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2025 Abstracts

I.01

Statin-Cyclodextrin Hydrogels Accelerate Healing In Multiple Pre-Clinical Wound Models
Veronika Jurczuk1, Beatriz Abdo Abujamra1, Victoria Soto1, Fotios Andreopoulos3, Johan Mueller2, Marjana Tomic-Canic1, Stephen Davis1, Ivan Jozic1
1Dermatology, University of Miami, Miami, FL, United States 2Mölnlycke Healthcare, Gothenburg, Sweden. 3Biomedical Engineering, University of Miami, Miami, FL, United States
Statin-Cyclodextrin Hydrogels Accelerate Healing in Multiple Pre-Clinical Wound Models

Veronika Jurczuk1, Beatriz Abdo Abujamra1, Victoria Soto1, Fotios Andreopoulos3, Johan Mueller2, Marjana Tomic-Canic1, Stephen Davis1, Ivan Jozic1 1Dermatology, University of Miami, Miami, FL; 2Mölnlycke Healthcare, Gothenburg, Sweden; 3Biomedical Engineering, University of Miami, Miami, FL

Chronic wounds present an enormous socioeconomic burden due to limited efficacy of currently available treatments, resulting in limb amputation of approximately 30% patients with a chronic wound and a 5-year mortality rate matching that of the pooled mortality rate for all reported cancer. Our group has previously demonstrated that both cholesterol and lipid-raft associated proteins are elevated in chronic wound patients. Thus, the current study aimed to develop and assess the therapeutic potential of a topical hydrogel therapy that targets cholesterol synthesis utilizing HMG-CoA-reductase inhibitors (statins).
Layered gelatin-hyaluronic acid (GA-HA) hydrogels with cyclodextrin (CD) inclusion complexes formed with mevastatin and simvastatin were used to target cholesterol in wounds. Hydrogel stability was previously assessed via swelling and degradation assays, while toxicity and migration assays were assessed using primary human keratinocytes and fibroblasts. Hydrogels were tested in several pre-clinical in vivo models including: (a) splinted db/+ mice (n = 10, 2 wounds/animal × 5 animals), (b) splinted acute db/db mice (n = 10, 2 wounds/animal × 5 animals), (c) chronic db/db mice (n = 10, 2 wounds/animal × 5 animals), and d) the full thickness porcine model (n = 4 wounds/treatment × 3 animals). The effectiveness of the statin/CD hydrogels was assessed by measuring re-epithelization (H&E), collagen deposition (Mason Trichrome), barrier restoration (TEWL), angiogenesis (CD31 staining), inflammation (CD45 staining), macrophage polarization (Arg1 and iNOS staining), cytokine profiling using sandwich ELISAs and bulk RNAseq.
By altering the concentration of hydrogel crosslinker, degrees of swelling and degradation were adjusted to allow for burst vs sustained release of cargo. In vitro, the layered hydrogels improved keratinocyte migration compared to EGF while demonstrating no cytotoxicity in either keratinocytes or fibroblasts, confirming biocompatibility. Moreover, topical treatment of hydrogels in all in vivo models promoted re-epithelialization, collagen deposition, angiogenesis, and dampened the inflammatory response and increased levels of pro-resolving M2 macrophage when compared to vehicle and PDGF-treated subjects.
Interestingly, the more chronic the wound model assessed; the more effective statin/CD hydrogels appeared to be in all assessments included in our study.
We have demonstrated that statin/CD layered hydrogels provide a topical, transient, and sustained release of statins which promoted wound healing in various pre-clinical models by targeting multiple phases of wound healing: re-epithelialization, angiogenesis and inflammation.

I.02

Semtwist Analysis Of Biofilm Aggregates In Wound Tissue
Veronika Jurczuk1, Beatriz Abdo Abujamra1, Victoria Soto1, Fotios Andreopoulos3, Johan Mueller2, Marjana Tomic-Canic1, Stephen Davis1, Ivan Jozic1
1Dermatology, University of Miami, Miami, FL, United States 2Mölnlycke Healthcare, Gothenburg, Sweden. 3Biomedical Engineering, University of Miami, Miami, FL, United States
Semtwist Analysis of Biofilm Aggregates in Wound tissue

Surabhi Singh, Fabio Muniz De Oliveira, Cong Wang, Yi Xuan, Deeptankar DeMazumder, Manoj Kumar, Subhadip Ghatak, Chandan K. Sen, Sashwati Roy McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA

Biofilm infection (BFI) complicates wound healing. Field emission scanning electron microscopic (FE-SEM) studies show that the wound biofilm, shaped by iterative exchange between microbial and host-defense processes, is a polymicrobial aggregate including host cells. Characteristics of the wound biofilm aggregates are unique and quantifying BFI in human wounds remains challenging due to the lack of standardized approach. The objective of this work was to develop SEMTWIST, SEM-based Trainable Weka Intelligent Segmentation Technology, to robustly detect and quantify wound biofilm in complex human wound tissue matrix. To standardize this model, TWIST training for BFI quantification was conducted on 240 SEM images derived from sixty chronic wound-edge tissue biospecimens, with four technical replicates per specimen. The workflow involved pre-processing steps followed by segmentation to classify regions as BFI (e.g., EPS, bacterial aggregates) or non-BFI (e.g., host tissue landscape features). Segmentation accuracy was verified through toggle overlays in SEMTWIST-analyzed images. Subsequently, a subset of images was used to compare SEMTWIST-generated results with expert human assessments of BFI abundance, which were further validated using a molecular approach, PNA-FISH (probes targeting S. aureus and P. aeruginosa), bacterial culture and wheat germ agglutinin (WGA) staining methods. Correlation and Bland-Altman studies were performed to test accuracy and reliability.
SEMTWIST reliably identified varying grades of BFI: ≤ 10% (no biofilm), 10% to < 40% (low biofilm), 40% to < 70% (intermediate biofilm), and ≥ 70% (high biofilm), providing valuable information that can facilitate prompt diagnosis of BFI in clinical samples. SEMTWIST results when compared with human expert assessments, revealed variability among human individual raters, with the Bland-Altman plot showing a mean bias of 17.8 and wide limits of agreement (33.34 to 68.93). In contrast, when the average scores from human raters were compared with SEMTWIST results, the mean bias was reduced to 1.25, with 95% limits of agreement ranging from 43.40 to 47.11. These findings demonstrate that human ratings are useful only when assessments from at least three independent experts are considered. SEMTWIST exhibited superior performance and with specialized training using additional dataset, is expected to improve further. The wound culture and WGA staining results showed clear limitations. While eight samples were negative for bacterial species in culture, five of these culture-negative specimens were biofilm-positive as detected by SEMTWIST followed by PNA-FISH confirmation. Findings of this work underscore the unreliability of CFU-based culture techniques to detect BFI in chronic wound tissues.
Open source SEMTWIST is recognized as a robust tool to objectively quantify BFI burden in human chronic wound-edge tissues.

I.03

WITHDRAWN
Oluchi Aroh2, Alex Cheong1, Meghan Brennan1, Lindsay Kalan2,1 1Madison, WI; 2McMaster University, Hamilton, ON, Canada
Integrated Transcriptional Analysis of the Host And Microbiome in Diabetic Foot Ulcers

Oluchi Aroh2, Alex Cheong1, Meghan Brennan1, Lindsay Kalan2,1 1Madison, WI; 2McMaster University, Hamilton, ON, Canada

Background: Over 2 million Americans develop a diabetic foot ulcer (DFU) each year, costing > $1.3 billion and resulting in > 130,000 amputations annually. We have previously found that microbial transcription can serve as a marker of healing status and amputation risk but the link to host inflammation has not been explored.
Methods: Here we integrate the human and microbiome transcriptomes (metatranscriptome) from 180 DFU samples, corresponding to debrided tissue and deep wound swabs of 90 patients. Differential gene expression analysis was conducted to compare host gene activity in fast-healing versus slow-healing wounds. Weighted gene network analysis (WGCNA) was employed to identify gene modules associated with healing outcomes, while microbiome abundance data was used to identify microbial features associated with healing outcomes.
Additionally, a supervised machine learning approach based upon a random forest classification model and feature selection (Boruta) was used to identify the best set of representative microbiota predictive of healing outcomes. Spearman correlation analysis was then performed between host gene modules and microbiome abundance data for taxa identified using the methods above. Results: We find that host transcription alone is sufficient to identify fast-healing wounds, defined as those healed in less than 4 weeks. Genes involved in biological processes such as epidermal development, keratinocyte differentiation, and keratinization were upregulated in fast-healing wounds, while genes involved in defense response were upregulated in slow-healing wounds. Modules positively correlated with healed wounds were made up of genes involved in epithelial and epidermis cell differentiation, keratinocyte differentiation, and keratinization. Conversely, genes in modules correlated with unhealed wounds were made of genes involved in immune response, including immune response-regulating and activating signaling pathway, phagocytosis, response to bacterium, and response to virus. Species in the Corynebacterium, Pseudomonas, Brevibacterium, and Staphylococcus genera were significantly negatively correlated with genes in modules associated with unhealed wounds, while species in Streptococcus and Anaerococcus genera were positively correlated. Conversely, species in Corynebacterium, Staphylococcus, Pseudomonas, and Brevibacterium genera were positively correlated with genes in modules associated with healed wounds, while species in Streptococcus and Fastidospila genera were negatively correlated.
Conclusions: Immune activation and inflammation are key components of innate immunity triggered by microbes and infection. Together, these results show that human-microbe interactions differ in healing and non-healing DFUs. Future work will investigate specific microbial transcripts associated with host immune activation and inflammation index in an independent cohort of DFU samples collected through the diabetic foot consortium.

I.04

Using In Vivo Fluorescence Lifetime Imaging of NADH to Quantify the Effects of Age and Diabetes on Skin Wound Metabolism
Divya M. Gollapalli, Marcos R. Rodriguez, Kyle Quinn
Biomedical Engineering, University of Arkansas, Fayetteville, AR, United States
Using In Vivo Fluorescence Lifetime Imaging of NADH to Quantify the Effects of Age and Diabetes on Skin Wound Metabolism

Divya M. Gollapalli, Marcos R. Rodriguez, Kyle Quinn Biomedical Engineering, University of Arkansas, Fayetteville, AR

Chronic wounds are a large health care burden, but innovative diagnostic approaches have been lacking. Our group has demonstrated that label-free multiphoton microscopy (MPM) can serve as a non-invasive diagnostic tool for skin wounds. In that prior work, we highlighted how fluorescence lifetime imaging (FLIM) of the metabolic cofactor NADH could be used to assess keratinocyte function in vivo without the use of an exogenous fluorescent label. FLIM measures the time between excitation and emission, and the proportion of free and protein bound NADH can be extracted by modeling the time decay rate of NADH autofluorescence. The FLIM-derived fractions of free and bound NADH in the epithelium have been shown to change during wound healing and differ between young and old individuals. The goal of this study was to quantify the effects of diabetes, age, and their interaction on skin wound metabolism using FLIM.
Young (5 mo.) and aged (24 mo.) streptozotocin-induced diabetic (n = 18) and non-diabetic (n = 17) C57BL/6J mice (50/50 sex split) received full-thickness, excisional wounds on their dorsum. NADH FLIM images (755nm excitation/460nm emission) along the wound edge were collected in vivo with a 20×, 1.0 NA objective and time-correlated single photon counting from each mouse on days 1, 3, 5, 7, and 10 post-wounding using a multiphoton microscope. NADH lifetime decay within the epithelium was fit to a bi-exponential model to measure the lifetime decay constants (τ1 and τ2) and relative proportions of free (A1%) and protein bound (A2%) NADH.
All FLIM parameters significantly changed during the 10 days of longitudinal wound monitoring, including the lifetime of free NADH (τ1) (p < 0.0001), the lifetime of protein-bound NADH (τ2) (p < 0.0001), and the percentage of protein-bound NADH (A2%) (p < 0.0001). For all three parameters, values were the lowest during the initial inflammatory phases of healing (Days 1–3), and significantly increased at the later phases (Days 5–1, p < 0). This shift to longer lifetimes and more protein-bound NADH over post-wound time-points indicates a sensitivity to the transition from glycolytic to oxidative metabolism during healing. Overall, diabetic mice had a significantly higher percentage of protein-bound NADH (A2%) (p = 0.0158) and significantly longer free NADH lifetime (τ1) (p = 0.0243). Interestingly, age-related differences in the NADH lifetime were not reflected in A2% or τ1 parameters, but the lifetime of bound NADH (τ2) was significantly longer in the epithelium of aged mice (p = 0.0005). This finding may be related to age-related changes in which protein complexes bind with NADH.
These findings indicate that in vivo FLIM imaging of NADH autofluorescence is sensitive to changes from glycolytic to oxidative metabolism during wound healing. Through modeling of the lifetime decay response, this approach also may be capable of discriminating the effects of diabetes and aging on keratinocyte metabolism at the edge of excisional skin wounds.

I.05

Preserving Lives and Limbs: The Role of Cellular, Acellular, and Matrix-like Products (CAMPs) in Diabetic Foot Ulcer Care Using TriNetX Data
Abigail Royfman1, Sarah Lux1, Kennedy Couch1, Molynna Nguyen1, Mandy Wong1, Richard Simman2, 1
1University of Toledo College of Medicine, Toledo, OH, United States 2Jobst Vascular Institute/ProMedica, Toledo, OH, United States
Preserving Lives and Limbs: The Role of Cellular, Acellular, and Matrix-like Products (CAMPs) in Diabetic Foot Ulcer Care Using TriNetX Data

Abigail Royfman1, Sarah Lux1, Kennedy Couch1, Molynna Nguyen1, Mandy Wong1, Richard Simman2,1 1University of Toledo College of Medicine, Toledo, OH; 2Jobst Vascular Institute/ProMedica, Toledo, OH

The burden of diabetic foot ulcers (DFUs) on patients and healthcare systems is substantial, with complications often leading to amputation and significant morbidity. Despite advancements in DFU management, optimizing treatment to reduce these adverse outcomes remains a priority. This study aimed to compare the effectiveness of cellular, acellular, and matrix-like products (CAMPs) versus standard care (debridement) in lowering the risk of lower-extremity amputation and improving survival outcomes in patients with DFUs.
A retrospective cohort study was conducted using the TriNetX database. Patients with type 1 or type 2 diabetes and foot ulcers were included. Patients were stratified into two cohorts: those receiving debridement alone (n = 66,868) and those treated with CAMPs in addition to debridement (n = 5,136). Sixty different CAMPs were available in the database, each with at least 10 patients. Baseline demographic and clinical characteristics, such as age, sex, race, hemoglobin A1c, and comorbidities, were collected. Kaplan-Meier survival analysis and risk ratios were performed to assess outcomes over five years.
The analysis revealed significant differences in outcomes between the two cohorts. The CAMPs cohort had a mean age of 62.2 ± 12.3 years, 66% male, with 67.28% White and 12.77% Black or African American representation. The debridement-only cohort had a mean age of 61.8 ± 13.1 years, 64% male, and a racial composition of 66.8% White and 16.37% Black or African American. Both groups had similar hemoglobin A1c levels (CAMPs: 8.06 ± 2.22 vs. debridement-only: 8.33 ± 2.38). Hypertension prevalence was higher in the CAMPs group (90% vs. 80%), as was the use of blood glucose regulation agents (88% vs. 80%). Over five years, the CAMPs group demonstrated a significantly reduced risk of lower-extremity amputation (18.7% vs. 21.9%; p < 0.0001) and improved survival rates (72.2% vs. 69.7%; p < 0.0001) compared to the debridement-only group.
Patients treated with CAMPs alongside debridement exhibited lower risks of lower-extremity amputation and improved survival compared to those receiving debridement alone. The demographic and clinical similarities between groups underscore the potential of CAMPs as an effective adjunctive therapy for DFUs, supporting their broader adoption in clinical practice.

I.06

WITHDRAWN
Manoj Kumar1,2, Debarati Chattopadhyay3, Mithun Sinha4, Akshay Kapoor3, Kanhaiya Singh1, shomita Mathew-Steiner1, Chandan K. Sen1 1Department of Surgery, University of Pittsburgh, Pittsburgh, PA; 2Centre for Economic Studies & Planning, Jawaharlal Nehru University, New Delhi, India; 3All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India, Rishikesh, Uttarakhand, India; 4Indiana University School of Medicine, Indianapolis, IN
May Elevated Trans-Epidermal Water Loss at Wound Closure Site Predict Wound Recurrence?

Manoj Kumar1,2, Debarati Chattopadhyay3, Mithun Sinha4, Akshay Kapoor3, Kanhaiya Singh1, shomita Mathew-Steiner1, Chandan K. Sen1 1Department of Surgery, University of Pittsburgh, Pittsburgh, PA; 2Centre for Economic Studies & Planning, Jawaharlal Nehru University, New Delhi, India; 3All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India, Rishikesh, Uttarakhand, India; 4Indiana University School of Medicine, Indianapolis, IN

Clinically, wound closure is defined as sustained (2 weeks) coverage of wound without discharge. It does not address functionality of the repaired skin. Recent patient-based studies have shown that wounds may close in a way such that the criteria of coverage with no discharge are met but barrier function of the skin is not restored. Thus, such wounds do not achieve functional wound closure. Compromised barrier function in such structurally “closed” wounds is measured as high transepidermal water loss (TEWL) at the site of wound repair. The current study utilizes TEWL measurement of repaired skin at the site of wound closure using DermaLab TEWL open chamber unit (Cortex Technologies, Denmark) as a surrogate marker to study post-closure cutaneous functionality of human wounds.
A single-center, prospective, observational pilot study was performed to evaluate wound healing endpoint and recurrence by measuring TEWL post-closure at the site of wound repair. Patients with clinically-defined chronic wounds (such as pressure ulcers, diabetic ulcers, and trauma wounds) were enrolled. Non-invasive point-of-care TEWL measurements were obtained, from closed wound-site and contralateral healthy skin site, starting from confirmation of closure (post-closure, V0) continuing every 2 weeks for a maximum of five visits or until the wound recurred. Statistical analyses of the data involved logistic regression and likelihood ratio chi-square tests to assess differences in TEWL at visit 0 (V0) between the closed wound site and reference skin, with the TEWL score as the sole predictor of recurrence.
Of the 72 subjects that completed the study, 44 (61%) showed no recurrence and 28 (39%) had wounds that recurred over a period of 12 weeks. A significant association was found between the V0 (post-closure) TEWL score and the odds of wound recurrence 1.26, 1.34, both in univariate and after adjusting for covariates in multivariable logistic analysis. The likelihood ratio chi-square analysis demonstrated that the V0 TEWL score may be a significant universal predictor of recurrence across all wound types studied. Cases of closed wounds with subsequent recurrence showed an overall higher post-closure V0 TEWL score, compared to those who did not have a wound recurrence, across visits. The TEWL score cut-off value predictive of recurrence was 24.1 reference unit.
The outcome of this study on a wide range of chronic wounds leads to the hypothesis that post-closure TEWL at the site of wound healing is a reliable biomarker of wound recurrence. It also raises the question whether the clinical endpoint of wound closure should include re-establishment of skin barrier function as additional criterion. The current standard of care wound closure endpoint calls for re-epithelialization of the wound with no discharge for two consecutive weeks disregarding the functional parameter of restoration of skin barrier function at the wound-site.

I.07

Novel Approach Using Cellchat Analysis To Identify Dysregulation Of Intercellular Communications During Initiation Of Wound Chronicity
Parnian Jabbari1, Brandon Le2, 3, Manuela M. Martins-Green1
1Department of Molecular, Cell, and Systems Biology, University of California Riverside, Riverside, CA, United States 2Institute of Integrative Genome Biology, University of California Riverside, Riverside, CA, United States 3Department of Botany and Plant Sciences, University of California Riverside, Riverside, CA, United States
Novel Approach Using Cellchat Analysis to Identify Dysregulation of Intercellular Communications During Initiation of Wound Chronicity

Parnian Jabbari1, Brandon Le2,3, Manuela M. Martins-Green1 1Department of Molecular, Cell, and Systems Biology, University of California Riverside, Riverside, CA; 2Institute of Integrative Genome Biology, University of California Riverside, Riverside, CA; 3Department of Botany and Plant Sciences, University of California Riverside, Riverside, CA

New approaches using single cell RNA sequencing (scRNAseq) have allowed us to identify dysregulations in different cell types during initiation of wound chronicity. What is not yet known is how the various cells in the wound communicate with each to affect processes such as proliferation, migration and angiogenesis. Recently, a novel approach to analyze cell–cell communication has been developed that involves a bioinformatic program called CellChat.
In this study, we used CellChat analysis on our data from a recent scRNAseq to identify the crosstalk between different cell types in non-chronic wounds (NCW) and Chronic wounds (CW) during chronic wound initiation and determined how these cells communicate during normal healing and how those communications become dysfunctional during chronic wound initiation.
Of the 17 clusters identified with the scRNAseq, we found two clusters of fibroblasts (Fib 1 enriched in NCW, Fib 2 enriched in CW), 6 clusters of keratinocytes, one cluster each of vascular (VEC) and lymphatic endothelial cells (LEC) and several clusters of immune cells, all altered in CW. Both Kerat 1 and Fib 1 crosstalk with VEC in NCW through Notch4 and Netrin signaling, respectively, to stimulate angiogenesis. These cross talks are absent in CW, leading to absence of angiogenesis. In NCW, Fib 1 crosstalk with several kerats and promote their proliferation and migration by secreting fibronectin. Fibronectin signaling is missing in CW, impairing Kerats proliferation and migration. Fib 2 in NCW secrete IL-6 and CSF which are essential for inflammation in the early phases of wound healing. These signaling pathways are crucial for mobilization of neutrophils to the wound for proliferation of mast cells. Lack of this crosstalk in CW could explain the fewer number of neutrophils and mast cells we observe in CW. Channel Kerats, express both ligands and receptors for FGF and EPGN signaling, which could explain the high number of these kerats in CW. Kerats 2 promote proliferation of Fib 1 in NCW through Periostin signaling. In CW, expression of both ligands and receptors that run on Periostin are high in Fib 2 leading to higher number of these cells, which have dysregulation of several prohealing processes. In NCW kerats can promote the function of M1 macrophages through SEMA5 signaling leading to more efficient phagocytosis of pathogens and cellular debris. In CW, Notch receptors promote M2 polarization, potentially explaining the high number of these cells in CW. Also, M2 macrophages express the ligand for APP signaling which binds to its receptor, CD74 in M1 macrophages decreasing their phagocytic abilities in CW.
In conclusion, by using CellChat to compare cell-cell communication between normal healing and chronicity, we can identify cellular interactions important for healing but are missing in chronic wounds, or interactions detrimental to healing and are present in CW. These new insights may be useful to devise new strategies to treat CW.

I.08

High Fat Induced Diabetes Depletes Systemic Ly6C+ CD64+ Monocytes In Blood And Bone Marrow To Impair Wound Healing
Maria Gracia Mora Pinos, Katharina Fischer, Lulejeta A. Latifi, Fidel Saenz, Amelia B. Knochel, Abdelrahman M. Alsharif, Syed Maisam Jafri, Olivia Renchar, Brodi Stevens, Eamonn McKenna, Hudson C. Kussie, Andrew Hostler, Autumn Lester, Maia Granoski, Kellen Chen, Geoffrey C. Gurtner
Surgery, University of Arizona, Tucson, AZ, United States
High Fat Induced Diabetes Depletes Systemic Ly6C+ CD64+ Monocytes in Blood and Bone Marrow to Impair Wound Healing

Maria Gracia Mora Pinos, Katharina Fischer, Lulejeta A. Latifi, Fidel Saenz, Amelia B. Knochel, Abdelrahman M. Alsharif, Syed Maisam Jafri, Olivia Renchar, Brodi Stevens, Eamonn McKenna, Hudson C. Kussie, Andrew Hostler, Autumn Lester, Maia Granoski, Kellen Chen, Geoffrey C. Gurtner Surgery, University of Arizona, Tucson, AZ

Affecting half a billion people globally, diabetes causes sustained hyperglycemia that leads to pathologic changes across the body, such as impairing immune cells from responding to pathogens or promoting chronic inflammation that delays wound healing. In this study, we characterized the presence of Ly6C+/CD64+ monocytes in various tissues of wounded diabetic mice to better understand how diabetes alters the systemic immune ecology.
Full-thickness excisional wounds were created on the dorsum of C57/BL6 wildtype mice fed a normal diet (WT = 5) or fed a high fat diet for 16 weeks to induce diabetes (DB = 5). Skin wound tissue, blood, and bone marrow (BM) were collected at postoperative days (PODs) 0, 2, and 7. Tissue was processed for either single cell RNA sequencing (scRNA-seq) or flow cytometry, gating for Viability-, CD45+, CD11b+, and CD64+Ly6C+. The Fcgr1 gene encodes for CD64.
WT mice healed fully by POD 14, significantly faster than DB mice which healed by POD 22 (p < 0.0001). ScRNA-seq of healing wound tissue found that Ly6c2+Fcgr1+ monocytes represented 68% of all myeloid cells at POD 2 in WT and were not present in DB mice. Using flow cytometry, we observed that WT mice doubled the amount of Ly6C+CD64+ blood monocytes at POD2 (p < 0.0001) and quadrupled the expression by POD7 (p < 0.0001) compared to DB blood. Specifically, Ly6C+CD64+ monocytes predominated 44% of WT myeloid cells at POD0 and increased to 80% by POD2 (p = 0.0003) and POD7 (p = 0.0015). In contrast, these monocytes accounted for 60% of POD0 DB myeloid cells that decreased to 40% at POD2 (p < 0.05) and 18% at POD7 (p < 0.0001). We observed no significant difference of Ly6C+CD64+ monocytes between the experimental groups' BM at POD0 and POD2 (p = 0.13; p = 0.06 respectively). However, POD7 WT BM expressed Ly6C+CD64+ monocytes 60% higher than in DB BM (p = 0.022). The mean expression of WT BM Ly6C+CD64+ monocytes stayed consistent around 17.5% of all myeloid cells across time. On the other hand, DB BM significantly decreased monocyte expression from 24% at POD0 (p = 0.0002) and POD2 (p < 0.0001) compared to 10% at POD7. Our results show that Ly6C+CD64+ monocytes are dynamic and change through the healing process in various tissues. Normal, unimpaired wound healing demonstrates upregulation of Ly6C+CD64+ monocytes in the blood as soon as POD2 that is delayed until POD7 in the BM. Diabetes suppresses these monocytes in early wound healing, potentially due to chronic hyperglycemia inducing epigenetic alterations that promote differentiation toward a persistent inflammatory state. This persistent inflammation might exhaust monocyte immune cells in the blood and bone marrow to impair healing. A better understanding of monocyte differentiation during wound healing and how different tissues interact during diabetes could improve and inform diabetic care, both locally and systemically.

I.09

High Fat Induced Diabetes Depletes Systemic Ly6C+ CD64+ Monocytes In Blood And Bone Marrow To Impair Wound Healing
Cotten A, Jeanneau C, Decherchi P, About I.
Aix-Marseille Université, Centre National de la Recherche Scientifique, Institut des Sciences du Mouvement (UMR 7287), Marseille, France
Complement C5a Enhances Neuroregeneration In Vitro

Cotten A, Jeanneau C, Decherchi P, About I Aix-Marseille Université, Centre National de la Recherche Scientifique, Institut des Sciences du Mouvement (UMR 7287), Marseille, France
Background including aim/purpose: Central nervous system injury has no effective treatment to restore the lost functions. Complement C5a protein has been shown to play a major role in the regeneration of tissues such as bone, liver, and dentin-pulp complex. This work aimed at investigating the effect of Complement C5a on the axonal growth after a physical injury in vitro. Methods: Primary hippocampal neurons were isolated from embryonic Wistar Rats (E18.5) and characterized by immunofluorescence. Cell expression of C5a mRNA receptor (C5aR) was studied by RT-PCR while its membrane expression, localization and phosphorylation were investigated by immunofluorescence. The effect of C5a on the axonal length and speed were investigated using 3D-printed microfluidic devices after performing axonal physical injuries. Complement C5a was encapsulated into Polylactic-poly-glycolic acid microspheres to allow its sustained release and the microspheres were incorporated into a collagen scaffold. The C5a-loaded scaffold toxicity was studied using an MTT assay while the cell migration into the scaffold was evaluated using a home-made 3D-printed device.
Results: Immunofluorescence labelling demonstrated that our primary cultures contained only mature neurons (93%) and astrocytes (7%) but no oligodendrocytes or immature neurons were detected. The C5aR mRNA was expressed in the primary cultures as demonstrated by RT-PCR.
Immunofluorescence revealed a co-localization of the NF-L and the C5aR or the phosphorylated receptor (C5aR-P) only in the mature neurons. Applying a C5a gradient on the injured axons in the microfluidic devices increased both the axonal growth length and speed. Finally, we observed no toxicity and cells adhered and migrated into the scaffold after 7 days.
Discussion/conclusion: Our findings highlight a new role of C5a in axonal regeneration after a physical injury. A patent on the incorporation of C5a in a scaffold for tissue regeneration has been published and its use in the spinal cord injury treatment is currently under investigation in vivo.

K1.01

BETA-ADRENERGIC ANTAGONIST FOR THE HEALING OF CHRONIC DIABETIC FOOT ULCERS
Sara Dahle*2,3, Johanna Ghebrehiwet-Kuflom1,6, Mirabel Dafinone1,6, Rawlings E. Lyle3,4, Pallas Lim1, Anuj Budhiraja3,5, Alisha Mehta1,5, Harrison J. Shawa1, Catherine Tchanque-Fossuo1, Ramanjot Kaur1, Anthony Gallegos6, Rivkah Isseroff3,6
1Dermatology, VA Northern California Healthcare System, Sacramento, CA; 2Podiatry, VA Northern California Healthcare System, Sacramento, CA; 3Dermatology, VA Northern California Healthcare System, Sacramento, CA; 4School of Medicine, University of California, Davis, Sacramento, CA; 5College of Medicine, California Northstate, Elk Grove, CA; 6Dermatology, University of California, Davis, Davis, CA
Beta-Adrenergic Antagonist for the Healing of Chronic Diabetic Foot Ulcers

Sara Dahle2,3, Johanna Ghebrehiwet-Kuflom1,6, Mirabel Dafinone1,6, Rawlings E. Lyle3,4, Pallas Lim1, Anuj Budhiraja3,5, Alisha Mehta1,5, Harrison J. Shawa1, Catherine Tchanque-Fossuo1, Ramanjot Kaur1, Anthony Gallegos6, Rivkah Isseroff3,6 1Dermatology, VA Northern California Healthcare System, Sacramento, CA; 2Podiatry, VA Northern California Healthcare System, Sacramento, CA; 3Dermatology, VA Northern California Healthcare System, Sacramento, CA; 4School of Medicine, University of California, Davis, Sacramento, CA; 5College of Medicine, California Northstate, Elk Grove, CA; 6Dermatology, University of California, Davis, Davis, CA

Diabetic foot ulcers (DFUs) represent a significant and growing public health concern, affecting millions of individuals with diabetes. These chronic wounds often fail to heal despite standard care, leading to prolonged disability, increased healthcare costs, and in severe cases, amputation. Current therapies primarily focus on infection control and offloading, with limited options for improving healing rates. A promising therapy is topical timolol, a nonselective beta-blocker, which has shown potential to improve two critical factors in wound healing, circulation and reduce inflammation. However, evidence supporting its efficacy in the treatment of DFUs remains underexplored. This study aimed to assess the effectiveness and safety of topical timolol as an adjunct to standard care in promoting the healing of chronic DFUs.
This is a randomized, double-blinded, controlled study. The primary endpoint was complete wound healing by week 14, with secondary outcomes assessing healing by week 31, time to wound closure, wound size reduction, and adverse events. Safety was monitored by measuring serum timolol levels. Statistical analysis was performed using Fisher’s exact test, Kaplan–Meier survival analysis, and Cox proportional hazards modeling to evaluate healing outcomes and potential confounders.
From 2018 to 2023, 108 patients with chronic DFUs were enrolled in the study from VA Northern California Health Care System. Of these, 48 met the inclusion criteria and were randomized to either the standard of care (SOC) group (n = 27) or the SOC + timolol group (n = 21). At week 15, 38% (n = 8/21) of patients in the SOC + timolol group had healed compared to 33% (n = 7/27) in the SOC group (p = 0.77). By week 30, 71% (n = 15) of patients in the timolol group had achieved complete healing, compared to 48% (n = 13) in the SOC group (log-rank test, p = 0.081). Cox proportional hazards modeling revealed that patients receiving timolol had a significantly higher likelihood of healing by week 30 (hazard ratio [HR] = 2.88, p = 0.027). Body mass index (BMI) was identified as a significant covariate (HR = 1.06, p = 0.031). In terms of wound closure, the timolol group demonstrated a significantly shorter mean time to healing at 15 weeks (5.8 vs. 9.2 weeks, p = 0.015).
Additionally, wound size reduction was greater in the timolol group at both 14 and 31 weeks (p < 0.05). Adverse events were similar between groups, with no cardiac events reported in the timolol cohort.
Topical timolol was associated with improved wound healing rates and faster time to closure compared to standard care. These findings suggest that timolol may be a promising and effective adjunct treatment for chronic DFUs, with minimal adverse effects. This study contributes to the growing body of evidence supporting novel adjuncts for the treatment of chronic diabetic wounds.

K1.02

ENGINEERED EXOSOMES RESTORE EPIDERMAL-DERMAL CROSSTALK ENABLING FUNCTIONAL WOUND REPAIR
ANITA YADAV*, Anu Sharma, Mohini Moulick, Parmeshwar Gavande, Aparajita Nandy, Chandan K. Sen, Sashwati Roy, Subhadip Ghatak
Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA
Engineered Exosomes Restore Epidermal-Dermal Crosstalk Enabling Functional Wound Repair

ANITA YADAV, Anu Sharma, Mohini Moulick, Parmeshwar Gavande, Aparajita Nandy, Chandan K. Sen, Sashwati Roy, Subhadip Ghatak Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA

Functional wound repair requires restoration of barrier function at the wound site above and beyond the standard requirement of wound closure with no discharge. We tested the hypothesis that engineering fibroblast-derived exosomes (Exofib) with AG53 peptide (ASKAIQVFLLAG) and WNT5A cargo for targeted epidermal delivery achieves functional wound closure. Human diabetic wounds were analyzed from the scRNA-seq dataset (GEO:GSE165816). We leveraged the affinity of archaeal ribosomal RNA-binding protein L7Ae for conserved box C/D RNA sequences to engineer Exofib. Plasmids were designed to encapsulate Wnt5a in Exofib, utilizing two constructs. The first set had 3 murine Col1a2 promoter-driven plasmids with RFP and L7Ae cloned “in frame” with the C-terminus of CD63, CD9, and CD81(exosome markers). An oligonucleotide sequence corresponding to AG53 peptide with an IRES element was added for keratinocyte targeting. The second set had a C/D box sequence cloned into the 3′-UTR of Wnt5a. The plasmids were validated in vitro across multiple cell types; encapsulation of Wnt5a in Exofib was confirmed by qRT-PCR and characterized as per MISEV2023 guidelines. Diabetic db/db mice underwent stented excisional wounding and tissue nanotransfection with the plasmid cocktails. Wound-edge (WE) tissues were collected on d7 to assess Wnt5a encapsulation within Exofib and its uptake by keratinocytes using dSTORM nano-imaging. D14 and 21 WE tissues were collected to evaluate tissue repair by employing analytical histology and biomechanical testing of the repaired skin. scRNA sequencing of 11 human diabetic ulcer samples, analyzed with Seurat (res: 0.50), identified 6 fibroblast and 4 keratinocyte subclusters. Sub-cluster interactome analysis revealed compromised crosstalk between the keratinocyte subsets K2, K3, and fibroblasts subset F3 in non-healing diabetic ulcers with altered expression of YAP1 and WNT5A. In mice, a significant increase in WNT5A expression was observed in DP fibroblasts (COL1A2+, CD90+MYL9+SCA1) at d7 post-wounding (p < 0.010, n = 12), unlike uninjured skin or diabetic WE tissue. Furthermore, WNT5A was localized within the endosomal-originated Exofib. Such Exofib was selectively taken up by basal keratinocytes expressing FZD6 and FZD7 receptors (p < 0.001, n = 6) at the site of epidermal invagination into the dermis. In db/db mice, delivery of engineered Exofib significantly enhanced Wnt5a encapsulation. Uptake of such engineered Exofib by keratinocytes improved functional wound closure outcomes as evident by lowered TEWL (p < 0.001, n = 8) and improved skin biomechanics as evident from tensile strengths, viscoelasticity and Young's modulus.
This study has the potential for advancing exosome-based therapies and assigns promising therapeutic value to engineered Exofib in improving healing outcomes in diabetic wounds, thus providing a robust strategy that could be applied to other regenerative and inflammatory disease contexts.

K1.03

Staphylococcus aureus Biofilm Infection Impairs Macrophage Dead Cell Clearance Necessary for Wound Healing
Piya Das Ghatak*, Fabio Muniz De Oliveira, Anita Yadav, Subhadip Ghatak, Chandan K. Sen, Sashwati Roy
Surgery, University of Pittsburgh, South Park, PA
Staphylococcus aureus Biofilm Infection Impairs Macrophage Dead Cell Clearance Necessary for Wound Healing

Piya Das Ghatak, Fabio Muniz De Oliveira, Anita Yadav, Subhadip Ghatak, Chandan K. Sen, Sashwati Roy Surgery, University of Pittsburgh, South Park, PA

Biofilm infection (BFI), implicated in 60% chronic wounds, involves Staphylococcus aureus (SA) as a predominant bacterial species. SA-BFI derails resolution of inflammation, necessary for wound healing. Macrophages perform cellular debridement by phagocytosing dead or dying cells via efferocytosis. Optimal efferocytosis helps resolve inflammation enabling wound healing. This work tested the hypothesis that SA-BFI impairs efferocytosis thus prolonging the inflammatory phase.
To specifically address the biofilm component of SA pathogenicity we employed a hypobiofilm (ΔsarA) and hyperbiofilm (ΔrexB) isogenic mutant strains of wildtype SA USA300. Mechanistic studies were performed on monocyte-derived macrophages (MDM) isolated from human peripheral blood and subjected to conditioned media (CM) derived from ΔsarA and ΔrexB biofilm cultures. To study the response of wound macrophages in chronic wounds, a murine model was used. Efferocytosis index or the ratio of the number of macrophages that have phagocytosed apoptotic cells to the total number of MDMs was analyzed. Efferocytosis index was significantly compromised (p < 0.05, n = 5) in high biofilm forming ΔrexB CM treated macrophages compared to treatment with low biofilm forming ΔsarA CM. RNAseq studies identified Macrophage receptor with collagenous structure (MARCO) as a biofilm-inducible surface protein that was significantly upregulated (p < 0.05; n = 5) both at the mRNA and protein levels in MDM treated with ΔrexB CM compared to treatment of MDM with low biofilm forming ΔsarA CM. Blocking MARCO with a neutralizing antibody in ΔrexB CM treated MDMs restored biofilm-induced suppression of macrophage efferocytosis index pointing towards a key role of MARCO in such dysfunction. MDM treated with ΔrexB CM, compared to ΔsarA CM, demonstrated high levels (p < 0.001; n = 6) of pro-inflammatory CCL3. To dissect the independent significance of myeloid cell MARCO, MARCO was overexpressed in wound macrophages using an in vivo myeloid cell targeted gene expression vector in a mouse excisional wound model. Over expression of MARCO in myeloid cells in excisional wound resulted in compromised (p < 0.001; n = 6) phosphatidylserine+ dead cell clearance and a pro-inflammatory environment in the granulation tissue.
This study identifies a specific cellular and molecular mechanism by which SA-BFI may complicate wound inflammation and healing. Specifically, MARCO causes SA-BFI dependent impairment of wound-site efferocytosis and sharp rise in wound-site phosphatidylserine+ apoptotic cells. In SA-BFI affected wounds, MARCO is a candidate therapeutic target.

K1.04

EXPLORING FUNGI IN THE MICROBIOME OF DIABETIC FOOT ULCERS
Michelle R. Bode*1, Alex Cheong2, Meghan Brennan2, Lindsay Kalan1,2
1Biochemistry & Biomedical Sciences, McMaster University, Hamilton, ON, Canada; 2Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI
Exploring Fungi in the Microbiome of Diabetic Foot Ulcers

Michelle R. Bode1, Alex Cheong2, Meghan Brennan2, Lindsay Kalan1,2 1Biochemistry & Biomedical Sciences, McMaster University, Hamilton, ON, Canada; 2Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI

Diabetic foot ulcers (DFUs) afflict 19-32% of people with diabetes in their lifetime. The treatment of DFUs is challenging as current assessments are subjective, and the effectiveness of prescribed therapies is unclear until months after initiation. Historically, DFUs lacked reliable biomarkers to predict which wounds would heal with treatment, hindering the development of new therapies and strategies to identify patients at risk of amputation. While recent studies have identified bacterial biomarkers associated with delayed wound healing, few have explored fungal presence in DFUs, raising questions about the role of fungi in these chronic wounds. This study analyzed the “mycobiome” of DFUs to identify correlations between fungal community structures and patient outcomes. For 100 patients with DFUs, three sample types were collected: tissue debrided from the wound surface, swabs from the inner wound bed, and swabs of healthy skin. A 12-week monitoring period tracked wound outcomes (healed, nonhealing, or amputated) with longitudinal samples collected every 1–2 weeks. High-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus was used to determine the presence and taxonomic classification of fungi.
Fungi are present in 97% of baseline samples. Healed wounds exhibited greater fungal diversity, particularly in debrided tissue samples. There were significant differences in fungal diversity between healed and non-healed wounds, with nonhealing wounds displaying lower diversity than healed wounds (p = 0.029) and amputated wounds showing the lowest diversity (p = 0.0046). Fungal diversity was significantly higher in healthy skin compared to wound samples (p < 0.0001). Healthy skin samples did not differ from each other based on outcome group as was observed for wound samples.
Fungi are frequently present in DFUs, with higher diversity in wounds that healed compared to those that were ongoing or amputated. This trend is in alignment with higher fungal diversity in healthy skin samples; however, healthy skin did not show distinct differences between outcome groups. The composition of fungal communities in DFUs, particularly on the wound surface, was strongly correlated with wound healing outcomes. This study represents the first to directly examine compositional differences in fungal colonization between DFU microbiomes and adjacent healthy skin, including both the surface and deeper wound layers. The findings suggest the potential for discovering fungal biomarkers of wound healing, which, in conjunction with bacterial biomarkers, could improve prognostic accuracy, enable personalized treatments, reduce amputation rates, and facilitate the development of alternative therapies to improve patient outcomes.

K1.05

OXYLIPINS DERIVED FROM HOST-BIOFILM INTERACTIONS IMPAIR DFU HEALING BY TARGETING IMMUNE CHECKPOINT MOLECULES VIA CD8+CTLA4+T-CELL POPULATIONS
Sunil Kumar*1, Ankit Tandon1, Miguel Jorge1, Ethan Rinne1, Bryce Hockman2, Kaitlyn Depinet2, Beth Altenburger2, Jaimee Hann2, Gregory Westin1, Mithun Sinha1
1Surgery, Indiana University, Indianapolis, IN; 2Comprehensive wound center, Indiana University Health, Indianapolis, IN
Oxylipins Derived From Host-Biofilm Interactions Impair Dfu Healing By Targeting Immune Checkpoint Molecules Via CD8+CTLA4+T-Cell Populations

Sunil Kumar1, Ankit Tandon1, Miguel Jorge1, Ethan Rinne1, Bryce Hockman2, Kaitlyn Depinet2, Beth Altenburger2, Jaimee Hann2, Gregory Westin1, Mithun Sinha1 1Surgery, Indiana University, Indianapolis, IN; 2Comprehensive wound center, Indiana University Health, Indianapolis, IN

Diabetic foot ulcers (DFUs) are a leading cause of lower limb amputations, with current treatments primarily relying on pharmacological interventions and standard care, often yielding suboptimal outcomes. The role of bacterial biofilms and their interactions with the host immune system in the context of wound healing remains poorly understood. Wound immunology research is primarily centered around macrophages and the role of effector T cells is less researched.
To test our hypothesis, we collected wound samples (N = 27) from consenting subjects under a protocol approved by IRB. We used scanning electron microscopy (SEM) and 16S rRNA (NGS) sequencing for biofilm quantification. Metabolite profiling of 10(S)-HOME and 9(S)-HODE was performed using (LC-MS/MS). Visium CytAssist was performed to determine spatial transcriptomics on patient samples. T-cells from a healthy donor were assessed for their immunological role and correlated with a human keratinocyte-derived cell line to simulate wound conditions under oxylipin challenge. Additionally, a dendritic cell line was employed to confirm the type of immune response against bacterial oxylipins. The study incorporated techniques such as flow cytometry, in vivo mouse stented excisional models, RT-qPCR, and ELISA.
Our analysis revealed a marked increase in bacterial biofilm abundance in wound tissues compared to controls. NGS sequencing identified key microbial species within the wound environment, including Staphylococcus aureus, Finegoldia magna, Pseudomonas aeruginosa, and Anaerococcus vaginalis. Additionally, oxylipin profiling demonstrated significant upregulation of 10(S)-HOME and 9(S)-HODE in wound tissue relative to normal, unwounded skin. Treatment with 10(S)-HOME resulted in a notable inhibition of human keratinocyte proliferation compared to sham controls (n = 10, p < 0.0003). Furthermore, immune checkpoint molecules, such as CTLA4 was dysregulated in CD4+ T-cells derived from healthy donors following treatment with 10(S)-HOME (n = 7) and 9(S)-HODE (n = 7). In addition, Mettl-3 inhibitor (STM2457) confirm CISH as a target of m6A-modification machinery. Dendritic cells treated with 10(S)-HOME exhibited enhanced antigen presentation capacity, as evidenced by the upregulation of co-stimulatory molecules.
These findings indicate that host-pathogen interactions mediated by biofilm-derived oxylipins may adversely affect the healing outcomes of diabetic foot ulcers (DFUs). This impairment appears to be facilitated through the increased abundance of immune checkpoint molecule CTLA4. Consequently, CTLA4 and other intracellular checkpoints represent promising targets for the management of DFUs by specifically modulating immune cell subsets.
Additionally, other checkpoint genes, such as PD-1 and CISH, are currently under investigation. Further research and validation are imperative to elucidate the potential of activating CD8+ effector T cells towards DFU healing.

K1.06

Tr1 Cells and Heparan Sulfate-Mediated IL-2 Sequestration Regulate Scarring Outcomes
Mary Elizabeth Guerra*1,2, Navya Nanda2, Casey Baxter1, Sonya Keswani1, Jaime Tellez2,1, Hui Li1,2, Ling Yu1,2, Mateusz Wietecha3, Paul Bollyky4, Swathi Balaji1,2, Sundeep Keswani1,2
1Baylor College of Medicine, Houston, TX; 2Texas Children’s Hospital, Houston, TX; 3University of Illinois Chicago, Chicago, IL; 4Stanford University School of Medicine, Stanford, CA
Tr1 Cells and Heparan Sulfate-Mediated IL-2 Sequestration Regulate Scarring Outcomes

Mary Elizabeth Guerra1,2, Navya Nanda2, Casey Baxter1, Sonya Keswani1, Jaime Tellez2,1, Hui Li1,2, Ling Yu1,2, Mateusz Wietecha3, Paul Bollyky4, Swathi Balaji1,2, Sundeep Keswani1,2 1Baylor College of Medicine, Houston, TX; 2Texas Children’s Hospital, Houston, TX; 3University of Illinois Chicago, Chicago, IL; 4Stanford University School of Medicine, Stanford, CA

Humans display variation in scarring responses even when wounded identically. We have shown that T lymphocyte subsets play differential roles in scarring and that regulatory lymphocytes (Tr1) promote regenerative healing via interleukin (IL)-10. Tr1 are activated by heparan sulfate-bound IL-2, which they access via heparanase (HPSE). However it is unknown if there are differences in wound cytokine memory, specifically in IL-2 expression, that affect Tr1 function in wound repair and contribute to an individual’s scarring phenotype.
First, IL-2 expression was tested in different scarring models. Small (2 mm) and large (6 mm) stented wounds in C57BL/6J mice (at D3 and D7) that scar differently based on size, and scar tissue from low (LS) and high scarring (HS) human patients were co-labelled with heparan sulfate and IL-2 ± HPSE digestion to reveal their colocalization. Then, CD4+ total lymphocytes, T naïve (Tn), Treg (CD4+, CD25+, and FOXP3+) and Tr1 (CD4+, CD44+, CD25+, and FOXP3-) were isolated from C57 mice. T cell conditioned media (CM) was added to fibroblasts (Fb) from LS and HS patients and its effect on fibrosis genes was assessed. Tn or Tr1 were adoptively transferred into SCID mice with 6mm wounds. Wounds were assessed at D7 by 10x Visium RNA sequencing and analyzed using R and Seurat workflows and histology to determine the effect of T cells on Fb populations in the wounds. n = 3/group, data presented as mean±SD, p-values by t-test.
Large murine wounds and HS patient scars had greater expression of heparan sulfate and IL-2. HPSE treatment reduced expression of IL-2, suggesting IL-2 colocalizes with heparan sulfate. Tr1 CM had the highest levels of IL-10 and HS Fb treated with Tr1 CM had reduced profibrotic gene expression. In vivo, T cell subsets uniquely influenced the spatial distribution of cell populations within the wound. 10× Visium showed that Tr1-treated D7 wounds had fewer inflammatory cell-compartments and increased granulation-related genes, indicating earlier inflammation resolution and wound healing. Deconvolution analysis showed unique subtypes of Fb in Tr1 wounds, and wounds that were untreated or treated with total lymphocytes or Treg had more association with up-regulated genes in HS Fb compared to wounds treated with Tr1, suggesting Tr1 reduce fibrotic gene signature.
Heparan sulfate sequesters IL-2 in the wound, which may be a form of “cytokine memory,” as it has a direct correlation with scarring outcomes. Tr1 regulate the fibrotic gene signature of Fb, resulting in unique effects on phenotype and geospatial cellular niches within wounds. These findings provide a novel mechanistic basis for the regenerative effects of Tr1, which may explain why humans exhibit varied scarring responses. Cumulatively this supports the development of Tr1 as a novel antifibrotic therapy.

K2.01

Selective Histamine Receptor Activation Promotes Murine Diabetic Wound Healing and Promotes Repair in Partial-Thickness Porcine Wounds
Jordan R. Yaron*2,1, Shubham Pallod2,1, Sudhakar Godeshala2,1, Rushil Kolipaka2,1, Kaushal Rege2,1
1School for Engineering of Matter, Transport, & Energy, Arizona State University, Tempe, AZ; 2Biodesign Center for Biomaterials Innovation and Translation, Arizona State University, Tempe, AZ
Selective Histamine Receptor Activation Promotes Murine Diabetic Wound Healing and Promotes Repair in Partial-Thickness Porcine Wounds

Jordan R. Yaron2,1, Shubham Pallod2,1, Sudhakar Godeshala2,1, Rushil Kolipaka2,1, Kaushal Rege2,1 1School for Engineering of Matter, Transport, & Energy, Arizona State University, Tempe, AZ; 2Biodesign Center for Biomaterials Innovation and Translation, Arizona State University, Tempe, AZ

Histamine released by mast cells and, to a lesser degree, by other immune cells acts as an early-stage mediator of healing in the skin by stimulating vascular, fibrotic and immune responses through the activity of four G-protein coupled receptors (HRH1-4). We have shown that histamine can augment biomaterial-mediated excisional acute and diabetic wound healing and that selective agonists of the four histamine receptors differentially augments healing in acute incisional wounds in mice. Here, we investigate the effects of histamine receptor agonists on excisional wound healing in diabetic mice, and on the effect of histamine receptor agonists 1 and 4 in acute excisional wounds in pigs.
For mouse wounds, splinted excisional wounds (5-mm) were generated in genetically obese and diabetic (db/db) mice and treated with saline or agonists for HRH1 (2-pyridylethylamine), HRH2 (dimaprit), or HRH4 (4-methylhistamine), hereafter referred to as HRHn-ag (where n is 1, 2 or 4) (N = 3-5/group). For pig wounds, partial-thickness 12-mm diameter wounds were generated in Yucatan miniature swine and treated with HRH1-ag, HRH4-ag, or the combination of both agonists with and without soluble or autoclave-insolubilized silk fibroin films, or treated with saline, or carboxymethylcellulose (CMC) gel without or with PDGF-bb (Regranex analog) (N = 2 per group). Planimetry, TEWL, skin viscoelasticity, histopathology and IHC were performed to evaluate immune and reparative responses.
Diabetic wound closure was accelerated by HRH1-ag by day 4 post-injury, whereas HRH4-ag accelerated closure beginning on day 7 post-injury. HRH2-ag did not benefit diabetic wound closure. H&E staining confirmed significantly enhanced epithelialization by HRH1-ag and HRH4-ag. HRH1-ag (p < 0.001) and HRH4-ag (p < 0.05) treatment significantly promoted myofibroblast activation with enhanced alpha-smooth muscle actin expression. Only HRH1-ag enhanced epithelial-mesenchymal transition (measured by loss of E-cadherin) versus saline controls (p < 0.05). All agonists reduced CD86+ macrophage levels (p < 0.001), increased Arginase-1+ macrophage levels (p < 0.05), and stimulated increased CD31+ angiogenesis (p < 0.05). Ongoing studies in pigs indicate positive therapeutic responses by HRH1-ag in combination with soluble silk, and to a lesser degree to HRH4-ag with soluble or insoluble silk, including in regeneration of Rete ridges, reduction of scar area, and improvements in collagen alignment.
HRH1 and HRH4 activation promotes diabetic wound healing, and HRH1 agonism combined with soluble silk fibroin scaffolds combinatorially enhanced excisional wound healing in a large animal model. Histamine pathway modulation is a promising approach to stimulate enhanced tissue repair.

K2.02

Optimized Dosing and Delivery of Bacteriophage Therapy for Chronic Wound Infections
Yung-Hao Lin, Tejas Dharmaraj, Qingquan Chen, Arne Echterhof, Lucy J. Zhang, Tony H. W. Chang Tony H. W. Chang, Julie Pourtois, Cas Liuew, Zhiwei Li, Maryam Hajfathalian, Francis G. Blankenberg, Derek Amanatullah, Ovijit Chaudhuri, Paul Bollyky*
Stanford School of Medicine, Stanford, CA
Optimized Dosing and Delivery of Bacteriophage Therapy for Chronic Wound Infections

Yung-Hao Lin, Tejas Dharmaraj, Qingquan Chen, Arne Echterhof, Lucy J. Zhang, Tony H. W. Chang Tony H. W. Chang, Julie Pourtois, Cas Liuew, Zhiwei Li, Maryam Hajfathalian, Francis G. Blankenberg, Derek Amanatullah, Ovijit Chaudhuri, Paul Bollyky Stanford School of Medicine, Stanford, CA

Antimicrobial resistant bacteria and bacterial biofilms delay wound healing. Lytic bacteriophages, viruses that lyse (kill) bacteria, hold great promise for treating antimicrobial resistant pathogens and bacterial biofilms, including those associated with Pseudomonas aeruginosa and Staphylococcus aureus. However, the optimal dosing and delivery strategies for phage therapy remain unclear.
In a mouse wound infection model, we investigated the impact of dose, frequency, and administration route on the efficacy of phage therapy. Building on the insights from that work, we developed “HydroPhage,” a hyaluronan-based hydrogel system that uses dynamic covalent crosslinking to deliver high-titre phages over one week.
We find that topical but not intravenous delivery is effective in this model. High-doses of phage reduces bacterial burden more effectively than low-doses, and repeated dosing achieves the highest eradication rates. Phage cocktails promote bacterial killing and these are most effective when delivered simultaneously. HydroPhage eradicates infections five times more effectively than intravenous injection. We conclude that hydrogel-based sustained phage delivery enhances the efficacy of phage therapy and offers a practical, well-tolerated option for topical application.

K2.03

THE ROLE OF THE VITAMIN D RECEPTOR IN REGULATING EMT-LIKE GENE EXPRESSION IN KELOID KERATINOCYTES
Dorothy Supp*1,2, Jennifer Hahn1, Kevin McFarland3, Kelly A. Combs1
1Surgery, University of Cincinnati College of Medicine, Cincinnati, OH; 2Scientific Staff, Shriners Children’s Ohio, Dayton, OH; 3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
The Role of the Vitamin D Receptor in Regulating EMT-Like Gene Expression In Keloid Keratinocytes

Dorothy Supp1,2, Jennifer Hahn1, Kevin McFarland3, Kelly A. Combs1 1Surgery, University of Cincinnati College of Medicine, Cincinnati, OH; 2Scientific Staff, Shriners Children’s Ohio, Dayton, OH; 3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

Keloids are disfiguring fibroproliferative lesions that result from an abnormal wound healing process in susceptible individuals. Keloids are challenging to treat, and recurrence is common. Development of better therapeutic interventions will require a deeper understanding of the underlying molecular mechanisms. Previous studies implicated epithelial-mesenchymal transition (EMT) in keloid pathology and revealed abnormalities in vitamin D signaling in keloids, including reduced epidermal expression and nuclear localization of the vitamin D receptor (VDR) in keloids. However, VDR expression levels are normalized when keloid keratinocytes are cultured in vitro, complicating experiments aimed at understanding the effects of decreased VDR levels in these cells. The purpose of this study was to investigate the effects of VDR downregulation on gene expression in keloid keratinocytes in vitro. Keratinocytes were isolated and cultured from excised keloids collected with IRB approval. VDR gene silencing in keloid-derived keratinocytes was achieved using lentiviral shRNA transduction and selection of stably modified cells; control cells were modified using non-specific shRNA. Gene expression was analyzed by RNA sequencing (RNAseq) to identify genes differentially expressed upon VDR knockdown. Functional enrichment was analyzed using ToppGene Suite (https://toppgene.cchmc.org/).
Gene knockdown via shRNA decreased VDR expression by 80% in keloid keratinocytes, resulting statistically significant > 2-fold changes in expression of 54 genes.
Functional analysis revealed that this gene set is enriched in genes involved in Epithelium Development, Response to Lipid, and Response to Wounding. Genes upregulated in keloid keratinocytes upon VDR silencing included interleukins IL1B and IL24, genes involved in promoting EMT (VCAN, KRTAP2-3, GLIPR1, MYL9), and genes that were previously shown to be upregulated in keloids (EDN1, PTGS2, GLIPR1). Downregulated genes included inhibitors of Wnt signaling (APCDD1, TNFAIP8L1) and inhibitors of EMT (TP73, SCARA3).
Silencing VDR expression in keloid keratinocytes in vitro resulted in gene expression changes consistent with abnormalities observed in keloids in vivo, including abnormal Wnt signaling, increased inflammatory gene expression, and an EMT-like gene expression signature. The results suggest that the observed downregulation of VDR in keloids may play a direct role in promoting EMT and inflammation, contributing to keloid pathology. Therefore, approaches that increase expression of VDR in keloids may have therapeutic value for keloid suppression.

K2.04

PLACENTAL-DERIVED PROTEIN MATRIX ENHANCES ESTROGEN SIGNALING AND PROMOTES HEALING OF AGED SKIN WOUNDS
Helena Zomer*1, Payton Corey1, Julia Mitze2, Gustavo Zamora1, Calum McFetridge3, Peter McFetridge3, Paul Cooke1
1Physiological Sciences, University of Florida, Gainesville, FL; 2University of Veterinary Medicine, Hannover, Germany; 3Crayton Pruitt Family Biomedical Engineering, University of Florida, Gainesville, FL
Placental-Derived Protein Matrix Enhances Estrogen Signaling And Promotes Healing of Aged Skin Wounds

Helena Zomer1, Payton Corey1, Julia Mitze2, Gustavo Zamora1, Calum McFetridge3, Peter McFetridge3, Paul Cooke1 1Physiological Sciences, University of Florida, Gainesville, FL; 2University of Veterinary Medicine, Hannover, Germany; 3Crayton Pruitt Family Biomedical Engineering, University of Florida, Gainesville, FL

Eighty-five percent of all chronic wounds occur in people over 65. However, current strategies largely overlook the unique biology of aged skin, and novel, promising regenerative therapies are often tested in young models, leaving aged skin repair a major unmet clinical need. Age-related 17β-estradiol (E2) deficiency significantly impairs wound healing, but strategies to safely restore E2 signaling in wounds are lacking. The placenta is an endocrine organ enriched in bioactive molecules that drive tissue (re)generation. Although multiple placenta-derived products have been used for wound healing applications, the estrogenic effects of placental materials have never been investigated. This study tested whether a human placental matrix (hPM) could be used to modulate estrogen signaling and promote robust healing in estrogen-deprived wounds. Human dermal fibroblasts were treated with hPM to assess estrogen signaling pathways (qPCR) and in vitro wound closure (scratch assay). Base medium and E2 (2 μg/ml) were used as controls. The effects of hPM on healing were tested on ovariectomized females, aged males, and intact adult mice (controls). Wound outcomes were compared between hPM- and saline-treated groups. The hPM protein composition was analyzed by LC-MS/MS, and E2 levels were quantified by ELISA. Experiments included ≥ 3 biological replicates, with significance set at p < 0.05 (t-test).
hPM upregulated estrogen receptors (ESR1, ESR2) and aromatase (CYP19A1) in human dermal fibroblasts (n = 3-4, p < 0.05) and modulated gene expression similarly to E2. A cell scratch assay showed hPM accelerated wound closure in vitro 2.3-fold compared to controls (n = 3, p < 0.01). In a mouse model, hPM accelerated wound closure 1.4-fold in intact, ovariectomized females and aged males (n = 7-10/group, p < 0.05) and reduced visible scars by 50% at day 60 (n = 3-10/group, p < 0.05). Proteomic analysis revealed that hPM is enriched in proteins involved in wound healing and E2 pathways, but contains only minimal amounts of E2 (1 ng/ml).
hPM, a placental-derived protein matrix, exhibits estrogenic effects on human skin cells and enhances wound healing in estrogen-deficient mice. Future studies will determine whether these estrogenic effects are essential for its regenerative properties. By targeting local estrogen pathways without systemic side effects, hPM offers a promising therapy for chronic wounds in older adults.

K2.05

Tissue nanotransfection-based Cell Specific Epigenetic Editing In Vivo to Rescue Diabetic Ischemic Tissue
Kanhaiya Singh*, Sumit S. Verma, Surya Gnyawali, Chandan K. Sen
McGowan Institute for Regenerative Medicine, Department of Surgery, University of Pittsburgh, Pittsburgh, PA
Tissue Nanotransfection-Based Cell Specific Epigenetic Editing In Vivo to Rescue Diabetic Ischemic Tissue

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

One of major reasons for the clinical disappointments in VEGF “monotherapy” for rescuing ischemic tissue perfusion is the inability to respond to changes in ligand concentration due to impaired receptor signaling. Hence, therapeutic implications that can enable VEGF therapy has potential to enhance angiogenic outcomes in diabetic wounds. Our previous work (PMID: 35192691) using single cell RNA-sequencing has identified that genetically silenced phospholipase Cγ2 (PLCγ2) and metallothioneins (MT1E and MT2A) 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 and type 2 diabetic (T2DM) model db/db mice. Diabetic VWF+ dermal endothelial cells were isolated using flow sorting. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. To specifically demethylate PLCγ2 promoter in endothelial cells, a CRISPR/dCas9-based demethylation cocktail was utilized: (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 topical tissue nano-transfection (TNT) technology. TNT is an innovative in vivo gene transfer technology, that comprises a silicon chip, metal electrodes, and microsecond electric pulses to directly transfect target cells within the tissue. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging (Perimed Inc.), immunohistochemistry 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). TNT-based delivery of plasmids to demethylate the gene promoters of downstream signaling molecules led to significant improvements in VEGF therapy for cutaneous diabetic wounds, resulting in better perfusion and accelerated ischemic wound closure.

K2.06

TRANSCRIPTIONAL ANALYSIS OF AVENANTHRAMIDES (AVN) AND ?-GLUCAN IN WOUND HEALING
Abdelrahman M. Alsharif*1,2, Fidel Saenz1, Jonathan P. Yasmeh1, Katharina Fischer1, Andrew Hostler1, Amelia B. Knochel1, Lulejeta A. Latifi1, Maria Gracia Mora Pinos1, Eamonn McKenna1, Autumn Lester1, nicholas matthews1,2, Syed Maisam Jafri1,2, Erika Kness-Knezinskis1, Geoffrey C. Gurtner1,2, Kellen Chen1,2
1Department of Surgery , university of Arizona, Tucson, AZ; 2Biomedical Engineering, university of Arizona, Tucson, AZ
Transcriptional Analysis of Avenanthramides (AVN) and β-Glucan in Wound Healing

Abdelrahman M. Alsharif1,2, Fidel Saenz1, Jonathan P. Yasmeh1, Katharina Fischer1, Andrew Hostler1, Amelia B. Knochel1, Lulejeta A. Latifi1, Maria Gracia Mora Pinos1, Eamonn McKenna1, Autumn Lester1, nicholas matthews1,2, Syed Maisam Jafri1,2, Erika Kness-Knezinskis1, Geoffrey C. Gurtner1,2, Kellen Chen1,2 1Department of Surgery, university of Arizona, Tucson, AZ; 2Biomedical Engineering, university of Arizona, Tucson, AZ

Non-healing wounds affects 30 million individuals in the U.S and costs the government up to $96.8 billion annually. As such, there is a need for cost-effective, accessible interventions. We previously established that locally injected A. sativa-derived molecules, AVN or β-Glucan, accelerate healing and improve scar quality. However, how these treatments promote healing remains poorly understood. Our research efforts investigate how AVN and β-Glucan contribute to accelerated wound healing by examining their molecular and cellular effects within the wound environment.
Excisional wounds were created on the dorsal surface of C57/BL6 (WT) mice, followed by subcutaneous AVN or β-Glucan injections. The mice were then sacrificed on post-operative day 14. The tissues were processed for immunohistochemical analysis and single-cell suspensions for single-cell RNA sequencing. Bioinformatic analyses were conducted using tools such as Seurat.
In the AVN group, the myeloid cell population was significantly reduced (18%) compared to the control (42%), while other cell types showed similar proportions. Further investigation of myeloid subtypes found that macrophages and neutrophils made up 60% and 15% of the myeloid population, respectively, in control untreated wounds versus 40% and 5% in AVN treated. Monocytes and dendritic cells were more prevalent in AVN (10% and. 20%) vs (4% and 5%) in control respectively. Notably, AVN macrophages produced insulin-like growth factor 1 (Igf1), typically linked to macrophages that resolve inflammation, reduce chemokine ligand 12 (Cxcl12) expression, and support skeletal muscle regeneration.
Cells of various types were represented at similar levels in both the control and β-Glucan groups. Fibroblasts were specifically analyzed because they accounted for 40% of the upregulated genes in β-Glucan.
Fibroblasts in the β-Glucan group exhibited increased expression of markers like Plac8 and Ly6c1, suggesting potential immune infiltration. Furthermore, β-Glucan-treated cells demonstrated increased expression of Cox-2, a key enzyme regulated by NF-κB, an inflammation pathway. In contrast, fibroblasts in the control group demonstrated higher expression of Col7a1, which is required for dermal-epidermal junction. This highlights a potential link between Col7a1 lack of expression and scarring in β-Glucan.
This study investigated the changes in cellular populations from AVN and β-Glucan treatment. Notably, AVN reduced the myeloid population and inflammatory cell recruitment while promoting the production of M2-like macrophages (Igf1+). In contrast, β-Glucan enhanced angiogenesis, accompanied by increased immune cell infiltration and activation of inflammatory pathways (Cox-2+). Future studies should test potentially promoting Igf1 macrophages or Cox-2 fibroblasts to improve wound repair and tissue regeneration. These findings could lead to new therapies to promote human wound healing.

K3.01

BREAKING BOUNDARIES: TESTING MICROGRAVITY ON INTERNATIONAL SPACE STATION REVEALS IMPAIRMENTS IN HUMAN WOUND HEALING
Nathan Balukoff*1, Garrett Houk1, Yael Berton2, Rivka C. Stone1, Irena Pastar1, Paul Kamoun3, Vincent Ronfard2, Marjana Tomic-Canic1
1Wound Healing and Regenerative Medicine Research Program, Dr Philip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2CUTISS Innovation, Sophia Antipolis, France; 3SpacePharma, Exploration Park, FL
Breaking Boundaries: Testing Microgravity On International Space Station Reveals Impairments In Human Wound Healing

Nathan Balukoff1, Garrett Houk1, Yael Berton2, Rivka C. Stone1, Irena Pastar1, Paul Kamoun3, Vincent Ronfard2, Marjana Tomic-Canic1 1Wound Healing and Regenerative Medicine Research Program, Dr Philip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2CUTISS Innovation, Sophia Antipolis, France; 3SpacePharma, Exploration Park, FL

Understanding the factors influencing wound healing in microgravity and space-flight conditions is imperative to ensure the safety and health of all space-travelers and may potentially advance healing on Earth. While cuts and abrasions are common among astronauts, previous studies focused on understanding the effect on microgravity utilizing mouse wound models and simulations of microgravity, that is, 0G. We have recently performed the first of its kind study using human 2D and 3D cutaneous wound models and lab-on-a-chip technology (SpacePharma). Samples were launched on SpaceX’s 27th Commercial Resupply Services mission from Cape Canaveral, FL and were kept at the International Space Station (ISS).
Primary skin cells were cultured in space onboard the CRS-27 and ISS with microfluidic chips to enable automated media flow and Oxygen/CO2 supply. Primary human skin cells and 3-D reconstructed skin equivalents consisting of human keratinocytes and fibroblasts embedded in Matrigel were cultured and wounded prior the launch and exposed to microgravity for 7 days. The samples were then fixed in 4% PFA prior to return to Earth. In parallel, a duplicate experiment with the same biological material was performed on Earth, in the laboratory at Cape Canaveral, serving as a control. RNA isolation was performed post-crosslinking, and the effects of spaceflight, microgravity and space radiation were analyzed using Nanostring’s nCounter technology.
Differential gene expression was normalized to control Earth samples using the NCounter software.
The experiment was successfully executed. We identified that the wound healing response including the epithelial-to-mesenchymal transition, is attenuated in response to space stressors, with genes including ADPGK, GLI1, BLK, F5 and IGF5 found suppressed a MPTPS2, CASP3, PTCH1, MYLK, IRS1 induced. In addition, key fibrotic genes including HIF1A, RBX1, PTNPN11, HMGCS2 and VCAM1 were found activated in response to space conditions. The data overall suggests significant modification of wound healing signals that may hinder healing in non-Earth conditions.
Understanding cellular and wound healing response in microgravity conditions is a first step in revealing effects of space travel on the human barrier repair. This and future studies are crucial for advancements in regenerative medicine and design of therapeutics tailored for wound healing in Space.

K3.02

Expression of MARCKSL1 in Dendritic Cells Modulates Fibroblast Differentiation and Promotes Scar Formation
Kento Takaya*, Yukari Nakajima, Noriko Aramaki-Hattori, Keisuke Okabe, Shigeki Sakai, Kazuo Kishi
Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
Expression of MARCKSL1 in Dendritic Cells Modulates Fibroblast Differentiation and Promotes Scar Formation

Kento Takaya, Yukari Nakajima, Noriko Aramaki-Hattori, Keisuke Okabe, Shigeki Sakai, Kazuo Kishi Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan

Wounds in developing embryos possess regenerative capabilities up to a specific developmental stage, after which they transition to forming scars, akin to adult organisms. This transition offers a unique opportunity to investigate mechanisms for scar suppression by analyzing the cellular and molecular changes occurring before and after this critical period. Utilizing a unique mouse embryonic wound healing model we established, single-cell analysis revealed a significant presence of dendritic cells (DCs) in embryonic day 17 (E17) wounds that form scar tissues as adults. These DCs express myristoylated alanine-rich C-kinase substrate-like protein 1 (MARCKSL1), a gene implicated in regeneration and cellular migration. However, the precise role of MARCKSL1 in scar formation and fibrosis remains underexplored. This study aimed to elucidate the function of DCs in scar formation and the impact of MARCKSL1 expression on this process.
(1) Wound stimulation and MARCKSL1 expression in DCs; We examined whether wound stimuli, simulating TGF-β1 activity, could induce MARCKSL1 expression in mouse DCs. Additionally, MARCKSL1 expression was analyzed in wound sites of both adult and fetal mouse wound. (2) Interaction between MARCKSL1-positive DCs and fibroblasts; Co-culture experiments were conducted to assess whether TGF-β1-stimulated DCs influence fibroblast differentiation into myofibroblasts, key contributors to scar formation. The effect of this interaction on actin dynamics and cell migration was also evaluated. Functional analysis was performed using DCs transfected with MARCKSL1-specific shRNA. (3) Role of MARCKSL1 regulation on wound healing; the role of MARCKSL1 in scarring was further investigated by administering a MARCK inhibitor (MANS peptide) to mouse wound models and observing scarring outcomes. The wound healing response was also assessed in MARCKSL1-deficient mice (C57BL/6-Marckstm2.1Pjb/J). MARCKSL1-positive DCs were specifically found to accumulate at the wound margins at E17, a developmental stage associated with the onset of scar formation. In vitro experiments revealed that TGF-β1 stimulation significantly upregulated MARCKSL1 expression in mouse DCs. Co-culture of TGF-β1-stimulated DCs with fibroblasts facilitated fibroblast differentiation into myofibroblasts, characterized by enhanced cytoskeletal organization and migratory activity. However, this differentiation was notably suppressed in MARCKSL1-depleted DCs, demonstrating the gene’s critical regulatory role. In vivo, mice treated with MANS peptides or MARCK knocked out exhibited significantly reduced scar formation compared to untreated controls or wild-type counterparts.
The expression of MARCKSL1 in wound-associated DCs plays a critical role in modulating fibroblast activity and driving scar formation. These findings position MARCKSL1 as a promising therapeutic target for innovative interventions aimed at reducing scarring and improving wound healing outcomes.

K3.03

Comprehensive Multiomics Profiling Reveals Novel Insights Into Wound Healing And Scar Formation In The Post-Hysterotomy Rat Uterus
Chris Erickson*2, Anisha Apte3, Efua H. Bolouvi3, Amanda McWhirter3, Shaun Bevers1, Kirk Hansen1, Angelo D’Alessandro2, Rosanne Kho4, Carlos Zgheib3, Kenneth W. Liechty3
1Surgery, University of Colorado, Aurora, CO; 2University of Colorado, Aurora, CO; 3University of Arizona, Tucson, AZ; 4University of Arizona, Phoenix, AZ
Comprehensive Multiomics Profiling Reveals Novel Insights Into Wound Healing And Scar Formation In The Post-Hysterotomy Rat Uterus

Chris Erickson2, Anisha Apte3, Efua H. Bolouvi3, Amanda McWhirter3, Shaun Bevers1, Kirk Hansen1, Angelo D’Alessandro2, Rosanne Kho4, Carlos Zgheib3, Kenneth W. Liechty3 1Surgery, University of Colorado, Aurora, CO; 2University of Colorado, Aurora, CO; 3University of Arizona, Tucson, AZ; 4University of Arizona, Phoenix, AZ

Scaring following classical hysterotomy can lead to hemorrhage, adhesions, impaired fertility, abnormal placentation and uterine rupture in subsequent pregnancies. Molecular events of how scars form and why they lead to complications are largely understudied, hindering our ability to treat these wounds. To expand our working knowledge of these injuries, we performed multiomics profiling of post-hysterotomy rat uterus.
8-10 weeks old nongravid (not pregnant) female rats underwent hysterotomy. Injured, uninjured, and sham uteri were collected (n = 6 each) 7 days post-injury. Tissue was processed for untargeted mass spectrometry proteomics (cell and ECM fractions) and metabolomics. One-way ANOVA with q-value < 0.05 determined significantly different omics among groups. Enrichment of the significant omics mentioned below was performed with Metascape and Metaboanalyst. 2560 cell proteins, 100 ECM proteins, and 194 metabolites were identified. Injured uteri cell proteome enriched for cell motility and contractility, apoptosis, and endopeptidase activity; while proteins involved in ovulation, host defense, and cell secretion were decreased. There was robust ECM response in injured uteri with elevated elastin assembly, minor collagens, wound healing proteins (TNC, TGFBI), and inflammatory healing regulators (ANXA1/2); while syndecans (SDC1/2) were reduced. These proteomics changes persisted in the uninjured uteri although to a lesser degree. Injured uteri had substantially elevated linoleic acid metabolism (prostaglandins, neural membranes), and decreased amino acid and purine metabolism. Interestingly uninjured uteri expressed a unique metabolome with elevated lactose and trehalose degradation (sources of glucose).
Hysterotomy scar remains a major clinical challenge, hindered by our lack of understanding of these injuries. Current methods to attenuate scar complications include suturing techniques and surgical resection, outcomes that vary by surgical skill and patient response. Reports by others have demonstrated reductions in scar formation following biomaterial and/or stem cell treatments in small animals, but results are mixed in clinical studies. A broader understanding of the basic mechanisms of scar formation are much needed to develop effective preventative treatments. Here we present unreported proteomic and metabolic changes in the rat uterus following hysterotomy, and begin a multiomics reference database to study this injury. Increases in cell motility and binding; tissue turnover; and resolution of coagulation in injured uteri were expected. Unexpected findings were elevated elastin fiber formation, tenascin C, gliogenesis (LAMB1/2 with ANXA1/7), and ECM organizational proteins concurrent with oxylipins and arachidonic acid derivatives (linoleic acid pathway), which collectively indicate a robust wound healing response. How these pathways and specific omics markers can be modulated to avert scar formation is the focus of ongoing research.

K3.04

SPATIO-TEMPORAL CHARACTERIZATION OF EXCISIONAL WOUND HEALING IN A SWINE MODEL
Hsin-ya Yang*1, Kan Zhu2, Anthony Gallegos1, Cynthia Recendez2, Guillermo Villa-Martinez1, Ksenia Zlobina3, Manasa Kesapragada3, Moyasar A. Alhamo1, Marcella Gomez3, Athena Soulika1,4, Elham Aslankoohi5, Marco Rolandi5, Min Zhao2, Rivkah Isseroff1,6
1Dermatology, University of California, Davis, Sacramento, CA; 2Ophthalmology and Vision Science, University of California, Davis, Sacramento, CA; 3Applied Mathematics, University of California, Santa Cruz, Santa Cruz, CA; 4Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA; 5Electrical Engineering, University of California, Santa Cruz, Santa Cruz, CA; 6Dermatology Section, VA Northern California Health Care System, Sacramento, CA
Spatio-Temporal Characterization of Excisional Wound Healing In A Swine Model

Hsin-ya Yang1, Kan Zhu2, Anthony Gallegos1, Cynthia Recendez2, Guillermo Villa-Martinez1, Ksenia Zlobina3, Manasa Kesapragada3, Moyasar A. Alhamo1, Marcella Gomez3, Athena Soulika1,4, Elham Aslankoohi5, Marco Rolandi5, Min Zhao2, Rivkah Isseroff1,6 1Dermatology, University of California, Davis, Sacramento, CA; 2Ophthalmology and Vision Science, University of California, Davis, Sacramento, CA; 3Applied Mathematics, University of California, Santa Cruz, Santa Cruz, CA; 4Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA; 5Electrical Engineering, University of California, Santa Cruz, Santa Cruz, CA; 6Dermatology Section, VA Northern California Health Care System, Sacramento, CA

The four phases of wound healing – hemostasis, inflammation, proliferation and maturation/remodeling have been studied intensively, yet characterization of the continuous progress for the “ground truth” of these overlapping phases is lacking. Domestic swine have evolved as the preferred model for wound healing studies due to the similarity of the architecture and healing processes to human skin. In this study, we utilized the swine excisional wound model for simultaneously and sequentially monitoring multiple cellular and molecular responses in the wound tissue over the full course of healing.
Twelve full-thickness, excisional wounds at 20mm in diameter were created bilaterally on the dorsa of 6 young, Yorkshire female pigs. Blood, wound images, and wound biopsies at different locations (3.5 mm samples from wound edges and center) were collected for histology and RNAseq from post-op day 1–21.
All wounds were fully epithelized by day 19–21, based on the wound images.
Histological analysis of the wound edge sections shows that the neo-epithelium extended from the wound edge from day 1 to 9 and exceeded the sampled area towards the wound center after day 9. Using CibersortX analysis (https://cibersortx.stanford.edu/) of the RNA sequencing data from the daily wound biopsies, neutrophil infiltration peaked at the wound center between day 2 and 5. Immunohistochemical analysis for macrophages demonstrated an increase of the M1 subset (cells with CD68 and iNOS signals) by day 3, whereas M2 macrophages (cells with CD68 and Arg 1 signals) began to rise on day 7. This trend is supported by the M1/M2 ratio, which peaked between days 3 and 5 (p < 0.01 compared to the day 1 ratio) and subsequently decreased in the later stages of healing. At the later phase of wound proliferation and maturation, collagen deposition was examined by picrosirius red staining. The area ratio of the type III to type I collagen peaked from day 9 to day 19 and decreased to the basal level on day 21. Innervation of peripheral nerves was identified by the immunostaining of PGP9.5 (ubiquitin carboxy-terminal hydrolase L1), a protein highly expressed in neurons. A consistent increase in the nerve staining was observed on day 7, suggesting the regeneration of epidermal nerves in the healing wounds. To ensure that the frequent sampling did not stress the animals and interfere with healing, the stress hormone cortisol and epinephrine were monitored in the serum samples by HPLC. Both hormones showed a trend of slight elevation in the first week and restored to the basal level after day 7, suggesting the animals were not stressed with the handling during the experiment.
In conclusion, the spatio-temporal characterization of the full course of healing offers valuable insights into acute healing processes and establishes baseline data for cellular and molecular events, which can serve as a reference for comparing future experimental datasets.

K3.05

A VISUAL NARRATIVE PROTOCOL FOR USING R TO ANALYZE A SINGLE-CELL TRANSCRIPTOMICS DATASET OF MOUSE SKIN WOUND HEALING
Shalyn Keiser*, Mateusz S. Wietecha
Oral Biology, College of Dentistry, University of Illinois Chicago, Chicago, IL
A Visual Narrative Protocol For Using R to Analyze A Single-Cell Transcriptomics Dataset of Mouse Skin Wound Healing

Shalyn Keiser, Mateusz S. Wietecha Oral Biology, College of Dentistry, University of Illinois Chicago, Chicago, IL

The process of wound healing is regulated by complex interactions between different cell types across space and time. Through the profiling of individual cells within their complex environment, single-cell transcriptomics methods enable the investigation of cellular heterogeneity, cell communication networks, and cell-cell interactions involved in the wound healing process. However, many single-cell analysis tools are run within a computer coding environment, and their more widespread use by wound healing scientists is thwarted by the apparent lack of bioinformatics expertise.
We present a step-by-step, visual workflow showing how to use a graphical R coding environment called RStudio to perform a basic single-cell analysis of a publicly available and previously published temporal mouse excisional skin wound healing single-cell RNA-sequencing dataset. We present a standard pipeline for dataset download, quality control, visualizations and cell type annotations using Seurat, and cell-cell interaction analysis using CellChat. This visual and guided protocol enables scientists with no bioinformatics background to download a previously published wound healing dataset, perform critical quality control steps, run a standard single-cell analysis workflow including dataset visualizations and cell type annotations, run cell subtype analyses, run module scoring analyses, run cell-cell interaction analyses, and perform integrative analyses of multiple datasets. Narrative explanations are provided for each step in the protocol and graphical results from every line of code are presented to safely guide the user through the workflow.
The goal of this visual introduction to a single-cell analysis pipeline is to enable more wound healing scientists to use bioinformatics tools directly in their own laboratories in order to facilitate deeper analyses of their own single-cell datasets as well as more widespread re-analyses of previously published single-cell datasets.

K3.06

Potent ionic calcium (Sigma Anti-Bonding Calcium Carbonate) enhances the wound-healing process in C57/BL6 mouse.
Syed H. Mehdi, Dongjoon Lee, Donghoon Yoon*
Internal Medicine, Univ. of Arkansas for Medical Sciences, Little Rock, AR
Potent Ionic Calcium (Sigma Anti-Bonding Calcium Carbonate) Enhances the Wound-Healing Process in C57/BL6 Mouse

Syed H. Mehdi, Dongjoon Lee, Donghoon Yoon Internal Medicine, Univ. of Arkansas for Medical Sciences, Little Rock, AR

Wound healing includes replacing destroyed/damaged tissues with newly generated tissue. Once the injury occurs, the epidermis and dermis form a protective barrier against the external environment. The wound is repaired through sequential steps with overlaps; hemostasis, inflammation, cell proliferation, and tissue remodeling (maturation and cell differentiation). Wound healing is a calcium-mediated process. Calcium regulates inflammatory cell infiltration, migration/proliferation, and differentiation of dermal fibroblast and keratinocytes. Sigma Anti-Bonding Calcium Carbonate (SAC) is a chemically modified calcium carbonate with a unique weak bonding that becomes SAC water-soluble and stable at room temperature. In the body, SAC behaves as ionic calcium but also helps dissociate protein-bound calcium into ionized calcium. In turn, SAC significantly increases active ionized calcium in the body. Collectively, we hypothesized that the topical treatment of SAC enhances wound healing.
To study the effects of the SAC on wound healing, we took 32, 8–10-month-old, C57/BL6 mice and singly housed them. Two bilateral circular wounds were created on the dorsum using a 8-mm punch biopsy. About 0.8mg of Control or SAC cream was applied to each circular wound twice a day for wound treatment. The SAC cream contains 0.12% SAC in a control dermatology cream provided by the company. The wound size was measured using CellSens software (Olympus Co.). In addition, skin biopsies were taken before the wound was created and after the wound closed. The skin biopsy was fixed in 4% paraformaldehyde and underwent hematoxylin & eosin immunohistology staining (Vector, H-3502) and collagen staining (Abcam, ab270993). The skin tissue was measured using a Zeiss AXIO Imager M2 microscope. The MTT cell proliferation assays were performed using HDFa and HaCaT cells in 24, 48, and 72 h at 0.032, 0.32, and 1.6mg/ml SAC.
We found that the SAC treatment rapidly reduced the wound size in the early healing phase and closed wounds earlier than the control. Furthermore, SAC increased in epidermal thickness during wound healing, which eventually returned to its basal level. SAC-derived epidermis and dermis showed an increased synthesis of loricrin and collagen, suggesting that SAC-promoted skin layers are functional. The in vitro experiments in adult human dermal fibroblast (HDFa) and keratinocyte (HaCaT) showed that i) SAC does not induce cytotoxicity at any tested concentrations, ii) SAC enhanced HDFa and HaCaT growth in a dose-dependent manner, and iii) SAC promotes HaCaT migration than CaCl2.
SAC cream improves wound healing by enhancing re-epithelialization and proliferation of epithermal keratinocytes and dermal fibroblasts with no toxicity. The SAC effect is transient as the effects gradually return to normal once the treatment is terminated. These results suggested that SAC cream can be a promising therapeutic intervention for skin injury healing.

K4.01

INTEGRATING PATIENT GENOMICS AND WOUND MICROBIOMES INTO A STRUCTURAL EQUATION MODEL FOR HEALING TIME
Jacob Ancira*1,2, Khalid Omeir1,2, Rebecca Gabrilska1, Craig Tipton2, Clint Miller3, Joseph Wolcott3, Todd D. Little1, Caleb Phillips1
1Texas Tech University, Lubbock, TX; 2MicroGenDX, Lubbock, TX; 3Southwest Regional Wound Care Center, Lubbock, TX
Integrating Patient Genomics and Wound Microbiomes Into a Structural Equation Model for Healing Time

Jacob Ancira1,2, Khalid Omeir1,2, Rebecca Gabrilska1, Craig Tipton2, Clint Miller3, Joseph Wolcott3, Todd D. Little1, Caleb Phillips1 1Texas Tech University, Lubbock, TX; 2MicroGenDX, Lubbock, TX; 3Southwest Regional Wound Care Center, Lubbock, TX

Chronicity and variation among patients in healing outcomes are multifaceted. Yet, there has been little progress integrating the many potentially important variables into a unified model. This is unfortunate as being able to identify more difficult to treat wounds early on might indicate the need for personalized intervention. Here, with the goal of providing such a predictive framework, a structural equation model (SEM) integrating patient data from electronic medical records, next generation sequencing wound microbiomes, and patients’ genomic information was developed to model the chronic wound environment in relation to healing.
The study was based on 127 chronic wound patients whose wound microbiomes were characterized at initial visit using 16S sequencing, and their genome was characterized at 6.2 million single nucleotide polymorphisms (SNPs) using genotyping arrays and imputation. Developing on work recently reported by our team for the integration of microbiomes into SEM, candidate human genomic regions influential to healing differences were identified through a novel sliding window approach, and identified regions were modeled as latent variables using a novel parceling algorithm. These genomic latent variables, a wound microbiome latent variable, and various patient and wound data were evaluated for model fit in the SEM.
The final SEM model included four genomic regions, a microbiome latent construct associated with faster healing, Psuedomonas aerogenosa, smoking, wound volume, slough, exudate, edema, percent granulation, and wound type. The model explained 71% of variation in healing time with the microbiome contributing the largest proportion of variance explained. None of the genomic latent variables on their own significantly accounted for variation among wound healing time, but through their covariance with microbiome and wound variables increased explanatory power by 15%. The importance of this covariance indicates the genomic regions influence wound bed morphology and physiology.
The modeling strategy presented here provides a framework to simultaneously integrate patient and wound data, wound microbiome, and genomics, into a unified predictive model. The addition of patient genomics into a model of healing, which is presented for the first time in this work, expands upon other recent work demonstrating that patient genetic differences are relevant in chronic wounds. This model is also flexible to future integration of other data types, such as gene expression and blood biomarker data. Future work will also focus on building larger and independent cohorts to assess model prediction performance.

K4.02

DISCARDED WOUND DRESSINGS IDENTIFY PREDICTIVE BIOMARKERS FOR CHRONIC WOUND HEALING
Veronika Jurczuk*1, Victoria Soto1, Lilian Valadar Tose2, Christian Gonzalez1, Victoria Garcia1, Oliver Umland3, Sujad Younis4, Natasa Strbo4, Matthew Hardy5, Hadar Lev-Tov1, Robert S. Kirsner1, Francisco Fernandez Lima2, Ivan Jozic1
1Dermatology, University of Miami, Miami, FL; 2Chemistry and Biochemistry, Florida International University, Miami, FL; 3Diabetes Research Institute, Miami, FL; 4Microbiology and Immunology, University of Miami, Miami, FL; 5Miami VA Medical Center, Miami, FL
Discarded Wound Dressings Identify Predictive Biomarkers for Chronic Wound Healing

Veronika Jurczuk1, Victoria Soto1, Lilian Valadar Tose2, Christian Gonzalez1, Victoria Garcia1, Oliver Umland3, Sujad Younis4, Natasa Strbo4, Matthew Hardy5, Hadar Lev-Tov1, Robert S. Kirsner1, Francisco Fernandez Lima2, Ivan Jozic1 1Dermatology, University of Miami, Miami, FL; 2Chemistry and Biochemistry, Florida International University, Miami, FL; 3Diabetes Research Institute, Miami, FL; 4Microbiology and Immunology, University of Miami, Miami, FL; 5Miami VA Medical Center, Miami, FL

Chronic wounds represent a significant and growing challenge in global healthcare, affecting millions of individuals. Despite advances in wound care, therapeutic options remain limited, with varying efficacy among patients. The goal of this study is to identify an aggregate set of predictive biomarkers for wound healing utilizing discarded wound dressings – a non-invasive, innovative approach to obtaining liquid biopsies. Biomarkers offer a promising pathway to develop more targeted and personalized therapies, enabling earlier intervention and optimized treatment strategies to enhance healing outcomes.
Wound dressings from patients (n = 23) with venous leg ulcers (VLUs) and diabetic foot ulcers (DFUs) were collected weekly over a 4-week period, in collaboration with the Diabetic Foot Consortium, with patients stratified as healers or non-healers, based on the reduction in wound size by week 4. Each dressing was mechanically separated into center (wound bed or WB) and perimeter (wound edge or WE), and the solubilized exudate was processed to isolate the total, cellular, and exosomal proteomes by differential centrifugation. Samples from each source and region were subject to proteomic profiling by PASEF-TIMS-TOF mass spectrometry to identify potential biomarkers associated with healing status (healers vs non-healers), wound region (WE vs. WB), and wound etiology (DFU vs. VLU). After normalization, Fisher’s t-test was performed to assess statistical significance using Perseus software and gene ontology terms imported into Bioconductor.
Proteomic analysis of wound exudate revealed distinct peptides differentiating healers (38 peptides) from non-healers (417 peptides), WB (16 peptides) from WE (56 peptides), and VLUs (592 peptides) from DFUs (43 peptides). Non-healers-specific peptides were associated with several targetable biological processes, like cholesterol homeostasis, endosomal transport regulation, immune response, and nucleosome assembly. Notably, in addition to human peptides, bacterial and fungal peptides were identified, highlighting the complex interplay of host and microbial factors in wound healing and the ability to detect wound infection by proteomic profiling of exudate.
This study underscores the transformative potential of proteomic analysis of wound exudate in guiding personalized therapeutic approaches and enhancing treatment efficacy for patients with VLUs and DFUs. The identification of differentially expressed proteins holds promise as predictive biomarkers which open new avenues for studies aimed at developing therapies tailored to specific wound etiologies and targeting shared pathways across different etiologies. These predictive biomarkers also pave the way for the creation of innovative “smart dressings”—advanced wound care solutions capable of detecting physiological changes over time to enable more precise, patient-specific treatments and promote optimal wound healing outcomes.

K4.03

PAI-1 in tissue engineered exosomes critical for normalizing diabetic preclinical wounds
Jasmina Abdalla*, Tamara Mestvirishvili, Bibi S. Subhan, Piul S. Rabbani
Hansjörg Wyss Department of Plastic Surgery, New York University Langone Health, New York, NY
PAI-1 in Tissue Engineered Exosomes Critical for Normalizing Diabetic Preclinical Wounds

Jasmina Abdalla, Tamara Mestvirishvili, Bibi S. Subhan, Piul S. Rabbani Hansjörg Wyss Department of Plastic Surgery, New York University Langone Health, New York, NY

Tissue engineering using human multipotent stromal cells (MSCs) and their exosomes, secreted membranous nanovesicles, promotes faster wound closure through neovascularization in animal models of delayed wound healing and early clinical studies. However, the mechanisms behind the evidence of effect are mostly unknown. Here, we sought to identify mechanistic candidates behind the neovascularization and their mode of activity as exosome cargo in the wound bed.
We isolated exosome preps from cultured human bone marrow MSCs (BMSCs) via differential ultracentrifugation and analyzed protein content using angiogenic protein arrays, mass spectrometry and EnrichR toolkits. We used the type 2 diabetic Leprdb/db mouse model (db/db) for severely delayed wound healing. We administered fluorescently labeled BMSC exosomes 24 hours post excisional 10mm diameter stented wounds. We collected timepoint tissues for immunofluorescent staining and monitored the wounds to closure by planimetry to evaluate the exosome induced changes.
The angiogenesis protein array showed that plasminogen activator inhibitor-1 (PAI-1) was the most abundant in the exosomes, alongside tissue inhibitor of metalloproteinase-1 (TIMP1), thrombospondin-1(TSP1), CD105, VEGF, urokinase plasminogen activator (uPA, a PAI-1 substrate). Higher resolution and unbiased proteomic analysis confirmed the abundance of PAI-1, and pathway enrichment analysis with FDR<0.05 showed that exosomes package cytoplasmic, extracellular and cytoskeleton proteins. Gene ontology classification showed high correlation with regulation of innate immune response. We detected fluorescent-labeled exosomes accumulated in the cytoplasm of endothelial cells in the granulation tissue at post-operative day 7. Compared to untreated db/db wounds, exosome-applied wounds had 7.8±2-fold larger granulation tissue area, with dense F4/80+ macrophages (MØs) in proximity to CD31+ endothelial cells. Using the ImageJ Colocalization macro to avoid subjective bias, we found 58% F4/80+ MØs co-express PAI-1 and 54% express Arg1, a pro-repair MØ marker, compared to 18.7% and 17.1%, respectively, in untreated db/db wounds. The PAI-1 signal in MØs may be due to direct exosome uptake or indirectly after exosome application. Exosome administration reduced the pathologic time to closure by 53% (vs untreated db/db and wild type wounds), but a PAI-1 inhibitor negated this effect. Untreated and exosome+PAI-1 inhibitor-treated db/db wounds have no significant difference in time to closure.
Our results demonstrate that PAI-1 in exosomal format has critical roles in wound healing, in addition to its appreciated extracellular anti-proteolytic roles. PAI-1 induces highly vascularized granulation tissue with pro-repair MØs. We find at least one core mechanism that is induced by BMSC exosome applications on chronic wounds, a necessary step to guide rational therapy design.

K4.04

Pre-diabetes and diabetes downregulate pro-healing monocytes in mice and humans
Katharina S. Fischer*, Maria Gracia Mora Pinos, Abdelrahman M. Alsharif, Amelia B. Knochel, Lulejeta A. Latifi, Fidel Saenz, Mansi Singh, Filiberto Quintero, Ben Litmanovich, Sultana M. Mojadidi, Javier Gonzalez, Dharshan Sivaraj, Autumn Lester, Syed Maisam Jafri, Andrew Hostler, Jonathan P. Yasmeh, Hudson C. Kussie, Kellen Chen, Geoffrey C. Gurtner
Surgery, University of Arizona, Tucson, AZ
Pre-diabetes and diabetes downregulate pro-healing monocytes in mice and humans

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

Diabetes is a huge and growing public health concern affecting nearly 12% of the population. Diabetes dysregulates cellular function leading to the development of a variety of disease states including chronic wounds and increases morbidity and mortality for virtually all surgical procedures. Why this occurs is not well understood. Here, we comprehensively catalog all circulating cells in human and murine diabetic wounds and identify a unique subpopulation of pro-healing monocytes that are selectively and specifically deleted in diabetes.
Full-thickness excisional wounds were created on the dorsum of C57/BL6 wildtype non-diabetic (N-DB) mice, WT mice fed with a high-fat diet (Pre-DB), and leptin-receptor deficient (DB) mice. Wound tissue and blood were explanted at post-operative days (PODs) 0, 2, 7, 14, and 30 and processed for single cell RNA sequencing (scRNA-seq) and flow cytometry. We processed human non-diabetic, pre-diabetic, and diabetic wounds for scRNA-seq.
Both prediabetes and diabetes significantly impaired wound healing compared to healthy WT mice. In the early wound healing phase, myeloid cells predominated all groups at 67% of all cells.
In N-DB mouse wounds, a unique subpopulation of Ly6c2+/Fcgr1+ monocytes proliferated to 68% of the myeloid population at POD 2 compared to 9% in Pre-DB or DB. In blood, LY6C2+/FCGR1+ monocytes comprised > 85% of myeloid cells at PODs 2 and 7, significantly doubled over Pre-DB mice. These levels resolved by POD 14–30. Human non-diabetic wounds also demonstrated an 82% upregulation of CD14 (represented by Ly6c2 in mice)+/FCGR1A+monocytes, compared to 5% in Pre-DB/DB wounds.
Both Pre-DB mice and Pre-DB humans overexpressed proinflammatory Dusp2+/Cxcl2+/Nfkbia+ macrophagescompared to N-DB wounds (mice: 70% vs. < 10% N-DB & DB at PODs 2 and 7; human: 90% DB and 61% Pre-DB vs 1% N-DB).
Both DB mice and DB humans overexpressed C1qa+/C1qb+/Timp2+ macrophages compared to N-DB (mice: 43% vs. < 13% N-DB/Pre-DB POD7; human: 14.6% DB vs. 0% N-DB).
We identified and confirmed upregulation of specific Ly6c2 (represented by CD14 in humans)+/Fcgr1+ monocytes during early normal wound healing across species, time, and tissue type. During pre-diabetic to diabetic development, myeloid cells differentiate temporally from Ly6c2+/Fcgr1+ to Dusp2+/Cxcl2+/Nfkbia+ to C1qa+/C1qb+/Timp2+ subpopulations, which are present both locally in healing tissue and systemically. Harnessing immunotherapy techniques to modulate myeloid populations could lead to new therapies to improve clinical outcomes for pre-diabetic and diabetic patients.

K4.05

INTEGRATED MULTI-OMICS ANALYSES IDENTIFY ACTIVATED T CELLS AND PD-1/PD-L1 SIGNALING AS TISSUE BIOMARKERS OF HEALING ACROSS FOUR DFU PATIENT COHORTS
Rivka C. Stone, Sophie M. Bilik*, Caroline Dodson, Katelyn Rivas, Jamie L. Burgess, Nathan Balukoff, Anthony Griswold, Andrew Sawaya, Irena Pastar, Natasa Strbo, Maria Morasso, Marjana Tomic-Canic
Dermatology and Cutaneous Surgery, University of Miami, Miami, FL
Integrated Multi-Omics Analyses Identify Activated T Cells and PD-1/PD-L1 Signaling as Tissue Biomarkers of Healing Across Four DFU Patient Cohorts

Rivka C. Stone, Sophie M. Bilik, Caroline Dodson, Katelyn Rivas, Jamie L. Burgess, Nathan Balukoff, Anthony Griswold, Andrew Sawaya, Irena Pastar, Natasa Strbo, Maria Morasso, Marjana Tomic-Canic Dermatology and Cutaneous Surgery, University of Miami, Miami, FL

Diabetic foot ulcers (DFUs) are a serious complication of diabetes, and amputations from non-healing DFUs carry high rates of morbidity and mortality. This study aimed to identify biomarkers for early intervention of hard-to-heal ulcers, enabling timely and targeted treatments to improve patient outcomes.
This study utilized a multi-omics approach to explore the immune response that impairs wound closure and drives DFU pathogenesis. To start, Ingenuity Pathway Analysis of bulk RNA-sequencing profiles from a cohort of DFU Healers and Non-healers (N = 10) identified markedTh1 and Th2 activation in DFU Healers (p = 3.8 × 10−15; activation Z score = +4.8), and CIBERSORTx imputation analysis estimated higher activated T cells in Healers. To validate and explore this further, GeoMx digital spatial profiling of CD3+ T cells was performed in tissue from a second DFU cohort (N = 23), identifying elevated PD-1 and PDL-1 in upper dermal T cell infiltrates of DFU Healers (p < 0.001). PrimeFlow cytometry performed in a third DFU cohort (N = 4) and confirmed higher proportions of CD3+PD-1+ and CD3+PD-L1+ T cells in DFU Healers compared with DFU Non-healers (PD-1, 59% vs. 27%; PD-L1, 71% vs. 6%). Finally, sub-clustering analysis of published single cell RNA-sequencing profiles from a fourth DFU cohort (6 Healers and 4 Non-healers) confirmed T cell activation in Healers, and further highlighted upregulation of PDCD1 (PD-1) and CD274 (PD-L1) in CD4+ T cells.
Taken together, integrated multi-omics analyses of wound tissue from 47 DFUs (25 Healers and 22 Non-healers) in four patient cohorts support PD-1 and PD-L1 as tissue biomarkers of DFU healing, and highlight the importance of T cell activation in modulating wound inflammation to achieve successful DFU closure.

K4.06

KEY CELL AND MOLECULAR PATHWAYS THAT ARE DYSREGULATED WHEN WOUND CHRONICITY IS INITIATED
Parnian Jabbari*, Manuela M. Martins-Green
Department of Molecular, Cell, and Systems Biology, University of California Riverside, Riverside, CA
Key Cell and Molecular Pathways That are Dysregulated When Wound Chronicity Is Initiated

Parnian Jabbari, Manuela M. Martins-Green Department of Molecular, Cell, and Systems Biology, University of California Riverside, Riverside, CA

Wound healing involves a complex set of overlapping phases that include homeostasis, inflammation, proliferation and remodeling. During these stages, fibroblasts, endothelial cells, keratinocytes and immune cells need to proliferate and migrate to form the granulation tissue and close the wound. Deviations from the normal healing process can lead to the development of chronic wounds (CWs). It has been shown that CW have high levels of oxidative stress (OS), prolonged inflammation and lack of vascularization. Studies characterizing the differences between CWs and healing wounds (NCWs) during initiation of healing or chronicity are scarce. Such comparisons cannot be conducted in patients with CW because they do not seek medical care until the chronicity is already advanced. Therefore, studies to understand chronic wound development require an animal model that consistently develop chronic wounds that mimic those in humans. We have developed such a model by increasing OS in diabetic mice. Using this model, we sought to understand how major cell and molecular mechanisms differ between CWs and NCWs early after injury by identifying signaling pathways that can be manipulated to reverse chronicity.
For this purpose, we compared CWs and NCWs at the transcription level using a NanoString metabolomic panel with samples collected at 12, 24, 48, and 72 h post-surgery (n = 5/time point). Of the several pathways significantly different between NCW and CW, we focused on those affecting: (1) Energy production (2) proliferation and (3) angiogenesis, all key processes important for healing.
ATP production by mitochondrial is essential to healing as cells require energy for high levels of proliferation and migration associated with wound healing. We found that ATP levels decrease with time after injury in CWs, whereas they increase with time in NCWs. To produce ATP, mitochondria require proper oxygen delivery and angiogenesis in tissues. In response to hypoxia in the wound microenvironment, hypoxia-inducible factors (HIF) increase the transcription of proangiogenic genes. HIF have α and β subunits that upon binding form the active transcription factor. The α subunit has three isoforms that compete for binding to the β subunit. Of the three isoforms of HIFα, HIF1α and HIF2α can increase angiogenesis, while HIF3α cannot. CWs showed higher levels of HIF3α. This can decrease expression of genes such as FGF and VEGF and impair angiogenesis. FGF1 can also promote fibroblast proliferation. We found that FGF1 levels were lower during CW initiation. Another molecule important in cell proliferation is PIP3 pathway that works through activating Akt. PIP3 levels were lower at all points after injury in CW, disrupting proliferation and migration of cells.
In conclusion, key healing processes such as angiogenesis, cell proliferation, migration, and energy production are dysfunctional in CWs, impairing formation of the granulation tissue and re-epithelialization.
WHS Session L1: Inflammation, Biofilm Infection, and Immunity

L1.01

Oral delivery of CNP-miR146 attenuates inflammatory response in acute TNBS colitis mouse model of inflammatory bowel disease
Efua H. Bolouvi*1, Anisha Apte1, Annette Mai1, Skopp Skopp1, Sudipta Seal3, Carlos Zgheib1,2, Kenneth W. Liechty1,2
1Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Arizona Tucson School of Medicine, Banner Children’s at Diamond Children’s Medical Center, Tucson, AZ; 2Ceria Therapeutics, Inc, Tucson, AZ; 3Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL
Oral delivery of CNP-miR146 attenuates inflammatory response in acute TNBS colitis mouse model of inflammatory bowel disease

Efua H. Bolouvi1, Anisha Apte1, Annette Mai1, Skopp Skopp1, Sudipta Seal3, Carlos Zgheib1,2, Kenneth W. Liechty1,2 1Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Arizona Tucson School of Medicine, Banner Children’s at Diamond Children’s Medical Center, Tucson, AZ; 2Ceria Therapeutics, Inc, Tucson, AZ; 3Advanced Materials Processing and Analysis Center, Nanoscience Technology Center, University of Central Florida, Orlando, FL

Inflammatory bowel disease (IBD) is a complex and relapsing chronic disease affecting 1 in 100 individuals nationally. We have previously shown that enema delivery of CNP-miR146a attenuated inflammatory response in the colitis model with a marked reduction in inflammatory markers, decreased clinical disease activity and weight loss, and restoration of colonic architecture. Moreover, we have shown that CNP-miR14a did not degrade when exposed to simulated gastrointestinal (GI) conditions in vitro study demonstrating that it is stable in the harsh conditions of the GI tract. Thus, based on these findings, we hypothesize that oral delivery of CNP-miR146a will attenuate inflammatory processes in 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis.
The Mice cohort consists of three groups of Balb/c mice strains. The control group (males, n = 8) was induced with a 50% ethanol enema. The colitis group (n = 11) was induced with 2.5% TNBS enema (75mg/kg) and received oral treatment with either PBS (n = 5) or CNP-miR146a (n = 6). The mice were monitored for clinical signs of colitis including weight loss of at least 10%. After 48 hours, the controls were treated with 200ul of oral PBS, whereas the TNBS-colitis groups received oral gavage of 200ul of either PBS or 25ng CNP-miR146a for the next three days. Five days after colitis induction, the mice were euthanized, and their colons were harvested for gene expression using quantitative polymerase chain reaction. The data was analyzed using one-way ANOVA with an alpha value of 0.05 for statistical significance.
CNP-miR146a treated TNBS colitis group demonstrated statistically significant reduction in weight loss from day 4 to 5 compared to the TNBS colitis + PBS group (p = 0.0075 and p = 0.0125, respectively). Moreover, the CNP-miR146a treated group had a reduction in the gene expression level of pro-inflammatory cytokine IL-6 and inducible nitric oxide synthase (NOS2). The level of microRNA-146a was increased in the CNP-miR146a treated group without statistical significance (p = 0.43).
Oral delivery of CNP-miR146a demonstrated promising results in the TNBS-induced colitis mouse model with reduction in inflammatory and oxidative stress markers implicated in inflammatory bowel disease pathophysiology.

L1.02

WITHDRAWN
Christina Garvey Griffith1, Kelsey Hauser2, Leon Grayfer1 1Biological Sciences, George Washington University, Washington; 2Noblis, Reston, VA
Insight From an Amphibian Model Into Macrophage-Mediated Scarless Wound Repair

Christina Garvey Griffith1, Kelsey Hauser2, Leon Grayfer1 1Biological Sciences, George Washington University, Washington; 2Noblis, Reston, VA

Xenopus laevis frogs can repair wounded skin without scarring, but these regenerative capacities wane with age. X. laevis is thus a promising model for parsing out the mechanisms of scarless regeneration versus scar deposition. Notably, mammalian macrophages (Mfs) are integral to wound healing, coordinating the initial inflammatory responses to the ensuing remodeling and repair. Across vertebrates, Mf biology is dictated by two colony-stimulating factor-1 (CSF1) receptor ligands; CSF1 and interleukin-34 (IL34). While IL34 is critical to the development of epidermal Mfs known as Langerhans cells, the contribution of CSF1- and IL34-derived Mfs to skin wound repair remains unexplored. Pertinently, using the X. laevis model we showed that CSF1- and IL34-Mfs are functionally distinct, formulating our hypothesis that these subsets play distinct roles during frog skin wound repair at distinct stages of animal life.
Juvenile (2–4 mo.) and adult frogs (2+ yrs.) were wounded dorsally and assessed over two months by histological, in situ hybridization and gene expression analyses to discern differences in wound repair. To define the roles of IL34- and CSF1-Mfs in scarless wound repair, animals were administered with recombinant® CSF1 and rIL34 and assessed as above.
While juveniles and adult frogs exhibited comparable wound csf1 gene expression, wounded juveniles possessed considerably greater il34 transcripts, correlating with substantial myofibroblast activation and presumably accounting for their faster wound closure. By contrast, adult frogs exhibited minimal changes to wounded skin il34 expression, which may account at least in part for their considerably slower wound contraction and impaired healing.
Strikingly, by 2 months juveniles successfully repaired their skin without scars, including complete regeneration of their cutaneous glands. By contrast, wounded adult skins continued to exhibit granulation and poorly reformed glands. Compared to control and rIL34-administered animals, wounded and rCSF1-injected juveniles showed more rapid wound repair combined with much more pronounced collagen deposition. Animals administered with rIL34 showed greater repair and collagen deposition than control animals but less so than the rCSF1-treated frogs.
The greater wound repair capacity of juvenile frogs may depend at least in part on IL34-Mfs/Langerhans cells and exogenous rIL34 further enhances this process. Surprisingly, exogenous CSF1 is even more effective at enhancing juvenile scarless wound repair, and we postulate that this may be due to the enrichment of CSF1-Mfs resulting in changes to kinetics inflammation and regeneration during the juvenile repair process. Moreover, we anticipate that CSF1- and IL34-Mfs play distinct roles in these kinetics of inflammatory and reparatory processes during juvenile frog wound repair while adult frogs rely more exclusively on CSF1-Mfs for their wound closure.

L1.03

CHARACTERIZATION OF INFLAMMATORY MYELOID POPULATIONS IN A MODEL OF DELAYED WOUND HEALING AND IDENTIFICATION OF IRF5 AS A POTENTIAL TARGET FOR NEW WOUND HEALING THERAPIES.
Amanda M. Beneat*1,2, Brooke Rothberg1, Bailey Roberts1,3, Betsy Barnes1, Alisha Oropallo2,3
1Feinstein Institute of Medical Research, Mineola, NY; 2Comprehsive Wound Healing Center, Northwell Health, New Hyde Park, NY; 3Surgery, Northwell Health, Manhasset, NY
Characterization of Inflammatory Myeloid Populations in a Model of Delayed Wound Healing and Identification of IRF5 as a Potential Target for New Wound Healing Therapies

Amanda M. Beneat1,2, Brooke Rothberg1, Bailey Roberts1,3, Betsy Barnes1, Alisha Oropallo2,3 1Feinstein Institute of Medical Research, Mineola, NY; 2Comprehsive Wound Healing Center, Northwell Health, New Hyde Park, NY; 3Surgery, Northwell Health, Manhasset, NY

Chronic wounds affect over 40 million people annually and failure of these wounds to heal can lead to costly procedures, recurrent infections, limb loss, and even death. Therefore early treatment of chronic wounds is crucial to preventing morbidity and mortality in these patients. Wound healing begins with inflammation, which involves immune cell trafficking to the wound, and differentiation into an inflammatory (M1) subtype. The tissue remodeling phase of healing is marked by a phenotypic switch of immune cells to an anti-inflammatory sub-type of macrophages (M2-like). Chronic wounds are believed to be stalled in the inflammatory phase, making modulation of these cells a potential target for novel therapies. Interferon regulatory factor 5 (IRF5) promotes M1-like macrophage polarization while loss promotes M2. Therefore, IRF5 is a potential target for inhibition to allow for macrophage reprogramming in a chronic wound.
Immune profile characterization of normal and chronic wounds, a model of delayed wound healing was created by administering LPS via intraperitoneal injections in C57Bl/6 mice every 3 days starting 9 days pre-wound. The wound area was calculated using the MolecuLight DX camera at the time of wound creation and at all experimental endpoints. Wounds were excised at days 0, 3, 7, and 10 for flow cytometric analysis of immune cell populations. To determine the effect of a loss of IRF5 on the immune profile of a wound, Irf5-/- and Irf5fl/fl-LysM-Cre+ mice were used to assess wound healing. Finally, an inhibitor of IRF5 (N5-1) was used to reprogram macrophages and promote healing.
An overall increase of lymphocytes was seen in the LPS-treated group compared to the untreated group, which persisted even after a peak was noted in the controls. Retention of M1-like macrophages specifically was seen in LPS-treated mice, corresponding to wound healing delays seen on wound area measurement. Partial recovery of wound healing at day 10 in the LPS group corresponded to a more anti-inflammatory cell population. Irf5-/- and Irf5fl/fl-LysM-Cre+ cohorts had notably decreased M1-like macrophages; however healing delays were also noted. Early experiments with IRF5 inhibition at the onset of wounding appear to phenocopy IRF5 knockout mouse models.
This study proposes a reproducible model of delayed wound healing that focuses on inflammation. Our results also show that M1-like macrophage retention (at a late stage) and/or absence (at an early stage) lead to delayed healing, which suggests that inflammation is needed to initiate healing, but the failure of phenotypic switching to anti-inflammatory cells can lead to delays in wound closure. Future studies will therefore focus on the administration of our lab-generated IRF5 inhibitor only once wound healing delays are seen (approximately day 3), to promote this phenotypic switching and accelerate wound healing.

L1.04

Skin myeloid cell dysfunction governs vascularized composite allograft wound healing and rejection
Javier Gonzalez1,2, Alex Lellouch1,3, Curtis Cetrulo1,3, Maxime Jeljeli*1,3
1Surgery, Cedars Sinai, Los Angeles, CA; 2Surgery, University of Arizona, Tucson, AZ; 3Surgery, Harvard University, Boston, MA
Skin Myeloid Cell Dysfunction Governs Vascularized Composite Allograft Wound Healing and Rejection

Javier Gonzalez1,2, Alex Lellouch1,3, Curtis Cetrulo1,3, Maxime Jeljeli1,3 1Surgery, Cedars Sinai, Los Angeles, CA; 2Surgery, University of Arizona, Tucson, AZ; 3Surgery, Harvard University, Boston, MA

Vascularized composite allotransplantation (VCA) is a life-changing procedure for patients with severe traumatic injuries. Unlike a simple organ transplant, VCA grafts involve various tissues, with the skin being the most immunogenic component with its own wound healing dynamics, that correlates with the risk of graft rejection. The immune mechanisms responsible for aberrant dermal wound healing and graft rejection in VCA remain elusive. This study investigates the role of maladaptive activation of macrophages and myeloid-derived suppressor cells (MDSC), in skin transplantation to identify potential biomarkers and therapeutic targets for improving graft survival.
We used full-skin transplantation models in mice to study phenotypic and functional alterations in macrophages and MDSCs in response to surgically induced inflammation (syngeneic: C57BL/6 (H-2b) donor → C57BL/6 (H-2b) recipient) and combined with MHC mismatch (allogeneic: BALB/c (H-2d) donor → C57BL/6 (H-2b)). A sham group was used for baseline comparison. Myeloid cells phenotype in the skin, spleen, and bone marrow were analyzed at day 12 post-transplant using Flow Cytometry. ELISA and RT-qPCR were used to assess the cytokine production capacity of spleen macrophages and the ability of MDSC to block T cell activation.
On day 12 post-transplant, skin wound healing was evident in the syngeneic group while the allogeneic group exhibited signs of graft rejection. Phenotypic analysis of dermal immune cells revealed a similar increase in dermal macrophages in both groups. However, macrophages in the allogeneic group displayed a shift toward a more inflammatory phenotype, with increased expression of Ly6C, CD80, MHC II and decreased expression of DC-SIGN, MerTK and CD200R. Functionally, allogeneic macrophages produced more IL-6 and less IL-10 compared to syngeneic macrophages upon LPS stimulation. In the allogeneic group, despite an increased frequency of MDSCs in the skin, functional assays showed a reduced MDSC immunosuppressive activity, with lower IL-10 production and impaired suppression of T cell proliferation. Gene expression analysis revealed distinct immune-regulatory profiles between the groups. MDSC from the syngeneic group displayed an upregulation of Arg1, IL-10, Mertk, and Ddit3, indicating enhanced anti-inflammatory and stress-response pathways whereas MDSC from the allogeneic group showed an upregulation of IL-6 but a downregulation of Arg1, IL-10, and Mertk, reflecting a pro-inflammatory and immunosuppressive imbalance compared to the syngeneic group.
Our findings reveal that MHC mismatch may drive myeloid cell dysfunction in skin rejection, leading to heightened inflammation and impaired immunosuppressive activity in allogeneic conditions. Investigating these mechanisms further could inform novel therapies to enhance VCA tolerance. Moreover, these immune alterations show promise as biomarkers for early detection of skin rejection.

L1.05

ONE-CARBON METABOLISM TUNES LIPID METABOLISM TO FACILITATE PRO-HEALING EFFEROCYTOSIS IN MACROPHAGES
Julia Drolet*, Kentaro Takahashi, Jasmine R. Jackson, Norifumi Urao
Pharmacology, SUNY Upstate Medical University, Syracuse, NY
One-Carbon Metabolism Tunes Lipid Metabolism to Facilitate Pro-Healing Efferocytosis in Macrophages

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

Macrophages play a pivotal role in tissue homeostasis and wound healing responses by clearing apoptotic cells through efferocytosis. Impaired efferocytosis is associated with conditions with non-resolving inflammation such as in metabolic syndrome, which is characterized by lower availability of the one-carbon (1C) metabolism metabolites—serine, glycine, and methionine.
To investigate the role of 1C metabolism in efferocytosis, we used oxygen controlled live cell imaging of bone marrow derived macrophages (BMDMs) co-cultured with apoptotic Jurkat cells stained with a pH-sensitive dye. BMDMs from mice fed high fat diet with L-NAME supplemented water were studied as a model of metabolic syndrome. We found that treatment of BMDMs with serine, an amino acid and key 1C metabolite, enhanced efferocytosis under hypoxia (1% oxygen) and in pro-inflammatory (M1) BMDMs, which displayed reduced efferocytosis compared to naïve or anti-inflammatory states. Lipid droplet accumulation inversely correlated with efferocytosis, and serine supplementation reduced lipid droplet formation under both hypoxic and M1-polarized conditions. Through manipulation of culture conditions, we confirmed that serum-derived lipids were incorporated into lipid droplets. Inhibition of s-adenosyl methionine (SAM) synthesis, a 1C metabolism product and the methyl donor for methylation reactions, increased lipid droplet accumulation and reduced efferocytosis. Conversely, methionine supplementation reduced lipid droplet accumulation suggesting the importance of SAM in regulating lipid droplets and efferocytosis. In BMDMs cultured from a mouse model of metabolic syndrome, we found reduced efferocytosis, lipid droplet accumulation, and dysregulated lipid metabolism-associated gene expression. Topical serine treatment failed to enhance wound closure in diabetic mice, highlighting cell intrinsic dysregulation in diabetic wound environments.
In summary, 1C metabolism regulates macrophage lipid metabolism to facilitate efferocytosis in wound macrophages. In metabolic syndrome, limited availability of 1C metabolites and intrinsic dysregulation of lipid metabolism in macrophages may impair efferocytosis, contributing to chronic inflammation and delayed wound healing.

L1.06

INFLAMMATORY RESPONSE AND TISSUE REMODELING IN EX VIVO HUMAN SKIN PERFUSION MODEL FOLLOWING NITROGEN MUSTARD EXPOSURE
Naresh Mahajan, Alexa Rivera del Rio Hernandez, José A. Arellano, Fuat Baris Bengur, Shawn Loder, Ethan Banks, Samantha Bosco, Francesco M. Egro, Jeffrey Gusenoff, J. Peter Rubin, Asim Ejaz.
Plastic Surgery, University of Pittsburgh, Pittsburgh, PA
Preclinical Human Skin Xenograft Mouse Model to Study Spatial Biology of Wound Tissue

Pradipta Banerjee, Fabio Muniz Oliveira, Piya DasGhatak, Surabhi Singh, Subhadip Ghatak, Chandan K. Sen, Sashwati Roy Surgery, University of Pittsburgh, Pittsburgh, PA

Background Humanized mouse models with human skin graft in a murine environment are particularly valuable for studying human-specific wound healing mechanisms in a spatially resolved manner and understanding the underlying molecular mechanisms. One of the challenges associated with this model is to assess the successful integration of the graft and what human versus mouse cells are involved with the healing process. Aim To assess the integration and performance of human graft in situ and microenvironmental adaptations critical to interpreting data from such models, we employed spatial omics technologies.
Methods Human skin biospecimens were obtained from Indiana University Health Biorepository. The skin specimens were prepared by removing the adipose layer followed by a sterilization protocol. Full-thickness human skin specimens were cut and grafted on the back of NSGS (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) mice. After 12 weeks of engraftment, a punch wound was created on the xenograft and monitored for 7d post-wounding (PW). To test the feasibility of human cell-based therapies for wound healing, human xenograft wounds were treated with 2.5 × 10⁶ freshly isolated human peripheral blood mononuclear cells (PBMC) on the day of wounding. These cells were applied topically by injecting under an occlusive dressing covering the wound. Spatial transcriptomics (Xenium), and proteomics (Phenocycler Fusion 2.0, PFC 2.0) were performed to study the spatial biology of the wound tissue over time.
Results Successful integration of human skin onto NSGS mice was demonstrated by the presence of human epidermis and dermis with intact human blood vessels in the human grafts (n = 6) via Xenium and PCF2.0, using custom-developed human and mouse-specific molecular target panels. Presence of human-specific cells in the graft region, and at the junctions of the human graft with mice skin, cells from both species were distinctly visible indicating a chimeric region. The human graft wounds treated with human peripheral blood mononuclear cells (PBMCs) showed a significant improvement (p < 0.05; n = 5/group) in wound closure by 7d PW compared to the placebo-treated control group. Spatial proteomics analysis confirmed a significant (p < 0.05; n = 4) increase in the number of blood vessels compared to the placebo group. Blood flow was imaged.
Conclusion. In the human skin graft mice model, the human and mouse-specific molecular target panels are critical for spatial biology studies providing insight into tissue composition. The study on PBMC treatment for human graft wounds holds significant value as it establishes proof of concept evidence that human cell-based therapies can be effectively evaluated in this preclinical model. This humanized mouse approach provides a platform to investigate the spatial biology mechanisms of human wound healing in a temporal manner.

WHS Session L2: Extracellular Matrix and Regenerative Healing

L2.01

CELL SPECIFIC EXOSOME-MEDIATED EPIDERMAL-DERMAL CROSSTALK IN FUNCTIONAL WOUND CLOSURE Mohini
Mohini Moulick*, Anita Yadav, Anu Sharma, Parmeshwar Gavande, Aparajita Nandy, Chandan K. Sen, Sashwati Roy, Subhadip Ghatak
Department of surgery, University of Pittsburgh, Pittsburgh, PA 15232-1864, PA
Cell Specific Exosome-Mediated Epidermal-Dermal Crosstalk in Functional Wound Closure

Mohini Moulick, Anita Yadav, Anu Sharma, Parmeshwar Gavande, Aparajita Nandy, Chandan K. Sen, Sashwati Roy, Subhadip Ghatak Department of surgery, University of Pittsburgh, Pittsburgh, PA 15232-1864, PA

Cutaneous repair relies on coordinated cellular interactions, particularly between epidermal keratinocytes and dermal papillary (DP) fibroblasts (COL1A2+, CD90+MYL9+SCA1). We tested the hypothesis that epidermal TRPS1 silences YAP1 in DP fibroblasts to facilitate epidermal regeneration via a non-canonical WNT5A pathway mediated by fibroblast-derived exosomes (Exofib). Keratinocyte-derived exosomes (Exok) and Exofib in mice were genetically labeled with Krt14 and Col1A2 promoter-driven plasmids with in-frame eGFP and mNeonGFP reporter respectively using tissue nanotransfection (TNT). Wound-edge (WE) Exok and Exofib were isolated and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and exosome antibody array assay as per MISEV2023 guidelines and reported in the EV-track consortium that received an EV-metric score of 100% (ID: EV220292). The keratinocyte-specific Trps1 conditional knockout transgenic mice were generated by breeding Trps1fl/fl mice with Krt14cre mice. Wound inducible epidermal TRPS1 was significantly enriched in Exok isolated from d7 WE tissue (59.41 ± 17.95%; n = 6). Reporter study identified nuclear localization of epidermal TRPS1 in DP fibroblasts (Pearson’s r, 0.75; p = 0.003, n = 4). Such nuclear localization was not observed in Krt14-Trps1−/− mice (n = 12) at d7 post-injury. However, a significant increase of YAP1 was observed in DP fibroblasts at d7 and compromised biomechanical properties (tensile strength, viscoelasticity and Young’s modulus) of the repaired skin (p < 0.001). scRNA-seq of human diabetic WE samples (n = 14) revealed that unlike healing wounds, non-healing wounds have compromised communication between keratinocyte subsets K2/K3 and fibroblast subset F3 with altered expression of key molecules like TRPS1 and YAP1. Experimental inhibition of Exoκ uptake by DP fibroblasts using "eat me not" Exoκ-GFP-CD47-RFP resulted in significantly increased YAP1 expression in DP fibroblasts, with compromised biomechanical properties and deficient epidermal appendages such as placode (K79+K75-K6-), germ (K79+K75+K6-), peg (K79+K75+K6+), bulbous peg (K79-K75+K6+) (n = 10). Additionally, CRISPR/Cas9 mediated DP fibroblast-specific YAP1 knockout (n = 10) was associated with reduced scarring, enhanced wound collagen deposition in "basket-weave" fashion and restoration of skin appendages, underscoring the therapeutic potential of targeting TRPS1-YAP1-signaling.
This work lays the foundation of a novel paradigm that addresses the molecular dynamics of keratinocyte-fibroblast crosstalk post-injury. Notably, it identifies a pivotal role of cargo packaging within exosomes to direct dermal remodeling, a process often disrupted in non-healing diabetic wounds. This discovery has significant implications for the therapeutic targeting of keratinocyte-fibroblast interactions to improve diabetic wound healing.

L2.02

Mechanically activated myofibroblasts drive macrophages into distinct transcriptional and functional states through direct cell contact
Li Diao*1, Ronen Schuster2, Fereshteh Younesi1,3, Boris Hinz1,3
1LTRR, Unity Health Toronto, Toronto, ON, Canada; 2CytoReason, Tel Aviv, Israel; 3Faculty of Dentistry, University of Toronto, Toronto, ON, Canada
Mechanically Activated Myofibroblasts Drive Macrophages into Distinct Transcriptional and Functional States Through Direct Cell Contact

Li Diao1, Ronen Schuster2, Fereshteh Younesi1,3, Boris Hinz1,3 1LTRR, Unity Health Toronto, Toronto, ON, Canada; 2CytoReason, Tel Aviv, Israel; 3Faculty of Dentistry, University of Toronto, Toronto, ON, Canada

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. We further hypothesized that mechanically activated MFs control distinct MΦ states in contact–dependent and contact–independent signaling processes.
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 and MFs on the respective substrates in setups that allowed either direct contact or communication restricted to the exchange of soluble factors. Cells obtained from all experimental combinations were separately analyzed using immunofluorescence 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. 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 both 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 profile. (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 IL-17, TNF, NF-κB, and C-type lectin signaling. Immunofluorescence and flow cytometry analysis 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 6–8-fold more pronounced in co-cultures with direct contact.
Direct contact with MFs generate a unique MΦ polarization state that features a combination of proinflammatory and profibrotic signaling pathways. The recognition of new profibrotic MΦ polarization states in direct contact with MFs offers novel therapeutic targets for the prevention and treatment of fibrosis.

L2.03

FAK AS A CENTRAL MEDIATOR OF PROVISIONAL MATRIX ASSEMBLY DURING STROMAL REPAIR
Emily Davis*1, Marina Uroz1,2, Christopher Chen1,2, Jeroen Eyckmans1,2
1Biomedical Engineering, Boston University, Brookline, MA; 2Biomedical Engineering, Wyss Institute, Boston, MA
FAK as a Central Mediator of Provisional Matrix Assembly During Stromal Repair

Emily Davis1, Marina Uroz1,2, Christopher Chen1,2, Jeroen Eyckmans1,2 1Biomedical Engineering, Boston University, Brookline, MA; 2Biomedical Engineering, Wyss Institute, Boston, MA

Fibroblasts play a central role in fibrous tissue repair by migrating into the wound bed, depositing a provisional matrix, and contracting the wound margins to promote closure. While the role of wound contraction during closure has been well-studied, the interplay between cell-matrix adhesion and contraction in coordinating matrix assembly during wound closure remains poorly understood.
In this study, we investigated how cell contractility and cell-matrix adhesion affect wound closure using Normal Human Dermal Fibroblasts Neonatal (NHDFneos) and Normal Human Lung Fibroblasts (NHLFs) in a 3D microtissue model of stromal wound healing. Briefly, NHDFneos and NHLFs were embedded in a type I collagen gel and seeded into microTug (μTUG) devices composed of microwells with micropillars to form 3D microtissues. Twenty-four hours after the microtissues formed, full-thickness wounds were created using a pulsed nanosecond Nd:Yag Laser. Prior to injury, microtissues were treated with contractility or adhesion modulators and wound closure dynamics were assessed for 24 hours post-injury using time-lapse microscopy. Traction force microscopy (TFM) on cells seeded on collagen type I coated polyacrylamide gels (Young’s modulus = 5 kPa) was used to measure contractility. Immunofluorescent staining against phosphorylated-paxillin quantified focal adhesion size as a metric for cell-matrix adhesion.
At baseline, NHDFneos fully healed microtissue wounds within 24 h, while NHLF microtissues failed to close the gaps. TFM revealed that NHLFs exhibited higher contractility and larger adhesions compared to NHDFs, prompting the hypothesis that elevated contractility levels may impair gap closure by NHLFs. Interestingly, treatment with myosin II and ROCK inhibitors (blebbistatin and Y-27632 respectively), lowered the contractility of NHLFs, but did not restore wound closure. Given that cellular contractility regulates cell-matrix adhesion via Focal Adhesion Kinase (FAK) activity, we investigated the role of FAK signaling in gap closure. While treatment with PF-573228, an inhibitor of FAK phosphorylation, did not rescue gap closure in NHLF microtissues, injured NHLF microtissues treated with low dose FC11, a FAK degrader, healed within 24 h post injury (N = 4, n = 22 tissues per treatment group). Interestingly, FC11 lowered contractility and the size of focal adhesions to levels comparable to NHDFneos. A course-grain computational model of healing microtissues revealed that a balance between contractility and adhesion is required for effective matrix assembly and gap closure.
Together, these findings highlight that the interplay between contractility and adhesion, regulated through FAK, is critical for matrix assembly in stromal wound healing, and may inform potential mechanotherapeutic strategies to treat impaired tissue repair.

L2.04

APPLICATION OF HORIZONTAL AND VERTICAL TENSION ON ABDOMINAL HUMAN SKIN SHOWS DISTINCTIVE ANISOTROPHY
Emily Davis*1, Marina Uroz1,2, Christopher Chen1,2, Jeroen Eyckmans1,2
1Biomedical Engineering, Boston University, Brookline, MA; 2Biomedical Engineering, Wyss Institute, Boston, MA
Application of Horizontal and Vertical Tension on Abdominal Human Skin Shows Distinctive Anisotrophy

Adrian Rodrigues, Anmar Abu-Romman, Brigid Coles, Thomas Mustoe, Seok Jong Hong, Robert Galiano Northwestern University, Wilmette, IL

The tensile strength of skin is highly dependent on the density and maturity of the interlining dermal collagens, with the crosslinking of elastin fibers influencing the strength with a smaller role. Traditionally, skin has shown directional variations in strength under dynamic loading, and therefore has been described as an anisotropic substance. However, the extent of directional load variation that is parallel (horizontal) and perpendicular (vertical) to Langer’s lines, using various skin widths on non-fixed, freshly excised human skin, has not been well studied. After human abdominoplasty, panni of the lower abdomen were transferred out of the surgical suite for tensile testing.
Rectangular mappings were then drawn on the skin of the panni with the following three dimensions: 0.5 cm × 7.0 cm, 1.0 cm × 7.0 cm, and 1.5 cm × 7.0 cm, with some of the mappings oriented vertically (perpendicular to Langer’s lines) and others oriented horizontally (parallel to Langer’s lines). Skin mappings were then excised from each pannus, producing rectangular skin specimens that were subjected to uniaxial tensometry—as it related to Langer’s collagen axis in the lower abdomen—with testing undergoing maximum load in N (Newtons) for each distinctive width with an Instron model tensometer.
Maximum load of skin pulled parallel (horizontally) to Langer’s tension lines showed an exponential gain in maximum load with each skin width tested. However, skin pulled perpendicular (vertically) to Langer’s collagen orientation showed diminishing returns, with 1.0 and 1.5 cm widths being very similar to each other. The tensile strength of skin is related to the direction of the underling collagen trajectory. When tensile velocities surpass the skin’s ability to align its collagen parallel to the vectoring load, the skin exhibits anisotropic properties. Strength increases exponentially when the load aligns with collagen fibers of increasing width, while diminishing returns occur when the load is applied perpendicular to the collagen network.

L2.05

Developing a Neonatal Hyaluronan-Enriched Nanofibrillar Scaffold to Modulate Adult Fibroblast Activity
Whitney A. Ponwith*, Tugba Ozdemir
Nanoscience and Biomedical Engineering, South Dakota Mines, Rapid City, SD
Developing a Neonatal Hyaluronan-Enriched Nanofibrillar Scaffold to Modulate Adult Fibroblast Activity

Whitney A. Ponwith, Tugba Ozdemir Nanoscience and Biomedical Engineering, South Dakota Mines, Rapid City, SD

Hyaluronan (HA) helps skin stay hydrated, elastic, and capable of repair. Over time, its levels decline in the both the dermis and epidermis, reducing regeneration potential and altering the extracellular matrix (ECM). While the mechanisms leading to loss of HA are unknown, changes in fibroblast activity are thought to play major roles in HA composition and architecture in aging skin. Harvesting neonatal fibroblast derived HA offers potential benefits anti-aging products. This study developed synthetic nanofibrillar membranes functionalized with Hyaluronan Binding Peptides (HABP) to promote HA retention and ECM assembly. We hypothesize that HABP functionalized nanofibers can harvest neonatal HA through fibroblast culture and matrix decellularization to create a neonatal HA rich matrix that alters adult fibroblast activity. The goal of this study is to engineer regenerative scaffolds for reversing aging.
Preliminary Surface Characterization: Initial studies on 2D silica glass surfaces coated with HABP or scrambled HABP (scrHABP) evaluated HA retention and fibroblast behavior. Nanofiber Synthesis and Functionalization: Biodegradable nanofibers were fabricated using coaxial electrospinning with a poly(lactic-glycolic acid) (PLGA, 50:50) core of varying molecular weights (10–14, 30–50, > 100k Da) and a poly(caprolactone) (PCL) shell. Polyblends of PLGA (Low MW: 7000–17,000 Da, High MW: 54,000–64,000 Da) and PCL (MW=80,000 Da) were functionalized with HABP after electrospinning. PCL:PLGA ratios (100:0, 60:40, 50:50, 30:70, and 0:100), were tuned to degrade within a month, aligning with adult wound healing.
Biological Characterization: After 7 days of neonatal fibroblast culture, the nanofibers will be decellularized using mild detergent followed by extensive washing with phosphate buffer saline. The deposited ECM from neonatal and adult fibroblasts will be characterized for HA, Type I collagen, and Type III collagen, and tenascin-C. The resulting neonatal HA-enriched nanofibers will be seeded with adult dermal fibroblasts. The alpha smooth muscle actin expression will be assessed using immunofluorescence imaging.
To verify the efficacy of HABP, flat glass substrates were functionalized with HABP and adult and neonatal fibroblasts were seeded and their behavior and morphology was analyzed before producing nanofibers. Preliminary work demonstrated enhanced HA retention compared to APTMS controls (p < 0.0001, n = 3) via Alcian Blue staining. NHDFs cultured on HABP coated surfaces displayed larger cell areas (p < 0.0001, n = 3) and reduced circularity (p < 0.01, n = 3 for NHDFS, p < 0.0001, n = 3 for AHDFs), suggesting enhanced cell spreading.
HABP coated surfaces significantly enhance HA retention and influence fibroblast behavior, promoting a more proliferative and regenerative environment. These findings support the potential of HABP functionalized nanofibers as advanced scaffolds for regenerative and anti-aging applications.

L2.06

miR-29a Mediated Dysregulation of Collagen Synthesis and Extracellular Matrix Remodeling in Pressure Ulcers
Efua H. Bolouvi*, Maria Emilia Mora, Anisha Apte, Jimena Canchis Angulo, Jacob Diermayr, YONA KLEINERMAN, Stacy Skopp, Alyssa San Agustin, Kenneth W. Liechty, Carlos Zgheib
Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Arizona Tucson School of Medicine, Banner Children’s at Diamond Children’s Medical Center, Tucson, AZ
miR-29a Mediated Dysregulation of Collagen Synthesis and Extracellular Matrix Remodeling in Pressure Ulcers

Efua H. Bolouvi, Maria Emilia Mora, Anisha Apte, Jimena Canchis Angulo, Jacob Diermayr, Yona Kleinerman, Stacy Skopp, Alyssa San Agustin, Kenneth W. Liechty, Carlos Zgheib Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Arizona Tucson School of Medicine, Banner Children’s at Diamond Children’s Medical Center, Tucson, AZ

Pressure ulcers (PUs) are chronic wounds that contribute significantly to patient morbidity and impose a substantial burden on healthcare systems. These wounds are characterized by disruptions in tissue architecture, notably within the extracellular matrix (ECM), and a reduction in collagen content, an essential component that provides structural integrity and regulates cellular functions critical for tissue repair. Collagen plays a pivotal role in maintaining tissue stability and facilitating wound closure. Evidence suggests that microRNA-29a (miR-29a) is a key regulator of ECM remodeling and collagen synthesis. We hypothesize that in PUs, impaired miR-29a expression disrupts collagen synthesis, contributing to the pathogenesis of these chronic wounds. A murine model of pressure ulcers was generated using cyclic ischemia (120 minutes) and reperfusion (60 minutes) for 10 cycles to mimic human pathology. Skin tissues were harvested from the pressure injury mice and non-injured mice were used as controls. Gene expressions of miR-29a, Col1a2 (Type I collagen), and Col3a1 (Type III collagen) were analyzed using quantitative polymerase chain reaction. Histological and immunohistochemical analyses were performed to assess collagen deposition and ECM integrity. Unpaired student T-test was used for analysis with a p-value threshold of < 0.05 for statistical significance.
The level of miR-29a expression was significantly upregulated (p = 0.0014) in the pressure injury skin compared to uninjured skin. Histological analysis revealed fragmented and sparse collagen deposition in the injured tissues compared to the controls. This disrupted ECM remodeling weakened tissue structure, impairing the healing process with a reduction in Col3a1 (p = 0.0001) and Col1a2 (p < 0.0001) gene expression.
This study shows that elevated expression of miR-29a, which is associated with suppressed collagen gene expression, could be the main driver of ECM dysregulation seen in pressure ulcers. These findings point to miR-29a as a pivotal mediator in chronic wound pathology. Targeting miR-29a offers a potential therapeutic strategy to restore ECM integrity and enhance healing in pressure ulcers.

L3.01

Radiation triggers a clinically relevant pro-fibrotic p53-mediated DNA damage response in an ex vivo human skin model
Caroline Dodson*1,3, Sophie M. Bilik1, Gabrielle DiBartolomeo1, Hannah Pachalis1, Seth Thaller2, Irena Pastar1, Marjana Tomic-Canic1,3, Anthony Griswold3, Rivka C. Stone1,3
1Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2Plastic, Aesthetic and Reconstructive Surgery, University of Miami Miller School of Medicine, Miami, FL; 3Human Genetics and Genomics, University of Miami Miller School of Medicine, Miami, FL
Radiation Triggers a Clinically Relevant Pro-Fibrotic p53-Mediated DNA Damage Response in an Ex Vivo Human Skin Model

Caroline Dodson1,3, Sophie M. Bilik1, Gabrielle DiBartolomeo1, Hannah Pachalis1, Seth Thaller2, Irena Pastar1, Marjana Tomic-Canic1,3, Anthony Griswold3, Rivka C. Stone1,3 1Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL; 2Plastic, Aesthetic and Reconstructive Surgery, University of Miami Miller School of Medicine, Miami, FL; 3Human Genetics and Genomics, University of Miami Miller School of Medicine, Miami, FL

Radiation-induced skin fibrosis (RISF) is a severe complication of radiation exposure, leading to functional impairment and reduced quality of life for patients. Radiation induces a chronic inflammatory state that promotes fibrosis through DNA damage, production of reactive oxygen species and activation of inflammatory pathways. To investigate the cutaneous response to radiation, we developed a human ex vivo model of RISF using skin from surgical procedures. Having previously confirmed a pro-fibrotic tissue response in this model, we further explored the mechanistic basis for RISF.
Skin was irradiated with 3.5–6 Gy, maintained at an air-liquid interface, and collected over a 15-day period. RNA-sequencing was performed in paired samples of irradiated and control skin from four human donors across 8 timepoints. Key findings were validated using qPCR and immunostaining, and were explored in skin transcriptome datasets from breast cancer patients post-irradiation.
Immunofluorescence of irradiated skin displayed robust phosphorylated gamma-H2AX staining, reflecting extensive DNA double-strand breaks. Ingenuity Pathway Analysis of transcriptomic profiles highlighted TP53 as a key upstream regulator of the radiation-triggered DNA damage response (DDR) (z-score = 4.328, p = 1.81E-45), with activation of p53 signaling as early as 24 hours post-radiation (z-score = 2.309, p = 5.63E-07) that was sustained over 15 days. QPCR validated the increased expression of p53 target genes CDKN1A, TRIAP1, NOTCH1 and SPATA18 in a dose-response manner in an expanded pool of 6 donors following radiation. Protein levels of the p53 target and regulator MDM2 increased in irradiated skin compared with controls, reflecting auto-regulation of the p53 response. Interestingly, transcriptomic profiles of irradiated skin from breast cancer patients also displayed activated TP53 (p = 5.64E-04) and upregulated MDM2 (p = 6.56E-03) when compared with non-irradiated skin from the contralateral breast, supporting the clinical-translational relevance of the p53-DDR mechanism.
Taken together, this ex vivo human skin model captures a pro-fibrotic p53-mediated DDR following radiation injury in a dose-dependent manner that mirrors the clinical cutaneous radiation response. As pathologically sustained p53 signaling is a known driver of tissue fibrosis, further mechanistic exploration of the p53-DDR using this model can pave the way for targeting the early molecular drivers of RISF to improve patient quality of life following radiotherapy.

L3.02

Diabetes Increases Fibrosis in Response to Liver Injury in Mice
Syed Maisam Jafri*, Maria Gracia Mora Pinos, Katharina S. Fischer, Abdelrahman M. Alsharif, Fidel Saenz, Hudson C. Kussie, Dharshan Sivaraj, Andrew Hostler, William Hahn, Filiberto Quintero, Maia Granoski, Eamonn McKenna, Kenneth W. Liechty, Kellen Chen, Geoffrey C. Gurtner
Surgery, University of Arizona, Tucson, AZ
Diabetes Increases Fibrosis in Response to Liver Injury in Mice

Syed Maisam Jafri, Maria Gracia Mora Pinos, Katharina S. Fischer, Abdelrahman M. Alsharif, Fidel Saenz, Hudson C. Kussie, Dharshan Sivaraj, Andrew Hostler, William Hahn, Filiberto Quintero, Maia Granoski, Eamonn McKenna, Kenneth W. Liechty, Kellen Chen, Geoffrey C. Gurtner Surgery, University of Arizona, Tucson, AZ

Chronic and fibrotic liver diseases affect 1.5 billion individuals worldwide. Current liver injury and fibrosis murine models are pharmacologic based and thus non-physiologic. A case study reported that Extracorporeal Shock Wave Lithotripsy (ESWL), a treatment for biliary and renal calculi, induced significant liver fibrosis in a female patient, suggesting tissue injury from shockwave exposure. ESWL utilizes cavitation to cause energetic microbubble collapse, with ultrasound contrast microbubble administration enhancing the tissue damage. Our study explored ESWL, with and without ultrasound contrast, as a potential non-pharmacological method for inducing liver fibrosis in mice to provide a novel platform for studying liver wound healing and fibrosis.
Wildtype (WT) and diabetic (DB) murine livers were subjected to ESWL using a Dornier SIGMA lithotripter. Mice were anesthetized with isoflurane and received 250 shockwaves at minimal intensity (1/6), delivered at a rate of 60 shocks per minute, with lens pressure set to level 4. OPTISON ultrasound contrast microbubbles were administered via tail vein. Liver tissue was collected immediately (day 0) and on day 21 post-ESWL for histological, pathological, and molecular analysis. Picrosirius Red, Thrombospondin-1 (THBS1), and F4/80 staining and imaging were used to evaluate collagen changes, fibrotic markers, and immune response.
ESWL induced significant liver fibrosis in 3 WT and 3 DB mice as evidenced by hemorrhage, hepatocyte ballooning, and collagen deposition. In response to ESWL without contrast, WT mice exhibited 37.6% liver damage at day 0, which increased to 73.6% by day 21 (p < 0.0001). In WT mice treated with ESWL + contrast, liver injury increased from 53.6% at day 0 to 95.1% by day 21 (p < 0.0001). WT THBS1 levels were upregulated by day 21, indicating activation of fibrotic pathways (p < 0.0001). DB mice treated with ESWL + contrast demonstrated 86.3% tissue damage at day 0, which increased to 97.9% by day 21 (p < 0.0001). F4/80 immunostaining showed macrophage recruitment increased from 17% at day 0 to 41.2% at day 21 in DB mice treated with ESWL + contrast (p < 0.01). Picrosirius Red staining showed collagen fiber elongation and thickening by day 21 in WT and DB (p < 0.01). ESWL generated stronger fibrotic responses than those observed in validated liver fibrosis models using injection of thioacetamide or CCl4. Overall, ESWL effectively induces liver fibrosis in mice, with contrast microbubbles significantly enhancing fibrogenic outcomes. This model provides a valuable non-pharmacologic platform to study the formation of liver fibrosis over time. Using this model, we observed that diabetic mice developed liver fibrosis with a stronger inflammatory reaction compared to wildtype mice. Future work should be performed to validate this model against human fibrosis and interrogate the importance of chronic, persistent inflammation on the temporal development of diabetic versus non-diabetic fibrosis.

L3.03

INNOVATIVE PEPTIDE-BASED TECHNOLOGY FACILITATES RAPID CLOSURE AND PROVIDES ANTIBACTERIAL PROTECTION OF SURGICAL INCISIONS
Brunno Caetano*, Trudy-Ann Grant, Bishnu P. Joshi, Nivedha Suresh, Rebecca Salamone, Tarak Bakhda, Manav Mehta, Ana Tellechea
Gel4Med, Lowel, MA
Innovative Peptide-Based Technology Facilitates Rapid Closure and Provides Antibacterial Protection of Surgical Incisions

Brunno Caetano, Trudy-Ann Grant, Bishnu P. Joshi, Nivedha Suresh, Rebecca Salamone, Tarak Bakhda, Manav Mehta, Ana Tellechea Gel4Med, Lowel, MA

Surgical wound complications, such as surgical wound dehiscence (SWD), represent a leading cause of morbidity following surgery. Prone to occur in high-risk patients, SWD involves the separation of the margins of a closed incision, resulting in increased mortality, prolonged hospital stays, and excess healthcare costs. To address this challenge, we developed a synthetic peptide-based flowable matrix (PFM) specifically designed for surgical wounds to control bioburden and simultaneously promote wound healing. Mammalian cell viability, cell attachment, and cell spreading were evaluated after 24–72 h of exposure to PFM, iodine or silver wound dressings, or a hydrolyzed collagen wound product. Efficacy against Gram-positive and Gram-negative bacteria was assessed using time-kill assays. In vitro, efficacy against 72 h-aged Pseudomonas aeruginosa (PAO1) biofilms was compared to marketed antimicrobial wound products. PFM wound healing efficacy was tested in a swine model of Methicillin-resistant Staphylococcus aureus (MRSA)-inoculated dorsal full-thickness incisional wounds. After the bacterial inoculation period, incisions were treated with PFM or an antibiotic-infused collagen surgical product, followed by incision closure. On days 3, 7, and 21 post-incision, sites were excised for histopathological evaluation. In vitro studies demonstrated PFM’s higher cell viability by > 2.5 orders of magnitude (p < 0.0001, n = 3) compared to hydrolyzed collagen, iodine, and silver wound products. Superior cell attachment and cell spreading were also observed. PFM exhibited bactericidal efficacy against ≥ 6 log10 CFU of PAO1 and MRSA at 5, 10, 15, 30, and 60 min, and after 24 hours (p < 0.0001, n = 3). PFM showed superior efficacy against mature PAO1 biofilms when compared to marketed antimicrobial dressings (p < 0.0001, n = 6). In vivo, PFM achieved incisional wound healing with full re-epithelialization, no signs of infection, no bacteria present, and total material resorption with complete tissue apposition by 7 days (n = 5). In contrast, sites treated with the collagen-based surgical product showed exuberant incision lines and presence of residual material, resulting in incomplete tissue apposition on post-operative day 7 (n = 5). On day 21, wounds treated with PFM remained fully closed, with minimal inflammation along the incisional line and complete tissue apposition (n = 5).
PFM demonstrates superior cell compatibility, as well as antibacterial and antibiofilm efficacy compared to hydrolyzed collagen and antimicrobial surgical wound products. In a swine model of infected incisional wounds, PFM resulted in rapid tissue integration, complete tissue apposition, and full reepithelization within 7 days. Together, the data supports PFM’s potential to better manage surgical incisions and prevent surgical wound complications. Clinical studies are needed to validate these findings.

L3.04

DEGRADATION OF VERSICAN IN KELOIDS.
Noriko Aramaki*, Keisuke Okabe, Kazuo Kishi
Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
Degradation of Versican in Keloids

Noriko Aramaki, Keisuke Okabe, Kazuo Kishi Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan

Keloids are known to cause prolonged inflammation and excessive production and accumulation of extracellular matrix. Versican, a large chondroitin-sulphate proteoglycan is degraded at the specific sites by Adamts (a disintegrin and metalloproteinase with thrombospondin motifs) and produce the degradation product called versikine. Although there have been reports of increased production of versican in keloids, there have been no reports on how its degradation and degradation products are involved in the pathogenesis of keloids. Therefore, we investigated the function of versican degradation and its degradation product, versikine, in keloids.
A total of 12 keloid specimens from 10 cases and 3 hypertrhophic scars specimens were collected. RNA was extracted from some of the tissues and the expression of Adamts-4,5 was measured by real-time PCR. In addition, experiments were conducted using fibroblasts derived from normal skin and keloid tissue to investigate the effect of versikine for fibroblasts. Moreover, proteins were extracted from keloid tissue and blot-overlay analysis was performed to determine whether versikine in keloids binds to SHAP (serum-derived hyaluronic acid-associated protein) and hyaluronic acid (HA) to form a complex.
Immunohistochemical staining revealed strong expression of versikine in keloids, but hardly any versikine was noted in the surrounding normal tissue and hypertrophic scar samples. Expression of Adamts-4,5 was significantly elevated in the keloids compared to areas around the keloids, normal skin, or hypertrophic scars. In addition, when keloids were compared to surrounding normal skin from the same sample, Adamts-4,5 expression was significantly elevated in the keloids from all 12 samples. Additional experiments on fibroblasts derived from normal skin and keloid tissue showed proliferative ability increased significantly when versikine was added. The blot-overlay analysis revealed that in keloid versikine bound to SHAP- HA to form a complex.
Our data suggest that versican production and degradation are upregulated in keloid tissue and that versikine promotes cell proliferation in both keloid and normal fibroblasts.
Versikine-SHAP-HA complexes have been reported to induce inflammation. Excess versikine in keloids may contribute to prolonged inflammation by binding to SHAP-hyaluronan and forming a complex. It is suggested that controlling the degradation of versican may lead to the treatment of keloids.

L3.05

PKM2 and Hsp27 Phosphorylation in Response to Mechanical Signaling Regulate Energy Metabolism and Fibrotic Responses in Dermal Fibroblasts
Jaime Tellez*, Mary Elizabeth Guerra, Pranav Bommekal, Casey Baxter, Nabila N. Anika, Navya Nanda, Sonya S. Keswani, Zara Chandrashekar, Hui Li, Ling Yu, Sundeep Keswani, Swathi Balaji
Pediatric Surgery, Baylor College of Medicine, Houston, TX
PKM2 and Hsp27 Phosphorylation in Response to Mechanical Signaling Regulate Energy Metabolism and Fibrotic Responses in Dermal Fibroblasts

Jaime Tellez, Mary Elizabeth Guerra, Pranav Bommekal, Casey Baxter, Nabila N. Anika, Navya Nanda, Sonya S. Keswani, Zara Chandrashekar, Hui Li, Ling Yu, Sundeep Keswani, Swathi Balaji Pediatric Surgery, Baylor College of Medicine, Houston, TX

Fibroblasts (FB) respond to changes in mechanical forces in the wound milieu by reorganizing the actin cytoskeleton. Heat shock protein (Hsp27) phosphorylation is known to govern actin dynamics and contractility. This is fueled by oxidative phosphorylation and glycolysis that regulate lactate and ATP. Pyruvate kinase (PK) is the rate-limiting enzyme in glycolysis. Recent cancer studies showed substrate stiffness increases glycolysis in epithelial cells via architectural actomyosin cytoskeleton, which couples cell metabolism to the mechanical properties of the surrounding tissue. However, the role of PKM1/M2 isoforms and Hsp27 in fibroblast responses during wound healing is unknown. We hypothesize that fibroblasts regulate their metabolism and fibrotic phenotype in response to mechanical signaling via phosphorylation changes in PKM2 and Hsp27-actin cytoskeleton dynamics. FB were isolated from 8wk C57BL/6J mice, then cultured on either flexible silicone membranes (flex – 930 kPa Young’s modulus), or tissue culture plastic dishes (TCP – 1107 KPa) with ± TGFB (10 ng/ml, 24 h). Total and phosphorylated PKM1/M2 and Hsp27 were analyzed using immunoblotting. Hsp27 intracellular expression and alignment with actin were assessed by immunofluorescence. Lactate, ADP/ATP balance, and a-SMA (qRT-pcr; immunoblotting) were analyzed. Lastly, the effects of TEPP (100uM) that regulates p-PKM2 and glycolysis were tested. n = 3/group, p values by ANOVA.
Total PKM2 (3.2 fold) was higher, and total Hsp27 (1.5 fold) was lower in cells on TCP compared to flex membranes, along with higher p-PKM2 (1.3 fold) and p-Hsp27 (2 fold). Hsp27 staining was more perinuclear in cells on flex and co-localized with a-SMA in cells on TCP. Higher PKM2 and a ratio of lower total Hsp27 and higher p-Hsp27 in TCP is conducive to fibrosis. TGFB treatment led to an increase in both total PKM2 (5 fold) and p-PKM2 (1.5 fold) dimerization in cells on TCP compared to flex, but had little to no effect on p-PKM2 in cells on flex.
Interestingly, p-Hsp27 was more induced by TGFB in cells on flex membrane than on TCP (6 vs. 3 fold). TGFB induced more lactate and reduced ADP/ATP ratio in cells on TCP compared to flex. While TEPP treatment was effective in reducing the effect of TGFB on lactate similarly in both conditions, it was ineffective in correcting ADP/ATP ratio in cells on TCP and only effectively increased the ratio in cells on flex membrane. Lastly, the cells on flex membrane had reduced a-SMA than those on TCP, and TGFB induced more a-SMA in cells on flex than on TCP (2 vs. 1.5 fold). TEPP treatment reduced a-SMA in cells on flex but not TCP.
Our data shows that fibroblasts display distinct PKM2 and Hsp27 activation in different microenvironmental stiffness conditions, with implications for energy metabolism, lactate, ADP/ATP, and a-SMA expression. They further indicate that fibroblasts are more amenable to treatment with TEPP to regulate glycolysis and fibrosis markers in less stiff environments.

L3.06

Role of IL-2 on Tr1 Activation and Fibroblast Function in Dermal Wound Healing
Casey Baxter*1, Navya Nanda1, Sonya Keswani1, Anjali Degala1, Pranav Bommekal1, Jaime Tellez1, Hui Li1, Ling Yu1, Mary Elizabeth Guerra1, Paul Bollyky2, Sundeep G. Keswani1, Swathi Balaji1
1Pediatric Surgery, Baylor College of Medicine, Houston, TX; 2Immunology, Stanford University School of Medicine, Palo Alto, CO
Role of IL-2 on Tr1 Activation and Fibroblast Function in Dermal Wound Healing

Casey Baxter1, Navya Nanda1, Sonya Keswani1, Anjali Degala1, Pranav Bommekal1, Jaime Tellez1, Hui Li1, Ling Yu1, Mary Elizabeth Guerra1, Paul Bollyky2, Sundeep G. Keswani1, Swathi Balaji1 1Pediatric Surgery, Baylor College of Medicine, Houston, TX; 2Immunology, Stanford University School of Medicine, Palo Alto, CO

We have shown that regulatory Tr1 lymphocytes produce high levels of interleukin-10 (IL-10) and promote regenerative dermal wound repair. The activation and function of Tr1 is influenced by IL-2. Tr1 cells both endogenously express low levels of IL-2 as well as sequester it from the ECM. The dose-dependent effects of IL-2 and IL-10 on Tr1 cells and dermal fibroblasts, the primary effectors of fibrosis and scarring, are not fully understood. We hypothesize that IL-2 regulates Tr1 activation, proliferation, and IL-10 production in a dose-dependent manner, directly influencing fibroblast fibrotic phenotype and ECM production.
CD4+ T lymphocytes were enriched from splenocytes of C57BL/6J mice. Tr1 cells (CD4+ CD44+ CD25+ FOXP3-) were sorted and activated in vitro with 1:1 cell to CD3/CD28 Dynabeads and treated with IL-2 at 0, 25, 50, 100, 200, and 400 U/mL on days 0, 3 and 6 of culture. Proliferation, IL-2 and IL-2 receptor gene expression (qRT-PCR), and IL-10 production (ELISA) were analyzed on day 6. Dermal fibroblasts from C57BL/6J mice (mDFb) were treated with either IL-2 doses or with conditioned media (CM) from mouse Tr1, Tnaive, or regulatory FOXP3+ Treg for 24 hours and assessed for gene expression of fibrosis-associated markers, Col1a1, Col3a1, TGFb1, and CTGF. n = 3/group, p values by t-test.
Tr1 cells exhibited proliferation and IL-10 production without exogenous IL-2 addition, indicating an intrinsic IL-2 production, which was confirmed by ELISA. We observed IL-2 dose-dependent Tr1 responses, including highest proliferation noted at 400 U/mL IL-2 on day 6, while IL-10 production significantly peaked at 200 U/mL IL-2 (p < 0.05) and decreased at 400 U/mL. Similarly, IL-2 receptor gene expression increased at 200 U/mL with a decrease at 400 U/mL. mDFb treated with doses of IL-2 did not show significant differences in fibrosis gene expression. However, Tr1 CM-treated mDFb showed an increased Col3a1/Col1a1 ratio (a pattern associated with a pro-regenerative phenotype) compared to mDFb treated with Tnaive CM. This effect was maintained even with the addition of IL-10 neutralizing antibody, suggesting a role for additional factors in mediating this response. IL-6 gene expression, associated with an inflammatory phenotype, was increased in mDFb treated with Tr1 CM and Tnaive CM. Further analysis showed high levels of TGF-b in CM from both Tr1 and Tnaive, possibly driving this effect.
These findings show a dose-dependent effect of IL-2 on Tr1 cell proliferation and IL-10 production. Tr1 CM-treated mDFb displayed a pro-healing and pro-regenerative phenotype, even in the absence of IL-10, suggesting additional pathways in which Tr1 cells influence fibroblast phenotype besides IL-10 mediated pathways. Our findings underscore the potential of our research in developing novel Tr1 cell based therapeutic strategies for regenerative wound healing, offering exciting possibilities for wound healing and inflammatory condition treatments.

L4.01

EFFICACY OF MICROPOROUS ANNEALED PARTICLES IN ABDOMINAL WALL RECONSTRUCTION IN A RABBIT MODEL
Stephanie Deshayes*1, Donald Griffin1,2, Daria Andriyanova1, Mitchell McMahon1, Westbrook Weaver1
1Tempo Therapeutics, San Diego, CA; 2University of Virginia, Charlotsville, VA
Efficacy of Microporous Annealed Particles in Abdominal Wall Reconstruction in a Rabbit Model

Stephanie Deshayes1, Donald Griffin1,2, Daria Andriyanova1, Mitchell McMahon1, Westbrook Weaver1 1Tempo Therapeutics, San Diego, CA; 2University of Virginia, Charlotsville, VA

Midline laparotomies are used in millions of patients every year including gastro-intestinal cancer procedures, hysterectomies and other ovarian cancer procedures, spinal fusions, and abdominal trauma surgeries. Currently, patients with certain comorbidities (e.g., diabetes, Body Mass Index (BMI) > 25, or are undergoing primary tumor resections) experience suture failure and hernia formation at the site of surgery at a rate of 30% within a 24-month window after surgery. These events cause major complications and even death in some patients. Surgical meshes, a commercial alternative or addition to sutures, has not been successful in reducing these rates of failure.
The purpose of this study was to evaluate the efficacy of the MAP Surgical Matrix, a novel biomaterial, to augment standard suture closure in a preclinical model. Specifically, to evaluate the ability of the biomaterial to strengthen the healed tissue at the site of incision.
An abdominal wall incision ( 5 cm) was created on the linea alba of New Zealand White rabbits (n = 7 per group) and closed using Prolene sutures. MAP Surgical Matrix was then applied directly over the completely closed incision line. After test article application, the subcutaneous tissue and intradermal skin were closed over the abdominal wall incision. 14, 28 and 42 days after treatment, the abdominal wall was collected, and wound healing and tissue strength were assessed through histological analysis and tensiometry, respectively. MAP Surgical Matrix was compared to sutures alone (control). Collected tissue was also analyzed by histological analysis (H&E and Picrosirius red).
MAP Surgical Matrix demonstrated superior wound healing outcomes compared to the control group. Specficallly, the material produced stronger abdominal midline incisions when used as adjunct to suture closure—40% increase in recovery strength was observed for MAP Surgical Matrix compared to sutures alone 42 days after treament. This improvement was corroborated by histological observation of additional collagen deposition within and over the scaffold covering the suture line. Lastly, tissue strength continuously increased overtime for MAP Surgical Matrix, while sutures alone plateaued at 42 days. By histology, complete cellular infiltration and mature vascularization were observed within MAP with minimal inflammation, and the majority of the scaffold was degraded by 42 days. Importantly, there was no observable signs of scaffold-initiated inflammation.
These results suggest that MAP Surgical Matrix has the potential to improve abdominal wall repair and reduce the incidence of incisional hernias, offering a promising approach for surgical wound management. Importantly, the scaffold’s ability to evade the foreign body response will minimize the risk of implant-mediated inflammation which is currently a major concern for surgical meshes.

L4.02

Lignin Composites Rescue Hif-1? and VEGF Levels to Improve Diabetic Wound Neovascularization
Mary Elizabeth Guerra*1,2, Oluyinka Olutoye1,2, Lane Yutzy3, Jaime Tellez1,2, Casey Baxter1,2, Hui Li1,2, Ling Yu1,2, Jangwook Jung3, Swathi Balaji1,2
1Baylor College of Medicine, Houston, TX; 2Texas Children’s Hospital, Houston, TX; 3Louisiana State University, Baton Rouge, LA
Lignin Composites Rescue Hif-1α and VEGF Levels to Improve Diabetic Wound Neovascularization

Mary Elizabeth Guerra1,2, Oluyinka Olutoye1,2, Lane Yutzy3, Jaime Tellez1,2, Casey Baxter1,2, Hui Li1,2, Ling Yu1,2, Jangwook Jung3, Swathi Balaji1,2 1Baylor College of Medicine, Houston, TX; 2Texas Children’s Hospital, Houston, TX; 3Louisiana State University, Baton Rouge, LA

Impaired neovascularization underlies the diabetic phenotype of delayed wound healing. Engineered biomaterials that can alleviate pericellular ischemia and reduce excessive reactive oxygen species (ROS) which impede healing can promote diabetic wound resolution. We engineered novel lignin (a natural antioxidant from lignocellulose)-based composites with antimicrobial, ROS-scavenging, and sustained oxygen-releasing properties. We hypothesize that they enhance neovascularization and attenuate inflammation and ROS to promote diabetic wound healing.
The effect of lignin composites on accelerating angiogenesis in high glucose conditions was tested. Groups included untreated control, gelatin methacrylate (GelMA), GelMA + thiolated lignosulfonate-antioxidant-nanoparticles (TLS), GelMA + TLS + calcium peroxide-based O2-generating nanoparticles (CPO). Capillary network formation of microvascular endothelial cells (MVECs) on lignin composites was measured using Angiogenesis Analyzer and bulk RNA sequencing was conducted. GSEA and Weighted Gene Co-expression Network Analysis (WGCNA) were used to perform correlation network analysis. Hif-1α and VEGF expression were analyzed. Excisional 6 mm wounds in db/db (type II diabetic) mice were treated with lignin composites and examined for wound healing, angiogenesis, VEGF and Hif-1α expression, and inflammation at D7. 10 Visium spatial transcriptomics was performed on D7 wounds and analyzed using R and Seurat workflows. n = 3/treatment, p values by ANOVA, Tukey’s post hoc tests.
MVECs cultured on CPO composites with antimicrobial, antioxidant, and O2 release showed increased capillary network formation and rescued Hif-1α and VEGF expression under high glucose conditions. The co-expression profiles of associated genes analyzed using unsupervised learning revealed distinct patterns, and a module containing the highest number of genes, 5073, was selected for further investigation. CPO composites facilitated the expression of Hif-1a in the pool of top 25 hub genes. In enrichment analysis, VEGF-associated pathways were significantly upregulated. In full thickness db/db skin wounds, CPO lignin composites promoted faster wound healing, granulation tissue deposition, and CD31+ capillary lumen density at D7 (p < 0.05). CPO-treated wounds had an increase in VEGF and decrease in Hif-1a expression, with a unique staining pattern noted in encroaching epidermis, suggesting angiogenesis is promoted by CPO lignin composites in diabetic hypoxic wounds. CPO wounds had increased CD206+ arginase+ macrophages. 10 Visium data indicate shifts in fibroblast and macrophage subtypes as well as altered geospatial distributions and associations among these cell types in wounds treated with CPO.
Engineered lignin composites with antimicrobial, antioxidation and oxygen-producing capacities enhance neovascularization and improve diabetic wound healing, representing new frontiers in improving diabetic wound healing by engineered biomaterials.

L4.03

TISSUE NANOTRANSFECTION GENE DELIVERY ENABLES IN VIVO SKIN REPROGRAMMING BY PRESERVING MITOCHONDRIAL BIOENERGETICS AND CYTOSKELETAL INTEGRITY
Aparajita Nandy*, Anu Sharma, Mohini Moulick, Anita Yadav, Subhadip Ghatak, Chandan K. Sen, Yi Xuan
Department of surgery, Mc Gowan Institute of Regenerative Medicine, Pittsburgh, PA
Tissue Nanotransfection Gene Delivery Enables in Vivo Skin Reprogramming by Preserving Mitochondrial Bioenergetics and Cytoskeletal Integrity

Aparajita Nandy, Anu Sharma, Mohini Moulick, Anita Yadav, Subhadip Ghatak, Chandan K. Sen, Yi Xuan Department of surgery, Mc Gowan Institute of Regenerative Medicine, Pittsburgh, PA

Tissue reprograming relies on tissue bioenergetics and preservation of cytoskeletal structures that conserve inherent cellular plasticity. Non-viral gene transfer circumvents the known risks of viral gene transfer. Among, non-viral gene transfer options, tissue nanotransfection (TNT) relies on electromotive forces and the establishment of nanopores to deliver reprogramming factors. This work tests TNT versus widely known bulk electroporation (BEP) and liposome-based delivery (LBD; Invivofectamine™) for their suitability in achieving skin reprograming.
TNT, BEP and LBD non-viral gene delivery approaches were employed to deliver expression plasmids (0.05 μg/μL) into epidermal keratinocytes. For both BEP and TNT, square-wave electric pulses were applied at 100 V with an interval of 0.1 s over 10 pulses. The efficiency of gene delivery to keratinocytes was evaluated using laser capture microdissection to isolate the epidermis, followed by qRT-PCR to measure the abundance of the delivered gene. Propagation of the delivered gene was further evaluated by measuring its abundance in keratinocyte-derived exosomes (Exok), and statistically correlated to cellular bioenergetics. Mitochondrial function was analyzed using Seahorse XF Analyzer to evaluate changes in metabolic activity. Cytoskeletal scarring and integrity was visualized using confocal microscopy.
Compared to LBD and BEP, TNT achieved the highest gene delivery efficiency (P < 0.001; n = 6), and increased encapsulation of delivered gene in Exok. Mitochondrial functional analysis revealed marked improvements in basal respiration, and ATP production (P < 0.001; n = 15) following TNT compared to resting cells. BEP and LBD both suppressed mitochondrial bioenergetics to a point lower than that exhibited by resting cells and associated with significantly higher levels of PINK1, PARKIN, and optineurin in keratinocytes (P < 0.05; n = 4), pointing towards stress-induced mitophagy that was not observed in response to TNT. Additionally, live/dead cell assays showed predominance of necrotic cells in BEP-treated group, further underscoring the superior biocompatibility of TNT. Super resolution airy scan microscopy and dSTORM nanoimaging revealed that TNT preserved cytoskeletal integrity with minimal scarring, unlike LBD and BEP that caused moderate and severe disruptions, respectively, as evidenced by actin filament organization and significantly lower levels of stress-induced cytoskeletal markers such as phosphorylated Myosin Light Chain and Cofilin (P < 0.01; n = 4).
Gene delivery methods often cause cytotoxicity in cells but successfully introduce genes into those that survive. However, in these cells gene delivery approach may adversely influence mitochondrial bioenergetics. TNT outperformed BEP and LBD by achieving superior gene delivery, preserving mitochondrial bioenergetics, and minimizing cytoskeletal disruption, making it a scalable option for regenerative therapies.

L4.04

IN VIVO STUDY OF VERIFYING THE ENHANCING EFFECT TO EARLY PHASE WOUND HEALING OF INTERMITTENT COLD PLASMA TREATMENT COMPARED TO THE CONVENTIONAL DRESSING MATERIALS IN THE FULL-THICKNESS DEFECT PORCINE WOUND MODLELS
Hoon Kim*
Plastic and Reconstructive Surgery, Konyang University Hospital, Daejeon, Korea (the Republic of)
In Vivo Study Of Verifying The Enhancing Effect to Early Phase Wound Healing of Intermittent Cold Plasma Treatment Compared to the Conventional Dressing Materials in the Full-Thickness Defect Porcine Wound Modlels

Hoon Kim Plastic and Reconstructive Surgery, Konyang University Hospital, Daejeon, Korea (the Republic of)

Cold plasma has been reported to have beneficial effect on wound healing with generating reactive oxygen and nitrogen species as well as redox signaling, although its exact mechanism is mostly unknown. The aim of this study is to compare the accelerating wound healing effect of cold plasma with the conventional dressing materials of gauze and foam in vivo. Total of 32 wounds in 16 guinea pigs were created with full-thickness skin defect of 2.5 2.5 cm. Each wound was on both side of the back, 2.5 cm from the mid-supine line. 8 wounds of each 4 groups were treated with gauze dressing group (G1), foam dressing group (G2), cold plasma (PRAMEDISON, SJ global, Korea) 3.5 kilovolt (kv) group (G3) and cold plasma 5.0 kv group (G4). All the dressings were changed every 48 h. Cold plasma was applied 2 times a day for 10 min with 6 h-interval. Wound dimension was measured every 2 days and histological examinations including hematoxylin and eosin (H&E) stain, Masson’s trichrome (MT) stain and immunohistochemical (IHC) studies including CD31 and ki-67 were performed on day 15 of sacrifice.
Cold plasma treated groups (G3 and G4) revealed significant decrease of wound area compared to G1 at day 9 (G3: p < 0.05, G4: p < 0.01) and G2 at days 3-15, except day 7 (p < 0.05 or p < 0.01), although complete wound healing was not achieved by 15 days in all four groups. Histological analysis displayed that cold plasma groups (G3 and G4) decreased defected lengths of epithelium and increased re-epithelialization and vessel numbers compared to G2, respectively. Immunohistochemical studies also revealed that cold plasma groups (G3 and G4) increased Ki-67 on epithelium and CD31 on granulation tissue compared to G1 and G2, respectively.
Intermittent cold plasma treatment facilitates accelerating wound healing with decreasing wound area and increasing proliferation of epithelial cells, angiogenesis, Ki-67 and CD31 on epithelial margin and granulation tissue compared to gauze or foam dressings in full-thickness skin defect porcine wound models.

L4.05

Metformin Attenuates UVB-Induced Skin Damage and Hyperpigmentation in an Ex Vivo Human Skin Perfusion Model
Aniekanabasi Ufot, Naresh Mahajan, Alexa Rivera del Rio Hernandez, José A. Arellano, Fuat Baris Bengur, Shawn Loder, Ethan Banks, Samantha Bosco, Francesco M. Egro, Jeffrey Gusenoff, J. Peter Rubin, Asim Ejaz
Plastic Surgery, University of Pittsburgh, Pittsburgh, PA
Metformin Attenuates UVB-Induced Skin Damage and Hyperpigmentation in an Ex Vivo Human Skin Perfusion Model

Aniekanabasi Ufot, Naresh Mahajan, Alexa Rivera del Rio Hernandez, Samantha Bosco, Francesco M. Egro, Jeffery Gusenoff, J. Peter Rubin, Asim Ejaz Plastic Surgery, University of Pittburgh, Pittsburgh, PA

Ultraviolet B (UVB) radiation is a significant environmental stressor that can cause DNA damage, inflammation, hyperpigmentation, and epidermal structural changes. Chronic UVB exposure leads to cumulative skin damage, contributing to photoaging and carcinogenesis, while acute high-dose exposure results in immediate epidermal injury and inflammation. Although melanin production is protective, it is often dysregulated, causing hyperpigmentation disorders therefore identifying effective therapeutic agents to mitigate UVB-induced skin damage is critical. Metformin, an antidiabetic drug with antioxidant, anti-inflammatory, and DNA repair properties, has emerged as a potential skin protectant. This study investigates metformin’s protective effects in an ex vivo human skin perfusion model exposed to acute and chronic UVB.
Full-thickness human skin flaps, obtained as surgical waste, were maintained on a perfusion system and exposed to UVB radiation. The chronic UVB group received 200 mJ/cm2 UVB daily for seven days, while the acute UVB group received a single high dose of 1400 mJ/cm2. Each group was subdivided into metformin-treated and non-metformin groups, with a non-UVB-exposed as control. Topical metformin was applied immediately after each UVB exposure in the treated groups. Skin biopsies were collected at 0, 24, 48, 72, 96, 120, and 144 h. Histological analysis was performed using Hematoxylin and Eosin (H&E) staining for epidermal and dermal integrity, Fontana-Masson for melanin deposition, and gamma-H2AX (γH2AX) for DNA double-strand breaks. Both acute and chronic UVB exposure induced significant epidermal thinning, separation of the dermo-epidermal junction, and increased melanin production. Fontana-Masson staining showed a marked increase in melanin deposition in both UVB groups, with the chronic exposure showing cumulative pigmentation over time. Metformin-treated skin demonstrated significantly reduced melanin levels in comparison. H&E staining showed widespread epidermal separation and structural disorganization in UVB groups, particularly at later time points (96–144 h). In contrast, metformin-treated skin exhibited improved epidermal integrity and reduced separation, suggesting enhanced tissue resilience. γH2AX staining revealed elevated DNA damage in the acute UVB group from 24 h post-exposure through 144 h, while chronic UVB exposure showed progressive DNA damage. Metformin-treated skin showed fewer γH2AX gamma-H2AX-positive cells, indicating protection against UVB-induced DNA strand breaks. Control biopsies remained intact with minimal pigmentation and DNA damage.
Metformin demonstrated a protective role in mitigating UVB-induced skin damage, hyperpigmentation, and genetic injury in an ex vivo skin perfusion model. Metformin offers a promising therapeutic strategy for preventing UV-induced skin disorders, and this model serves as a valuable platform for further research on UVB-induced damage and protective agents.

L4.06

MITOCHONDRIAL TRANSPLANTATION AS A STRATEGY TO ACCELERATE ACUTE WOUND HEALING .
Parmeshar singh*1, Fanjaniaina Andriamifidy Henintsoa2, Jacob Kazmi1, Daniel Grande2,3, Alisha Oropallo3,4, Lance Becker1,3, Amanda Beneat,1
1Department of Emergency Medicine, Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY; 2Department of Orthopaedic Research, Feinstein Institute for Medical Research,Northwell Health, Manhasset, NY; 3Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY; 4Department of Vascular Surgery, Comprehensive Wound Healing Center, Northwell Health, New Hyde Park, NY
Mitochondrial Transplantation as a Strategy to Accelerate Acute Wound Healing

Parmeshar singh1, Fanjaniaina Andriamifidy Henintsoa2, Jacob Kazmi1, Daniel Grande2,3, Alisha Oropallo3,4, Lance Becker1,3 1Department of Emergency Medicine, Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY; 2Department of Orthopaedic Research, Feinstein Institute for Medical Research,Northwell Health, Manhasset, NY; 3Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY; 4Department of Vascular Surgery, Comprehensive Wound Healing Center, Northwell Health, New Hyde Park, NY

Acute wounds have a profound impact on the U.S. healthcare system, contributing significantly to both clinical and economic burdens. In all stages of wound repair, mitochondria play a critical role-both through supporting metabolism of various cell types and engaging in cellular signaling cascades. These functions highlight the potential for mitochondrial targeted therapies to enhance wound repair. Mitochondrial targeted treatments which aim to alleviate mitochondrial dysfunction, reduce oxidative stress, and promote healing in acute wounds are currently under clinical investigations. Mitochondrial transplantation (MTx) is one such approach and involves transferring exogenous mitochondria into recipient cells. This technique has shown promise in various acute and chronic conditions. Human dermal fibroblasts (HDFs) are activated in response to injury and migrate to the wound site for tissue repair. Upon injury, they produce new extracellular matrix (ECM) components, secrete growth factors, and regulate inflammation, angiogenesis, and tissue remodeling. In this study, we investigated the effects of mitochondria isolated from a healthy 15-year-old human donor’s synovial fluid-derived stem cells on acute wound closure in adult HDFs using an in vitro scratch assay.
Adult dermal fibroblasts were cultured in fibroblast growth media. At 95% confluency, cells were seeded onto a type I collagen coated 96-well plate to form a monolayer. After 24 h, a uniform scratch was created using the Incucyte® WoundMaker tool. Mitochondria were isolated through differential centrifugation using a commercial kit (Thermofisher). Isolated mitochondria were then co-cultured with HDFs. Cell migration and wound confluence were monitored for 24 h using the Incucyte® imaging system. Wound width was quantified using Incucyte® analysis software and expressed as mean ± standard deviation (n = 4) for comparison between MTx and control HDFs. Mitochondrial uptake was validated by staining exogenous mitochondria with MitoTracker Red CMXRos dye and cells with Green Mask F-Actin. Mitochondrial uptake by HDFs was visualized using a confocal microscope.
Confocal microscopy confirmed mitochondrial uptake by HDFs, demonstrating that mitochondrial transplantation in dermal fibroblasts can occur through simple in vitro co-incubation. After 24 h of co-incubation, the in vitro wound scratch assay revealed complete wound closure in samples treated with donor mitochondria. Results showed a significant improvement in wound confluence, cell migration, and closure rates in the mitochondrial transplantation group compared to controls (p < 0.05).
This study demonstrates that mitochondrial transplantation accelerates acute wound closure by enhancing cell migration. This finding paves the way for future studies to further explore its applications. Further research is warranted to elucidate the mechanistic role of exogenous mitochondria in improving cellular migration of HDFs. WHS Session O: Rapid Fire Poster Presentations

O.01

DIFFERENTIATION OF RESIDUAL AND CONTRALATERAL LIMB BY BARRIER FUNCTION QUANTIFICATION AMONG PERSONS WITH UNILATERAL TRANSTIBIAL AMPUTATIONS
Alice Teper*1,2, Noah L. Tostenson1,2, Molly Baumann2,3, Julian Acasio3,4, Bradford Hendershot3,4, Walter L. Childers2,3
1Henry M. Jackson Foundation, Fort Sam Houston, TX; 2Center for the Intrepid/Brooke Army Medical Center, San Antonio, TX; 3Extremity Trauma and Amputation Center of Excellence, San Antonio, TX; 4Walter Reed National Military Medical Center, Bethesda, MD
Differentiation of Residual and Contralateral Limb by Barrier Function Quantification Among Persons With Unilateral Transtibial Amputations

Alice Teper1,2, Noah L. Tostenson1,2, Molly Baumann2,3, Julian Acasio3,4, Bradford Hendershot3,4, Walter L. Childers2,3 1Henry M. Jackson Foundation, Fort Sam Houston, TX; 2Center for the Intrepid/Brooke Army Medical Center, San Antonio, TX; 3Extremity Trauma and Amputation Center of Excellence, San Antonio, TX; 4Walter Reed National Military Medical Center, Bethesda, MD

Skin-related health problems are common among prosthesis users [1] due to increased mechanical stress at the skin-socket interface [2]. Poor skin health causes pain, reduces mobility, and can require time out of socket that limits functional independence. Skin barrier function, measured by transepidermal water loss (TEWL) and surface electrical capacitance (SEC), may serve as an early indicator for skin breakdown as changes in these measurements have been related to pressure ulcer formation and prevention [3]. If skin breakdown could be identified prior to visual presentation, this will improve future outcomes as prosthetic adjustments could be made earlier, and not require them to stop using their prosthesis. The objective of this study was to evaluate the capability of skin barrier function measurements to differentiate between the residual and contralateral limbs of individuals with unilateral transtibial amputation.
The study protocol was approved by the San Antonio Institutional Review Board in compliance with all applicable Federal regulations governing the protection of human subjects. TEWL and SEC were assessed in seven (six male/one female) past/present Service members with unilateral transtibial amputation (37.6 ± 8.3 y) using a Tewameter and Corneometer (C+K, Acaderm Inc, Menlo Park, CA), respectively. Points of interest on the anterior distal tibia, midpatellar tendon, and interosseous space were marked on both residual and contralateral limbs. The Tewameter was gently placed and held against the skin for 30 seconds; the Corneometer was gently placed on the skin for a single reading. Measurements of TEWL and SEC were repeated three times at each location. Percent differences in TEWL and SEC from contralateral to residual limb were calculated by dividing the difference between the residual and contralateral limb by the contralateral limb. A Related-Samples Wilcoxon Signed Rank Test compared residual and contralateral limb raw measurements (p < 0.05).
TEWL values were larger (p = 0.001) within the residual limb (15.7±6.6 g/m2/h) compared to the contralateral limb (9.2±4.9 g/m2/h), indicating poorer barrier function. The mean percent difference per participant was 104% at the distal tibia, 58% at the interosseous space, and 113% at the midpatellar tendon. SEC values (unitless on this system) were similar (p = 0.39) in the residual limb (33.4 ± 17.5) and the contralateral limb (26.8 ± 19.5).
TEWL was able to more consistently differentiate between the residual and contralateral limb, and thus provides a viable metric of skin barrier function for proactive management of residual limb skin health through clinical intervention. Future research will increase sample size and define the relationship between measurements of skin health to positive and negative clinical outcomes.
References
1. Highsmith. Tech and Innov. 2016.
2. Sanders. JRRD. 1993.
3. Schario. J Tissue Viability. 2017

O.02

ELU511: A NOVEL SMALL-MOLECULE BACTERIOSTATIC WNT SIGNALING INHIBITOR THAT PROMOTES REGENERATIVE REPAIR AND IMPROVES TENSILE STRENGTH FOLLOWING FULL-THICKNESS CUTANEOUS INCISIONAL WOUNDS IN YORKSHIRE PIGS
Daniel Holsworth*, Sarika Saraswati, John Delgado, Michael Stone
Eluciderm Inc, San Diego , CA
ELU511: A Novel Small-Molecule Bacteriostatic Wnt Signaling Inhibitor That Promotes Regenerative Repair And Improves Tensile Strength Following Full-Thickness Cutaneous Incisional Wounds In Yorkshire Pigs

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

The canonical Wnt signaling pathway is quiescent in many mammalian organs but is activated upon injury. Specifically, Wnt signaling promotes fibrotic wound healing (including scarring) following acute cutaneous injury. Studies with superhealer mice have shown that inhibiting this pathway leads to regeneration of healthy tissue. Eluciderm, Inc. has developed ELU511, a potent small-molecule Wnt signaling inhibitor that mimics the abilities of superhealer mice. ELU511 promotes regenerative cutaneous repair and increases tensile strength in acute full-thickness suture wounds; it has also exhibited bacteriostatic properties that facilitate rapid wound healing by helping to combat the spread of infection. ELU511’s patented formulation as a topical therapeutic offers easy “serum” application to a wound site. In this study, we utilized a porcine model to analyze repair following a surgical suture wound. Six (6) full-thickness 5 cm 0.5 cm fusiform wounds spaced 3 cm apart in a staggered pattern were created on the backs of Yorkshire pigs. ELU511 (0.1%, 200uL) was applied topically every other day up to and including Day 21. The animals were sacrificed on Day 22, and histopathological analyses were performed on excised tissues. Topical application of the novel small-molecule Wnt signaling inhibitor, ELU511, significantly promoted regeneration of tissue, as evidenced by the presence of restored skin architecture with adnexal structures and re-built, well-organized cross-linked collagen; results were assessed using Trichrome Blue staining and polarized microscopy. Wounds treated with ELU511 demonstrated improved cosmesis and displayed a 105-fold improvement in tensile strength compared to saline-treated wounds.
Creating drugs to enhance and accelerate wound healing presents a significant challenge, let alone drugs that promote healing via regeneration of healthy tissue. To date, no true wound healing drugs of any kind have been identified. Here we report the discovery and development of a novel Wnt signaling inhibitor that not only regenerates healthy tissue, but also substantially improves the tensile strength of skin and exhibits bacteriostatic properties. In addition, because it is formulated as a topical serum, it is easy to apply to a wound site. ELU511 has strong pharmacological potential to be the first state-of-the-art therapeutic for the enhanced healing of all surgical wounds.

O.03

Circulating Proteome as a Prognostic Biomarker Tool for Diabetic Wound Healing Course.
Monika A. Niewczas*1,2, Youngshin Keum1, Maria Tracy1, Jessica Gilman3, Georgios Theocharidis3,2, Zaipul Md Dom1,2, Aristidis Veves3,2, Alyssa Suarez1
1Joslin Diabetes Center, Boston, MA; 2Harvard Medical School, Boston, MA; 3Beth Israel Deaconess Medical Center, Boston, MA
Circulating Proteome as a Prognostic Biomarker Tool for Diabetic Wound Healing Course

Monika A. Niewczas1,2, Youngshin Keum1, Maria Tracy1, Jessica Gilman3, Georgios Theocharidis3,2, Zaipul Md Dom1,2, Aristidis Veves3,2, Alyssa Suarez1 1Joslin Diabetes Center, Boston, MA; 2Harvard Medical School, Boston, MA; 3Beth Israel Deaconess Medical Center, Boston, MA

Diabetic foot ulcers (DFUs) represent a severe complication of diabetes, often leading to significant morbidity and healthcare burden. Clinical legacy measures do not fully explain the DFU course. Newly developed, robust high-throughput affinity technologies capable of probing circulating proteome shall offer insights into the disease process.
Our prospective cohort comprised 100 subjects with DFU at baseline, participants of the Diabetic Foot Consortium study. The average age of participants was 58 ± 12 years with a predominance of male subjects (83%). The study group was followed for 12 weeks for their prospective ascertainment of the diabetic wound healing course. Baseline serum specimens were subjected to the proximity extension affinity proteomics. Logistic regression was used to assess the odds ratios (OR) and p-values for the association of protein levels with clinical outcomes; complemented by machine learning models.
Sixty four percent of subjects failed to heal their ulcer within the follow-up (non-healers). In the analyses of 5187 measured proteins, there were 259 proteins differentiating subjects whose wound successfully closed vs. those whose wound did not. The top proteins included zinc finger proteins (ZNF252P-AS1 with odds ratios (OR) and 95% confidence intervals (95%CI): 2.57 (1.49, 4.41); p = 0.0006; and ZNF746, ZFYVE19, ZNF131, respectively); coagulation factors (F7: 2.37 (1.40, 4.02; p = 0.001); and F9, respectively), single chemokine (CCL28: 1.74 (1.07, 2.85); p = 0.027) among others. There was no enrichment in any class of cytokine or cytokine receptors, as examined by the Kegg roster: hsa04060. A biostatistical logistic regression model with top 5 proteins had a discrimination metrics of c = 0.84. Discriminative ability of machine learning models were comparable or superior (c = 0.90 for random forest, c = 0.83 for generalized boosting model, c = 0.95 for elastic net and c = 0.92 for neural network, respectively). This study identifies a roster of candidate protein biomarkers, including F7, ZFYVE19, ANKZF1, and CCL28, with a robust model accuracy to differentiate the DFU course. These findings lay the groundwork for an immediate next step of the refinement of the proteomics biomarker signature supporting future therapeutic development strategies in the field.

O.04

Fluoxetine Delivery for Wound Treatment Through an Integrated Bioelectronic Device – Pharmacokinetic Parameters and Safety Profile in Swine
Anthony Gallegos*1, Hsin-ya Yang1, Guillermo Villa-Martinez1, Houpu Li2, Ksenia Zlobina3, Itidal Bazzi1, Samhitha Sathyanarayanan1, Moyasar A. Alhamo1, Wan-Shen Hee2, Prabhat Baniya2, Narges Asefifeyzabadi2, Tiffany Nguyen2, Maryam Tebyani2, Alexie Barbee2, Kaelan Schorger2, Sydnie Figurres2, Gordon H. Keller2, Marcella Gomez3, Athena Soulika1,4, Min Zhao5, Mircea Teodorescu2, Marco Rolandi2, Rivkah Isseroff1,6
1Dermatology, University of California Davis, Sacramento, CA; 2Electrical and Computer Engineering, University of California Santa Cruz, Santa Cruz, CA; 3Applied Mathematics, University of California Santa Cruz, Santa Cruz, CA; 4Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA; 5Ophthalmology and Vision Science, University of California Davis, Sacramento, CA; 6Dermatology Section, VA Northern California Health Care System, Sacramento, CA
Fluoxetine Delivery for Wound Treatment Through an Integrated Bioelectronic Device—Pharmacokinetic Parameters and Safety Profile in Swine

Anthony Gallegos1, Hsin-ya Yang1, Guillermo Villa-Martinez1, Houpu Li2, Ksenia Zlobina3, Itidal Bazzi1, Samhitha Sathyanarayanan1, Moyasar A. Alhamo1, Wan-Shen Hee2, Prabhat Baniya2, Narges Asefifeyzabadi2, Tiffany Nguyen2, Maryam Tebyani2, Alexie Barbee2, Kaelan Schorger2, Sydnie Figurres2, Gordon H. Keller2, Marcella Gomez3, Athena Soulika1,4, Min Zhao5, Mircea Teodorescu2, Marco Rolandi2, Rivkah Isseroff1,6 1Dermatology, University of California Davis, Sacramento, CA; 2Electrical and Computer Engineering, University of California Santa Cruz, Santa Cruz, CA; 3Applied Mathematics, University of California Santa Cruz, Santa Cruz, CA; 4Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA; 5Ophthalmology and Vision Science, University of California Davis, Sacramento, CA; 6Dermatology Section, VA Northern California Health Care System, Sacramento, CA

Wound infections are a significant medical challenge, complicating healing and often leading to outcomes such as chronic wounds or systemic infections. The rise of microbial resistance to antibiotics has further exacerbated this problem, rendering many traditional treatments ineffective and creating an urgent need for alternative strategies. Exploring non-antibiotic approaches, such as repurposing existing drugs with known safety profiles, offers a promising avenue to mitigate wound infections. Selective serotonin reuptake inhibitors (SSRIs) have emerged as potential non-antibiotic candidates with studies that have demonstrated their ability to limit growth and biofilm formation in Gram-negative bacteria, in addition to their demonstrated pro-healing activity. We hypothesize that topical delivery of an SSRI drug can reach effective concentrations in the wound without systemic perturbation. We compared direct delivery of the SSRI fluoxetine by repeated topical bolus dosing to delivery from an iontophoresis bandage device with an actuator for spatially and temporally controlled drug delivery. Full thickness 20 mm diameter circular wounds were created on the backs of Yorkshire pigs, and fluoxetine was delivered to the wound surface either by the fluoxetine delivery device or directly with daily bolus application. Wound tissues and blood were harvested after treatment for analysis of fluoxetine and serotonin by HPLC. Device delivery of fluoxetine resulted in a maximum concentration of 12.3 ug fluoxetine per mL tissue, compared to 2.93 ug/mL following bolus dosing. Tissue levels of fluoxetine were strongly correlated with the cumulative dose for both treatment modalities (device, n = 8, R = 0.862, p = 0.0089; bolus, n = 6, R=0.956, p = 0.022), and tissue fluoxetine levels were higher after treatment with the experimental device than after bolus dosing across the range of doses tested (p = 0.0041). The half-life of fluoxetine in the wound tissue was 1.43 days. Fluoxetine was not detected in the plasma of any animal, and plasma serotonin levels were not affected by topical fluoxetine wound treatment. Fluoxetine delivery using the device produced tissue concentrations above the MIC for some clinically important strains of A. baumannii, while bolus dosing failed to produce tissue concentrations above the MIC. We demonstrated that the experimental device can effectively deliver topical fluoxetine to the wound with minimal risk of off-target effects. The experimental device produces higher tissue concentrations of fluoxetine at lower cumulative doses compared to bolus dosing, which may lead to greater efficacy and an improved safety profile when using the device. The integrated bioelectronic device with fluoxetine delivery may simplify wound treatment by reducing the burden for daily drug application, possibly increasing adherence to a prescribed treatment regimen. In the future, we hope to further optimize the device design for commercialization and clinical use.

O.05

NEUTROPHIL EXTRACELLULAR TRAPS INHIBIT ACTIVATION OF PRO-HEALING GENE PROGRAMS IN HUMAN KERATINOCYTES AND EX VIVO WOUNDS
Victoria Frerichs2, Karina Imisheva1, Dario Garcia2, Rivka C. Stone2, Irena Pastar2, Pablo de Rivero Vaccari2, Marjana Tomic-Canic2, Andrew Sawaya2
1Dermatology, University of Miami, Miami, FL; 2Neurological Surgery and The Miami Project to Cure Paralysis, University of Miami, Miami, FL
Neutrophil Extracellular Traps Inhibit Activation of Pro-Healing Gene Programs in Human Keratinocytes And Ex Vivo Wounds

Victoria Frerichs2, Karina Imisheva1, Dario Garcia2, Rivka C. Stone2, Irena Pastar2, Pablo de Rivero Vaccari2, Marjana Tomic-Canic2, Andrew Sawaya2 1Dermatology, University of Miami, Miami, FL; 2Neurological Surgery and The Miami Project to Cure Paralysis, University of Miami, Miami, FL

Diabetic foot ulcers (DFUs) are a debilitating complication of diabetes affecting over 6.5 million people in the US, often resulting in lower limb amputations and mortality. We have previously shown increased neutrophil extracellular traps (NETs) and inflammasome activation in DFUs and validated it in preclinical diabetic mouse wound models. However,the mechanisms by which NETs affect keratinocyte function in the process of diabetic wound healing remains poorly understood.
Keratinocyttye migration was quantified in both in vitro scratch assays and ex vivo human wound models. Expression of wound healing relevant genes in keratinocytes was verified with qPCR.
We found that NETs isolated from human neutrophils significantly inhibited wound healing and keratinocyte migration in ex vivo human wound models and in vitro keratinocyte scratch assays. In addition, NETs suppressed wound healing relevant genes (i.e., EGF, K16, and ITGA6) in keratinocytes treated with NETs, contributing to impairment in their migration. Moreover, NETs induced inflammasome activation in NETs-treated keratinocytes that resulted in suppression of previously stated wound healing relevant genes, which was restored by blocking inflammasome activation with inflammasome inhibitor IC100.
Our data provide mechanistic insights regarding complex cellular crosstalk that contributes to impairment of healing, revealing novel therapeutic targets that can be used to restore immune balance and promote healing of chronic wounds.

O.06

3D cell printed muscle tissue for functional muscle recovery
YOUNG-JOON JUN*1,2
1plastic surgery, The Catholic university of Korea, Seoul, Other, Korea (the Republic of); 2The korean wound management society, SEOUL, Korea (the Republic of)
3D Cell Printed Muscle Tissue for Functional Muscle Recovery

Young-Joon Jun1,2 1plastic surgery, The Catholic university of Korea, Seoul, Other, Korea (the Republic of); 2The Korean wound management society, SEOUL, Korea (the Republic of)

Reported tissue engineered skeletal muscle repair constructs remodeled into a fibrotic tissue and showed limited functional improvement in volumetric muscle loss (VML) model. Therefore hydrogel-based 3D engineered muscles and the decellularized extracellular matrix (dECM) have been considered for VML treatment, but they also have shown limited efficacy. The authors established the skeletal muscle construct composed of cell-laden dECM bioink generated with a granule-based printing reservoir.
To mimic the hierarchical architecture of vascularized muscles, coaxial nozzle printing method was used. Human umbilical vein endothelial cell (HUVEC) and human skeletal muscle(hSKM) were printed with the muscle and vascular dECM bioink. In vitro studies revealed well aligned and striated muscle fibers with high cell viability without hypoxia of the 3D cell printed muscle constructs. About 15 6 4 mm sized vascularized skeletal muscle constructs were implanted to 40% defect of tibialis anterior (TA) muscle of Sprague-Dawley rats.
After 4 weeks, the coaxial printing group showed a significantly improved TA muscle weight than other control groups and achieved recovery at 78.6 ± 3.2% of the contralateral native TA muscle. Masson’s trichrome staining demonstrated very few fibrotic tissues with well-organized de novo muscle fibers in the coaxial printing group. In situ force production showed that the coaxial printing group yielded an isometric torque of 87.2 ± 3.44 N mm/kg, which corresponds to 85% of the uninjured muscle, superior than other groups.
Our present results suggest that a 3D cell printing and tissue-derived bioink-based approach could effectively generate biomimetic engineered muscles to improve the treatment of VML injuries.

O.07

A 3D tissue culture model to assess the ability of negative pressure wound therapy to rescue fibroblasts from quiescence
Amy K. McNulty*, Kerry Sokol
MS, Solventum, St. Paul, MN
A 3D Tissue Culture Model to Assess the Ability of Negative Pressure Wound Therapy to Rescue Fibroblasts From Quiescence

Amy K. McNulty, Kerry Sokol MS, Solventum, St. Paul, MN

Many in vitro studies have been conducted to assess the mechanisms of action of negative pressure wound therapy (NPWT). An issue with many of these studies is that the cells are tested in models whereby they are actively proliferating and showing high cellular activity. In contrast, chronic wound metabolism and proliferation may be depressed.1 The purpose of the following study was to develop a fibroblast 3D tissue culture model which may be more consistent with chronic wounds. The model was then used to test NPWT use and assess the effects on fibroblast cell proliferation and energetics.
Fibroblasts (n = 200,000) were encapsulated in a fibrinous matrix composed of 6900 mg fibrinogen clotted with 70IU of thrombin in 6-well plate inserts. Wells were fed with CO2-free medium with 10% fetal bovine serum (FBS) and 50 units/mL penicillin (P) and 50 ug/mL streptomycin (S). This medium was changed 3 times a week for up to 3 weeks. At various time points hanging wells were placed in a specially designed bioreactor and treated with negative pressure using a reticulated, open cell foam* as the tissue interface. An NPWT unit† was set to deliver 125 mmHg continuously for 23–24 h. CO2-free medium with 5% FBS and P/S was delivered to the bioreactor at 750 uL/h to simulate rates of wound fluid expression in the wound. At the end of the NPWT period, wells were removed and replaced with fresh CO2-free medium with 10% FBS and P/S for 1–1.5 h. After incubation, proliferation and adenosine triphosphate (ATP) levels were measured using cell viability and cell proliferation assays, respectively. The cell viability average optical density (OD) for control cell cultures decreased with culture age. The average for control cultures less than 15 days was 1.196 ± 0.15 while greater than 15 days was 0.440 ± 0.094. The ATP relative light unit values also decreased by over 80% with control cell culture age. NPWT treatment showed a higher increase in cell viability OD (3- to 14-fold higher) versus control for cultures greater than 15 days old. There also was a greater percentage improvement in energy status of the older cultures (216%) vs younger cultures (67%) when treated with NPWT. Fibroblasts encapsulated in a fibrinous matrix and fed with CO2 free media showed decreased cellular activity over time. The cells became more quiescent with lower proliferation and less ATP. Treatment with NPWT had a proportionally greater effect on cellular proliferation and energy status in the older vs younger cultures. The ability to rescue tissues from quiescence may be a mechanism of action of NPWT which requires further exploration.

O.08

ACCELERATING WOUND HEALING: AN IN-VIVO MODEL COMPARISON OF COLLAGEN-BASED DERMAL MATRICES FOR FULL-THICKNESS DEFECT RECONSTRUCTION
Jayson W. Jay*, Barbara Nsiah, Katie Bush
AVITA Medical, Houston, TX
Accelerating Wound Healing: An In-Vivo Model Comparison of Collagen-Based Dermal Matrices For Full-Thickness Defect Reconstruction

Jayson W. Jay, Barbara Nsiah, Katie Bush AVITA Medical, Houston, TX

Dermal matrices (DM) are used for wound healing, skin grafting, and tissue regeneration in full-thickness cutaneous injuries. These matrices, derived from various biological sources or designed from synthetic materials, play a crucial role in burn, plastic, and reconstructive surgery by providing a temporary scaffold for tissue repair, offering structural support and modulating cellular responses. However, differences in DM composition result in various challenges related to integration, inflammation, durability, infection risk, and cost. Moreover, most current DMs can only sustain a viable autograft approximately 3 weeks after placement, adding time to definitive closure. To address these gaps, current research has been focused to improve DM integration, improve cellular infiltration, and enhance angiogenesis to support early viable autografting, thereby accelerating time to closure.
The purpose of the current study was to evaluate the wound healing effects of collagen-based DMs: bovine tendon collagen with glycosaminoglycans (ColGAG), fish skin graft (FSG), and a novel bovine dermal collagen matrix (BDCM) consisting of type I/III collagens.
Full-thickness wounds, down to fascia approximately 5 mm deep, measuring 16 cm2 were created on the dorsum of female Yorkshire pigs. Wounds were randomly assigned to receive either ColGAG (N = 10), FSG (N = 6), or BDCM (N = 8). Dermal matrices were mechanically affixed to the wound edges, bolstered securely in place, and integration was allowed for 7 days. At Day 7, wounds were autografted with a 3:1 meshed split-thickness graft combined with an autologous skin cell suspension. Autograft take and percent re-epithelialization were visually evaluated at Day 14. Contraction was evaluated at Day 42. Trichrome analysis on Day 7 showed a collagen fiber network abundant in cells and complex microvasculature in BDCM. ColGAG-treated wounds showed similar results with lower cellular density. In contrast, while dense clusters of inflammatory cells were present in the FSG wounds, no intact three-dimensional matrix was observed. By Day 14, graft survival was highest in the BDCM group (96.9 ± 7.0%) compared to ColGAG (85.0 ± 34.1%) and FSG (68.0 ± 28.0%). Additionally, by this time, all BDCM-treated wounds demonstrated at least 75% re-epithelialization, while 60% of ColGAG-treated wounds and 40% of FSG-treated wounds achieved at least 75% closure. At Day 42 BDCM-treated wounds had a significantly lower percent contraction compared to FSG- and Col-GAG treated wounds (p < 0.05).
The novel BDCM creates a favorable environment for robust cellular infiltration and formation of well-vascularized dermal bed that is ready for autografting within seven days, in a porcine wound healing model.

P1.01

Microporous Annealed Particle Hydrogel Attenuates Contraction of Split-Thickness Skin Grafted burn wounds
Isabelle Bergman*1, Jayson W. Jay1, Alekhya Gurram2, Rochak Khatri2, Jyothika Annareddy2, juquan song1, Julia Kleinhapl1, Kan Nakamoto3, Steven E. Wolf1, Amina El Ayadi2
1Surgery, University of Texas Medical Branch, Galveston, TX; 2School of Medicine, University of Texas Medical Branch, Galveston, TX; 3Anesthesiology, University of Texas Medical Branch, Galveston, TX
Microporous Annealed Particle Hydrogel Attenuates Contraction of Split-Thickness Skin Grafted Burn Wounds

Isabelle Bergman1, Jayson W. Jay1, Alekhya Gurram2, Rochak Khatri2, Jyothika Annareddy2, juquan song1, Julia Kleinhapl1, Kan Nakamoto3, Steven E. Wolf1, Amina El Ayadi2 1Surgery, University of Texas Medical Branch, Galveston, TX; 2School of Medicine, University of Texas Medical Branch, Galveston, TX; 3Anesthesiology, University of Texas Medical Branch, Galveston, TX

Hypertrophic scars subsequent to severe burns are associated with sub-optimal wound healing. Current interventions have shown limited efficacy in accelerating wound re-epithelialization and reducing scar formation. Existing biomaterials can help with wound coverage in the acute phase but achieve modest efficiency in terms of tissue regeneration. This study explores the potential of a novel biomaterial termed microporous Annealed particles (MAP) hydrogel on burn wound healing and scarring. We hypothesize that the unique characteristics of MAP scaffolds- specifically their microporosity, injectability, and immunomodulatory properties – can enhance cellular migration and create a microenvironment conducive to skin regeneration.
The Red Duroc Pig model of burn wound healing and scarring that closely mimics the human condition was used in this study. Split-thickness skin (STSG) grafts were placed on burned excised wounds treated with MAP hydrogel, a non-porous hydrogel (PEG hydrogel), or non-treated control. We assessed the efficacy of MAP hydrogel in accelerating wound healing and restoring skin function throughout the wound healing process up to 120 days after injury. The Dermalab system was used to assess skin hydration, erythema, elasticity, and transepidermal water loss. 3D imaging was used to estimate wound contraction and scar formation. Data were analyzed using two-way ANOVA and mixed analysis with significance set at p < 0.05.
MAP hydrogel significantly reduced wound contracture compared to PEG hydrogel on days 60, 90, and 120 (all p < 0.001). MAP hydrogel reduced erythema, indicative of inflammation, on day 28 after injury (p < 0.01). Transepidermal water loss was also reduced in MAP hydrogel and STSG-treated wounds (all p < 0.0001), indicating a preservation of the skin barrier. Epidermal thickness was also reduced on day 120 in the MAP-treated wounds compared to STSGs and PEG controls (p < 0.05 and p < 0.01, respectively).
MAP hydrogel accelerates wound healing and reduces scarring, as demonstrated by reduced wound contraction and decreased epidermal thickness in the later stages of healing. These data suggest that MAP hydrogel creates a pro-regenerative microenvironment that promotes tissue repair.

P1.02

TEMPORAL GENDER DIFFERENCES IN IMMUNE RESPONSES AT THE BURN WOUND SITE
Shannon M. Clayton*2,1, Abigail Graham2, Chinarose Carlos2, Maliha Newsome2, Guadelupe Lugo2, Niayab Ahad2, Laasya Gadamsetti2, Adriana Xochitl Vasquez2, Athena Soulika1,2
1Dermatology, UC Davis, Sacramento, CA; 2Research, Shriners Northern CA, Sacramento, CA
Temporal Gender Differences in Immune Responses at the Burn Wound Site

Shannon M. Clayton2,1, Abigail Graham2, Chinarose Carlos2, Maliha Newsome2, Guadelupe Lugo2, Niayab Ahad2, Laasya Gadamsetti2, Adriana Xochitl Vasquez2, Athena Soulika1,2 1Dermatology, UC Davis, Sacramento, CA; 2Research, Shriners Northern CA, Sacramento, CA

The skin of both humans and rodents is sexually dimorphic. Female mice have increased hypodermal fat and epidermal thickness, while males have increased dermal thickness and collagen. Previous research suggests that both skin development and immunity is influenced by sex.
It has been documented that excision wounds heal faster in females compared to males. To elucidate whether burn wound healing varies between sexes, we induced scald burn injury (10% TBSA) in 8–10-week-old male and female mice. Mice were euthanized on days 3 (early inflammation), 7 (late inflammation), 14 (proliferative phase), 28 (wound closure, late proliferative phase), and 42 (2 weeks post closure/remodeling) post injury (pi). Tissues from each time point were examined histologically and via bulk RNAseq. Tissues from day 3, 7, and 14pi were also analyzed via flow cytometric analysis.
Contrary to what expected, female mice exhibited decreased re-epithelialization on days 3 (p = 0.0047) and 7 (p = 0.029) post injury (pi), compared to males. However, there were no differences in re-epithelialization rates at later timepoints between the two sexes. Interestingly, further analysis of the wound histology showed that there was no difference in the length of the neo-epithelium on days 3 and 7, but female mice had larger wound bed lengths compared to males. These suggest that contraction may be more intense in male wound healing.
Flow cytometric analysis revealed that on day 3pi, a time point associated with early inflammatory responses, male mice have increased total number of gd T cells (CD45+CD11b-CD3+gdTCR+) (p = 0.011), and decreased numbers of inflammatory macrophages (CD45+CD11b+Ly6G-MHCII+Ly6C+) (p = 0.028), compared to their female counterparts. By day 7pi, a time point that is associated with late stages of the inflammatory phase, the numbers of gd T cells were similar between two sexes, but at this timepoint, males had increased inflammatory macrophages (p = 0.00014) compared to females. On day 14pi, a time point that is characterized by increased proliferation, but the wound is still not healed, the wounds of male mice had increased numbers of neutrophils (CD45+CD11b+Ly6G+) (p = 0.0016), and decreased numbers of gd T cells (p = 0.0010), while there were no differences in the numbers of inflammatory macrophages between the sexes. Bulk RNA-seq analysis revealed that wounded skin of male mice had increased Il6 transcripts as well as genes involved in response to cytokines, acute inflammatory response, and positive regulation of IL-1b starting on day 14pi.
These results suggest that the burn wounds of male and female mice exhibit different inflammatory profiles and wound closure mechanics over time. Additional studies are necessary to clarify the differences in burn wound healing between the two sexes.

P1.03

EXPLORING THE IMPACT OF BURN EXUDATE ON CELLULAR REGENERATION AND WOUND HEALING IN HUMAN SKIN
Mahsa Pipelzadeh*1, Ali Khodadadi2, Anna Fucs1, Desmond J. Tobin1,3
1Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin, Ireland; 2Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (the Islamic Republic of); 3Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
Exploring the Impact of Burn Exudate on Cellular Regeneration And Wound Healing in Human Skin

Mahsa Pipelzadeh1, Ali Khodadadi2, Anna Fucs1, Desmond J. Tobin1,3 1Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin, Ireland; 2Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (the Islamic Republic of); 3Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland

The role of burn blister fluid (exudate) in human skin wound healing is poorly understood, with clinical opinions on its therapeutic potential remaining controversial. This study aims to investigate the effects of burn exudate on wound healing using ex vivo and in vitro models of human skin scald burns A novel ex vivo human skin scald burn model was developed using skin from patients undergoing elective abdominoplasty. The skin was exposed to boiling water (100C) for 30 s using a custom-insulated pipe and analyzed via immunohistochemistry (IHC) methods. The model was validated by comparing burn and non-burn skin to examine cellular and extracellular matrix (ECM) dynamics over a 5-day period. These dynamics were analyzed in three conditions: media supplemented with burn exudate, human serum, and serum-free media using specific antibodies.
Key protein markers analyzed included Ki67 (proliferation), K14 and K16 (keratinocyte differentiation), collagen IV (basement membrane integrity), and myofibroblast markers (α-SMA and VWF), epithelial-mesenchymal transition (EMT)-associated markers, such as E-cadherin, N-cadherin, Snail, and Slug.
For the in vitro method, primary human dermal fibroblasts from healthy abdominal skin were seeded in 96-well plates (10,000 cells/well) with 10% FBS. Mitomycin (10 μg/mL) was applied for 2 hours to inhibit cell division, followed by scratch creation in the monolayer. Wound closure was monitored over 72 h in an Incucyte incubator under the same above three conditions Proteomic analysis was performed on exudate samples taken from 50 superficial 2nd degree burn volunteers. Samples were collected 24 hours post-burn from non-haired areas and haired areas. Mass spectrometry identified proteins involved in wound healing, with comparisons made to plasma samples from healthy donors to highlight differences in protein expression
Ex vivo studies demonstrated that burn exudate enhanced keratinocyte proliferation (Ki67), upregulated regenerative markers (K14, K16), and restored basement membrane integrity (collagen IV). EMT markers, including Snail and Slug, were upregulated, while E-cadherin was downregulated, indicating a shift toward a mesenchymal state. These changes were more pronounced in exudate-supplemented media, reflecting dynamic cellular responses.
In vitro, fibroblasts treated with burn exudate exhibited faster wound closure compared to controls.
Proteomic analysis identified burn exudate-specific proteins, which are believed to be involved in wound healing and regeneration, including angiostatin (angiogenesis), kinins (protease inhibition), and filaggrin (ECM stability).
Burn exudate contains bioactive proteins that promote cellular regeneration, EMT, and ECM remodeling in ex vivo human skin. The identification of specific proteins within burn exudate that enhance wound healing could potentially lead to the development of novel therapeutic agents aimed at accelerating burn wound healing.

P1.04

SILK PROTEIN-BASED SPRAY-ON DRESSING FOR ACUTE BURN WOUND MANAGEMENT
Kristo Nuutila*1, Yushu Wang4, Anders Carlson2, Sean Christy1, David Larson3, Chan Rodney2, David Kaplan4
1USAISR, San Antonio, TX; 2Metis Foundation, San Antonio, TX; 3UT Health San Antonio, San Antonio, TX; 4Tufts University, Boston, MA
Silk Protein-Based Spray-On Dressing for Acute Burn Wound Management

Kristo Nuutila1, Yushu Wang4, Anders Carlson2, Sean Christy1, David Larson3, Chan Rodney2, David Kaplan4 1USAISR, San Antonio, TX; 2Metis Foundation, San Antonio, TX; 3UT Health San Antonio, San Antonio, TX; 4Tufts University, Boston, MA

There is a critical unmet need to improve treatment of combat related burns in the battlefield and during transportation. Fast and efficient wound protection and closure is desired to avoid painful dressing changes, risk of infection and scarring. Therefore, the purpose of this study is to introduce a silk protein-based spray to treat burn-injured soldiers in the battlefield. The spray contains antibiotics to prevent infection and coats the injured area with a robust matrix mat to provide protection. Further, once applied, the treatment does not need to be removed as it will resorb in situ, thus avoiding new damage to the burn site as the materials integrate into the wound bed.
The coating containing silk fibroin, polyethylene glycol (PEG) and gentamicin was formulated, and its characteristics were optimized in vitro. Subsequently, the coating was used in a porcine deep partial-thickness (DPT) burn model. Up to 16 standardized DPT burns were created on the dorsum of four anesthetized pigs. Analgesia was provided prior to all surgical procedures with buprenorphine SR. Burns on each animal were randomized into four experimental groups: (1) silk PEG; (2) silk PEG + gentamicin; (3) silver sulfadiazine cream (SSD); (4) no treatment. The animals were followed for 3, 7, 14 or 21 days. After euthanasia, the burns were excised for histologic analyses. Non-invasive imaging was utilized to assess burns macroscopically.
Coating containing 6% silk, 20% PEG and 1 mg/ml gentamicin solution exhibiting sprayable film matrix formation, adhesive and robust mechanical properties was chosen for the animal studies. Antibacterial testing showed the mixture to be effective against E. coli and S. aureus by killing 99.5%–99.95% of the bacteria in vitro. Furthermore, cell assays demonstrated that the silk coating did not have a negative effect on cell viability and migration. The porcine study indicated that the silk PEG + gentamicin treatment mitigated burn wound progression in comparison to other groups. The burns treated with silk PEG + gentamicin, silk PEG, SSD or left untreated had depths of 550, 920, 900, and 930 μm, respectively. The results also showed that the silk treated burns were fully re-epithelialized by day 21 post burn.
Spray techniques provide rapid protection to large and geometrically complex areas of the body using a fieldable formulation. This novel spray-on silk coating was characterized, and its feasibility tested in a relevant burn model.

P1.05

Synergistic Effect of Mesenchymal Stem Cells and ?-Terpineol-Loaded Hydrogel on Full-Thickness Burn Wounds: A Promising Therapeutic Approach
Fatima Jameel*1, Maria Khalid2, Asmat Salim1, Irfan Khan3, Muhammad Raza Shah2
1Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan; 2HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan; 3Center for Regenerative Medicine and Stem Cell Research, The Aga Khan University, Stadium Road, P. O. Box 3500, Karachi 74800, Karachi, Pakistan

P1.06

BLT1 antagonism does not affect burn wound haling
Fariba Mohebichamkhorami*1,2, Maliha Newsome1, Guadelupe Lugo1, Athena Soulika1,2
1Shriners Children’s Northern California, Sacramento, CA; 2Dermatology, University of California, Sacramento, CA
BLT1 Antagonism Does Not Affect Burn Wound Haling

Fariba Mohebichamkhorami1,2, Maliha Newsome1, Guadelupe Lugo1, Athena Soulika1,2 1Shriners Children’s Northern California, Sacramento, CA; 2Dermatology, University of California, Sacramento, CA

5-Lipoxygenase (5-LO) is the rate limiting enzyme for the biosynthesis of leukotrienes. Leukotrienes are lipid mediators that participate in immune responses and are comprised of LTB4 and the cysteinyl leukotrienes (LTC4, LTD4, and LTE4). LTB4 is known to promote neutrophil migration to the affected site and increase local inflammatory responses, while cysLTs are known to promote vasoconstriction, and to recruit leukocytes to the affected site.
Our preliminary results show that in murine burn wounds LTB4 increase drastically early after injury, while cysLTs are detected only in low levels. LTB4 acts via a high affinity receptor (BLT1) to recruit neutrophils and promote inflammatory responses and a low affinity receptor (BLT2) which exerts context-dependent effects and has been shown to play both pro-inflammatory and reparative effect. Previous studies have shown that BLT1 antagonism promotes excision wound healing, but it is unclear whether this is the case in burn wounds as well.
We thus set to examine the effect of BLT1 antagonism in burn wound healing. Our hypothesis predicted that BLT1 antagonism will shorten the inflammatory phase and will promote burn wound healing.
We induced scald burn injury on dorsa of C57BL/6 mice (wild type; WT) covering 1 cm2. To antagonize BLT1, we employed the selective and potent non-competitive BLT1 antagonist, CP-105,696. Mice were randomly divided in two groups receiving daily intraperitoneal injections of either 5 mg/kg CP-105,696, dissolved in carboxymethylcellulose (group1) or vehicle (carboxymethylcellulose only; group 2). Injections were initiated right before burn injury and up to day 9 post-injury, the end of the experiment, and a time point that represents the late proliferative phase of wound closure in control mice. As additional controls we also employed 5LO-/- mice. Tissues were isolated and examined histologically for wound morphology and wound closure, and immunohistochemically for the presence of neutrophils at the wound site.
Contrary to excision wounds, our data show non-statistically significant changes in wound areas of mice treated with CP-105,696 compared with vehicle in burn wound healing. Furthermore, there were no statistically significant differences in neutrophil numbers at the wound site. All these suggest that BLT1 does not play a role in burn wound healing. Additional studies using other commercially available BLT1 and/or BLT2 antagonists are necessary to discern the function of LTB4 in burn wounds.

P2.01

IMMUNE-RELATED GENES LINKED WITH WOUND MICROBIOTA AS POTENTIAL BIOMARKERS FOR DIABETIC FOOT ULCER HEALING
Juan Cortes-Troncoso*1, Jessica M. Eager1, Lindsay Kalan2, Amy Campbell3, Jacquelyn S. Michaelis4, Amit Rao5, Michael S. Weingarten6, Elizabeth A. Grice3, Alisha Oropallo5, David S. Margolis7, Ahmet Sacan1, Kara L. Spiller1
1School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA; 2Department of Medical Microbiology & Immunology, School of Medicine and Public Health, University of Wisconsin Madison, Madison, WI; 3Departments of Dermatology and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 4Institute for Advanced Computer Studies, University of Maryland, College Park, MD; 5Northwell Health Department of Surgery, Comprehensive Wound Healing Center and Hyperbarics, Northwell Health, Lake Success, NY; 6Department of Surgery, Drexel University College of Medicine, Philadelphia, PA; 7Departments of Dermatology and Biostatistics, Epidemiology and Biostatistics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
Immune-Related Genes Linked With Wound Microbiota as Potential Biomarkers for Diabetic Foot Ulcer Healing

Juan Cortes-Troncoso1, Jessica M. Eager1, Lindsay Kalan2, Amy Campbell3, Jacquelyn S. Michaelis4, Amit Rao5, Michael S. Weingarten6, Elizabeth A. Grice3, Alisha Oropallo5, David S. Margolis7, Ahmet Sacan1, Kara L. Spiller1 1School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA; 2Department of Medical Microbiology & Immunology, School of Medicine and Public Health, University of Wisconsin Madison, Madison, WI; 3Departments of Dermatology and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 4Institute for Advanced Computer Studies, University of Maryland, College Park, MD; 5Northwell Health Department of Surgery, Comprehensive Wound Healing Center and Hyperbarics, Northwell Health, Lake Success, NY; 6Department of Surgery, Drexel University College of Medicine, Philadelphia, PA; 7Departments of Dermatology and Biostatistics, Epidemiology and Biostatistics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

Diabetes foot ulcers (DFU) are challenging to treat because the mechanisms behind impaired DFU healing are complex and unknown. The varying effectiveness of treatments highlights the need for accurate biomarkers to predict wound healing and guide optimal treatment decisions. Chronic inflammation and dysfunctional macrophage behavior impact healing in DFUs.
Additionally, wound microbiota is a potential factor that tunes immune cell phenotypes in chronic wounds and is a strong factor that impairs wound healing. Hence, this study aimed to compare changes in inflammation- and macrophage phenotype-related genes over time in human healing and non-healing DFUs, to investigate the microbiome’s influence as a potential mediator, and to uncover possible biomarkers of healing outcomes.
Using a targeted panel of 227 human genes and 16S ribosomal RNA amplicon sequence to analyze microbial species, paired samples in two-time points from 27 subjects were analyzed to understand the changes over time and their correlation with wound microbial species.
After using the Fisher method to combine p-values across all eight normalization methods and correcting for multiple hypothesis testing, we discovered eight differentially expressed genes (DEGs) or gene sets between healing and non-healing groups at the initial sample collection time point, and 61 DEGs 4 weeks later when the healing subjects were within 8 weeks of healing. In addition, when we analyzed correlations between microbial species or diversity metrics and human gene expression values using the samples collected at week 0, were found 5 genes to be significantly correlated with microbial species in the non-healing group, while 16 genes were found to be significantly correlated with microbial species within the healing group. There were no genes that were significantly correlated with both healing and non-healing DFUs. Finally, utilizing machine learning based on logistic regression models with feature selection, we explored the potential of genes to predict healing outcomes when using samples collected at the week 0 time points only. Although no genes predicted healing outcome individually, the ratio of C3AR1 to CCL22 emerged as a unique biomarker that can predict healing outcome 20 weeks later. These findings were validated in an independent cohort of 51 subjects in which the area under the ROC curve was 0.71. When individual clinical factors, such as wound area, were considered, the area under the curve (AUC) increased to 0.77. Furthermore, when adjusting the predictive value of C3AR1/CCL22 for all factors including gender, race, amputation status, hypertension, hyperlipidemia, chronic kidney disease (CKD), Charcot’s foot, wound area, peripheral vascular disease (PVD), cardiovascular disease, and myocardial infarction (MI), the AUC increased to 0.81. These results provide insight into the heterogeneity of human DFU healing and suggest C3AR1/CCL22 as a biomarker that can be useful for guiding treatment decisions.

P2.02

SINGLE-CELL MULTI-OMICS OF MOUSE DIABETIC WOUND HEALING REVEALS STROMAL CELL CROSSTALK DYSFUNCTION
Mateusz S. Wietecha*1, Jingbo Pang2, Avin Hafedi1, Shalyn Keiser1, Timothy J. Koh2
1Oral Biology, College of Dentistry, University of Illinois Chicago, Chicago, IL; 2Kinesiology and Nutrition, College of Applied Health Sciences, University of Illinois Chicago, Chicago, IL
Single-Cell Multi-Omics of Mouse Diabetic Wound Healing Reveals Stromal Cell Crosstalk Dysfunction

Mateusz S. Wietecha1, Jingbo Pang2, Avin Hafedi1, Shalyn Keiser1, Timothy J. Koh2 1Oral Biology, College of Dentistry, University of Illinois Chicago, Chicago, IL; 2Kinesiology and Nutrition, College of Applied Health Sciences, University of Illinois Chicago, Chicago, IL

Type II diabetes mellitus is one of the most prevalent diseases associated with diabetic foot ulcers and impaired wound healing. The diabetic mouse model with dysfunctional leptin receptor (db/db) has been used in basic and translational studies due to its phenotypes (hyperphagia, hypometabolism, obesity, diabetes), including for delayed diabetic wound healing research, although a characterization of its temporal cellular dynamics has not been performed.
Excisional wounds were harvested from wild-type (WT) and diabetic (db/db) mice at 3, 6, and 10 days post-injury. Epithelium was discarded and stromal cells from digested dermal tissue were isolated, purified for live cells, and tagged using a panel of 18 antibodies against surface markers of major stromal cell types, including immune cells (neutrophils (CD45,CD11b,Ly6G), monocytes/macrophages (CD45,CD11b,F4/80), T lymphocytes (CD45,CD3,CD4) and non-immune cells (endothelial cells (CD31), fibroblasts (CD26,CD140a)). The cells were processed for 10X Genomics single-cell RNA-sequencing, followed by stringent quality control measures, resulting in a final dataset of 49963 high-quality cells. Downstream bioinformatics data analysis was conducted in R (4.4.1) using RStudio (2024.09.0) interface and the single-cell analysis package Seurat (5.1.0) and the cell-cell communication analysis package CellChat (2.1.2). Cell clustering was performed at multiple resolutions followed by cell typing analyses using both protein- and RNA-level data. Temporal cell abundance and pseudobulk analyses were performed to evaluate healing progression. Sub-clustering analyses were performed to analyze cell subtypes. Ligand-receptor analyses were performed to analyze putative cell-cell communication pathways. FACS analyses were performed on additional samples to validate major bioinformatics findings.
We identified 8 major cell types (in decreasing abundance): Fibroblasts, Macrophages, Neutrophils, Endothelial cells, Smooth muscle cells, T cells, Muscle progenitor cells, Skeletal muscle cells. Pseudobulk and gene co-expression analyses showed a marked delay in wound healing progression in db/db vs WT mice. Correlation of protein- and RNA-level data revealed high concordance between cell markers and their gene expressions in major cell types. Protein-level data was useful for cell typing and subtyping, identifying 4 fibroblast, 4 macrophage, and 2 neutrophil subtypes. These stromal cell subtypes showed dysregulated dynamics across the timecourse of healing in db/db vs WT mice. CellChat analyses revealed striking differences in cell-cell communication dynamics between fibroblasts, macrophages and neutrophils across healing, with the SPP1 pathway being especially dysregulated between these cells in db/db vs WT mice.
We present a comprehensive multi-omic characterization of mouse skin diabetic wound healing at the single-cell level, revealing temporal dysfunctions in major cell type abundances and stromal cell crosstalk dynamics.

P2.03

PREDICTING WOUND HEALING: A MACHINE-LEARNING PARTIAL LEAST SQUARES DISCRIMINANT ANALYSIS MODEL UTILIZING MICROBIOME, METABOLOME, AND CLINICAL MARKER DATA SETS
Catherine B. Anders*, Hannah L. Smith, Jeremy Boyd, Michael Davis, Margaret M. Doucette, Mary Cloud B. Ammons
Research, Boise VA Medical Center, Boise, ID
Predicting Wound Healing: A Machine-Learning Partial Least Squares Discriminant Analysis Model Utilizing Microbiome, Metabolome, and Clinical Marker Data Sets

Catherine B. Anders, Hannah L. Smith, Jeremy Boyd, Michael Davis, Margaret M. Doucette, Mary Cloud B. Ammons Research, Boise VA Medical Center, Boise, ID

Type 2 Diabetes affects more than 37 million people in the United States and is the number one cause of lower-limb amputation in adults due to diabetic foot ulcers (DFU). The chronic wound microenvironment consists of a complex milieu of host cells, microbial species, and metabolites. While much is known about the wound microbiome, our knowledge of the metabolic landscape and its influence on microbial diversity and wound healing is limited. Furthermore, the integration of these complex datasets into a predictive model with relevance to clinical outcome is almost non-existent. Here, we present a multiomics data analysis coupled with machine-learning cross validation of microbiome and metabolome profiles from human chronic wounds. The model was then integrated with patient metadata to determine predictive correlation to clinical outcome.
Microbial ribosomal RNA (rRNA) and total metabolites were extracted from 45 DFU debridement samples collected from 13 patients at the Boise VA Medical Center. Of 45 samples analyzed, 25 samples were isolated from wounds that that failed respond to standard treatment while the remailing 20 samples were taken from wounds that progressed to healing and remained closed for > 30 days. 16S rRNA sequencing and global metabolomics were performed and clinical metadata was collected from patient records. Healing outcome was modeled as a function of three blocks of features (N = 21 clinical, 634 microbiome, and 865 metabolome) using multiblock sparse partial least squares discriminant analysis (sPLS-DA) which performs feature selection using LASSO regularization. Seven-fold cross-validation with 100 repeats was used to find the amount of regularization associated with the smallest predictive error.
The final model selected a total of 527 features (N = 16 clinical, 91 microbiome, and 420 metabolome), and was able to predict the clinical outcome with an overall error rate of 5.4%.
These results indicate that the integration of wound microbiome and metabolomics data with patient clinical metadata can be utilized to predict clinical outcomes regarding wound healing and with low error rates. Furthermore, the biomarkers selected within the model may offer novel insights into wound microenvironment composition and improve treatment efficacy in difficult to heal wounds.

P2.04

Rising Antimicrobial Resistance in Chronic Wounds
Elina Zhivov*, Jelena Marjanovic, Tammy Gonzalez, Jamie L. Burgess, Cheyanne Head, Marjana Tomic-Canic, Hadar Lev-Tov, Irena Pastar
Dermatology , University of Miami, Miami, FL
Rising Antimicrobial Resistance in Chronic Wounds

Elina Zhivov, Jelena Marjanovic, Tammy Gonzalez, Jamie L. Burgess, Cheyanne Head, Marjana Tomic-Canic, Hadar Lev-Tov, Irena Pastar Dermatology, University of Miami, Miami, FL

Antimicrobial resistance (AMR) remains one of the most critical global public health threats, with an estimated 4.95 million deaths attributed to bacterial AMR in 2019 including resistance in the context of wound care. In addition to widely spread antibiotic resistance in chronic wounds, emerging evidence suggests diminishing efficacy of silver based prodcuts, particularly in chronic wound management. We analyzed the prevalence of methicillin and mupirocin resistance genes in patients with chronic venous leg ulcers (VLU, n = 24) with no clinical signs of infection. These targets were chosen as methicillin-resistant Staphylococcus aureus is one of the most common wound pathogens with the highest increase related to AMR attributable deaths, while mupirocin is the most prescribed topical wound antimicrobial. Our findings revealed a methicillin resistance rate of 66.67% and an alarming mupirocin resistance rate of 83.33% among the total number of patients tested.
Next, we conducted a single-center, randomized controlled trial to compare an antimicrobial silver-based dressing with a Dialkylcarbomoyl chloride (DACC)-based dressing, designed to eliminate bacteria through irreversible binding, in modifying bacterial load in VLUs. Thirty-one subjects were randomized to either group to receive 4 weeks of treatment, followed by a 2-week follow-up. Tissue samples were collected at weeks 0, 2, and 3, and bacterial load was quantified using 16s rRNA qPCR. Isolated bacterial DNA samples from week 0 and week 3 were sent for shotgun metagenomic sequencing. RNA analysis focused on inflammatory markers CCL4, CXCL9, CXCL12, CCL13, ICAM-1, and VCAM-1.
We found that individuals over 65 years old had a significantly higher bacterial load before intervention, which is concerning given the 80% increase in AMR-related deaths in adults over 70. The silver-based dressing group showed a significantly higher bacterial load by third treatment visit compared to the DACC group. This correlated with healing outcomes, with higher healing rates in the DACC group. Healers, designated based on 50% or more of wound size reduction, had higher baseline tissue inflammation, which reduced over time, while non-healers showed suppressed low levels of inflammation persisting throughout the treatment, specifically in the silver dressing group. These findings were further supported by patient outcome surveys, which revealed significant improvements in wound quality of life, EQ-5D-5L scores, and periwound pain levels in the DACC group.
Our findings highlight the growing AMR challenge in chronic wounds and the need for antimicrobial stewardship, accurate infection diagnosis, and novel treatment strategies to prevent further resistance.

P2.05

BRIDGING CLINICAL INSIGHTS AND LABORATORY INNOVATION: LEUKOCYTE AND PLATELET RICH PLASMA GELS IN DIABETIC FOOT ULCER MANAGEMENT
Aleksandra Olszewska*1, Cecile Dreiss1, John Hogwood2, James Rickard3, Simon Pitchford1, Ben Forbes1
1Institute of Pharmaceutical Sciences, King’s College London, London, United Kingdom; 2Medicines and Healthcare products Regulatory Agency, South Mimms, United Kingdom; 3Biotherapy Services, London, United Kingdom
Bridging Clinical Insights and Laboratory Innovation: Leukocyte and Platelet Rich Plasma Gels in Diabetic Foot Ulcer Management

Aleksandra Olszewska1, Cecile Dreiss1, John Hogwood2, James Rickard3, Simon Pitchford1, Ben Forbes1 1Institute of Pharmaceutical Sciences, King’s College London, London, United Kingdom; 2Medicines and Healthcare products Regulatory Agency, South Mimms, United Kingdom; 3Biotherapy Services, London, United Kingdom

Diabetic foot ulcers (DFUs) are a life-altering complication affecting 10%–30% of diabetic patients, significantly impairing quality of life and contributing to substantial global healthcare costs. This research addresses gaps in DFU management through a dual approach: (1) an investigation into the characteristics (co-morbidities and medication use) of the population presenting with DFU in England, and (2) laboratory characterization of a novel autologous leukocyte and platelet-rich plasma gel, RAPID™ L-PRP Biodynamic Haematogel.
An observational study preliminary analysis of 50 individuals with DFUs was conducted to evaluate demographic factors, comorbidities, and medication use. Participants completed a 26-questions questionnaire including the EQ-5D-5L score to assess quality of life. Laboratory studies aimed to characterize and optimize the novel RAPID™ L-PRP gel for DFU treatment. Gels were assessed for their formation and degradation as well as growth factor release and strength based on the activation methods (calcium chloride and/or autologous thrombin). Data were collected via thromboelastography (TEG) and scanning electron microscopy (SEM).
The observational study identified a majority of older males (76% aged 60–80 years) with recurrent DFUs (25% experiencing ≥ 5 incidents) and prolonged diabetes duration (38% with 10–20 years; 18% with 20–30 years) as the primary affected demographic. The mean quality-of-life score was 6.06/12, reflecting moderate mobility impairment. Multiple comorbidities were common (mean: 5 per patient), including hypertension (58%) and neuropathy (54%), with 22% of patients prescribed statins, 6% aspirin and 3% clopidogrel—medications known to impact platelet function. Laboratory data showed notable VEGF (685.8 ± 185.9 pg/mL) and PDGF (322.4 ± 154.3 pg/mL) release at 3 hours post-gel formation. When comparing calcium chloride and autologous thrombin activation structural differences were observed when calcium chloride was used, producing stable gels with reduced degradation rates. Thromboelastographic data showed enhanced gel strength and rapid formation when calcium chloride and autologous thrombin were combined (angle: 81.93 ± 0.45, n = 9; strength: 79.7 ± 1.49 mm). SEM imaging confirmed a denser fibrin network in calcium-activated gels.
By integrating clinical insights with laboratory experimentation, this study bridges the gap between patient needs and innovative therapeutic solutions. The clinical data outline key demographic and lifestyle factors influencing DFU outcomes, while the laboratory findings highlight the potential of tailored platelet-based therapies to correct the variability in each individual’s clotting profile to produce a consistent gel better addressing complexities of chronic wound healing. Together, these contributions lay a foundation for advancing DFU management, emphasizing personalized approaches that consider both patient-specific characteristics and cutting-edge biotechnological interventions.

P2.06

Ethacrynic Acid as a Therapeutic Agent for Reducing Hypertrophic Scar Formation via Inflammatory Pathway Modulation.
David Dolivo, Adrian Rodrigues, Thomas Mustoe, Robert Galiano, Seok Jong Hong*
Northwestern University, Wilmette, IL
Ethacrynic Acid as a Therapeutic Agent for Reducing Hypertrophic Scar Formation via Inflammatory Pathway Modulation

David Dolivo, Adrian Rodrigues, Thomas Mustoe, Robert Galiano, Seok Jong Hong Northwestern University, Wilmette, IL

Fibrosis refers to a pathological form of tissue repair where damaged tissue is replaced with nonfunctional scar tissue instead of regenerating functional pre-injury tissue. Dermal fibrosis commonly results from conditions like hypertrophic scars, keloids, and burn wounds, yet therapeutic options are limited. Mucosal wounds heal with minimal scarring, undergo faster healing, and exhibit less inflammation compared to cutaneous wounds. We have previously shown that occluded skin wounds (OSW), highly occluded by polyurethane dressings to mimic a moist mucosal environment, showed faster healing with less scarring compared to non-occluded skin wounds (NOSW). Transcriptomic analyses of the epidermises of these wounds revealed that many genes were significantly dysregulated in NOSW compared to OSW, a pattern that we refer to as the reduced hydration (RH) signature.
We used the RH to query the Connectivity Map (CMap), a tool that can predict small molecule therapeutics to reverse an input gene expression signature, and identified chemically diverse small molecule candidates predicted to ameliorate the RH response. We screened the candidate drugs for their ability to antagonize TNF-α-induced proinflammatory gene expression of HaCaT cells in monolayer culture. We determined whether these drugs were sufficient to antagonize the RH-induced epidermal response utilizing in vitro stratified keratinocyte cultures (SKCs), which recapitulate the human epidermis and contain multiple distinct strata, contain layers of keratinocytes displaying distinct differentiation markers. We investigated whether candidate drugs could ameliorate skin fibrosis in our well-characterized rabbit ear hypertrophic scar model.
We used the 699 DEGs as a query signature, which represents the changes induced by the RH condition, and subjected it to analysis by CMap, predicting nine candidate drugs with potential to reverse the RH signature. Among the candidates, ethacrynic acid (ECA) reduced pro-inflammatory gene expression in keratinocytes in vitro and in a 3-D epidermal model simulating NOSW conditions. In a rabbit ear model of hypertrophic scars, topical ECA reduced fibrosis by downregulating pro-inflammatory damage-associated molecular patterns (DAMP) proteins, such as S100A8 and S100A12, in the epidermis and the myofibroblast marker α-SMA in the dermis.
Our hypothesis is that excessive or prolonged expression of pro-inflammatory cytokines in the epidermis, caused by increased tansepidermal water loss (TEWL) and subsequent RH in NOSW, induces fibrosis and scar formation in the dermis. Our results highlight that ECA reduces hypertrophic scar formation by antagonizing pro-inflammatory pathways.

P3.01

CELLULAR VS. ACELLULAR MATRIX PRODUCTS FOR DIABETIC FOOT ULCER TREATMENT: A RANDOMIZED CLINICAL TRIAL
Harrison J. Shawa1, Ramanjot Kaur1, Catherine Tchanque-Fossuo1, Hadar Lev-Tov1,2, Kaitlyn West1, Pallas Lim1, Nuen Yang1, Mirabel Dafinone1, Rawlings E. Lyle1, Chin Chang1, David Rocke1, Sara Dahle*1, Rivkah Isseroff1
1Dermatology, Veterans Affair and UC Davis, Mather, CA; 2Dermatology, University of Miami, Miami, FL
Cellular Vs. Acellular Matrix Products For Diabetic Foot Ulcer Treatment: A Randomized Clinical Trial

Harrison J. Shawa1, Ramanjot Kaur1, Catherine Tchanque-Fossuo1, Hadar Lev-Tov1,2, Kaitlyn West1, Pallas Lim1, Nuen Yang1, Mirabel Dafinone1, Rawlings E. Lyle1, Chin Chang1, David Rocke1, Sara Dahle1, Rivkah Isseroff1 1Dermatology, Veterans Affair and UC Davis, Mather, CA; 2Dermatology, University of Miami, Miami, FL

The healing rates for chronic diabetic foot ulcers (DFU) are relatively low with the current standard of care (SOC), prompting the use of advanced wound care therapies. The purpose of the study was to determine whether a cellular matrix (CM) product resulted in better healing rates than acellular matrix (ACM) product, given associated differences in cost.
This was a federally-funded, non-industry, randomized, single-blinded, three-arm, controlled trial (cellular vs. acellular matrix to standard care) carried out in the VA Northern California Health Care System (ClinicalTrials.gov NCT01450943). Patients (aged ≥ 18 years) with a full thickness, nonhealing DFU > 0.5 < 25 cm2 who met inclusion/exclusion criteria were enrolled. After 2-week run-in period, patients were randomized to either SOC, or SOC + CM or SOC+ ACM arm. SOC included weekly sharp debridement and offloading; treatment groups had weekly SOC and either CM or ACM applied for up to 8 visits until the primary endpoint (12 weeks), after which only SOC was provided until the secondary endpoint (28 weeks). Images of wounds were scored for healing by team member blinded to treatment. Of 169 eligible patients, 138 were enrolled and 117 were randomized. For 12 weeks, patients received SOC, SOC + CM, or SOC + ACM. The primary outcome was the percentage of wounds healed by 12 weeks. Of the 117 subjects, 41 were in the CM group, 48 in the ACM group, and 28 in the SOC group. Complete re-epithelialization of the ulcer by 12 weeks occurred in 59% of the 117 total subjects: 49% in the CM group, 69% in the ACM, and 57% in the SOC group, however, these outcomes were not significantly different, p > 0.05). At 28 weeks, the percentages were 25 (61%) in the CM group, 27 (56%) in the ACM group, and 18 (64%) in the SOC group healed (not statistically different Chi-square test, p = 0.78). No differences were found in wound recidivism or adverse event occurrence between groups. CM are far more expensive than SOC or ACM, so CMs should be routinely used only if there is sufficient evidence of their improved healing performance relative to other modalities. In both the primary and secondary outcomes in this trial, the chi-square test among the three groups showed no significant difference. Minimizing using products that do not demonstrate evidence of improvement in the healing of full-thickness DFUs can reduce the economic burden.
Ultimately, improving SOC provides the best outcome for DFU treatment, but if selecting matrices, ACM appears to be the more cost-effective approach for healing DFUs.

P3.02

Development of a porcine hard-to-heal wound model and evaluation of a bromelain-based enzymatic debriding agent
Yaron Shoam*1, Adam J. Singer2
1Plastic and Reconstructive Surgery, Soroka Medical Center, Ben-Gurion University, Beer-Sheba, Israel; 2Emergency Medicine, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY
Development of a Porcine Hard-to-Heal Wound Model and Evaluation of a Bromelain-Based Enzymatic Debriding Agent

Yaron Shoam1, Adam J. Singer2 1Plastic and Reconstructive Surgery, Soroka Medical Center, Ben-Gurion University, Beer-Sheba, Israel; 2Emergency Medicine, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY

Most animal models used in wound management research focus primarily on the stages of wound healing. As a result, there is very limited focus on modeling wound eschar, which is essential for studying wound debridement. We describe here the development of a novel porcine eschar model and compare the debridement efficacy of various concentrations of a novel bromelain-based enzymatic agent with collagenase.
Full thickness excisional wounds were created on pigs and injected intradermally with various doses of doxorubicin. Wounds were monitored for a period of 46 days for the development of eschar and wound closure. After determining the optimal concentration and dose of doxorubicin resulting in delayed-healing wounds and eschars composed of necrotic tissue and slough, these conditions were used to create additional wounds on another set of animals. The resulting wounds covered with eschar were treated with various concentrations of bromelain-based enzymatic debriding agent (BBD, EscharEx formulation) or collagenase. The primary endpoint was greater than 95% removal of the eschar on the wound surface. Two distinct areas were observed after injection of doxorubicin: a central area of exudate and slough representing the hard-to heal wound bed, and a peripheral area of full-thickness mummified necrosis, which is a direct result of the model establishment. Complete removal of the central eschar was achieved in 100% of the wounds after 5 treatments with 5% BBD. Complete removal of the central eschar with collagenase ointment (SANTYL) was achieved in 82% of the wounds after 16 treatments.
We describe a novel porcine model for creating eschars similar to hard-to-heal wounds in humans. BBD was more effective and acted faster than a commercially available collagenase in removing eschars in this wound model. These results are consistent with the recently published results from the ChronEx study in patients with venous leg ulcers, demonstrating the superiority of BBD vs. collagenase ointment in the incidence and time to complete debridement.

P3.03

BROMELAIN BASED DEBRIDEMENT - MOLECULAR MODE OF ACTION
Dafna Geblinger*, Eilon Asculai
MediWound Ltd, Yavne, Israel
Bromelain Based Debridement — Molecular Mode of Action

Dafna Geblinger, Eilon Asculai MediWound Ltd, Yavne, Israel

Debridement is a crucial first step in the treatment of various wound etiologies, including burns (thermal, chemical, and electrical) and chronic wounds (such as venous leg ulcers [VLU], diabetic foot ulcers [DFU], and pressure ulcers). The composition of the eschar can vary depending on the underlying etiology and wound characteristics, as well as the phase of wound healing. It may include denatured collagen and other extracellular matrix (ECM) proteins, foreign materials, exudate components, and microbial bioburden.
To gain a better understanding of the molecular mechanisms underlying the clinical efficacy of Bromelain-Based Debridement (BBD, the active ingredient in NexoBrid and EscharEx), a mixture of proteolytic enzymes enriched in bromelain derived from the stem of pineapple, the study evaluates BBD’s ability to degrade various components of eschar and non-viable tissues.
Kinetic studies were performed using Franz cells with membranes composed of various ECM proteins to examine the degradation rates of different proteins by BBD. A faster degradation rate of a specific protein indicated a higher affinity of BBD proteases for that protein. Additionally, a separate study assessed the competition between different substrates and known inhibitors at the BBD proteases active sites. In this study, the substrate that required the highest inhibitor concentration to effectively block its degradation was determined to be the one to which BBD had the highest affinity.
These studies demonstrated that BBD degrades denatured collagen (i.e., gelatin) about twice as fast and more effectively than it does native collagen, which suggests a significantly higher affinity for gelatin compared to native collagen. Furthermore, BBD proteases effectively degrade various ECM proteins, including elastin and fibrin. Additionally, BBD demonstrates collagenase activity with a stronger affinity for collagen types I and III compared to types II and IV.
This high affinity for gelatin, the predominant component of burn eschar, facilitates the rapid and effective debridement of severe burns by BBD, as observed in clinical trials.
Fibrin is a key component of the chronic wound bed matrix. Excessive fibrin accumulation in chronic wounds can potentially impede effective healing. In clinical trials, BBD has been proven to provide rapid and effective debridement of venous leg ulcers and diabetic foot ulcers.
The findings highlight the multiple mechanisms underlying the potent efficacy of BBD in debridement of burns and chronic wounds. The enzymatic mixture composition is an important and unique attribute of BBD that enables fast and effective debridement of wounds exhibiting various forms of denatured proteins and non-vital tissues.

P3.04

PHOTOGRAPHIC GUIDANCE FOR PYODERMA GANGRENOSUM: OPTIMIZING PHOTOGRAPHY IN THE ERA OF DECENTRALIZED CLINICAL TRIALS
Morgan Vague*1, Martha Oberg2, Emile Latour1, Angelo Marzano3, Willy Huang5, Robert McLafferty6, Robert Kelly7, Elizabeth White-Chu6, Arash Mostaghimi4, Alex Ortega-Loayza1
1Dermatology, Oregon Health and Science University, Portland, OR 97239, OR; 2Washington State University College of Medicine, Spokane, WA; 3Derma, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4Dermatology, Harvard Medical School, Boston, MA; 5Dermatology, Duke University School of Medicine, Durham, NC; 6Oregon Health and Science University, Portland, OR; 7Dermatology, St. Vincent’s Hospital, Melbourne, Victoria, Australia
Photographic Guidance for Pyoderma Gangrenosum: Optimizing Photography in the Era of Decentralized Clinical Trials

Morgan Vague1, Martha Oberg2, Emile Latour1, Angelo Marzano3, Willy Huang5, Robert McLafferty6, Robert Kelly7, Elizabeth White-Chu6, Arash Mostaghimi4, Alex Ortega-Loayza1 1Dermatology, Oregon Health and Science University, Portland, OR 97239, OR; 2Washington State University College of Medicine, Spokane, WA; 3Derma, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4Dermatology, Harvard Medical School, Boston, MA; 5Dermatology, Duke University School of Medicine, Durham, NC; 6Oregon Health and Science University, Portland, OR; 7Dermatology, St. Vincent’s Hospital, Melbourne, Victoria, Australia

Pyoderma gangrenosum (PG) is a rare, neutrophilic ulcerative dermatosis characterized by recurrent, painful, and often rapidly progressing skin ulcers. Increased healthcare utilization and delayed wound healing make effective management challenging. Photo documentation provides essential insights into disease progression and treatment outcomes. However, a standardized criterion for wound photography in PG remains lacking, hindering clinical care and research. While some progress has been made with established protocols for documenting other inflammatory conditions, the absence of uniform standards for PG photography could lead to variance in how clinical care is delivered and the validity of clinical research outcomes. Herein, we examine the first steps of determining standardization of photography for PG.
This study was reviewed and approved by the OHSU IRB # 27328. Twelve patients with ulcerative PG from a single-center study were included. Each patient was positioned in a room with natural window lighting, with a black backdrop behind each wound.
Photographs of the PG wounds were captured using three devices: an iPhone 12, an iPhone 15 Pro Max, and a Canon EOS Rebel T7. For each device, images were taken at distances of 15 cm, 30 cm, and 45 cm from the wound. The images were subsequently randomized within a Qualtrics survey platform. Five experts in the management of PG and other chronic wounds were shown 6 images corresponding to 18 different wounds for a total of 180 images. They were instructed to select the 2 images most useful for clinical assessment. Results were analyzed using descriptive and agreement statistics to assess which camera type and image distance were most helpful in evaluating PG.
There was no intra-rater agreement among raters regarding the optimal image or camera type when photographing ulcerations of PG (Fleiss’ Kappa = 0.001, 95% CI: 0.030 to 0.032). However, rater survey feedback suggested distances of 15cm and 30cm were better than 45 cm. Rater feedback highlighted survey fatigue due to the high volume of images and the study design.
The lack of consensus for an optimal image distance or camera type underscores the need for determining standards using photography for PG. Compared to dermatology inpatient photography (60 cm) or hidradenitis suppurativa (50 cm), PG poses more challenges. These results highlight the need for future studies to assess how to incorporate appropriate photography methods to optimize care and research on PG.

P3.05

INTRAVENOUS FIBONECTIN (FN)-DERIVED cNP8 PEPTIDE PHASE 1 STUDY
Susan Szambelan2, Richard A. Clark*1
1NeoMatrix Therapeutics, East Setauket, NY; 2Clinical Research, NeoMatrix Therapeutics, Inc., Stony Brook, NY
Intravenous Fibonectin (FN)-Derived cNP8 Peptide Phase 1 Study

Susan Szambelan2, Richard A. Clark1 1NeoMatrix Therapeutics, East Setauket, NY; 2Clinical Research, NeoMatrix Therapeutics, Inc., Stony Brook, NY

Burns are dynamic injuries characterized by progressive extension of the depth and size of the burn over the course of several days. NeoMatrix Therapeutics (NMT) discovered a natural, partially elastase-resistant, P46 peptide. Next P46 was engineered to a peptide (cNP8) that was completely resistant to elastase, and validated cNP8’s ability to speed wound closure with a single intravenous (IV) infusion initiated 8–48 h post-burn.
This was a randomized, double-blind, placebo-controlled, single ascending dose study to evaluate the safety, tolerability, and PK profile of cNP8 in healthy male and female subjects between 18 and 65 years that met Inclusion and Exclusion Criteria. Each subject was randomized to receive either a single dose of cNP8 or placebo. Each cohort included 6 subjects dosed with cNP8 and 2 subjects dosed with placebo. The first two subjects in each cohort were dosed as sentinel subjects (one with cNP8 and one with placebo) and monitored for advers events. If no serious events occurred in the acute post-dose period (24 h), the remaining 6 subjects were dosed from lowest to highest cNP8 dose (0.001, 0.003, 0.01 and 0.03 mg/kg). Subjects received a single, approximately 30-minute IV infusion of cNP8 or placebo. Samples for serum chemistries and hematology, urinalysis, vital signs, and ECGs were obtained at several time points during the study. Blood samples for cNP8 plasma pharmacokinetic (PK) analysis were collected at pre-dose; 15, 30, and 45 min following the start of study drug infusion; and 1–12 h following the start of infusion. The pharmacokinetic profile of cNP8 will be calculated for standard PK parameters using non-compartmental methods and the actual sampling times for each subject. Summary statistics including, number (n), mean, standard deviation, median, and range (minimum, maximum) will be tabulated for the observed values and the change (or percent change) from baseline for all continuous parameters for vital signs, ECG, and laboratory tests will be reported.
All 32 subjects who entered the study received study drug, completed the study per protocol, and were included in the safety analysis. There were no deaths, SAEs, or subject discontinuations due to AEs in this study. In total, 7 subjects experienced a total of 9 AEs of which 4 were assessed to be AEs reported on/after initiation of study treatment or any pre-existing AE/condition that worsened in severity following initiation of study treatment. At post-dose timepoints, mean hematology, serum chemistry, and urinalysis parameters remained within reference ranges. After single IV cNP8 infusions of 0.001, 0.003, 0.01, and 0.03 mg/kg, mean concentrations of cNP8 peaked around 0.5 h post-infusion, which is the end of infusion. After Cmax, rapid clearances were observed across all dose groups, particularly within the first 2 h. These results support the favorable safety and PK profiles of cNP8 in healthy subjects.

P3.06

BETHLEM MYOPATHY: A UNIQUE PRESENTATION WITH DIFFUSE SPONTANEOUS KELOIDS
Anuj Budhiraja*1, Harrsion Shawa2, Alisha Mehta1, Mirabel Dafinone3, Catherine Tchanque-Fossuo4, Maija Kiuru4, Sara Dahle5, Rivkah Isseroff4
1California Northstate University, Elk Grove, CA; 2Dermatology, Washington University in St. Louis, St Louis, MO; 3University of Nevada Reno, Reno, NV; 4Dermatology, University of California, Davis, Sacramento, CA; 5Podiatry, VA Northern California Health Care System, Mather, CA
Bethlem Myopathy: A Unique Presentation With Diffuse Spontaneous Keloids

Anuj Budhiraja1, Harrsion Shawa2, Alisha Mehta1, Mirabel Dafinone3, Catherine Tchanque-Fossuo4, Maija Kiuru4, Sara Dahle5, Rivkah Isseroff4 1California Northstate University, Elk Grove, CA; 2Dermatology, Washington University in St. Louis, St Louis, MO; 3University of Nevada Reno, Reno, NV; 4Dermatology, University of California, Davis, Sacramento, CA; 5Podiatry, VA Northern California Health Care System, Mather, CA

Bethlem myopathy, a rare genetic disorder affecting approximately 1 in 129,870 individuals, is caused by mutations in the COL6 genes. Cutaneous manifestations of Bethlem myopathy are poorly characterized and include hyperkeratosis and atrophic scars. This study aims to explore the pathogenic link between Bethlem myopathy and diffuse, spontaneous keloid formation. We describe a case of a patient with diffuse, spontaneous keloid formation unresponsive to treatment. Clinical evaluation included dermatologic and genetic assessments. DNA from keloid fibroblasts underwent whole genome sequencing on the Illumina HiSeq 4000 platform, with raw reads aligned to the GRCh38 reference genome and variants identified using the DRAGEN FPGA pipeline. Bioinformatic analyses explored candidate genes implicated in keloid formation.
The patient was diagnosed with BM based on clinical presentation and confirmation of a COL6A3 mutation via genetic testing. Genetic and bioinformatic analyses identified 12 potential keloid-implicated genes, suggesting a multifactorial model for keloid pathogenesis. These findings highlight a novel pathogenic link between COL6A3 mutations and keloid formation in Bethlem myopathy.
This study identifies a multifactorial pathogenic relationship between Bethlem myopathy and keloid formation. These findings expand the clinical phenotype of BM to include keloid formation and emphasize the importance of recognizing cutaneous manifestations for accurate diagnosis and management of this rare genetic disorder.

P4.01

FIBROBLAST BIOLOGICAL AGE DIRECTS ECM REMODELING AND CONTRACTION IN TISSUE ENGINEERED DERMAL MODELS
Autumn Campbell*1, Divya Sridharan2, Pragyan Paramita1, Syed Ashraf3, Dorothy Supp4, Mahmood Khan2, Heather Powell1,5
1Materials Science and Engineering, The Ohio State University, Columbus , OH; 2Emergency Medicine, The Ohio State University, Columbus, OH; 3Health Sciences, The Ohio State University, Columbus, OH; 4Surgery, University of Cincinnati, Cincinnati, OH; 5Biomedical Engineering, The Ohio State University, Columbus, OH
Bethlem Myopathy: A Unique Presentation With Diffuse Spontaneous Keloids

Anuj Budhiraja1, Harrsion Shawa2, Alisha Mehta1, Mirabel Dafinone3, Catherine Tchanque-Fossuo4, Maija Kiuru4, Sara Dahle5, Rivkah Isseroff4 1California Northstate University, Elk Grove, CA; 2Dermatology, Washington University in St. Louis, St Louis, MO; 3University of Nevada Reno, Reno, NV; 4Dermatology, University of California, Davis, Sacramento, CA; 5Podiatry, VA Northern California Health Care System, Mather, CA

Bethlem myopathy, a rare genetic disorder affecting approximately 1 in 129,870 individuals, is caused by mutations in the COL6 genes. Cutaneous manifestations of Bethlem myopathy are poorly characterized and include hyperkeratosis and atrophic scars. This study aims to explore the pathogenic link between Bethlem myopathy and diffuse, spontaneous keloid formation. We describe a case of a patient with diffuse, spontaneous keloid formation unresponsive to treatment. Clinical evaluation included dermatologic and genetic assessments. DNA from keloid fibroblasts underwent whole genome sequencing on the Illumina HiSeq 4000 platform, with raw reads aligned to the GRCh38 reference genome and variants identified using the DRAGEN FPGA pipeline. Bioinformatic analyses explored candidate genes implicated in keloid formation. The patient was diagnosed with BM based on clinical presentation and confirmation of a COL6A3 mutation via genetic testing. Genetic and bioinformatic analyses identified 12 potential keloid-implicated genes, suggesting a multifactorial model for keloid pathogenesis. These findings highlight a novel pathogenic link between COL6A3 mutations and keloid formation in Bethlem myopathy. This study identifies a multifactorial pathogenic relationship between Bethlem myopathy and keloid formation. These findings expand the clinical phenotype of BM to include keloid formation and emphasize the importance of recognizing cutaneous manifestations for accurate diagnosis and management of this rare genetic disorder.

P4.02

Role of pH and bile acids in modulating protease activity and implications for peristomal skin damage
Alexander Leininger2, Monika Pawlikowski2, Iman Hamid2, Amaan Faruqi2, Ebaad Rehman2, Zohaib Farooq2, Madhavan Narayanan2, Jayant Joshi1, Abram D. Janis*1, Jayashree Sarathy2
1Hollister Incorporated, Libertyville, IL; 2Biological Sciences, Benedictine University, Lisle, IL
Role of pH and Bile Acids in Modulating Protease Activity and Implications for Peristomal Skin Damage

Alexander Leininger2, Monika Pawlikowski2, Iman Hamid2, Amaan Faruqi2, Ebaad Rehman2, Zohaib Farooq2, Madhavan Narayanan2, Jayant Joshi1, Abram D. Janis1, Jayashree Sarathy2 1Hollister Incorporated, Libertyville, IL; 2Biological Sciences, Benedictine University, Lisle, IL

Peristomal Skin Complications (PSC) are a significant concern in > 40% of ostomy patients, often resulting from prolonged exposure to enzymatically active effluent containing pancreatic proteases (trypsin (trp), chymotrypsin (ctrp), and elastase (ela)) and unconjugated primary BA, like chenodeoxycholic acid (CDCA). Our previous work demonstrated that CDCA induces oxidative stress and cytokine production in human colonic epithelial cells. Further, altered luminal pH and bile acid profiles, common in inflammatory bowel disease, may worsen skin damage. Based on these findings, we hypothesize that CDCA modulates pancreatic protease activity in a pH- and dose-dependent manner, and thereby may contribute to stoma-associated epithelial damage. Protease activity was measured across a pH range (4.0–8.3) using an Invitrogen EnzCheckTM fluorescence-based assays for trp, ctrp, and ela, individually and in combination. The effect of CDCA (50–250 μM) on protease activity at different pH values were then quantified, enzyme kinetics parameters (Km and Vmax) analyzed using Michaelis-Menten models and reported as percentages relative to their controls. Human colonic T84 cells were treated with CDCA (25–250 μM) and protease cocktails (10%–25%) at varying pH to evaluate cell death and apoptosis via Annexin-V (AV) and propidium iodide (PI) staining. Examination of the effect of pH on protease activity revealed that it was pH-dependent, with maximum activity at pH 8.3. Reducing pH resulted in significant activity decreases (pH 8.3: 100%, 7.4: 85%, 6.0: 65%, 5.0: 58%, and 4.0: 49%). Protease cocktails consistently exhibited the highest activity at pH 8.3. Next, we identified that CDCA dose-dependently inhibited protease activity, with a pronounced 70% reduction at pH 4.0 and 250 μM CDCA. At all pH values, the Lineweaver-Burk plot shows that CDCA reduced both Vmax and Km (50%) values, indicative of mixed, non-competitive inhibition. CDCA induced dose-dependent cell death and apoptosis in T84 cells, which were exacerbated by proteases (e.g., pH 7.4, CDCA 100 μM: 12 ± 4%; CDCA + 10% protease cocktail: 18 ± 4%). Interestingly, this apoptosis was slightly mitigated at lower pH (e.g., pH 5.0, CDCA + 10% protease cocktail: 15 ± 4%). Thus, at acidic pH and physiological dose, CDCA plays a role in reducing enzymatic activity and mitigating cell damage. Our findings suggest that mildly acidic conditions could protect peristomal skin by decreasing protease activity, potentially reducing the incidence and severity of PSC. Incorporating pH-lowering strategies may serve as a novel therapeutic approach to mitigate skin irritation and improve quality of life for ostomy patients. Ongoing studies in skin cell models will further elucidate these interactions and inform targeted interventions.

P4.03

Natural Biomaterial Dressing Advances Pressure Wound Healing after Spinal Cord Injury
Suneel Kumar*, Dhruv Patel, Greeshma Manjunath, Dnyaneshwari Rananavare, Shashank Madhavan, Noshir Langrana, Francois Berthiaume
Biomedical Engineering, Rutgers University, Piscataway, NJ
Natural Biomaterial Dressing Advances Pressure Wound Healing after Spinal Cord Injury

Suneel Kumar, Dhruv Patel, Greeshma Manjunath, Dnyaneshwari Rananavare, Shashank Madhavan, Noshir Langrana, Francois Berthiaume Biomedical Engineering, Rutgers University, Piscataway, NJ

Pressure wounds are among the secondary complications after spinal cord injury (SCI) that hinder an individual’s social, psychological, and physical well-being. Studies have shown that pressure wounds below the SCI site are especially prone to heal slowly or not heal at all, further spotlighting the severity of these wounds. While many preventive measures and therapeutic options are available for those facing pressure wounds, they have high recurrence rates and can prove to be costly to treat for many patients. A low-cost, easy-to-use treatment modality that promotes wound healing progression under such suboptimal circumstances is necessary. We developed and proposed the utilization of natural biomaterials-based wound dressing applied to pressure wounds to accelerate the progression of wound healing in SCI mice. Polyelectrolyte complexes (PEC) films were made from a combination of medical-grade chitosan (98% deacetylated), polygalacturonic acid (PgA), and polyethylene glycol (PEG) solutions. Both chitosan and PgA solutions were prepared in separate tubes and placed on a nutator overnight. Following nutation, both solutions were combined in a 60:40 volumetric ratio and sonicated. The pH of the individual samples and after combinations were recorded. Then 0.1 M of PEG solution was added, and an additional 30 seconds of sonication was performed before pouring the solution into a circular dish to dry. PEC films were then sterilized in the UV Crosslinker for 15 min on each side. The dressing was investigated in experimentally induced full-thickness wounds on male and female mice with complete SCI (T10-11). The circular piece of dressing was placed over the wound and then covered with Tegaderm dressing. The wounds were photographed on days 0, 3, 7, and then weekly. At the healing endpoint, mice were sacrificed and wound tissue including the scar area was excised and processed for histology and immunohistochemical studies. Our data suggested that PEC and PEC-PEG dressings significantly enhance wound closure in male and female mice after SCI in terms of time and the rate of wound closure. However, female mice’s healing time window was faster overall. Histology analysis revealed significant differences in wound width, epidermis and dermis thickness, number of macrophages, blood vessel formation, cell proliferation, and extracellular matrix deposition (p < 0.05) when the biomaterials were used as treatment compared to non-treated SCI control wounds. The presence of PEG in PEC dressing enhances the blood vessel formation in both male and female mice in comparison to PEC alone. In contrast, the same biomaterial dressing does not affect the healing of similar skin wounds in non-SCI mice. Our findings demonstrate that these natural biomaterials combined into wound dressings significantly improve skin wound healing mechanisms and present a promising treatment option for promoting pressure wound healing in SCI patients.

P4.04

PEPTIDE-BASED BIOMIMETIC MATRIX OFFERS ANTIMICROBIAL PROTECTION AND RAPID WOUND CLOSURE OF STALLED DIABETIC FOOT ULCERS
Ana Tellechea*1, Brunno Caetano1, Trudy-Ann Grant1, Bishnu P. Joshi1, Nivedha Suresh1, Rebecca Salamone1, Daljit Kaur1, Tarak Bakhda1, Sumedha Raut1, Manav Mehta1, Sara Rose-Sauld2,3, Jennifer Skolnik2,3, Adam Landsman2,3
1Gel4Med, Lowel, MA; 2Massachusetts General Hospital, Boston, MA; 3Harvard Medical School, Boston, MA
Peptide-Based Biomimetic Matrix Offers Antimicrobial Protection and Rapid Wound Closure of Stalled Diabetic Foot Ulcers

Ana Tellechea1, Brunno Caetano1, Trudy-Ann Grant1, Bishnu P. Joshi1, Nivedha Suresh1, Rebecca Salamone1, Daljit Kaur1, Tarak Bakhda1, Sumedha Raut1, Manav Mehta1, Sara Rose-Sauld2,3, Jennifer Skolnik2,3, Adam Landsman2,3 1Gel4Med, Lowel, MA; 2Massachusetts General Hospital, Boston, MA; 3Harvard Medical School, Boston, MA

Approximately 60% of diabetic foot ulcers (DFUs) become infected, substantially increasing morbidity and raising the rate of lower extremity amputations up to 90%. Current approaches have serious limitations, and the rise of multidrug-resistant organisms (MDROs) and biofilms further complicates treatment. To address this issue, we developed a biomimetic matrix (BMM) that prevents infection while promoting healing. BMM is a synthetic self-assembling cationic peptide matrix designed to (i) provide antibacterial protection through a mechanism that evades microbial resistance and (ii) encourage tissue regrowth via a flowable 3D scaffold with cell attachment sites that eliminates dead space. Preclinical: Efficacy against several clinically relevant bacterial and fungal pathogens was tested by in vitro assays. Efficacy against 72 h-aged Pseudomonas aeruginosa (PAO1) biofilms was evaluated ex vivo [porcine skin]. MRSA-inoculated murine wounds treated with BMM were assessed by microbiology. In a swine model of full-thickness excisional wounds, BMM healing efficacy was tested vs. silver and collagen wound products using the Tissue Analytics platform and histopathology. Clinical: In a prospective case series, eight patients with Wagner 1–3 DFUs that failed to respond to previous treatments were selected to receive BMM and monitored for wound healing progress. Preclinical: In vitro, BMM eliminated ≥ 6 log10 CFU of Gram-positive and Gram-negative clinical isolates, as well as sporulating and non-sporulating fungal pathogens within 24 h (p < 0.0001, n = 3). Notably, a single BMM application eradicated mature PAO1 biofilms in pig skin explants by 24 h (p < 0.0001, n = 3). Rapid bioburden reduction was confirmed in MRSA-inoculated murine full-thickness wounds (p = 0.02, n = 10). BMM-treated full-thickness excisional wounds in swine showed greater closure rates (p = 0.01, n = 5) and reduced inflammation compared to commercial collagen and silver dressings, singularly achieving full re-epithelialization and healthy granulation tissue repletion by day 14. Clinical: All patients (n = 8) responded positively with granulation tissue formation and progress towards wound closure; one DFU fully closed with a single BMM application. Six weeks after treatment [1–3 applications], an average percent area reduction of 64% was achieved in DFUs originally measuring 7.5 cm2 and present for 21.5 months on average. Odor, drainage, inflammation, and wound depth were noticeably reduced. No adverse effects were observed. BMM demonstrates broad-spectrum activity against MDROs and biofilms. In the clinically relevant swine model, BMM shows superior re-epithelialization, granulation tissue formation, and inflammation resolution when compared to silver and collagen wound products. Clinical outcomes confirm rapid healing progression of challenging DFUs, suggesting benefits in hard-to-heal wound management. Further studies are needed to validate and expand our findings.

P4.05

Topical Osteopontin Accelerates Diabetic Wound Healing
Eda Kurtulmus1, Aditi Dantuluri1, Georg Weber2, Daria Narmoneva*1
1Biomedical Engineering, University of Cincinnati, Cincinnati, OH; 2Cancer Institute, College of Pharmacy, University of Cincinnati, Cincinnati, OH
Topical Osteopontin Accelerates Diabetic Wound Healing

Eda Kurtulmus1, Aditi Dantuluri1, Georg Weber2, Daria Narmoneva1 1Biomedical Engineering, University of Cincinnati, Cincinnati, OH; 2Cancer Institute, College of Pharmacy, University of Cincinnati, Cincinnati, OH

The occurrence of ulcers in diabetes constitutes a substantial worsening of the prognosis. Due to the metabolic alterations in diabetic patients, their healing is delayed, with poor vascular supply and infection being significant factors that contribute to impaired healing. The early type 1 (Th1) inducer cytokine Osteopontin (OPN) has been studied in various modalities of wound healing and may be supportive of the process. The objective of this study is to determine the effect of topical treatment with OPN or OPN peptide containing its key integrin binding domain (OPNp), locally administered in a hydrogel formulation, on diabetic wound healing in the db/db mouse model. Controlled release (up to 80%–90% over 7–10 days) of both OPN as well as OPNp was confirmed in vitro. 6 mm dorsal excisional full-thickness skin wounds were treated with OPN or OPNp (embedded in the nanofiber hydrogel) or hydrogel alone (2 wounds per animal, n = 6 animals per group for the OPN experiment and n = 3 animals/group for the OPNp experiment). The immune cell profile was analyzed by flow cytometry from whole blood 3 days after wounding. Wounds were photographed sequentially during healing and harvested at days 7 and 21. Upon termination of the experiment on day 21, tissue was harvested. Wound closure, morphology and vascularization were quantified using H&E and lectin staining. We observed significantly faster wound closure in the OPN-treated wounds as compared to the hydrogel controls, while a similar trend in the OPNp-group fell short of significance (possibly due to lower power). OPN treatment improved wound bed vascularization and formation of hair follicles, indicative of regenerative healing. The OPNp administration increased the CD4+ fraction of T-cells (labeled with anti-CD3), while there was no significant change in the myeloid cell composition (marked by CD11b, CD45, and Gr1). Studies are ongoing to further quantify the effects of OPN and OPNp treatment on wound morphology and immune cell infiltration. These results support the promise of OPN as a therapeutic agent to improve diabetic wound healing via mechanisms to be fully elucidated (induction of the cellular immune response may be a factor). While the OPNp peptide did not significantly accelerate the closure of the wounds (implying that the Integrin-binding domain alone may not be sufficient), it did modulate the immune response as reflected in an increased CD4+ subset of T-cells in the blood. Further investigations will optimize the OPN domains to be delivered for wound healing support in diabetes.

P4.06

ANTIBACTERIAL AND IMMUNOMODULATORY FOAM SCAFFOLDS FOR THE TREATMENT OF BURNS
Kristo Nuutila*1, Anders Carlson2, Sean Christy1, David Larson3, Mohamadmahdi Samandari4, Ali Tamayol4, Tannin Schmidt4
1USAISR, San Antonio, TX; 2Metis Foundation, San Antonio, TX; 3UT Health San Antonio, San Antonio, TX; 4UConn, Farmington, CT
Antibacterial and Immunomodulatory Foam Scaffolds For The Treatment of Burns

Kristo Nuutila1, Anders Carlson2, Sean Christy1, David Larson3, Mohamadmahdi Samandari4, Ali Tamayol4, Tannin Schmidt4 1USAISR, San Antonio, TX; 2Metis Foundation, San Antonio, TX; 3UT Health San Antonio, San Antonio, TX; 4UConn, Farmington, CT

The management of high-energy blast injuries and burns is complicated by high rates of soft tissue contamination and prolonged delays to bedside care. Such burns often result in infections, as such the timely delivery of antimicrobials, along with debridement and wound covering with healing agents is crucial. Yet, this is not feasible in an austere battlefield environment and poorly healed burns can significantly reduce quality of life. Therefore, immediate treatment of burns using antimicrobial and immunomodulatory scaffolds is a significant but unmet need. The purpose of this study was to develop a simple and robust strategy for in situ fabrication and delivery of microporous gelatin methacryloyl (GelMA) scaffolds releasing minocycline, a broad-spectrum antibiotic, and proteoglycan 4 (PRG4), an immunomodulatory protein. Scaffold was fabricated by combining GelMA with lithium phenyl-2,4,6- trimethylbenzoylphosphinate photoinitiator (LAP), followed by foaming using a micromesh- equipped double-syringe. The foam was then deposited in situ and crosslinked with blue light. The concentration of the components was optimized in vitro using mechanical testing, stability evaluation, degradation assessment, release kinetics, biocompatibility testing, antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), and anti- inflammatory effects on RAW macrophages. Subsequently, the GelMA foam was tested in a porcine deep partial-thickness (DPT) burn model. Up to 20 standardized DPT burns were created on the dorsum of four anesthetized pigs. Analgesia was provided prior to all surgical procedures with buprenorphine SR/ER. Burns on each animal were randomized into four experimental groups: (1) GelMA; (2) GelMA + minocycline; (3) GelMA + PRG4; (4) GelMA + minocycline + PRG4; (5) Silvadene. The animals were followed for 3, 7, 14 or 21 days. After euthanasia, the burns were excised for histologic analyses. Non-invasive imaging was utilized to assess burns macroscopically. An optimized precursor containing 15% GelMA, 0.4 mg/ml minocycline, 1mg/ml PRG4, and 0.67% LAP was identified with 100 μm pore size, 67% porosity, 5 kPa compression modulus, biodegradability, long term stability (> 14 days), a sustained release of PRG4 (> 14 days), and a rapid release of minocycline (24 h). The optimized composite was biocompatible with human dermal fibroblasts and murine myoblast, and significantly reduced in inflammatory markers (TNF-α) secretion from activated macrophages. The scaffolds were able to reduce MRSA-bacterial load by > 5. Porcine studies confirmed scaffolds prevented infection, and improved quality of healing. We developed a highly translational robust strategy for the immediate treatment of burn wound via in situ fabrication of adherent macroporous scaffolds enabling cell migration and regeneration, while releasing compounds to prevent infection and reduce inflammation associated scarring.