Dates
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8:00 AM - 8:15 AM
Geoffrey C. Gurtner, MD, FACS; Elizabeth A. Grice, PhD; Katherine Radek, PhD
8:15 AM - 9:15 AM
Randy W. Schekman, PhD
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9:15 AM - 9:30 AM
9:30 AM - 11:00 AM
Victor Nizet, MD; Marvin Whitely, PhD; Irena Pastar, PhD
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11:00 AM - 11:15 AM
11:15 AM - 12:45 PM
Geoffrey C. Gurtner, MD, FACS; Sundeep G. Keswani, MD; Latha Satish, PhD
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12:45 PM - 2:00 PM
Vickie Driver, DPM; Mitchell Sanders, PhD; Gary Robinson, PhD
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2:00 PM - 3:30 PM
Rivkah Isseroff, MD; Daniel Kaplan, MD; Sangeeta Chavan, PhD
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3:30 PM - 3:45 PM
3:45 PM - 5:15 PM
Kara L. Spiller, PhD; Kaitlyn Sadtler, PhD; Karen L. Christman, PhD, FAHA
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5:15 PM - 6:00 PM
6:30 PM - 9:30 PM
7:30 AM - 9:00 AM
9:00 AM - 9:15 AM
9:15 AM - 10:30 AM
10:30 AM - 10:45 AM
10:45 AM - 11:45 AM
Ryoichi Mori, MD; Munezumi Fujita, MD
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11:45 AM - 12:00 PM
12:00 PM - 1:30 PM
1:30 PM - 1:45 PM
1:45 PM - 4:00 PM
Geoffrey C. Gurtner, MD, FACS; Alan Wells, MD DMSc; Ulrich auf dem Keller, PhD
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4:00 PM - 4:15 PM
4:15 PM - 5:15 PM
Geoffrey C. Gurtner, MD, FACS; Alan Wells, MD DMSc; Ulrich auf dem Keller, PhD
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Adrian Barbul, MD; Sundeep Kerwani, MD
Swathi Balaji, PhD; Latha Satish, PhD
Timothy W. King, MD, PhD; Kanhaiya Singh, PhD
Boris Hinz, MD; Erin O'Brien, MS
5:15 PM - 7:45 PM
WHS Exhibit Booth #
7:30 AM –9:00 AM
9:00 AM - 9:15 AM
9:15 AM - 10:15 AM
Brian Eliceiri, PhD; Alan Wells, MD, DMsC
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10:15 AM - 10:30 AM
10:30 AM - 11:30 AM
Oral presentations will feature the highest scoring abstracts submitted to the WHS.
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Daria A. Narmoneva, PhD; Moderator TBD
Jeffrey W. Shupp, MD; Robel Beyene, MD
Anie Philip, PhD; Maha Alsharqi, PhD
Jaideep Banerjee, PhD; Amitava Das, PhD
11:30 AM - 2:00 PM
12:15 PM - 2:00 PM
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2:00 PM - 2:15 PM
2:15 PM - 3:15 PM
Kyle P. Quinn, PhD; Katherine Gallagher, MD
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3:15 PM - 3:30 PM
3:30 PM - 4:20 PM
Piul Rabbani, MD
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4:20 PM - 4:30 PM
4:30 PM - 4:45 PM
4:45 PM - 5:15 PM
Heather Powell, PhD; Angela Gibson, MD, PhD
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5:15 PM - 5:30 PM
5:30 PM - 6:30 PM
Antonio De Maio, PhD, FCCSI; Julie Jameson, PhD
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6:30 PM - 8:00 PM
**POSTER PRESENTERS SHOULD ATTEND THIS ENTIRE EVENT**
8:00 PM - 9:00 PM
(Omni Hotel Bar)
7:30 AM –9:00 AM
9:00 AM - 9:15 AM
9:15 AM - 10:15 AM
Mayumi Ito, PhD; Joshua Currie, PhD
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10:15 AM - 10:30 AM
10:30 AM - 11:30 AM
Oral presentations will feature the highest scoring abstracts submitted to the WHS.
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Richard Schugart, PhD; Lisa Tucker-Kellogg, PhD
Harvey N. Himel, MD, MPH; Moderator TBD
Mithun Sinha, MD; Norifumi Urao, MD, PhD
Praveen R. Arany, DDS PhD; Mohamed Ibrahim, MD
11:30 AM - 12:30 PM
Ivan Jozic, PhD
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12:30 PM
12:30 PM - 2:00 PM
M1.01.
BACKGROUND: Burn injuries often heal through abnormal wound healing mechanisms and result in hypertrophic scar (HTS) with dyschromia. The mechanism behind HTS dyschromia has not been studied and treatments are lacking. In previous work, we showed that regions of hypo-pigmented scar contain inactive melanocytes. Normal skin cells make melanin through keratinocyte secretion and binding of alpha-melanocyte stimulating hormone (α-MSH) to melanocyte melanocortin receptors to initiate melanogenesis by tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), and dopachrome tautomerase (DCT). In previous work, we showed that α-MSH expression is absent in hypo-pigmented scar. A nude mouse model of xenografted porcine dyschromic HTS was developed to test if supplying hypo-pigmented cells with α-MSH can re-pigment the scar.
METHODS: Dyschromic HTSs were created in Duroc pigs. Epidermal cells were derived from regions of hyper-, hypo-, or normally pigmented scar or skin. Dermal fibroblasts (DFs) were isolated separately. Wounds were created on nude mice and a grafting dome was placed. DFs were seeded on day 0. Epidermal cells were seeded on day 3. The dome was removed on day 7 and hypo-pigmented xenografts were treated with synthetic α-MSH delivered with microneedling. On day 10, the xenografts were excised. Sections were stained using H&E. RNA was isolated and qRT-PCR was performed for TYR, TYRP1, and DCT.
RESULTS: HTSDFs formed a dermis similar in structure and cellularity to HTS dermis from porcine HTS. When hyper-, hypo-, and normally-pigmented epidermal cells were seeded, a fully stratified epithelium was formed. In the xenograft, epidermal thickness by H&E staining was 0.11 vs. 0.06 μm in normal pig skin. Treated hypo-pigmented xenografts showed increased pigmentation and had increased transcription of TYR, TYRP1, and DCT compared to controls (TYR: 2.7 vs 0.3; TYRP1: 2.6 vs 0.3; DCT: 0.7 vs 0.3 fold change from control; n=3).
CONCLUSIONS: The developed nude mouse scar xenograft model can be used to study treatments for dyschromia. Hypo-pigmented regions of burn scar can be stimulated to make melanin by synthetic α-MSH.
M1.02.
BACKGROUND: The ethiology of chronic wound initiation and development is not well understood and to date therapeutic agents have only been successful in improving prognosis in 50 to 60% of the patients. Because development of wound chronicity is complex, a multiomic approach is needed to understand the difference in gene expression between non-chronic and chronic wounds.
METHODS: We performed an RNASeq analysis using skin tissue from non-chronic and chronic wounds in db/db-/- mice at days (D) 1, D10 and D20 after stimulation of chronicity. A Weighted Gene Correlation Network Aalysis (WGCNA) was performed in R on 9983 genes identified in the RNAseq.
RESULTS: Genes were grouped into 25 modules based on expression values. 16 modules were significantly correlated to chronic wounds (p value <0.01) at D1(mild chronicity), 12 modules at D10 (moderate chronicity) and 16 modules at D20 (severe chronicity). Gene Ontology analysis for modules associated with mild chronicity showed enrichment for receptor tyrosine kinase (RTK) genes, specifically the ephrin receptor (EphR), the largest family of RTK, which uses PDZ domain-containing proteins, such as DKK1, whose transcript was also enriched at this stage. This protein is important in regulating gene expression and antagonizes the canonical Wnt signaling pathway by reducing β-catenin which could explain why we find massive deregulation of gene expression on D1 post-wounding. In moderate chronicity, biological processes that release sequestered Ca++ from the endoplasmic reticulum (ER) were enriched. Ca++ release may come from irreversible depolymerization of Calsequestrin1 due to Ca++ depletion in the ER. This may explain why the wound becomes dysfunctional. By severe chronicity, Hedgehog (Hh) signaling through the Smoothened receptor (SMOR) and granzyme-mediated cell death pathways, were found enriched.
CONCLUSIONS: The negative regulation of Smoothened mediated Hh signaling, a pathway studied extensively for cancer treatment, maybe accompanied by mutation of the SMOR which confers acquired resistance to drugs. This suggests that Hh and its signaling pathway through SMOR, could be a good target to understand how chronic wounds become resilient to treatment.
M1.03.
M1.04.
BACKGROUND: Skin fibrosis manifests clinically at the site of a dermal wound or sclerodermatic lesion, depending on the etiology of the condition in question. Though fibrotic disease states differ somewhat in their causes and manifestations, several common factors underlie the development of fibrosis, including myofibroblast differentiation and deposition of collagen, and research of recent decades has borne substantial progression in our understanding of the pathophysiology of fibrosis. Nevertheless, there remains a dearth of available fibrotic therapies due in large part to positive feedback loops between the macroscale pathological changes in the mechanical and biochemical properties of fibrotic tissues and the signal transduction pathways driving these tissue processes, as well as the pleiotropy and functional redundancy of the major pathways governing fibrotic progression. Thus, investigation of novel pathways driving the etiologies of fibrotic diseases is of paramount importance. We have previously demonstrated that in vivo knockdown of the atypical voltage-gated sodium channel Nax (Scn7a) results in amelioration of inflammation and tissue fibrosis in several animal models.
METHODS: We utilized a common murine scleroderma model induced by subcutaneous injection of bleomycin in both wild type mice and mice lacking functional Nax. We assessed skin thickening and changes in skin phenotypes using histological analysis and compared them between mice of these genotypes. Since previous analysis of this model has also demonstrated development of lung fibrosis, and since pulmonary expression of Nax has previously been described, we also analyzed the development of fibrosis in the lung using histological analysis and immunohistochemistry to stain for collagen and α-SMA .
RESULTS: Preliminary data demonstrate that mice lacking functional Nax resist development of scleroderma as assessed by dermal thickening. Mice lacking functional Nax also demonstrate a lesser degree of lung fibrosis as assessed by histological analysis of collagen deposition, qualitative tissue cellularity, and immunohistochemical expression of α-SMA.
CONCLUSIONS: Taken together, these data suggest that Nax may be causally involved in tissue fibrosis, and that targeting of Nax may represent a novel therapeutic modality for fibrotic tissue pathologies.
M1.05.
BACKGROUND: The study of fibrosis has typically followed the pattern of utilizing a model with a consistent injury and following up at specified time points after injury. The tissue status and molecular profile at these various time points are then often compared to the uninjured tissue and differences are considered to be related to the fibrotic process. However, substantial biological variance has been observed in injury models in terms of actual fibrotic outcomes. This variance, termed “biological variance”, can lead to the masking of the key regulators of fibrosis or an attenuation in statistical power in studies seeking to reduce scarring. While biological variance can hinder research and translation, it can also be used as a powerful tool to reduce complex dataset and reveal the core regulatory networks which are active during active fibrosis.
METHODS: A rabbit corneal lamellar keratectomy model was used to injure 20 corneas on 10 rabbits. The scar formation process initially presents as a dense light reflecting cellular haze which develops between days 5 and 14 after injury. The haze can be followed and quantified via an automated image analysis technique, and the haze formation rate can be calculated from two successive follow-up points during the formation process. Corneas presenting with no haze growth were segregated from those with high levels of haze growth prior to analysis of mRNA via RNASeq. Reads were mapped to the most recent version of the OryCun 2.0 assembly, and a differential gene expression analysis was performed.
RESULTS: Approximately 15,000 genes out of the approximately 33,000 known genes were mapped to the rabbit genome. Of these 15,000, only 399 were differentially expressed between corneas with active fibrosis versus those without. Two growth factors: placental growth factor (PGF, p = 0.0002) and vascular endothelial growth factor A (VEGF-A, p = 5×10-5) were the only so-named growth factors differentially regulated, along with a cluster of genes highly associated with cells derived from the myeloid lineage. None of the corneas presented with angiogenesis from the limbus.
CONCLUSIONS: Common pro-fibrotic factors such as TGF-beta and CTGF were not found to be differentially expressed during the later, active fibrotic phase, whereas two growth factors typically associated angiogenesis and vasculogenesis were highly differentially expressed, but without angiogenic activity. Results from other studies indicate a potential pleiotropic role for these factors and our data support these findings.
M1.06.
BACKGROUND: Disruption of the skin barrier results in delayed wound healing, hypertrophic scar formation, and the development of various skin diseases. Perturbation of the skin barrier causes up-regulation of inflammatory cytokines. Nax (SCN7A) is an atypical voltage-gated sodium channel and acts as a sodium concentration-sensitive channel in the epidermis. Both atopic dermatitis (AD) and psoriasis are chronic inflammatory skin diseases denoted by marked infiltration of inflammatory cells. Though the causes of AD and psoriasis are multifactorial, defective skin barrier function is a major contributor to the pathogenesis of both diseases.
METHODS: We have established AD-like and psoriasis-like models in rabbit ear skin by application of house dust mite extract and imiquimod, respectively, and evaluated the effects of RNAi-mediated Nax knockdown on inflammatory phenotypes in these models. Epidermal hyperplasia, keratinocyte hyperproliferation, and inflammatory cell infiltration were determined by histology and immunohistological analysis. Erythema, scaling, and papule formation were assessed by visual analysis. Expression of S100A9 was quantified by immunohistological analysis.
RESULTS: We found that in vivo knockdown of Nax using RNAi reduced epidermal hyperplasia and keratinocyte hyperproliferation in AD-like rabbit ear skin. Knockdown of Nax also decreased skin erythema, scaling, and papule formation in psoriasis-like rabbit ear skin. Infiltration of inflammatory cells induced by house dust mite extract was reduced in dermatitis skin upon knockdown of Nax. Expression of S100A9, a downstream pro-inflammatory gene target of Nax and a mediator of AD and psoriasis, was also decreased by Nax-RNAi. We also showed that inhibition of Nax relieved dermatitis symptoms in vivo.
CONCLUSIONS: Taken together, our data indicate that inhibition of Nax relieved dermatitis symptoms in vivo, suggesting that Nax is a novel therapeutic target for AD and psoriasis, which currently have limited therapeutic options.
M1.07.
M1.08.
BACKGROUND: Heart failure (HF) remains a significant health issue leading to approximately 1 million hospitalizations and 300,000 deaths annually in the United States. While acute myocardial injury results in cell death and the progression of replacement fibrosis, chronic myocardial insult causes interstitial fibrosis. Our current understanding of replacement versus interstitial fibrosis is limited without any molecular and contextual understanding.
METHODS: Canonical Wnt/beta-catenin signaling has been linked to fibrosis in multiple organ injury models. To investigate Wnt signaling pathway in the context of interstitial and replacement fibrosis, we generated a transgenic mouse to activate beta-catenin in activated fibroblasts, the major cellular drivers of fibrosis.
RESULTS: Canonical Wnt signaling activation significantly promoted maladaptive interstitial fibrosis, a decline in cardiac function, and an increase in the heart failure marker ANF following pressure overload injury; however, no significant functional outcomes or increased fibrosis were observed following Wnt activation in the setting of ischemic injury. Wnt activation resulted in increased numbers of CD45-positive immune cell populations, specifically higher percentage of macrophages and cytotoxic T-cells, following TAC in comparison to MI injury. In vitro co-culture of Wnt-overexpressing fibroblasts with activated CD11b presenting immune cells promoted fibroblast proliferation and collagen synthesis.
CONCLUSIONS: Therefore, we hypothesize that Wnt signaling activation promotes interstitial fibrosis via recruitment of specific inflammatory cells. Genomic analysis further supports this by demonstrating distinct chemokine gene expression patterns in fibroblasts resulting from Wnt activation in these injury models.. Our future goal is to elucidate the role of Wnt signaling in modulating the fibroblast-immune cell cross-talk in specific injury contexts.
P.IRD1.
BACKGROUND: Amniotic tissues have a long history in treating chronic wounds; however, there have been few studies to evaluate the in situ effect of these tissues on the wound microenvironment. Hypothermically stored amniotic membrane (HSAM º) has been shown to contain growth factors and cytokines, extracellular matrix and cells; in vitro, HSAM has been shown to promote proliferation and migration of fibroblasts and keratinocytes1. The goal of this study was to evaluate VLUs treated with a HSAM.
METHODS: In this prospective, single arm study, 15 female patients with venous leg ulcers which had been present for 8 months to 35 years were treated with both HSAM from male donors along with standard of care multilayer compression therapy for 12 weeks. Over the course of the study wound exudate was collected and evaluated using a proteomic microarray and biopsies were collected from the wound bed one week after the first, second, and fourth application of HSAM in order to evaluate tissue for the presence of remaining HSAM tissue.
RESULTS: At 4 weeks 60% (9/15) of subjects achieved a 50% or greater reduction in wound size, by 8 weeks this increased to 73% (11/15) of subjects. At 12 weeks 53% (8/15) of subjects achieved 100% re-epithelialization. 28% of biopsies were found to be positive for male DNA (presumably from HSAM graft present in the wound one week after treatment) determined by detection of the Y chromosome. Proteomic analysis of wound exudate revealed that wounds on a healing trajectory had significantly higher levels of MMP-10 and significantly lower levels of IL-1ra, IL-1a, IL-9, IL-2, MCP-1, and TNF-b compared to stalled wounds. Additionally, we observed significant correlations of TIMP-2, MMP-10, EGF, IL-1ra, IL-7, IL-8 all with the percent reduction in wound size.
CONCLUSIONS: In summary, this study provides a novel approach to investigating VLUs treated with advanced therapies.
P.IRD2.
BACKGROUND: Proteases play a central role in wound management. Proteases are instrumental in the degradation and remodeling of the extra cellular matrix (ECM) which is one of the key elements of tissue repair and plays a role in the different stages of wound healing including influx of leukocytes, angiogenesis and re-epithelialization. The present study was aimed at testing the effects of four such proteases: Ficin, Actinidin, Bromelain and Wrightin on LPS-induced cytokine release by wound macrophages ex vivo and and keratinocyte function in vitro.
METHODS: To study the effect of these proteases on expression of antimicrobial peptides (AMPs), differentiation markers and cell migration HaCaT keratinocytes were used. Day 3 and day 10 wound macrophages were isolated from PVA sponges implanted in mice and treated individually with different concentrations of the proteases.
RESULTS: Significant decrease in LPS induced pro-inflammatory cytokines (TNF-α and IL-1β) were noted in d3 wound macrophages that were subjected to co-treatment with the proteases (n = 4; *p<0.05). Moreover, protease treatment (Ficin, Actinidin, Bromelain and Wrightin) to d10 wound macrophages resulted in a significant increase in the release of LPS-induced VEGF. Antimicrobial peptides (AMPs) act to form a chemical shield on the surface of the skin, they are also thought to trigger and coordinate multiple components of the innate and adaptive immune system. Ficin (10μg/ml; 24h) and Bromelain (1μg/ml; 24h) treatment induced the expression of the AMPs, β-defensin-1, and S100A9 as well as differentiation markers Involucrin and Filaggrin (n = 4; *p<0.05). Interestingly, Bromelain also exhibited significant cell migration when HaCaT keratinocytes were pretreated for 24 hours (n = 3-4; *p<0.05).
CONCLUSIONS: Taken together, findings of this work reposition proteases as potential agents that may be effective in resolving wound inflammation, bolstering host defenses and promoting keratinocyte migration.
P.IRD3.
BACKGROUND: The benefits of amniotic-based products across various regenerative applications are owed to a multitude of factors including cytokines, growth factors, and extracellular matrix components that synergistically function to accelerate the healing process. Innately immune-privileged, these materials are also associated with anti-inflammatory, anti-microbial, and immune-modulatory functions essential for both effective wound healing and minimization of scarring. The purpose of the present study was to elucidate the full range of anti-inflammatory and immunomodulatory capabilities of both a single-layer dehydrated amniotic membrane allograft (DAMA) and a tri-layer placental allograft membrane (TPAM) such that their contributions towards improved wound healing outcomes could be better understood.
METHODS: The anti-inflammatory functionalities of DAMA and TPAM were evaluated via cytokine analysis following PBMC activation with lipopolysaccharide in the presence of DAMA or TPAM extracts. An M1/M2 macrophage polarization assay was implemented to explore immune-modulation specifically through the assessment of CCL22, CCL18, PDGF, IL-13, TNF-alpha, IFN-gamma, IL-6, and RANTES. Macrophage polarization was conducted using both direct physical contact of the cells with DAMA/TPAM as well as soluble DAMA/TPAM extracts alone. Polarization outcomes were compared to non-stimulated macrophages as well as those in contact with non-immune-privileged collagen constructs. Finally, the effect of DAMA/TPAM extracts on T cell stimulation was explored using standard assays.
RESULTS: Cytokine analysis indicates that amniotic extracts possess a significant ability to reduce pro-inflammatory molecule secretion including TNF-alpha, IL-1beta, IL-10, MIP-1a, and IL-6 by over 90% in PBMC compared to controls (n=4 per group; p<0.05). Macrophage polarization analysis suggests that exposure to both DAMA and TPAM (n= 3 per group) modulates an M2-preferred macrophage response as a consequence of both soluble factors alone as well as physical contact with constructs. T cell responses were also favorably altered through exposure to amniotic extracts.
CONCLUSIONS: The ability of DAMA and TPAM to constructively influence both inflammatory and immunomodulatory processes therefore likely constitutes a substantial part of their overall effectiveness in wound remediation.
P.IRD4.
BACKGROUND: Traditional wound assessment methods rely on visual assessments by the care provider and cannot predict the progress of healing. While myriads of studies have suggested that a survey of wound pH environment could indicate wound healing activities, it is not clear whether wound alkalinity can be used as a prognostic indicator of non-healing wounds. The objective of this study was to develop a device that can assess the pH environment across wounds which, unfortunately, cannot be reliably done using currently available systems.
METHODS: A disposable device, DETEC™ pH, was developed and characterized by its ability to map wound alkalinity by pressing a freshly recovered wound dressing against its test surface. Wound alkalinity was used to prognosticate short-term wound size reduction rates by comparing the wound’s alkalinity and size reduction rates (per week) following pH measurement.
RESULTS: The device had high accuracy and specificity in determining the alkalinity of simulated wound fluids soaked onto wound dressing. The type of wound dressing had no significant effect on detection sensitivity. The device tested discarded wound dressings from human patients to quickly determine alkaline and acidic wounds. Finally, statistical analyses of wound size reduction rates in wounds with various alkalinities confirmed its strong influence on, at least, short-term wound healing activity.
CONCLUSIONS: Without directly contacting the patient, this device provides a quick assessment (within a minute) of wound alkalinity to prognosticate immediate and short-term wound healing activities. DETEC™ pH may serve as an aid for wound care specialists during routine wound assessment in predicting wound healing progress which can inform the decision-making process ultimately auguring well for chronic wound treatment. DETEC™ pH can also be used as an aid for home health-care nurses, healthcare providers, or in skilled nursing facilities to screen non-healing wounds outside clinics and alert doctors or nurses for on-time diagnosis and treatment.
P.IRD5.
BACKGROUND: Mesenchymal stem cells or more recently termed “medicinal signaling cells” (MSCs) role in native tissue healing and their availability throughout tissues in the form of pericytes has recently been established1. Regardless of mechanism, MSCs have been shown to promote resolution of wound inflammation, increased angiogenesis, and regulation of extracellular matrix remodeling in vivo2. Hypoxia has been shown to affect MSC responses including proliferation, differentiation, and secretory prolife3. Relevance of hypoxia is especially interesting when considering that diabetic wounds are known to be hypoxic in nature. In the current study, we have evaluated the response of bone marrow (bm) MSCs to dehydrated amnion chorion membranes (dACMº) in vitro under normal and hypoxic conditions.
METHODS: bm-MSCs were treated with either assay media (AM) or media conditioned with dACM (CM) and evaluated for proliferation and migration. CM was obtained by incubating dACM grafts in basal media for 3-5 days at 4°C at a ratio of 1 cm2to 1 mL media. To characterize the role of hypoxia, bm-MSCs treated with CM were cultured under normoxic (18.6% O2, 5% CO2) and hypoxic (2.5% O2, 5% CO2) conditions for 3 days, then supernatants were analyzed using Quantibody arrays (RayBiotech).
RESULTS: dACM CM promoted bm-MSC proliferation and migration (1.3 and 3.5-fold change compared to AM, respectively) in normoxic conditions. When evaluating secreted factors, we found dACM CM treatment resulted in significant increased secretion of chemokine ligand 3 (CCL3), CCL5, interleukin 6 (IL-6), and IL-8 from MSCs under normoxic and hypoxic conditions. Increased trends were also seen in insulin-like growth factor-binding protein 3 (IGFBP-3), IGFBP-4, and IGFBP-5, though not statistically significant.
CONCLUSIONS: These results demonstrate that dACM promotes bm-MSCs proliferation, migration, and stimulates secretion of factors related to inflammation and angiogenesis in vitro. º NuShield®, Organogenesis®, Canton, MA
1. References: 1)Caplan, A. I. Mesenchymal stem cells: Time to change the name! Stem Cells Translational Medicine6, 1445-1451 (2017). 2) Lee, D. E., et.al. Mesenchymal stem cells and cutaneous wound healing: Novel methods to increase cell delivery and therapeutic efficacy. Stem Cell Research and Therapy7, 1-8 (2016).3) Das, R., Jahr, H., van Osch, G. J. V. M. & Farrell, E. The Role of Hypoxia in Bone Marrow-Derived Mesenchymal Stem Cells: Considerations for Regenerative Medicine Approaches. Tissue Engineering Part B: Reviews16, 159-168 (2010).
P.IRD6.
BACKGROUND: Enzymatic debridement is an effective way to remove necrotic tissue from wounds. It offers benefit over passive autolytic debridement by providing an active exogenous protease for rapid completion of wound preparation, and advancement to healing. We have identified a novel enzyme, SN514, that produces rapid debridement of burn eschar. We compared efficiency of SN514 versus autolytic debridement in a porcine wound eschar model.
METHODS: Deep partial-thickness wounds were generated on the dorsum of anesthetized pigs using a dermatome. A brief chemical burn was then performed in each wound, and the wounds were dressed and eschar allowed to form over two days. Wounds were treated daily for ten days with SN514 gel, vehicle gel, petrolatum, medical honey (MH), or hydrocolloid dressing (HD). Clinical scores, including eschar amount and type, exudate, and granulation assessment, were used to compare debridement effectiveness of different treatments. Wound images were recorded to determine the remaining necrotic area by color analysis.
RESULTS: An SN514 gel exhibited significantly faster debridement compared to vehicle judging by clinical scoring of amount and type of necrotic tissue, as well as based on color analysis (p<0.05 after 3-9 days of treatment). SN514 treatment also increased granulation tissue formation and exudate versus vehicle. In comparison to petrolatum, HD, and MH, SN514 gel decreased amount and type of necrotic tissue, and increased granulation tissue and exudate. Based on color analysis, SN514 gel produced highly significant debridement (p<0.001) vs. petrolatum after 24 hours treatment. Linear fits indicated SN514 debrided >5X faster than petrolatum and HD, whereas MH debrided at less than half the rate of petrolatum. MH also exhibited decreased granulation tissue and exudate versus petrolatum, and increased necrotic tissue by clinical scoring. Visually wounds appeared largely debrided by SN514 within a few days.
CONCLUSIONS: SN514 provides rapid enzymatic debridement of necrotic tissue, and merits evaluation in human chronic wounds.
P.IRD7.
BACKGROUND: Wounds frequently become infected or contaminated with pathogens such as bacteria. Highly effective but less adversely reactive agents are expected in wound cleansing.
METHODS: A soap is made from alkaline and fatty and fatty acids and in this experiment, several potassium salts of fatty acids were tested.
RESULTS: Potassium oleate (C18:1K), a type of fatty acid potassium and the major component of a soap, caused >4 log colony-forming unit (CFU)/mL reductions in the numbers of Staphylococcus aureus and Escherichia coli within 10 min and a >2 log CFU/mL reduction in the number of Clostridium difficile within 1 min. C18:1K (proportion removed: 90.3%) was significantly more effective at removing Staphylococcus aureus biofilms than the synthetic surfactant detergents sodium lauryl ether sulfate (SLES) (74.8%, p < 0.01) and sodium lauryl sulfate (SLS) (78.0%, p < 0.05). In the WST (water-soluble tetrazolium) assay, mouse fibroblasts (BALB/3T3 clone A31) in C18:1K (relative viability vs. control: 102.8%) demonstrated a significantly higher viability than those in SLES (30.1%) or SLS (18.1%, p < 0.05). In a lactate dehydrogenase (LDH) leakage assay, C18:1K (relative leakage vs. control: 108.9%) was found to be associated with a significantly lower LDH leakage from mouse fibroblasts than SLES or SLS (720.6% and 523.4%, respectively; p < 0.05).
CONCLUSIONS: Potassium oleate demonstrated bactericidal effects against various species including Staphylococcus aureus, Escherichia coli, Bacillus cereus and Clostridium difficile, removed significantly greater amounts of Staphylococcus aureus biofilm material than SLES and SLS and maintained fibroblast viability; therefore, it might be useful for wound cleaning and peri-wound skin components.
P.IRD8.
BACKGROUND: Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of trophoblast -free chorionic membrane (VLCM) and compare it to the cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed.
METHODS: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by live/dead cell fluorescent staining. Inhibition of TNFα secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and pro-angiogenic properties, respectively. An in vivo rabbit abdominal adhesion model was used to evaluate the anti-fibrotic properties.
RESULTS: CM was shown to be non-immunogenic. Tissue architecture, growth factors and cell viability of fresh CM were maintained in VLCM and VCCM. In vitro studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Further, VLCM prevents formation of post-surgical adhesions in a rabbit abdominal surgical adhesion model.
CONCLUSIONS: Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, pro-angiogenic and anti-fibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.
P.IRD9.
P.IRD10.
BACKGROUND: Placental membranes, amnion and chorion, have anti-fibrotic activity. Recent studies have shown that exosomes function as conduits for intercellular transfer and contain all the necessary components to induce resolution of fibrosis. Exosomes are nano-sized vesicles produced by many cell types that carry bioactive cargos of proteins, lipids, DNA and miRNA. In this study we tested a hypothesis that anti-fibrotic activity of placental membranes is mediated by exosomes.
METHODS: Amnion and chorion-derived exosomes from conditioned media were isolated by sequential ultracentrifugation. Particle size and morphology of isolated exosomes were analyzed using Dynamic Light Scattering and transmission electron microscopy. Exosomal anti-fibrotic activity was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Effect of exosomes on proliferation of LX-2 or TGFβ1 activated LX-2 cells was assessed using alamarBlue assay and EdU staining. Migration of LX-2 cells was evaluated in a scratch wound assay. The expression of fibrotic markers was analyzed by qPCR and immunofluorescent staining.
RESULTS: Exosomes isolated from amnion- or chorion-conditioned media had an average size of 75nm and adapted spherical shapes. Exosomes significantly inhibited the proliferation of TGFβ1 activated LX-2, but had no effect on the proliferation of resting LX-2 cells. In a scratch wound assay exosomes reduced the migration of LX-2. Furthermore, exosomes reduced the gene expression of pro-fibrotic COL1A1, ACTA, and TGFβ1 and increased the expression of anti-fibrotic HGF and IL-1β in LX-2.
CONCLUSIONS: Exosome effects on LX-2 suggest its potential use in a cell-free treatment of fibrotic diseases including keloid and hypertrophic scars. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic and chorionic tissues.
P.IRD11.
BACKGROUND: Recent studies from our lab have demonstrated the efficacy of electroceutical intervention in disrupting bacterial biofilm infection. Chronic wounds are a favorable niche for polymicrobial (bacterial and fungal) biofilm infections. Fungal invasion into deep tissues is a major contributor of delayed wound healing. Members belonging to genus Candida, especially Candida albicans, are the most prevalent fungi in wounds. Candida albicans (CA), an opportunistic pathogen, has been reported to form multispecies biofilms with bacteria and impede wound healing in diabetic foot ulcers. It is also a causative agent for cutaneous candidiasis, oral thrush or candidemia in immunocompromised or neutropenic patients. Objective: The objective of the current work is to test whether electroceutical dressing based therapy may productively manage CA infection.
METHODS: A US FDA-cleared WED, based on electrochemistry enabled by silver and zinc printed on polyester fabric to generate weak electric field, was tested. Planktonic growth, metabolism, efflux pump activity, cell wall alterations, morphology switch and biofilm formation in Ketoconazole resistant C albicans (ATCC 64124; isolated from human mouth swab) were investigated in response to WED.
RESULTS: Yeast cells cultured in presence of WED showed a two-fold reduction in planktonic growth accompanied with impaired metabolism. Effect of external electric field on CA efflux pump activity was measured using Nile red. An increase in Nile red accumulation within 30 min to 3h of treatment with WED or with a combination of WED and Ketoconazole, indicated impaired efflux pump activity. Ultrastructure studies, differential staining of cell wall components and secondary stress assays proved compromised cell wall integrity in CA treated with WED. Yeast cells failed to form pathogenic hyphae in the presence of WED, when cultured in hyphae inducing conditions. Confocal Laser Scanning microscopy demonstrated severe defects during in vitro biofilm formation by C albicans in presence of WED. All aforementioned results were obtained from four independent experiments.
CONCLUSIONS: The reported anti-fungal properties of WED, taken together with previously reported anti-bacterial biofilm function, position this electroceutical dressing as an attractive counter measure to manage mixed species polymicrobial infections as commonly noted in chronic wounds.
P.AW01.
BACKGROUND: Approximately, 86% of post-surgical patients experience pain. More importantly, 80% of post-surgical patients receive opioids for pain control. To improve patients’ quality of life and reduce opioid usage, detecting biomarkers for appropriate and effective wound pain management is important to implement.
METHODS: We compared longitudinal transcriptomic profiles from RNA sequencing data and extracted genes encoding secreted proteins from in situ hybridization in two rat models: a peripheral inflammatory rat model using 4% carrageenan injection and a surgical incision rat model with a 1 cm incision. Both manipulations were performed on the plantar surface of the hind paw. Thermal hyperalgesia was assessed by a plantar test using Hargreaves’ method.
RESULTS: We found 9,098 incision-specific alterations in gene expressions compared with the inflammation model. We classified the genes into clusters by the expression patterns. In the incision model, in the clusters of gene expression with peaks at 1 or 6 hours, over 50% of differentially expressed genes (DEG) encoded secreted proteins. By comparison in the inflammation model in clusters with peaks between 1 and 8 hours, less than 50% of DEGs were genes encoding secreted proteins. In the clusters that have the peaks of gene expression at 1 hour after incising or inducing inflammation, two chemokines, Cxcl1 and Cxcl2 were found to be shared between both models. However, their expression levels in the incision model were much higher than in the inflammation model. At timepoints of worst thermal hyperalgesia in both models, the most highly expressed gene fold-change in the incision model at 24 hours was Klk6 and in the inflammation at 4 hours after inducing inflammation was Il6.
CONCLUSIONS: This abstract presents the secretomic profile and corresponding gene expression changes associated with thermal hyperalgesia in the surgical incision model and the peripheral incision model. These initial findings have the potential to identify biomarkers of wound pain.
P.AW02.
BACKGROUND: Ileostomy output is enzymatically active due to the presence of pancreatic enzymes, and exposure can lead to the disruption of the skin barrier and development of peristomal skin complications (PSC). Historical work (PH Andersen et al. Contact Dermatitis 1994; 30(3):152-8) investigated the effect of intestinal enzymes on skin barrier function (transepidermal water loss [TEWL], diffuse reflectance spectroscopy [DRS], & pH) using Hilltop chambers on sites on the backs of healthy volunteers for cumulative irritation studies at 5 & 12-days and found that a daily application of a mixture of 0.06mg/mL trypsin, 0.80mg/mL lipase and bile salts at pH 8 caused severe erythema and epidermal barrier disruption. We hypothesized that the duration of exposure could be decreased by applying serial 1-hour applications within a single day and that the same enzyme solution concentration could increase the severity of skin barrier disruption if applied shortly after preparation, rather than following a freeze/thaw.
METHODS: A single-day pilot study of 5 one-hour trypsin and lipase (pH 7.4) exposures on abdominal skin in 12 healthy adult volunteers was performed using the same endpoints, as well as visual grading for erythema and denudation, to determine if this study design could be as effective as the prior 5 & 12-day study and feasible for rapid modeling of PSC in ileostomates.
RESULTS: The first accelerated, hourly enzymatic exposure led to a slight increase in grade of erythema, TEWL, DRS and pH values compared to baseline. Unexpectedly, the subsequent second through fifth exposures did not further increase response, as hypothesized from the historical 12 day study results.
CONCLUSIONS: The differences between the observations here and the historical work by Anderson et al suggests that chronic exposure to enzymes, moisture, or both may be necessary to recreate the same level of barrier disruption, pH change, and erythema. The pilot lacked a sham control and longer exposures to test this hypothesis. Future model development efforts may include additional relevant exocrine pancreatic enzymes, characterization of the relative contribution of moisture associated skin damage, and/or use of bench and porcine models to extend the range of damage that can be studied.
P.AW03.
BACKGROUND: Prolonged antimicrobial and osteoinductive activities of polycaprolactone, PCL nanofiber membrane ,NFM, for biomedical application is possible by tethering the antimicrobial and osteoinductive molecules with PCL NFM. The effect of MgO NP in which tethered with PCL on the antimicrobial activities is not known; leading to the proposed study. The evaluation of the antimicrobial properties of PCL with and without MgO nanoparticles using Staphylococcus aureus is the purpose of this study. 100, 200 and 400mg/ml of MgO nanoparticles were immobilized with PCL in which supported by acrylic molds.
METHODS: Methods: Several methods were used in this study such as the Agar Disc Diffusion Method, Microbial Penetration Test, and Growth Kinetics test. The antibacterial activity of these tests were compared with the commonly used antibiotic disc (Gentamicin) against the Staphylococcus aureus. The concentration of MgO was obtained from a previous study according to the cytotoxity test that has already been done. All the experiments were repeated three times.
RESULTS: Results: It is demonstrated that Gentamicin have inhibition zones in bacterial cultured (TSA) and turbidity tests show no signs of bacterial growth. PCL-MgON showed no inhibition zones, yet no growth under the molds had seen, and no bacterial growth according to the turbidity test which supported in vitro and lead us for further in vivo study.
CONCLUSIONS: Conclusion: Following OUHSC guidelines, the animal study of 10 rats were processed. Rats having superficial wound were dressed with MgO NP bandages with known concentrations and a control group (regular wound dressing). Rats were checked after one, two, three, and four days. No inflammation was detected and the sample group with MgO NP bandage showed a faster healing process than the control group in which supported the goal of this study.
P.AW04.
P.AW05.
BACKGROUND: Negative pressure wound therapy (NPWT) systems include a material usually foam or gauze at the wound/device interface. The purpose of this study was to compare the three most common NPWT systems (V.A.C.VIA™, PREVENA™ and PICO™) with a new device without foam or gauze (Platform Wound Dressing™ (PWDTM)) in the treatment of full-thickness wounds. The PWD is a single component impermeable polyurethane dressing for NPWT whose embossed surface eliminates the need for foam or gauze. A strong effort was made to avoid bias. The study was done in a GLP laboratory with the presence of an independent observer.
METHODS: In pigs, three types of wounds were studied; full thickness excisions, open incisions and closed incisions. N number for each treatment group was 8. The pigs were euthanized on day 9 and all wounds were processed for histology and excisions for immunohistochemistry.
RESULTS: The devices produced similar results with only a few significant differences. In the excisions, the PWD reduced wound area more than the VIA and the PICO (PWD: 41.4 ± 5.3 %; VIA: 18.4 ± 7.0 %; PICO: 25.1 ± 8.1 %). In the excisional wounds, re-epithelialization was the same whereas in open incisions PREVENA was better than the PWD (PWD: 24.7 ± 4.0 %; PREVENA: 50.4 ± 10.0 %). Histology showed that in open incisions there was less inflammation in the PREVENA treated in comparison to the PWD and the PICO treated wounds (PREVENA: moderate; PWD: marked; PICO: moderate). Immunohistochemical analyses showed that the PWD treated excisions had significantly more blood vessels (von Willebrand Factor) than the VIA treated ones (PWD: 8.1 ± 0.5 %; VIA: 5.5 ± 1.0 %). In addition, the PICO caused less T-cell activation (CD3) than the other two (PICO: 0.1 ± 0.0 %; PWD: 0.4 ± 0.1 %; VIA: 0.5 ± 0.2 %).
CONCLUSIONS: The devices, with foam, with gauze or without any of the two and just an embossed membrane performed equally in general.
P.AW06.
BACKGROUND: We aimed to determine the efficacy of in vitro Duroc fibroblasts as a model to understand mechanisms of fibroproliferative responses to injury and to identify novel genes involved in wound healing.
METHODS: Fibroblasts from 8 Duroc and 8 Yorkshire pigs were cultured for 48 hours ± TGF-ß. Tissue samples were taken from Duroc uninjured skin and burn wounds 3 days after injury. RNA was sequenced using Illumina Hi-Seq sequencers. Differentially regulated genes from in vitro fibroblasts were compared to genes from in vivo wounds with at least 1.5 fold-change and false discovery rate of 5%.
RESULTS: Duroc 3-day burns had 2866 differentially expressed genes compared to uninjured skin. Comparatively, TGF-ß induced differential expression of only 149 genes in Duroc fibroblasts but not Yorkshire cells. 46 (31%) of the genes were differentially expressed in Duroc early wounds with 18 (12%) demonstrating concordant expression change in the same direction in both the fibroblasts and early wounds (Table 1). Many of these novel genes have not been studied in the context of wound healing.
CONCLUSIONS: In vitro Duroc fibroblasts cultured with TGF-ß do not fully represent the in vivo porcine cutaneous wound healing process, supporting the contribution of other cells to the early response to injury. 18 genes concordant between TGF-ß- treated Duroc fibroblasts and Duroc 3 day burn wounds represent novel targets.
| Duroc fibroblasts + TGF-ß | Duroc wounds at 3 days | |
| Gene | Log FC | Log FC |
| SYNPO2L | 0.912 | 5.445 |
| LDB3 | 0.85 | 4.795 |
| CMYA5 | 1.224 | 4.425 |
| P2RY6 | 1.221 | 2.011 |
| HCLS1 | 1.06 | 1.468 |
| ARHGAP45 | 0.968 | 1.287 |
| CAMK4 | 0.726 | 1.222 |
| ULBP1 | 0.8 | 1.036 |
| Duroc fibroblasts + TGF-ß | Duroc wounds at 3 days | |
| Gene | Log FC | Log FC |
| TPST2 | 0.548 | 0.6451 |
| SPOCK1 | -0.602 | -1.373 |
| ZNF385D | -0.819 | -1.123 |
| TFCP2L1 | -0.836 | -0.8748 |
| CHAD | -0.917 | -0.8422 |
| SERPINF1 | -0.516 | -0.6822 |
| COL4A5 | -0.731 | -0.6536 |
| MATN2 | -1.288 | -0.6247 |
| Duroc fibroblasts + TGF-ß | Duroc wounds at 3 days | |
| Gene | Log FC | Log FC |
| MAP2 | -1.073 | -0.6196 |
| PBX1 | -0.581 | -0.4921 |
P.AW07.
BACKGROUND: Necrotizing soft tissue infections often result in large soft tissue defects following debridement. When these infections affect a limb, they often result in amputation or require large reconstructions. Many of these patients are poor surgical candidates for large reconstructions. Reconstruction with a dermal regeneration template followed by skin grafting has been described extensively in the literature; however, the use of Biodegradable Temporizing Matrix followed by skin grafting for limb salvage following necrotizing soft tissue infections has not. Our study aims to describe the efficacy and safety of this novel application.
METHODS: A retrospective chart review of patients who underwent reconstruction following necrotizing soft tissue infection of an extremity with Biodegradable Temporizing Matrix (BTM) was performed. Only patients who were reconstructed using BTM followed by a skin graft were included. Several data points were analyzed including wound size and location of wound, number of debridements prior to reconstruction, length of time between stages, rate of revision surgeries, wound healing complications, and recurrent infections. Demographic analysis of the patients was also performed.
RESULTS: Five patients to date have undergone reconstruction with BTM after a necrotizing soft tissue infection of a limb. Four of the 5 patients had complete limb salvage. The remaining patient was saved a higher level of amputation with the use of BTM. The average number of debridements prior to reconstruction was 6.2. The average size of the reconstructed wounds was 635 cm2. The average length of time from presentation to skin grafting was 235 days. The average time from BTM placement to skin grafting was 25 days. The average number of days from first debridement to BTM was 178.9.
CONCLUSIONS: BTM followed by autologous skin graft in the setting of necrotizing soft tissue infection is a safe option in the reconstructive surgeon’s armamentarium. In our limited experience, BTM has shown great promise as a limb salvage technique despite life threatening infection.
P.AGS01.
BACKGROUND: Dupuytren’s Contracture (DC) is the buildup of scar tissue in the hand that causes one or more digits to flex, reducing utility. Common treatments are surgical removal or an enzyme treatment. After treatment this contracture has a high recurrence rate. There is evidence that recurrent nodule cells proliferate slower than primary nodule cells; additionally, fibrotic conditions appear to have telomere dysfunction; therefore, short telomeres may play a role in the pathology of recurrent DC. Telomeres shorten when primary cells are grown in culture. We used this rationale to study phenotypic differences of DC cells aged in vitro as a comparison group for future in vivo studies.
METHODS: Cells of different population doublings (PD) from a single DC patient (Early and Late PD) were thawed and cultured using standard culture conditions. A scratch wound migration assay was conducted using serum-free media and varying concentrations of platelet derived growth factor (PDGF) up to 100 ng/ml. A vinculin stain to visualize focal adhesions was conducted to test whether focal adhesion size in the presence or absence of TGF-β, contributed to in vitro aging phenotype.
RESULTS: Both Early and Late PD cell cultures had a PDGF dose-dependent increase in migration. Late PD cells showed lower migratory ability overall. Early PD cells had more super mature focal adhesions in control conditions than TGF-β conditions; conversely, late PD cells had more super mature focal adhesions in TGF-β conditions than control conditions.
CONCLUSIONS: The effect of in vitro aging on this cell type indicated a change from migratory to stationary. Our results suggest that collagen matrix compaction and contraction will be regulated differently by Early and Late PD cultures. Our future goal is to test this using the anchored collagen matrix model. Research with other patients’ in vitro aged DC cells is ongoing. These studies may provide insights into effects telomere shortening may have in the recurring DC condition.
P.ANGIO1.
BACKGROUND: The regression of blood vessels during wound resolution creates a significant number of apoptotic endothelial cells (EC) that must be cleared. In considering the fate of apoptotic EC, we uncovered the possibility that fibroblasts might unexpectedly serve as phagocytes and be involved in EC removal. The current study investigated whether dermal fibroblasts engulf apoptotic ECs and whether this uptake alters the phenotype of dermal fibroblasts.
METHODS: In in vitro studies, human dermal fibroblasts (HDF) were incubated with apoptotic green fluorescent protein-human dermal microvascular endothelial cells (GFP-HDMEC) for 8 or 24 hours. In in vivo studies, apoptotic mouse dermal microvascular endothelial cells (MDMEC) were injected subcutaneously in mouse skin and the tissue was harvested after 1 day.
RESULTS: In vitro, confocal microscopic analysis showed green apoptotic particles inside vimentin-positive fibroblasts after 8 and 24 hours. Flow cytometry showed that the percentage of HDF that engulf apoptotic GFP-HDMEC was 4.0% and 4.9% after 8 and 24 hours, respectively. In functional analyses, HDF incubated with apoptotic HDMEC showed an increase in the rate of cell migration and collagen gel contraction after 8 and 24 hours, as well as increased expression of α-smooth muscle actin (alpha-SMA) and collagen type III after 8 hours. In vivo studies demonstrated that, compared to control, the injection of apoptotic MDMEC led to an increase in alpha-SMA-positive myofibroblasts in subcutaneous tissue at 24 hours. Moreover, the alpha-SMA-positive myofibroblasts observed in apoptotic MDMEC-treated tissue were shown to have engulfed apoptotic MDMEC.
CONCLUSIONS: In conclusion, dermal fibroblasts are capable of engulfing apoptotic EC both in vivo and in vitro. Following the engulfment of apoptotic endothelial cells, fibroblasts acquire a pro-fibrotic phenotype. Our studies demonstrate the novel finding that fibroblasts may engulf apoptotic cells in wounds, and suggest that this function may have significant implications for tissue repair.
P.ANGIO2.
BACKGROUND: Bone marrow-derived mesenchymal progenitor cells “fibrocytes” have been postulated to differentiate into fibroblasts, leading to fibrosis and scar formation in wounds. However, the roles of fibrocytes for granulation tissue formation in wounds remain obscure, especially for the contribution of fibrocytes to vessels formation in granulation tissues during the course of basic fibroblast growth factor (bFGF) induced angiogenesis in wounds.
METHODS: To clarify the specific role of fibrocytes for vessel formation in granulation tissues , we conducted collagen gel culture spayed by bFGF-treated granulation tissues.
RESULTS: The collagen gel culture spayed by bFGF-treated granulation tissues demonstrated an increase in the number of capillary-like structures that were almost composed of CD34+procollagen I+ fibrocytes. The result was confirmed by PCR analysis, in which the same treated gel showed an increase in CD34 and collagen type I mRNA expression. Furthermore, we found an increase in the expression of chemockine ligand 12 (CXCL12) and chemockine receptor 4 (CXCR4) in the gel culture spayed by bFGF-treated granulation tissues .
CONCLUSIONS: These results suggest that bFGF has a novel role in inducing CD34+procollagen I+ fibrocytes in granulation tissues via CXCL12/CXCR4 signaling, which might contribute to vessel formation in wounds following bFGF treatment.
P.BIO01
BACKGROUND: The growing demand for cell and tissue-based products, rich in growth factors, multiple cell types, and extracellular matrix, stems from their ability to accelerate the wound healing process. An autologous source of tissue contains regenerative properties for patients without the risk of immunogenicity. However, limitations reside in the quantity of harvestable tissue and donor site morbidities. This study investigates the use of micronized collagen-chondroitin-sulfate (MCCS) matrix reconstituted with saline as an extender to autologous tissue in the treatment of full-thickness skin defects.
METHODS: In a preclinical pilot study, sixteen 2cm diameter full-thickness skin wounds were created in the back skin of a Yorkshire pig. Full-thickness autologous skin was harvested and chopped to a size of 1-2mm. MCCS particles range between 200-2000痠 in size. The treatment groups were (a) untreated control (b) minced full-thickness skin grafts (c) minced full-thickness skin grafts + MCCS or (d) MCCS alone (all N=4). Approximately 15% of the weight of the excised wound was replaced with treatment. Dressing changes and wound images were captured every 3-4 days, for up to 10 days. Tissues were harvested for histological analysis.
RESULTS: At Day 10, the granulation bed in the untreated control group was composed of a continuous layer of maturing dense collagen. Minced tissue and MCCS remained intact, viable and infiltrated with fibroblasts and macrophages. Furthermore, all 3 treatment groups enhanced full-thickness wound healing by disrupting formation of a maturing granulation bed, thereby minimizing scarring. Finally, minced autologous tissue treated groups had greater re-epithelialization, enhanced vascularization, and fewer ulcers than all other treatments.
CONCLUSIONS: Results suggest that MCCS can be successfully used as an extender to autologous tissue. This study suggests that autologous minced tissue in combination with MCCS provides essential cytokines and growth factors to create a beneficial environment for wound healing while minimizing donor site morbidities, without sacrificing healed tissue quality.
P.BIO02
BACKGROUND: Adhesion formation is the tissue repair following tissue trauma. Excess adhesions on a significant area can cause malfunction such as intestinal obstruction, contracture. As a result, significant effort has been made toward development of suitable methods for reducing adhesion formation. The film barrier between two organs has been widely used in clinical settings. In this study, we assess the effectiveness of porous polylactide film in reducing intra-abdominal adhesions in a rat model.
METHODS: Polylactide, poly(L-lactide) form of molecular weight 100,000 or greater was used. Methylene chloride and ethyl alcohol were used as the solvent for composition of porous polylactide film. Thickness and pore appearance of a film was observed using a scanning electron microscope. In the rat model, abdominal wall and cecum were abraded to induce adhesion, and the film was inserted between two organs. Presence and degree of adhesion were compared.
RESULTS: The thickness of porous polylactide film was 10-20 μm. The 10 μm or less pores were distributed irregularly on the surface and inner part of film. In the rat model, incidence of adhesions was 23.1% in the film group compared with 92.9% in the control group. Porous polylactide film was found to significantly reduce severity of adhesions.
CONCLUSIONS: Porous polylactide film can potentially be used as a possible material for reduction of adhesion formation. By the addition of antibiotics or anti-inflammatory agent with pores, the analgesic effects, antibacterial action, and drug sustained-release effect can be expected.
P.BIO03.
BACKGROUND: The purpose of this study is to design the skin-contact and absorptive layers of a tri-layered wound dressing with optimal material and geometric properties needed to deliver gentamicin sulfate (GS) effectively while absorbing excess moisture in chronic wounds.
METHODS: Poly-l-lactide (PLA) microfibers were fabricated using a large-scale melt extruder and knitted to create the contact layer. Parts A and B of FlexFoam-it! IV, V, and 6 (Smooth-On, Inc. Mancungie, PA) were mixed together and cured to create the Polyurethane (PU) absorptive layer. Two methods of GS incorporation into the contact layer via soaking and diffusion were compared to determine the optimal antibacterial and biocompatible response. Elution was determined by a dialysis membrane method, where contact layers were submerged in Phosphate-Buffered Saline with the release measured via fluorescent spectrometry using o-Pthaldialdehyde. Contact layers were placed into cultures of Pseudomonas putida. Antibacterial efficacy was determined by measuring the Zone of Inhibition after 24 hours and the optical density of liquid broth over five days. Porosity of the PU foam layer fabricated from kits IV, V, and 6 was measured using a Capillary Flow Porometer and a Brunaeur-Emmett-Teller instrument. Absorption rate was determined by measuring the increase in weight after applying the PU foam to a water bath over a 7-day period. Absorption of human serum samples collected from donors from The Blood Connection of Greenville, SC, used to mimic wound exudate viscosity, was also measured.
RESULTS: The elution profile for all conditions released antibiotic over the minimum inhibitory concentration (MIC) against P. putida. The antibacterial experiments showed that GS exhibited both cidal and static trends by killing bacteria and inhibiting growth, respectively. PU foam exhibits sustainable absorption over 7 days without saturation.
CONCLUSIONS: This study explores antibacterial activity and absorptive properties needed for a holistic approach to chronic wound healing. Controlling GS release through geometry and incorporated mechanism while absorbing excess moisture can allow chronic wounds to respond with a normal inflammatory response needed for healing.
P.BIO04.
BACKGROUND: The use of acellular dermal matrices (ADM) in breast reconstruction has grown significantly since first used 2005. Success relies on rapid ADM biointegration. Macrophages regulate tissue vascularization and undergo transition from the pro-inflammatory M1 phenotype, to the tissue repair M2 phenotype during healing. Tissue vascularization requires M1 and M2 macrophages in the proper sequence. We investigated incorporating immunomodulatory cytokines into ADMs to promote M1-to-M2 transition to improve biointegration. We hypothesized that sustained release of the M2-promoting cytokine interleukin-4 (IL4) would control inflammation dynamics, accelerating vascularization in the ADM, improving outcomes.
METHODS: IL4 was adsorbed onto ADM samples, and release studies determined the elution profile. Next, a mouse dorsal skinfold chamber model was used to determine the effects of IL4-treated ADM on biointegration (n=8 IL4, n=8 control). Real-time measurements of vascular integration and oxygen saturation were made over 21 days. Macrophage function (M2:M1 ratio), and vascular ingrowth (CD31) were also evaluated histologically.
RESULTS: ADM samples sustained release of cytokine concentrations to 11 days in vitro. Repeated in vivo oxygen sensing imaging demonstrated that IL4-treatment of ADM drove a 34% increase in vascular coverage and a 24% increase in oxygen saturation levels within the ADM at day 21, compared to control ADM. Supporting analysis of CD31 staining revealed a significant 3-fold increase in vessels within IL4-treated ADM compared to control.
CONCLUSIONS: The use of ADM as an immunomodulatory drug delivery vehicle has great clinical potential. Our in vivo model provides the ability to serially assess ADM integration. This novel approach can improve the clinical outcomes of ADM use in implant-based breast reconstruction through modulating the immune response to facilitate rapid and efficient biointegration. Future work includes increasing the complexity of a designed pharmacologic strategy of improved ADM integration through spatial and temporal control of a multifactorial signaling cytokine profile.
P.BIO05.
BACKGROUND: Soft tissue defects treated with dermal substitutes often require a two-stage procedure to achieve epidermal closure. A single-stage procedure, in contrast, will reduce patient morbidity, minimize hospital stay and reduce overall cost of care. However, single-stage procedures could result in autograft loss due to lack of contact between the graft and wound bed leading to insufficient vascularization. We have previously demonstrated that channels in a collagen-chondroitin-6-sulfate (CC6S) matrix allow blood/inflammation to pass through the matrix to enhance autograft survival. Here, we investigate the impact of matrix channel diameter on vascular ingrowth.
METHODS: Anesthetized rats (n=18) had 2x2cm2 matrix intra-abdominally bilaterally implanted onto ventral peritoneum with excoriated serosa. Matrix was perforated with either 1 and 3mm diameter channels or 1mm diameter channels with varied spacing. At days 8 and 22 post-implantation, four animals/timepoint were perfused with a vascular imaging agent (AltaBlue), followed by abdominal CT and micro-CT. The remaining animals were sacrificed for histological analysis.
RESULTS: The dermal matrix insulates the abdominal contents from the acute inflammation that arise from the excoriated peritoneum. Channels create openings for inflammatory mediators and vessels to access the matrix outer surface resulting in increased remodeling in and around the channels. At Day 8, micro-CT analysis confirmed presence of distinct feeder vessels branching from the muscle through the channels (both 1 and 3mm) toward the peritoneal surface, unlike solid regions of the matrix. Histologically, 1mm channels contained a thin, patent vessel rimmed by maturing fibrous connective tissue whereas the 3mm channels usually contained a vessel that was dilated. In terms of ability to support blood flow, both channels performed similarly. At Day 22, matrix was remodeled such that the defining features became indistinguishable from native tissue.
CONCLUSIONS: Both 1 and 3mm channels provide adequate space for vascularization. However, to optimize dermal healing outcome (low scarring), preservation of matrix collagen is critical. Therefore, smaller channels are likely to be more beneficial since they will maximize collagen content while enhancing vascularity and STSG survival.
P.BIO06.
BACKGROUND: For patients who are at risk of developing bacterial infections and biofilms that are hard to heal, it is important that garments and bedclothes that can come into direct contact with the patient and body fluids such as sweat, have antimicrobial properties. This study is on the use of covalently bonding an organo-selenium to cotton fiber to block possible growth or biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa, the two main bacteria associated with wound infection, in textile material.
METHODS: Cotton fibers were prepared that contained covalently-attached organo-selenium, which was shown to catalyze superoxide formation on the fibers. These fibers were placed in a growth medium and treated with Staphylococcus aureus and Pseudomonas aeruginosa, in vitro. After 24 hours of growth, the amounts of bacteria growing on the fiber were determined by a colony-forming unit (CFU) assay.
RESULTS: Using colony-forming unit (CFU) assays, over 7 logs of inhibition (100%) was found for both Staphylococcus aureus and Pseudomonas aeruginosa bacterial strains on the fibers coated with 1% and 8% concentrations of organo-selenium compound, when compared with the control (untreated) fibers. All experiments were done at least in triplicate.
CONCLUSIONS: The organo-selenium treated fibers completely inhibited Staphylococcus aureus and Pseudomonas aeruginosa biofilm formation on the fibers, while untreated fibers showed biofilm formation. The treated fibers are suitable for the manufacture of undergarments, socks and bedclothes.
P.BIO07.
BACKGROUND: The mechanical effect of the biopolymer matrix can have a significant effect on cell behavior. Considerable progress has been made in studying the behavior of cells in a low-density collagen matrix, but the behavior of cells inside a dense collagen matrix, remains poorly understood. Meanwhile, the use of high-density biopolymer matrices made of collagen in tissue-engineering structures is of great interest, since they can create stable structures, including 3D bioprinting, for further clinical use. The aim of this work is to elucidate how differences in the mechanics of the matrix affect the behavior of human dermal cells during in vitro cultivation in a 3D collagen matrix.
METHODS: Skin dermis cells were cultured in collagen gels, the rigidity of which was manipulated by changing the concentration of type I collagen. The differences in cell survival and proliferative activity were studied depending on the stiffness of the matrix. We also studied the dependence of the morphology and dynamics of dermal cells on the stiffness of the matrix using time-lapse photography.
RESULTS: The results indicate that human dermal cells retain high viability and proliferative activity in a high density collagen gel. The speed of movement of cells in the collagen hydrogel and the distance covered decreases with an increase in concentration of collagen from 5 to 10 mg/ml, but does not change with a further increase in collagen concentration to 40 mg/ml.
CONCLUSIONS: The combined use of biophysical approaches and methods of cell biology will allow us to determine the mechanical strength characteristics of the resulting structures and their effect on cellular activity as part of tissue-engineering structures.
P.BW01.
BACKGROUND: Stem cells implanted hydrogel constructs have emerged as a promising alternative for treating traumatic skin damages. Although, stem cells have regenerative competence but, the microenvironment of injured tissue is not conducive for the transplanted cells due to prevalent oxidative stress, causing reduced potential restorative outcome of treatment. While, antioxidants are believed to help control oxidative stress and accelerate the wound healing mechanism. Gelatin is emanated by hydrolysis denaturation of collagen under high temperature condition. Gelatin Meth-acrylamide hydrogel (GelMa), a modified form of Gelatin is photo-cross linkable, with tune-able mechanical properties, and possess number of applications in tissue engineering.
METHODS: Present study arbitrated sufficiency of human umbilical cord derived Wharton Jelly MSCs (WJMSCs) primed with an antioxidant concoction (Vit C, Vit E, Vit D3) encapsulated in GelMa hydrogel. This research involved, random distribution of SD rats (n=10 each) in different groups (Untreated Burn; GelMa Hydrogel; WJMSCs embedded in GelMa; Antioxidant Cocktail pretreated WJMSCs embedded in GelMa). A deep dermal burn wound (1.5×1.5 cm2) was created, and all the specified treatments were applied. Healed skin after maximum re-epithelization (28 days) was excised and subjected to H and E staining, immunohistochemistry evaluation with epidermal (CK10, Loricrin) and dermal (Col I, Col III, Vimentin) antibodies, qRT-PCR analysis for several wound healing markers.
RESULTS: Antioxidants cocktail preconditioned WJMSCs with GelMa group portrayed faster wound closure as compared to untreated group. Histological investigations demonstrated organized dermal and epidermal content in preconditioned WJMSCs with GelMa. While, gene and proteomic expression assessment also confirmed that preconditioning of WJMSCs with antioxidants cocktail stimulated long-term cell survival of WJMSCs, and improved vascularization in conjugation with GelMa scaffold for PCNA, BCl-xL, VEGF, HGF, FGF-7, Col III markers.
CONCLUSIONS: Antioxidant primed WJMSCs GelMa hydrogel composite served as a potent therapeutic replacement for in vivo burns.
P.BW02.
BACKGROUND: For the acute management of the burn patient, the depth of injury does not only dictate the extent and complexity of care required but also determines the long-term functional and aesthetic outcomes. This fact underscores the continuing concerted efforts at improving the accuracy of burn-depth diagnosis, a science that has remained largely dependent on the surgeon’s clinical acumen despite the plethora of sophisticated techniques, such as temperature color mapping, injection of vital dyes and radioactive isotopes, ultrasonography, infrared thermography, laser Doppler imaging, and most recently, the laser Doppler line scanner.
METHODS: Even when this most important determination of burn depth is accomplished with accuracy by human or machine, continual provisions must be made to accommodate the phenomenon of burn wound conversion if suboptimal outcomes are to be avoided. Burn injury may continue to progress over the first few days, leading to the conversion of superficial burns to deep burns, which influence treatment requirement and outcome
RESULTS: On the premise that burn wound conversion can influence the overall patient outcome, clinicians and researchers aim to better understand the pathophysiology of wound conversion and identify novel therapies that can mitigate it.
CONCLUSIONS: The purpose of this review is to examine the mechanisms known to contribute to burn wound conversion and the current therapeutic measures to mitigate it. Additionally, this review will highlight clinical scenarios in which burn wound conversion may be playing an even more significant role than previously thought. Our discussion will focus on the unanswered questions surrounding the extent to which such variables as nature of burn, site of injury, age of patient, presence of comorbidities, and type of wound intervention may contribute to the onset and duration of burn wound conversion.
P.BW03.
BACKGROUND: A number of tissue sealants are available as adjunctive measures to improve hemostasis and healing of surgical wounds.1,2 A self-assembling peptide hemostat (SAPH*) is in development to control bleeding during surgical procedures while not interfering with normal wound healing.3 The purpose of this preliminary study was to examine the effect of SAPH on second-degree burn progression and wound healing in a porcine model.4, 5 This model was selected due to the similarities between pig and human skin.6
METHODS: Thirty-two (32) second-degree burn wounds were created. Sixteen (16) wounds were randomly assigned to one of two treatment groups: A) SAPH or B) Saline. All wounds were covered with polyurethane film dressing (PFD)^ and four wounds from each treatment group were assessed on days 2, 3, 5 and 7. A trained dermatopathologist blindly assessed biopsies for burn progression, healing and bacterial load.
RESULTS: The depth of inflammation (measured and graded) in SAPH treated wounds was less than that in Saline control burns. On day 2, tissue necrosis in SAPH treated burns appeared to have been reduced when compared to Saline control. On Day 5, the percentage of re-epithelialization was higher in the group of burns that had been treated with SAPH when compared to the group treated with Saline. On days 5 and 7, the total bacterial count in the SAPH treated wounds was lower than that in the Saline treated group.
CONCLUSIONS: These results highlight the potential for SAPH to reduce burn progression and enhance healing. Additional studies are needed to substantiate these findings.*Arch Therapeutics, Inc. Framingham, Massachusetts. ^3M, St. Paul, Minnesota.
P.BW04.
BACKGROUND: Thermal injury of the face results in ectropion (epithelial-ocular junction), eversion of the lip (epithelial-oral junction), skin contracture, and excessive scar formation. The resultant facial disfiguration along with features like oral incompetence burdens the subject socially, emotionally, and psychologically. The goal of this work was to test the healing outcomes of a glycerin-based wound dressing Elasto-Gel on a preclinical porcine model of maxillofacial burn trauma.
METHODS: Approximately 50% facial surface of the female domestic Yorkshire pig was subjected fourth degree burn involving the bone. Wounds were treated with placebo dressing (Acticoat) or Elasto-Gel once a week for 84 days. Progression of burn wound healing was followed using noninvasive imaging such as laser speckle microperfusion imaging, harmonic ultrasound with Doppler (HUSD), and computed tomography with angiography for 3D reconstruction of face and vasculature.
RESULTS: Heat transfer consistent with modelling IED related burn resulted in fourth degree burn with bone involvement showing severe deficits including ectropion, eversion of the oral mucosa, overt contracture and excessive scarring. These characteristics closely match human clinical outcomes. Affected pigs exhibited oral incompetence during eating. Contracture and scarring were markedly evident at d84 post burn. Elasto-Gel significantly accelerated the rate of wound closure during the acute phase (p<0.05). The later phase of healing was characterized by accelerated regression of blood vessels upon Elasto-Gel treatment consisted with improved healing response(p<0.05). Elasto-Gel treated wounds showed significantly less scar area at all time-points (d21, d42, d63 and d84; p<0.05). Scar stiffness as measured by HUSD elastography was also found to be significantly reduced in the Elasto-Gel treated wound area at the zygomatic arch at d21 and d77 post treatment (p<0.05).
CONCLUSIONS: The glycerin-based wound dressing Elasto-Gel improved healing outcomes in preclinical porcine model of maxillofacial burn trauma.
P.BW05.
BACKGROUND: Probiotics are microorganisms, that when administered in adequate amounts, confer a benefit to the host. We have shown previously that prophylactically administering live cultures of probiotic L. plantarum (Lp) to infected burn wounds protected the animals from infection and significantly reduced the deposition of type I collagen. We hypothesize that the heat-killed probiotic Lp (HKLp) will provide a similar immunomodulatory response and a better healing response as can be achieved from live cultures.
METHODS: Full-thickness scald burn mouse model yielding a 28 % total body surface area burn was utilized. The Luminex assay was used to determine the levels of granulocyte-colony stimulating factor (G-CSF). Mouse macrophage cell line RAW 264.7 and immortalized mouse NIH3T3 fibroblasts were used to study the effects of HKLp. Bone marrow-derived neutrophils obtained from mice exposed to various treatments were subjected to flow cytometry-based neutrophil oxidative burst assay (DHR), E.coli, and pHrodo red phagocytosis assays, NETosis assay, and Annexin V apoptosis assay. IL-6, IL-10 and TNF levels were measured using ELISA analysis.
RESULTS: We found an increased expression of the cytokine, G-CSF in both plasma (903.ą25pg/ml), and tissue homogenates (7147ą 1233. pg/ml) harvested from scald burned mice administered live cultures of Lp when compared to burn alone animals. G-CSF is known to play a role in host defense during microbial infection and to promote wound healing, and our findings on Lp induced elevation is likely the mechanism of protection from infection and improved wound healing. In-vitro studies using RAW 264.7 macrophages and NIH3T3 fibroblasts showed an increased production of G-CSF when stimulated with HKLp (2e8 CFU). Macrophages produced copious amounts of G-CSF (22,554ą676 pg/ml) compared to fibroblasts (18 ą1 pg/ml). In a co-culture model of macrophages and fibroblasts, HKLp stimulated macrophages augmented the production of G-CSF in fibroblasts (182,744.ą 34,992 pg/ml). An increased presence of G-CSF in wound fibroblasts may increase the recruitment of neutrophils from bone marrow to the wound site. Bone marrow-derived neutrophils exposed to HKLp changed the overall neutrophil response by priming the neutrophils for increased phagocytosis, increased production of reactive oxygen species (ROS), induced neutrophil extracellular traps (NETosis) and cell death (apoptosis). HKLp induced neutrophils increased the production of IL-10 and a moderate increase in IL-6 and TNF.
CONCLUSIONS: Overall, our findings suggest that HKLp induces a strong inflammatory response by modulating neutrophils and macrophages, which may thereby change the fibroblast response and prepare the wound bed for better healing outcomes.
P.BW06.
BACKGROUND: Unlike in cases involving wounds in the extremities, applying a splint for a wound on an individual’s trunk or the neck (including areas such as the neck, shoulder, chest, buttock, axilla, and abdomen) is considerably difficult. This may result in the grafted skin not being fixed firmly at the site of the wound on the trunk or the neck. We hypothesized that negative-pressure dressing could help circumvent this issue and allow the grafted skin to remain firmly attached at the site of the wound on the trunk or the neck, and allow relatively easy removal of exudates.
METHODS: From 2013 to 2017, 57 patients with wounds on the trunk or the neck underwent STSGs. Prospective and retrospective data were used for analysis in this study. The size of wounds (maximum length and width, cm) in these patients ranged between 100 cm2 and 400 cm2.Patients were divided into two groups based on the treatment method used. Group 1 included 27 adult patients, requiring STSG, who were amenable to negative-pressure dressing.
RESULTS: In group 1, no serious adverse effects were observed. No major graft lossoccurred. After postoperative day 7, the average survival score for the skin graft was more than 80 inall patients. In group 2, major graft loss occurred in 5 patients. Therefore, a second STSG was performed.
CONCLUSIONS: Negative-pressure wound therapy using meshed STSG seems to be a convenient method for graft fixation in patients with defects of various origins in non-ideal positions. Owing to the flexibility and elasticity of the transparent adhesive film that is utilized during this procedure, patients can be more mobile and feel less discomfort. This method increases the percentage of graft survival and apparently reduces the requirement for second STSG operations. Therefore, the length of hospitalization is shortened as well.
P.CW01.
BACKGROUND: Every year, many patients undergo lower extremity amputation due to chronic diabetic wounds, a life-threatening complication of diabetes mellitus that lacks pharmacologic treatment options. Diabetic skin exhibits increased oxidative stress and decreased expression of the transcription factor Nuclear Factor Erythroid 2-related Factor 2 (NRF2), which activates a cytoprotective pathway in response to increased oxidative stress. Exogenous activation of NRF2 improves wound healing in diabetic mice, and NRF2 -/- diabetic mice heal more slowly than NRF2+/+ diabetic mice, suggesting that NRF2 plays a critical role in diabetic wound healing.
METHODS: An excisional wound healing model was used to determine the effects of the novel NRF2 activators ADJ-310 and PRL-295 on wound healing in Lepr db/db mice. Two full-thickness 8mm dorsal skin wounds were made on each mouse, and wounds were treated daily for 15 to 21 days with either a novel activator, the positive control activator Bardoxolone-methyl, or vehicle alone (either 40% DMSO in PBS, or 10% DMSO in F-127 pluronic gel). Wound closure rates were compared by measuring the open wound area on each day, normalized to the area on Day 0.
RESULTS: In the first study, ADJ-310-treated wounds were smaller than control wounds at every time point from Day 1 to 15, with statistically significant differences on days 1 and 2, suggesting an improved “jump-start” in healing. In a second study comparing ADJ-310, PRL-295, and Bardoxolone-methyl, treated wounds were smaller than controls on every day between Day 0 and 8, after which they could not be measured.
CONCLUSIONS: These results indicate that our novel NRF2 activators improve diabetic wound healing, demonstrate comparable efficacy to pre-existing classes of activators, and serve as an effective tool to study the effects of NRF2 activation. Our investigation highlights the potential of NRF2 activation as a topical therapeutic that could be a practical, feasible measure to improve diabetic wound healing.
P.CW02.
P.CW03.
BACKGROUND: Pressure sore is commonly occurred on bed ridden patients. Common causes of bed-ridden state are medical problems, dementia and central nervous system injury. The elderly patients of pressure sore caused by medical problems are not usually expected to recover physical status, therefore these patients are often excluded on indications of flap surgery. Grade III or IV pressure sores of the patients need flap surgery for complete healing. When surgeons consider whether to do surgery on the elderly patients with multiple morbidities, they should choose between the risk of long general anesthesia, burden of the flap surgery and the benefit of the flap surgery of pressure sore
METHODS: The patients over 65 of age from May 2010 to August 2018 on our clinic were enrolled in this study. The patients with grade III or IV pressure sore who underwent the only debridement of infected tissue were included in the control group and patients who underwent complete coverage of the pressure sore were included in the case group. The age, sex, flap location, and size were analyzed. Serum hemoglobin, neutrophil fraction, albumin, sodium, potassium and C-reactive protein level were evaluated pre and postoperatively in table 1. Numbers of albumin replacement and transfusion per a week were compared on both group. The numbers of 7~1 day before surgery and 7~13 days after surgery were compared
RESULTS: A total of 63 patients was analyzed. 53 patients were treated with flap coverage and 10 patients with the only debridement. The age, sex, and flap size were similar on both groups. The average age was 75.2 on the flap coverage group and 77.5 on only the debridement group. Among the flap coverage group, 47 patients were treated with local myocutaneous flap and the other 6 patients were treated with local fasciocutaneous flap. The most common site of pressure sores was sacrum and ischium. On the debridement group, there was no statistically significant laboratory change after surgery. On the flap coverage group, postoperative neutrophil fraction and CRP level improvement showed significant change. The numbers of albumin replacement and transfusion showed no significant differences comparing before and after surgery.
CONCLUSIONS: Pressure sore coverage with mycutaneous or fasciocutaneous flaps can lead to early recovery and ambulation on rehabilitating patients. In addition, the flap surgery may resolve infection on elderly co-morbid patients with severe pressure sore.
P.CW04.
BACKGROUND: Negative-pressure wound therapy with instillation (NPWTi) is an adjunctive treatment modality for complex and infected wounds. However, commercial devices are not readily available in many countries and are expensive. The objective of this study was to introduce an NPWTi method that can be easily applied where commercial NPWTi devices are not available and to report the clinical outcomes of the NPWTi method for the adjunctive treatment of complex wounds.
METHODS: This prospective clinical experimental study included 51 patients who had wounds for which operative debridement was performed between January 2017 and March 2019. An NPWT device was applied with an intravenous (IV) line for continuous instillation of 0.9% normal saline plus 1% povidone-iodine solution for chronic wounds. The outcomes measured were number of operating room visits, time to final surgical procedure, number of infected wounds, time to resolution of infection, type of reconstruction operation, and occurrence of complications.
RESULTS: The average number of operations performed was 2.5ą0.8, and the time to final surgical procedure was 28.4ą15.4 days. The number of infected wounds was 35 (69%), and the time to resolution of infection was 15ą14.6 days. All wounds were closed or covered. Partial graft failure occurred in two cases, but they were completely healed by secondary healing in 2 weeks.
CONCLUSIONS: A continuous-instillation NPWT system with IV line could be an adjunctive modality in treating complex wounds at institutions where commercial NPWTi systems are not readily available.
P.CW05.
P.CW06.
BACKGROUND: The present study aimed to identify the polymorphisms associated with pressure ulcer.
METHODS: This was a cross-sectional study approved by the research ethics committee of the Graduate School of Medicine, The University of Tokyo, and was conducted in a long-term care facility in Ishikawa prefecture, Japan. Inclusion criteria was the hospital stay for 6 months or longer. The main outcome was the history of pressure ulcers within the last 6 months. Swabs were obtained from the oral cavity of patients and total genomic DNA was extracted. Subsequently, real-time PCR was performed to identify 8 single nucleotide polymorphisms (SNPs) of 5 genes including vascular endothelial growth factor C (VEGFC), hypoxia inducible factor 1 subunit alpha (HIF1A), vitamin D receptor (VDR), heat shock protein 90 alpha family class A member 1 (HSP90AA1), and myostatin (MSTN). The association of SNPs with pressure ulcers was assessed by Chi-square test or Fisher’s exact test.
RESULTS: Total 201 patients were recruited, and 53 patients had history of pressure ulcers within the last 6 months. T allele of VEGFC rs1485766 was significantly associated with Category II pressure ulcers (p=0.050), and T allele of HIF1A rs11549465 was associated with Category III or IV pressure ulcers (p = 0.028).
CONCLUSIONS: These results suggested that SNPs of VEGFC and HIF1A are genetic risks for pressure ulcers.
P.CW07.
BACKGROUND: The Notch pathway is implicated in many cellular processes, a better understanding of this signaling cascade in mouse and human skin could lead us to the development of a therapeutic intervention for the treatment of cutaneous wounds. Based upon our previous work that inhibiting Notch inhibits wound healing, our hypothesis is that topical application of the Notch ligand, JAG1, to ex vivo excisional wounds of mice increases wound healing rates as compared to untreated wounds. We also wanted to study if age and gender have a repercussion in the results of this activation.
METHODS: We cultured ex vivo skin biopsies of 6-mm in diameter with another wound of 3-mm in the center from 10-12 week-old (young) and at least one-year-old (aged) mice. We included both male and female mice from control and skin specific (K14) knockouts of Notch1 and Notch2. Topical application of JAG1 (1μg/ml, 7nM), vehicle (PBS), PBS+DAPT (Notch signaling inhibitor) or JAG1+DAPT were applied every two days. Photomicrographs were taken and histological and protein analysis performed. The tissue growth was calculated as the difference of the area at each timepoint minus the area at day 0 of treatment.
RESULTS: Notch activation by JAG1 resulted in a significant tissue growth of the ex vivo organotypic cultures from mice Notch1+/+ Notch2+/+, young and aged, males and females; compared to K14-Notch1 or K14-Notch2 knockouts. Treatment with DAPT inhibited the growth in mice where JAG1 had a positive effect. JAG1 also better preserved the epidermis strata of Notch1+/+ Notch2+/+ aged mice after 28 days in culture.
CONCLUSIONS: Activation of the Notch pathway by JAG1 increases the rate of tissue growth of cutaneous wounds in an ex vivo murine wound-healing model, but not in Notch1 or Notch2 skin knockouts, indicating that signaling through both Notch1 and Notch2 are crucial in wound healing. This increase was observed in young and aged mice, males and females, being significantly higher in females.
P.CW08.
BACKGROUND: During 8-16 hour aeromedical evacuation (AE), casualties transported on vacuum spine boards (VSB) are at risk for pressure injury. 5-10% are reported to develop pressure injuries (PI). During prolonged field care (PFC), casualties are high-risk due to extended time on unpadded litters or ground, with minimal PI prevention capabilities. PURPOSE: Test the ability of two interventions (foam dressing (FD) or liquid membrane (LM) to mitigate risk factors associated with skin pressure injuries under conditions simulating long-distance AE or PFC.
METHODS: Using a skin microclimate model 72 subjects are being recruited with stratified randomization (% body fat) into six groups: Groups 1&2: AE mattress on stretcher with 30° backrest with/without FD; Groups 3&4: VSB (30° reverse Trendelenburg) with/without FD; Group 5&6: PFC Talon litter with/without LM. Dependent variables: Sacral skin injury (cutaneous IL1-α), skin perfusion (transcutaneous PO2), skin microclimate (temperature, epidermal/subepidermal moisture) and sacral interface pressure. Skin damage scores are estimated using Kokate’s equation, which integrates pressure, temperature and time.
RESULTS: Preliminary results (N = 31). 8 male/23 female: Height: 66 ± 5 inches; Weight 142 ± 23 lbs. BF% 25.3 ± 9.0. BF% Category (Under 4; Within 18; Above 9). Peak skin interface pressure 45 ± 16 mm Hg, with highest on Talon with LM (Median 55 – IQR 50); Skin temperature 35.6 ± 1.1°C (Time 0: 34.0 ± 1.0°C/Time 120: 36.1± 0.8°C), average skin temperature increase (baseline – 120 minutes): 2.9 ± 1.3°C. Loaded sacral TcPO2 42 ± 33 mm Hg (unloaded 68 ± 19 mm Hg), with lowest values on Talon (No LM: Median 8 mm Hg; With LM Median 1 mm Hg). Eight subjects had low (< 5 mm Hg) TcPO2 (7 on Talon – 3 with/4 without LM).
CONCLUSIONS: Preliminary data demonstrate baseline pressure injury risk for three surfaces used under military conditions. This is the first study to describe these conditions and risk beyond skin interface pressure. The temperature increase is an independent risk factor, with every 1°C increase contributing 14 times greater risk of injury than a 1 mm Hg increase. Increased risk for subcutaneous injury will be assessed using epidermal/subepidermal moisture and IL1α.
P.CW09.
BACKGROUND: As the leading cause of non-traumatic, lower limb amputations, diabetic ulcers cost the United States economy an estimated $58 billion annually and are a significant quality of life issue for patients. While normal wound healing proceeds through a well-described process, diabetic ulcers stall at the transition from inflammation to tissue repair/regeneration. Our central hypothesis is that macrophage phenotypic plasticity is essential to this transition and that a disrupted metabolic environment combined with the bioburden of colonizing bacterial biofilms causes this stalled healing process in diabetic wounds.
METHODS: This research fellowship project focused on comparing how macrophages from healthy/pre-diabetic/diabetic donors respond when grown in co-culture with common, wound-associated bacterial biofilms. Briefly, monocytes were isolated from donor blood, differentiated into resting macrophages in culture, and then polarized into five, functionally-defined phenotypes. During polarization, macrophages were co-cultured with biofilms composed of Pseudomonas aeruginosa, Staphylococcus aureus, or Clostridium perfunges, previously established on 0.2痠 pore tissue culture inserts. Biofilms were harvested for quantification of colony-forming units (CFUs) and macrophages were characterized by an analyte matrix of cell surface markers, immunomodulating proteins (cytokines, chemokines, and growth factors), expressed myeloid genes, and metabolites (including lipids). Biological triplicates for each co-culture combination were characterized by this systems biology approach.
RESULTS: We observed similar patterns of early inflammatory markers in both healthy and diabetic macrophages; however, late inflammatory markers were prolonged and exaggerated in diabetic macrophages. We also observed that bacterial biofilm co-culture significantly inhibited macrophage polarization, both pro-inflammatory and anti-inflammatory, in both healthy and diabetic macrophages.
CONCLUSIONS: These findings indicate that prolonged inflammation observed in non-healing wounds may result from inherent macrophage dysfunction in diabetics and that the burden of colonizing bacterial biofilms may dramatically inhibit macrophage functional shift to tissue repair as required for normal wound healing.
P.CW10.
P.CW11.
P.EP01.
BACKGROUND: Randomized studies have shown that Negative Pressure Wound Therapy applied to closed incisions (iNPWT) reduces surgical site infection (SSI) or surgical site complications (SSC), by around 50%1. However, the expense of single-use electromechanical pumps (EMP) (US$250-500) may limit the cost effectiveness of iNPWT 2,3 . This pilot study tested a novel, low-cost-of-manufacture, non-electrical device that uses solid-state oxygen management technology (OxMT) to create and sustain a vacuum in an iNPWT dressing
METHODS: The study was conducted in pigs (n=2) using 12 stapled incision wounds (38mm long, 10mm depth) per animal. All wounds were covered with the same (pre-weighed) iNPWT dressing (length 5cm) connected either to the OxST device or a conventional EMP. Wounds (3) were harvested for each of the OxMT and EMP systems on days 1, 3, 5 and 7 for: total bacterial burden, H&E histology and exudate handling. Pressure sensors were connected to each of the (12) OxMT dressings.
RESULTS: Over the duration of the study mean negative pressure across all OxMT dressings was -81.1 mmHg (n=9, lost 3 sensors). Exudate in the iNPWT dressings was minimal (mean 0.3015 mL n=24). Histological analysis was similar for new epithelial growth, epithelial thickness, white cell infiltration, granulation tissue and angiogenesis. Total bacterial counts for the OxMT treated wounds were significantly lower over the 7 days than the EMP treated wounds, with the difference at day 7 being approximately 1.5 Log CFU/ml.
CONCLUSIONS: This preliminary study on porcine incisions demonstrates that a novel, low cost iNPWT device and dressing system, utilizing a solid-state oxygen management technology, delivers comparable negative pressure therapy to existing electrical devices. Additionally, there are indications that the OxMT technology may have a beneficial effect on limiting bacterial viability in the wound. Additional studies are warranted to substantiate these findings. EMP – PICO NPWT pump Smith & Nephew Inc OxMT – Aatru iNPWT system – Aatru Medical LLC
P.EP02.
BACKGROUND: Reduction of blood loss from patients is one of the most important considerations during surgical procedures, especially during emergency events.1-2 A new composition that provides improved surgical hemostasis while not impeding the wound healing process could greatly reduce patient’s recovery time and improved overall patient outcomes.3 A self-assembling peptide hemostat (SAPH*) is in development to control bleeding during surgical procedures while not interfering with normal wound healing.4 The purpose of this preliminary study was to examine the effect of SAPH on full thickness wound healing in a porcine model.5 Porcine skin is morphologically and biochemically most similar to humans, and it is ideal to evaluate wound healing therapies.6
METHODS: Thirty-two (32) full-thickness wounds were created using a 10mm punch biopsy. Twelve (12) wounds were randomly assigned to one of two treatment groups: SAPH or Saline control. Eight (8) wounds were assigned to a commercial skin substitute (SS)^. Wounds were treated immediately after they were created. All treated wounds were covered with a polyurethane dressing. Histological assessment was performed blind by a dermatopathologist on days 4, 6, 8 and 11.
RESULTS: On Day 8 and 11, the percent of re-epithelialization was highest in wounds that had been treated with SAPH when compared to those treated with SS and Saline control. In addition wounds treated with SAPH had higher granulation scores and lower inflammatory scores than the other two treatment groups (Day 8).
CONCLUSIONS: This study indicates that SAPH may enhance healing of full thickness wounds. Further studies are warranted to substantiate these conclusions.*Arch Therapeutics, Inc. Framingham, Massachusetts.^Integra Life Sciences Corporation. Plainsboro Township, New Jersey.
P.EP03.
BACKGROUND: The skin is considered a primary barrier organ to maintain bodily homeostasis and prevent infection, as well as to prevent water loss. The healing of skin wounds is a complicated, orchestrated process that involves sensation of environmental cues and serves to re-establish epithelial integrity and maintain the homeostatic tissue environment. When loss of epidermal barrier function results from wounding, cells at the wound site are exposed to the wound environment. In particular, the epidermis and dermis proximal to the wound site experience heightened levels of extracellular sodium resulting from the concentration of solutes as a consequence of transepidermal water loss. Our lab has previously demonstrated that increases in sodium concentration stimulate the poorly characterized, concentration-dependent sodium channel Nax (SCN7A) resulting in modulation of downstream pro-inflammatory gene expression that is relevant to inflammatory skin conditions in vivo. However, the role of Nax in critical processes underlying excisional wound healing such as inflammation and re-epithelialization are not well-characterized.
METHODS: Here we utilize murine excisional wound models in the dorsum, scalp, and tail to compare the wound healing processes between wild type mice and knockout mice lacking expression of functional Nax using histological and immunohistological analysis.
RESULTS: Preliminary data suggest that Nax-knockout mice undergo delayed wound healing compared to wild type mice expressing functional Nax as assessed by gross histological parameters including epithelial gap.
CONCLUSIONS: These data suggest that function of Nax leads to progression of wound healing processes including re-epithelialization, in vivo. Future experiments will serve to establish the cell types within which Nax is active during wound healing and the molecular processes underlying the phenotypes driven by the absence of Nax, to work towards a more complete understanding of the role of Nax and changes in the microenvironment during processes critical to wound healing.
P.EXC01.
BACKGROUND: Dupuytren’s contracture (DC) is a condition where connective fibrous tissue continuously grows on the palm of the hand and attaches to the tendon sheaths, resulting in a scar-like deformity. This will progress until movement of fingers is completely immobilized, severely decreasing quality of life. The cell believed to be responsible for DC is the myofibroblast. Myofibroblasts, when placed within an anchored collagen matrix creates migration and contraction properties, similar to what is seen in DC. Migration is focused on lamellopodia movement, while contraction focuses on stress fibers. Both properties are regulated by small G-proteins labeled Rac and Rho, respectively. Compaction requires migration and tension while contraction requires tension only. We focused on the study of Rac and Rho regulation on myofibroblast compaction and contraction in collagen matrices in vitro. We asked whether Rac and Rho regulation would affect anchored collagen tension.
METHODS: Anchored collagen cell matrices were cultured for 3 days to allow for tension generation. On day 4, we added Rac and Rho inhibitors, waited one hour to allow for maximum inhibitory effects, and measured matrix height utilizing optical coherence tomography (OCT). OCT measurement was then repeated for 3 more consecutive days. OCT was used to monitor collagen tension development under anchored conditions. Cells were released from all OCT measured collagen matrices for western blot analysis of Rac and Rho inhibitors.
RESULTS: Inhibiting Rho and Rac reduced compaction; however, Rho inhibition was more effective, suggesting the stress fiber organization of actin was more important to compaction than the migration-related actin organization.
CONCLUSIONS: The in vitro model is useful in studying the effect of inhibitors on an anchored collagen matrix model that focuses on compaction and contraction. OCT allows for the monitoring of tension in an anchored model without the need for release or sacrifice. Rac and Rho proteins may be key targets in inhibiting DC and improving quality of life, reducing the need for costly and invasive surgical procedures.
P.EXC02.
BACKGROUND: Autologous keratinocyte sheets have been introduced as a clinical treatment for burn wounds for over 30 years. However, this method has never been translated to clinical practice with some complications associated like graft rejection and blistering. The reason for this negative outcome is using an enzymatic method for sheet detachment that breaks the extra cellular matrix (ECM) and affects their quality. In this study, we are proposing non-enzymatic method for detaching the sheets by using the temperature responsive dishes (TRD).
METHODS: Keratinocytes were cultured either in TRDs and detached by temperature reduction to 20°C (T-KS), or in conventional dishes and detached by enzyme Dispase at 37°C (D-KS). Sheets were subjected to immunoblotting to measure ECM proteins (Collagen IV and Laminin 5). We also studied the behavior of these keratinocyte sheets on in vitro burn wound healing model created with addition of burn exudate. Cells proliferation and activation of MAPK pathway were measured. The quality of the sheets was assessed by measuring sheet thickness, strength and size. Later, we also tested the efficacy of these keratinocyte sheets on in vivo burn wound healing model. Several histopathological evaluations were measured to assess the efficacy of the sheets as a treatment for burn injury.
RESULTS: The extra cellular matrix proteins (Collagen IV and Laminin 5) were significantly preserved in T-KS, while measured proteins were reduced when sheets treated with enzyme Dispase (D-KS). The activation of MAPK was higher in T-KS compared to D-KS when grafted onto in vitro skin model. The proliferation capacity was significantly increased in T-KS when grafted onto in vitro burn wound healing model. The sheet thickness, strength and size were significantly higher in T-KS when compared to D-KS. The epithelization percentage after grafting KS in vivo on third degree grafted burn wound was significantly higher on wounds treated with T-KS compared to control or D-KS treated wounds. The dermal epidermal junction was well formed between grafted sheets and wound bed and that was concluded with significantly higher number of hemidesmosomes and lamina densa percentage in T-KS treated wounds. T-KS treated wounds tend to have more angiogenesis percentage and less Keratinocyte growth factor expression.
CONCLUSIONS: Effects of non-enzymatically detached keratinocyte sheets are superior to those of enzymatically detached sheets on ovine grafted burn wound healing. Ovine grafted burn wound healing model closely mimics the clinical situation. Non-enzymatic detachment of cultured keratinocytes may potentially enable their translation to clinical practice.
P.FS01.
BACKGROUND: Radiation-induced skin fibrosis is an unwanted side effect of radiation therapy (RT) with the potential to alter skin form and function and profoundly impact quality of life. Subcutaneous injections of deferoxamine (DFO) can improve skin vascularity and promote subsequent fat graft retention in irradiated sites. Here, we asked whether topical administration of DFO prior to irradiation could mitigate radiation-induced skin fibrosis.
METHODS: CD1 Nude mice underwent; 1) irradiation without DFO, 2) irradiation with therapeutic DFO, 3) irradiation with continuous (prophylactic and therapeutic) DFO, or 4) no irradiation and no DFO (control group) (n=5/group). DFO was administrated using a transdermal drug delivery system (TDDS) secured to the mouse scalp and changed every 24 hours. 30 Gy was delivered to the mouse scalps via external beam radiation. A 4-week recovery period followed to allow for radiation-induced skin fibrosis to develop. Prophylactic DFO treatment started 2 weeks before irradiation and continued for 6 weeks post irradiation. Therapeutic DFO started 4 weeks after irradiation and continued for 2 weeks post irradiation. Laser Doppler analysis was used to measure skin perfusion immediately following irradiation and 6 weeks after irradiation, at which point the skin was harvested for histological assessment of vascularization and fibrosis.
RESULTS: Immediately following RT, skin perfusion was significantly improved in mice receiving prophylactic DFO, and 6 weeks after RT, mice receiving prophylactic and therapeutic DFO had the greatest perfusion and greatest immunohistological evidence of vascularity. DFO treatment reduced dermal thickness and collagen content followed RT and resulted in collagen fiber network patterns that closely resembled those found in non-irradiated skin. The greatest benefits were found in mice receiving prophylactic and therapeutic DFO.
CONCLUSIONS: Topical administration of DFO, both before and after RT, improves skin vascularity and reduces skin fibrosis, highlighting a potential therapeutic role for DFO TDDS in patients undergoing irradiation for treatment of their cancer.
P.EXC02.
BACKGROUND: Wound healing is a complex interaction of resident cells, recruited cells, and the extracellular matrix. Monocytes recruited to the wound site during the inflammatory phase of wound healing differentiate into macrophages, including M1 and M2 phenotypes; however, the exact monocytic origin and role of these different macrophage phenotypes in wound healing and scarring remain unclear. This study aims to identify the distribution of the mononuclear phagocyte subtypes during wound healing and subsequent development of scarring in the dermal fibrotic mouse model.
METHODS: To create the dermal fibrotic model, split thickness human skin grafts were grafted onto the dorsum of athymic nude mice. Blood, spleen and scar tissues were collected at day 0 (n=3, before grafting), 3, 7, 14, 30, 60, 90, and 180 (n=6, post-grafting) during the scarring process to analyze the presence of monocytes and M1, M2a, M2b, M2c, and M2d macrophages.
RESULTS: Increased skin thickness was observed, peaking on day 30, in both epidermis (p<0.05) and dermis. An increase in dermal overall cellularity on day 180 (p<0.05) and dermal vascularity on day 14 (p<0.05) was seen. On day 30, a significant increase was seen in CD11b+/Ly6C++/CCR2+ monocytes (p<0.05); whereas CD11b/Ly6C-/CCR2- monocytes peaked on day 60 (p<0.05), as compared to normal skin (day 0). In the hypodermis, M1 macrophages had an initial peak on day 7 and a second peak between days 30 and 60. In the upper dermis, M1 macrophages peaked on day 60 (p<0.01) compared to day 0. An increase in M2b macrophages appeared in the hypodermis on day 30, although not significant. Other M2 subtypes are currently being analyzed.
CONCLUSIONS: It is our hypothesis that by understanding the temporal distribution of monocyte and macrophage phenotypes in dermal scarring, one can identify and target the ‘pro-fibrotic’ macrophage phenotype as a potential therapeutic target in dermal scarring and possibly other forms of fibrosis.
P.FS03.
BACKGROUND:
Angiotensin-converting enzyme (ACE) inhibitors are used as antihypertensive medicines and are effective in the treatment of keloid and hypertrophic scars. We compared the differences in scar development and the effects of systemic ACE inhibitor administration on scar tissue in a normotensive rat, the Wistar Kyoto rat (WKY), and a hypertensive rat, the spontaneously hypertensive rat (SHR).
METHODS: Using an 8-mm punch, we created two full thickness skin defects in 16 WKY and 16 SHR, for a total of 64 defects. We established control WKY (n = 16), captopril-treated WKY (n = 16), control SHR (n = 16), and captopril-treated SHR (n = 16) groups and started captopril (100 mg/g per day) treatment on day 21 in the appropriate groups. The systolic blood pressure (BP) of all groups was measured at 0, 3, and 5 weeks. Histopathology was quantified using the scar area, fibroblast and capillary count. The expression of heat shock protein (HSP) 47, type I and III collagens, alpha-smooth muscle actin (α-SMA), Ki67, and vascular endothelial growth factor (VEGF) was investigated by immunohistochemistry.
RESULTS: The scar area and fibroblast count were significantly higher in control SHR than in control WKY. The scar area, fibroblast count, capillary count were significantly smaller in captopril-treated SHR compared with control SHR. Immunostaining for α-SMA, Ki67, and VEGF also showed prominent decreases in treated SHR vs. control SHR.
CONCLUSIONS: In this study, large scars tended to develop more often in hypertensive rats than in normotensive rats. The ACE inhibitor significantly reduced the scars in the hypertensive rats, while it did not reduce the scars effectively in the normotensive rats. Based on these results, blood pressure might affect scar development in this rat model. Moreover, the ACE inhibitor would be effective at reducing scars that develops only in hypertensive conditions.
P.FS04.
BACKGROUND: We have been researching for the complete regeneration of the skin. The Scar have been treated in various ways, for the purpose of improving the condition, due to the loss of texture, lack of appendages, and changes in color. One of the treatments is to make resurfacing by fractional laser. In this study, we devised a treatment that combines ablative CO2 fractional laser with cell transplantation and examined whether it is possible to treat the scar.
METHODS: A full-thickness skin defect was created in the dorsal skin with 8-week-old male C57Bl / 6 mice, and the scar were confirmed after 2-3 months. The hairless scar site was irradiated with a fractional laser (DEKA), and the cells collected and prepared from the skin of GFP fetal mice were seeded and sealed with a film. After 4 weeks, the tissue was collected to confirm whether the cells were transplanted. In addition, the control group, fibroblast group, fibroblast long-term culture sphere group, and fresh whole skin sphere group were seeded and evaluated.
RESULTS: GFP positive cells were observed in the collected tissue sections. GFP positive cells were found scattered in the dermal papilla layer and subcutaneous. The fibroblast sphere long-term culture group was the closest to normal skin.
CONCLUSIONS: Although the introduction of cells was not uniform between dermis and subcutaneous, cells could be transplanted. With the advancement of technology, it is now possible to create holes with a certain interval depth with little thermal damage, and we believe that cells can be introduced. In the future, it is expected that the cells introduced treatment will be developed by researching the setting of the laser for the human scar.
P.FS05.
BACKGROUND: Wnt proteins secrete glycoproteins involved in many cellular processes to maintain developmental and adult homeostasis. It is also involved in various processes of skin development and the formation of dermis and skin appendages such as hair. However, the expression and role of Wnt protein in wound healing is still unclear. Our previous study showed that after skin wounding early stage fetuses (embryonic day 13; E13) completely regenerate without any scars, but the later stage fetuses (after E14) repair their wound with scars. To elucidate the mechanism of complete skin regeneration, we analyzed the role of Wnt-related protein expression and localization during mouse fetal wound healing.
METHODS: E13, 15 and 17 mouse fetuses were wounded and collected 24 hours later, and frozen sections and paraffin sections were prepared. Wnt-related molecules were detected by in situ hybridization, immunostaining, and real-time polymerase chain reaction. In addition, Wnt enhancers / inhibitors were administered to the wound sites of E15 fetus, collected 48 hours later, and scars were observed with a stereomicroscope and scored by an objective method.
RESULTS: Our results showed that some of the Wnt subtypes during the wound healing process were significantly associated with the presence or absence of scar formation. Wnt inhibitors suppressed scar formation in E15 mice.
CONCLUSIONS: Our results suggested that Wnt signaling is involved in wound regeneration and scar formation. Thus, Wnt signaling can be a therapeutic target in the development of scar treatment.
P.FS06.
BACKGROUND: The anchored collagen matrix model is commonly used to study the myofibroblast phenotype characteristic of fibroses, scars, and contractures. Resident cells in the matrix reorganize and secrete collagen, then generate tension to produce a stiff, concentrated tissue that facilitates cell differentiation and proliferation. Classically, monitoring tissue reorganization required careful tissue thickness or height measurement using light microscopy. This approach presumed the tissue was homogeneous because the technique only provided information at a single point of a large 3D tissue. We recently introduced the use of optical coherence tomography (OCT) to monitor tissue-level remodeling during matrix maturation, thereby providing a non-invasive visualization of the overall cross-sectional contours of the developing tissue. We also showed that tension generation was correlated with matrix compaction (height reduction over time). Our goal was to use OCT to study collagen compaction and contraction in a non-homogeneous matrix.
METHODS: Rat tail type I collagen was mixed with fibroblasts and plated rapidly on 35mm tissue culture-treated dishes and allowed to polymerize. Control matrices were plated in prewarmed conditions. Matrices were cultured submerged in standard culture conditions. Cross-sectional contour was monitored by OCT daily for 7 days. In some cases, matrices were released from anchorage and contraction measured to determine amount of tension present.
RESULTS: We observed a characteristic interface between the lower, compacted matrix and the upper, noncompacted matrix; each phase was characterized by contraction differences, when the matrix was released from anchorage.
CONCLUSIONS: We demonstrate that OCT is an efficient imaging tool to measure localized tension generation in a multiphasic (non-homogeneous) tissue, and that morphological features of the collagen matrix are affected by altered mechanical tension. Using OCT for tissue model characterization will allow investigators to monitor multiple changes in the same tissue during development or perturbation without losing samples to fixing and staining procedures.
P.FS07.
BACKGROUND: Scar formation is an inevitable consequence of surgery and there are many techniques to correct scar such as elliptical excision, Z-plasty and W-plasty. But for depressive scar caused by deep tissue injury, there are no general consensus. Because acellular dermal substitute is world widely used for management of soft tissue defect, we consider acellular dermal substitute application for revision of depressive scar.
METHODS: Between March 2018 and July 2019, a retrospective review of patients who had undergone depressive scar revision using acellular dermal substitute (MegaDerm®, L&C BIO Inc., Seoul, Korea) was performed. In the surgical procedure, after excision of scar, acellular dermal substitute was inserted under the muscle or subcutaneous layer. We enrolled 15 patients and evaluated using the Vancouver Scar Scale (VSS).
RESULTS: The patient group consisted of 9 men and 6 women between age of 18 and 77 (A mean age = 46.7). After surgery, depressive scar was improved in all patients and filling effect persisted without relapse of a depression. Although 1 case was seen skin partial necrosis, all patients were satisfied with their appearance. The mean total VSS score improved from 6.57 to 3.29.
CONCLUSIONS: A small number of patients had a short follow-up period, so further studies that included a larger number and long follow-up period was needed. Despite these limitations, we get a favorable result from patients on appearance of the scar. It allows patient’s high satisfaction, good skin-quality and low complication. So we recommend considering acellular dermal substitute application as an option for management of depressive scar.
P.IB01.
BACKGROUND: Diabetic Foot Ulcers (DFU) are common and dangerous complications of diabetes, emerging in between 19 and 34% of patients with the disease. The presentation of a DFU alone corresponds to a 2.5-fold increase in five-year mortality risk among diabetic individuals, owing in part to the fact that over half of DFU become infected and that an estimated 20% of moderate and severe DFU infections necessitate amputation. Many such infections are attributed to the bacterium Staphylococcus aureus, but this species is commonly cultured from DFU even in the absence of clinical signs of infection and can also be found in the normal microbiota of the human skin and nasal epithelium.
METHODS: Having previously shown that different strains of S. aureus promote divergent healing outcomes in DFU, we now present a genome-wide association study (GWAS) for the genetic mechanisms underlying S. aureus virulence. Our method leverages complete genome sequences for 224 longitudinally collected S. aureus isolates from DFU against clinical metadata, metagenomic community profiles, and in vitro phenotypic measures matched to these isolates.
RESULTS: Applying machine learning methods to these data, we uncover genetic variants in S. aureus which may boost its ability to delay healing and cause chronic infection. Our results reflect the first large-scale comparative genomic analysis, to our knowledge, of variable S. aureus virulence.
CONCLUSIONS: Our identified variants could aid in early risk diagnoses and appropriate treatment decisions, but could also potentially serve as therapeutic targets for novel DFU treatments.
P.IB02.
BACKGROUND: Six million people in the US seek treatment for chronic wounds annually. Biofilms play a key role in delayed healing of chronic wounds. We have developed a 20 µm thick bioresorbable antibiofilm polymeric matrix containing approximately 0.1 mg/in2 silver and 0.8 mg/in2 gallium ions and demonstrated its efficacy at killing P. aeruginosa (PA) encased in biofilms. Bacteria in biofilms cannot distinguish gallium (Ga3+) from iron (Fe3+) ions because of their similar ionic radii. Intake of Ga3+ disrupts iron-dependent redox processes because it cannot be reduced in physiological conditions. We hypothesized that uptake of Ga3+ by bacteria may sensitize them to low non-toxic concentrations of silver.
METHODS: Robust biofilms containing 108 PA CFU were established on 10×10 mm gauze specimens over 48h, rinsed with saline to remove planktonic bacteria, and confirmed with crystal violet staining. These gauze-biofilm specimens were placed on blood agar and treated for 24h with a matrix containing silver, gallium, or identical doses of silver and gallium paired (test matrix). Post treatment, specimens were rinsed, homogenized and plated to determine CFU. For in vivo evaluation, gauze-biofilm specimens were placed in 6 mm diameter splinted mice wounds (day 0) for 24h, resulting in colonization of biofilm in wounds (107 CFU). Twelve wounds were treated with test matrix and covered with Telfa® and Tegaderm® on days 1, 2, 3, 5, and 7. Negative control wounds received only Telfa® and Tegaderm®. Wounds were harvested on day 9, homogenized, and plated to determine CFU. Test matrix was further subjected to a panel of biocompatibility testing per ISO 10993.
RESULTS: In vitro experiments revealed that silver and gallium worked synergistically to kill biofilm bacteria. Microfilm matrix containing silver or gallium alone reduced biofilm bacteria by ≤1 Log10 CFU, whereas test matrix killed 5.2±0.4 Log10 CFU. In-vivo experiments showed test matrix reduced CFU below threshold of clinical infection (5 Log10 CFU/g tissue) compared to clinically infected negative control (p<0.05) by day 9. Furthermore, test matrix exhibited no acute systemic toxicity in mice.
CONCLUSIONS: Silver and gallium delivered through microfilm matrix killed PA encased in biofilms at potentially non-toxic doses.
P.IB03.
BACKGROUND: A variety of wound matrix materials that are designed to help heal both acute and chronic wounds are currently available. Since wounds often encounter opportunistic microbes that can delay healing, the beneficial effects of these materials may be altered. The aim of this study was to examine the ability of a novel dressing consisting of purified native cross-linked extracellular matrix with polyhexamethylene biguanide (PHMB) to control bacterial load in deep dermal wounds infected with Methicillin Resistant Staphylococcus aureus (MRSA) in a porcine model. This novel dressing (PCMP) utilizes collagen Type I and PHMB to create a protective biocompatible scaffold.
METHODS: Two versions of PCMP were tested (regular, and thicker (PCMP-XT). Pigs (n=6) were used due to their skin similarities to humans1. On each animal, twenty-four deep dermal wounds (22mmX22mmX3mm) were created with a dermatome and inoculated with MRSA (106 LogCFU/ml). Wounds were covered for 72 hours with a polyurethane dressing to allow for biofilm formation.2-4 Wounds were then debrided and sets of wounds were randomly assigned to one of the following six following treatment groups: 1) PCMP*, 2) PCMP-XT˟, 3) Antimicrobial dermal repair scaffold: ADRS°, 4) Antimicrobial hydrofiber wound dressing: AHWD^, 5) Antimicrobial wound gel (AWG)~, and 6) untreated control. Groups of wounds were biopsied and assessed for MRSA counts on days 4, 8 and 11 after treatment application.
RESULTS: All treatment groups significantly reduced MRSA counts as compared to baseline and untreated control. PCMP and PCMP-XT treated wounds showed a significant reduction in counts as compared to ADRS and AHWD. PCMP-XT also showed lower MRSA counts as compared to AWG treated wounds. Overall, PCMP-XT was the most effective treatment group to reduce MRSA counts.
CONCLUSIONS: Our results suggest that wound matrix dressings that contain antimicrobials may have added beneficial effects when used in the clinic. These results may have important clinical implications.
*PuraPly AM™ Antimicrobial Wound Matrix (Organogenesis Inc., Canton, MA) X PuraPly AM-XT™ Antimicrobial Wound Matrix (Organogenesis Inc., Canton, MA) °Primatrix® Ag (Integra Lifesciences, Plainsboro NJ) ^Aquacel-Ag™ (ConvaTec Inc, Bridgewater, NJ) ~BlastX™ Wound Gel (Next Science Ltd, Jacksonville, FL)
P.IB04.
BACKGROUND: Chronic and acute wound infections can be difficult to treat and very costly.1 Cationic antimicrobial cyclic lipopeptides have been developed to provide broad spectrum activity.2
METHODS: The purpose of this study was to examine the effect of two novel cyclic lipopeptides on wounds infected with either Methicillin Resistant Staphylococcus aureus (MRSA) or Pseduomonas aeruginosa (PA) using porcine infection models.3,4 We also determined their effect on healing. For the microbiology study (n=4), 36 full thickness wounds (10mm) were created. Two animals were inoculated with MRSA and the other two with PA. Prior to treatment all wounds were covered with a polyurethane film dressing for 24 hours to allow biofilm formation.3 Wounds were then divided and treated daily with either: DMSO+Peptide 2605-4 (20mg/ml) against MRSA or DMSO+Peptide 2612-8.1 (20mg/ml) against PA; DMSO Vehicle Control; Mupirocin or silver sulfadiazine positive controls; or untreated control. The wound healing study (n=2) were wounded and treated as described above. Microbiological assessments were performed on days 2 and 5 post infection. Histological wound assessments were performed blinded on days 5, 7 and 10.
RESULTS: For both MRSA and PA analysis, the peptide exhibited comparable results against the respective positive controls while showing significantly lower counts than baseline, untreated and Vehicle control wounds. By day 5, MRSA infected wounds treated with DMSO+Peptide had lower bacterial counts when compared against baseline wounds and untreated control. PA infected wounds treated with DMSO+Peptide were significantly lower than all other wounds, including the positive control. Peptide 2605-4 significantly inhibited re-epithelialization as compared to DMSO and untreated controls.
CONCLUSIONS: These results show that the lipopeptides can have different antimicrobial and wound healing effects. Therefore, it is important when studying the effects of antimicrobial therapies, one also needs to evaluate their effects on healing. This data may have important clinical implications. This study was supported by US Army grant W81XWH-15-1-0658
P.IB06.
BACKGROUND: Oregano is an aromatic herb that has demonstrated antimicrobial, antioxidant, and anti-inflammatory properties.1,2 However, the essential oil component of oregano contains terpene compounds that are strong sensitizers.3,4 Water-soluble oregano extract retains phenolic compounds and is obtained after distillation of the essential oil.5 A previous randomized, double-blind, controlled study using 3% water-soluble oregano extract in a petrolatum vehicle on post-surgical wounds demonstrated a decrease in S. aureus culture positivity as compared to petrolatum, with no increase in pruritis, allergic contact dermatitis, or other adverse effects.6
METHODS: Minimum inhibitory concentration (MIC) was determined for 10 microorganisms commonly implicated in skin pathology (B. subtilis, C. difficile, C. acnes, E. faecalis, E. coli, M. Furfur, MRSA, MSSA, P. aeruginosa, and S. pyogenes). This was done by growing the microorganisms on agar plates containing various dilutions of water-soluble oregano extract, and recording the lowest concentration that inhibited visible growth.
RESULTS: MIC was successfully determined for 9/10 organisms: B. subtilis (0.10%), C. difficile (1.0%), C. acnes (0,50%), E. faecalis (1.0%), E. coli (10.0%), M. Furfur (0.50%), MRSA (0.75%), MSSA (0.75%), and S. pyogenes (1.0%).
CONCLUSIONS: Our results support the antimicrobial properties of water-soluble oregano extract. Specifically, in vitro inhibition of MRSA, MSSA, and S. pyogenes growth may indicate a role for water-soluble oregano extract in wound care. This would decrease the risk of allergic contact dermatitis associated with common wound-healing ointments, which is attributed to ingredients such as lanolin, bacitracin, and neomycin.7
References: 1. Baricevic D, et al. The biological/pharmacological activity of the Origanum Genus. In Kintzios SE. Oregano. London and New York:Taylor & Francis; 2002: 177-214. 2. Eng W, et al. Development of an oregano-based ointment with anti-microbial activity including activity against methicillin-resistant Staphlococcus aureus. J Drugs Dermatol. 2010 Apr;9(4):377-80. 3. Hausen B, et al. Degradation products of monterpene are the sensitizing agent in tea tree oil. Am J Cont Derm. 1999; 10(2):68-77.4. de Groot A, et al. Essential Oils, Part IV: Contact Allergy. Dermatitis. 2016;27(4):170-175. 5. Havkin-Frenkel D. (Bakto Natural Preservatives, LLC., US). Recovery of residual plant components after distillation of essential oils. US Patent 0254149 A1, October 16, 2008. 6. Ragi J, et al. Oregano extract ointment for wound healing: a randomized, double-blind, petrolatum-controlled study evaluating efficacy. J Drugs Dermatol. 2011 Oct;10(10):1168-72.7. Jacobs, S, James, W. Bacitracin after clean surgical procedures may be risky. J Am Acad Dermatol. 2004; 51(6):1036.
P.IB07.
BACKGROUND: There is a need to improve current assessment procedures used to track and diagnose the bacterial colonization of chronic, non-healing wounds towards infection. Real-time, rapid detection of antimicrobial susceptibility has the potential to significantly reduce healing times and morbidity in patients with chronic wounds. Current infection detection methods rely heavily on clinician experience and lab culturing. These methods are highly subjective and time consuming.
METHODS: In this work we developed a point-of-care biosensor that can detect not only the presence of bacteria, but also metabolic activity towards the release of virulence factors related to the spread of infection. We used an impedance-based approach to provide clinicians with a quantitative tool to detect the onset of increased bacterial growth or antimicrobial susceptibility. The sensors consist of poly-l-lactide/multi-walled carbon nanotube composites arranged into a nonwoven nanofiber mat via solution blow spinning (SBS).
RESULTS: 1. 1. Produced SBS nanofibers with various chemical agents 2. 2. Validated in-vitro efficacy of bacterial quantification against Pseudomonas putida. Conductivity values up to 474 S/cm were generated to detect the increased bacterial growth and antimicrobial susceptibility of Pseudomonas putida (P. putida). Bacteria were cultured in the presence of glucose supplemented media and the impedance spectra were recorded over a 24-hour period. Functions for continuous and endpoint bacterial growth were derived from impedance data with respect to bacterial concentration and nanofiber composition. Additionally, AgNO3 (10, 100, 1000 mM) was used to determine the antimicrobial susceptibility of P. Putida to include a therapeutic effect along with the diagnostic sensor. Zone of inhibition testing was employed to determine the Antimicrobial efficacy of various AgNO3 concentrations.
CONCLUSIONS: Further development of this technology has the potential to provide clinicians with a tool for rapid bacteria quantification and therapeutic decision making.
P.IB08.
BACKGROUND: Some wounds remain chronically infected despite aggressive antibiotic therapy. In part, drugs fail to clear chronic wound infections because some pathogen populations adopt a metabolic state that renders them tolerant to antibiotics. Antibiotic tolerance is associated with pathogens that grow as biofilms, where cells inside the biofilm have low metabolic activity because they lack access to oxygen. Thus, an important area of research is identifying drugs that kill antibiotic-tolerant pathogen populations living in hypoxic environments such as those inside biofilms.
METHODS: Nitrate respiration is a widespread mode of anaerobic energy generation used by many pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Having identified nitrate respiration as a relevant therapeutic target, we tested the ability of chlorate to kill pathogen populations that use this metabolism. We previously showed that the P. aeruginosa nitrate reductase, Nar, reduces nontoxic chlorate (a nitrate analog) to generate toxic chlorite within the cytoplasm. We showed that chlorate specifically kills hypoxic/anoxic P. aeruginosa biofilm populations; these same populations exhibited tolerance to the aminoglycoside, tobramycin.
RESULTS: Our current work expands on these promising findings. By subjecting a library of P. aeruginosa transposon mutants (TnSeq) to chlorate, we identified genes that are important for the pathogen’s defense against chlorate, as well as genes that can be mutated to confer chlorate resistance. These experiments suggest protein oxidation and misfolding contribute to chlorate-mediated cell death. Additionally, mutations that disrupt nitrate reductase (nar genes) are the primary mechanism of chlorate resistance, so acquiring resistance might hinder pathogen growth and biofilm formation in wounds. We are also testing chlorate’s ability to resolve chronic wound infections in a diabetic mouse model system. We are using novel imaging techniques to generate 3D reconstructions of fluorescently labeled bacteria within these chronic wounds. This approach will allow us to identify which biofilm populations are killed by different drugs (e.g. antibiotics vs. chlorate) in vivo.
CONCLUSIONS: Together, this work explores chlorate’s potential as a prodrug for killing antibiotic-tolerant pathogen populations in chronic wounds.
P.IB09.
P.IB10.
BACKGROUND: To demonstrate that marginal surgical amputation or debridement can be performed, instead of amputation of the foot or leg, in well selected cases of complicated diabetic foot.
METHODS: Five cases of diabetic patients complicated with skin infection, soft tissues and osteoarticular structures, in which more extensive amputations are traditionally performed, and who underwent amputation and surgical debridement in the healthy margin. of the involved tissues, prospectively, seeking to preserve the limb.
RESULTS: Limitation of damage and recovery of the rest of the limb was achieved without the need for major amputations in 100% of the cases shown, with closure of the lesions in less than 20 weeks after the cleaning surgery. For the closure of the same, no vacuum system type VAC or Hyperbaric Oxygen Therapy, PRP or Stem Cells were used.
CONCLUSIONS: Debridement and marginal surgical cleaning is an excellent therapeutic option in well selected cases of infected diabetic foot, without the need for major amputations. It requires extensive experience and adequate follow-up of cases to decide which and when is the best time to perform such surgical interventions.
P.IM01.
P.IM02.
BACKGROUND: We have previosuly reported the ability of dermal fibroblasts to express a number of pro-inflammatory cytokines after exposure to lipopolysaccharide (LPS) in vitro. These studies have now been extended to show that disruption of a confluent monolayer of cells in the classic Dulbecco wound model is also a potent inducer of these cytokines.
METHODS: Non-transformed human dermal fibroblasts (GM23971 & GM23967) were obtained from the NIGMS cell bank at the Coriell Institute. The cells were grown to confluence in 100 mm2 dishes in DMEM medium containing 10% FBS and penicillin/streptomycin. The cell monolayers were scraped in a grid profile using a micropipette tip and total RNA extracted at various times using a Monarch® Nucleic Acid Purification Kit (New England Biolabs). Quantitation of mRNA was by qRT-PCR using an iTaq Universal SYBR Green One-Step Kit (Bio-Rad Laboratories) with primers synthesized by IDT for interleukin-1 (IL-1), interleukin-8 (IL-8) and GPDH. Values are expressed as the ∆∆CT value using GAPDH as the housekeeping gene. Positive controls were non-scraped cells treated with LPS for 24 hrs. @ 1.0 µg/mL.
RESULTS: An inital study showed that 24 hrs. after scraping the cell monolayer, IL-8 mRNA levels had increased to 5.4 relative to non-scraped, with LPS treatment increasing the message levels by 14.7. A second study at 18, 24 and 30 hrs. post-scraping showed IL-8 values of 5.87, 7.25, & 5.21 respectively; and a third study at times of 8, 18 and 24 hrs. showed values of 7.9, 725, & 8.38 respectively, whereas IL-1 levels were 1.3, 4.82 & 4.37 at these times.
CONCLUSIONS: These data illustrate the capacity of fibroblasts to induce expression of at least two pro-inflammatory cytokines following a disruption of a confluent monolayer. Disruption of the cell monolayer therefore represents a second stimulus to express these pro-inflammatory mediators, along with exposure to LPS, suggesting a potential contribution of fibroblasts to the inflammatory phase of wound healing.
P.IM03.
BACKGROUND: Monocytes and macrophages play a critical role in all phases of skin wound healing. Dogma suggests that monocyte/macrophage accumulation in skin wounds is driven primarily by recruitment of blood monocytes. The purpose of this study is to determine whether proliferation of monocytes and/or macrophages contributes to their accumulation during skin wound healing.
METHODS: Male C57Bl/6 mice (N=4-12/group) were subjected to excisional wounding with an 8mm biopsy punch. Cell proliferation was determined by immunofluorescent staining of wound cryosections and flow-cytometry of cells isolated from wounds. Protein levels were measured by a multiplex assay in wound homogenates. Differences between groups were considered significant if P≤0.05.
RESULTS: Proliferating monocytes/macrophages (F4/80+Ki67+) were observed in wound cryosections, peaking on day 5 post-wounding. Surprisingly, cell cycle analysis revealed that wounding increased both the number and percentage of pro-inflammatory Ly6C+ monocytes/macrophages in the S/G2/M phases, peaking on Day 6 post-wounding. In contrast, the more mature F4/80+Ly6C- cells were found predominantly in the G0 phase with a significantly lower percentage of cells in S/G2/M phases compared to Ly6C+ cells in response to injury. In the peripheral blood, monocytes were very rarely found in the S/G2/M phase, suggesting that the wound environment triggered Ly6C+ monocyte proliferative response. Furthermore, wound monocytes/macrophages expressed surface receptors of several potential regulators of proliferation, suggesting cells can respond to those factors during wound healing. Our current focus is to identify the critical regulator(s) of wound monocyte proliferation.
CONCLUSIONS: Our novel findings indicate that proliferation may contribute to monocyte/macrophage accumulation in wounds and, contrary to findings in other pathophysiological conditions, monocytes proliferate during skin wound healing at a much higher rate than mature macrophages.
P.IM04.
BACKGROUND: Microarray analysis showed that the expression levels of 22 microRNAs (miRNAs) in diabetic-derived neutrophils were more than double those in non-diabetic-derived neutrophils, while those of 80 miRNAs were decreased to less than half in fold change analysis. Furthermore, we have suggested that miRNAs might be involved in the dysfunction of diabetic-derived neutrophils, and retention kinetics of neutrophil and chronic inflammation might be initiated by miRNAs-regulated genes. Accordingly, miRNAs play an important role in regulation of diabetic-derived neutrophils. By the way, there are reports that circular RNA (circRNA) have function as miRNA sponges or as miRNA reservoirs to regulate miRNA availability for cancer and type 2 diabtes mellitus. Therefore, in order to clarify the molecular mechanism of regulation of inflammation in diabetic-derived neutrophils, we identified specific circRNAs in diabetic-derived neutrophils using microarrays.
METHODS: Neutrophils from pooled BM of 3 db or 3 non-db mice were isolated using anti-Ly-6G microbead kit (MACS), and total RNA was extracted by miRNeasy mini kit (QIAGEN). Microarray analysis was performed on a total of 6 pools (3 pools of 3 db BM samples and 3 pools of 3 non-db BM samples) using Arraystar Mouse Circular RNA Array (Arraystar).
RESULTS: Microarray analysis showed that diabetic-derived neutrophils expressed 3 circRNAs signficantly higher than non-diabetic-derived neutrophils, and 6 circRNAs showed a significant decrease, and these circRNAs differentially expressed have binding sites of miRNAs, which differentially expressed in diabetic-derived neutrophils.
CONCLUSIONS: These results suggest that circRNAs may be involved in the dysfunction of diabetic-derived neutrophils.
P.NOV01.
P.NOV02.
BACKGROUND: Negative pressure wound therapy (NPWT) with a reticulated open cell foam (ROCF) has impacted the practice of healing wounds. While effective in stimulating granulation tissue formation, the ease-of-use of the therapy can be improved due to challenges including rapidly obtaining a good seal and performing dressing changes at 2-3 day intervals. The goal of this study was to evaluate genomic responses due to a novel dressing from a porcine study.
METHODS: Four domestic porcine were used to acquire samples from full thickness skin excisional wounds (3.0 x 7.5 cm) treated with either NPWT ROCF1, NPWTi-d2 , NPWT ROCF with novel dressing3, or NPWTi-d with novel dressing4. Biopsy samples were taken at study termination day 7. Porcine PCR wound healing arrays were performed to determine differences in gene expression (>2 fold difference; p<0.05).
RESULTS: The novel dressing demonstrated greater upregulation in genes important in signaling (WNT1-inducible signaling pathway protein 1-like, (2.19)) and collagen (Collagen type 1, alpha 1, (2.04)) while down regulating genes for extracellular matrix remodeling enzymes (Matrix metallopeptidase 9 (MMP9), (-5.89)) as compared to NPWT with ROCF. The novel dressing with instillation showed an increase in endothelin 1 (2.82) while down regulating MMP9 (-3.86) as compared to NPWT ROCF and when NPWTi-d was compared to NPWTi-d with novel dressing, prostaglandin synthase-2 was decreased (-3.34). Interestingly, if the novel dressing was left in place for all 7 days of treatment, without a dressing change, the only difference was a greater down regulation of MMP9 (-9.74).
CONCLUSIONS: Following 7 days of treatment in a porcine model, a novel NPWT dressing has, relative to control ROCF dressings, been shown to increase extracellular matrix constituents and WNT1-inducible signaling pathway protein 1 while down regulated MMP9 which might lead to an increase in structural constituents important to wound healing.
P.NOV03.
BACKGROUND: Chronic wounds represent a significant clinical burden and result in substantial morbidity and mortality in patients who suffer from them. Thus, therapeutic modalities that promote the closure and enhance the subsequent quality of chronic wounds are of critical interest. Placental allografts have long been utilized in the clinic, but most of these products have utilized tissue processed from the amnion alone. Further, chemical processing steps and sterilization procedures are likely to damage some of the pro-regenerative factors within the tissue. Recent clinical success has prompted us to investigate the efficacy and mechanisms underlying wound healing promoted by aseptically-processed allografts containing both amnion and chorion tissue.
METHODS: We utilized a delayed wound healing model consisting of splinted excisional wounds applied to the dorsa of db/db mice, to which placental-derived, aseptically processed dressings, or no placental dressings, were applied. Gross wound healing was observed and quantified by photography and histology. Immunohistological analysis was performed to stain for Ki67 and CD31 to quantify proliferative cells and vascularization, respectively, at the wound site. Immunofluorescence using macrophage markers was used in order to evaluate macrophage phenotypes at the wound site. Quantification of expression of fibrosis markers was performed by qRT-PCR analysis of expression of pro-fibrotic genes.
RESULTS: We demonstrate that application of dehydrated, aseptically-processed human amnion/chorion allograft (dHACA) resulted in increased rate of wound closure and formation of granulation tissue compared to application of dehydrated human amnion allograft (dHAA) or wounds to which only normal dressings were applied, as assessed by histological analysis. Application of dHACA resulted in enhanced Ki67 and CD31 staining in the dermis, denoting increased rates of granulation tissue formation and neovascularization, respectively. Analysis of wound macrophage markers suggested that dHACA application resulted in promotion of a shift from M1 to M2 macrophage phenotype, denoting enhanced progression of wound healing beyond dHAA-applied and control wounds analyzed at the same time point. Analysis of expression of pro-fibrotic genes Acta2, Ccn2, and Col1a1, encoding α-SMA, CTGF, and type I collagen, respectively, demonstrated that application of dHACA resulted in reduced expression of pro-fibrotic genes.
CONCLUSIONS: Taken together, these data suggest that aseptically-processed, amnion/chorion allografts effectively promote wound closure in a chronic wound murine model via increasing the rate of progression of the wound healing cascade while antagonizing pro-fibrotic gene expression
P.NOV04.
BACKGROUND: Disruption of dermal architecture is a central characteristic of scarring. Despite decades of effort devoted to “engineering” skin tissue, reconstitution of dermal architecture remains an elusive goal, and can only be achieved by autologous skin grafting. Recently, we developed a method to harvest small, full-thickness micro skin tissue columns (MSTCs) with minimal donor site morbidity. Treating wounds with randomly applied MSTCs accelerated wound closure but failed to restore native dermal architecture. We hypothesize that orientation of MSTCs along the epidermal-dermal axis could lead to a more faithful recapitulation of native skin’s mechanical and structural properties.
METHODS: We developed a magnet-based self-assembly approach to achieve this at scale. In brief, a thin magnetic coating is applied to the epidermal surface of the donor site prior to harvest, the MSTCs are then oriented with an external magnetic field and placed into the wound bed. Various scaffolding and adhesive materials were screened for their ability to enable the assembled MSTCs to withstand mechanical stresses in the wound environment. The healing response following implantation of these MSTC assemblies was tested in the porcine model. Tissue biopsies were taken a regular time points and Trichrome staining was used to label dermal collagen.
RESULTS: Our approach was able to orient MSTCs en masse, forming densely-packed constructs with high fidelity in epidermal-dermal polarity that closely resembles the microarchitecture of natural skin. This method is scalable to large areas, and integrity of the constructs could be maintained in vivo using clinically available adhesive dressings. Porcine excision wounds treated with oriented MSTCs had a significantly higher collagen content after 4 weeks in comparison to wounds treated with randomly scattered MSTCs.
CONCLUSIONS: This technology has the potential to restore full-thickness skin via preservation of skin’s native architecture to ensure proper function – the “holy grail” that has long eluded the skin tissue regeneration field, and provide a new, practical modality for treating deep skin wounds where dermal restoration is important.
P.NOV05.
BACKGROUND: Wound care and the associated morbidity and mortality continue to pose major challenges in medicine and society, especially chronic non-healing wounds. In recent years, alternative medicine has become increasingly used to treat chronic wounds due to its natural origins, weaker side effects, and low cost compared to synthetic pharmaceutical drugs. “Healing Paste” is a topical Chinese herbal medicine that has been used in clinics to treat chronic wounds based on descriptive evidence. It is a mixture of the following 5 herbs: Bai Ji (Blettilla striata), Mo Yao (Myrrh), Ru Xiang (Frankincense), San Qi (Pseudoginseng), and Xue Jie (Draconis Sanguis).
METHODS: The objective of this study was to assess the effects of individual components of “Healing Paste” on keratinocyte and fibroblast proliferation and migration, and microbial growth.
RESULTS: Scratch migration assays demonstrated significant increases in keratinocyte migration 24-hours after treatment with Bai Ji (p < 0.001) and San Qi (p < 0.0001) compared to controls. In addition, chamber migration analysis revealed the significant effects of Bai Ji and San Qi on fibroblast migration (all p < 0.0001) compared to controls. Bai Ji also significantly increased fibroblast proliferation throughout a 7-day timing course (p < 0.01) and keratinocyte proliferation throughout a 5-day timing course (p < 0.001). Agar well diffusion assays revealed the inhibitory effects of a mix of all 5 components on methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa growth 24-hours after treatment (all p < 0.0001). Individually, Mo Yao, Ru Xiang, San Qi, and Xue Ji significantly inhibited methicillin-sensitive S. aureus growth (all p < 0.0001); Bai Ji, Mo Yao, Ru Xiang, and Xue Ji significantly inhibited methicillin-resistant S. aureus growth (all p < 0.001); and Ru Xiang and San Qi significantly inhibited Pseudomonas aeruginosa growth (all p < 0.0001).
CONCLUSIONS: Our results suggest that the mechanism in which “Healing Paste” promotes chronic wound healing includes induction of keratinocyte and fibroblast proliferation and migration and inhibition of microbial growth. This study provides scientific evidence for the use of “Healing Paste,” which will further be investigated using mouse models.
P.NOV06.
BACKGROUND: Debridement is a fundamental step to prepare wounds for healing. Among different options, enzymatic debridement does not require surgical training and is easy to perform. As papain/urea ointment (PUO) is no longer available in the U.S., there is a need for a new efficient enzymatic debrider. We identified SN514 protease as a candidate for rapid debridement of necrotic tissue. SN514 gel was evaluated for removal of thermal burn eschar in a porcine model and compared to PUO and vehicle.
METHODS: Partial-thickness burns were introduced by applying heated brass rods to the dorsum of Yorkshire-cross pigs. Thermal burn wounds were covered with dry dressings and wraps, and the burns were allowed to progress and form eschar over five days. Daily SN514 gel application was then initiated. SN514 was compared to vehicle or PUO. Clearance of burn eschar over time was tracked by scaled clinical scoring, imaging, and quantitation of eschar area. Histological analysis confirmed the partial-thickness nature of necrotic tissue in burn wounds and removal of necrotic tissue.
RESULTS: Based on measured area of eschar, SN514 gel showed a highly significant debridement effect vs. vehicle (p<0.05 at 3-4 days treatment, p<0.0002 for later time points) Clinical scores of eschar thickness demonstrated significant debridement occurred after just two days of treatment (p<0.0002 to study end). Modest peri-burn erythema was observed; however, peri-burn erythema lessened as treatment of burns was stopped after completion of debridement. Modest erythema was also observed when SN514 was applied directly to intact skin. Histology performed on burn samples at study end confirmed the removal of burn eschar by SN514. Comparing SN514 gel to PUO, a favorable trend for debridement occurred over early days of treatment, and SN514 overall exhibited similar debridement efficiency to PUO through study end.
CONCLUSIONS: SN514 is a promising drug candidate that could provide rapid enzymatic debridement for burns and chronic wounds.
P.NOV07.
BACKGROUND: Lack of access to adequate wound care supplies can have a detrimental effect on the general patient population’s ability to heal a wound which many times progresses to infection, especially among poor and disadvantaged patients. In the US annually 8.2 million Medicare beneficiaries have a chronic non-healing wound at a cost of over $31 billion dollars annually. More than 7 million elderly people live below the poverty level. Many times lack of adequate bandaging supplies will lead to failure of typical topical wound care treatments as well and represent the a nidus for infection and a pathway of potential progression to life threatening infection.
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METHODS: Traditional bandage systems will be compared to one which is designed to be reusable on the same person and supports soft tissue, muscles, and joints and aids in mobility and recovery.
RESULTS: This modern approach will be shown to protect, support and assist in wound healing in a hypoallergenic fashion versus traditional cotton gauze wraps at a cheaper price.
CONCLUSIONS: Treating patients using a microbial-resistant compression and securement device can have a positive impact on wound healing and infection modulation in poor and disadvantaged patients and can restore patients to a healing state and decrease the potential progression to infection when compared to traditional bandage systems due in part to their ability to be reused.
P.NOV08.
BACKGROUND: Wound healing normally progresses through four overlapping phases (hemostasis, inflammation, proliferation, and remodeling). Despite many attempts to develop advanced therapies to accelerate one or more of these phases, autologous skin grafting remains the “gold standard” for wound repair, but is limited by donor site morbidity. We recently developed the approach of using autologous, full-thickness micro skin tissue columns (MSTCs) to enhance wound healing. The small size of MSTCs (< 1mm diameter) allows donor sites to heal quickly with minimal morbidity, which eliminates the main drawback of autologous grafting. In this study, we investigated how treatment with MSTCs alters the healing process.
METHODS: 3cm-diameter full-thickness excision wounds were produced on 4 adult female Yorkshire swine. Autologous MSTCs corresponding to approximately 20% of the excised wound mass were spread evenly over the wound beds. Control wounds were not treated with MSTCs. 10mm biopsies were taken from wound centers after 1 or 2 weeks. Tissue sections were stained with H&E for general histology, trichrome for collagen, and immunohistochemistry for various cellular markers. At least 5 independent wound sites were sampled for each treatment/control group at each time point.
RESULTS: Histology at 1 week was consistent with epidermal “heads” of MSTCs migrating towards the skin surface. After 2 weeks, control wounds were characterized by incomplete re-epithelialization, an exuberant infiltration of CD45+ leucocytes, high density of α-SMA+ myofibroblasts, and low collagen content. In contrast, most MSTC-treated wounds were completely re-epithelialized, with wound beds containing about 4-fold less leucocytes, 2-fold fewer myofibroblasts and 2-fold higher collagen content. FABP4 staining showed a significant presence of adipocytes within MSTC-treated wound, but much less in controls.
CONCLUSIONS: Our data shows that MSTC treatment led to quicker resolution of the inflammation phase, and faster progression to the proliferation and remodeling phases of wound healing.
P.NOV09.
BACKGROUND: Lecture 1: Making more with less in India: India and other developing countries lack the resources of proper podiatry care. In these setting innovative methods need to used to heal the diabetic foot lesions. In this lecture, the speaker elaborates his 2-decade experience in managing the diabetic foot in low resource parts of the world. This includes the invention of his basic research (a patented system) called The Samadhan System of offloading the diabetic foot, which is basically a 1 USD cost offloading method, used to heal plantar foot lesions. Contrary to the multidisciplinary approach to diabetic foot care in developed world, the speaker has developed a one-man team approach (OMTA -published in literature of Academy of Physicians in Wound Healing, USA), designed for the low resource parts of the world, where the team of experts simply do not exist. The speaker has developed his own medical college (developed from donated funds) which offers free, structured diabetes education to patients⋯the speaker elaborates this inspiring journey spanning 2 decades. The speaker also elaborates his other innovations like The Samadhan foot stand⋯.(word Samadhan means solution). This is a simple 1 USD foot examination stand which facilitates quick examination of the feet to detect the foot lesions easily and early. Over the years, the speaker has used mobile phones for patient education⋯. he elaborates in his lecture.. how he has used mobile phone video clips for diabetes education and how the diabetes patients bring mobile phone video clips from their homes to give a glimpse of their diet, exercise and other life style methods, to be noticed, appreciated and commented upon by the Physician/Nurses for the betterment of the patient. Lecture 2: Charcot versus Osteomyelitis: Charcot foot and osteomyelitis have similar clinical presentation of red, hot, swollen foot. Skills needed are careful history taking, ordering the right investigation and then interpreting correctly to diagnose if the case is of osteomyelitis or Charcot foot or has both!
METHODS: ..
RESULTS: ..
CONCLUSIONS: ..
P.NOV10.
P.REG01.
BACKGROUND: In this study, we focused on the relationship between the structure of the hair follicle and the skin texture during the fetal development in order to achieve the complete skin regeneration.
METHODS: Embryonic 14.5, 15.5, 16.5, 17.5 and 18.5 day of mouse back skin were stained with CD31 and keratin 17 (K17) using whole-mount preparation. In addition, using the gene transfer device①GFP + non-targeting-siRNA②GFP + K17-siRNA③GFP + K16-siRNA + K17-siRNA was introduced into the lateral skin of embryonic day 15.5 ICR mouse fetus by electroporation to knock down the expression. Thereafter, the side skin was peeled and cultured, and the texture was observed 48 hours after introduction. After the observation, the collected tissues were used for ① quantitative PCR ② 3D texture measurement using PRIMOS-CR ③ immunostaining for PCNA (proliferative cell nuclear antigen).
RESULTS: During the fetal development, expression of K17 was observed in the epithelium corresponding to the skin groove from the embryonic day 15.5, and the expression gradually increased and decreased with the completion of the skin groove. It may suggest that the generation process of skin texture is an epithelial hyperproliferative state. Knockdown of K17 in fetal skin suppressed the formation of texture at the developmental stage (embryonic day 15.5 to 17.5). Moreover, when K17 expression was knocked down, the number of PCNA-positive epidermal cells was decreased compared to control.
CONCLUSIONS: Our results suggest that epithelial cell proliferation via K17 may play an important role in the formation of texture at the developmental stage.
P.REG02.
BACKGROUND: Radiation therapy (RT) remains an essential curative and palliative treatment for a range of malignancies. The skin, however, is extremely radiosensitive and over 95% of patients receiving RT develop moderate to severe skin reactions. Acute damage may progress to hypovascularity and fibrosis with time which severely limits subsequent surgical reconstructions. Fat grafting is a technique able to both restore volume and improve the quality of irradiated tissue. Decellularized adipose tissue extracellular matrices (DAMs) such as Renuva® are alternatives to fat grafts, initially developed to address contour deformities. Here, we explored whether Renuva® can also promote a regenerative response to radiation-induced soft tissue injury.
METHODS: Adult Crl:Nu-Foxn1nu mice (n=6) were treated with 30 Gy external beam radiation to the scalp, followed by 4 weeks to allow for radiation-induced skin fibrosis to develop. The irradiated mouse scalps were then injected with 200μl of: 1) 100% fat; 2) 50:50 Renuva®:fat; or 3) 100% Renuva®. Graft retention was monitored over 8 weeks radiographically and skin and fat were harvested for histological assessment of fibrosis and vascularity. The skin also underwent mechanical strength testing and isolation of fibroblast subpopulations by flow cytometry.
RESULTS: Grafts of 50:50 Renuva®:fat underwent the least resorption and had the greatest cellularity upon histological analysis. In contrast, grafts of Renuva® alone had poor retention and evidence of tissue necrosis. The irradiated skin overlying the mice grafted with 50:50 Renuva®:fat grafts was less stiff and fibrotic than the skin overlying 100% fat grafts. In addition, there were more CD26+ papillary fibroblasts and adipocyte precursors, and less pro-fibrotic reticular fibroblasts in the skin overlying the 50:50 Renuva®:fat grafts.
CONCLUSIONS: Renuva®, when injected with fat, helps retain tissue volume and improves the biomechanical properties of irradiated skin, possible through expansion of progenitor and/or regenerative fibroblast subpopulations.
P.REG03.
BACKGROUND: For over 50 years, human cryopreserved skin allografts (HCAs) have been used for temporary coverage of major burns when autograft donor sites are insufficient. HCAs have also been shown to promote healing of diabetic foot ulcers and prevent fluid loss and infections. Their mechanism of action, however, remains elusive.
METHODS: HCA and human acellular dermal matrix (ADM) grafts were implanted subcutaneously in C57BL/6 (WT) mice and explanted after 1, 3, 7, 14, and 28 days (n= 5 per group). A sham surgery served as a control. Immunofluorescent staining (IF) of tissue sections was used to determine the immune reaction against HCA and ADM as well as vascularization and pro-angiogenic signaling within the grafts and the overlaying mouse skin. HCA and ADM grafts were also implanted into WT mice parabiosed to GFP-positive mice to analyze the infiltration of circulating cells by IF and flow cytometry. HCA grafts explanted after 14 days were processed for droplet-based microfluidic single cell RNA sequencing (scRNA seq) of infiltrating cells using the 10X Genomics platform. Data were log-normalized and partitioned using UMAP based density mappings.
RESULTS: Subcutaneous pockets with implanted grafts healed without clinically apparent rejection. Immune cell infiltration, characterized by F4/80, CD11c, and Myeloperoxidase staining, was greater in HCA compared to ADM on post-implantation day 3, whereas no differences were seen on day 7. CD31 staining showed significantly greater vascularization in HCA on day 7 compared to ADM and sham. HCA also demonstrated higher VEGF expression. A greater number of circulating GFP+ cells were found in HCA compared to ADM and sham by IF and FACS. scRNA seq identified 11 distinct cell clusters out of which 2 were defined as macrophage sub-populations, which highly expressed classical M2 macrophage genes (Mrc1, Arg1, Retnla, Cd163). One M2 subpopulation also highly expressed Col3a1 and pro-angiogenic Hif1a.
CONCLUSIONS: Our data indicate that the immune reaction to HCA is associated with macrophage polarization towards an M2 phenotype. We have identified a sub-population of pro-angiogenic, collagen-3 expressing macrophages, which may be responsible for increased vascularization after HCA implantation in our xenograft model and may underlie the beneficial clinical effects of HCA transplantation.
P.REG04.
P.REG05.
P.REG06.
