Comparison of Long-Term Skin Quality and Scar Formation in Partial-Thickness Burn Wounds Treated with Suprathel® and epicitehydro® Wound Dressings.

Background and Objectives: Scar formation after burn trauma has a significant impact on the quality of life of burn patients. Hypertrophic scars or keloids can be very distressing to patients due to potential pain, functional limitations, or hyper- or hypopigmentation. In a previous study comparing Suprathel® and the new and cheaper dressing epicitehydro®, we were able to show that pain reduction, exudation, and time until wound-healing of partial-thickness burn wounds were similar, without any documented infections. No study exists that objectively measures and compares skin and scar quality after treatment with Suprathel® and epicitehydro® at present. Materials and Methods: In this study, the scar quality of 20 patients who had been treated with Suprathel® and epicitehydro® was objectively assessed using the Cutometer®, Mexameter®, and Tewameter®, as well as subjectively with the Patient and Observer Scar Assessment Scale, 3, 6, and 12 months after burn injury. Results: In all performed measurements, no significant differences were detected in scar formation after treatment of partial-thickness burn wounds with the two dressings. Conclusions: Both the newer and less expensive wound-dressing epicitehydro® and the well-known wound-dressing Suprathel® resulted in stable wound closure and showed good cosmetic results in the follow-up examinations.

The topical use of non-thermal dielectric barrier discharge (DBD): nitric oxide related effects on human skin.

Dielectric barrier discharge (DBD) devices generate air plasma above the skin containing active and reactive species including nitric oxide (NO). Since NO plays an essential role in skin physiology, a topical application of NO by plasma may be useful in the treatment of skin infections, impaired microcirculation and wound healing. Thus, after safety assessments of plasma treatment using human skin specimen and substitutes, NO-penetration through the epidermis, the loading of skin tissue with NO-derivates in vitro and the effects on human skin in vivo were determined. After the plasma treatment (0-60 min) of skin specimen or reconstructed epidermis no damaging effects were found (TUNEL/MTT). By Franz diffusion cell experiments plasma-induced NO penetration through epidermis and dermal enrichment with NO related species (nitrite 6-fold, nitrate 7-fold, nitrosothiols 30-fold) were observed. Furthermore, skin surface was acidified (~pH 2.7) by plasma treatment (90 s). Plasma application on the forearms of volunteers increased microcirculation fourfold in 1-2 mm and twofold in 6-8 mm depth in the treated skin areas. Regarding the NO-loading effects, skin acidification and increase in dermal microcirculation, plasma devices represent promising tools against chronic/infected wounds. However, efficacy of plasma treatment needs to be quantified in further studies and clinical trials.

Blue light inhibits transforming growth factor-ß1-induced myofibroblast differentiation of human dermal fibroblasts.

Transforming growth factor-ß1 (TGF-ß1) is the major promoter of phenotypic shift between fibroblasts and myofibroblasts accompanied by the expression and incorporation of α-smooth muscle actin (α-SMA). This differentiation is crucial during normal wound healing and wound closure; however, myofibroblasts are considered as the main effecter cell type in fibrosis, for example in scleroderma and hypertrophic scarring. As blue light has exerted antiprolific and toxic effects in several cell types, we investigated whether blue light irradiations with a light-emitting diode array (420 nm) were able to affect proliferation and differentiation of human dermal fibroblasts (HDF). We found that repeated irradiation with non-toxic doses significantly inhibits TGF-ß1-induced differentiation of HDF into myofibroblasts shown by α-SMA immunocytochemistry and Western blotting. Additionally, used doses reduced proliferation and myofibroblast contractibility measured by resazurin and collagen gel contraction assays. It could be demonstrated that blue light mediates cell toxicity by oxidative stress due to the generation of singlet oxygen. We postulate that irradiations at non-toxic doses induce low-level oxidative stress and energy-consuming cellular responses, which both may effect proliferation stop and interfere with myofibroblast differentiation. Thus, targeting differentiation, proliferation and activity of myofibroblasts by blue light may represent a useful strategy to prevent or reduce pathological fibrotic conditions.

Intraoperative use of enriched collagen and elastin matrices with freshly isolated adipose-derived stem/stromal cells: a potential clinical approach for soft tissue reconstruction.

BACKGROUND: Adipose tissue contains a large number of multipotent cells, which are essential for stem cell-based therapies. The combination of this therapy with suitable commercial clinically used matrices, such as collagen and elastin matrices (i.e. dermal matrices), is a promising approach for soft tissue reconstruction. We previously demonstrated that the liposuction method affects the adherence behaviour of freshly isolated adipose-derived stem/stromal cells (ASCs) on collagen and elastin matrices. However, it remains unclear whether freshly isolated and uncultured ASCs could be directly transferred to matrices during a single transplantation operation without additional cell culture steps. METHODS: After each fat harvesting procedure, ASCs were isolated and directly seeded onto collagen and elastin matrices. Different time intervals (i.e. 1, 3 and 24 h) were investigated to determine the time interval needed for cellular attachment to the collagen and elastin matrices. Resazurin-based vitality assays were performed after seeding the cells onto the collagen and elastin matrices. In addition, the adhesion and migration of ASCs on the collagen and elastin matrices were visualised using histology and two-photon microscopy. RESULTS: A time-dependent increase in the number of viable ASCs attached to the collagen and elastin matrices was observed. This finding was supported by mitochondrial activity and histology results. Importantly, the ASCs attached and adhered to the collagen and elastin matrices after only 1 h of ex vivo enrichment. This finding was also supported by two-photon microscopy, which revealed the presence and attachment of viable cells on the upper layer of the construct. CONCLUSION: Freshly isolated uncultured ASCs can be safely seeded onto collagen and elastin matrices for ex vivo cellular enrichment of these constructs after liposuction. Although we observed a significant number of seeded cells on the matrices after a 3-h enrichment time, we also observed an adequate number of isolated cells after a 1-h enrichment time. However, this approach must be optimised for clinical use. Thus, in vivo studies and clinical trials are needed to investigate the feasibility of this approach.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for the septic mortality of burn patients. Objective: High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections and will be investigated in this study. Methods: Antibacterial effects of a light-emitting diode array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540 J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: aBL reduced the bacterial number [2.51-3.56 log10 colony-forming unit (CFU)/mL], whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. aBL reduced biofilm formation by 60-66%. PRT or CA treatment showed reductions by 25% or 13%. Here, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extend than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for septic mortality of burn patients. High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections. Methods: Antibacterial effects of an light-emitting diode (LED) array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: The aBL reduced the bacterial number (2.51-3.56 log10 CFU/mL), whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. The aBL reduced biofilm formation by 60%-66%. PRT or CA treatment showed reductions by 25% or 13%. In this study, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extent than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: The aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

The Antimicrobial Efficacy of Topically Applied Mafenide Acetate, Citric Acid and Wound Irrigation Solutions Lavanox and Prontosan against Pseudomonas aeruginosa.

Since burn wound infections caused by Pseudomonas aeruginosa (PA) lead to major complications and sepsis, this study evaluates the antimicrobial efficacy of the wound irrigation solutions Prontosan (PRT), Lavanox (LAV), citric acid (CA) and mafenide acetate (MA) using microbiology assays and an ex vivo skin wound model. In suspension assays, all the solutions showed significant reductions in bacterial number (log10 reduction: CA 5.77; LAV 4.91; PRT 4.74; MA 1.23). The biofilm assay revealed that PRT and LAV reduced biofilm formation by ~25% after a 15 min treatment, while PRT was most effective after a 24 h treatment (~68%). The number of PA in biofilms measured directly after a 15 min treatment was reduced most effectively with CA and LAV (log10 reductions ~2.5), whereas after a 24 h treatment, all solutions achieved only 1.36-1.65 log10 reductions. In the skin wound model, PRT and LAV provided the highest bacterial reduction after a 15 min treatment (log10 reduction 1.8-1.9), while MA was more effective after a 22 h treatment (log10 reduction 3.6). The results demonstrated the antimicrobial efficacy of all solutions against PA. Further investigation is needed to explore the potential clinical applications of a combination or alternating use of these solutions for infection prophylaxis and treatment of wound infections caused by PA.

Characterization of novel nitrite-based nitric oxide generating delivery systems for topical dermal application.

Topical application of nitric oxide (NO) has been shown to exert beneficial effects in the therapy of chronic wounds, impaired microcirculation, and skin infections. Nitrite acidified by ascorbic acid has been widely used in many studies as NO-donor system, unfortunately with inflammatory and toxic effects on the treated skin due to unregulated excessive NO generation, low pH and possible toxic side products. Here we describe an essentially modified nitrite based NO generating system that avoid the mentioned unwanted side effects on human skin by using a pH-stable acetate/acetic acid buffer with a skin neutral pH of 5.5 and sodium ascorbate. In order to overcome the shortcoming of lower NO yields due to the higher pH-value and low nitrite concentrations, we have determined additionally the influence of copper ions. To investigate the influence of different NO release and penetration kinetics on NO-induced toxicity, we have developed a fibroblast assay using cell culture plates with gas permeable bottoms. The results show clearly that the donor system can achieve a sustained NO generation without generating high peaks. Furthermore, the presence of Cu(2+) ions enhances manifold NO generation of pH/ascorbate-induced nitrite decomposition, a mechanism comprising the reduction of Cu(2+) ions to Cu(1+) by ascorbate. Finally, we have found that apart from the NO dose the NO release kinetics had a significant influence of cell toxicity. Thus, application of comparable NO amounts within a time interval of 600s led to the development of variable cell toxicities, which predominantly depended on the NO concentration values generated in the first 200s. In summary, we here describe a novel nitrite-based NO-donor system that can provide well defined NO concentrations at skin neutral pH-values for side effect poor topical dermal application, i.e. in the therapy of chronic wounds and impaired microcirculation.

Redox-mediated mechanisms and biological responses of copper-catalyzed reduction of the nitrite ion in vitro.

During ischemia nitrite may be converted into nitric oxide (NO) by reaction with heme-carrying proteins or thiol-containing enzymes. NO acts as a regulator of vasodilation and protector against oxidative stress-induced tissue injuries. As a result of ischemia-induced oxidative stress, hypoxia and/or acidosis bivalent copper ions (Cu(2+)) can dissociate from their physiological carrier proteins. Reduced by the body's own antioxidants, the resultant Cu(1+) might represent an effective reductant of nitrite. Here we have evaluated in vitro copper-dissociation from copper/BSA (bovine serum albumin) complexes under ischemic conditions. Furthermore, using physiological concentrations, we have characterized the capacity of antioxidants and bivalent copper ions to serve as Cu(1+)-agitated catalytic sites for nitrite reduction and also the biological responses of this mechanism in vitro. We found that as a consequence of an acidic milieu and/or oxidative stress the copper-binding capacity of serum albumin strongly declined, leading to significant dissociation of copper ions into the ambient solution. At physiologically relevant pH-values Cu(2+) ions in combination with physiologically available copper reductants (i.e., ascorbate, glutathione, Fe(2+)) significantly enhanced nitrite reduction and subsequent non-enzymatic NO generation under hypoxic but also normoxic conditions. Our data demonstrate for the first time that upon ischemic conditions carrier protein-dissociated copper ions combined with appropriate reductants may serve as Cu(1+)-driven catalytic sites for nitrite reduction, leading to the formation of biologically relevant NO formation. Thus, in addition to the action of heme proteins, copper-catalyzed non-enzymatic NO formation from nitrite might represent a further physiologically relevant vasodilating and NO-dependent protective principle to ischemic stress.

Enrichment of Bone Tissue with Antibacterially Effective Amounts of Nitric Oxide Derivatives by Treatment with Dielectric Barrier Discharge Plasmas Optimized for Nitrogen Oxide Chemistry.

Cold atmospheric plasmas (CAPs) generated by dielectric barrier discharge (DBD), particularly those containing higher amounts of nitric oxide (NO) or NO derivates (NOD), are attracting increasing interest in medical fields. In the present study, we, for the first time, evaluated DBD-CAP-induced NOD accumulation and therapeutically relevant NO release in calcified bone tissue. This knowledge is of great importance for the development of new therapies against bacterial-infectious complications during bone healing, such as osteitis or osteomyelitis. We found that by modulating the power dissipation in the discharge, it is possible (1) to significantly increase the uptake of NODs in bone tissue, even into deeper regions, (2) to significantly decrease the pH in CAP-exposed bone tissue, (3) to induce a long-lasting and modulable NO production in the bone samples as well as (4) to significantly protect the treated bone tissue against bacterial contaminations, and to induce a strong bactericidal effect in bacterially infected bone samples. Our results strongly suggest that the current DBD technology opens up effective NO-based therapy options in the treatment of local bacterial infections of the bone tissue through the possibility of a targeted modulation of the NOD content in the generated CAPs.

The LipoDerm Method for Regeneration and Reconstruction in Plastic Surgery: A Technical Experimental Ex Vivo Note.

The combination of adipose-derived stem cells (ASCs) and dermal scaffolds has been shown to be an approach with high potential in soft tissue reconstruction. The addition of dermal templates to skin grafts can increase graft survival through angiogenesis, improve regeneration and healing time, and enhance the overall appearance. However, it remains unknown whether the addition of nanofat-containing ASCs to this construct could effectively facilitate the creation of a multi-layer biological regenerative graft, which could possibly be used for soft tissue reconstruction in the future in a single operation. Initially, microfat was harvested using Coleman's technique, then isolated through the strict protocol using Tonnard's technique. Finally, centrifugation, emulsification, and filtration were conducted to seed the filtered nanofat-containing ASCs onto Matriderm for sterile ex vivo cellular enrichment. After seeding, a resazurin-based reagent was added, and the construct was visualized using two-photon microscopy. Within 1 h of incubation, viable ASCs were detected and attached to the top layer of the scaffold. This experimental ex vivo note opens more dimensions and horizons towards the combination of ASCs and collagen-elastin matrices (i.e., dermal scaffolds) as an effective approach in soft tissue regeneration. The proposed multi-layered structure containing nanofat and dermal template (Lipoderm) may be used, in the future, as a biological regenerative graft for wound defect reconstruction and regeneration in a single operation and can also be combined with skin grafts. Such protocols may optimize the skin graft results by creating a multi-layer soft tissue reconstruction template, leading to more optimal regeneration and aesthetic outcomes.

Can Cold Atmospheric Plasma Be Used for Infection Control in Burns? A Preclinical Evaluation.

Wound infection with Pseudomonas aeruginosa (PA) is a serious complication and is responsible for higher rates of mortality in burn patients. Because of the resistance of PA to many antibiotics and antiseptics, an effective treatment is difficult. As a possible alternative, cold atmospheric plasma (CAP) can be considered for treatment, as antibacterial effects are known from some types of CAP. Hence, we preclinically tested the CAP device PlasmaOne and found that CAP was effective against PA in various test systems. CAP induced an accumulation of nitrite, nitrate, and hydrogen peroxide, combined with a decrease in pH in agar and solutions, which could be responsible for the antibacterial effects. In an ex vivo contamination wound model using human skin, a reduction in microbial load of about 1 log10 level was observed after 5 min of CAP treatment as well as an inhibition of biofilm formation. However, the efficacy of CAP was significantly lower when compared with commonly used antibacterial wound irrigation solutions. Nevertheless, a clinical use of CAP in the treatment of burn wounds is conceivable on account of the potential resistance of PA to common wound irrigation solutions and the possible wound healing-promoting effects of CAP.

Low-Dose Blue Light (420 nm) Reduces Metabolic Activity and Inhibits Proliferation of Human Dermal Fibroblasts.

Hypertrophic scarring in burn wounds is caused by overactive fibroblasts and myofibroblasts. Blue light reveals wavelength- and dose-dependent antibacterial and antiproliferative effects and may serve as a therapeutic option against wound infection and fibrotic conditions. Therefore, we evaluated in this study the effects of single and multiple irradiations with blue light at 420 nm (BL420) on the intracellular ATP concentration, and on the viability and proliferation of the human skin fibroblast (HDFs). In addition, possible BL420-induced effects on the catalase expression and differentiation were assessed by immunocytochemical staining and western blot analyses. Furthermore, we used RNA-seq analyses to identify BL420-affected genes. We found that BL420 induced toxicity in HDFs (up to 83%; 180 J/cm2). A low dose of 20 J/cm2 reduced the ATP concentration by ~50%. Multiple irradiations (4 × 20 J/cm2) inhibited proliferation without visible toxicity and reduced catalase protein expression by ~37% without affecting differentiation. The expression of about 300 genes was significantly altered. Many downregulated genes have functions in cell division/mitosis. BL420 can strongly influence the fibroblast physiology and has potential in wound therapy. However, it is important to consider the possible toxic and antiproliferative effects, which could potentially lead to impaired wound healing and reduced scar breaking strength.

Gas Flow-Dependent Modification of Plasma Chemistry in µAPP Jet-Generated Cold Atmospheric Plasma and Its Impact on Human Skin Fibroblasts.

The micro-scaled Atmospheric Pressure Plasma Jet (µAPPJ) is operated with low carrier gas flows (0.25-1.4 slm), preventing excessive dehydration and osmotic effects in the exposed area. A higher yield of reactive oxygen or nitrogen species (ROS or RNS) in the µAAPJ-generated plasmas (CAP) was achieved, due to atmospheric impurities in the working gas. With CAPs generated at different gas flows, we characterized their impact on physical/chemical changes of buffers and on biological parameters of human skin fibroblasts (hsFB). CAP treatments of buffer at 0.25 slm led to increased concentrations of nitrate (~352 µM), hydrogen peroxide (H2O2; ~124 µM) and nitrite (~161 µM). With 1.40 slm, significantly lower concentrations of nitrate (~10 µM) and nitrite (~44 µM) but a strongly increased H2O2 concentration (~1265 µM) was achieved. CAP-induced toxicity of hsFB cultures correlated with the accumulated H2O2 concentrations (20% at 0.25 slm vs. ~49% at 1.40 slm). Adverse biological consequences of CAP exposure could be reversed by exogenously applied catalase. Due to the possibility of being able to influence the plasma chemistry solely by modulating the gas flow, the therapeutic use of the µAPPJ represents an interesting option for clinical use.

Comparative Clinical Study of Suprathel® and Jelonet® Wound Dressings in Burn Wound Healing after Enzymatic Debridement.

Following the enzymatic debridement of deep dermal burns, the choice of wound dressing is crucial for providing an adequate environment and suitable conditions for rapid wound healing. As Suprathel® and fatty gauze (Jelonet®) are the most commonly used dressings in burn centers, the aim of this study is to compare Suprathel® and Jelonet® in the treatment of deep dermal burns after enzymatic debridement with respect to wound healing, patient comfort, and pain. A total of 23 patients with deep dermal burns of the hand or foot (mean total body surface area of 4.31%) were included in this prospective, unicentric, open, comparative, and intra-individual clinical study. After enzymatic debridement, wounds were divided into two areas: one was treated with Suprathel® and the other with Jelonet®. Suprathel® remained on the wounds without dressing changes while Jelonet® was regularly changed. Wound healing, infection, bleeding, exudation, time for dressing changes, and pain were documented (from days 2 to 48) after injury. Satisfactory results were obtained in 22 cases; only one patient had to undergo a second debridement followed by skin grafting. No significant difference in time to final wound healing could be observed (18-19 d). Patients reported significantly less pain during the dressing changes for Suprathel® compared to Jelonet®. Furthermore, the wound areas treated with Suprathel® showed significantly less exudation and bleeding. Wound infections rarely occurred in both groups. In conclusion, the authors found that both wound dressings could be used to achieve safe and rapid wound healing after the enzymatic debridement of deep dermal burns of the hands and feet. However, compared to Jelonet®, Suprathel® showed superior results in terms of patient comfort and pain reduction.

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans.

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.

The role of photolabile dermal nitric oxide derivates in ultraviolet radiation (UVR)-induced cell death.

Human skin is exposed to solar ultraviolet radiation comprising UVB (280-315 nm) and UVA (315-400 nm) on a daily basis. Within the last two decades, the molecular and cellular response to UVA/UVB and the possible effects on human health have been investigated extensively. It is generally accepted that the mutagenic and carcinogenic properties of UVB is due to the direct interaction with DNA. On the other hand, by interaction with non-DNA chromophores as endogenous photosensitizers, UVA induces formation of reactive oxygen species (ROS), which play a pivotal role as mediators of UVA-induced injuries in human skin. This review gives a short overview about relevant findings concerning the molecular mechanisms underlying UVA/UVB-induced cell death. Furthermore, we will highlight the potential role of cutaneous antioxidants and photolabile nitric oxide derivates (NODs) in skin physiology. UVA-induced decomposition of the NODs, like nitrite, leads not only to non-enzymatic formation of nitric oxide (NO), but also to toxic reactive nitrogen species (RNS), like peroxynitrite. Whereas under antioxidative conditions the generation of protective amounts of NO is favored, under oxidative conditions, less injurious reactive nitrogen species are generated, which may enhance UVA-induced cell death.

Enhancement of Nitric Oxide Bioavailability by Modulation of Cutaneous Nitric Oxide Stores.

The generation of nitric oxide (NO) in the skin plays a critical role in wound healing and the response to several stimuli, such as UV exposure, heat, infection, and inflammation. Furthermore, in the human body, NO is involved in vascular homeostasis and the regulation of blood pressure. Physiologically, a family of enzymes termed nitric oxide synthases (NOS) generates NO. In addition, there are many methods of non-enzymatic/NOS-independent NO generation, e.g., the reduction of NO derivates (NODs) such as nitrite, nitrate, and nitrosylated proteins under certain conditions. The skin is the largest and heaviest human organ and contains a comparatively high concentration of these NODs; therefore, it represents a promising target for many therapeutic strategies for NO-dependent pathological conditions. In this review, we give an overview of how the cutaneous NOD stores can be targeted and modulated, leading to a further accumulation of NO-related compounds and/or the local and systemic release of bioactive NO, and eventually, NO-related physiological effects with a potential therapeutical use for diseases such as hypertension, disturbed microcirculation, impaired wound healing, and skin infections.

Phototherapy of Pseudomonas aeruginosa-Infected Wounds: Preclinical Evaluation of Antimicrobial Blue Light (450-460 nm) Using In Vitro Assays and a Human Wound Skin Model.

Objective: To determine effective treatment strategies against bacterial infections of burn wounds with Pseudomonas aeruginosa, we tested different treatment regimens with antibacterial blue light (BL). Background: Infections of burn wounds are serious complications and require effective and pathogen-specific therapy. Hereby, infections caused by P. aeruginosa pose a particular challenge in clinical practice due to its resistance to many antibiotics and topical antiseptics. Methods: LED-based light sources (450-460 nm) with different intensities and treatment times were used. Antibacterial effects against P. aeruginosa were determined by colony-forming unit (CFU) assays, human skin wound models, and fluorescence imaging. Results: In suspension assays, BL (2 h, 40 mW/cm2, 288 J/cm2) reduced bacterial number (>5 log10 CFU/mL). Applying 144 J/cm2, using 40 mW/cm2 for 1 h was more effective (>4 log10 CFU) than using 20 mW/cm2 for 2 h (>1.5 log10 CFU). BL with low irradiance (24 h, 3.5 mW/cm2, 300 J/cm2) only revealed bacterial reduction in thin bacteria-containing medium layers. In infected in vitro skin wounds only BL irradiation (2 h, 40 mW/cm2, 288 J/cm2) exerted a significant antimicrobial efficacy (2.94 log10 CFU/mL). Conclusions: BL treatment may be an effective therapy for P. aeruginosa-infected wounds to avoid radical surgical debridement. However, a significant antibacterial efficacy can only be achieved with higher irradiances and longer treatment times (min. 40 mW/cm2; >1 h), which cannot be easily integrated into regular clinical treatment protocols, for example, during a dressing change. Further studies are necessary to establish BL therapy for infected burns among tissue compatibility and interactions with previous therapeutic agents.

Whole body UVA irradiation lowers systemic blood pressure by release of nitric oxide from intracutaneous photolabile nitric oxide derivates.

RATIONALE: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. OBJECTIVE: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. METHODS AND RESULTS: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm(2)) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled (15)N-nitrite, whole body UVA irradiation (20 J/cm(2)) induced significant levels of (15)N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11+/-2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R(2)=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26+/-7%), with increased flow mediated vasodilation of the brachial artery (+68+/-22%), and with decreased forearm vascular resistance (-28+/-7%). CONCLUSIONS: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.