What is slough? Defining the proteomic and microbial composition of slough and its implications for wound healing.

Slough is a well-known feature of non-healing wounds. This pilot study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Ten subjects with slow-to-heal wounds and visible slough were enrolled. Aetiologies included venous stasis ulcers, post-surgical site infections and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analysed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound aetiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation and immune processes. To predict variables associated with wound healing, protein, microbial and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anaerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterise wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilised biomarker.

American Burn Association Clinical Practice Guidelines on the Treatment of Severe Frostbite.

This Clinical Practice Guideline addresses severe frostbite treatment. We defined severe frostbite as atmospheric cooling that results in a perfusion deficit to the extremities. We limited our review to adults and excluded cold contact or rapid freeze injuries that resulted in isolated devitalized tissue. After developing population, intervention, comparator, outcomes (PICO) questions, a comprehensive literature search was conducted with the help of a professional medical librarian. Available literature was reviewed and systematically evaluated. Recommendations based on the available scientific evidence were formulated through consensus of a multidisciplinary committee. We conditionally recommend the use of rapid rewarming in a 38 to 42°C water bath and the use of thrombolytics for fewer amputations and/or a more distal level of amputation. We conditionally recommend the use of "early" administration of thrombolytics (≤12 hours from rewarming) compared to "later" administration of thrombolytics for fewer amputations and/or a more distal level of amputation. No recommendation could be formed on the use of vascular imaging studies to determine the use of and/or the time to initiate thrombolytic therapy. No recommendation could be formed on the use of intravenous thrombolytics compared to the use of intra-arterial thrombolytics on fewer amputations and/or a more distal level of amputation. No recommendation could be formed on the use of iloprost resulting in fewer amputations and/or more distal levels of amputation. No recommendation could be formed on the use of diagnostic imaging modalities for surgical planning on fewer amputations, a more distal level of amputation, or earlier timing of amputation.

Pooled safety analysis of STRATA2011 and STRATA2016 clinical trials evaluating the use of StrataGraft® in patients with deep partial-thickness thermal burns.

OBJECTIVE: This analysis includes pooled safety data from 2 clinical trials (NCT01437852; NCT03005106) that evaluated the safety and efficacy of StrataGraft in patients with deep partial-thickness (DPT) burns. METHODS: The study enrolled 101 adult patients with thermal burns covering 3-49% of total body surface area. Patients were followed for up to 1 year. The pooled safety events included: adverse events (AEs), adverse reactions (ARs), serious AEs (SAEs), discontinuation, and deaths; immunological responses (reactivity to panel reactive antibodies [PRA] and human leukocyte antigen [HLA] class 1 alleles); and persistence of allogeneic DNA from StrataGraft. RESULTS: Eighty-seven (86.1%) patients experienced 397 AEs. Thirty patients (29.7%) experienced ARs; 16 patients (15.8%) experienced SAEs. The most frequent AEs were pruritus (n = 31; 30.7%), and blister, hypertension, and hypertrophic scar (n = 11 each; 10.9%); the most common AR was pruritus (n = 13; 12.9%). One patient discontinued the study; 2 patients experienced SAEs (unrelated to StrataGraft) leading to death. PRA and HLA allele reactivity was ≤ 25% at Month 3, with no persistent allogeneic DNA from StrataGraft. CONCLUSIONS: StrataGraft was well tolerated by patients, with a safety profile similar to autograft. StrataGraft may offer a safe alternative to autograft for DPT burns.

Ex Vivo Human and Porcine Skin Effectively Model Candida auris Colonization, Differentiating Robust and Poor Fungal Colonizers.

Candida auris proliferates and persists on the skin of patients, often leading to health care-associated infections with high mortality. Here, we describe 2 clinically relevant skin models and show that C. auris grows similarly on human and porcine skin. Additionally, we demonstrate that other Candida spp., including those with phylogenetic similarity to C. auris, do not display high growth in the skin microenvironment. These studies highlight the utility of 2 ex vivo models of C. auris colonization that allow reproducible differentiation among Candida spp., which should be a useful tool for comparison of C. auris clinical isolates and genetically mutated strains.

Contrasting recruitment of skin-associated adipose depots during cold challenge of mouse and human.

KEY POINTS: Several distinct strategies produce and conserve heat to maintain the body temperature of mammals, each associated with unique physiologies, with consequences for wellness and disease susceptibility Highly regulated properties of skin offset the total requirement for heat production  We hypothesize that the adipose component of skin is primarily responsible for modulating heat flux; here we evaluate the relative regulation of adipose depots in mouse and human, to test their recruitment to heat production and conservation We found that insulating mouse dermal white adipose tissue accumulates in response to environmentally and genetically induced cool stress; this layer is one of two adipose depots closely apposed to mouse skin, where the subcutaneous mammary gland fat pads are actively recruited to heat production In contrast, the body-wide adipose depot associated with human skin produces heat directly, potentially creating an alternative to the centrally regulated brown adipose tissue ABSTRACT: Mammalian skin impacts metabolic efficiency system-wide, controlling the rate of heat loss and consequent heat production. Here we compare the unique fat depots associated with mouse and human skin, to determine whether they have corresponding functions and regulation. For humans, we assay a skin-associated fat (SAF) body-wide depot to distinguish it from the subcutaneous fat pads characteristic of the abdomen and upper limbs. We show that the thickness of SAF is not related to general adiposity; it is much thicker (1.6-fold) in women than men, and highly subject-specific. We used molecular and cellular assays of ß-adrenergic-induced lipolysis and found that dermal white adipose tissue (dWAT) in mice is resistant to lipolysis; in contrast, the body-wide human SAF depot becomes lipolytic, generating heat in response to ß-adrenergic stimulation. In mice challenged to make more heat to maintain body temperature (either environmentally or genetically), there is a compensatory increase in thickness of dWAT: a corresponding ß-adrenergic stimulation of human skin adipose (in vivo or in explant) depletes adipocyte lipid content. We summarize the regulation of skin-associated adipocytes by age, sex and adiposity, for both species. We conclude that the body-wide dWAT depot of mice shows unique regulation that enables it to be deployed for heat preservation; combined with the actively lipolytic subcutaneous mammary fat pads they enable thermal defence. The adipose tissue that covers human subjects produces heat directly, providing an alternative to the brown adipose tissues.

Robbing Peter to Pay Paul: Chlorhexidine gluconate demonstrates short-term efficacy and long-term cytotoxicity.

Wound cleansing agents are routine in wound care and preoperative preparation. Antiseptic activity intends to prevent contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. We evaluated the cytotoxicity of five clinically used wound cleaning agents (saline, povidone iodine, Dove® and Dial® soaps, and chlorhexidine gluconate [CHG]) using both an ex vivo and in vivo human skin xenograft mouse model, in contrast to classical in vitro models that lack the structural and compositional heterogeneity of human skin. We further established an ex vivo wound contamination model inoculated with ~100 cells of Pseudomonas aeruginosa or Staphylococcus aureus to evaluate antimicrobial efficacy. Scanning electron microscopy and confocal microscopy were used to evaluate phenotypic and spatial characteristics of bacterial cells in wound tissue. CHG significantly reduced metabolic activity of the skin explants, while all treatments except saline affected local cellular viability. CHG cytotoxicity persisted and progressed over 14 days, impairing wound healing in vivo. Within the contamination model, CHG treatment resulted in a significant reduction of P. aeruginosa wound surface counts at 24 h post-treatment. However, this effect was transient and serial application of CHG had no effect on both P. aeruginosa or S. aureus microbial growth. Microscopy revealed that viable cells of P. aeruginosa reside deep within wound tissue post-CHG application, likely serving as a reservoir to re-populate the tissue to a high bioburden. We reveal concerning cytotoxicity and limited antimicrobial activity of CHG in human skin using clinically relevant models, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

Accelerated complete human skin architecture restoration after wounding by nanogenerator-driven electrostimulation.

BACKGROUND: Electrostimulation (ES) therapy for wound healing is limited in clinical use due to barriers such as cumbersome equipment and intermittent delivery of therapy. METHODS: We adapted a human skin xenograft model that can be used to directly examine the nanogenerator-driven ES (NG-ES) effects on human skin in vivo-an essential translational step toward clinical application of the NG-ES technique for wound healing. RESULTS: We show that NG-ES leads to rapid wound closure with complete restoration of normal skin architecture within 7 days compared to more than 30 days in the literature. NG-ES accelerates the inflammatory phase of wound healing with more rapid resolution of neutrophils and macrophages and enhances wound bed perfusion with more robust neovascularization. CONCLUSION: Our results support the translational evaluation and optimization of the NG-ES technology to deliver convenient, efficient wound healing therapy for use in human wounds.

A phase 3, open-label, controlled, randomized, multicenter trial evaluating the efficacy and safety of StrataGraft® construct in patients with deep partial-thickness thermal burns.

OBJECTIVE: This phase 3 study evaluated StrataGraft construct as a donor-site sparing alternative to autograft in patients with deep partial-thickness (DPT) burns. METHODS: Patients aged ≥18 years with 3-49% total body surface area (TBSA) thermal burns were enrolled. In each patient, 2 DPT areas (≤2000cm2 total) of comparable depth after excision were randomized to either cryopreserved StrataGraft or autograft. Coprimary endpoints were: the difference in percent area of StrataGraft treatment site and autograft treatment site autografted at Month 3 (M3), and the proportion of patients achieving durable wound closure of the StrataGraft site without autograft at M3. Safety assessments were performed in all patients. Efficacy and safety follow-up continued to 1 year. RESULTS: Seventy-one patients were enrolled. By M3, there was a 96% reduction in mean percent area of StrataGraft treatment sites that required autografting, compared with autograft treatment sites (4.3% vs 102.1%, respectively; P<.0001). StrataGraft treatment resulted in durable wound closure at M3 without autografting in 92% (95% CI: 85.6, 98.8; n/n 59/64) of patients for whom data were available. The most common StrataGraft-related adverse event was pruritus (15%). CONCLUSIONS: Both coprimary endpoints were achieved. StrataGraft may offer a new treatment for DPT burns to reduce the need for autografting. CLINICAL TRIAL IDENTIFIER: NCT03005106.

Priority effects dictate community structure and alter virulence of fungal-bacterial biofilms.

Polymicrobial biofilms are a hallmark of chronic wound infection. The forces governing assembly and maturation of these microbial ecosystems are largely unexplored but the consequences on host response and clinical outcome can be significant. In the context of wound healing, formation of a biofilm and a stable microbial community structure is associated with impaired tissue repair resulting in a non-healing chronic wound. These types of wounds can persist for years simmering below the threshold of classically defined clinical infection (which includes heat, pain, redness, and swelling) and cycling through phases of recurrent infection. In the most severe outcome, amputation of lower extremities may occur if spreading infection ensues. Here we take an ecological perspective to study priority effects and competitive exclusion on overall biofilm community structure in a three-membered community comprised of strains of Staphylococcus aureus, Citrobacter freundii, and Candida albicans derived from a chronic wound. We show that both priority effects and inter-bacterial competition for binding to C. albicans biofilms significantly shape community structure on both abiotic and biotic substrates, such as ex vivo human skin wounds. We further show attachment of C. freundii to C. albicans is mediated by mannose-binding lectins. Co-cultures of C. freundii and C. albicans trigger the yeast-to-hyphae transition, resulting in a significant increase in neutrophil death and inflammation compared to either species alone. Collectively, the results presented here facilitate our understanding of fungal-bacterial interactions and their effects on host-microbe interactions, pathogenesis, and ultimately, wound healing.

Coming to Consensus: What Defines Deep Partial Thickness Burn Injuries in Porcine Models?

Deep partial thickness burns are clinically prevalent and difficult to diagnose. In order to develop methods to assess burn depth and therapies to treat deep partial thickness burns, reliable, accurate animal models are needed. The variety of animal models in the literature and the lack of precise details reported for the experimental procedures make comparison of research between investigators challenging and ultimately affect translation to patients. They sought to compare deep partial thickness porcine burn models from five well-established laboratories. In doing so, they uncovered a lack of consistency in approaches to the evaluation of burn injury depth that was present within and among various models. They then used an iterative process to develop a scoring rubric with an educational component to facilitate burn injury depth evaluation that improved reliability of the scoring. Using the developed rubric to re-score the five burn models, they found that all models created a deep partial thickness injury and that agreement about specific characteristics identified on histological staining was improved. Finally, they present consensus statements on the evaluation and interpretation of the microanatomy of deep partial thickness burns in pigs.

Determining clinically meaningful thresholds for innovative burn care products to reduce autograft: A US burn surgeon Delphi panel.

Reducing the amount of donor skin needed for definitive wound closure can improve outcomes in patients with severe burns. This Delphi Consensus Panel (DCP) aimed to achieve expert consensus on the percentage reduction in donor skin for autograft that constitutes a clinically meaningful benefit. A two-round DCP of fifteen US burn surgeons was conducted via a web-based survey platform. Fourteen panelists (93.3%) completed both rounds. In Round 2, consensus, defined as ≥70% agreement, was achieved for five of the seven consensus statements. All panelists agreed that a clinically meaningful reduction in the amount of donor skin required would facilitate wound management and decrease donor site morbidity experienced by patients. Furthermore, based on three treatment scenarios, consensus was achieved for a clinically meaningful reduction in the amount of donor skin required for autograft for the adult population in deep partial-thickness and full-thickness burns. Findings from this DCP indicate that an innovative cellular and/or tissue product that would reduce the needed amount of donor skin, by the identified thresholds, has the potential to improve the outcomes for patients with severe burn injuries in a meaningful way.

Modeling early thermal injury using an ex vivo human skin model of contact burns.

BACKGROUND: Early mechanisms underlying the progressive tissue death and the regenerative capability of burn wounds are understudied in human skin. A clinically relevant, reproducible model for human burn wound healing is needed to elucidate the early changes in the human burn wound environment. This study reports a reproducible contact burn model on human skin that explores the extent of tissue injury and healing over time, and defines the inter-individual variability in human skin to enable use in mechanistic studies on burn wound progression and healing. METHODS: Using a customized burn device, contact burns of various depths were created on human skin by two operators and were evaluated for histologic depth by three raters to determine reproducibility. Early burn wound progression and wound healing were also evaluated histologically after the thermally injured human skin was cultured ex vivo for up to 14 days. RESULTS: Burn depths were reproducibly generated on human skin in a temperature- or time-dependent manner. No significant difference in operator-created or rater-determined depth was observed within each patient sample. However, significant inter-individual variation was identified in burn depth in ten patient samples. Burn-injured ex vivo human skin placed into culture demonstrated differential progression of cell death and collagen denaturation for high and low temperature contact burns, while re-epithelialization was observed in superficial burn wounds over a period of 14 days. CONCLUSION: This model represents an invaluable tool to evaluate the inter-individual variability in early burn wound progression and wound healing to complement current animal models and enhance the translation of preclinical research to improvements in patient care.

Optical imaging of collagen fiber damage to assess thermally injured human skin.

Surgery is the definitive treatment for burn patients who sustain full-thickness burn injuries. Visual assessment of burn depth is made by the clinician early after injury but is accurate only up to 70% of the time among experienced surgeons. Collagen undergoes denaturation as a result of thermal injury; however, the association of collagen denaturation and cellular death in response to thermal injury is unknown. While gene expression assays and histologic staining allow for ex vivo identification of collagen changes, these methods do not provide spatial or integrity information in vivo. Thermal effects on collagen and the role of collagen in wound repair have been understudied in human burn models due to a lack of methods to visualize both intact and denatured collagen. Hence, there is a critical need for a clinically applicable method to discriminate between damaged and intact collagen fibers in tissues. We present two complementary candidate methods for visualization of collagen structure in three dimensions. Second harmonic generation imaging offers a label-free, high-resolution method to identify intact collagen. Simultaneously, a fluorophore-tagged collagen-mimetic peptide can detect damaged collagen. Together, these methods enable the characterization of collagen damage in human skin biopsies from burn patients, as well as ex vivo thermally injured human skin samples. These combined methods could enhance the understanding of the role of collagen in human wound healing after thermal injury and potentially assist in clinical decision-making.

Evolution of ischemia and neovascularization in a murine model of full thickness human wound healing.

Translation of wound healing research is limited by the lack of an appropriate animal model, due to the anatomic and wound healing differences in animals and humans. Here, we characterize healing of grafted, full-thickness human skin in an in vivo model of wound healing. Full-thickness human skin, obtained from reconstructive operations, was grafted onto the dorsal flank of NOD.Cg-KitW41J Tyr + Prkdcscid Il2rgtm1Wjl /ThomJ mice. The xenografts were harvested 1 to 12 weeks after grafting, and histologic analyses were completed for viability, neovascularization, and hypoxia. Visual inspection of the xenograft shows drying and sloughing of the epidermis starting at week four. By week 12, the xenograft appears healed but has lost 63.05 ± 0.24% of the initial graft size. There is histologic evidence of epidermolysis as early as 2 weeks, which progresses until week 4, when new epidermis appears from the wound edges. Epidermal regeneration is complete by week 12, although the epidermis appears hypertrophied. An initial increase of infiltrating immune mouse cells into the xenograft normalizes to baseline 6 months after grafting. Neovascularization, as evidenced by positive staining for the proteins human CD31 and alpha smooth muscle actin, is present as early as 2 weeks after grafting at the interface between the xenograft and the mouse tissue. CD31 and alpha smooth muscle actin staining increased throughout the xenograft over the 12 weeks, leading to greater viability of the tissue. Likewise, there is increased Hypoxia Inducible Factor 1-alpha expression at the interface of viable and nonviable tissue, which suggest a hypoxia-driven process causing early graft loss. These findings illustrate human skin wound healing in an ischemic environment, providing a timeline for use of full thickness human skin after grafting in a murine model to study mechanisms underlying human skin wound healing.

Standards in Biologic Lesions: Cutaneous Thermal Injury and Inhalation Injury Working Group 2018 Meeting Proceedings.

On August 27 and 28, 2018, the American Burn Association, in conjunction with Underwriters Laboratories, convened a group of experts on burn and inhalation injury in Washington, DC. The goal of the meeting was to identify and discuss the existing knowledge, data, and modeling gaps related to understanding cutaneous thermal injury and inhalation injury due to exposure from a fire environment, and in addition, address two more areas proposed by the American Burn Association Research Committee that are critical to burn care but may have current translational research gaps (inflammatory response and hypermetabolic response). Representatives from the Underwriters Laboratories Firefighter Safety Research Institute and the Bureau of Alcohol, Tobacco, Firearms and Explosives Fire Research Laboratory presented the state of the science in their fields, highlighting areas that required further investigation and guidance from the burn community. Four areas were discussed by the full 24 participant group and in smaller groups: Basic and Translational Understanding of Inhalation Injury, Thermal Contact and Resulting Injury, Systemic Inflammatory Response and Resuscitation, and Hypermetabolic Response and Healing. A primary finding was the need for validating historic models to develop a set of reliable data on contact time and temperature and resulting injury. The working groups identified common areas of focus across each subtopic, including gaining an understanding of individual response to injury that would allow for precision medicine approaches. Predisposed phenotype in response to insult, the effects of age and sex, and the role of microbiomes could all be studied by employing multi-omic (systems biology) approaches.

Indeterminate-Depth Burn Injury-Exploring the Uncertainty.

In the management of indeterminate-depth burns (IDB), common challenges include the ability to predict time to healing and regenerative potential, risk of burn wound progression, and timing of excision. Several technologies exist to aid in determination of the depth of a burn injury, yet surgeons continue to rely on the naked eye-visual assessment-as the standard of care. Newer and improved imaging technologies are closing in on the goal of inexpensive, accurate, noninvasive modalities for depth determination. Likewise, management of IDB is becoming more sophisticated as newer wound healing technologies continue to be developed. By describing what is meant by "indeterminate" depth burns, and their associated challenges, we hope to stimulate interest in research to develop new therapies and management strategies. The ultimate goal is to treat IDB without the need for autografts.

Optimization of interstrand interactions enables burn detection with a collagen-mimetic peptide.

Collagen is an abundant component of the extracellular matrix and connective tissues. Some collagen-mimetic peptides (CMPs) that do not form homotrimers can anneal to damaged tissue. Here, through a computational screen, we identify (flpHypGly)7 as an optimal monomeric CMP for heterotrimer formation. We find that (flpHypGly)7 forms stable triple helices with (ProProGly)7 but not with itself. The nonnatural amino acid HflpOH, which is (2S,4S)-4-fluoroproline, is not toxic to human fibroblasts or keratinocytes. Conjugation of (flpHypGly)7 to a fluorescent dye enables the facile detection of burned collagenous tissue with high specificity. The ubiquity of collagen and the prevalence of injuries and diseases that disrupt endogenous collagen suggests widespread utility for this approach.

An open-label, prospective, randomized, controlled, multicenter, phase 1b study of StrataGraft skin tissue versus autografting in patients with deep partial-thickness thermal burns.

OBJECTIVE: This open-label, controlled, randomized study assessed the safety, tolerability, and efficacy of StrataGraft tissue compared to autograft in the treatment of deep partial-thickness (DPT) burns. METHODS: Thirty subjects with DPT thermal burns (3%-43% total body surface area) were treated with StrataGraft tissue as follows: cohort 1, ≤220 cm2 refrigerated tissue; cohort 2, ≤440 cm2 refrigerated tissue; and cohort 3, ≤440 cm2 cryopreserved tissue. On each subject, two comparable areas of DPT burn were randomized to receive StrataGraft tissue or autograft. Coprimary end points were the percent area of the StrataGraft tissue treatment site undergoing salvage autografting by Day 28 and wound closure of treatment sites by 3 months. RESULTS: By Day 28, no StrataGraft tissue treatment sites underwent autografting. By 3 months, 93% and 100% of the StrataGraft tissue and autograft treatment sites achieved complete wound closure, respectively. No significant differences in observer total and overall opinion POSAS scores between StrataGraft tissue and autograft treatment sites were observed at any timepoint. The most common adverse event was pruritus (17%). CONCLUSIONS: StrataGraft tissue treatment of DPT thermal burns reduced the need for autograft, resulted in wound closure and treatment-site cosmesis comparable to that of autograft, and was well tolerated.