlapping. A total of 10 images from 2 female and 4 male patients were provided from 5 published case reports with a mean (SD) age of 47 (12) years. GPT-4 demonstrated competence with initially assessing and providing global recommendations for hypothetical treatment approaches as well as highlighting negative pressure wound therapy settings, wound healing stages, and possible flap coverage, among other factors, but in the majority of cases diverted from offering exact specifications for management of the patient when questions were open ended. Binary prompting or offering a range of treatment options in conjunction with providing patient information, wound characteristics, and medical history was a superior approach.
Conclusion: ChatGPT demonstrated a comprehensive understanding of wound care and management. Although prone to possible hallucinations and varying performance based on prompting scheme, ChatGPT and its successor, GPT-4, have immense potential in serving as an adjunct to patients and providers. Fine tuning a chatbot using relevant articles and cases may be a valuable investment for more profound strides in AI-based involvement for wound care.

L4.02

DNA Methylation Profiling for the Prediction of Recurrences and Prognosis in Patients with Diabetic Foot Ulcers
Sik Namgoong
Plastic Surgery, Stanford University, Irvine, CA, United States
DNA Methylation Profiling for the Prediction of Recurrences and Prognosis in Patients with Diabetic Foot Ulcers

Sik Namgoong Plastic Surgery, Stanford University, Irvine, CA

Background: Diabetic foot ulcers (DFUs) are recalcitrant to healing. However, the molecular mechanism causing this dysfunction is not fully understood. DNA methylation profiles change during the proliferation, differentiation, and development of an organism, resulting in tissue or disease identification. To elucidate the biomarkers for DFU prognosis, we hypothesized that differences in DNA methylation patterns could provide important therapeutic targets in the treatment of DFUs.
Methods: We collected 48 blood samples from 36 DFU patients treated at Korea University Guro Hospital from October 2019 to November 2021. The Illumina MethylationEPIC (850k) DNA methylation microarray was used to determine the pattern between differentially methylated regions (DMRs) in DFU patients with good or poor prognoses. We then selected and visualized the DMRs in the form of heatmaps, and enriched terms associated with these DMRs were identified. By using the DMR list in two processes, Kyoto Gene and Genome Encyclopedia (KEGG) and gene ontology (GO) analysis, gene-concept network, GSEA, and decision tree were performed.
Results: In total, 92 DMRs and 108 DMRs (|Log2 fold change| > 0.1 and P < 0.03) were hypermethylated and hypomethylated, respectively. In the good prognosis sample, 69 and 156 DMRs were hypermethylated and hypomethylated, respectively. In the KEGG analysis, the MAPK signaling pathway was commonly detected as the highest pathway. In the decision tree, MORN1 hypomethylation and NCOR2 hypermethylation were crucial classifiers by recurrence.
Conclusion: Collectively, MORN1 and NCOR2 genes may be used as biomarkers for predicting the recurrences and prognosis in DFU patients. In DFUs, the clues of recurrence and prognosis prediction may be provided through DMRs and the molecular mechanisms related to inflammation.

L4.03

Racial Disparities In Incidence Of And Access To Hypertrophic And Keloid Scar Management
Krish V. Shah1, Stuti P. Garg2, Robert D. Galiano2
1Case Western Reserve University School of Medicine, Cleveland, OH, United States 2Northwestern University Feinberg School of Medicine, Chicago, IL, United States
Racial Disparities In Incidence Of And Access To Hypertrophic And Keloid Scar Management

Stuti P. Garg2, Krish Shah1, Robert Galiano2 1Case Western Reserve University School of Medicine, Cleveland, OH; 2Northwestern University Feinberg School of Medicine, Chicago, IL

Background: Incidence of hypertrophic and keloid scars is influenced by inherent skin attributes such as skin color. Both are clinically challenging to forecast and manage. This study aims to understand differences in occurrence and access to treatment to normalize skin color as consideration of scar management following surgery or trauma in order to achieve optimal results.
Methods: Using the All of Us Research Program’s Data and Research Center (DRC), the incidence of hypertrophic and keloids scars segmented by race was retrospectively analyzed. Proportions of scars for Black and White self-identifying patients were compared to the rates of access to treatment, defined as either topical or revision protocol to address scars by race. Chi-squared test and Mann–Whitney U test were used to determine statistical significance between groups.
Results: 10,910 total scars were included. Hypertrophic and keloid scars represented 16.0% and 12.6% of these scars across all races. Scars in Black patients represented 31.9% of hypertrophic scars and 34.5% of keloids while overall scars from Black patients only represented 17.4% of the total (p<0.05). Black patients only represented 20.2% of all scars treated topically and 20.0% of scars treated with a revision protocol. There was a statistically significant difference between the proportion of hypertrophic and keloid scars represented by Black patients and the proportion of scars treated (p<0.05).
Conclusion: Black patients have a disproportionately lower rate of access to treatment as compared to the rate of incidence of hypertrophic and keloid scars. As Black patients are more likely to have worse perceptions and lower probability of improvement for these scar types, it is important to increase access to treatment options. This study highlights the need to address patient race when considering the healing process of wounds.

L4.04

Evaluating TEWL as a Predicitve Marker for Reulceration in Healed Chronic Diabetic Foot Ulcers
Rawlings E. Lyle1, Pallas Lim2, Mirabel Dafinone2, Sara Dahle3, Rivkah Isseroff4
1School of Medicine, University of California, Davis, Sacramento, CA, United States 2Dermatology Section, VA Northern California Health Care System, Mather, CA, United States 3Podiatry Section, VA Northern California Health Care System, Mather, CA, United States 4Department of Dermatology, University of California, Davis, Sacramento, CA, United States
Evaluating TEWL as a Predicitve Marker for Reulceration in Healed Chronic Diabetic Foot Ulcers

Rawlings E. Lyle1, Pallas Lim2, Mirabel Dafinone2, Sara Dahle3, Rivkah Isseroff4 1School of Medicine, University of California, Davis, Sacramento, CA; 2Dermatology Section, VA Northern California Health Care System, Mather, CA; 3Podiatry Section, VA Northern California Health Care System, Mather, CA; 4Department of Dermatology, University of California, Davis, Sacramento, CA

Background: Chronic diabetic foot ulcers (DFUs) present a significant healthcare challenge due to their high recurrence rate and associated morbidity. Identifying reliable predictive markers for ulcer reulceration is critical for improving patient outcomes. This study investigates the role of transepidermal water loss (TEWL) as such a marker. TEWL’s potential relevance stems from its reflection of skin barrier function, which is crucial in wound healing and prevention of complications.
Methods: Nineteen patients, averaging 69 years of age (±6.4 years), including 50% with a smoking history, were enrolled. These individuals had an average Charlson comorbidity index of 5.7 (±1.5) and a mean diabetes duration of 21 years (±6.7 years). Wound chronicity varied from 8 to 53 weeks with a mean of 20.8 weeks. TEWL was measured using a Vapometer™ device at the healed wound site, 5.0 cm from the wound site, the identical site on the contralateral leg, and 5.0 cm from the contralateral site at three to four monthly visits. Reulceration occurred at a mean of 78.7 weeks (±60.6 weeks) after initial healing, and the time from the last TEWL reading to reulceration ranged from 4 to 146 weeks.
Results: A significant difference was observed in TEWL values at the healed ulcer site between patients whose ulcers remained healed and those who re-ulcerated within 3 months to 3 years post-study. Patients who did not re-ulcerate had a mean TEWL at the healed wound site of 22.85, compared to 27.04 in patients who subsequently re-ulcerated (p<0.001). The difference in TEWL between the wound and contralateral sites was 4.46 in patients who did not re-ulcerate and 7.61 in those who re-ulcerated (p=0.005). Significance and analysis were determined by T-tests and generalized linear modeling.
Conclusions: The results indicate that TEWL, especially the previously unrecognized variance between the wound and contralateral sites, may serve as a reliable indicator for predicting the risk of re-ulceration in patients with healed DFUs. However, the generalizability of these findings is limited due to the small sample size, suggesting a need for further research with larger cohorts. Future studies should aim to establish TEWL norms in healthy individuals for better comparative analysis and the development of effective preventative strategies for diabetic patients, considering the high 5-year mortality rate associated with DFUs.

L4.05

Discarded Wound Dressings: An Untapped Source Of Predictive And Monitoring Biomarkers Of Multiple Types Of Wounds
Victoria Soto1, Sujad Younis2, Natasa Strbo2, Matthew Hardy3, Jason Levine4, Monica Perez4, Juan O. Bravo1, Hadar Lev-Tov1, Robert S. Kirsner1, Ivan Jozic1
1Dermatology, University of Miami School of Medicine, Miami, FL, United States 2Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, United States 3Miami VA Medical Center, Miami, FL, United States 4Division of Endocrinology, Metabolism and Diabetes, University of Miami Miller School of Medicine, Miami, FL, United States
Discarded Wound Dressings: An Untapped Source Of Predictive And Monitoring Biomarkers Of Multiple Types Of Wounds

Victoria Soto1, Sujad Younis2, Natasa Strbo2, Matthew Hardy3, Jason Levine4, Monica Perez4, Juan O. Bravo1, Hadar Lev-Tov1, Robert S. Kirsner1, Ivan Jozic1 1Dermatology, University of Miami School of Medicine, Miami, FL; 2Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL; 3Miami VA Medical Center, Miami, FL; 4Division of Endocrinology, Metabolism and Diabetes, University of Miami Miller School of Medicine, Miami, FL

Purpose: The purpose of our study was to utilize discarded wound dressings in order to identify potential predictive and diagnostic biomarkers of different wound types by the proteomic analysis of cells and exosomes captured from wound exudate.
Methods: We solubilized biomaterial from discarded wound dressings from patients with pyoderma gangrenosum, diabetic foot ulcer, venous leg ulcer and acute wounds left to heal by secondary intention. Patients were followed weekly for 4 weeks and change in wound size measured by EKARE inSight imaging equipment at each visit, at which point the wound dressing was collected. Cells from each dressing were then isolated, number of viable cells quantified and immunophenotyping using flow cytometry. Differential centrifugation was then used to isolate exosomes from each specimen, with exosomes purified using magnetic beads and validated by western blotting and FACS flow cytometry. Next, all samples were lysed and normalized to total protein, followed by trapped ion mobility spectrometry analysis of cellular and exosomal proteome. Lastly, we utilized mass-to-charge ratio (m/z) to identify and quantify relative peptide species, followed by analysis of hierarchical clustering of samples and functional annotation of identified clusters.
Results: Although we were able to isolate viable cells and exosomes from multiple types of wound dressings, foam and alginate dressings yielded highest levels of viable cells (5.8-7.9 x 107 cells). Next, from immunophenotyping, we were able to determine those wounds which exhibited higher levels of gamma/delta T-cells (CD45+CD3+TCRg/d+) also exhibited trends towards reductions in wound size. Numbers of dendritic (CD45+-CD11c+), macrophages -CD11+), neutrophils (-CD66b+) or either CD4+/CD8+ T-cells did not correlate with the surrogate healing outcome at week 4, nor where their relative numbers able to predict healing outcome from week 1 samples. Next, we were able to validate previous proteomic profiling reports of wound exudate that associated with poor healing outcomes (CTSG, LCN2, DEF1, MMP9, HBA, HBB, CAMP, VTN, among others). Lastly, our data suggests that healing wounds exhibit higher levels of ALB, SERPING1, CERU, CO3, POSTN, FN1, FGA (among others) in their week 1 dressings, and may act to serve as potential predictive biomarkers of healing wounds.
Conclusions: Together, our innovative approach to proteomic profiling of various types of wound dressings has demonstrated reproducible and encouraging results and we anticipate that higher recruitment (expected n=30 from each wound type), will shed light on variability in healing outcomes and identify other potential biomarkers.

L4.06

Detection of Predictive Human Genetic Markers Of The Wound Microbiome
Rebecca Gabrilska1, Khalid Omeir2, Ashley Noe2, Jacob Ancira2, Clint Miller3, Craig Tipton2, Kendra Rumbaugh1, Joseph Wolcott3, Nicole Phillips4, Caleb Phillips2
1Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, United States 2Biological Sciences, Texas Tech University, Lubbock, TX, United States 3Southwest Regional Wound Care Center, Lubbock, TX, United States 4Microbiology, Immunology & Genetics, University of North Texas Health Sciences Center, Fort Worth, TX, United States
Detection of Predictive Human Genetic Markers Of The Wound Microbiome

Rebecca Gabrilska1, Khalid Omeir2, Ashley Noe2, Jacob Ancira2, Clint Miller3, Craig Tipton2, Kendra Rumbaugh1, Joseph Wolcott3, Nicole Phillips4, Caleb Phillips2 1Surgery, Texas Tech University Health Sciences Center, Lubbock, TX; 2Biological Sciences, Texas Tech University, Lubbock, TX; 3Southwest Regional Wound Care Center, Lubbock, TX; 4Microbiology, Immunology & Genetics, University of North Texas Health Sciences Center, Fort Worth, TX

Background: Chronic wounds are a burden to millions of patients and healthcare providers worldwide. With rising incidence and prevalence, there is an urgent need to address these non-healing wounds with novel approaches. Impaired wound healing has been shown to be associated with the wound microbiota, as multiple bacterial species are known to contribute to infection and delays in closure. Whether these microbial communities are shaped by host genetics is less understood. Microbiome genome wide association studies (mbGWAS) can provide insight into host genetic factors that may influence bacterial community structure. Previous work reports that the alpha diversity of the chronic wound microbiome is significantly associated with specific human genomic loci and healing.
Methods: To compare wound microbiomes to human genomes, we performed a two-stage mbGWAS using a cohort of 458 patients. Briefly, patients with lower extremity, non-healing wounds were consented from the Southwest Regional Wound Care Center in Lubbock, TX. Buccal swabs and wound samples were collected then genotyped and sequenced for bacterial taxa, respectively. After quality control and genome imputation, mbGWAS was then performed to test association of the relative abundances of multiple wound-relevant bacteria with patient genotype.
Results: We identified bacterial taxa that are repeatedly significantly associated with single nucleotide polymorphisms (SNPs) in the host genome. The significance threshold was set at the Bonferroni adjusted p value of <0.05. Hundreds of unique SNPs across multiple unique bacterial species were repeatedly associated, and these species included both common and uncommon members of wound microbiomes.
Conclusions: Using an mbGWAS design, we reveal human genomic markers that significantly associate to specific bacterial taxa, suggesting a genetic predisposition to colonization or infection by members of the wound microbiome. Future studies will involve translating these mbGWAS findings to functionally validate likely causal SNPs. Identification of correlated biomarkers may provide new mechanistic insight into microbe-host interactions and may serve as predictive risk factors to guide personalized management for chronic wound patients.

O1.01

Impact of Extracellular Matrix Graft Composition on Degradation Dynamics and Scaffold Functionality
Katrina Harmon, Miranda Burnette, Justin Avery, Kelly Kimmerling, Katie Mowry
Research & Development, Organogenesis, Birmingham, AL, United States
Impact of Extracellular Matrix Graft Composition on Degradation Dynamics and Scaffold Functionality

Katrina Harmon, Miranda Burnette, Justin Avery, Kelly Kimmerling, Katie Mowry Research & Development, Organogenesis, Birmingham, AL

Extracellular matrix (ECM) grafts are emerging as a promising treatment option for chronic wounds due to their scaffold properties. Here, two porcine small intestinal submucosa-derived cross-linked collagen matrices with PHMB (PCMP,2layers; PCMP-XT,5layers) were compared to ovine forestomach matrix (OFM^,1layer) derived from propria submucosa and assessed for their durability in a simulated wound environment and functionality as a scaffold by their ability to inhibit proteases and support cell attachment and proliferation. Matrices were evaluated for structure using scanning electron microscopy (SEM) and histology, ability to modulate matrix metalloproteinases (MMPs) using fluorometric assays, and graft durability using an in vitro degradation model comprised of simulated wound fluid plus collagenases type I and II (SWF+). Throughout degradation, scaffold functionality was assessed using an in vitro primary human dermal fibroblast cell attachment model.
Grafts differed in matrix structure, composition, and thickness. SEM demonstrated a more fibrous structure in OFM and PCMP-XT, which was confirmed following histological evaluation. When evaluating tissue thickness, PCMP-XT was significantly thicker than PCMP, while PCMP and OFM were comparable. All matrices resulted in inhibition of collagenases, gelatinases, and stromelysins, but PCMP and PCMP-XT were overall more inhibitory compared to OFM. In an in vitro SWF+ degradation model, OFM degraded rapidly (<3 hours) and was therefore omitted from further analysis. Both PCMP and PCMP-XT significantly degraded in the SWF+ model within 7 days (p<0.0001), with PCMP having a significantly higher degradation rate (p<0.05). To assess scaffold functionality, intact (non-degraded) and degraded grafts were seeded with primary human fibroblasts and evaluated for attachment and proliferation over 7 days. Cells readily attached to both matrices. Cells seeded on PCMP resulted in significant proliferation on days 3 and 7 (p<0.01), while cells seeded on PCMP-XT demonstrated significant proliferation at day 7 (p<0.05). To assess the ability of matrices to support cell attachment through degradation, 3-and 5-day SWF+ degraded scaffolds were evaluated for cell proliferation for 7 days. PCMP and PCMP-XT exhibited robust cell attachment on 3- and 5-day degraded scaffolds, with significant proliferation on both by day 7.
Interestingly, 5-day degraded PCMP resulted in overall more proliferation compared to 3-day degraded samples on day 7 (p<0.01), while proliferation was comparable for both 3- and 5-day degraded PCMP-XT groups (p<0.0001).
Together, these findings demonstrate that differing compositions of ECM grafts result in varying MMP inhibition and durability. Throughout degradation, both PCMP and PCMP-XT demonstrated a maintenance of scaffold properties by supporting fibroblast attachment and proliferation.
Puraply® AM; Puraply® AM-XT, Organogenesis, Canton, MA Endoform, Aroa Biosurgery, San Diego, CA

O1.02

The Balance Between Cell Contractility And Adhesion Modulates Provisional Matrix Assembly During Wound Closure
Emily Davis, Marina Uroz, Christopher Chen, Jeroen Eyckmans
Biomedical Engineering, Boston University, Brookline, MA, United States
The Balance Between Cell Contractility And Adhesion Modulates Provisional Matrix Assembly During Wound Closure

Emily Davis, Marina Uroz, Christopher Chen, Jeroen Eyckmans Biomedical Engineering, Boston University, Brookline, MA

Upon injury of fibrous tissues, resident fibroblasts migrate into the wound bed, depositing a fibrous provisional matrix and pull the margins of the wound closer to accelerate wound closure. While cellular contractility and cell adhesion are two processes regulating wound healing, it remains unclear how these processes coordinate provisional matrix assembly during stromal closure. To address this question, we manipulated cell contractility and cell-matrix adhesion of Normal Human Dermal Fibroblasts Neonatal (NHDFneos) and Normal Human Lung Fibroblasts (NHLFs) and assessed wound closure in a 3D microtissue model of stromal wound healing. Briefly, NHDFneos and NHLFs were embedded in a type I collagen gel and seeded into μTug devices composed of wells with micropillars to form 3D microtissues. 24 hours after the tissues formed, a Nd:Yag Laser was used to create full-thickness wounds in the center of the tissue. Both NHDFneo and NHLF tissues were treated with contractility or adhesion modulators before injury and wound closure dynamics were assessed for 24 hours post-injury using time-lapse microscopy. Traction force microscopy was performed on the individual cell types seeded on type I collagen coated 5 kPa polyacrylamide gels under different treatment conditions using a Nikon-Eclipse-Ti microscope. Immunofluorescent staining against phosphorylated-paxillin was performed to visualize and quantify size of focal adhesions. In some experiments, staining against cellular fibronectin was performed to visualize provisional matrix assembly of fibers in tissues 24 hours post-injury.
At baseline, NHDFneos healed the wounds in the tissues within 24 hours post-injury, while NHLF tissues failed to close the wounds. On 2D substrates, NHDFneos exhibited lower contractility and smaller adhesions compared to NHLFs. Treatment with manganese (Mn) increased focal adhesion size and contractility of NHDFneos as well as inability to heal full-thickness wounds in tissues. Mn-treated NHDFneo tissues revealed clustering of cellular fibronectin with little to no fibrillogenesis throughout the tissue post-injury, while untreated tissues assembled fibronectin fibers throughout the remodeled tissue post-injury. Conversely, treatment of NHLFs with a low dose FC11, a FAK inhibitor, lowered contractility and the size of focal adhesions to comparable levels as untreated NHDFneos. NHLF tissues treated with FC11 healed within 24 hours post-injury while untreated tissues failed to heal (N = 3, n = 20 tissues per group). Interestingly, NHLFs treated with blebbistatin and Y-27632, a myosin II and ROCK inhibitor respectively, exhibited lower contractility, but continued to fail healing tissues. Together, our data support the hypothesis that the balance between contractility and adhesion levels modulates wound closure in our microtissue system, which may inform new mechanotherapeutic strategies for treating impaired wound healing.

O1.03

Mechano-Immuno-Fibrotic Wound Healing Pathways Regulate The Cellular And Molecular Ecology Of Foreign Body Response
Andrew Hostler, William Hahn, Jenne Stensland, Katharina Fischer, Abdelrahman Alsharif, Maria Gracia Mora Pinos, Hudson C. Kussie, Autumn Lester, Fidel Saenz, Jonathan P. Yasmeh, Eamonn McKenna, maisam Jafri1, Maia Granoski, Jose Vasquez, Amelia B. Knochel, Aaron Mason, Kellen Chen, Geoffrey C. Gurtner
Surgery, University of Arizona College of Medicine -Tucson, Tucson, AZ, United States
Mechano-Immuno-Fibrotic Wound Healing Pathways Regulate The Cellular And Molecular Ecology Of Foreign Body Response

Andrew Hostler, William Hahn, Jenne Stensland, Katharina Fischer, Abdelrahman Alsharif, Maria Gracia Mora Pinos, Hudson C. Kussie, Autumn Lester, Fidel Saenz, Jonathan P. Yasmeh, Eamonn McKenna, maisam Jafri, Maia Granoski, Jose Vasquez, Amelia B. Knochel, Aaron Mason, Kellen Chen, Geoffrey C. Gurtner Surgery, University of Arizona College of Medicine -Tucson, Tucson, AZ

Background: Implantable biomedical devices have revolutionized medicine, with >70 million device implantations annually that benefit millions of patients. 30% of devices will undergo premature failure during their lifetime, primarily due to an immune-mediated physiologic reaction known as the foreign body response (FBR).
Biophysiochemical incompatibilities between host tissues and device biomaterials, regulated through immune signaling, activates the wound healing pathway resulting in fibrosis and collagenous tissue encapsulation of the device. Herein, we characterized the cellular and molecular ecology of human FBR capsule tissue. Methods: Breast implant FBR tissue was collected with an IRB protocol from patients (n=14) undergoing standard of care surgical explanation of biomedical devices. Baker capsular contracture grading (B1: n=5; B2: n=3, B3: n=2, B4: n=4) was evaluated. Histological staining and analysis using hematoxylin and eosin (H&E), Masson’s trichrome, and picrosirius red was performed to assess tissue morphology, collagen deposition, and collagen architecture. Immunofluorescent staining and analysis was performed using CD68 (macrophage marker), aSMA (myofibroblast marker), and FAK (mechanical signaling marker).
Results: Severe FBR, Baker grades 3 & 4 (B3/B4), demonstrated significantly increased capsule thickness on H&E staining compared to mild FBR B1/B2 (p=0.0006). Analyzing collagen architecture with software algorithms CT-FIRE and CurveAlign, we found severe FBR significantly increased collagen fiber length compared to mild FBR (p=0.0408), while also creating a trend of increased collagen fiber angle and density (p=0.1920 and p=0.1774) and decreased mean distance (p=0.1093). Severe FBR caused an increase in CD68+ (macrophage) and aSMA (myofibroblast) layering within the FBR capsule, shown by significantly increased co-localized expression of CD68 and aSMA (p<0.01) and co-localized CD68 and FAK (p=0.0465). More severe FBR also increased aSMA and immune cell numbers (p<0.009).
Conclusions: We uncovered that increased immune-mediate crosstalk between macrophages and myofibroblasts within the FBR capsule correlated with higher degree Baker capsules, characterized by elevated fibrosis and collagen deposition. Interestingly, more severe FBR in humans also caused more colocalized macrophage-myofibroblast cellular layering, as well as elevated mechanoresponsive myeloid cells. Since myeloid cells circulate through the blood stream and home to sites of wound injury, targeting these myeloid cells could lead to new therapies to prevent FBR.

O1.04

Senescent-Associated Extracellular Matrix Production And Delayed Wound Healing By Senescent Dermal Fibroblasts
Anish Srinivas Vasan1, Emma Lejeune1, Wilson Wong1, Daniel S. Roh2, Christopher Chen1, Jeroen Eyckmans1
1Biomedical Engineering, Boston University, Boston, MA, United States 2School of Medicine, Boston University, Boston, MA, United States
Senescent-Associated Extracellular Matrix Production And Delayed Wound Healing By Senescent Dermal Fibroblasts

Anish Srinivas Vasan1, Emma Lejeune1, Wilson Wong1, Daniel S. Roh2, Christopher Chen1, Jeroen Eyckmans1 1Biomedical Engineering, Boston University, Boston, MA; 2School of Medicine, Boston University, Boston, MA

Background: After skin injury, a sub-population of the dermal fibroblasts undergo senescence during the early stages of the healing process. While the transient appearance of senescent cells is required for normal wound healing, the accumulation of persistent senescent cells is associated with delayed and chronic wound healing. The mechanisms by which senescent fibroblasts and their associated extracellular matrix (ECM) impair wound healing are still elusive. Given the role of fibroblasts to produce a provisional matrix during tissue closure, we hypothesize that senescent human dermal fibroblasts produce a senescent-associated ECM that has a distinct composition and tissue architecture compared to non-senescent fibroblasts which delays wound closure.
Methods: To address this hypothesis, we leveraged our 3D wound-on-chip microtissue platform to study the effect of senescent cells on stromal tissue repair and ECM architecture. Microfabricated devices consisting of 4 micropillars were cast in a 96-well plate using polydimethylsiloxane (PDMS). We seeded normal or senescent human dermal fibroblasts from neonatal foreskin in a rat-tail collagen type I matrix and allowed them to self-assemble into anchored microtissues. Senescence was induced in the normal fibroblasts through multiple exposures to hydrogen peroxide and confirmed with positive stains for p21, γ-H2AX, and senescent-associated-β-galactosidase. Microtissues were then ablated to create full-thickness wounds using a pulsed nanosecond laser. The provisional ECM was visualized with immunofluorescence labeling and confocal microscopy.
Results: We found that microtissues composed of senescent human dermal fibroblasts exhibited significantly delayed wound closure (41 ± 6.27 hours, n=9, N=3) when compared to normal human dermal fibroblasts from neonatal foreskin (26.2 ± 3.53 hours, n=9, N=3). Senescent fibroblasts deposited more fibronectin, collagen type I, collagen type III, and tenascin-C in the provisional matrix than normal fibroblasts. Interestingly, confocal microscopy also revealed that senescent microtissues contained larger, aligned fiber bundles of all four ECM components compared to the fine, mesh-like deposition in normal fibroblast microtissues. Notably, tenascin-C was deposited in small globules in normal fibroblast microtissues, highlighting the stark organizational differences between these fibroblast phenotypes.
Conclusion: Together, these data demonstrate that senescent cells produce a senescent-associated matrix with a tissue architecture that is distinct from the ECM produced by non-senescent fibroblasts. This may contribute to the delayed healing observed in senescent microtissues. Our 3D microtissue model provides a robust platform to further study the role of stromal cell ECM deposition during tissue repair, with control over cell populations and ECM density and composition.

O1.05

Lineage Tracing Of Vasculogenic Fibroblasts In Vivo and Their Significance In The Rescue Of Diabetic Ischemic Tissue
Kanhaiya Singh1, Sedat Kacar2, Sumit S. Verma1, Manishekhar Kumar1, Sashwati Roy1, Chandan K. Sen1
1McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, United States 2Indiana University, Indianapolis, IN, United States
Lineage Tracing Of Vasculogenic Fibroblasts In Vivo And Their Significance In The Rescue Of Diabetic Ischemic Tissue

Kanhaiya Singh1, Sedat Kacar2, Sumit S. Verma1, Manishekhar Kumar1, Sashwati Roy1, Chandan K. Sen1 1McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA; 2Indiana University, Indianapolis, IN

Our recent work reported on the identification of the vasculogenic fibroblast (VF) that is capable of generating new blood vessels during tissue repair (Pal et al. Nat Com, 2023). While these cells are physiologic and injury-inducible, the generation of VF is blunted under conditions of diabetes. Such barrier may be overcome by the inhibition of fibroblast miR-200b using tissue nanotransfection technology (PMIDs: 28785092, 34837085, 35819852, 36380587). To understand the mechanisms underlying VF function and significance, in this work we developed miR-200b-429fl/fl COL1A2creER GT(ROSA)mT/mG mice. In these mice, miR-200b regions in fibroblasts (COL1A2+ cells) are knocked out in response to tamoxifen. To gain insight into the significance of VF in vivo, ischemic hind limb studies were performed. Tamoxifen treatment not only led to Cre excision of miR-200b-429 in these mice, but also induced the constitutive expression of ROSA driven tdTomato in fibroblasts with onset of permanent GFP expression. Significant lowering of miR-200b was noted in laser capture dissected dermal fibroblast tissue elements from the wound-edge of tamoxifen treated mice (n=4). Tamoxifen treatment led to significantly enhanced perfusion in animals subjected to ischemic injury as determined by laser speckle perfusion imaging (Perimed Inc.) (n=8). This rescue was associated with increased abundance of GFP+CD31+ VF (n=5) which accounted for 26% of all CD31+ vascular cells in the laser captured blood vessels (n=9). Thus, targeted lowering of miR-200b abundance within fibroblasts resulted in substantial augmentation of VF in vivo which was associated with improved blood flow.

O1.06

Wound Alkalinity Measurement To Prognosticate The Healing Activities of DFUs
Jon Senkowsky2, Shuxin Li1, Wenjing Hu1, Liping Tang1
1Progenitec, Arlington, TX, United States 2Texas Health Physician’s group, Arlington, TX, United States
Wound Alkalinity Measurement To Prognosticate The Healing Activities of DFUs

Jon Senkowsky2, Shuxin Li1, Wenjing Hu1, Liping Tang1 1Progenitec, Arlington, TX; 2Texas Health Physician’s group, Arlington, TX

PURPOSE: The aim of this study was to determine whether and when wound alkalinity can be measured to prognosticate nonhealing DFUs and to predict healing outcome. METHODS: Wound alkalinity was monitored during the routine visits by assessing discarded wound dressings, via a disposable device – DETEC® pH, from 60 DFU patients to prognosticate healing outcome (probability of complete healing in 12 weeks). RESULTS: To determine the optimal time frame, the test sensitivity, specificity, pre- and post-test healing outcome of these patients were correlated with their wound alkalinity measurements from their 1st, 2nd and 3rd visits. Interestingly, we found that alkalinity assessment from the 2nd visit (7-21 days following the first visit) had the highest sensitivity (77.5%) and specificity (80.0%) to predict a non-healing outcome. The pre-test and post-test risk of a wound not healing was 88.9% and 96.9%, respectively, representing an 8.0% increased risk of not healing. The risk of non-alkaline wounds not healing was 69.2%, representing a 19.7% reduced risk of not healing. The improved predictability of the 2nd visit over the first visit can be explained by the wound healing processes. Specifically, following initial wound treatment, such as topical debridement, it takes at least 7 days for the wound to reach its homeostasis. Those patients with alkaline wounds during their 2nd visit suggest that the wounds did not initiate inflammatory responses after the standard of care and the wounds may have a higher chance of non-healing than those with non-alkaline wounds. Further analysis was carried out to determine the optimal time frame of the 2nd visit for prognostic healing outcome by dividing all DFUs patients (48 patients) based on their visit time frame following the first visit (<7 days: 24 patients: 7-21 days: 24 patients). Our analyses show that, as compared to the <7 days group, the alkalinity determination for the 7-21 days group had highest sensitivity (87.5% vs 72.2%) and specificity (62.5% vs. 50.0%) to prognosticate the wound healing outcome. For 7-21 days group, while the pre-test and post-test risk of not healing was 66.7% and 82.4%, respectively, which represented a 15.7% increased risk of not healing. The risk of not healing when the wounds were non-alkaline was 28.6% which represents a 38.1% reduced risk of not healing. The results support that the 2nd visit (7-21 days after the initial diagnosis/ treatment) would be the best time for alkalinity determination with the best non-healing prognostic capability. Using a logistic regression model, we determined that, with all p-values>0.4, age, gender, race, initial wound size, wound location, and infection status were found to have negligible influence on predicted healing outcome based on wound alkalinity. Conclusions: Our analyses show that alkalinity status can prognosticate healing outcome (p=0.0436).

O1.07

Anthocyanins From Black Soybean Seed Coat Prevent Radiation-Induced Skin Fibrosis By Downregulating Tgf-? And Smad3 Expression
Jaehoon Choi
Department of plastic and reconstructive surgery, Keimyung University School of Medicine, Daegu, Daegu, Korea (the Republic of)
Anthocyanins From Black Soybean Seed Coat Prevent Radiation-Induced Skin Fibrosis By Downregulating Tgf-β And Smad3 Expression

Jaehoon Choi Department of plastic and reconstructive surgery, Keimyung University School of Medicine, Daegu, Daegu, Korea (the Republic of)

Background: The aim of this study was to evaluate the protective effects of anthocyanins from the black soybean seed coat against radiation injury in dermal fibroblasts and mouse skin.
Methods: Dermal fibroblasts treated with 50 and 100 μg/mL anthocyanins were irradiated with single doses of 20 Gy. Cell viability, intracellular reactive oxygen species (ROS) production, and mRNA expression were measured. A total of 60 mice were used for an in vivo study. A dose of 100 μg/mL anthocyanins was administered daily for 5 days before or after radiation therapy. Following irradiation (45 Gy), mice were inspected for gross pathology twice per wk for 8 weeks. At 4 and 8 weeks post-irradiation, dorsal skin was harvested for histopathologic examination and protein isolation.
Results: In dermal fibroblasts, treatment with 50 and 100 μg/mL anthocyanins significantly reduced radiation-induced apoptosis at 72 h and intracellular reactive oxygen species generation at 48 h. Furthermore, 100 μg/mL anthocyanins markedly decreased Smad3 mRNA expression and increased Smad7 mRNA expression at 72 h post-irradiation. In mice, treatment with 100 μg/mL anthocyanins resulted in a significant reduction in the level of skin injury, epidermal thickness, and collagen deposition after irradiation. Treatment with 100 μg/mL anthocyanins significantly decreased the number of α-SMA-, TGF-β-, and Smad3-positive cells after irradiation.
Conclusion: Our study demonstrated that black soybean anthocyanins inhibited radiation-induced fibrosis by downregulating TGF-β and Smad3 expression. Therefore, anthocyanins may be a safe and effective candidate for the prevention of radiation-induced skin fibrosis.

O1.08

Granzyme B Mediates Degradation Of Hemidesmosome Proteins In Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis
Michael M. Lane1, Faith Liu1, Alexandre Aubert1, Valerio Russo1, Karen Jung1, Touraj Khosravi2, Hongyan Zhao1, Layla Nabai1, Richard Crawford2, Elizabeth Phillips3, David Granville1
1Pathology and Laboratory Medicine, The University of British Columbia, Port Coquitlam, BC, Canada. 2Dermatology and Skin Science, The University of British Columbia, Vancouver, BC, Canada. 3Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, United States
Granzyme B Mediates Degradation Of Hemidesmosome Proteins In Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis

Michael M. Lane1, Faith Liu1, Alexandre Aubert1, Valerio Russo1, Karen Jung1, Touraj Khosravi2, Hongyan Zhao1, Layla Nabai1, Richard Crawford2, Elizabeth Phillips3, David Granville1 1Pathology and Laboratory Medicine, The University of British Columbia, Port Coquitlam, BC, Canada; 2Dermatology and Skin Science, The University of British Columbia, Vancouver, BC, Canada; 3Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN

Background: Stevens-Johnson syndrome (SJS) /Toxic epidermal necrolysis (TEN) are life-threatening, immune-mediated, cutaneous adverse drug reactions characterized by the separation of the epidermal and dermal layers of the skin, resulting in severe blistering and peeling. Serine protease Granzyme B (GzmB) was recently found to contribute to the sub-epidermal blistering in bullous pemphigoid through the cleavage of α6/β4 integrin, collagen VII, and collagen XVII at the dermal-epidermal junction. In the present study, the role of GzmB in SJS/TEN was investigated.
Hypothesis: GzmB accumulation at the dermal-epidermal junction contributes to sub-epidermal blistering in SJS/TEN through the cleavage of α6/β4 integrin, collagen VII, and/or collagen XVII.
Methods: Skin biopsies collected from SJS/TEN patients (n=8) and healthy participants (n=8) were analyzed using immunohistochemistry to assess protein levels of GzmB and its substrates, α6/β4 integrin, collagen VII, and collagen XVII. ELISA was used to quantify GzmB levels in blister fluid from patients with SJS/TEN (n=6) and was compared that of bullous pemphigoid patients (n=2). Western blotting was used to identify fragments of Collagen XVII in SJS/TEN blister fluid samples (n=6).
Results: Epidermal and dermal GzmB levels were significantly elevated in SJS/TEN compared to healthy skin. SJS/TEN sections exhibited reduced α6/β4 integrin, collagen VII, and collagen XVII at the dermal-epidermal junction compared to healthy skin. Increased levels of GzmB as well as fragments of collagen XVII (around 120 kDa and 97 kDa, as previously observed in GzmB cleavage assays in vitro) were detected in all SJS/TEN blister fluid samples, suggesting that the extracellular proteolytic activity of GzmB is sustained in SJS/TEN.
Conclusions: The present study provides a novel pathological mechanism of action in SJS/TEN whereby elevated levels of extracellular GzmB mediates degradation of dermal-epidermal junction proteins, leading to separation of the epidermis from the dermis

P1.01

SLI-F06, A Fibromodulin-Based Therapeutic Peptide, Enhances Wound Healing In Diabetic Rodent And Pig Models
Pin Ha2, Zhaohan Zeng1, Chenshuang Li3, Joshua Yang2, Evan Yen4, Eric Yen4, Sang Yub Kim2, Elisabeth Leeflang1, Andrew Vardanian2, Kang Ting5, Chia Soo2, ZHONG ZHENG1
1Scarless Laboratories Inc., Cherry Hill, NJ, United States 2University of California, Los Angeles, Los Angeles, CA, United States 3University of Pennsylvania, Philadelphia, PA, United States 4Arcadia High School, Arcadia, CA, United States 5American Dental Association Forsyth Institute, Cambridge, MA, United States
SLI-F06, A Fibromodulin-Based Therapeutic Peptide, Enhances Wound Healing In Diabetic Rodent And Pig Models

Pin Ha2, Zhaohan Zeng1, Chenshuang Li3, Joshua Yang2, Evan Yen4, Eric Yen4, Sang Yub Kim2, Elisabeth Leeflang1, Andrew Vardanian2, Kang Ting5, Chia Soo2, ZHONG ZHENG1 1Scarless Laboratories Inc., Cherry Hill, NJ; 2University of California, Los Angeles, Los Angeles, CA; 3University of Pennsylvania, Philadelphia, PA; 4Arcadia High School, Arcadia, CA; 5American Dental Association Forsyth Institute, Cambridge, MA

SLI-F06 is a fibromodulin-derived peptide that effectively promoted wound healing and reduced scar size without discernable adverse effects in multiple preclinical and clinical acute wound studies. It functions through, in part, enhancing fibroblast migration, myofibroblast differentiation, and contraction. Since chronic wounds are deficient in cell migration and wound contraction, we sought to test SLI-F06’s ability to heal cutaneous wounds in challenging diabetic models. Because over 90% of adult diabetic patients in the US exhibit type 2 diabetes mellitus (DM) rather than type 1, we used a NONcNZO10/LtJ type 2 DM mouse model for our first set of wound studies. Two excisional, full-thickness wounds (6 mm diameter) were created on the back of each mouse. To simulate human-type repair in the loose-skin mouse, we sewed silicone rings to each wound periphery to “splint” the wound to minimize excessive wound margin contraction. Then, we injected SLI-F06 (25 mg/ml) intradermally at four points around the wound edge (25 ml/point; 100 ml total) every other day for 14 days. We documented wound area healing by digital photography and fitted the data to a quantile mixed-effect model with a 95% confidence interval. These data demonstrated that the SLI-F06 application significantly accelerated the wound healing of diabetic NONcNZO10/LtJ mice (P < 0.0001). Moreover, multiple comparisons applying Sidák correction revealed that SLI-F06 markedly enhanced wound healing from Day 1 to 13 (adj. P < 0.05, N = 12). Particularly, during Days 2-8, the quantile mixed-effect model revealed an overall 65.6% (> 30%) faster median wound healing rate with SLI-F06 treatment. Furthermore, the proportion of wounds with complete closure at day 14 post-injury was increased considerably (Mantel-Cox test P = 0.0156; N = 12) at 83.3% in the SLI-F06 group vs. 41.7% in control. Next, we used a streptozotocin-induced diabetic Yorkshire pig model that simulates certain characteristics of later-stage human type 2 non-insulin dependent DM, such as increased triglycerides and glucose intolerance. We excised 1.5 x 1.5-cm square wounds and topically applied 110 ml/cm2 of 25 mg/ml SLI-F06 in a hydroxypropyl cellulose excipient twice/week. Excitingly, topical SLI-F06 administration not only accelerated wound healing as assessed by the healed wound area, but also increased the proportion of wounds with complete closure (SLI-F06: 15.0 day vs. control: 18.0 day; Mantel-Cox test P = 0.0300, N = 7). These promising preclinical data strongly support the effectiveness of SLI-F06 in diabetic wound management, which can significantly improve the quality of life of diabetic patients suffering from chronic wounds that can lead to amputations and death.

P1.02

Linking Human Genetics And Wound Infection With Transcriptome-Wide Associations
Khalid Omeir1, Rebecca Gabrilska2, Jacob Ancira1, Ashley Noe1, Clint Miller3, Craig Tipton1, Kendra Rumbaugh2, Joseph Wolcott3, Nicole Phillips4, Caleb Phillips1
1Biological Sciences, Texas Tech University, Lubbock, TX, United States 2Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, United States 3Southwest Regional Wound Care Center, Lubbock, TX, United States 4Microbiology, Immunology & Genetics, University of North Texas Health Science Center, Fort Worth, TX, United States
Linking Human Genetics And Wound Infection With Transcriptome-Wide Associations

Khalid Omeir1, Rebecca Gabrilska2, Jacob Ancira1, Ashley Noe1, Clint Miller3, Craig Tipton1, Kendra Rumbaugh2, Joseph Wolcott3, Nicole Phillips4, Caleb Phillips1 1Biological Sciences, Texas Tech University, Lubbock, TX; 2Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX; 3Southwest Regional Wound Care Center, Lubbock, TX; 4Microbiology, Immunology & Genetics, University of North Texas Health Science Center, Fort Worth, TX

Introduction: Microbes are believed to be key contributors to wound chronicity, yet our understanding about the reasons for inter-patient variation in the species infecting chronic wounds remains unclear. Previous investigations reveal that human genetic variation may partially explain differences in infection and may have to do with how our genetic variation encodes variation in wound bed cellular phenotypes. Because the wound bed is comprised of multiple cell and tissue types, we conducted a microbiome transcriptome-wide association study to investigate how patient genetic variation determines tissue-specific expression and in turn relates to infection. Significant and repeatable gene expression associations may provide insights into the mechanistic details of chronic wound bacterial infection.
Methods: In a deidentified fashion, consenting patients provided buccal swabs and wound debridement samples. The buccal swabs were genotyped at 660k loci, and chronic wound bacterial microbiota were characterized via 16S sequencing. Whole genome imputation was performed to increase genomic coverage and improve subsequent gene expression imputation. Single nucleotide polymorphisms (SNPs) were used to predict patients’ gene expression levels and splicing patterns for artery, blood, fibroblast, skeletal muscle, skin, subcutaneous fat, and nerve tissue. A balanced and race-stratified two-cohort design of 458 patients was used. Across tissues, there was an average of 12,217 expressed genes and 30,100 splice variants considered. The effect of these genes on transformed relative abundances of 68 bacterial species was evaluated.
Results and Conclusion: The genetically regulated expression of more than 200 differentially expressed genes and splice variants were found to be significantly associated with bacterial relative abundances and reproducible across both cohorts (Bonferroni corrected p<0.05). Fifty-six species had significantly associated genes (median number of significant associations per bacterium = 4.5; first quartile = 1; third quartile = 10). Notably, significant genes had roles in cell adhesion and attachment, cell migration, cytokinesis, cytoskeletal integrity, and cytoskeletal dynamics. Findings inform how individual genetic variation translates to differences in bacterial colonization of chronic wounds. Future work will focus on predictive value of SNPs and gene expression for specific infection types.

P1.03

Does Opat Improve Long Term Outcomes Following Hospitilization For Foot Infections?
Arthur Tarricone1, Mario Reyes1, Mehmet Suludere1, Lee Rogers2, Lawrence A. Lavery1
1Plastic Surgery , University of Texas Southwestern, Dallas, TX, United States 2Department of Orthopedics, University of Texas Health Science Center, San Antonio, TX, United States
Does Opat Improve Long Term Outcomes Following Hospitilization For Foot Infections ?

Arthur Tarricone1, Mario Reyes1, Mehmet Suludere1, Lee Rogers2, Lawrence A. Lavery1 1Plastic Surgery, University of Texas Southwestern, Dallas, TX; 2Department of Orthopedics, University of Texas Health Science Center, San antonio, TX

Background: Diabetic foot ulcers are a common complication of diabetes that is associated with both high morbidity and mortality. It is estimated that the United States alone spends up to $13 billion USD each year on DFUs, with individual costs rising to $15,000 per year. Much of this expenditure has been attributed to lengthy hospital stays and inpatient care, with the need prolonged antibiotic therapy. Outpatient antibiotic therapy (OPAT) is a service that provides intravenous antimicrobial medication in the outpatient setting and has been shown to reduce hospital stay and overall medical costs. This study aims to determine the benefit of OPAT therapy in a cohort with soft tissue and bone foot infections.
Methods: Retrospective cohort study of consecutive patients hospitalized for foot infection. We collected demographic data, comorbidities and one-year outcomes including healing, surgical interventions, number of surgeries, length of stay, re-infection, and re-hospitalization. Patients were grouped by discharge with OPAT program or standard of care. All patients were followed for a minimum of one year. Statistics were performed using Stata Be7. Continuous variables were reported as mean ± standard deviation and categorical variables were reported as their n. Odds ratios were written as O.R, followed by their associated confidence interval [CI]. Significance was determined as p≤0.05.
Results: A total of 382 patients with hospitalized lower extremity infections were analyzed in this cohort. There were 202 subjects with osteomyelitis and 180 subjects with soft tissue infections. OPAT was used in 99 patients. The primary outcomes examined in this study included reinfection, amputation, mortality, rehospitalization of the same foot, and length of stay. The overall events included 168 reinfections, 206 amputations, 9 deaths, 165 rehospitalizations for the target limb. OPAT was significantly associated with reinfection OR = 2.2 [CI: 1.3 – 3.5] and rehospitalization of the same foot O.R = 2.4 [CI: 1.5-3.9].
A sub analysis was further conducted for only the patients with diabetes (n = 294). An association was seen between OPAT and reinfection O.R = 2.1 [CI:1.3-3.6] and rehospitalization for the target limb O.R = 2.4 [CI: 1.4- 4.0] as well. Sub analysis was also performed for patients with osteomyelitis (n=202). OPAT was protective of index amputation O.R = 0.3 [CI: 0.1-0.6] and associated with rehospitalization of index limb O.R = 2.1 [CI: 1.1- 3.7].
Conclusion: OPAT is associated with reinfection and rehospitalization of the target limb in this cohort. These findings were strongly related to patients with diabetes while no significant findings were observed in patients without diabetes.

P1.04

Impact Of Diagnostic Delay On Healing Outcomes And Healthcare Utilization In Patients With Pyoderma Gangrenosum
Olivia M. Haddadin1, Katherine M. Erickson2, Emile Latour1, Jonathan Sisley1, Alex Ortega-Loayza1
1OHSU, Portland, OR, United States 2NYMC, New York, NY, United States
Impact Of Diagnostic Delay On Healing Outcomes And Healthcare Utilization In Patients With Pyoderma Gangrenosum

Olivia M. Haddadin1, Katherine M. Erickson2, Emile Latour1, Jonathan Sisley1, Alex Ortega-Loayza1 1OHSU, Portland, OR; 2NYMC, New York, NY

Pyoderma gangrenosum (PG) is a rare neutrophilic dermatosis classically causing painful skin ulcerations with a large impact on quality of life. PG diagnosis is often challenging as the clinical presentation can mimic other ulcerative skin disorders with no specific biological marker. Misdiagnosis and delayed diagnosis can induce mismanagement. However, the impact of the delay of PG diagnosis on healing outcomes and subsequent healthcare utilization has not yet been studied and the purpose of our study was to determine this impact. We conducted a prospective study supplemented by retrospective data for 100 patients with ulcerative PG from the Oregon Health and Science University Pyoderma Gangrenosum Study patient registry. Prospective data included healing time and hospitalizations while ulcer onset, date of diagnosis, and emergency room (ER) visits were collected retrospectively. Total healing time was defined from the date of diagnosis to wound closure. The patients were divided into two groups based on a delay of diagnosis ≤3 months and >3 months based on the definition of a chronic wound. Healing time was evaluated with a Kaplan-Meier analysis.
Twenty-six percent of patients had a diagnostic delay of ≤3 months. The average ulcer size (available for 84% of patients) was 56cm2 (95% CI 10 to 103) for ≤3 months and 71cm2 (95% CI 50 to 92) for >3 months diagnostic delay. The average pain in a numeric rating scale was 3.6 and 4.4 out of 10 for each group respectively (p=0.213). There was a statistically significant difference in median time to healing between the groups: 14.0 months (95% CI 9.4 to 26.6) with a delay of diagnosis ≤3 months and 21.7 months (95% CI 10.2 to 62.9) with a delay of diagnosis >3 months (log-rank P = 0.014). The difference in number of ER visits was also statistically significant with a mean of 0.5 visits for a delay of diagnosis ≤3 months and 1.5 visits for a delay of diagnosis >3 months (P = 0.038). Moreover, the number of hospitalizations was significantly higher for patients with a diagnostic delay >3 months [2.3 hospitalizations versus 1.4 hospitalizations (P = 0.034)].
Our study demonstrated that a delay of PG diagnosis >3 months by medical providers prolongs healing time and significantly increases the number of ER visits and hospitalizations. As a result, delayed diagnosis may lead to higher costs and an increased burden on the healthcare system influencing treatment time and treatment costs for patients. Given average pain is similar in both groups, patients with a longer disease course as a result of delayed diagnosis have prolonged suffering. Delayed healing time could also lead to increased time lost from work.
In conclusion, this study clearly shows the negative consequences of a delayed PG diagnosis supporting why it is paramount to increase awareness of this underdiagnosed disease among medical providers and the necessity of specific markers to aid in a timely diagnosis.

P1.05

LCM-Directed Profiling Of Acute And Chronic Wounds Identifies Proteomic And Lipidomic Signatures Of Healing And Non-Healing Wounds
Veronika Jurczuk1, Lilian Valadar Tose2, Leticia L. Rodriguez3, Beatriz Abdo Abujamra1, Maria Boulina3, Sinan K. Jabori4, Devinder Singh4, Sara Danker4, Francisco Fernandez Lima2, Ivan Jozic1
1Dermatology, University of Miami School of Medicine, Miami, FL, United States 2Chemistry and Biochemistry, Florida International University, Miami, FL, United States 3Diabetes Research Institute, University of Miami, Miami, FL, United States 4Division of Plastic and Reconstructive Surgery, University of Miami, Miami, FL, United States
LCM-Directed Profiling Of Acute And Chronic Wounds Identifies Proteomic And Lipidomic Signatures Of Healing And Non-Healing Wounds

Veronika Jurczuk1, Lilian Valadar Tose2, Leticia L. Rodriguez3, Beatriz Abdo Abujamra1, Maria Boulina3, Sinan K. Jabori4, Devinder Singh4, Sara Danker4, Francisco Fernandez Lima2, Ivan Jozic1 1Dermatology, University of Miami School of Medicine, Miami, FL; 2Chemistry and Biochemistry, Florida International University, Miami, FL; 3Diabetes Research Institute, University of Miami, Miami, FL; 4Division of Plastic and Reconstructive Surgery, University of Miami, Miami, FL

Purpose: The purpose of our study was to utilize unbiased proteomic and lipidomic approaches to identify potential biomarkers of healing and non-healing wounds.
Methods: For acute wounds, we topically treated ex vivo human skin with deuterium oxide (D2O), whose incorporation into newly synthesized macromolecules was assessed at 24, 48, 72 and 96hr post treatment (n=4). Similarly, for chronic wounds we utilized diabetic foot ulcer (DFU) and venous leg ulcer (VLU) specimens (n=3). Harvested tissues were cryosectioned & freeze dried, with areas of interest dissected using LCM microscopy and sequential sections subjected to either lipidomic or proteomic analysis using time of flight secondary ion mass spectrometry (ToF-SIMS) or trapped ion mobility spectrometry (timsToF-Ultra) with a high-spatial resolution LMIG analytical beam (respectfully). Mass-to-charge ratio (m/z) was utilized to identify various lipid and peptide species using Spectronaut 18, thus allowing for hierarchical clustering of samples, followed by STRING-DB and DAVID analysis for functional annotation of identified clusters of different lipidomic and proteomic species.
Results: TOF-SIMS detected fatty acids 16:0 and 16:1 (255.2, 253.2 m/z), 18:0, 18:1 and 18:2 (283.3, 281.2, 279.2 m/z), and cholesterol sulfate (465.3 m/z) in both acute and chronic wounds with the latter exhibiting close to 200-fold increase in epidermis of both DFUs and VLUs. Conversely, long chain fatty acids (24:0, 25:0, 26:0 and 28:0), as well as ceramides were almost devoid from the healing epidermis and were primarily localized to granular and cornified layers away from the healing epidermis in acute wounds and throughout the entirety of both DFUs and VLUs. On the other hand, proteomic analyses identified a number of proteins that exhibited differential expression in either healing epidermis or dermis in comparison to their counterparts away from the wound (including but not limited to: ALDOC, ANXA3, ATPA, BAF, CALD1, CALM1, CAV1, DESP, EPIPL, FAK2, G3PT, GELS, H11, K1C24, KRT36, K2C6A, PDIA1, POSTN, RL12, ROA1, SPB5, VINC) with a relative difference in abundance >2.0 fold. Moreover, we were able to identify a number of differentially expressed peptides between acute and chronic wounds, within both epidermal and dermal compartments (A1AG, CALM2, CCN1, DCD, FIBB, FNDC1, H2A1J, HBB, K1C9, K22E, MACF1, OR2T2, O2T35, PGS2, RLA2, S10A9, VIME, among others).
Conclusions: Together our data identify a novel method for detection of proteomic and lipidomic changes in acute and chronic wounds. Moreover, identified signatures serve as targets for development of novel topical formulations for accelerated wound closure, which we are actively pursuing.

P1.06

Staphylococcus Epidermidis Fitness In The Chronic Wound Microenvironment Is Driven By Antimicrobial Resistance Traits
Jamie L. Burgess2, Miroslav Dinic1, Rebecca Verpile2, Tammy Gonzalez2, Jingjing Ming2, Jelena Marjanovic2, Carmen Beliz2, Lisa Plano3, Seth Thaller4, Hadar Lev-Tov2, Marjana Tomic-Canic2, Irena Pastar2
1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia. 2Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami, Miami, FL, United States 3Department of Microbiology and Immunology, University of Miami, Miami, FL, United States 4Department of Surgery, University of Miami, Miami, FL, United States
Staphylococcus Epidermidis Fitness In The Chronic Wound Microenvironment Is Driven By Antimicrobial Resistance Traits

Jamie L. Burgess2, Miroslav Dinic1, Rebecca Verpile2, Tammy Gonzalez2, Jingjing Ming2, Jelena Marjanovic2, Carmen Beliz2, Lisa Plano3, Seth Thaller4, Hadar Lev-Tov2, Marjana Tomic-Canic2, Irena Pastar2 1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; 2Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami, Miami, FL; 3Department of Microbiology and Immunology, University of Miami, Miami, FL; 4Department of Surgery, University of Miami, Miami, FL, United States

A perturbed microbiome with persistent wound infection by biofilm forming bacteria is characteristic of chronic wounds, including venous leg ulcers (VLUs). Staphylococcus epidermidis is the most abundant skin-resident bacteria primarily known to be beneficial to the host but is also recognized as an “accidental” pathogen in certain environments. As the role of S. epidermidis in the wound chronicity is still unclear, we performed an in-depth characterization of S. epidermidis isolated from chronic VLUs. We utilized a human ex vivo wound model to show that healthy, commensal strains of S. epidermidis were incapable of surviving the human ex vivo wound environment. However, S. epidermidis strains isolated from VLUs showed a biofilm-dependent induction of IL-1β, IL-8 and IL-6 and inhibition of wound re-epithelialization, correlating with the healing outcome of the corresponding VLU patients. Using whole genome sequencing, we found both commensal and VLU isolates of S. epidermidis had similar signatures for biofilm formation and adhesion, but only the VLU isolates demonstrated higher biofilm formation and extracellular matrix binding. A majority of the VLU patients tested (n=24) had a high prevalence of mupirocin and methicillin resistance genes, contributing to the emergence of treatment resistant virulent lineages in patients with non-healing ulcers. Our data emphasizes the need to develop therapeutics targeting bacterial attachment or mechanical removal by debridement rather than a bactericidal approach to infection in VLUs.

P2.01

Characterizing Vascular Leakage After Angiopoietin-1 Sirna Knockdown In Dermal Microvascular Endothelial Cells Derived From Post-Burn Hypertrophic Scar
Esteban Molina1, Lauren Moffatt2, Jeffrey W. Shupp3, Bonnie Carney2
1Georgetown University School of Medicine, Clifton, VA, United States 2Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington, DC, United States 3The Burn Center, Department of Surgery, MedStar Washington Hospital Center, Washington, DC, United States
Characterizing Vascular Leakage After Angiopoietin-1 Sirna Knockdown In Dermal Microvascular Endothelial Cells Derived From Post-Burn Hypertrophic Scar

Esteban Molina1, Lauren Moffatt2, Jeffrey W. Shupp3, Bonnie Carney2 1Georgetown University School of Medicine, Clifton, VA; 2Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington; 3The Burn Center, Department of Surgery, MedStar Washington Hospital Center, Washington DC, United States

Recent data demonstrate that dermal microvascular endothelial cells (DMVECs) from hypertrophic scars (HTSs) have increased angiopoietin-1 (ANGPT-1) gene and protein expression compared to normal skin (NS) DMVECs and lower permeability of FIT-C dextran using a transwell assay. This study evaluated the effect of ANGPT-1 knockdown on endothelial cell permeability to characterize the role of endothelial dysfunction in HTS.
Full-thickness burns which formed HTSs were created in Duroc pigs. Punch biopsies were taken at day 84 post-burn and stained with Verhoeff Van Geison stain (VVG). HTS and areas of NS were excised and digested in collagenase. Fibroblast-DMVEC co-cultures were saved in cryostorage. Ulex europaeus agglutinin 1 lectin was used to sort for DMVECs by magnetic-activated cell sorting. For the knockdown, DMVECs were grown in 6-well plates with n=3 wells per experimental (treatment, control, scramble) group. Treatment group was treated with siRNA for ANGPT-1. At 24 hours post-transfection, RNA was isolated. Gene expression of ANGPT-1 was quantified using qRT-PCR. In the permeability assay, DMVECs from HTS were seeded onto 12-well transwell plates. Trans-endothelial electrical resistance (TEER) was assessed on day 1 to assess the formation of a monolayer. On day 3 post-seeding, transwells were treated with either siRNA for ANGPT-1 (n=8), H2O as control (n=7), or scramble siRNA (n=7). On day 4, 24 hours post-transfection, FIT-C was added to the apical well and dwelled for 2 hours. FITC-dextran that diffused into the bottom well was measured by spectrophotometry. Student’s t-test was used to evaluate gene expression between knockdown vs control group with p<0.05 considered significant. Transwell concentrations between experimental groups were analyzed using one-way ANOVA with Tukey's correction for multiple comparisons. Punch biopsies from scars showed significantly increased blood vessel density compared to normal skin on VVG stain (p<0.01). DMVEC identity was confirmed by immunofluorescence staining for Von-Willebrand factor. ANGPT-1 gene expression showed downregulation >4-fold for siRNA knockdown group compared to control group (p=0.21) while scramble did not show ANGPT-1 knockdown. TEER on day 1 averaged 17.28±4.5 Ω/cm2 for n=12 wells and 28.97±5.3 Ω/cm2 for n=12 wells. siRNA knockdown group showed increased permeability to FIT-C dextran compared to scramble siRNA (-23.39±3.9 vs 4.24±1.6; p=0.0002).
Knockdown using siRNA for ANGPT-1 in HTS DMVECs led to an increase in endothelial permeability compared to the scramble group. Additional siRNA targets outside of the angiopoietin group will be tested in future work to identify candidate drug targets for treating endothelial dysfunction in scars.

P2.02

Endothelial Cell-Derived Extracellular Vesicles And Their Micrornas Downregulate Fibrotic Pathways In Fibroblasts
Heidi Yuan, Anna Salapatas, Trevor R. Leonardo, Chen Han, Devina Koshal, Mateusz S. Wietecha, Lin Chen, Sriram Ravindran, Luisa A. DiPietro
University of Illinois Chicago, Chicago, IL, United States
Endothelial Cell-Derived Extracellular Vesicles And Their Micrornas Downregulate Fibrotic Pathways In Fibroblasts

Heidi Yuan, Anna Salapatas, Trevor R. Leonardo, Chen Han, Devina Koshal, Mateusz S. Wietecha, Lin Chen, Sriram Ravindran, Luisa A. DiPietro University of Illinois Chicago, Chicago, IL

Background: An adequate wound healing response requires the coordination of intercellular signals between multiple cell types. During the proliferative and remodeling phases of healing, endothelial cells and fibroblasts are likely to communicate. We hypothesize that this communication occurs through the release of endothelial cell-derived extracellular vesicles (ECEVs), which contain microRNAs that transcriptionally regulate fibrotic gene signatures and influence fibroblast behavior.
Methods: ECEVs were collected from primary human dermal microvascular endothelial cells using Exoquick-TC and validated using Nanoparticle Tracking Analysis (NTA), Western blot (WB), and transmission electron microscopy (TEM). Bulk RNA sequencing was performed on human dermal fibroblasts treated with ECEVs. ECEVs were also sequenced to determine microRNA content. The relative expression of select gene targets and ECEV microRNAs was validated with qRT-PCR of fibroblasts treated with ECEVs for 24 hours. Fibroblast function was assessed following transfection of a microRNA candidate that had increased expression in ECEV-treated fibroblasts.
Results: Ingenuity Pathway Analysis (IPA) was used to identify top Canonical Pathways affected in fibroblasts treated with ECEVs. The microRNA target filter in IPA was then applied to determine the pathway-specific transcriptional targets of the top 20 microRNAs found in ECEVs. The analysis identified Pulmonary Fibrosis Idiopathic, Hepatic Fibrosis, and Wound Healing as the top 3 downregulated pathways in ECEV-treated fibroblasts, suggesting that ECEVs exert anti-fibrotic effects on fibroblasts. Among these three pathways, 11 common ECEV microRNAs were predicted to target genes related to fibroblast proliferation, differentiation into myofibroblasts, contraction, and formation of collagen. Five genes (ACTA2, MAP2K6, PDGFA, TGFBR3, COL1A2) demonstrated reduced relative expression in ECEV-treated fibroblasts when validated using qRT-PCR (p<0.05). MiR-126-3p was the most highly expressed microRNA in ECEVs, comprising over 20% of total microRNA content. Intracellular levels of miR-126-3p in fibroblasts were also elevated (p<0.05) following ECEV treatment, and transfection of fibroblasts with mimics of miR-126-3p resulted in impaired migration and proliferation (p<0.05).
Conclusions: ECEVs contain microRNAs that are predicted to repress pro-fibrotic gene signatures in fibroblasts. Preliminary in vitro validation suggests that the transfer of miR-126-3p from ECEVs to fibroblasts is one mediator of this anti-fibrotic effect. In the context of wound healing, this endothelial cell-fibroblast communication may be a means of precluding early ECM deposition or contraction, allowing proper vascular remodeling in the wound bed.

P2.03

Radiation Triggers An Early Scarring Response Involving Epithelial-Mesenchymal Transition And Upregulation Of Pro-Fibrotic Genes In A Human Ex Vivo Skin Model
Sophie M. Bilik2, Caroline Dodson2, Gabrielle DiBartolomeo2, Seth Thaller1, Rivka C. Stone2
1Division of Plastic, Aesthetic, and Reconstructive Surgery, University of Miami Miller School of Medicine, Miami, FL, United States 2Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, United States
Radiation Triggers An Early Scarring Response Involving Epithelial-Mesenchymal Transition And Upregulation Of Pro-Fibrotic Genes In A Human Ex Vivo Skin Model

Sophie M. Bilik2, Caroline Dodson2, Gabrielle DiBartolomeo2, Seth Thaller1, Rivka C. Stone2 1Division of Plastic, Aesthetic, and Reconstructive Surgery, University of Miami Miller School of Medicine, Miami, FL; 2Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL

Radiation-induced skin fibrosis (RISF) is a severe consequence of cutaneous injury following irradiation, producing functional impairment and reduced quality of life for patients. Epithelial-mesenchymal transition (EMT) plays a global role in fibrosis by pathologically sustaining extracellular matrix production after tissue injury. To elucidate the early cutaneous response to radiation, a human ex vivo skin model was developed. Skin from human subjects (n=7) was irradiated at a dose of 3.5Gy, maintained at an air-liquid interface, and collected over a 15-day period. Tissue fibrosis was captured by measurement of dermal collagen bundles in histologic sections using automated quantitative assessment. Dermal collagen thickness increased following radiation, supporting evolving fibrosis in this model. Radiation treatment also triggered the induction of fibrosis-associated genes actin alpha 2 smooth muscle (ACTA2), collagen alpha-1 chain (COL1A1), connective tissue growth factor (CTGF), and fibronectin 1 (FN1). In addition, an EMT-like response was detected via immunohistochemical analysis of irradiated skin, which demonstrated decreased E-cadherin and increased vimentin levels in comparison to non-irradiated controls. In conclusion, a scarring response involving global processes of fibrosis was successfully induced in human skin ex vivo, providing a platform for further study of molecular and cellular events underlying RISF and leading to the development of targeted therapies for early intervention.

P2.04

Cancer And Lymphatic Marker Foxc2 Drives Wound Healing And Fibrotic Tissue Formation
Maia Granoski1, William Hahn1, Katharina Fischer1, Hudson Kussie1, Dharshan Sivaraj2, Andrew Hostler1, Eamonn McKenna1, Jonathan P. Yasmeh1, Robert Erickson1, Marlys Witte1, Geoffrey C. Gurtner1, Kellen Chen1
1Surgery, University of Arizona, Tucson, AZ, United States 2Stanford University, Palo Alto, CA, United States
Cancer and Lymphatic Marker FOXC2 Drives Wound Healing And Fibrotic Tissue Formation

Maia Granoski1, William Hahn1, Katharina Fischer1, Hudson Kussie1, Dharshan Sivaraj2, Andrew Hostler1, Eamonn McKenna1, Jonathan P. Yasmeh1, Robert Erickson1, Marlys Witte1, Geoffrey C. Gurtner1, Kellen Chen1 1Surgery, University of Arizona, Tucson, AZ; 2Stanford University, Palo Alto, CA

Background: Wound repair is a complex process that engages many different physiological systems as the tissue progresses through a series of interdependent phases, including inflammation and remodeling. The FOXC2 transcription factor has been tied to tissue development during embryogenesis, and has been clinically associated with aggressive basal-like human breast cancers. Systemic dysregulation of FOXC2 expression has also been found to promote defects in lymphatic remodeling and hyperplastic lymphedema-distichiasis (LD). Since chronic lymphedema is a forerunner of several malignancies and cancers have been known to arise from poorly healing chronic wounds, we examined the effect of Foxc2 dysfunction on skin wound healing.
Methods: We used our splinted excisional wounding model that mimics human-like wound healing on wildtype and Foxc2 +/- mice, which demonstrate incomplete lymphatic vasculature, lymphatic dysfunction, and enhanced cancer metastasis. Wound size was measured over the course of 18 days. Tissue was explanted from both groups at post-operative day (POD) 14 and 18 and stained with Masson’s Trichrome to assess scar formation, Picrosirius Red for dermal integrity, or immunofluorescence to assess macrophage (F4/80) cell populations. Results: Wildtype mice had completely healed wounds by POD 14, while Foxc2+/- mice did not heal until POD18 (p=0.0104). On PODs 8 (p<0.0001), 10 (p<0.0001), 12 (p<0.0001), and 14 (p=0.0002), the wound size of Foxc2+/- mice was significantly larger than that of wildtype mice. Scar area of healed Foxc2+/- mice (POD 18) was significantly larger than that of healing Foxc2+/- mice (POD14; p=0.0098) and that of healed wildtype mice (POD 14; p=0.0294). Collagen fibers in the healing scar of Foxc2+/- mice were narrower (p=0.0117) and more highly aligned (p=0.0110), indicating significantly more fibrosis in Foxc2+/- mice compared to wildtype mice. Collagen fibers in both groups became significantly longer (p=0.0116) and wider (p=0.0020) over time, indicating a temporal evolution of fibrosis during the remodeling phase of wound healing. Foxc2+/- mice also had significantly lower numbers of F4/80 cells (p=0.0014) compared to wildtype mice, indicating poor immune cell infiltration at the wound site.
Conclusion: We found that FOXC2, which is tied to cancer metastasis and lymphatic dysregulation, also impairs wound healing and promotes fibrotic tissue architecture, linking these disease states together. With FOXC2 proposed as a potential therapeutic target for cancer metastasis, its pleiomorphic downstream systemic effects should be considered and weighed against the increased risk of developing nonhealing wounds, potentially by inhibiting the immune system. Further delineation of the microenvironment, cellular events, and molecular signals during normal and Foxc2-associated abnormal wound healing will improve clinical therapies targeting this marker.

P2.05

Inhibition Of Cyp24a1, An Enzyme Involved In Vitamin D Metabolism, Alters Profibrotic Gene Expression In Keloid-Derived Keratinocytes
Dorothy Supp1, Jennifer Hahn1, Kelly A. Combs1, Heather Powell2
1Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, United States 2Materials Science Engineering and Biomedical Engineering, The Ohio State University, Columbus, OH, United States
Inhibition Of CYP24A1, An Enzyme Involved In Vitamin D Metabolism, Alters Profibrotic Gene Expression In Keloid-Derived Keratinocytes

Dorothy Supp1, Jennifer Hahn1, Kelly A. Combs1, Heather Powell2 1Surgery, University of Cincinnati College of Medicine, Cincinnati, OH; 2Materials Science Engineering and Biomedical Engineering, The Ohio State University, Columbus, OH

Introduction: Keloids are disfiguring fibroproliferative lesions that can occur following injury to skin in susceptible individuals. Keloids are challenging to treat and recurrence after treatment is common. A deeper understanding of the molecular mechanisms driving keloid formation is necessary for development of more effective therapies for keloid suppression. We previously identified reduced expression and decreased nuclear localization of the vitamin D receptor (VDR) in keloid epidermis, implicating vitamin D signaling in keloid pathology. Here we report that CYP24A1 is overexpressed in keloid keratinocytes compared with normal keratinocytes. The CYP24A1 gene encodes 24 hydroxylase, a vitamin D metabolizing enzyme that degrades 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. The CYP24A1 gene is itself induced by vitamin D in a feedback loop that regulates 1,25-D3 levels. In this study, we investigated the effects of CYP24A1 inhibition in normal and keloid-derived keratinocytes.
Methods: Normal and keloid keratinocytes (N=3 donors each) were cultured +/- 1,25-D3 and +/- inhibitors of CYP24A1, which included ketoconazole, a non-specific inhibitor of cytochrome P-450 enzymes, and VID-400, a specific inhibitor of CYP24A1. Proliferation was measured using an MTT assay, and gene expression was analyzed by quantitative PCR. Statistical analyses were performed using t test (2 groups) or One Way ANOVA (>2 groups) using SigmaPlot 15.0.
Results: CYP24A1 mRNA was expressed at 3.5X higher levels in keloid keratinocytes compared with normal keratinocytes. Ketoconazole inhibited proliferation of keloid and normal keratinocytes, but VID-400 had no significant effect on keratinocyte proliferation. The two inhibitors had different effects on expression of vitamin D target genes in keratinocytes. For example, ketoconazole treatment alone reduced expression of CYP24A1 in normal and keloid keratinocytes, whereas VID-400 treatment increased CYP24A1 expression. Both inhibitors decreased expression of the profibrotic genes Periostin (POSTN) and Hyaluronan synthase 2 (HAS2), previously shown to be upregulated in keloid-derived cells. Combined treatment of keloid-derived keratinocytes with 1,25-D3 and ketoconazole or VID-400 increased the effects of 1,25-D3 treatment on vitamin D target genes and profibrotic gene expression, although the effects were gene- and cell type-specific.
Conclusions: The data suggest that reduction of 1,25-D3 inactivation with inhibitors of CYP24A1 may reduce profibrotic gene expression in keloid-derived cells. Because vitamin D has numerous anti-inflammatory and antifibrotic activities, inhibitors of CYP24A1 may serve as adjunctive therapies to suppress keloid-associated gene expression changes.

P2.06

Extracorporeal Shock Wave Therapy Alleviate Radiation-Induced Skin Fibrosis By Downregulating Tgf-? Expression
Department of Plastic and Reconstructive Surgery, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwonsi, Changwonsi, Korea (the Republic of)
Extracorporeal Shock Wave Therapy Alleviate Radiation-Induced Skin Fibrosis By Downregulating TGF-β Expression

Sangwoo Park Department of Plastic and Reconstructive Surgery, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwonsi, Changwonsi, Korea (the Republic of)

Background: The aim of this study was to evaluate the protective effects of extracorporeal shock wave therapy (ESWT) against radiation injury in dermal fibroblasts and mouse skin.
Methods: Dermal fibroblasts were treated with ESWT (1000 impulses at 4Hz and 0.1mJ/mm2) after irradiation (20 Gy). Cell viability, cell migration, and mRNA and protein expression were measured. A total of 24 mice were used for an in vivo study. Mice were treated with ESWT (200 impulses at 4Hz and 0.25mJ/mm2) daily for 2 weeks after irradiation (45 Gy). At 8 weeks post-irradiation, dorsal skin was harvested for histopathologic examination and protein isolation.
Results: In dermal fibroblasts, the viability of irradiated cells was significantly increased after treatment with ESWT, relative to irradiated, nontreated cells (p=0.005). ESWT significantly reduced TGF-β1 protein expression at 48h post-irradiation (p=0.024). ESWT significantly increased cell migration at 24h post-irradiation (P =0.002). In mice, the irradiated skin treated with ESWT exhibited decreased collagen deposition compared with the control. ESWT significantly reduced TGF-β1 (p=0.001) and phospho-Smad3 (p=0.004) protein expression after irradiation. ESWT significantly decreased the number of TGF-β1- and α-SMA-positive cells after irradiation (p<0.001).
Conclusions: Our study demonstrated that ESWT inhibited radiation-induced fibrosis by downregulating TGF-β1 expression. Therefore, ESWT may be a safe and effective candidate for the prevention of radiation-induced skin fibrosis.

P3.01

Innate Immune Interactions Define Key Mechanisms Of Exosome-Induced Healing Of Chronic Wounds
Kody P. Mansfield, Dianny Almanzar, Bibi S. Subhan, Jasmina Abdalla, Juan Troncoso, Lesly Honore, Piul S. Rabbani
Hansjörg Wyss Department of Plastic Surgery, NYU Langone, Brooklyn, NY, United States
Innate Immune Interactions Define Key Mechanisms Of Exosome-Induced Healing Of Chronic Wounds

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

Chronic wounds, thus far, are managed clinically but are not treated for underlying pathology. Specifically, in the population with diabetes, chronic wounds do not manifest the repair or regenerative properties of skin and thus, are one of the leading causes of non-traumatic lower limp amputations. Major hallmarks of these chronic wounds include neuropathy, peripheral artery defects, and immune dysfunction. We found that local administration of secreted nanoscale vesicles, called exosomes, harvested from cultured primary human bone marrow multipotent stromal cells, accelerate wound closure in a genetic animal model of type 2 diabetic delayed wound healing. The cellular and molecular sequelae of exosome-based treatment, however, remain to be investigated and is at the heart of our study. Cutaneous administration of exosomes at post-operative day 1 (POD1) significantly decreased time to closure and wound burden of type 2 diabetic wounds, in a dose dependent manner compared to PBS-treated control diabetic wounds. In order to characterize exosome-induced healing mechanisms, we employed multiplexed immunofluorescence imaging and single cell RNA sequencing. Our analysis revealed a pro-healing phenotype, likely modulated through interactions between innate immunity and cutaneous vasculature. Immunofluorescence imaging of 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. We also observed increased presence of undifferentiated monocytes/macrophages and macrophages in the diabetic wound tissue by 1.6-fold and 6.2-fold of that in control wounds, respectively. In vitro studies confirmed that exosomes were capable of modulating primary murine macrophages to a pro-healing-like phenotype. Further confirming our hypothesis of innate immune activation, exosome treatment failed to rescue the delayed healing in clodronate liposome-treated, macrophage-depleted mice. These macrophage-depleted but exosome-treated wounds also lack the development of highly vascularized granulation tissue indicating that macrophage induction is paramount for exosome-induced wound healing. Interestingly, even the endogenous control of macrophage plasticity throughout the sequential phases of skin healing remains elusive even in the context of non-pathological or typical healing wounds. Our results demonstrate that macrophage modulation is at least one avenue through which exosomes mediate wound closure benefit. Our results begin to address the obvious need of studies of cellular events engaged following exosome-induced wound healing, a critical barrier in the safe clinical translation of this tissue engineering approach.

P3.02

Topical Fluoxetine Dosage and Administration Chronobiology Affects Cutaneous Wound Healing
Moyasar A. Alhamo1, Anthony Gallegos1, Hsin-ya Yang1, Elham Aslankoohi2, Marcella Gomez3, Marco Rolandi2, Rivkah Isseroff1
1UC Davis Health, Sacramento, CA, United States 2UC, Santa Cruz, CA, United States 3UC, Santa Cruz, CA, United States
Topical Fluoxetine Dosage and Administration Chronobiology Affects Cutaneous Wound Healing

Moyasar A. Alhamo1, Anthony Gallegos1, Hsin-ya Yang1, Elham Aslankoohi2, Marcella Gomez3, Marco Rolandi2, Rivkah Isseroff1 1UC Davis Health, Sacramento, CA; 2UC, Santa Cruz, CA; 3UC, Santa Cruz, CA

Cutaneous wound healing requires the coordinated processes of cell proliferation and migration. Fluoxetine (FLX), a selective serotonin reuptake inhibitor widely used for treatment of mood disorders, has also been shown to have immunomodulatory properties. Moreover, research from our group has shown that FLX enhances the migration of keratinocytes and promotes healing in murine wounds. Therefore, it is reasonable to propose that applying FLX topically during the inflammatory phase of wounds may both modulate wound inflammation and improve re-epithelialization. However, the optimal FLX dosage and administration time remain to be elucidated. Our investigation focused on assessing local wound gene expression and re-epithelization, with the hypothesis that dosage and administration timing may modulate key healing parameters. Using a pig wound model, twelve circular wounds measuring 20 mm diameter each were created on the dorsal skin of young female Yorkshire pigs. FLX was applied topically at either high dose (0.45 mg/wound/day) or low dose (0.025 mg/wound/day) during specific time intervals (days 0-2, 3-6, or 7-9). Despite observing a decreased mRNA expression of the macrophage 1(NOS2)/macrophage 2 (ARG-1) ratio in the high-dose FLX groups during days 0-2, 3-6, or 7-9 compared to low-dose groups, we found that the low-dose FLX group exhibited the highest wound re-epithelization at 49.2% of original wound size on days 3-6, surpassing the high-dose groups (on days 0-2, 3-6, or 7-9). Similarly, higher mRNA expression of the early neuronal marker (DXC), essential for neuronal migration, was observed in low-dose groups (on days 0-2, 3-6, or 7-9) compared to high-dose groups. Together, despite a decreased macrophage 1(NOS2)/macrophage 2 (ARG-1) ratio in high-dose FLX groups suggesting down modulation of the inflammatory response under that condition, the low-dose FLX group demonstrated improved wound re-epithelization at 49.2% when the drug was applied during days 3-6 post wounding, outperforming the high-dose groups across various time intervals. Additionally, increased expression of the early neuronal marker (DXC) in low-dose groups throughout different time points suggests a potential association between lower FLX doses and enhanced neuronal migration into the wound. This line of inquiry provides valuable insights into the impact of FLX dosage and administration chronobiology on the wound healing program.

P3.03

Skint-1 Regulates Gamma Delta T Cell Activity During Wound Repair
Sujad Younis1, Katelyn Rivas1, Jelena Marjanovic2, Laura Padula1, Jamie L. Burgess2, Ayrimah Malcolm-Parker2, Irena Pastar2, Marjana Tomic-Canic2, Natasa Strbo1
1Microbiology and Immunology, University of Miami, Miami, FL, United States 2University of Miami, Miami, FL, United States
SKINT-1 Regulates Gamma Delta T Cell Activity During Wound Repair

Sujad Younis1, Katelyn Rivas1, Jelena Marjanovic2, Laura Padula1, Jamie L. Burgess2, Ayrimah Malcolm-Parker2, Irena Pastar2, Marjana Tomic-Canic2, Natasa Strbo1 1Microbiology and Immunology, University of Miami, Miami, FL; 2University of Miami, Miami, FL

Butyrophilin (BTN)-related Skint-1 (Selection and upkeep of intraepithelial T cells 1) molecule is expressed on epidermal keratinocytes and thymic epithelial cells. Skint-1 specifically drives the development of the subtype of gamma delta T (GDT) cells, dendritic epidermal T cells (DETCs) progenitors in the thymus. Importantly, homeostasis and activation of resident epithelial GDT cells are under the control of butyrophilin (BTN) and butyrophilin-like (BTNL) molecules, in humans and mice. However, it is not well understood how Skint-1 influence DETCs function in the epidermis in response to wounding. Interactions between commensal microbiota and the multiple cell types involved in cutaneous wound healing regulate the immune response and promote barrier restoration. In that regard, we have recently shown that Perforin-2 (P-2), novel anti-microbial protein expressed in the skin, is critical for the clearance of intracellular pathogens. We postulated that P-2 contributes to GDT cell activation and recruitment in response to wounding through regulation of Skint-1.
We induced full thickness wounds on the dorsal skin of 2- or 10-month-old C57/BL6 WT and P-2 knock-out (KO) mice and assessed healing at days 3 and 6 post wounding. We analyzed re-epithelialization and keratinocyte activation using histomorphometry and keratin-6 staining. We expressed Skint-1 on the HEK-293 cells and co-cultured skin GDT cells in the presence of anti-Skint-1 antibody and measured activation of GDT cells by multicolor flow cytometry.
We found that in aged 10-month-old mice, wound re-epithelialization was significantly delayed at day 6 in P-2 KO mice compared to aged WT and young P-2 KO (p<0.05). The healing delay was accompanied by significantly decreased keratin-6 expression at the wound edge (p<0.05).
Immunofluorescence staining and flow cytometric analysis revealed significant reduction in GDT cells in the wound bed in aged P-2 KO mice (p<0.05). The GDT cells in the P2 KO background also displayed lower expression of activation markers, including significantly lower mRNA and protein levels of Skint-1 on day 6 post-wounding (p<0.05). In vitro co-culture experiments confirmed that epithelial Skint-1 induces activation of GDT cells.
Our data demonstrate impaired keratinocyte activation and GDT cell infiltration in the absence of P-2 that is more pronounced in aged mice, indicating a novel mechanism relying on the P-2 and Skint-1 in modulating the inflammatory response upon wounding.

P3.04

Comprehensive Transcriptional Characterization Of Macrophage And Monocyte Dysregulation In Diabetic Wound Healing
Katharina S. Fischer, Abdelrahman Alsharif, Mansi Singh, Filiberto Quintero, Amelia B. Knochel, Maria Gracia Mora Pinos, Fidel Saenz, Ben Litmanovich, Sultana M. Mojadidi, Javier Gonzalez, Dharshan Sivaraj, Hudson C. Kussie, Andrew Hostler, Maia Granoski, Jonathan P Yasmeh, Autumn Lester, Kellen Chen, Geoffrey C Gurtner
Surgery, University of Arizona, Tucson, AZ, United States
Comprehensive Transcriptional Characterization Of Macrophage And Monocyte Dysregulation In Diabetic Wound Healing

Katharina S. Fischer, Abdelrahman Alsharif, Mansi Singh, Filiberto Quintero, Amelia B. Knochel, Maria Gracia Mora Pinos, Fidel Saenz, Ben Litmanovich, Sultana M. Mojadidi, Javier Gonzalez, Dharshan Sivaraj, Hudson C. Kussie, Andrew Hostler, Maia Granoski, Jonathan P. Yasmeh, Autumn Lester, Kellen Chen, Geoffrey C. Gurtner University of Arizona, Tucson, AZ

Background: Diabetes is a huge and growing public health concern. Monocytes (Mo) and macrophages (Mp) play major roles in all phases of wound healing and are particularly affected by diabetes-induced transcriptomic impairments. We characterize the role of Mos and Mps in genetically altered and high-fat diet induced diabetic wound healing models. Methods: Full-thickness excisional wounds were created on the dorsum of C57/BL6 (WT) mice, WT mice fed with a high-fat (HF) diet, and leptin-receptor deficient (DB) mice. Tissue was explanted at post-operative days (PODs) 0, 2, 7 and 30 and processed for high-throughput single cell RNA sequencing. Mps and Mos were identified with the canonical cell markers Cd45, Cd68 and Ly6c. Human tissue was collected from non-diabetic, and diabetic wounds and processed for scRNA-seq.
Results: Both genetic and pathophysiologic diabetes significantly impaired wound healing compared to healthy WT mice. In the early wound healing phase, myeloid cells predominated across all groups (67%), followed by fibroblasts (15%) and epithelial cells (9%). DB demonstrated a distinct anti-inflammatory, proliferating M2 Mp cluster expressing C1qa, C1qb and Timp2 at POD 7, at 43% (vs 13% in WT, 0% in HF). HF mice overexpressed a pro-inflammatory M1 Mp cluster, defined by the genes Dusp2, Cxcl2 and Nfkbia at PODs 2 (71%, vs 2% in both WT and DB) and 7 (67%, vs 11% in WT and 5% in DB). In human diabetic wounds, we saw an upregulation of the same pro-inflammatory DUSP2+, CXCL2+, NFKBIA1+ cells by 90% in the pre-diabetic and diabetic samples (vs 1% in the non-diabetic human).
In non-diabetic WT mice, we identified a proliferating Mo cluster upregulated at POD 2 expressing Ly6c2, Vcan1 and Chil31 at 68% (vs 9% in DB and HF). In human samples, non-diabetic wounds also demonstrated an 82% upregulation of this cell population, compared to only 5% in pre-diabetic and diabetic wounds.
Conclusion: To fully understand the changes in the cellular ecology of diabetic wound healing, we investigated two popular models that imitate the human diabetic physiology. Both DB and HF models demonstrated delayed wound healing similar to diabetic patients, but we discovered that these two models exhibited very different Mo/ Mp responses. An upregulation of Mo in the pro-inflammatory wound healing phase is essential to initiate normal, non-diabetic wound healing in both humans and mice. DB mice exhibit a Mp polarization profile with anti-inflammatory, proliferating M2 Mps driving the transition into chronic wounds. Contrarily, in HF mice, enhanced and prolonged inflammation from M1 Mp proliferation delays wound closure. Confirming the human relevance of our findings, pre-diabetic and diabetic human samples showed a similar dominance of M1 Mps representing over >80% of the Mo/Mp populations. These findings also demonstrate that the HF model causes delays in wound closure through similar cellular mechanisms as seen in human diabetes.

P3.05

Excessive Levels Of Neutrophils Cause Macrophage Dysfunction In Fibrotic Volumetric Muscle Loss (Vml) Injury
Ricardo Whitaker, Kara L. Spiller
School of Biomedical Engineering, Sciences and Health Systems, Drexel University, Philadelphia, PA, United States
Excessive Levels Of Neutrophils Cause Macrophage Dysfunction In Fibrotic Volumetric Muscle Loss (VML) Injury

Ricardo Whitaker, Kara L. Spiller School of Biomedical Engineering, Sciences and Health Systems, Drexel University, Philadelphia, PA

Volumetric Muscle Loss (VML) is a debilitating condition defined by the rapid loss of muscle mass, leading to permanent impairment. The current standard of care is inefficient due to the poor understanding of the molecular/cellular processes governing VML repair. Macrophages are crucial for muscle repair due to its interaction with neighboring, and changes in macrophage phenotype have been observed following critical size VML injuries. The goal of this study is to uncover mechanisms driving macrophages towards a dysfunctional phenotype following VML.
We employed a murine model of VML using subcritical size injuries (Regenerative), and critical size injuries (Fibrotic). We utilized flow cytometry to evaluate immune cell trafficking and macrophage phenotype in muscle over 28 days, and for 7 days in the spleen and bone marrow. Multiplex gene expression (NanoString) was used to characterize FACS-sorted macrophages and whole muscle tissue over 3 days. Systemic cytokines were quantified via multiplex cytokine analysis (Luminex) over 7 days. Finally, tissue healing was assessed via histology on Day 28.
Fibrotic muscle macrophages demonstrated a higher expression of inflammatory and lower expression of reparative markers over 28 days. Noticeably, similar results were observed in the spleen over 7 days. Hierarchical clustering of muscle macrophages also uncovered hybrid macrophage phenotypes, particularly at Day 7. At the gene level, Fibrotic macrophages in muscle presented a broad gene downregulation as early as Day 1 compared to Regenerative injury. These results demonstrated that macrophage dysfunction appears soon after injury and prior to onset of fibrosis. Whole muscle and blood analyses uncovered a higher expression of neutrophil chemokines and systemic G-CSF after Fibrotic injuries, accompanied by an increase in neutrophil influx to the muscle. These results suggest an active role of neutrophils in the progression of this pathology, and perhaps in modulating macrophage phenotype. To evaluate neutrophil-macrophage crosstalk, we partially depleted neutrophils so that neutrophil accumulation in the muscle following Fibrotic injuries resembles levels observed in Regenerative group. Partial neutrophil depletion decreased macrophage expression of inflammatory markers/genes and increased reparative marker/genes expression. However, no improvement in muscle physiology was observed, suggesting that neutrophil depletion improves macrophage phenotype, but is not enough to leverage muscle healing.
Here we have, for the 1st time, observed systemic changes in immune behavior following VML injury. Moreover, we uncovered neutrophils as critical macrophage modulators. These results can instruct the development of better therapeutics.

P3.06

Transcriptomic Predictors Of Oral Mucositis Severity In Head And Neck Cancer Patients
Taichi Goto1, Patricia Corby2, Alexander Lin3, John Lukens3, Stephen Sonis4, Leorey N. Saligan1
1Symptoms Biology Unit, NIH/NINR, Bethesda, MD, United States 2Department of Radiation Oncology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, United States 3Department of Radiation Oncology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States 4Brigham and Women’s Hospital and the Dana-Farber Cancer Institute, Boston, MA, United States
Transcriptomic Predictors Of Oral Mucositis Severity In Head And Neck Cancer Patients

Taichi Goto1, Patricia Corby2, Alexander Lin3, John Lukens3, Stephen Sonis4, Leorey N. Saligan1 1Symptoms Biology Unit, NIH/NINR, Bethesda, MD; 2Department of Radiation Oncology, University of Pittsburgh, School of Medicine, Pittsburgh, PA; 3Department of Radiation Oncology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA; 4Brigham and Women’s Hospital and the Dana-Farber Cancer Institute, Boston, MA

BACKGROUND: A common side effect of radiation therapy for head and neck cancer (HNC) is oral mucositis (OM), characterized by erythema, edema, and ulcers. Severe OM affects more than two-thirds of HNC patients, which can lead to malnutrition, dehydration, and risk of infection. Preliminary evidence suggests the role of transcriptome changes in pain heterogeneity associated with OM, but the literature lacks evidence if transcriptomic factors play a role in the development and worsening of OM. The present study aimed to identify differentially expressed genes (DEGs) before cancer treatment (baseline) that may be associated with the worsening of OM during radiation therapy (RT), and 2) to determine the gene sets associated with the identified DEGs.
METHODS: This is a sub-analysis of a clinical trial (ARMOR-Trial NCT03843554), where HNC patients who were expected to receive at least 5000 cGy of RT to the oral or oropharyngeal mucosa were recruited. This study used data from 26 participants in a control group that received regular oral care. Total RNA extracted from whole blood at baseline (before RT) was subjected to RNA-seq analysis. Based on changes in OM severity from baseline to RT completion assessed by the World Health Organization’s Oral Toxicity Scale, the participants were classified into mucositis (change ≧ 1, n = 20) or no mucositis groups (change < 1, n = 6). DEGs were identified between the two groups with a p-value < 0.05 and |log2 fold change| > 1. Gene Set Enrichment Analysis (GSEA) determined gene sets associated with the identified DEGs.
RESULTS: A total of 161 (132 upregulated and 29 downregulated) DEGs were identified. GSEA showed 13 activated biological processes, including “peptide metabolic process (normalized enrichment score (NES) = 1.73, p = .006),” “regulation of hormone levels (NES = 1.76, p = .008),” and “homeostatic process (NES = 1.58, p = .045).” GSEA also showed four activated and one suppressed molecular function, including activated “cytokine activity (NES = 1.78, p = .007)” and suppressed “enzyme binding (NES = -1.94, p = .003),” and four activated and one suppressed cellular components, including activated “bounding membrane of organelle (NES = 1.74, p = .015)” and suppressed “somatodendritic compartment (NES = -1.71, p = .023).”
CONCLUSIONS: We profiled the transcriptomic signatures before RT that are related to worsening OM severity in HNC patients. The GSEA suggested that some homeostasis-related biological activities, including metabolism, hormone regulation, and cytokine activities were associated with the worsening of OM in this population, which is informative for further investigations.

P4.01

5-Lipoxygenase Exerts Sex-Dependent Effects On Burn Wound Healing
Shannon M. Clayton1, Kristina Sanchez3, Maliha Newsome2, Niayab Ahad2, Athena Soulika1
1Dermatology, UC Davis, Sacramento, CA, United States 2Shriners Hospital for Children Northern California, Sacramento, CA, United States 3Anatomy, Physiology and Cell Biology, UC Davis, Sacramento, CA, United States
5-Lipoxygenase Exerts Sex-Dependent Effects On Burn Wound Healing

Shannon M. Clayton1, Kristina Sanchez3, Maliha Newsome2, Niayab Ahad2, Athena Soulika1 1Dermatology, UC Davis, Sacramento, CA; 2Shriners Hospital for Children Northern California, Sacramento, CA; 3Anatomy, Physiology and Cell Biology, UC Davis, Sacramento, CA

5-lipoxygenase (5-LO) is the rate-limiting enzyme for the synthesis of bioactive lipid mediators. These mediators are members of the eicosanoids, which are bioactive lipids, derived from polyunsaturated fatty acids that promote and/or regulate inflammation. Previous research suggests that following in vitro stimulation, 5-LO activity is increased in female neutrophils and monocytes of humans and rodents, compared with that of males, indicating a sex bias.
To elucidate whether the sex bias in 5-LO activity influences burn wound healing, we generated global Alox5 (Arachidonate 5-lipoxygenase, the gene that encodes for 5-LO) knockout mice (Alox5-/-) in C57BL/6 background. Alox5-/- and littermate Alox5+/+ control mice underwent burn injury, and wound tissues were isolated and examined 3, 7, and 14 days later.
Our data show a sex-specific effect of 5-LO activity in burn wound healing. Female Alox5-/- mice exhibited increased re-epithelialization compared to their controls, on day 14 post injury (pi). Furthermore, flow cytometric analysis on day 7pi revealed increased total numbers of wound immune (CD45+) cells, but no differences in their make-up, in female Alox5-/- mice compared to their controls. This suggests that 5-LO activity regulates overall immune cell recruitment in burn wounds of female mice; however, whether these cells are directly involved in the wound healing process is not currently elucidated.
On the other hand, Alox5-/- male mice showed decreased re-epithelization on days 7 and 14 pi, compared to their controls. In contrast with female mice, infiltration levels of male Alox5-/- wounds on day 7 pi were not different than those of their controls. This suggests that the observed delay in re-epithelialization may be independent of the inflammatory environment and could instead be mediated via 5-LO-induced effects on local skin cells.
Collectively, these results suggest that 5-LO activity affects burn wound healing in a sex-dependent manner. We hypothesize that sex-specific 5-LO-induced changes in the eicosanoid profile play a key role in the differential burn wound healing process. In this study, we will present eicosanoid wound profiles over time and in relation to sex and genotype. Differences in collagen production and transcriptome signatures will also be presented. Our study will help elucidate sex-specific 5-LO evoked mechanisms in burn wound healing.

P4.02

Angiography: A Limb Salvage Strategy Following Lower Extremity Thermal Injury In Patients With Diabetes Mellitus And Peripheral Artery Disease
Desiree Pinto1, Isabel Snee1, Saher Sabri2, Lauren Moffatt1, Taryn Travis1, Jeffrey W. Shupp2, Shawn Tejiram1
1Firefighter’s Burn and Surgical Research Laboratory, Washington, DC, United States 2The Burn Center, MedStar Washington Hospital Center, Washington, DC, United States
Angiography: A Limb Salvage Strategy Following Lower Extremity Thermal Injury In Patients With Diabetes Mellitus And Peripheral Artery Disease

Desiree Pinto1, Isabel Snee1, Saher Sabri2, Lauren Moffatt1, Taryn Travis1, Jeffrey W. Shupp2, Shawn Tejiram1 1Firefighter’s Burn and Surgical Research Laboratory, Washington; 2The Burn Center, MedStar Washington Hospital Center, Washington, DC

Background: Patients with diabetes mellitus (DM) or peripheral arterial disease (PAD) are at high risk of wound complications and amputations following lower extremity thermal injury. Algorithms to improve healing and limb salvage in chronic wounds from DM or PAD include angiography to facilitate peripheral blood flow analysis and revascularization. However, the role of angiography in patients with acute thermal injuries and DM or PAD have yet to be elucidated. This study describes a regional burn center’s experience incorporating angiography into the acute management of lower extremity thermal injuries in patients with DM or PAD.
Methods: Patients admitted with a lower extremity partial or full thickness thermal injury and history of DM or PAD between 2021 and 2023 were retrospectively reviewed. Patients with an abnormal arterial-brachial index (ABI), which prompted angiography evaluation, were included. Vascular disease identified and interventions performed during angiography were obtained. Clinical outcomes, including graft loss and amputations were evaluated.
Results: There were 23 patients with a lower extremity thermal injury, history of DM or PAD, and an abnormal ABI that underwent lower extremity angiography. Most patients were male (65.2%), had a thermal injury to their foot (82.6%) and had uncontrolled DM (median hemoglobin A1c of 10.2 mmol/mol). The median total body surface area (TBSA) was 2.0%. Vascular disease was identified in 18 patients (79%) by angiography. The tibial arteries were the most common site of vascular disease (87%), followed by the popliteal (14%) and superficial femoral arteries (14%). Incomplete pedal arch was identified in 5 patients (22%), with microvascular disease as the primary cause (60%). Vascular disease was amendable to re-vascularization attempts using balloon angioplasty in 9 patients (50%), with an 88.8% success rate. After re-vascularization over half of patients (55.6%) healed without grafting, while most patients without re-vascularization required further grafting (71.4%). When grafting was required, the rate of graft loss necessitating prolonged wound care or re-operation was less for patients who underwent re-vascularization (7.1% vs 14.3%). Amputations were necessary in 33.3% of patients who underwent re-vascularization compared to 14.3% of patients who did not have re-vascularization. There is not enough power to reach statistical significance.
Conclusions: This study is the first to describe the role of angiography in the acute management of lower extremity thermal injuries in patients with DM or PAD. We demonstrated angiography’s ability to improve vascularization in the lower extremity to promote wound healing without grafting and minimize graft loss. Further studies are needed to better understand the impact of angiography on limb salvage.

P4.03

Evaluation Of Subcutaneous Combination Ibuprofen And Resolvin D2 Therapy To Mitigate Burn Progression
Marc Thompson1, Sergio Garcia1, Lucy Shaffer1, Michelle Holik1, David Larson1, Logan Leatherman1, Valeta Sanders1, Anna Ochoa1, LTC Julie Rizzo2, Robert Christy1, Christine Kowalczewski1
1US Army Institute of Surgical Research, JBSA Ft. Sam Houston, TX, United States 2Trauma Surgery, Brooke Army Medical Center, JBSA Ft Sam Houston, TX, United States
Evaluation Of Subcutaneous Combination Ibuprofen And Resolvin D2 Therapy To Mitigate Burn Progression

Marc Thompson1, Sergio Garcia1, Lucy Shaffer1, Michelle Holik1, David Larson1, Logan Leatherman1, Valeta Sanders1, Anna Ochoa1, LTC Julie Rizzo2, Robert Christy1, Christine Kowalczewski1 1US Army Institute of Surgical Research, JBSA Ft. Sam Houston, TX; 2Trauma Surgery, Brooke Army Medical Center, JBSA Ft Sam Houston, TX

Introduction: Thermal injuries are common to all military conflicts. Difficulties treating partial-thickness burns in the field are further complicated by their propensity to progress/convert to deep-partial or full-thickness burns within the first 24 hours of injury. It is hypothesized that burn conversion (deepening) occurs as a function of irregular perfusion linked to inflammation, which ultimately causes progressive necrosis in the wound. This study hypothesizes that subcutaneous administration of inflammation mitigating Ibuprofen and Resolvin D2, in a porcine model of burn progression, will prevent partial-thickness thermal burns from progressing to deep partial/full-thickness injuries, potentially improving wound healing outcomes.
Materials and Methods: To create partial-thickness burns in isoflurane anesthetized Yorkshire swine (also provided Buprenorphine SR for pain management), a 100C heated 6 cm diameter brass cylinder was applied to the dorsum, to create ten burns per animal. An N of 6 animals were used; all 6 received the following treatments (2 wounds per treatment/animal): 1. Gauze (standard of care control), 2. Saline (vehicle control) 3. Ibuprofen (0.1 mg/mL) 4. Resolvin (0.05 mg/mL) 5. Combination treatment of Ibuprofen and Resolvin D2 at the same doses.
Treatments were injected 3 hours post-injury and once again 24 hours later to simulate a prolonged field care (PFC) scenario. Before the burn injury and at pre-assigned time points post-burn (days 0, 1, 3, 7, 14, 21, and 28), wounds were imaged (Digital, Silhouette, MolecuLight, Moor Laser Doppler Imaging) and biopsies harvested. Half of the biopsy was evaluated histologically by H&E and immunohistochemistry (IHC) to determine the depth (normalized % of dermal depth) of damaged tissue and wound healing over the experimental period. Remaining biopsy samples were preserved for proteomic analysis.
Results: A one-way Analysis of Variance (ANOVA) was employed to determine that, 24 hours after injury, combination therapies of Ibuprofen and Resolvin D2 prevented burns from progressing to deep partial-thickness burns (55.8 ±21.4%) (p<0.0001), whereas the military standard of care (Gauze) did not (66.6 ±21.9%). In addition, all experimental treatments reduced the lateral size (surface area) of burn injuries compared to Gauze, albeit not to a statistically significant degree.
Conclusions: Therapies already available in the field (Ibuprofen) and those less so (Resolvin D2) have previously displayed the ability to mitigate injury severity by lessening the degree of inflammation. We have shown here that while individual therapies may improve wound healing outcomes compared to the current standard of care, it may be the case that therapies used in concert, delivered directly to the injury, with varying mechanisms of action, can provide even greater efficacy.

P4.04

ELU42, A Small Molecule Wnt Signaling Inhibitor, Significantly Accelerates Wound Closure And Promotes Regenerative Repair Following Cutaneous And Third-Degree Burn Injury In Yorkshire Pigs
Daniel Holsworth, Sarika Saraswati, John Delgado, Michael Stone
Eluciderm Inc, San Diego , CA, United States
ELU42, A Small Molecule WNT Signaling Inhibitor, Significantly Accelerates Wound Closure And Promotes Regenerative Repair Following Cutaneous And Third-Degree Burn Injury In Yorkshire Pigs

Daniel Holsworth, Sarika Saraswati, John Delgado, Michael Stone Eluciderm Inc, San Diego, CA

BACKGROUND: The canonical WNT signaling pathway is quiescent in many mammalian organs and becomes activated in response to injury. WNT signaling promotes fibrotic wound healing (including scarring) following acute cutaneous injury. Topical “spray-on” application of a proprietary WNT signaling inhibitor accelerated wound closure and promoted regenerative cutaneous repair in acute and third-degree burn wounds.
METHODS: In this study, we utilized two (2) porcine models to analyze wound repair. Six (6) full-thickness 3 x 3 cm2 acute and ten (10) third-degree burn excisional wounds were created on the backs of Yorkshire pigs. ELU42, a novel, potent, aqueously soluble, topical “spray-on” small molecule WNT signaling inhibitor, was applied three (3) days a week (Mon, Wed, Fri; 200mL) up to Day 30. The animals were allowed to heal for another 30 days before being sacrificed at Day 60. Histopathological analyses were performed on excised tissues.
RESULTS: In full-thickness acute and third-degree excisional wounds, topical application of the novel small molecule WNT signaling inhibitor, ELU42, significantly promoted wound closure, and also promoted regeneration of tissue, as evidenced by the presence of restored skin architecture with adnexal structures and restoration of well-organized granulation tissue. A statistically significant increase in rete peg formation at the dermal–epidermal junction was also identified. Significance calculations were performed using a two-way Analysis of Variances (ANOVA) using the Prism10 software (Graph-pad prism). P<0.05 was considered significant.
CONCLUSIONS: Until now, studies using small molecule WNT signaling inhibitors were limited for therapeutic usage due to their poor aqueous solubility. We have created ELU42, a water-soluble small molecule WNT signaling inhibitor, in spray-on form. It is a non-toxic potential drug, and does not require a sterile environment when applying to traumatic soft tissue (acute) wounds and third-degree burns. Our study presents a stable, potent, bioavailable, small molecule WNT signaling inhibitor that has strong pharmacological potential for use as a therapeutic for the regenerative repair of cutaneous wounds.

P4.05

The Use Of Therapeutics Peptides With Full-Thickness Skin Columns To Improve Healing Of Excisional Wounds
Kristo Nuutila1, Anders Carlson2, Sean Christy2, David Larson2, Chan Rodney2, Thomas Darling3, Ira M. Herman4
1USAISR, San Antonio, TX, United States 2The Metis Foundation, San Antonio, TX, United States 3USUHS, Bethesda, MD, United States 4TUFTS University, Boston, MA, United States
The Use Of Therapeutics Peptides With Full-Thickness Skin Columns To Improve Healing Of Excisional Wounds

Kristo Nuutila1, Anders Carlson2, Sean Christy2, David Larson2, Chan Rodney2, Thomas Darling3, Ira M. Herman4 1USAISR, San Antonio, TX; 2The Metis Foundation, San Antonio, TX; 3USUHS, Bethesda, MD; 4TUFTS University, Boston, MA

Introduction: In split thickness skin grafting (STSG) a very thin layer of skin containing the epidermis and a small portion of the dermis is grafted. This is done to create a quick coverage on the receiving site but also to minimize donor site morbidity. One of the drawbacks is the absence of adnexal structures and thus the grafted wounds lack basic skin functions such as durability, thermoregulation, maintenance of hydration and lubrication. An alternative technique to solve this problem is fractional autologous skin grafting using full-thickness skin columns (FTSC).
Harvesting occurs orthogonally by taking numerous individual skin columns containing the epidermis down through the dermis including the skin appendages. Bioactive peptides have been shown to rescue hair follicular unit survivals. The purpose of this study is to investigate whether transplantation of FTSCs in combination with bioactive peptides would better reconstitute skin function due to the positive effect of the peptides on adnexal structures within FTSCs.
Materials and Methods: Up to 16 standardized full-thickness excisional wounds were created on the dorsum of two anesthetized pigs. Analgesia was provided prior to all surgical procedures and the animals were monitored for pain twice every 24 hours for the first 72 hours. FTSC biopsies were harvested from donor sites located on the cranial-dorsum at a ratio of up to sixteen 1.5 mm-diameter skin columns/1cm2. The wounds were randomized to receive either FTSC + bioactive peptide hydrogel (carboxymethyl cellulose), FTSC + scrambled peptide hydrogel, FTSC only or left untreated. Healing was monitored for up to 28 days. The wounds were excised and fixed in formalin for histologic analyses. In addition, non-invasive imaging systems were utilized to assess both wound healing and quality of healing.
Results: By day 14, the FTSC + bioactive peptide, FTSC + scrambled peptide, FTSC only and untreated wounds were 71%, 55%, 88% and 27% re-epithelialized respectively. The FTSC + bioactive peptide and the FTSC only treated wounds were significantly more re-epithelialized than the untreated wounds (p < 0.05). By day 28 all the FTSC transplanted wounds were fully re-epithelialized and the difference to the untreated wounds was statistically significant (p < 0.05). The results demonstrated that wound treated with the FTSC + bioactive peptide had more mature epidermis at day 28 post transplantation. The FTSC + bioactive peptide treated wounds had significantly more rete ridges and more mature epidermis in comparison to the other groups. In terms of wound contraction, no significant differences were observed. Conclusions: FTSC can be transplanted in a hydrogel to close a full-thickness wound. Furthermore, it was shown that transplantation of FTSC in combination with bioactive peptides increased quality of wound healing.

P4.06

Immediate Application Of Topical Anti-Inflammatory Agents On Burn Wounds And Their Effect On Healing
Jamie Neelon1, Irene Yau2, Kristo Nuutila3
1General Surgery, Brooke Army Medical Center, San Antonio, TX, United States 2General Surgery, William Beaumont Army Medical Center, El Paso, TX, United States 3USAISR, San Antonio, TX, United
Immediate Application Of Topical Anti-Inflammatory Agents On Burn Wounds And Their Effect On Healing

Jamie Neelon1, Irene Yau2, Kristo Nuutila3 1General Surgery, Brooke Army Medical Center, San Antonio, TX; 2General Surgery, William Beaumont Army Medical Center, El Paso, TX; 3USAISR, San Antonio, TX

Introduction: Though recent advances in the treatment of burns have considerably improved overall survival rates, they have also highlighted several long-term sequelae related to the injury. Hypertrophic scars, for example, can impair function, reduce quality of life, and require multiple procedures as well as physical therapy. The purpose of this study was to investigate the effects of topical application of anti-inflammatory drugs on burn wound progression, overall wound healing, and quality of healing.
Materials and Methods: 15 deep-partial thickness burns were created on the dorsum of four anesthetized swine using a custom burn device at 100C. Analgesia was provided prior to all surgical procedures with buprenorphine and the animals were monitored for pain twice every 24 hours for the first 72 hours. The burn wounds were randomized to receive amiloride, celecoxib, dexamethasone or minocycline formulated in a hydrogel. Silver sulfadiazine cream and blank hydrogel acted as controls. The animals were followed for 90 days and the wounds were assessed on days 3, 14, 28 and 90 post-burn. Assessments were performed using digital photographs (macroscopic healing, contraction), laser-Speckle imagery (blood perfusion), 3D camera (scarring, pigmentation), and histology (burn wound depth, epidermal thickness, rete ridges).
Results: 15 deep-partial thickness burns were evaluated. It was shown that on day 3, burn depth varied from 155 μm (celecoxib) to 222 μm (blank hydrogel) but no statistically significant differences were observed. In terms of wound healing, the results showed that by day 14 post-burn, percent wound closure ranged from 45% (dexamethasone) to 84% (celecoxib) but no significant differences were observed. By day 28 post-burn all the wounds were fully healed. Quality of healing was studied on day 90 post-burn. Wound contraction varied from 28% (celecoxib) to 43% (minocycline) but no significant differences were seen. No differences were observed in the thickness of epidermis or number of rete ridges.
Conclusions: This study concluded that topical application of amiloride, celecoxib, dexamethasone or minocycline formulated in a hydrogel did not mitigate burn wound progression, promote wound healing or increase quality of healing when compared to controls.