ARTICLE: Evolution of Skin Barrier Science for Healthy and Compromised Skin.

Skin is a complex organ comprised of multiple cell types and microstructures that work in concert to serve critical functions and support the body’s homeostasis. It is the outermost, cornified layer of our body that is primarily responsible for the permeability barrier, protecting against external aggressors and preventing water loss from within. The understanding of the organization, functionality, and underlying mechanisms of the skin barrier has evolved greatly through the years. The formation of an intact and well-maintained stratum corneum (SC), where the permeability barrier resides, relies heavily on the differentiation of epidermal keratinocytes and the synthesis, release, localization, and binding of lipids that include principally ceramides, cholesterol, and free fatty acids. The in-depth research on SC barrier, its disruption in the pathogenesis of diseases, as well as on barrier responses to environmental insults, has enabled the development of modern therapeutics and topical care routines. Among them, ceramide-containing moisturizers have clinically demonstrated the ability to support the management of skin conditions such as atopic dermatitis and psoriasis by reducing the disease severity and recurrence and improving the patients’ perception of overall skin quality and health. This review focuses on the contributions of various barrier constituents to skin barrier function in health and pathological conditions, and how topical interventions containing essential barrier lipids support barrier restoration and provide relief. J Drugs Dermatol. 20(4 Suppl):s3-9. doi:10.36849/JDD.S589A.

Ciprofloxacin-loaded keratin hydrogels reduce infection and support healing in a porcine partial-thickness thermal burn.

Infection is a leading cause of morbidity and mortality in burn patients. Current therapies include silver-based creams and dressings, which display limited antimicrobial effectiveness and impair healing. The need exists for a topical, point-of-injury antibiotic treatment that provides sustained antimicrobial activity without impeding wound repair. Fitting this description are keratin-based hydrogels, which are fully biocompatible and support the slow-release of antibiotics. Here we develop a porcine model of an infected partial-thickness burn to test the effects of ciprofloxacin-loaded keratin hydrogels on infection and wound healing. Partial-thickness burns were inoculated with either Pseudomonas aeruginosa or Methicillin-resistant Staphylococcus aureus, resulting in infections that persisted for >2 weeks that exceeded 10(5) and 10(6) cfu per gram of tissue, respectively. Compared to silver sulfadiazine, ciprofloxacin-loaded keratin hydrogel treatment significantly reduced the amount of P. aeruginosa and S. aureus in the burn by >99% on days 4, 7, 11, and 15 postinjury. Further, burns treated with ciprofloxacin-loaded keratin hydrogels exhibited similar healing patterns as uninfected burns with regards to reepithelialization, macrophage recruitment, and collagen deposition and remodeling. The ability of keratin hydrogels to deliver antibiotics to fight infection and support healing of partial-thickness burns make them a strong candidate as a first-line burn therapy.

Biomolecular Analysis of Beta Dose-Dependent Cutaneous Radiation Injury in a Porcine Model.

While cutaneous radiation injury (CRI) is generally referenced as a consequence of a nuclear attack, it can also be caused by less dangerous events such as the use of dirty bombs, industrial radiological accidents, or accidental overexposure of beta (ß) particle or gamma (γ) radiation sources in medical procedures. Although the gross clinical consequences of these injuries have been well documented, relatively little is known about the molecular changes underlying the progression of pathology. Here we describe a porcine model of cutaneous radiation injury after skin was exposed to strontium-90 b particle at doses of 16-42 Gy and characterize the anatomical and molecular changes over 70 days. The results show that irradiated sites displayed dosedependent increases in erythema and moist desquamation that peaked between days 35 and 42. Dose-dependent histopathological changes were observed, with higher doses exhibiting increased inflammation and epidermal hyperplasia beyond day 35. Furthermore, immunohistochemistry showed that exposure to 37 Gy ß-particle radiation decreased epidermal cell proliferation and desmosomal junction proteins at day 70, suggesting compromised epidermal integrity. Metabolomic analysis of biopsies revealed dose- and time-dependent changes as high as 252-fold in several metabolites not previously linked to CRI. These alterations were seen in pathways reflecting protein degradation, oxidative stress, eicosanoid production, collagen matrix remodeling, mitochondrial stress, cell membrane composition and vascular disruption. Taken together, these data show that exposure to high doses of ß particle damaged the molecular processes underlying skin integrity to a greater extent and for a longer period of time than has been shown previously. These findings further understanding of radiation-induced skin injury and serve as a foundation for the development and testing of potential therapeutics to treat CRI.

In Situ Delivery of Fibrin-Based Hydrogels Prevents Contraction and Reduces Inflammation.

While early excision and grafting has revolutionized burn wound care, autologous split-thickness skin grafts are sometimes unavailable. Tissue-engineered skin substitutes have generated great interest but have proven inadequate. Therefore, the development of novel biomaterials to replace/augment skin grafting could improve burn patient outcomes. Herein, we establish the effects of debridement on deep-partial thickness burns and subsequently examine the effects of 3 different hydrogels on healing. Burns were created on the dorsum of pigs and 4 days after, the eschar was either left intact or debrided for treatment with collagen, PEGylated fibrinogen (PEG-fibrin) or PEGylated autologous platelet-free plasma (PEG-PFP) hydrogels. Wounds were photographed, scored, and biopsied for histology on postburn days 7, 10, 14, and 28. Compared with nondebrided wounds, debridement improved wound color and suppleness but accelerated contraction. Debridement also significantly reduced the number of neutrophils in the wound bed at days 10 and 14 postburn. Treatment with any hydrogel transiently mitigated contraction, with the PEG-fibrin group displaying less contraction on day 28. All hydrogels were visible histologically for up to 10 days, with significant cellular and blood vessel infiltration observed in PEG-fibrin hydrogels. Collagen and PEG-fibrin hydrogels reduced neutrophils and macrophages in surrounding granulation tissue on day 7, while PEG-fibrin hydrogels contained less immune cells. These data suggest that a single hydrogel application at the time of debridement has immunomodulatory properties that aid in wound healing. Ultimately, these hydrogels may be combined with other biomaterials, cells, or biologics for replacing/augmenting skin substitutes.

An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue.

BACKGROUND: Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue. FINDINGS: A modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue. CONCLUSIONS: We describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host tissue. The samples used in this study demonstrate that this staining technique has laboratory and clinical applicability. This modification only adds minutes to traditional Gram stain with reusable reagents, and results in a cost- and time-efficient technique for identifying bacteria in any clinical biopsy containing connective tissue.

Ciprofloxacin-Loaded Keratin Hydrogels Prevent Pseudomonas aeruginosa Infection and Support Healing in a Porcine Full-Thickness Excisional Wound.

Objective: Cutaneous wound infection can lead to impaired healing, multiple surgical procedures, and increased hospitalization time. We tested the effectiveness of keratin-based hydrogels (termed "keratose") loaded with ciprofloxacin to inhibit infection and support healing when topically administered to porcine excision wounds infected with Pseudomonas aeruginosa. Approach: Using a porcine excisional wound model, 10 mm full-thickness wounds were inoculated with 106 colony-forming units of P. aeruginosa and treated on days 1 and 3 postinoculation with ciprofloxacin-loaded keratose hydrogels. Bacteria enumeration and wound healing were assessed on days 3, 7, and 11 postinjury. Results: Ciprofloxacin-loaded keratose hydrogels reduced the amount of P. aeruginosa in the wound bed by 99.9% compared with untreated wounds on days 3, 7, and 11 postinjury. Ciprofloxacin-loaded keratose hydrogels displayed decreased wound contraction and reepithelialization at day 7 postinjury. By day 11, wounds treated with ciprofloxacin-keratose hydrogels contained collagen-rich granulation tissue and myofibroblasts. Wounds treated with ciprofloxacin-loaded keratose hydrogels exhibited a transient increase in macrophages in the wound bed at day 7 postinjury that subsided by day 11. Innovation: Current therapies for wound infection include systemic antibiotics, which could lead to antibiotic resistance, and topical antimicrobial treatments, which require multiple applications and can delay healing. Here, we show that ciprofloxacin-loaded keratose hydrogels inhibit cutaneous wound infection without interfering with key aspects of the healing process including granulation tissue deposition and remodeling. Conclusions: Ciprofloxacin-loaded keratose hydrogels have the potential to serve as a point-of-injury antibiotic therapy that prevents infection and supports healing following cutaneous injury.

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function.

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5ß1 integrins. However, on vitronectin-adherent cells, α5ß1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvß3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5ß1 integrin activation and fibronectin fibril extension.

Recombinant fibronectin matrix mimetics specify integrin adhesion and extracellular matrix assembly.

Tissue engineering seeks to create functional tissues and organs by integrating natural or synthetic scaffolds with bioactive factors and cells. Creating biologically active scaffolds that support key aspects of tissue regeneration, including the re-establishment of a functional extracellular matrix (ECM), is a challenge currently facing this field. During tissue repair, fibronectin is converted from an inactive soluble form into biologically active ECM fibrils through a cell-dependent process. ECM fibronectin promotes cell processes critical to tissue regeneration and regulates the deposition and organization of other ECM proteins. We previously developed biomimetics of ECM fibronectin by directly coupling the heparin-binding fragment of the first type III repeat of fibronectin (FNIII1H) to the integrin-binding repeats (FNIII8-10). As adhesive substrates, fibronectin matrix mimetics promote cell growth, migration, and contractility through a FNIII1H-dependent mechanism. Here, we analyzed fibronectin matrix mimetic variants designed to include all or part of the integrin-binding domain for their ability to support new ECM assembly. We found that specific modifications of the integrin-binding domain produced adhesive substrates that selectively engage different integrin receptors to, in turn, regulate the amount of fibronectin and collagen deposited into the ECM. The ability of fibronectin matrix mimetics to direct cell-substrate interactions and regulate ECM assembly makes them promising candidates for use as bioactive surfaces, where precise control over integrin-binding specificity and ECM deposition are required.

Fibronectin matrix mimetics promote full-thickness wound repair in diabetic mice.

During tissue repair, fibronectin is converted from a soluble, inactive form into biologically active extracellular matrix (ECM) fibrils through a cell-dependent process. ECM fibronectin promotes numerous cell processes that are critical to tissue repair and regulates the assembly of other proteins into the matrix. Nonhealing wounds show reduced levels of ECM fibronectin. To functionally mimic ECM fibronectin, a series of fibronectin matrix mimetics was developed by directly coupling the matricryptic, heparin-binding fragment of the first type III repeat of fibronectin (FNIII1H) to various sequences from the integrin-binding domain (FNIII8-10). The recombinant proteins were produced as glutathione-S-transferase (GST)-tagged fusion proteins for ease of production and purification. Full-thickness, excisional wounds were produced in genetically diabetic mice, and fibronectin matrix mimetics were applied directly to the wounds. A significant enhancement of wound closure was observed by day 9 in response to GST/III1H,8-10 versus GST-treated controls (73.9%±4.1% vs. 58.1%±4.7% closure, respectively). Two weeks after injury, fibronectin matrix mimetic-treated wounds had developed a multi-layered epithelium that completely covered the wound space. Furthermore, significant increases in granulation tissue thickness were observed in response to treatment with GST/III1H,8-10 (4.05±0.93-fold), GST/III1H,8,10 (2.91±0.49-fold), or GST/III1H,8(RGD) (3.55±0.59-fold) compared with GST controls, and was accompanied by dense collagen deposition, the presence of myofibroblasts, and functional vasculature. Thus, the recombinant fibronectin matrix analogs normalized the impairment in repair observed in this chronic wound model and may provide a new approach to accelerate the healing of diabetic wounds.

Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate.

Therapeutic protein engineering combines genetic, biochemical, and functional information to improve existing proteins or invent new protein technologies. Using these principles, we developed an approach to deliver extracellular matrix (ECM) fibronectin-specific signals to cells. Fibronectin matrix assembly is a cell-dependent process that converts the inactive, soluble form of fibronectin into biologically-active ECM fibrils. ECM fibronectin stimulates cell functions required for normal tissue regeneration, including cell growth, spreading, migration, and collagen reorganization. We have developed recombinant fibronectin fragments that mimic the effects of ECM fibronectin on cell function by coupling the cryptic heparin-binding fragment of fibronectin's first type III repeat (FNIII1H) to the integrin-binding domain (FNIII8-10). GST/III1H,8-10 supports cell adhesion and spreading and stimulates cell proliferation to a greater extent than plasma fibronectin. Deletion and site-specific mutant constructs were generated to identify the active regions in GST/III1H,8-10 and reduce construct size. A chimeric construct in which the integrin-binding, RGDS loop was inserted into the analogous site in FNIII8 (GST/III1H,8(RGD)), supported cell adhesion and migration, and enhanced cell proliferation and collagen gel contraction. GST/III1H,8(RGD) was expressed in bacteria and purified from soluble lysate fractions by affinity chromatography. Fibronectin matrix assembly is normally up-regulated in response to tissue injury. Decreased levels of ECM fibronectin are associated with non-healing wounds. Engineering fibronectin matrix mimetics that bypass the need for cell-dependent fibronectin matrix assembly in chronic wounds is a novel approach to stimulating cellular activities critical for tissue repair.