Helium Generated Cold Plasma Finely Regulates Activation of Human Fibroblast-Like Primary Cells

Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2′,7′-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a valid therapeutic approach for liver and gut lesions.     

Microbiological interactions with cold plasma

There is a diverse range of microbiological challenges facing the food, healthcare and clinical sectors. The increasing and pervasive resistance to broad‐spectrum antibiotics and health‐related concerns with many biocidal agents drives research for novel and complementary antimicrobial approaches. Biofilms display increased mechanical and antimicrobial stability and are the subject of extensive research. Cold plasmas (CP) have rapidly evolved as a technology for microbial decontamination, wound healing and cancer treatment, owing to the chemical and bio‐active radicals generated known collectively as reactive oxygen and nitrogen species. This review outlines the basics of CP technology and discusses the interactions with a range of microbiological targets. Advances in mechanistic insights are presented and applications to food and clinical issues are discussed. The possibility of tailoring CP to control specific microbiological challenges is apparent. This review focuses on microbiological issues in relation to food‐ and healthcare‐associated human infections, the role of CP in their elimination and the current status of plasma mechanisms of action.     

Inactivation of spores by nonthermal plasmas

Bacterial and fungal spore contamination in different industries has a greater economic impact. Because of the remarkable resistance of spores to most physical and chemical microbicidal agents, their inactivation need special attention during sterilization processes. Heat and chemical sporicides are not always well suited for different sterilization/decontamination applications and carries inherent risks. In recent years, novel nonthermal agents including nonthermal plasmas are emerging as effective sporicides against a broad spectrum of bacterial and fungal spores. The present review discusses various aspects related to the inactivation of spores using nonthermal plasmas. Different types of both low pressure plasmas (e.g., capacitively coupled plasma and microwave plasma) and atmospheric pressure plasmas (e.g., dielectric barrier discharges, corona discharges, arc discharges, radio-frequency-driven plasma jet) have been successfully applied to destroy spores of economic significance. Plasma agents contributing to sporicidal activity and their mode of action in inactivation are discussed. In addition, information on factors that affect the sporicidal action of nonthermal plasmas is included.     

Plasma treatments of dressings for wound healing: a review

This review covers the use of plasma technology relevant to the preparation of dressings for wound healing. The current state of knowledge of plasma treatments that have potential to provide enhanced functional surfaces for rapid and effective healing is summarized. Dressings that are specialized to the needs of individual cases of chronic wounds such as diabetic ulcers are a special focus. A summary of the biology of wound healing and a discussion of the various types of plasmas that are suitable for the customizing of wound dressings are given. Plasma treatment allows the surface energy and air permeability of the dressing to be controlled, to ensure optimum interaction with the wound. Plasmas also provide control over the surface chemistry and in cases where the plasma creates energetic ion bombardment, activation with long-lived radicals that can bind therapeutic molecules covalently to the surface of the dressing. Therapeutic innovations enabled by plasma treatment include the attachment of microRNA or antimicrobial peptides. Bioactive molecules that promote subsequent cell adhesion and proliferation can also be bound, leading to the recruitment of cells to the dressing that may be stem cells or patient-derived cells. The presence of a communicating cell population expressing factors promotes healing.     

Cold Atmospheric Plasma (CAP) Changes Gene Expression of Key Molecules of the Wound Healing Machinery and Improves Wound Healing In Vitro and In Vivo

Cold atmospheric plasma (CAP) has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time) in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.     

Differential gene expression in Escherichia coli following exposure to nonthermal atmospheric pressure plasma

Aim:  Nonthermal atmospheric-pressure plasmas offer significant advantages as an emerging disinfection approach. However the mechanisms of inactivation, and thus the means of optimizing them, are still poorly understood. The objective of this study, therefore, was to explore differential gene expression on a genome-wide scale in Escherichia coli following exposure to a nonthermal atmospheric-pressure argon plasma plume using high-density oligonucleotide microarrays.
Methods and Results:  Plasma exposure was found to significantly induce the SOS mechanism, consisting of about 20 genes. Other genes involved in regulating response to oxidative stress were also observed to be up-regulated. Conversely, the expression of several genes responsible for housekeeping functions, ion transport, and metabolism was observed to be down-regulated.
Conclusions:  Elevated yet incomplete induction of various DNA damage repair processes, including translesion synthesis, suggests substantial DNA damage in E. coli . Oxidative stress also appeared to play a role. Thus it is proposed that the efficacy of plasma is due to the synergistic impact of UV photons and oxygen radicals on the bacteria.
Significance and Impact of the Study:  This study represents the first investigation of differential gene expression on a genome-wide scale in an organism following plasma exposure. The results of this study will help enable the design of safe and effective plasma decontamination devices.     

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.     

Role of the PI3K/AKT (mTOR and GSK3β) signalling pathway and photobiomodulation in diabetic wound healing

Activated phosphatidylinositol 3 kinase/Protein kinase B (PI3K/AKT) signalling with increased or reduced mTOR and GSK3β activity influences the wound repair process. Diabetic wounds, usually ulcerated, are characterised by reduced growth factors and cellular performance. The occurrence of diabetic ulcers is linked to peripheral arterial disease, neuropathy, and wound contamination. Lasers or light emitting diodes (LEDs) provide photon energy with therapeutic benefits (Photobiomodulation-PBM), and has been broadly commended to quicken diabetic wound healing. PBM is efficient in the visible red and near-infrared electromagnetic spectrum, and fluencies ranging from 2 to 6 J/cm². However, cellular and molecular mechanisms induced by PBM are not fully understood. In this review we discuss PBM and the PI3K/AKT pathway with specific focus on the mTOR and GSK3β downstream activity in diabetic wound healing.     

细菌生物膜形成与慢性难愈性创面相关性的研究进展

感染是影响慢性难愈性创面愈合最常见的原因,由于多种细菌混合感染、耐药性产生、生物膜的形成使其治疗成为难题.其中,细菌生物膜形成是导致创面的难以愈合的重要因素之一.本文就慢性难愈合创面中细菌生物膜的形成机制、特征、生态学、对伤口愈合的影响以及可能的治疗对策等作一综述.     

A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapies

Diabetes mellitus is an epidemic multisystemic chronic disease that frequently is complicated by complex wound infections. Innovative topical antimicrobial therapy agents are potentially useful for multimodal treatment of these infections. However, an appropriately standardized in vivo model is currently not available to facilitate the screening of these emerging products and their effect on wound healing. To develop such a model, we analyzed, tested, and modified published models of wound healing. We optimized various aspects of the model, including animal species, diabetes induction method, hair removal technique, splint and dressing methods, the control of unintentional bacterial infection, sampling methods for the evaluation of bacterial burden, and aspects of the microscopic and macroscopic assessment of wound healing, all while taking into consideration animal welfare and the '3Rs' principle. We thus developed a new wound infection model in rats that is optimized for testing topical antimicrobial therapy agents. This model accurately reproduces the pathophysiology of infected diabetic wound healing and includes the current standard treatment (that is, debridement). The numerous benefits of this model include the ready availability of necessary materials, simple techniques, high reproducibility, and practicality for experiments with large sample sizes. Furthermore, given its similarities to infected-wound healing and treatment in humans, our new model can serve as a valid alternative for applied research.     

Initial Characterization of the Pig Skin Bacteriome and Its Effect on In Vitro Models of Wound Healing

Elucidating the roles and composition of the human skin microbiome has revealed a delicate interplay between resident microbes and wound healing. Evolutionarily speaking, normal cutaneous flora likely has been selected for because it potentiates or, at minimum, does not impede wound healing. While pigs are the gold standard model for wound healing studies, the porcine skin microbiome has not been studied in detail. Herein, we performed 16S rDNA sequencing to characterize the pig skin bacteriome at several anatomical locations. Additionally, we used bacterial conditioned-media with in vitro techniques to examine the paracrine effects of bacterial-derived proteins on human keratinocytes (NHEK) and fibroblasts (NHDF). We found that at the phyla level, the pig skin bacteriome is similar to that of humans and largely consists of Firmicutes (55.6%), Bacteroidetes (20.8%), Actinobacteria (13.3%), and Proteobacteria (5.1%) however species-level differences between anatomical locations exist. Studies of bacterial supernatant revealed location-dependent effects on NHDF migration and NHEK apoptosis and growth factor release. These results expand the limited knowledge of the cutaneous bacteriome of healthy swine, and suggest that naturally occurring bacterial flora affects wound healing differentially depending on anatomical location. Ultimately, the pig might be considered the best surrogate for not only wound healing studies but also the cutaneous microbiome. This would not only facilitate investigations into the microbiome’s role in recovery from injury, but also provide microbial targets for enhancing or accelerating wound healing.     

封闭负压引流对兔糖尿病溃疡创面组织愈合影响的观察

目的:探讨封闭负压引流(VAC)对兔糖尿病溃疡创面组织愈合的影响及其可能机制。方法:采用四氧嘧啶法建立兔糖尿病溃疡模型,设空白对照组和实验组(对照组创面采用常规包扎治疗处理,实验组创面则采用VAC处理),观察和比较两组动物的创面肉眼观、愈合时间,在致伤前、致伤后3 d、7 d、14 d取创面软组织,检测和比较两组动物的创面组织含水量、血流量以及血浆ET-1和NO含量。结果:与对照组比较,实验组动物的创面肿胀及分泌物得到明显控制,创面坏死组织的清除与肉芽组织的生长明显加快,平均愈合时间明显缩短(P0.05);致伤后3 d、7 d和14 d,创面组织含水量与血浆ET-1含量明显下降(P0.05),创面组织血流量与血浆NO含量明显增加(P0.05)。结论:VAC对兔糖尿病溃疡创面组织的愈合可起到积极的促进作用,这可能与其增加血浆NO含量及降低ET-1的含量有关,其具体机制尚有待于进一步的研究。     

Progress in research on radiation combined injury in China

The significant feature of radiation combined injury is the occurrence of a combined effect. For decades our institute has focused on studying the key complications of radiation-burn injury, including shock, suppression of hematopoiesis and immunity, gastrointestinal damage and local refractory wound healing. Here we summarize recent advancements in elucidating the mechanisms of and potential treatments for radiation combined injury. Concerning the suppression and regeneration of hematopoiesis in radiation combined injury, mechanisms of megakaryocyte damage have been elucidated and a new type of fusion protein stimulating thrombopoiesis has been developed and is being tested in animals. With regard to the damage and repair of intestinal epithelium, the important molecular mechanisms of radiation combined injury have been clarified, and new measures to prevent and treat gastrointestinal tract injury are proposed. With respect to the difficulties encountered in wound healing, the underlying causes of radiation combined injury have been proposed, and some potential methods to accelerate wound closure are under study. Systemic experiments have been done to determine the appropriate time for eschar excision and skin grafting, and the results provided significant insight into clinical treatment of the injury. In the search for early therapeutic regimens for severe burns and radiation combined injury to prevent deterioration of injuries and to improve survival, cervical sympathetic ganglion block was used for the treatment of animals with radiation combined injury and had significant benefits. These research advancements have potential for application in on-site emergency rescue and in-hospital treatment of radiation combined injury.     

Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms

Background aimsMesenchymal stromal cells (MSC) can be isolated from the perivascular connective tissue of umbilical cords, called Wharton's jelly. These human umbilical cord perivascular cells (HUCPVC) might provide therapeutic benefits when treating skeletal or cutaneous malformations in neonatal patients.MethodsHUCPVC were isolated, and their proliferation rate, marker expression and multilineage differentiation potential determined. HUCPVC or their conditioned medium (HUCPVC-CM) was injected into the excisional wound of a mouse splinted-wound model. The effects of the treatment on wound closure were examined by morphohistochemical and gene expression analyses.ResultsHUCPVC expressed typical MSC markers and could differentiate into osteoblastic and adipogenic lineages. HUCPVC transplanted into the mouse wound accelerated wound closure. Immunohistologic analysis showed that the HUCPVC accelerated wound healing by enhancing collagen deposition and angiogenesis via paracrine mechanisms. Furthermore, treatment with HUCPVC-CM alone significantly enhanced wound closure. HUCPVC-CM increased the number of anti-inflammatory M2 macrophages expressing resistin-like molecule (RELM)-α/CD11b and promoted neovessel maturation. Quantitative polymerase chain reaction (PCR) analysis showed that HUCPVC-CM increased the expression of tissue-repairing cytokines interleukin (IL)-10, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-1 and angiopoietin-1 at the healing wound.ConclusionsOur results show that HUCPVC promotes wound healing via multifaceted paracrine mechanisms. Together with their ability to differentiate into the osteogenic linage, HUCPVC may provide significant therapeutic benefits for treating wounds in neonatal patients.     

Exosomes from adipose‐derived stem cells and application to skin wound healing

Skin wound healing is an intractable problem that represents an urgent clinical need. To solve this problem, a large number of studies have focused on the use of exosomes (EXOs) derived from adipose‐derived stem cells (ADSCs). This review describes the mechanisms whereby ADSCs‐EXOs regulate wound healing and their clinical application. In the wound, ADSCs‐EXOs modulate immune responses and inflammation. They also promote angiogenesis, accelerate proliferation and re‐epithelization of skin cells, and regulate collagen remodelling which inhibits scar hyperplasia. Compared with ADSCs therapeutics, ADSCs‐EXOs have highly stability and are easily stored. Additionally, they are not rejected by the immune system and have a homing effect and their dosage can be easily controlled. ADSCs‐EXOs can improve fat grafting and promote wound healing in patients with diabetes mellitus. They can also act as a carrier and combined scaffold for treatment, leading to scarless cutaneous repair. Overall, ADSCs‐EXOs have the potential to be used in the clinic to promote wound healing.     

Mechanisms of action of mesenchymal stem cells in cutaneous wound repair and regeneration

Mesenchymal stem cells (MSCs) are multipotent cells with the capacity for self-renewal and differentiation and have a broad tissue distribution. These characteristics make them candidate cells for wound healing and regeneration in a variety of disorders. Endogenous MSCs or exogenously delivered MSCs can traffic and migrate to injured tissue and participate in the healing of this tissue. The concentrated conditioned medium from MSCs can modulate wound repair without MSCs being present in the wound. The therapeutic effects of MSCs might be attributable to their ability to differentiate and transdifferentiate into tissue-specific cells, to fuse with the resident cells, to secrete a wide array of paracrine factors in order to stimulate the survival and functional recovery of the resident cells, or to regulate the local microenviroment or niche and immune response. These mechanisms are probably independent but not mutually exclusive. In many circumstances, a combination of these protective mechanisms might work together to affect cutaneous wound healing. This review gives a brief overview and discusses the mechanisms by which MSCs promote skin repair and regeneration, although the specific mechanisms in each type of cutaneous wound are still unclear and controversial. A comprehensive understanding of the mechanisms should allow us to find advanced and better treatment strategies for various skin diseases, even those that are currently incurable.     

生物活性透明质酸治疗激光所致皮肤创口炎症的临床疗效观察

目的:探讨生物活性透明质酸治疗激光所致皮肤创口炎症的临床疗效。方法:选择我院收治的40例激光皮肤除斑患者为研究对象,按就诊顺序将其分为实验组和对照组。实验组20例患者采用生物活性透明质酸涂抹激光治疗后的皮肤伤口,对照组20例患者采用30分冰袋冷敷皮肤激光治疗后的皮肤伤口,观察和比较两组患者伤口第1-7天的的红肿痛的改善情况。结果:(1)疼痛症状的改善情况:实验组第1天的显效率0%,有效20例,总有效率100%;对照组第1天的显效率0%,有效20例,总有效率100%,两组比较无统计学差异(P0.05)。(2)红肿的改善情况:实验组第1天显效20例,总有效率为100%;对照组第1天显效0例,有效20,总有效率为100%,两组总有效率比较无统计学差异(P0.05),但实验组的显效率明高于对照组,差异具有统计学意义(P0.01)。结论:生物活性透明质酸涂抹可以有效控制皮肤伤口炎症引发的红肿痛,且临床效果明显优于冰袋冷敷,方便可行,值得推广。     

Adipose-derived stem cells for wound healing

Wound healing is a complex but a fine-tuned biological process in which human skin has the ability to regenerate itself following damage. However, in particular conditions such as deep burn or diabetes the process of wound healing is compromised. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose-derived stem cells (ASCs) seem to possess the least limitations for clinical applications, and literature showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. In the present review, advantages and disadvantages of various stem cells which can be used for promoting wound healing are discussed. In addition, potential mechanisms of action by which ASCs may accelerate wound healing are summarised. Finally, clinical studies applying ASCs for wound healing and the associated limitations are reviewed.     

Effects of Cold Atmospheric Plasma (CAP) on ?-Defensins,Inflammatory Cytokines,and Apoptosis-Related Molecules in Keratinocytes In Vitro and In Vivo

Cold atmospheric plasma (CAP) has been gaining increasing interest as a new approach for the treatment of skin diseases or wounds. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. This study explored in vitro and in vivo whether CAP influences gene expression and molecular mechanisms in keratinocytes. Our results revealed that a 2 min CAP treatment using the MicroPlaSter ß in analogy to the performed clinical studies for wound treatment induces expression of IL-8, TGF-ß1, and TGF-ß2. In vitro and in vivo assays indicated that keratinocyte proliferation, migration, and apoptotic mechanisms were not affected by the CAP treatment under the applied conditions. Further, we observed that antimicrobial peptides of the ß-defensin family are upregulated after CAP treatment. In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes. The induction of ß-defensins in keratinocytes describes an absolutely new plasma strategy. Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of ß-defensin activation by CAP is not investigated so far.     

Recent trends and technological development in plasma as an emerging and promising technology for food biosystems

The rising need for wholesome, fresh, safe and “minimally-processed” foods has led to pioneering research activities in the emerging non-thermal technology of food processing. Cold plasma is such an innovative and promising technology that offers several potential applications in the food industry. It uses the highly reactive, energetic and charged gas molecules and species to decontaminate the food and package surfaces and preserve the foods without causing thermal damage to the nutritional and quality attributes of food. Cold plasma technology showed promising results about the inactivation of pathogens in the food industry without affecting the food quality. It is highly effective for surface decontamination of fruits and vegetables, but extensive research is required before its commercial utilization. Recent patents are focused on the applications of cold plasma in food processing and preservation. However, further studies are strongly needed to scale up this technology for future commercialization and understand plasma physics for getting better results and expand the applications and benefits. This review summarizes the emerging trends of cold plasma along with its recent applications in the food industry to extend shelf life and improve the quality of food. It also gives an overview of plasma generation and principles including mechanism of action. Further, the patents based on cold plasma technology have also been highlighted comprehensively for the first time.