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Wound healing requires a complex cascade of highly controlled and conserved cellular and molecular processes. These involve numerous cell types and extracellular matrix molecules regulated by the actin cytoskeleton. This microscopic network of filaments is present within the cytoplasm of all cells and provides the shape and mechanical support required for cell movement and proliferation. Here, an overview of the processes of wound healing are described from the perspective of the cell in relation to the actin cytoskeleton. Key points of discussion include the role of actin, its binding proteins, signaling pathways, and events that play significant roles in the phases of wound healing. The identification of cytoskeletal targets that can be used to manipulate and improve wound healing is included as an emerging area of focus that may inform future therapeutic approaches to improve healing of complex wounds. Refereed/Peer-reviewed

Refereed/Peer-reviewed Cellular mechanisms and/or microbiological interactions which contribute to chronic diabetes related foot ulcers (DRFUs) were explored using serially collected tissue specimens from chronic DRFUs and control healthy foot skin. Total RNA was isolated for next-generation sequencing. We found differentially expressed genes (DEGs) and enriched hallmark gene ontology biological processes upregulated in chronic DRFUs which primarily functioned in the host immune response including: (i) Inflammatory response; (ii) TNF signalling via NFKB; (iii) IL6 JAK-STAT3 signalling; (iv) IL2 STAT5 signalling and (v) Reactive oxygen species. A temporal analysis identified RN7SL1 signal recognition protein and IGHG4 immunoglobulin protein coding genes as being the most upregulated genes after the onset of treatment. Testing relative temporal changes between healing and non-healing DRFUs identified progressive upregulation in healed wounds of CXCR5 and MS4A1 (CD20), both canonical markers of lymphocytes (follicular B cells/follicular T helper cells and B cells, respectively). Collectively, our RNA-seq data provides insights into chronic DRFU pathogenesis.

This file contains 7 supplementary figures supporting that Flightless-I blocks p62-mediated recognition of LC3 to impede selective autophagy and promote breast cancer progression.

Clinical information of patients with breast cancer.

p62 is a receptor that facilitates selective autophagy by interacting simultaneously with cargoes and LC3 protein on the autophagosome to maintain cellular homeostasis. However, the regulatory mechanism(s) behind this process and its association with breast cancer remain to be elucidated. Here, we report that Flightless-I (FliI), a novel p62-interacting protein, promotes breast cancer progression by impeding selective autophagy. FliI was highly expressed in clinical breast cancer samples, and heterozygous deletion of FliI retarded the development of mammary tumors in PyVT mice. FliI induced p62-recruited cargoes into Triton X-100 insoluble fractions (TI) to form aggregates, thereby blocking p62 recognition of LC3 and hindering p62-dependent selective autophagy. This function of Flil was reinforced by Akt-mediated phosphorylation at Ser436 and inhibited by phosphorylation of Ulk1 at Ser64. Obstruction of autophagic clearance of p62-recruited cargoes by FliI was associated with the accumulation of oxidative damage on proteins and DNA, which could contribute to the development of cancer. Heterozygous knockout of FliI facilitated selectively autophagic clearance of aggregates, abatement of ROS levels, and protein oxidative damage, ultimately retarding mammary cancer progression. In clinical breast cancer samples, Akt-mediated phosphorylation of FliI at Ser436 negatively correlated with long-term prognosis, while Ulk1-induced FliI phosphorylation at Ser64 positively correlated with clinical outcome. Together, this work demonstrates that FliI functions as a checkpoint protein for selective autophagy in the crosstalk between FliI and p62-recruited cargoes, and its phosphorylation may serve as a prognostic marker for breast cancer.Significance: Flightless-I functions as a checkpoint protein for selective autophagy by interacting with p62 to block its recognition of LC3, leading to tumorigenesis in breast cancer.Cancer Res; 78(17); 4853–64. ©2018 AACR.

Supplementary materials and methods and supplementary figure legends.

Background: Electrospun fibers are widely studied in regenerative medicine for their ability to mimic the extracellular matrix (ECM) and provide mechanical support. In vitro studies indicated that cell adhesion and migration is superior on smooth poly(L-lactic acid) (PLLA) electrospun scaffolds and porous scaffolds once biofunctionalized with collagen. Methods: The in vivo performance of PLLA scaffolds with modified topology and collagen biofunctionalization in full-thickness mouse wounds was assessed by cellular infiltration, wound closure and re-epithelialization and ECM deposition. Results: Early indications suggested unmodified, smooth PLLA scaffolds perform poorly, with limited cellular infiltration and matrix deposition around the scaffold, the largest wound area, a significantly larger panniculus gape, and lowest re-epithelialization; however, by day 14, no significant differences were observed. Collagen biofunctionalization may improve healing, as collagen-functionalized smooth scaffolds were smallest overall, and collagen-functionalized porous scaffolds were smaller than non-functionalized porous scaffolds; the highest re-epithelialization was observed in wounds treated with collagen-functionalized scaffolds. Conclusion: Our results suggest that limited incorporation of smooth PLLA scaffolds into the healing wound occurs, and that altering surface topology, particularly by utilizing collagen biofunctionalization, may improve healing. The differing performance of the unmodified scaffolds in the in vitro versus in vivo studies demonstrates the importance of preclinical testing. Refereed/Peer-reviewed

Electrospun polymer fibers have garnered substantial importance in regenerative medicine owing to their intrinsic 3D topography, extracellular matrix microenvironment, biochemical flexibility, and mechanical support. In particular, a material's nano-topography can have a significant effect on cellular responses, including adhesion, proliferation, differentiation, and migration. In this study, poly(l-lactic acid) (PLLA), a biodegradable polymer with excellent biocompatibility was electrospun into fibers with either smooth or porous topologies. The scaffolds were further modified and biofunctionalized with 0.01% and 0.1% collagen to enhance bioactivity and improve cellular interactions. Human keratinocytes (HaCaTs) and fibroblasts (human foreskin fibroblasts-HFF) were cultured on the scaffolds using a modified co-culture technique, where keratinocytes were grown on the dorsal plane for 5 days, followed by flipping, seeding with fibroblasts on the ventral plane and culturing for a further 5 days. Following this, cellular adhesion of the skin cells on both the unmodified and collagen-modified scaffolds (smooth and porous) was performed using scanning electron microscopy (SEM) and immunofluorescence. Distinct outcomes were observed with the unmodified smooth scaffolds showing superior cell adhesion than the porous scaffolds. Modification of the porous and smooth scaffolds with 0.1% collagen enhanced the adhesion and migration of both keratinocytes and fibroblasts to these scaffolds. Further, the collagen-modified scaffolds (both porous and smooth) produced confluent and uniform epidermal sheets of keratinocytes on one plane with healthy fibroblasts populated within the scaffolds. Thus, presenting a vast potential to serve as a self-organized skin substitute this may be a promising biomaterial for development as a dressing for patients suffering from wounds Refereed/Peer-reviewed

Refereed/Peer-reviewed Fundamental knowledge about cell–surface interactions can be applied in the development of wound dressings and scaffolds to encourage wounds to heal. As surfaces produced with acid-functionalised monomers encourage keratinocyte adhesion, proliferation and migration, whilst amine functionalisation enhances fibroblast proliferation and migration in vitro, standard care wound dressings were plasma-coated with either acrylic acid or allylamine and applied to 6 mm excisional wounds on the backs of mice to test their effectiveness in vivo. At day 3, the rate of wound healing was increased in mice treated with dressings that were plasma-coated with allylamine compared to uncoated dressings, with a significantly reduced wound area. However, healing may be impaired following prolonged treatment with allylamine-functionalised dressings, with delayed re-epithelialisation and increased cellularisation of the wound site at later timepoints. Acrylic acid functionalisation, however, offered no early improvement in wound healing, but wounds treated with these dressings displayed increased collagen deposition at day 7 post wounding. These results suggest that plasma polymerisation may allow for the development of new dressings which can enhance wound closure by directing cell behaviour, but that the application of these dressings may require a timed approach to enhance specific phases of the wound healing response.

Silver-based nano-antibiotics are rapidly developing as promising alternatives to conventional antibiotics. Ideally, to remain potent against a wide range of drug-resistant and anaerobic bacteria, silver-based nano-antibiotics should easily penetrate through the bacterial cell walls and actively release silver ions. In this study, highly monodispersed, ultrasmall (3 nm), polycationic silver nanoclusters (pAgNCs) are designed and synthesized for the elimination of a range of common Gram-negative and Gram-positive pathogens and their corresponding established and matured biofilms, including those composed of multiple species. The pAgNCs also show greatly enhanced antibacterial efficacy against anaerobic bacteria such as

Abstract: Wound healing is a complex and dynamic process that requires intricate synchronization between multiple cell types within appropriate extracellular microenvironment. Wound healing process involves four overlapping phases in a precisely regulated manner, consisting of hemostasis, inflammation, proliferation, and maturation. For an effective wound healing, all four phases must follow in a sequential pattern within a time frame. Several factors might interfere with one or more of these phases in healing process, thus causing improper or impaired wound healing resulting in non-healing chronic wounds. The complications associated with chronic non-healing wounds, along with the limitations of existing wound therapies, have led to the development and emergence of novel and innovative therapeutic interventions. Nanotechnology presents unique and alternative approaches to accelerate the healing of chronic wounds by the interaction of nanomaterials during different phases of wound healing. This review focuses on recent innovative nanotechnology-based strategies for wound healing and tissue regeneration based on nanomaterials, including nanoparticles, nanocomposites and scaffolds. The efficacy of the intrinsic therapeutic potential of nanomaterials (including silver, gold, zinc oxide, copper, cerium oxide, etc.) and the ability of nanomaterials as carriers (liposomes, hydrogels, polymeric nanomaterials, nanofibers) and therapeutic agents associated with wound-healing applications have also been addressed. The significance of these nanomaterial-based therapeutic interventions for wound healing needs to be highlighted to engage researchers and clinicians towards this new and exciting area of bio-nanoscience. We believe that these recent developments will offer researchers an updated source for the use of nanomaterials as an advanced approach to improve wound healing.

Background: While many aspects of the wound repair process are understood, there is still limited understanding about why chronic wounds develop, how we predict, diagnose and improve healing outcomes, and why some wounds heal with scarring. Identifying current gaps in knowledge, as well as areas of strengths, will inform recommendations for future research-based themes and influence the targeting of resources. Aims: This scoping review aims to (i) map current knowledge and research activities into wound healing in Australia and (ii) understand strengths and identifying research gaps in science-focused wound research in Australia. Methods: Guided by the PRISMA-ScR framework, this scoping review will include original laboratory-based science studies on wounds conducted in Australia. Records will be searched in PubMed, CINAHL and Embase from January 2010 to March 2021. All screened titles and abstracts will be independently assessed by two reviewers using Rayyan®. A third reviewer will solve any conflicts that arise. This will be followed by full text screening and in-depth analysis after resolution of conflicts. Results and dissemination: Following data extraction from the final included articles, results will be grouped according to area of research and synthesised in a narrative review. Findings will be published in a peer-reviewed journal Refereed/Peer-reviewed

Chimeric antigen receptor (CAR)-T immunotherapy is a novel treatment that genetically modifies the patient’s own T cells to target and kill malignant cells. CAR-T cells demonstrated robust clinical activity against certain B-cell malignancies. However, identification of tumour-specific antigens expressed on multiple cancer types, especially on solid cancers, remains a major challenge. P2X purinoceptor 7 (P2X7) is an ATP gated cation channel that forms homotrimers and heterotrimers at the cell surface. When functioning normally, it controls ion transport in response to ATP. A dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from a range of tissues, while being undetectable on healthy cells. We generated prototype nfP2X7-targeting human CAR-T cells, which demonstrated effective antigen-specific cytotoxicity against twelve solid cancer types including breast, prostate, lung, colorectal, brain and skin in vitro. In preclinical xenograft mouse models of aggressive breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibited robust anti-tumour efficacy. These data indicate CAR-T cells targeting nfP2X7 have potential as a novel broad-spectrum cancer immunotherapy for solid tumours in humans.

Skin is an important barrier to pathogenic microorganisms and plays a critical role in a ctivation of innate immune responses. When the skin barrier is breached following wounding or burn injury, pathogens can invade and complicate healing with infection resulting in delayed healing and symptomatic scarring. Wound infection is a significant problem after burn injury and in patients with chronic wounds. Antimicrobial silver has had a significant role in wound antisepsis throughout history and, given the rise in community acquired antibiotic resistance, silver dressings are now commonly used to combat wound infection. The multi-modal mechanism of action, low potential for toxicity and formation of microbial resistance makes silver dressings suitable tools against a wide array of clinically important microbes. There are, however, a number of issues with silver dressings including a conflicting evidence base, the important environmental consideration of nanoparticle manufacture, and the significant cost of these products. One solution may be to adopt an ‘opened-but-unused’ means of wound care whereby bulk dressing materials are used piecemeal and stored in between dressing changes to increase the cost-effectiveness and reduced wastage. There is, however, little literature on this topic and so in vitro and clinical research must be performed to consider the efficacy of active ingredient dressings in wound care including silver dressings once opened and stored Refereed/Peer-reviewed

Biofilm-associated infections are a major cause of impaired wound healing. Despite the broad spectrum of anti-bacterial benefits provided by silver nanoparticles (AgNPs), these materials still cause controversy due to cytotoxicity and a lack of efficacy against mature biofilms. Herein,highly potent ultrasmall AgNPs were combined with a biocompatible hydrogel with integrated synergistic functionalities to facilitate elimination of clinically relevant mature biofilms in-vivo combined with improved wound healing capacity. The delivery platform showed a superior release mechanism, reflected by high biocompatibility, hemocompatibility, and extended antibacterial efficacy.In vivo studies using the S. aureus wound biofilm model showed that the AgNP hydrogel (200 µg/g)was highly effective in eliminating biofilm infection and promoting wound repair compared to the controls, including silver sulfadiazine (Ag SD). Treatment of infected wounds with the AgNPhydrogel resulted in faster wound closure (46% closure compared to 20% for Ag SD) and accelerated wound re-epithelization (60% for AgNP), as well as improved early collagen deposition. The AgNPhydrogel did not show any toxicity to tissue and/or organs. These findings suggest that the developed AgNP hydrogel has the potential to be a safe wound treatment capable of eliminating infection and providing a safe yet effective strategy for the treatment of infected wounds.https://creativecommons.org/licenses/by/4.0/ Refereed/Peer-reviewed

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/−, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/− embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Pericytes have the potential to be developed as a cell therapy for the treatment of wounds; however, the efficacy of any cell therapy relies on the successful delivery of intact and functioning cells. Here, the effect of delivering pericytes on wound repair was assessed alongside the development of a surface-functionalized pericyte patch. Plasma polymerization (PP) was used to functionalize the surface of silicone patches with heptylamine (HA) or acrylic acid (AA) monomers. Human pericytes were subsequently delivered to murine excisional wounds by intradermal injection or using the pericyte-laden patches and the comparative effects on wound healing, inflammation and revascularization determined. The AA surface provided the superior transfer of the cells to de-epidermized dermis. Excisional murine wounds treated either with pericytes injected directly into the wound or with the pericyte-laden AA patches showed improved healing with decreased neutrophil infiltration and reduced numbers of macrophages in the wounds. Pericyte delivery also enhanced angiogenesis through a mechanism independent of VEGF signalling. Pericytes, when delivered to wounds, improved healing responses by dampening inflammation and promoting angiogenesis. Delivery of pericytes using PP-AA-functionalized patches was equally as effective as direct injection of pericytes into wounds. Pericyte-functionalized dressings may therefore be a clinically relevant approach for the treatment of wounds.

usc Silver nanoparticles (AgNPs) have attracted enormous interest because of their excellent antibacterial properties, low cytotoxicity and limited evidence for resistance. As a general trend, smaller nanoparticles are considered to have stronger antibacterial activity. In this work we investigate whether this trend is valid for the sub-10 nm region by designing and synthesising three types of sub-10 nm AgNPs (∼1.87, ∼2.93 and ∼6.53 nm) to reveal the influence of size, valence state and structure on the antibacterial potency of AgNPs. We found that NPs with a size of ∼2.93 nm having a high concentration of silver in the first valence state presented the highest bacterial killing potency as well as low cytotoxicity to mammalian cells. The new insights presented in this study open future avenues for the engineering of highly potent silver nanoantibiotics that can be incorporated into future advanced medical devices and therapies capable of protecting patients from infections. Refereed/Peer-reviewed

Refereed/Peer-reviewed Strategies that alter the pH of wounds to improve healing outcomes are an emerging area of interest. Currently, there is limited understanding of the effect of hydrogen (H+) on the functionality of skin cells during proliferation and migration, highlighting the need for research to determine the effect of pH during wound healing. This study aimed to determine the effect of acidification on the metabolic activity and migration of human immortalized keratinocytes (HaCaT) and human foreskin fibroblasts (HFF). In vitro models were used with phosphoric and citric acid buffers at a pH range between 3 and 7. Our results showed that cells were more viable in buffers with low rather than high ionic strength. A time-dependent effect of the acidification treatment was also observed with cell metabolic activity varying with treatment duration and frequency. Our results showed that a 24 h treatment and subsequent resting phase significantly improved cell proliferation and migration. This in vitro study is the first to establish a correlation between the role of acidic pH, molarity and treatment regimen in cellular activity. Our data demonstrated a positive effect of acidic pH on cell metabolic activity and migration rate, suggesting a clinical potential in indications such as wound healing.

The increasing emergence of antibiotic resistance coupled with the limited effectiveness of current treatments highlights the need for a novel treatment modality. Silver nanoparticles (AgNPs) are a promising alternative with broad-spectrum antibacterial activity. However, the clinical translation of AgNPs has been hampered primarily due to the delivery of unsafe levels of silver ions (Ag+) resulting in cellular toxicity and they are susceptible to degradation resulting in loss of efficacy. Here, we describe the development of a safe and effective, thermo-responsive AgNP hydrogel delivery system that provides long-lasting antibacterial effects in conjunction with the polymeric hydrogel serving as tissue regeneration scaffold for improved healing. Using a murine model of infection, we demonstrate that AgNP hydrogel not only provides superior bactericidal activity but also reduces the inflammatory response leading to accelerated wound closure compared to silver sulfadiazine. The AgNP hydrogel treated wounds demonstrated significantly smaller wound length and dermal gape with a concurrent increase in cellular proliferation and wound re-epithelization. Additionally, AgNP hydrogel decreased neutrophil infiltration, increased levels of anti-inflammatory M2 macrophages, and reduced the number of apoptotic cells. Therefore, this novel multifunctional AgNP hydrogel offers great potential for safe and effective delivery of Ag+ for the treatment of bacterial wound infections.

The increasing emergence of antibiotic resistance coupled with the limited effectiveness of current treatments highlights the need for the development of new treatment modalities. Silver nanoparticles (AgNPs) are a promising alternative with broad-spectrum antibacterial activity. However, the clinical translation of AgNPs have been hampered primarily due to the delivery of unsafe levels of silver ions (Ag+) resulting in cellular toxicity and their susceptibility to aggregation resulting in loss of efficacy. Here, we describe a safe and effective, thermo-responsive AgNP hydrogel that provides antibacterial effects in conjunction with wound promoting properties. Using a murine model of wound infection, we demonstrate that the applied AgNP hydrogel to the wound (12 µg silver) not only provides superior bactericidal activity but also reduces inflammation leading to accelerated wound closure when compared to industry-standard silver sulfadiazine (302 µg silver). The AgNP hydrogel-treatment significantly accelerated wound closure at day 4 post-infection (56 closure) compared to both blank hydrogel or Ag SD (74% and 91% closure respectively) with a concurrent increase in PCNA-positive proliferating cells corresponding with a significant 32% improvement in wound re-epithelization compared to the blank hydrogel. Treatment of infected wounds with AgNP hydrogel also decreased neutrophil infiltration, increased anti-inflammatory Ym-1 positive M2 macrophages, and reduced the number of caspase-1 positive apoptotic cells. Therefore, this novel multifunctional AgNP thermo-responsive hydrogel is potentially a safe and effective treatment at much lower concentration for the treatment of wound infections. Statement of significance: In this study, we describe the development of a multifunctional thermo-responsive hydrogel of ultrasmall silver nanoparticles (AgNPs) for controlled and optimized delivery of silver to infected wounds. The in vivo biological effects of the developed hydrogel showed significant S. aureus elimination from infected mouse wounds compared to a commercial antibacterial formulation. The developed AgNP hydrogel optimally regulates inflammatory responses to promote wound healing as indicated by increased cell proliferation and wound re-epithelization. Additionally, AgNP hydrogel shows significant potential in regulating neutrophil infiltration while increasing levels of anti-inflammatory M2 macrophages and reduces the number of apoptotic cells. Therefore, the multifunctional properties of the developed AgNP thermo-responsive hydrogel offers great clinical potential to control bacterial infections and promote wound healing. Refereed/Peer-reviewed

Cryopreservation is an essential part of tissue banking and effective cryopreservation methods are critical for the development of cost-effective cell therapy products. Cell sheets are an attractive subset of cell therapy types, and cryopreservation has the potential to further drive down costs of allogeneic cell sheet therapy. This is currently a challenge as adhered cell monolayers are more susceptible to membrane damage during the freezing process. In this article, we investigate the performance of a surface-modified dressing for the cryopreservation of cells and strategies to improve cell recovery. Cryopreservation of multipotent adult progenitor cells (MAPC®) was performed on cells following their attachment to a surface for different periods of time. MAPC cells, given just 1 h to attach, washed off and were not recovered on the surface following thawing. Cells attached for longer periods, elongated further, and were more susceptible to damage from cryopreservation. A temporal window was identified that could allow cryopreservation on adherent surfaces where cells had attached to a surface without full elongation. By functionalizing the surface with coupled hyaluronic acid, cell spreading was initially retarded, thereby widening this temporal window. This approach demonstrates a novel method for enhancing the recovery of cryopreserved cell sheets on surfaces. Refereed/Peer-reviewed

Persistent wound infections have been a therapeutic challenge for a long time. Current treatment approaches are mostly based on the delivery of antibiotics, but these are not effective for all infections. Here, we report the development of a sensitive pH-responsive hydrogel that can provide controlled, pH-triggered release of silver nanoparticles (AgNPs). This delivery system was designed to sense the environmental pH and trigger the release of AgNPs when the pH changes from acidic to alkaline, as occurs due to the presence of pathogenic bacteria in the wound. Our results show that the prepared hydrogel restricts the release of AgNPs at acidic pH (pH = 4) but substantially amplifies it at alkaline pH (pH = 7.4 and pH = 10). This indicates the potential use of the hydrogel for the on-demand release of Ag+ depending on the environmental pH. In vitro antibacterial studies demonstrated effective elimination of both Gram-negative and positive bacteria. Additionally, the effective antibacterial dose of Ag+ showed no toxicity towards mammalian skin cells. Collectively, this pH-responsive hydrogel presents potential as a promising new material for the treatment of infected wounds. Refereed/Peer-reviewed

The rapid and continuing emergence of antimicrobial resistance is challenging our ability to effectively fight bacterial infections. This challenge is particularly felt in clinical settings where patients experience severely infected chronic wounds. Despite the increased development and reliance on silver-based dressings, the evidence underpinning their clinical efficacy is inconclusive and there remain concerns and confusion about their use. In this review, we describe the rationale for the current use of silver and silver-based wound dressings and highlight the progress and challenges associated with developing more advanced nanoparticle-based dressings for the safe and effective delivery of silver. Specifically, we describe the development of silver nanoparticles (AgNPs) with unique characteristics that render them applicable for controlling bacterial infections while simultaneously promoting wound healing through mechanisms including the dampening of inflammation and promotion of wound re-epithelialisation. Finally, we describe the latest progress in the development of different types of hydrogels for the therapeutic delivery of AgNPs to infected wounds. The possibility of designing and establishing multimodal silver therapies for the safe and effective treatment of infected wounds would be of great benefit for wound care management and improved patient outcomes. Refereed/Peer-reviewed

Flightless I is an actin-binding member of the gelsolin family of actin-remodeling proteins that inhibits actin polymerization but does not possess actin severing ability. Flightless I functions as a regulator of many cellular processes including proliferation, differentiation, apoptosis, and migration all of which are important for many physiological processes including wound repair, cancer progression and inflammation. More than simply facilitating cytoskeletal rearrangements, Flightless I has other important roles in the regulation of gene transcription within the nucleus where it interacts with nuclear hormone receptors to modulate cellular activities. In conjunction with key binding partners Leucine rich repeat in the Flightless I interaction proteins (LRRFIP)1/2, Flightless I acts both synergistically and competitively to regulate a wide range of cellular signaling including interacting with two of the most important inflammatory pathways, the NLRP3 inflammasome and the MyD88-TLR4 pathways. In this review we outline the current knowledge about this important cytoskeletal protein and describe its many functions across a range of health conditions and pathologies. We provide perspectives for future development of Flightless I as a potential target for clinical translation and insights into potential therapeutic approaches to manipulate Flightless I functions. Refereed/Peer-reviewed

Background Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. Methods The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice (n = 8/group) and 100 μL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. Results Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. Conclusion These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.

p62 is a receptor that facilitates selective autophagy by interacting simultaneously with cargoes and LC3 protein on the autophagosome to maintain cellular homeostasis. However, the regulatory mechanism(s) behind this process and its association with breast cancer remain to be elucidated. Here, we report that Flightless-I (FliI), a novel p62-interacting protein, promotes breast cancer progression by impeding selective autophagy. FliI was highly expressed in clinical breast cancer samples, and heterozygous deletion of FliI retarded the development of mammary tumors in PyVT mice. FliI induced p62-recruited cargoes into Triton X-100 insoluble fractions (TI) to form aggregates, thereby blocking p62 recognition of LC3 and hindering p62-dependent selective autophagy. This function of Flil was reinforced by Akt-mediated phosphorylation at Ser436 and inhibited by phosphorylation of Ulk1 at Ser64. Obstruction of autophagic clearance of p62-recruited cargoes by FliI was associated with the accumulation of oxidative damage on proteins and DNA, which could contribute to the development of cancer. Heterozygous knockout of FliI facilitated selectively autophagic clearance of aggregates, abatement of ROS levels, and protein oxidative damage, ultimately retarding mammary cancer progression. In clinical breast cancer samples, Akt-mediated phosphorylation of FliI at Ser436 negatively correlated with long-term prognosis, while Ulk1-induced FliI phosphorylation at Ser64 positively correlated with clinical outcome. Together, this work demonstrates that FliI functions as a checkpoint protein for selective autophagy in the crosstalk between FliI and p62-recruited cargoes, and its phosphorylation may serve as a prognostic marker for breast cancer. Significance: Flightless-I functions as a checkpoint protein for selective autophagy by interacting with p62 to block its recognition of LC3, leading to tumorigenesis in breast cancer.Cancer Res; 78(17); 4853–64. ©2018 AACR.

Chronic wounds are a serious medical problem both in the hospital and community setting. The healing of chronic wounds is often compromised by colonisation of different bacterial pathogens leading to life-threatening infections. Bacterial infections are a critical contributing factor to chronic wounds and can lead to biofilm formation and inhibition of innate inflammatory responses, including the reduction of acute inflammation.Concerningly, the overuse of systemic antibiotics and the use of traditional therapeutics, including topical antimicrobials – iodine, chlorhexidine and silver – have both greatly contributed to the development of a global increase in antimicrobial resistance. This has therefore led to a renewed interest in natural and alternative antimicrobial treatment strategies in wound care for the treatment of infected wounds.This review summarises the pre-clinical and clinical evidence that exists for the use of natural remedies, namely essential and animal oils, as adjunctive therapeutic approaches for the treatment of infected wounds. It also discusses novel approaches in nanotechnology that are being used for the development of natural remedies aimed at improving the healing of infected chronic wounds. Refereed/Peer-reviewed

Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine important to wound healing. In non-healing wounds, it has been suggested that the expression of TNF-α is prolonged and elevated which contributes to impaired healing responses. Hence it is of great interest to develop biosensors towards the detection of TNF-α in non-healing wounds. In this study, we have developed a label-free optical TNF-α biosensor based on interferometric reflectance spectroscopy (IRS) technique using a functionalized nanoporous anodic alumina (NAA) thin film transducer. The biosensor is fabricated by functionalizing NAA pore walls with anti-TNF-α antibodies using silanization chemistry. Binding of TNF-α to the bioreceptors within the pores causes a change in effective optical thickness (EOT) of the NAA thin film. Thus, analyte detection is achieved by monitoring EOT evolution with time. Label-free detection of TNF-α was demonstrated in buffer solution and in complex media such as simulated wound fluid. A limit of detection of 0.13 μg/mL was achieved. This study provides proof-of-concept evidence which sets foundations for further development of biosensors as point-of-care (POC) diagnostic tools for chronic wound care. Refereed/Peer-reviewed

Skin has a remarkable capacity for regeneration; however, with an ever aging population, there is a growing burden to the healthcare system from chronic wounds. Novel therapies are required to address the problems associated with nonhealing chronic wounds. Novel wound dressings that can encourage increased reepithelialization could help to reduce the burden of chronic wounds. A suite of chemically defined surfaces have been produced using plasma polymerization, and the ability of these surfaces to support the growth of primary human skin cells has been assessed. Additionally, the ability of these surfaces to modulate cell migration and morphology has also been investigated. Keratinocytes and endothelial cells were extremely sensitive to surface chemistry showing increased viability and migration with an increased number of carboxylic acid functional groups. Fibroblasts proved to be more tolerant to changes in surface chemistry; however, these cells migrated fastest over amine-functionalized surfaces. The novel combination of comprehensive chemical characterization coupled with the focus on cell migration provides a unique insight into how a material's physicochemical properties affect cell migration. Refereed/Peer-reviewed

Australian native plants have a long history of therapeutic use in indigenous cultures, however, they have been poorly studied scientifically. We analysed the effects of 14 plant derived compounds from the species Pilidiostigma glabrum, Myoporum montanum, Geijera parviflora, and Rhodomyrtus psidioides for their potential wound healing properties by assessing their ability to induce or suppress Collagen I and Collagen III expression in human skin fibroblasts in culture. The compound 7-geranyloxycoumarin was able to significantly increase Collagen I (23.7%, p < 0.0002) expression in comparison to control. Significant suppression of Collagen III was observed for the compounds flindersine (11.1%, p < 0.02), and (N-acetoxymethyl) flindersine (27%, p < 0.00005). The implications of these finding is that these compounds could potentially alter the expression of different collagens in the skin allowing for the potential development of new wound healing therapies and new approaches for treating various skin diseases as well as photo (sun) damaged, and aged skin. Refereed/Peer-reviewed

Plasma polymerization was used to coat a melt electrospun polycaprolactone scaffold to improve cell attachment and organization. Plasma polymerization was performed using an amine containing monomer, allylamine, which then allowed for the subsequent immobilization of biomolecules, i.e., heparin and fibroblast growth factor-2. The stability of the plasma-polymerized amine-coating was demonstrated by X-ray photoelectron spectroscopy analysis and imaging time-of-flight secondary ion mass spectrometry revealed that a uniform plasma amine-coating was deposited throughout the scaffold. Based upon comparison with controls, it was evident that the combination scaffold aided cell ingress and the formation of distinct fibroblast and keratinocyte layers.

usc Objective: Activation of epidermal stem cells (EpSCs) from their quiescent niche is an integral component of wound reepithelialization and involves Wnt/β-catenin (β-Cat) signaling and remodeling of the actin cytoskeleton. The aim of this study was to investigate the effect of Flightless I (Flii), a cytoskeletal protein and inhibitor of wound healing, on EpSC activation during wound repair. Approach: Genetically modified Flii mice (Flii knockdown: Flii+/−, wild type: WT, Flii overexpressing: FliiTg/Tg) received two incisional wounds along the lateral axis of the dorsal skin. Indicators of EpSC activation (epidermal growth factor receptor 1 [EGFR1], leucine-rich repeats and immunoglobulin-like domains-1 [Lrig1], K14), Wnt/β-Cat signaling (Lgr6, Flap2, β-Cat, and axis inhibition protein 2 [Axin2]), and cell proliferation (proliferating cell nuclear antigen [PCNA]) were assessed using immunohistochemistry. β-Cat stabilization was examined using western blotting with cell cycling and differentiation of isolated CD34+ITGA6high EpSCs examined using real time-quantitative polymerase chain reaction after treatment with wound-conditioned media. Results:Flii+/− led to increased numbers of activated EpSCs expressing PCNA, elevated EGFR1, and decreased Lrig1. EpSCs in Flii+/− hair follicle niches adjacent to the wounds also showed expression of Wnt-activation markers including increased β-Cat and Lgr6, and decreased Axin2. EpSCs (CD34+ITGA6high) isolated from Flii+/− unwounded skin showed elevated expression of cell-cycling genes including ΔNp63, filaggrin (Fila), involucrin (Invo), cyclin D1 (Ccnd1), and cell-division cycle protein-20 (Cdc20); and elevated ΔNp63 and Invo after treatment with wound-conditioned media compared with WT and FliiTg/Tg counterparts. Innovation: Flii was identified as an inhibitor of EpSC activation that may explain its negative effects on wound reepithelialization. Conclusion: Flii may inhibit EpSC activation by interrupting Wnt/β-Cat signaling. Strategies that reduce Flii may increase activation of EpSCs and promote reepithelialization of wounds. Refereed/Peer-reviewed

Burns are characterised by significant local swelling and redness around the site of injury, indicative of acute inflammation. Whilst the inflammatory response is fundamental to the healing process, triggering a cascade of cytokines and growth factors to protect against the risk of infection, it is clear that prolonged inflammation can be detrimental and lead to scarring and fibrosis. Severe burns may display chronic, persistent inflammation long after the initial burn injury and may even result in multiple organ failure (MOF) due to systemic inflammatory response syndrome (SIRS). Excessive inflammation in the early stages of healing has been identified as a causative factor in the formation of scars which can be disfiguring, functionally restrictive and may require revisionary surgeries. Therefore, it is imperative that inflammation is effectively managed following burn injuries in order to optimise the benefits it provides whilst actively preventing the complications of inflammation including SIRS, multiple organ failure (MOF) and the development of scarring and fibrosis. Reviewing the current knowledge about the role of the inflammatory response in burns and the treatments available for the management of inflammation during wound healing, highlights the importance of continued research into understanding and developing new approaches to regulate inflammatory responses post-burn injuries.

Gene silencing using small interfering RNA has been proposed as a therapy for cancer, viral infections and other diseases. This study aimed to investigate whether layer-by-layer polymer surface modification could deliver small interfering RNA to decrease fibrotic processes associated with medical device implantation. Anti-green fluorescent protein labelled small interfering RNA was applied to tissue culture plates and polyurethane using a layer-by-layer technique with small interfering RNA and poly-L-lysine. In vitro studies showed that the level of down-regulation of green fluorescent protein was directly related to the number of coatings applied. This layer-by-layer coating technique was then used to generate Rhodamine-Flii small interfering RNA-coated implants for in vivo studies of small interfering RNA delivery via subcutaneous implantation in mice. After two days, Rh-positive cells were observed on the implants surface indicating cellular uptake of the Rhodamine-Flii small interfering RNA. Decreased Flii gene expression was observed in tissue surrounding the Rhodamine-Flii small interfering RNA coated implants for up to seven days post implantation, returning to baseline by day 21. Genes downstream from Flii, including TGF-β1 and TGF-β3, showed significantly altered expression confirming a functional effect of the Rhodamine-Flii small interfering RNA on gene expression. This research demonstrates proof-of-principle that small interfering RNA can be delivered via layer-by-layer coatings on biomaterials and thereby can alter the fibrotic process. Refereed/Peer-reviewed

Open Access Article. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence., The proinflammatory cytokine, tumor necrosis factor-α (TNF-α), is elevated in several diseases such as uveitis, rheumatoid arthritis and non-healing chronic wounds. Adding Infliximab, a chimeric IgG1 monoclonal antibody raised against TNF-α, to chronic wound fluid can neutralise human TNF-α, thereby providing a potential therapeutic option for chronic wound healing. However, to avoid the need for repeated application in a clinical setting, and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. Porous silicon (pSi) is a biodegradable high surface area material commonly employed for drug delivery applications. In this study, the use of pSi microparticles (pSi MPs) for the controlled release of Infliximab to disease environments, such as chronic wounds, is demonstrated. Surface chemistry and pore parameters for Infliximab loading are first optimised in pSi films and loading conditions are transferred to pSi MPs. Loading regimens exceeding 60 μg of Infliximab per mg of pSi are achieved. Infliximab is released with zero-order release kinetics over the course of 8 days. Critically, the released antibody remains functional and is able to sequester TNF-α over a weeklong timeframe; suitable for a clinical application in chronic wound therapy.

Epidermolysis bullosa (EB) consists of a spectrum of genodermatoses characterised by skin fragility and various degrees of skin and mucous membrane blistering. Minimal trauma and friction can cause extensive blistering in patients with EB, resulting in a number of complications. However, wound management is the main challenge for these patients because of a high risk of infection, fluid loss and potential development of aggressive squamous cell carcinoma (SCC). Indeed, patients with EB have an increased risk for developing skin cancers compared to the general population. In 2012, a home nursing programme was established in Australia to provide assistance to families or patients with severe forms of EB. Nursing care was provided to patients with severe EB during dressing changes in their homes over a period of 2 years. Both families of patients and nurses were surveyed periodically using a developed questionnaire to assess the benefits of this home nursing and its impact on the patients, their families and the nurses. Key findings included a perceived improvement in quality of life, a better provision of support and improved family life management. These findings are the first to highlight the benefits of this national home nursing programme for EB patients within Australia and demonstrate the continued need and benefit of home nursing for patients with severe skin blistering disorders.

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Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.

Background: The development of new therapies for hypertrophic scarring has been hampered by the lack of an appropriate animal model. The authors' objective was to establish a reproducible murine model of hypertrophic scarring by infusing bleomycin over a prolonged period to stimulate dermal fibroproliferation. Conclusions: The bleomycin infusion model stimulates dermal fibroproliferation, creating reproducible murine scars that are comparable to human hypertrophic scars in terms of histological features, collagen content and organization, cellularity, the presence of myofibroblasts, and expression of transforming growth factor β1. The bleomycin model represents a promising technique for studying scar formation and testing new antiscarring therapies. Methods: Osmotic pumps filled with 90 μl of 2.8 mg/ml bleomycin or a control solution (phosphate-buffered saline) were inserted subcutaneously under the dorsal skin of BALB/c mice. The pumps delivered their content at a constant rate of 0.11 μl/hour for 28 days before mice were euthanized or kept alive for a further 28 days and euthanized at day 56. The resulting lesions were analyzed using histological and immunohistochemical techniques. Results: The lesions displayed histopathological features of hypertrophic scar similar to those observed in humans and had increased cellularity, abnormal collagen I-collagen III ratios, elevated levels of the proscarring cytokine transforming growth factor β1, and increased numbers of myofibroblasts. The 28-day model displayed features analogous to those of a developing human hypertrophic scar, while the 56-day model was analogous to a mature hypertrophic scar. Refereed/Peer-reviewed

Background & Aim Although survival rates are increasing, burn injuries remain a challenge in the field of cutaneous wound healing. Stem cells have been shown to be a potential new therapy for burns and promote wound healing through a decrease in the inflammation and increase in collagen production. Multipotent adult progenitor cells (MAPC®) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of growth factors and cytokines which have effects on cellular activities. This study aimed to examine the effects of MAPC treatment and its secretome on burn injury repair using in vitro and in vivomodels. Methods, Results & Conclusion The effect of MAPC secretome on the capacity of keratinocytes, fibroblast and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay respectively. Secretome-treated fibroblasts were also immunostained for collagen 1 and 3 to investigate its effect on matrix production. Additionally, second degree burns were created on the dorsal surface of mice (n=8/group) and 5 × 105 MAPCs in 100µl PBS were administered via intradermal injection to the wound margins, 24h post-burn injury. The burns were imaged daily until day 7, for macroscopic wound area determination, when they were collected and analysed by H&E staining to determine dermal wound width and rate of re-epithelialisation. Masson's trichrome staining was also performed to investigate the total amount of collagen deposited within the burns. Cells treated with MAPC-secretome showed improved rate of scratch closure compared to controls. The proliferation of these cells was also increased when treated with MAPC-secretome. Moreover, fibroblasts treated with MAPC-secretome deposited more collagen 1 and 3. Burns intradermally injected with MAPCs showed a significant reduction in macroscopic wound area compared with controls at day 3 and day 7. Analysis of day 7 burn tissues showed significant decrease in the dermal wound width and increased rate of reepithelialisation. Masson's trichrome staining showed that collagen deposition in the wounds was significantly elevated following MAPC cell treatment. This study demonstrates the beneficial effects of MAPC cell therapy on burn injury repair including reduced time to healing and increased collagen deposition. The beneficial effect of MAPC cells may be due in part to the factors they secrete which improve skin cell proliferation and migration as well as collagen deposition.

Advances in wearable, highly sensitive and multifunctional strain sensors open up new opportunities for the development of wearable human interface devices for various applications such as health monitoring, smart robotics and wearable therapy. Herein, we present a simple and cost-effective method to fabricate a multifunctional strain sensor consisting of a skin-mountable dry adhesive substrate, a robust sensing component and a transdermal drug delivery system. The sensor has high piezoresisitivity to monitor real-time signals from finger bending to ulnar pulse. A transdermal drug delivery system consisting of polylactic-co-glycolic acid nanoparticles and a chitosan matrix is integrated into the sensor and is able to release the nanoparticles into the stratum corneum at a depth of ~60 &micro, m. Our approach to the design of multifunctional strain sensors will lead to the development of cost-effective and well-integrated multifunctional wearable devices.

A 72-year-old female with venous insufficiency presented to a hospital based multidisciplinary wound clinic after 20 years of recurrent episodes of venous leg ulcers. Examination showed bilateral leg ulcers with no evidence of arterial insufficiency, but complicated by considerable devitalised tissue, abnormally high bacterial load and the presence of multi-resistant organisms. The ulcers were initially treated with larvae to aid debridement and reduce the bacterial load, prior to skin grafting. Although ulcer free for a period of 4 months, further debridement was required when the skin condition deteriorated. Surgical intervention was chosen as the preferred method by the surgeons for a second acute care admission using hydrosugery, along with supplementary skin grafts and compression. Ongoing management, consisting of regular debridement, skin care and compression therapy, continues. Refereed/Peer-reviewed

Pericytes are cells that reside on the wall of the blood vessels and their primary function is to maintain the vessel integrity. Recently, it has been realized that pericytes have a much greater role than just the maintenance of vessel integrity essential for the development and formation of a vascular network. Pericytes also have stem cell-like properties and are seemingly able to differentiate into adipocytes, chondrocytes, osteoblasts and granulocytes, leading them to be identified as mesenchymal stem cells (MSCs). More recently it has been suggested that pericytes play a key role in wound healing, whereas the beneficial effects of MSCs in accelerating the wound healing response has been recognized for some time. In this review, we collate the most recent data on pericytes, particularly their role in vessel formation and how they can affect the wound healing process. Refereed/Peer-reviewed

Epidermolysis bullosa (EB) is a chronic inheritable disease that leads to severe blistering and fibrosis. Previous studies have shown that the actin cytoskeletal protein flightless I (Flii) impairs wound healing associated with EB. Using a mouse model of EB acquisita (EBA), the effect of "mopping up" Flii using Flii-neutralizing antibodies (FnAbs) before, during, and after blister formation was determined. FnAbs, incorporated into a cream vehicle and applied topically to the skin, penetrated into the basal epidermis and upper papillary dermis but were not detected in serum or other organs and did not alter neutrophil or macrophage infiltration into the blistered skin. Histological assessment of blister severity showed that treatment of early-stage blisters with FnAb cream reduced their severity and improved their rate of healing. Treatment of established blisters with FnAb cream also improved healing and restored the skin's tensile strength toward that of normal skin. Repeated application of FnAbs to EBA skin before the onset of blistering reduced the severity of skin blistering. Independent of when the FnAbs were applied, skin barrier function and wound healing were improved and skin fragility was reduced, suggesting that FnAbs could potentially improve healing of patients with EB. Refereed/Peer-reviewed

Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF-κB production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.