Silver oxysalts promote cutaneous wound healing independent of infection.

Chronic wounds often exist in a heightened state of inflammation whereby excessive inflammatory cells release high levels of proteases and reactive oxygen species (ROS). While low levels of ROS play a fundamental role in the regulation of normal wound healing, their levels need to be tightly regulated to prevent a hostile wound environment resulting from excessive levels of ROS. Infection amplifies the inflammatory response, augmenting levels of ROS which creates additional tissue damage that supports microbial growth. Antimicrobial dressings are used to combat infection; however, the effects of these dressing on the wound environment and healing independent of infection are rarely assessed. Cytotoxic or adverse effects on healing may exacerbate the hostile wound environment and prolong healing. Here we assessed the effect on healing independent of infection of silver oxysalts which produce higher oxidative states of silver (Ag2+ /Ag3+ ). Silver oxysalts had no adverse effect on fibroblast scratch wound closure whilst significantly promoting closure of keratinocyte scratch wounds (34% increase compared with control). Furthermore, dressings containing silver oxysalts accelerated healing of full-thickness incisional wounds in wild-type mice, reducing wound area, promoting reepithelialization, and dampening inflammation. We explored the mechanisms by which silver oxysalts promote healing and found that unlike other silver dressings tested, silver oxysalt dressings catalyze the breakdown of hydrogen peroxide to water and oxygen. In addition, we found that silver oxysalts directly released oxygen when exposed to water. Collectively, these data provide the first indication that silver oxysalts promote healing independent of infection and may regulate oxidative stress within a wound through catalysis of hydrogen peroxide.

An ex vivo porcine skin model to evaluate pressure-reducing devices of different mechanical properties used for pressure ulcer prevention.

Pressure ulcers are complex wounds caused by pressure- and shear-induced trauma to skin and underlying tissues. Pressure-reducing devices, such as dressings, have been shown to successfully reduce pressure ulcer incidence, when used in adjunct to pressure ulcer preventative care. While pressure-reducing devices are available in a range of materials, with differing mechanical properties, understanding of how a material's mechanical properties will influence clinical efficacy remains limited. The aim of this study was to establish a standardized ex vivo model to allow comparison of the cell protection potential of two gel-like pressure-reducing devices with differing mechanical properties (elastic moduli of 77 vs. 35 kPa). The devices also displayed differing energy dissipation under compressive loading, and resisted strain differently under constant load in compressive creep tests. To evaluate biological efficacy we employed a new ex vivo porcine skin model, with a confirmed elastic moduli closely matching that of human skin (113 vs. 119 kPa, respectively). Static loads up to 20 kPa were applied to porcine skin ex vivo with subsequent evaluation of pressure-induced cell death and cytokine release. Pressure application alone increased the percentage of epidermal apoptotic cells from less than 2% to over 40%, and increased cellular secretion of the pro-inflammatory cytokine TNF-alpha. Co-application of a pressure-reducing device significantly reduced both cellular apoptosis and cytokine production, protecting against cellular damage. These data reveal new insight into the relationship between mechanical properties of pressure-reducing devices and their biological effects. After appropriate validation of these results in clinical pressure ulcer prevention with all tissue layers present between the bony prominence and external surface, this ex vivo porcine skin model could be widely employed to optimize design and evaluation of devices aimed at reducing pressure-induced skin damage.

p63 control of desmosome gene expression and adhesion is compromised in AEC syndrome.

Ankyloblepharon, ectodermal defects, cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder caused by mutations in the p63 gene, essential for embryonic development of stratified epithelia. The most severe cutaneous manifestation of this disorder is the long-lasting skin fragility associated with severe skin erosions after birth. Using a knock-in mouse model for AEC syndrome, we found that skin fragility was associated with microscopic blistering between the basal and suprabasal compartments of the epidermis and reduced desmosomal contacts. Expression of desmosomal cadherins and desmoplakin was strongly reduced in AEC mutant keratinocytes and in newborn epidermis. A similar impairment in desmosome gene expression was observed in human keratinocytes isolated from AEC patients, in p63-depleted keratinocytes and in p63 null embryonic skin, indicating that p63 mutations causative of AEC syndrome have a dominant-negative effect on the wild-type p63 protein. Among the desmosomal components, desmocollin 3, desmoplakin and desmoglein 1 were the most significantly reduced by mutant p63 both at the RNA and protein levels. Chromatin immunoprecipitation experiments and transactivation assays revealed that p63 controls these genes at the transcriptional level. Consistent with reduced desmosome function, AEC mutant and p63-deficient keratinocytes had an impaired ability to withstand mechanical stress, which was alleviated by epidermal growth factor receptor inhibitors known to stabilize desmosomes. Our study reveals that p63 is a crucial regulator of a subset of desmosomal genes and that this function is impaired in AEC syndrome. Reduced mechanical strength resulting from p63 mutations can be alleviated pharmacologically by increasing desmosome adhesion with possible therapeutic implications.

A statistical analysis of murine incisional and excisional acute wound models.

Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation.

Direct evidence that PKCα positively regulates wound re-epithelialization: correlation with changes in desmosomal adhesiveness.

Non-healing wounds cause considerable patient morbidity and represent a significant economic burden. Central to wound repair is re-epithelialization, a crucial process involving the modulation of cell adhesion to allow keratinocyte migration to cover the exposed underlying tissues. The cellular mechanisms regulating the earliest stages of re-epithelialization are unclear. We present the first direct evidence that protein kinase Cα (PKCα) plays an important role in regulating wound re-epithelialization. In PKCα(-/-) mice re-epithelialization is delayed, while in novel bitransgenic mice over-expressing constitutively active PKCα it is accelerated. These effects are not due to changes in keratinocyte proliferation, apoptosis or intrinsic cell motility. Instead, they correlate with changes in desmosomal adhesiveness, delay being preceded by retained desmosomal hyper-adhesiveness and acceleration with a rapid switch to desmosomal Ca(2+) -dependence. We demonstrate mechanistic conservation in acute human wounds where PKCα localizes to wound edge desmosomes, which become Ca(2+) -dependent. However, in chronic wounds PKCα remains cytoplasmic and desmosomes fail to switch from the hyper-adhesive state. These results throw new mechanistic light on the earliest stages of wound re-epithelialization and suggest activation of PKCα as a new therapeutic strategy for non-healing wounds.

Anti-biofilm effects and healing promotion by silver oxynitrate-based dressings.

Microbial growth within a wound often manifests as biofilms, which can prevent healing and is difficult to eradicate. Novel silver dressings claim to combat wound infection, but anti-biofilm efficacy and effects on healing independent of infection are often unclear. Using in vitro and in vivo S. aureus and P. aeruginosa biofilm models, we report the efficacy of a dressing which produces Ag1+ ions; an Ag1+ dressing containing ethylenediaminetetraacetic acid and benzethonium chloride (Ag1+/EDTA/BC), and a dressing containing silver oxynitrate (Ag Oxysalts) which produces Ag1+, Ag2+ and Ag3+ ions, against wound biofilms, and their effects on healing. Ag1+ dressings had minimal effect on in vitro and murine (C57BL/6j) wound biofilms. In contrast, Ag Oxysalts and Ag1+/EDTA/BC dressings significantly reduced viable bacteria within in vitro biofilms and demonstrated a visible reduction in bacteria and EPS components within murine wound biofilms. The dressings had different effects on the healing of biofilm-infected and uninfected wounds, with Ag Oxysalts dressings having a greater beneficial effect on re-epithelialisation, wound size and inflammation than the control treatment and the other silver dressings. The different physicochemical properties of the silver dressings result in varied effects on wound biofilms and healing which should be considered when selecting dressings to treat biofilm-infected wounds.

Desmosomes: adhesive strength and signalling in health and disease.

Desmosomes are intercellular junctions whose primary function is strong intercellular adhesion, known as hyperadhesion. In the present review, we discuss how their structure appears to support this function as well as how they are assembled and down-regulated. Desmosomal components also have signalling functions that are important in tissue development and remodelling. Their adhesive and signalling functions are both compromised in genetic and autoimmune diseases that affect the heart, skin and mucous membranes. We conclude that much work is required on structure-function relationships within desmosomes in vivo and on how they participate in signalling processes to enhance our knowledge of tissue homoeostasis and human disease.

Wound dressings: curbing inflammation in chronic wound healing.

Chronic wounds represent an economic burden to healthcare systems worldwide and a societal burden to patients, deeply impacting their quality of life. The incidence of recalcitrant wounds has been steadily increasing since the population more susceptible, the elderly and diabetic, are rapidly growing. Chronic wounds are characterised by a delayed wound healing process that takes longer to heal under standard of care than acute (i.e. healthy) wounds. Two of the most common problems associated with chronic wounds are inflammation and infection, with the latter usually exacerbating the former. With this in mind, researchers and wound care companies have developed and marketed a wide variety of wound dressings presenting different compositions but all aimed at promoting healing. This makes it harder for physicians to choose the correct therapy, especially given a lack of public quantitative data to support the manufacturers' claims. This review aims at giving a brief introduction to the clinical need for chronic wound dressings, focusing on inflammation and evaluating how bio-derived and synthetic dressings may control excess inflammation and promote healing.

Infection and Inflammation in the Wound Environment: Addressing Issues of Delayed Wound Healing With Advanced Wound Dressings.

Impaired wound healing is estimated to affect about 2% of the US population, and a major goal of health care providers (HCPs) is to better understand delayed healing so they can effectively choose advanced wound dressings to manage these wounds. However, there are estimated to be more than 3000 dressing options available, making dressing selection an overwhelming burden. An expert panel of 7 HCPs experienced in diverse medical disciplines and 3 scientists convened to discuss the use of 2 families of dressings (silver-oxysalt [AgOx] dressings and oxidized regenerated cellulose/collagen [ORC/C] dressings) and delayed wound healing. Before the meeting, panelists reviewed 16 articles concerning the dressings, and 2 scientists presented on the topics of infection and inflammation in the wound environment, along with providing information about the dressing families, during the meeting. In addition, each HCP presented specific cases in which they had applied AgOx or ORC/C dressings and described how the dressing was used to manage stalled healing. After the meeting, the panelists and another HCP who was unable to attend the panel meeting provided insight and feedback for this publication, which provides an overview of the meeting. A major theme of this panel discussion was the need for a fundamental change in how HCPs approach wound care, especially for nonhealing wounds and underlying issues of infection and inflammation in the wound environment. Ultimately, the panel developed a decision-tree model for risk-stratifying patients based on their potential to have or develop these 2 underlying issues, followed by deciding on treatment options based on the status of infection and inflammation in the wound.

Facial clefting in Tp63 deficient mice results from altered Bmp4, Fgf8 and Shh signaling.

During embryogenesis, the transcription factor Tp63 is expressed in the basal cells of multiple epithelial tissues. In humans, mutations in TP63 have been identified in five distinct human developmental disorders that are characterized by limb abnormalities, ectodermal dysplasia, and facial anomalies. To dissect the molecular pathogenesis of the bilateral cleft lip and cleft palate that results from mutation of Tp63, we analysed Tp63 mutant mice. At E10.5, Tp63-deficient mice exhibited abnormal morphogenesis of the medial nasal, lateral nasal and maxillary processes. Analysis of key signaling molecules revealed that these defects result from increased Bmp4 signaling in the epithelia of the facial processes. Acting antagonistically on Fgf8 and Shh, this aberrant signaling led to a reduction in mesenchymal cell proliferation and increased cell death in specific regions of the facial processes. In addition, a proliferative defect in the mesenchyme of the maxillary processes at E11.5 resulted in absence of the anterior region of the palatal shelves and, subsequently, cleft palate. Our results are consistent with a role for Tp63 in the regulation of Bmp signaling controlling the growth, modelling and fusion events underlying facial development and shed new light on the complex abnormality of facial clefting.

Enhanced ectodysplasin-A receptor (EDAR) signaling alters multiple fiber characteristics to produce the East Asian hair form.

Hair morphology differs dramatically between human populations: people of East Asian ancestry typically have a coarse hair texture, with individual fibers being straight, of large diameter, and cylindrical when compared to hair of European or African origin. Ectodysplasin-A receptor (EDAR) is a cell surface receptor of the tumor necrosis factor receptor (TNFR) family involved in the development of hair follicles, teeth, and sweat glands. Analyses of genome-wide polymorphism data from multiple human populations suggest that EDAR experienced strong positive selection in East Asians. It is likely that a nonsynonymous SNP in EDAR, rs3827760, was the direct target of selection as the derived p.Val370Ala variant is seen at high frequencies in populations of East Asian and Native American origin but is essentially absent from European and African populations. Here we demonstrate that the derived EDAR370A common in East Asia has a more potent signaling output than the ancestral EDAR370 V in vitro. We show that elevation of Edar activity in transgenic mice converts their hair phenotype to the typical East Asian morphology. The coat texture becomes coarse, with straightening and thickening of individual hairs and conversion of fiber cross-sectional profile to a circular form. These thick hair fibers are produced by enlarged hair follicles, which in turn develop from enlarged embryonic organ primordia. This work shows that the multiple differences in hair form between East Asian and other human populations can be explained by the simplest of genetic alterations.

Genetic Pathways of Neuroregeneration in a Novel Mild Traumatic Brain Injury Model in Adult Zebrafish.

Mild traumatic brain injuries (mTBIs) are one of the most prevalent neurological disorders, and humans are severely limited in their ability to repair and regenerate central nervous system (CNS) tissue postinjury. However, zebrafish (Danio rerio) maintain the remarkable ability to undergo complete and functional neuroregeneration as an adult. We wish to extend knowledge of the known mechanisms of neuroregeneration by analyzing the differentially expressed genes (DEGs) in a novel adult zebrafish model of mTBI. In this study, a rodent weight drop model of mTBI was adapted to the adult zebrafish. A memory test showed significant deficits in spatial memory in the mTBI group. We identified DEGs at 3 and 21 days postinjury (dpi) through RNA-sequencing analysis. The resulting DEGs were categorized according to gene ontology (GO) categories. At 3 dpi, GO categories consisted of peak injury response pathways. Significantly, at 21 dpi, GO categories consisted of neuroregeneration pathways. Ultimately, these results validate a novel zebrafish model of mTBI and elucidate significant DEGs of interest in CNS injury and neuroregeneration.

Cutaneous Nod2 Expression Regulates the Skin Microbiome and Wound Healing in a Murine Model.

The skin microbiome exists in dynamic equilibrium with the host, but when the skin is compromised, bacteria can colonize the wound and impair wound healing. Thus, the interplay between normal skin microbial interactions versus pathogenic microbial interactions in wound repair is important. Bacteria are recognized by innate host pattern recognition receptors, and we previously showed an important role for the pattern recognition receptor NOD2 in skin wound repair. NOD2 is implicated in changes in the composition of the intestinal microbiota in Crohn's disease, but its role on skin microbiota is unknown. Nod2-deficient (Nod2-/-) mice had an inherently altered skin microbiome compared with wild-type controls. Furthermore, we found that Nod2-/- skin microbiome dominated and caused impaired healing, shown in cross-fostering experiments of wild-type pups with Nod2-/- pups, which then acquired altered cutaneous bacteria and delayed healing. High-throughput sequencing and quantitative real-time PCR showed a significant compositional shift, specifically in the genus Pseudomonas in Nod2-/- mice. To confirm whether Pseudomonas species directly impair wound healing, wild-type mice were infected with Pseudomonas aeruginosa biofilms and, akin to Nod2-/- mice, were found to exhibit a significant delay in wound repair. Collectively, these studies show the importance of the microbial communities in skin wound healing outcome.

Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63(+/L514F) ) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b(-/-) mice. Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome.

Exploring the "hair growth-wound healing connection": anagen phase promotes wound re-epithelialization.

When the skin is damaged, a variety of cell types must migrate, proliferate, and differentiate to reform a functional barrier to the external environment. Recent studies have shown that progenitor cells residing in hair follicles (HFs) are able to contribute to this re-epithelialization of wounds in vivo. However, the influence of the hair cycle on wound healing has not previously been addressed. Here, we have exploited spontaneous postnatal hair-cycle synchronicity in mice to systematically examine the influence of the different hair-cycle stages on murine skin wound healing. We report significant acceleration of healing during the anagen phase of HF cycling in vivo, associated with alterations in epithelial, endothelial, and inflammatory cell types. Intriguingly, gene profiling data reveal a clear correlation between the transcription of genes beneficial for wound healing and those upregulated during the anagen phase of the hair cycle in unwounded skin. These findings, which demonstrate a previously unappreciated association between HF cycling and wound healing, reveal numerous molecular correlates for further investigation.

Cooperation between the transcription factors p63 and IRF6 is essential to prevent cleft palate in mice.

Cleft palate is a common congenital disorder that affects up to 1 in 2,500 live human births and results in considerable morbidity to affected individuals and their families. The etiology of cleft palate is complex, with both genetic and environmental factors implicated. Mutations in the transcription factor-encoding genes p63 and interferon regulatory factor 6 (IRF6) have individually been identified as causes of cleft palate; however, a relationship between the key transcription factors p63 and IRF6 has not been determined. Here, we used both mouse models and human primary keratinocytes from patients with cleft palate to demonstrate that IRF6 and p63 interact epistatically during development of the secondary palate. Mice simultaneously carrying a heterozygous deletion of p63 and the Irf6 knockin mutation R84C, which causes cleft palate in humans, displayed ectodermal abnormalities that led to cleft palate. Furthermore, we showed that p63 transactivated IRF6 by binding to an upstream enhancer element; genetic variation within this enhancer element is associated with increased susceptibility to cleft lip. Our findings therefore identify p63 as a key regulatory molecule during palate development and provide a mechanism for the cooperative role of p63 and IRF6 in orofacial development in mice and humans.