Exposure of Bladder Cancer Cells to Blue Light (λ = 453 nm) in the Presence of Riboflavin Synergistically Enhances the Cytotoxic Efficiency of Gemcitabine.

Non-muscle invasive bladder cancer is a common tumour in men and women. In case of resistance to the standard therapeutic agents, gemcitabine can be used as off-label instillation therapy into the bladder. To reduce potential side effects, continuous efforts are made to optimise the therapeutic potential of drugs, thereby reducing the effective dose and consequently the pharmacological burden of the medication. We recently demonstrated that it is possible to significantly increase the therapeutic efficacy of mitomycin C against a bladder carcinoma cell line by exposure to non-toxic doses of blue light (453 nm). In the present study, we investigated whether the therapeutically supportive effect of blue light can be further enhanced by the additional use of the wavelength-specific photosensitiser riboflavin. We found that the gemcitabine-induced cytotoxicity of bladder cancer cell lines (BFTC-905, SW-1710, RT-112) was significantly enhanced by non-toxic doses of blue light in the presence of riboflavin. Enhanced cytotoxicity correlated with decreased levels of mitochondrial ATP synthesis and increased lipid peroxidation was most likely the result of increased oxidative stress. Due to these properties, blue light in combination with riboflavin could represent an effective therapy option with few side effects and increase the success of local treatment of bladder cancer, whereby the dose of the chemotherapeutic agent used and thus the chemical load could be significantly reduced with similar or improved therapeutic success.

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.

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.

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.

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.

Molybdenum as a Potential Biocompatible and Resorbable Material for Osteosynthesis in Craniomaxillofacial Surgery-An In Vitro Study.

Titanium and stainless steel are commonly known as osteosynthesis materials with high strength and good biocompatibility. However, they have the big disadvantage that a second operation for hardware removal is necessary. Although resorbable systems made of polymers or magnesium are increasingly used, they show some severe adverse foreign body reactions or unsatisfying degradation behavior. Therefore, we started to investigate molybdenum as a potential new biodegradable material for osteosynthesis in craniomaxillofacial surgery. To characterize molybdenum as a biocompatible material, we performed in vitro assays in accordance with ISO Norm 10993-5. In four different experimental setups, we showed that pure molybdenum and molybdenum rhenium alloys do not lead to cytotoxicity in human and mouse fibroblasts. We also examined the degradation behavior of molybdenum by carrying out long-term immersion tests (up to 6 months) with molybdenum sheet metal. We showed that molybdenum has sufficient mechanical stability over at least 6 months for implants on the one hand and is subject to very uniform degradation on the other. The results of our experiments are very promising for the development of new resorbable osteosynthesis materials for craniomaxillofacial surgery based on molybdenum.

Enhancement of human bladder carcinoma cell chemosensitivity to Mitomycin C through quasi-monochromatic blue light (λ = 453 ± 10 nm).

Human urothelial bladder carcinoma (uBC) is the second most tumor entity of the urogenital tract. As far as possible, therapy for non-muscle invasive uBC takes place as resection of the tumor tissue, followed by intravesical chemotherapy or immunotherapy. Because of the high recurrence rate of uBC, there is a need for improved efficiency in the treatment. In the present in vitro study we have evaluated a new approach to enhance the cytotoxic efficiency of Mitomycin C (MMC), which is commonly used for intravesical treatment of uBC on the relevant urothelial cancer cell line RT112. For that we used quasi-monochromatic blue light (453 ± 10 nm) at its non-toxic dose of 110 J/cm2 as an additive stimulus to enhance the therapeutic efficiency of MMC (10 µg/ml). We found, that blue light exposure of RT112 cells led to a very strong increase in intracellular production of reactive oxygen species (ROS) and to a significant reduction (p < 0.05) of all function parameters of mitochondrial respiration, including basal activity and ATP production. Although not being toxic when used as a single impact, together with MMC blue light strongly enhanced the therapeutic efficiency of MMC in the form of significantly enhanced cytotoxicity via apoptosis and secondary necrosis. Our results clearly show that blue light, most likely due to its ability to increase intracellular ROS production and reduce mitochondrial respiration, increased the cytotoxic efficiency of MMC and therefore might represent an effective, low-side-effect, and success-enhancing therapy option in the local treatment of bladder cancer.

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.

Superoxide dismutase and catalase significantly improve the osteogenic differentiation potential of osteogenetically compromised human adipose tissue-derived stromal cells in vitro.

Mesenchymal stromal cells (MSCs) play a pivotal role in modern therapeutic approaches of bone defect regeneration. A complication that is steadily increasing due to demographic population developments is an age-related disorder of the osteogenic differentiation potential (ODP) of MSCs. In the present in vitro study we have evaluated the impact of reactive oxygen species (ROS) and antioxidants on ODP of "normal healthy" as well as osteogenetically compromised human adipose tissue-derived stromal cells (ASC). We show that although ASC exhibit a marked ODP, a significant number of ASC cultures showed a retarded osteogenesis. These so-called "non-responder"-ASC cultures (NR-ASC) expressed an antigenic phenotype identical to that of the well osteogenically differentiating "responder"-ASCs (R-ASC), but NR-ASC significantly correlated with the older age of their donors as well as with a significantly higher intracellular generation of ROS. Furthermore, we found that treatment of the cell cultures with low concentration of H2O2 seemed to promote osteogenic differentiation but higher H2O2 concentration strongly reduced osteogenesis in all ASC cultures. Additionally, we found that exogenously applied superoxide dismutase (SOD) or catalase completely restored the impaired ODP of NR-ASC, whereas ascorbate, glutathione, N-acetylcysteine or a water soluble vitamin E derivate did not show any relevant effects. Our results suggest that aging-related increases in oxidative stress, especially due to H2O2, can severely impair the ODP of ASC whereas SOD or catalase almost completely corrected this disorder. Our findings point to novel osteo-therapeutic aspects of catalase and SOD and open up new approaches to ASC-based regeneration of bone defects.

Evaluation of pro-angiogenic properties of an inorganic silica gel fibre fleece.

Hard-to-heal wounds represent an increasing health and economic burden on society. At present, therapy options for hard-to-heal wounds are often unsatisfactory, and the development of more effective wound treatments is urgently needed. We have shown that orthosilicic acid-releasing silica fibre fleece (SIFIB), via its pronounced anti-inflammatory properties, exhibited a significantly enhanced effect on wound closure kinetics in a porcine wound model in vivo. In this present study, we have examined in vitro the impact of the pro-angiogenic potential of SIFIB. Using an in vitro angiogenesis assay we describe for the first time how an inorganic biodegradable silica-based material significantly improved endothelial microvessel-like structure formation. We further demonstrate that the molecular mechanism of this pro-angiogenic activity of SIFIB is based on a significantly increased and tumour necrosis factor (TNF)α-dependent VEGF protein expression. In conclusion, due to its positive effects on angiogenesis, our results further indicate that decomposition products of silica-based biodegradable inorganic materials might represent very relevant therapeutic components of modern wound dressings for the treatment of hard-to-heal wounds.

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.

Impact of growth factor content on proliferation of mesenchymal stromal cells derived from adipose tissue.

Autologous adipose tissue (AT) transfer has gained widespread acceptance and is used for a broad variety of regenerative clinical indications. It is assumed that the successful outcome of AT transfer essentially depends on the amount of autocrine-generated growth factors (GF). It is supposed that several GF enhance and improve the anatomic and functional integration of the transplanted AT grafts at the site of implantation. In the present study we have investigated for the first time the correlation between the concentration of GF of freshly isolated AT and the proliferation and migration capacity of mesenchymal stroma cells (MSCs) derived from the respective AT sample. We here show that the proliferation and migration capacity of MSCs strongly depends on the GF content of the AT the cells were isolated from but in an inversely proportional manner. The lower the GF content of an AT sample was, the higher was the proliferation and migration capacity of the respective MSC population contained in the AT and vice versa. Furthermore, we found that supplementation with recombinant GFs only in the case of AT samples with low but not with higher growth factor contents led to a significant enhancement of proliferation and migration of the AT-resident MSCs. As we further show, this inefficiency of GFs to enhance MSC proliferation and migration in AT samples with high GF contents indicates a GF-mediated negative feedback mechanism leading to an impaired GF signaling in MSC obtained from those AT samples. Our results might explain why the successful use of AT grafting is frequently limited by low and unpredictable survival rates, and we suggest to use the knowledge of GF content of harvested AT as a predictive clinical parameter for risk assessment of the therapeutic outcome of autologous AT transfer.

Blue light (λ=453 nm) nitric oxide dependently induces ß-endorphin production of human skin keratinocytes in-vitro and increases systemic ß-endorphin levels in humans in-vivo.

ß-Endorphin exerts a broad spectrum of physiological activity on mood, immune functions, pain management, reward effects, and behavioral stability. ß-Endorphin is produced in certain neurons within the central and peripheral nervous system but also in the skin, especially in response to ultraviolet radiation. In the present study we have investigated the impact of visible blue light at λ = 453 nm (BL) on ß-endorphin production of primary human skin keratinocytes (hKC) in-vitro as well as on systemic ß-endorphin formation of whole-body exposed subjects in-vivo. We found that BL irradiation significantly enhanced both keratinocytic ß-endorphin production of hKC cultures as well as systemic ß-endorphin concentrations in light exposed healthy subjects. Interestingly, in hKC cultures elevated ß-endorphin formation was paralleled by significantly increased levels of non-enzymatically generated nitric oxide (NO), whereas elevated systemic ß-endorphin values of BL-exposed subjects were accompanied by enhanced systemic concentration of bioactive NO-derivates. These findings point to a pivotal role of NO in the molecular mechanism of the observed BL-induced effects, and indeed, exogenously applied NO was able to significantly enhance ß-endorphin production in hKC cultures. Thus, our finding of BL-induced increases in systemic ß-endorphin concentration in-vivo can be plausibly explained by an event sequence comprising 1.) BL-driven non-enzymatic formation of NO in the exposed skin tissue, 2.) systemic distribution of cutaneously produced NO in the form of bioactive nitroso compounds, 3.) a subsequent NO-dependent induction of ß-endorphin synthesis in epidermal keratinocytes, and 4.) probably also a NO-dependent modulation of ß-endorphin synthesis in specialized neurons within the central and peripheral nervous system.

Blue light exposure decreases systolic blood pressure, arterial stiffness, and improves endothelial function in humans.

AIMS: Previous studies have shown that ultraviolet light can lead to the release of nitric oxide from the skin and decrease blood pressure. In contrast to visible light the local application of ultraviolet light bears a cancerogenic risk. Here, we investigated whether whole body exposure to visible blue light can also decrease blood pressure and increase endothelial function in healthy subjects. METHODS: In a randomised crossover study, 14 healthy male subjects were exposed on 2 days to monochromatic blue light or blue light with a filter foil (control light) over 30 minutes. We measured blood pressure (primary endpoint), heart rate, forearm vascular resistance, forearm blood flow, endothelial function (flow-mediated dilation), pulse wave velocity and plasma nitric oxide species, nitrite and nitroso compounds (secondary endpoints) during and up to 2 hours after exposure. RESULTS: Blue light exposure significantly decreased systolic blood pressure and increased heart rate as compared to control. In parallel, blue light significantly increased forearm blood flow, flow-mediated dilation, circulating nitric oxide species and nitroso compounds while it decreased forearm vascular resistance and pulse wave velocity. CONCLUSION: Whole body irradiation with visible blue light at real world doses improves blood pressure, endothelial function and arterial stiffness by nitric oxide released from photolabile intracutanous nitric oxide metabolites into circulating blood.

Molecular- and microarray-based analysis of diversity among resting and osteogenically induced porcine mesenchymal stromal cells of several tissue origin.

Mesenchymal stromal cells (MSCs) play a pivotal role in modern therapeutic approaches in bone-healing disorders. Although bone marrow-derived MSCs are most frequently used, the knowledge that many other adult tissues represent promising sources for potent MSCs has gained acceptance. In the present study, the osteogenic differentiation potential of porcine skin fibroblasts (FBs), as well as bone marrow- (BMSCs), adipose tissue- (ASCs) and dental pulp-derived stromal cells (DSCs) were evaluated. However, additional application of BMP-2 significantly elevated the delayed osteogenic differentiation capacity of ASC and FB cultures, and in DSC cultures the supplementation of platelet-rich plasma increased osteogenic differentiation potential to a comparable level of the good differentiable BMSCs. Furthermore, microarray gene expression performed in an exemplary manner for ASCs and BMSCs revealed that ASCs and BMSCs use different gene expression patterns for osteogenic differentiation under standard media conditions, as diverse MSCs are imprinted dependent from their tissue niche. However, after increasing the differentiation potential of ASCs to a comparable level as shown in BMSCs, a small subset of identical key molecules was used to differentiate in the osteogenic lineage. Until now, the importance of identified genes seems to be underestimated for osteogenic differentiation. Apparently, the regulation of transmembrane protein 229A, interleukin-33 and the fibroblast growth factor receptor-2 in the early phase of osteogenic differentiation is needed for optimum results. Based on these results, bone regeneration strategies of MSCs have to be adjusted, and in vivo studies on the osteogenic capacities of the different types of MCSs are warranted. Copyright © 2016 The Authors Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

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.

The effects of constant flow bioreactor cultivation and keratinocyte seeding densities on prevascularized organotypic skin grafts based on a fibrin scaffold.

Organotypic full-thickness skin grafts (OTSG) are already an important technology for treating various skin conditions and are well established for skin research and development. These obvious benefits are often impaired by the need of laborious production, their noncomplete autologous composition, and, most importantly, their lack of included vasculature. Therefore, our study focused on combining a prevascularized dermal layer with an epidermis to cultivate full-thickness skin grafts incorporating capillary-like networks. It has been shown that prevascularization accelerates ingrowth of tissue-engineered grafts, and it is a prerequisite to circumvent diffusion limits due to graft thickness. To obtain such a graft, we chose a dermal layer incorporating human umbilical vein endothelial cells (HuVEC) amid human dermal fibroblasts within a fibrin-based scaffold, seeded apically with human foreskin keratinocytes (hfKC). Our research investigated the used concept's feasibility, as well as the effect of hfKC addition on the development of a well-connected capillary-like network after approximately 21 days. In addition, we evaluated the utilization of a custom-made constant flow bioreactor for simplified cultivation of these grafts, therefore possibly easing graft production and presumably increasing their cost effectiveness. Skin grafts were assessed by conventional two-dimensional histology. In addition, software-assisted three-dimensional evaluation of the capillary-like structure networks was performed by two-photon laser scanning microscopy (TPLSM) and subsequent image processing was done with ImagePro(®) Analyzer 7.0 software, thereby evaluating its platform technology power in the field of prevascularized skin grafts. All samples showed a capillary-like structure network, but we could report a significant reduction of its total length after 14 days of tri-culture with 5×10(5)/cm(2) seeded hfKC, possibly indicating nutritional deficiencies for this particular high cell density experimental setup. Lower concentrations of hfKC did not affect the formation of the capillary-like structures significantly. The developed bioreactor simplified cultivation of prevascularized OTSG. However, a flow-dependent reduction of capillary-like structures in 1 and 5 mL/min flow conditions occurred. We conclude that our technique for creating prevascularized OTSG is feasible. In addition, TPLSM is well suited for analyzing the prevascularization process. We hypothesize that the handling benefits of our bioreactor can be preserved by using considerably lower flow rates while not impairing the forming of capillary-like structure networks.

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.

Signalling-dependent adverse health effects of carbon nanoparticles are prevented by the compatible solute mannosylglycerate (firoin) in vitro and in vivo.

The inhalation of combustion-derived nanoparticles leads to adverse health effects in the airways. In this context the induction of membrane-coupled signalling is considered as causative for changes in tissue homeostasis and pro-inflammatory reactions. The identification of these molecular cell reactions allowed to seek for strategies which interfere with these adverse effects. In the current study, we investigated the structurally different compatible solutes mannosylglycerate (firoin) from thermophilic bacteria and ectoine from halophilic bacteria for their capability to reduce signalling pathways triggered by carbon nanoparticles in target cells in the lung. The pre-treatment of lung epithelial cells with both substances decreased the particle-specific activation of mitogen-activated protein kinases and also the endpoints proliferation and apoptosis. Firoin applied into the lungs of animals, like ectoine, led to a significant reduction of the neutrophilic lung inflammation induced by particle exposure. The pro-inflammatory effect of carbon nanoparticles on human neutrophil granulocytes ex vivo was significantly reduced by both substances via the reduction of the anti-apoptotic membrane-dependent signalling. The data of this study together with earlier studies demonstrate that two structurally non-related compatible solutes are able to prevent pathogenic reactions of the airways to carbon nanoparticles by interfering with signalling events. The findings highlight the preventive or therapeutic potential of compatible solutes for adverse health effects caused by particle exposure of the airways.

Zinc regulates iNOS-derived nitric oxide formation in endothelial cells.

Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1ß. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation.