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.

Experimental evidence for Parthanatos-like mode of cell death of heat-damaged human skin fibroblasts in a cell culture-based in vitro burn model.

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.

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.

Which Moisturizer to Use in Scar Therapy after Burn Injuries? A Subjective and Objective Skin and Scar Evaluation after Topical Treatment with Dexpanthenol, Aloe Vera, and Plant Oil.

Background and Objectives: Good scar management in burn care is essential. Nevertheless, there are no consistent recommendations regarding moisturizers for scar management. Our aim was to investigate and compare the effects of commonly used products on normal skin and burn scars. Materials and Methods: A total of 30 skin-healthy (control group) and 12 patients with burn scars were included in this study. For an intraindividual comparison, each participant received creams consisting of dexpanthenol (P), aloe vera (A), and a natural plant oil (O) with instructions to apply them daily to a previously defined area for at least 28 days. Objective scar evaluation was performed with Visioscan®; Tewameter®; Cutometer®, and the Oxygen To See® device. Subjective evaluation was performed with an "application" questionnaire, the Patient and Observer Scar Assessment Scale (POSAS), and with the "best of three" questionnaire. Results: After (A) a high trend of amelioration of +30%, TEWL was detected on the scar area. Blood flow increased slightly on healthy skin areas after (A) application to +104%. The application of (A) on healthy skin demonstrated a positive effect on the parameters of scaliness (+22%, p < 0.001), softness (+14%, p = 0.046), roughness R1 (+16%, p < 0.001) and R2 (+17%, p = 0.000), volume (+22%, p < 0.001), and surface area (+7%, p < 0.001) within the control group. After (P), a significant improvement of the baseline firmness parameter of +14.7% was detected (p = 0.007). (P) also showed a beneficial effect on the parameters of R1 (+7%, p = 0.003), R2 (+6%, p = 0.001), and volume (+17%, p = 0.001). (O) lead to a statistically significant improvement of volume (+15%, p = 0.009). Overall, most study participants stated (A) to be the "best of three". Conclusions: (A) performed statistically best, and is a well-tolerated moisturizing product. However, further quantitative studies are needed to provide statistically significant clarification for uniform recommendations for scar therapy.

Sublesional fat grafting leads to a temporary improvement of wound healing in chronic leg ulcers: A prospective, randomised clinical trial.

Chronic wounds remain a therapeutic and financial challenge for physicians and the health care systems. Innovative, inexpensive and effective treatment methods would be of immense value. The sublesional fat grafting could be such treatment, although effectiveness and safety have only been assessed in a few randomised clinical trials. The fat graft was obtained by liposuction, washed with the Coleman method and then injected sublesional and into the wound margins after surgical debridement. For the control group, saline solution was used instead of fat. The primary endpoint was to determine the wound size reduction in both groups. The wounds were measured preoperatively, intraoperatively and 3, 7, 21 and 60 days after the intervention. A p-value of <0.05 was considered significant. Furthermore, histology and microbiology of the wounds and pain were assessed. A temporary effect of the treatment was observed after 14 and 21 days. The wound size reduction was significantly larger in the intervention group, whereas after 60 days, no significant difference was detected between both groups. No adverse events could be reported and the pain level was almost equal in the control and intervention group. Sublesional fat grafting temporarily enhanced healing of chronic wounds. The procedure was safe and the pain level was low. Repeated interventions could lead to complete wound closure, which should be determined in future studies.

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.

Establishment of the German Burn Registry - five years of prospective data collection.

BACKGROUND: From 1991-2014, all major burn centers of the German-speaking countries participated in a multicenter study in which essentially demographic data were collected. Individual patient data was located at the particular burn centers and only cumulated data were summarized annually for presentation. Retrospective statistical analysis of the entire data collection and identification of subgroups was not possible. In 2015 the German Burn Registry was established for prospective collection of individual patient data as a tool for quality management (QM) and for scientific analyses. METHODS: A working group was formed to ensure the development and administration of the registry. From the official start of the German Burn Registry at the beginning of the year 2015 prospective data collection was realized with an individualized, web-based data collection software in a pseudonymized way. Selected data analysis was performed for the first 5 years of data collection. Severely burned adults and all hospitalized children with burn injuries were documented in the registry. RESULTS: The German Burn Registry was successfully established. 64 burn divisions have already been registered. 18,891 patients were documented over 5 years, of which 58% where children (<16 years). Mean ABSI Score was 4 (children: 3, adults: 6) with a mortality rate of 3.8.0% (children: 0.2%, adults: 9.3%). Children were hospitalized for an average of 7 days, whereas adults were discharged after 18 days. CONCLUSION: A registry is mandatory for quality assurance in burn medicine, since realization of randomized studies is difficult due to the heterogeneity of burn injuries. The German Burn Registry already is one of the biggest burn registries in Europe. Several scientific projects, based on the registry database, are in working process or have already been published.

Comparative Study of the Osteogenic Differentiation Potential of Adipose Tissue-Derived Stromal Cells and Dedifferentiated Adipose Cells of the Same Tissue Origin under Pro and Antioxidant Conditions.

Adipose tissue-derived stromal cells (ASCs) play an important role in various therapeutic approaches to bone regeneration. However, such applications become challenging when the obtained cells show a functional disorder, e.g., an impaired osteogenic differentiation potential (ODP). In addition to ASCs, human adipose tissue is also a source for another cell type with therapeutic potential, the dedifferentiated fat cells (DFATs), which can be obtained from mature adipocytes. Here, we for the first time compared the ODPs of each donors ASC and DFAT obtained from the same adipose tissue sample as well as the role of oxidative stress or antioxidative catalase on their osteogenic outcome. Osteogenic potential of ASC and DFAT from nine human donors were compared in vitro. Flow cytometry, staining for calcium accumulation with alizarin red, alkaline phosphatase assay and Western blots were used over an osteogenic induction period of up to 14 days. H2O2 was used to induce oxidative stress and catalase was used as an antioxidative measure. We have found that ASC and DFAT cultures' ODPs are nearly identical. If ASCs from an adipose tissue sample showed good or bad ODP, so did the corresponding DFAT cultures. The inter-individual variability of the donor ODPs was immense with a maximum factor of about 20 and correlated neither with the age nor the sex of the donors of the adipose tissue. Oxidative stress in the form of exogenously added H2O2 led to a significant ODP decrease in both cell types, with this ODP decrease being significantly lower in DFAT cultures than in the corresponding ASC cultures. Regardless of the individual cell culture-specific ODP, however, exogenously applied catalase led to an approx. 2.5-fold increase in osteogenesis in the ASC and DFAT cultures. Catalase appears to be a potent pro-osteogenic factor, at least in vitro. A new finding that points to innovative strategies and therapeutic approaches in bone regeneration. Furthermore, our results show that DFATs behave similarly to ASCs of the same adipose tissue sample with respect to ODPs and could therefore be a very attractive and readily available source of multipotent stem cells in bone regenerative therapies.

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.

Characterization of Blue Light Treatment for Infected Wounds: Antibacterial Efficacy of 420, 455, and 480 nm Light-Emitting Diode Arrays Against Common Skin Pathogens Versus Blue Light-Induced Skin Cell Toxicity.

Objective: To determine effective treatment strategies against bacterial infections of chronic wounds, we tested different blue light (BL)-emitting light-emitting diode arrays (420, 455, and 480 nm) against wound pathogens and investigated in parallel BL-induced toxic effects on human dermal fibroblasts. Background: Wound infection is a major factor for delayed healing. Infections with Pseudomonas aeruginosa and Staphylococcus aureus are clinically relevant caused by their ability of biofilm formation and their quickly growing antibiotics resistance. BL has demonstrated antimicrobial properties against various microbes. Methods: Determination of antibacterial and cell toxic effects by colony-forming units (CFUs)/biofilm/cell viability assays, and live cell imaging. Results: A single BL irradiation (180 J/cm2), of P. aeruginosa at both 420 and 455 nm resulted in a bacterial reduction (>5 log10 CFU), whereas 480 nm revealed subantimicrobial effects (2 log10). All tested wavelengths of BL also revealed bacteria reducing effects on Staphylococcus epidermidis and Escherichia coli (maximum 1-2 log10 CFU) but not on S. aureus. Dealing with biofilms, all wavelengths using 180 J/cm2 were able to reduce significantly the number of P. aeruginosa, E. coli, and S. epidermidis. Here, BL420nm achieved reductions up to 99%, whereas BL455nm and BL480nm were less effective (60-83%). Biofilm-growing S. aureus was more BL sensitive than in the planktonic phase showing a reduction by 63-75%. A significant number of cell toxic events (>40%) could be found after applying doses (>30 J/cm2) of BL420nm. BL455nm showed only slight cell toxicity (180 J/cm2), whereas BL480nm was nontoxic at any dose. Conclusions: BL treatment can be effective against bacterial infections of chronic wounds. Nevertheless, using longer wavelengths >455 nm should be preferred to avoid possible toxic effects on skin and skin cells. To establish BL therapy for infected chronic wounds, further studies concerning biofilm formation and tissue compatibility are necessary.

The impact of non-toxic blue light (453 nm) on cellular antioxidative capacity, TGF-ß1 signaling, and myofibrogenesis of human skin fibroblasts.

Studies have demonstrated that blue light induces biological effects, such as cell death, and inhibition of proliferation and differentiation. Since blue light at longer wavelength (>440 nm) exerts less injurious effects on cells than at shorter wavelengths, (400-440 nm), we have investigated the impact of non-toxic (LED) blue light at 453 nm wavelength on human skin fibroblasts (hsFBs). We found that besides its decreasing effects on the proliferation rate, repeated blue light irradiations (80 J/cm2) also significantly reduced TGF-ß1-induced myofibrogenesis as shown by diminished α-SMA and EDA-FN expression accompanied by reduced protein expression and phosphorylation of ERK 1/2, SMAD 2/3, and p38-key players of TGF-ß1-induced myofibrogenesis. In parallel, catalase protein expression, intracellular FAD concentrations as well as NADP+/NADPH ratio were reduced, whereas intracellular reactive oxygen species (ROS) were increased. We postulate that as a molecular mechanism downregulation of catalase and photoreduction of FAD induce intracellular oxidative stress which, in turn, affects the signaling factors of myofibrogenesis leading to a lower rate of α-SMA and EDA-FN expression and, therefore, myofibroblast formation. In conclusion, blue light even at longer wavelengths shows antifibrotic activity and may represent a suitable and safe approach in the treatment of fibrotic skin diseases including hypertrophic scarring and scleroderma.

The effect of the macrophage migration inhibitory factor (MIF) on excisional wound healing in vivo.

Background: The macrophage migration inhibitory factor (MIF) has been determined as a cytokine exerting a multitude of effects in inflammation and angiogenesis. Earlier studies have indicated that MIF may also be involved in wound healing and flap surgery. Methods: We investigated the effect of MIF in an excisional wound model in wildtype, Mif-/- and recombinant MIF treated mice. Wound closure rates as well as the macrophage marker Mac-3, the pro-inflammatory cytokine tumor necrosis factor α (TNFα) and the pro-angiogenic factor von Willebrand factor (vWF) were measured. Finally, we used a flap model in Mif-/- and WT mice with an established perfusion gradient to identify MIF's contribution in flap perfusion. Results: In the excision wound model, we found reduced wound healing after MIF injection, whereas Mif deletion improved wound healing. Furthermore, a reduced expression of Mac-3, TNFα and vWF in Mif-/- mice was seen when compared to WT mice. In the flap model, Mif-/- knockout mice showed mitigated flap perfusion with lower hemoglobin content and oxygen saturation as measured by O2C measurements when compared to WT mice. Conclusions: Our data suggest an inhibiting effect of MIF in wound healing with increased inflammation and perfusion. In flaps, by contrast, MIF may contribute to flap vascularization.

Vitamin D deficiency may stimulate fibroblasts in Dupuytren's disease via mitochondrial increased reactive oxygen species through upregulating transforming growth factor-ß1.

Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-ß1 (TGF-ß1) and its downstream Smad signaling system is well established as a keyplayer during fibrogenesis. Vitamin D has been extensively studied as an anti-fibrotic agent in malignant chronic diseases. A number of studies have shown that myofibroblasts are main target cells of 1,25(OH)2D3 inhibitory action. The myofibroblast in the palmar aponeurosis of patients in different stages of Dupuytren's disease was found by electron microscopy to contain a large number of mitochondria. Mitochondria play a critical role in cell metabolism being the major source of reactive oxygen species (ROS) in cells. TGF-ß1 has been shown to increase mitochondrial ROS production in different cell types, which mediate fibrosis related gene expression and myofibroblast differentiation. TGF-ß1 increases mitochondrial ROS production in patients with Dupuytren's contracture potentially in consequence of Vitamin D deficiency, leading to myofibroblast differentiation. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies.

Macrophage Migration Inhibitory Factor - A Favorable Marker in Inflammatory Diseases?

BACKGROUND: Macrophage migration inhibitory factor (MIF) was firstly described in the 1960s as a pleiotropic cytokine affecting a variety of immune cells. Different physiological functions mainly involving inflammatory reactions such as chemokine-like function and regulating systemic stress responses have been reported. OBJECTIVE: In several clinical studies the use of MIF as a biomarker has been investigated promising support for diseases with an inflammatory aspect such as sepsis, systemic infections and autoimmune diseases. This article in detail reviews clinical data and evaluates the function as biomarker focusing on inflammatory and autoimmune diseases. CONCLUSION: Recent studies suggest MIF to be a marker for different inflammatory diseases and might serve as therapeutic target in the future.

Growth Differentiation Factor 5 Accelerates Wound Closure and Improves Skin Quality During Repair of Full-Thickness Skin Defects.

BACKGROUND: A fast and stable wound closure is important, especially for extended and unstable wounds found after burn injuries. Growth can regulate a variety of cellular processes, including those involved in wound healing. Growth differentiation factor 5 (GDF-5) can accelerate fibroblast cell migration, cell proliferation, and collagen synthesis, which are essential for wound healing. Nevertheless, no standardized evaluation of the effect of GDF-5 on the healing of full-thickness wounds has been published to date. METHODS: Five full-thickness skin defects were created on the backs of 6 minipigs. Three wounds were treated with GDF-5 in different concentrations with the help of a gelatin-collagen carrier, and 2 wounds served as control group. The first was treated with the gelatin carrier and an Opsite film (Smith & Nephew, Fort Worth, Texas), and the other was treated solely with an Opsite film that was placed above all wounds and renewed every second day. RESULTS: Growth differentiation factor 5 accelerates wound closure (10.91 [SD, 0.99] days) compared with treatment with the carrier alone (11.3 [SD, 1.49] days) and control wounds (13.3 [SD, 0.94] days). Epidermal cell count of wounds treated with GDF-5 revealed a higher number of cells compared with the control group. In addition, mean epidermal thickness was significantly increased in GDF-5-treated wounds compared with the control wounds. CONCLUSIONS: Because of its ability to improve skin quality, GDF-5 should be considered when developing composite biomaterials for wound healing.

Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide.

The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal "cold" atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 µl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 µM), nitrate (~1 mM) and H2O2 (~200 µM). Fibroblast viability was reduced by single DBD treatments (60-300 s; ~77-66%) or exposure to freshly DBD-treated PBS (60-300 s; ~75-55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in order to avoid possible unwanted side effects such as a delayed healing process.

Fatal Necrotizing Fasciitis following Episiotomy.

Introduction. Necrotizing fasciitis is an uncommon condition in general practice but one that provokes serious morbidity. It is characterized by widespread fascial necrosis with relative sparing of skin and underlying muscle. Herein, we report a fatal case of necrotizing fasciitis in a young healthy woman after episiotomy. Case Report. A 17-year-old primigravida underwent a vaginal delivery with mediolateral episiotomy. Necrotizing fasciitis was diagnosed on the 5th postpartum day, when the patient was referred to our tertiary care medical center. Surgical debridement was initiated together with antibiotics and followed by hyperbaric oxygen therapy. The patient died due to septic shock after 16 hours from the referral. Conclusion. Delay of diagnosis and consequently the surgical debridement were most likely the reasons for maternal death. In puerperal period, a physician must consider necrotizing fasciitis as a possible diagnosis in any local sings of infection especially when accompanied by fever and/or tenderness. Early diagnosis is the key for low mortality and morbidity.

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.

Adipose-derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine.

Chronic wounds represent a major socio-economic problem in developed countries today. Wound healing is a complex biological process. It requires a well-orchestrated interaction of mediators, resident cells and infiltrating cells. In this context, mesenchymal stem cells and keratinocytes play a crucial role in tissue regeneration. In chronic wounds these processes are disturbed and cell viability is reduced. Hydroxyectoine (HyEc) is a membrane protecting osmolyte with protein and macromolecule stabilising properties. Adipose-derived stem cells (ASC) and keratinocytes were cultured with chronic wound fluid (CWF) and treated with HyEc. Proliferation was investigated using MTT test and migration was examined with transwell-migration assay and scratch assay. Gene expression changes of basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases-2 (MMP-2) and MMP-9 were analysed by quantitative real-time polymerase chain reaction (qRT-PCR). CWF significantly inhibited proliferation and migration of keratinocytes. Addition of HyEc did not affect these results. Proliferation capacity of ASC was not influenced by CWF whereas migration was significantly enhanced. HyEc significantly reduced ASC migration. Expression of b-FGF, VEGF, MMP-2 and MMP-9 in ASC, and b-FGF, VEGF and MMP-9 in keratinocytes was strongly induced by chronic wound fluid. HyEc enhanced CWF induced gene expression of VEGF in ASC and MMP-9 in keratinocytes. CWF negatively impaired keratinocyte function, which was not influenced by HyEc. ASC migration was stimulated by CWF, whereas HyEc significantly inhibited migration of ASC. CWF induced gene expression of VEGF in ASC and MMP-9 in keratinocytes was enhanced by HyEc, which might partly be explained by an RNA stabilising effect of HyEc.