Cell Viability Assessment of PEDOT Conducting Polymer-Coated Microneedles for Skin Sampling.

Recently, transdermal monitoring and drug delivery have gained much interest, owing to the introduction of the minimally invasive microneedle (MN) device. The advancement of electroactive MNs electrically assisted in the capture of biomarkers or the triggering of drug release. Recent works have combined conducting polymers (CPs) onto MNs owing to the soft nature of the polymers and their tunable ionic and electronic conductivity. Though CPs are reported to work safely in the body, their biocompatibility in the skin has been insufficiently investigated. Furthermore, during electrical biasing of CPs, they undergo reduction or oxidation, which in practical terms leads to release/exchange of ions, which could pose biological risks. This work investigates the viability and proliferation of skin cells upon exposure to an electrochemically biased MN pair comprising two differently doped poly(3,4-ethylenedioxy-thiophene) (PEDOT) polymers that have been designed for skin sampling use. The impact of biasing on human keratinocytes and dermal fibroblasts was determined at different initial cell seeding densities and incubation periods. Indirect testing was employed, whereby the culture media was first exposed to PEDOTs prior to the addition of this extract to cells. In all conditions, both unbiased and biased PEDOT extracts showed no cytotoxicity, but the viability and proliferation of cells cultured at a low cell seeding density were lower than those of the control after 48 h of incubation.

PEDOT coated microneedles towards electrochemically assisted skin sampling.

Skin sampling is a diagnostic procedure based on the analysis of extracted skin tissues and/or the observation of biomarkers in bodily fluids. Sampling using microneedles (MNs) that minimize invasiveness is gaining attention over conventional biopsy/blood lancet. In this study, new MNs for electrochemically assisted skin sampling are reported, specifically tailored for combined skin tissue biopsy and interstitial fluid (ISF) extraction. To overcome risks associated with using metal MNs, a highly electroactive, mechanically flexible, and biocompatible organic conducting polymer (CP) coated onto plastic is chosen as an alternative. Two different variants of doped poly(3,4-ethylenedioxythiophene) are coated on polymethyl methacrylate and used in combination as a MN pair with subsequent testing via a variety of electrochemical techniques to (i) give real-time information of the MN penetration depth into the skin, and (ii) yield new information on various salts present in the ISF. The MN skin sampler shows the ability to extract ions from the hydrated excised skin as a step towards in vivo ISF extraction. The presence of ions was analyzed using X-ray photoelectron spectroscopy. This added chemical information in conjunction with the existing biomarker analysis increases opportunity for disease/condition detection. For example, in the case of psoriasis, information about salt in the skin is invaluable in combination with pathogenic gene expression for diagnosis.

Plasma-Functionalised Dressings for Enhanced Wound Healing.

Fundamental knowledge about cell-surface interactions can be applied in the development of wound dressings and scaffolds to encourage wounds to heal. As surfaces produced with acid-functionalised monomers encourage keratinocyte adhesion, proliferation and migration, whilst amine functionalisation enhances fibroblast proliferation and migration in vitro, standard care wound dressings were plasma-coated with either acrylic acid or allylamine and applied to 6 mm excisional wounds on the backs of mice to test their effectiveness in vivo. At day 3, the rate of wound healing was increased in mice treated with dressings that were plasma-coated with allylamine compared to uncoated dressings, with a significantly reduced wound area. However, healing may be impaired following prolonged treatment with allylamine-functionalised dressings, with delayed re-epithelialisation and increased cellularisation of the wound site at later timepoints. Acrylic acid functionalisation, however, offered no early improvement in wound healing, but wounds treated with these dressings displayed increased collagen deposition at day 7 post wounding. These results suggest that plasma polymerisation may allow for the development of new dressings which can enhance wound closure by directing cell behaviour, but that the application of these dressings may require a timed approach to enhance specific phases of the wound healing response.

The development of microfluidic-based western blotting: Technical advances and future perspectives.

Over the past two decades significant technical advancement in the field of western blotting has been made possible through the utilization of microfluidic technologies. In this review we provide a critical overview of these advancements, highlighting the advantages and disadvantages of each approach. Particular attention is paid to the development of now commercially available systems, including those for single cell analysis. This review also discusses more recent developments, including algorithms for automation and/or improved quantitation, the utilization of different materials/chemistries, use of projection electrophoresis, and the development of triBlots. Finally, the review includes commentary on future advances in the field based on current developments, and the potential of these systems for use as point-of-care devices in healthcare.

Influence of Acidic pH on Wound Healing In Vivo: A Novel Perspective for Wound Treatment.

There has been little understanding of acidification functionality in wound healing, highlighting the need to study the efficacy of wound acidification on wound closure and cellular activity in non-infected wounds. This study is focused on establishing the healing potential of wound acidification in non-infected wounds. Acidic buffers, constituting either phosphoric or citric acid, were employed to modify the physiological pH of non-infected full-thickness excisional murine wounds. Acidification of the wound by acidic buffers was found to be an effective strategy to improve wound healing. A significant improvement in wound healing parameters was observed as early as 2 days post-treatment with acidic buffers compared to controls, with faster rate of epithelialization, wound closure and higher levels of collagen at day 7. pH is shown to play a role in mediating the rate of wound healing, with acidic buffers formulated at pH 4 observed to stimulate faster recovery of wounded tissues than pH 6 buffers. Our study shows the importance of maintaining an acidic wound microenvironment at pH 4, which could be a potential therapeutic strategy for wound management.

Wound Healing from an Actin Cytoskeletal Perspective.

Wound healing requires a complex cascade of highly controlled and conserved cellular and molecular processes. These involve numerous cell types and extracellular matrix molecules regulated by the actin cytoskeleton. This microscopic network of filaments is present within the cytoplasm of all cells and provides the shape and mechanical support required for cell movement and proliferation. Here, an overview of the processes of wound healing are described from the perspective of the cell in relation to the actin cytoskeleton. Key points of discussion include the role of actin, its binding proteins, signaling pathways, and events that play significant roles in the phases of wound healing. The identification of cytoskeletal targets that can be used to manipulate and improve wound healing is included as an emerging area of focus that may inform future therapeutic approaches to improve healing of complex wounds.

Increased Expression of Flightless I in Cutaneous Squamous Cell Carcinoma Affects Wnt/ß-Catenin Signaling Pathway.

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/ß-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/ß-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased ß-catenin and a decrease in the expression of the downstream effector of ß-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/ß-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/-, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/- embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and ß-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Multifunctional ultrasmall AgNP hydrogel accelerates healing of S. aureus infected wounds.

The increasing emergence of antibiotic resistance coupled with the limited effectiveness of current treatments highlights the need for the development of new treatment modalities. Silver nanoparticles (AgNPs) are a promising alternative with broad-spectrum antibacterial activity. However, the clinical translation of AgNPs have been hampered primarily due to the delivery of unsafe levels of silver ions (Ag+) resulting in cellular toxicity and their susceptibility to aggregation resulting in loss of efficacy. Here, we describe a safe and effective, thermo-responsive AgNP hydrogel that provides antibacterial effects in conjunction with wound promoting properties. Using a murine model of wound infection, we demonstrate that the applied AgNP hydrogel to the wound (12 µg silver) not only provides superior bactericidal activity but also reduces inflammation leading to accelerated wound closure when compared to industry-standard silver sulfadiazine (302 µg silver). The AgNP hydrogel-treatment significantly accelerated wound closure at day 4 post-infection (56 closure) compared to both blank hydrogel or Ag SD (74% and 91% closure respectively) with a concurrent increase in PCNA-positive proliferating cells corresponding with a significant 32% improvement in wound re-epithelization compared to the blank hydrogel. Treatment of infected wounds with AgNP hydrogel also decreased neutrophil infiltration, increased anti-inflammatory Ym-1 positive M2 macrophages, and reduced the number of caspase-1 positive apoptotic cells. Therefore, this novel multifunctional AgNP thermo-responsive hydrogel is potentially a safe and effective treatment at much lower concentration for the treatment of wound infections. STATEMENT OF SIGNIFICANCE: In this study, we describe the development of a multifunctional thermo-responsive hydrogel of ultrasmall silver nanoparticles (AgNPs) for controlled and optimized delivery of silver to infected wounds. The in vivo biological effects of the developed hydrogel showed significant S. aureus elimination from infected mouse wounds compared to a commercial antibacterial formulation. The developed AgNP hydrogel optimally regulates inflammatory responses to promote wound healing as indicated by increased cell proliferation and wound re-epithelization. Additionally, AgNP hydrogel shows significant potential in regulating neutrophil infiltration while increasing levels of anti-inflammatory M2 macrophages and reduces the number of apoptotic cells. Therefore, the multifunctional properties of the developed AgNP thermo-responsive hydrogel offers great clinical potential to control bacterial infections and promote wound healing.

Treatment of murine partial thickness scald injuries with multipotent adult progenitor cells decreases inflammation and promotes angiogenesis leading to improved burn injury repair.

Stem cells have been shown to have potential as a new therapy for burns and promote wound healing through decreasing inflammation and increasing angiogenesis. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of cytokines which can affect cellular activities. This article aimed to examine the effects of MAPC cells treatment on burn injury repair using a mouse model of partial thickness burn injury. The immunomodulatory effect of MAPC cells was investigated in vitro using a simultaneous T-cell proliferation assay. Partial thickness burns were created on the dorsal surface of mice and MAPC cells were administered via intradermal injection to the wound margins 24 h post-burn injury. The burn tissues were analysed macroscopically to determine wound area and histologically assessed to determine wound width and rate of re-epithelialisation. Immunohistochemistry and ELISA were employed to assess cell proliferation, inflammation and angiogenesis and collagen deposition in the burn area. MAPC cells inhibit the proliferation of stimulated T cells in culture. Burns intradermally injected with MAPC cells showed a significant reduction in the macroscopic wound area, histologic wound width and had an increased rate of re-epithelialisation. Immunohistochemistry and ELISA analysis of burn tissues showed dampened inflammation evidenced by a reduction in neutrophilic infiltration and modulation of inflammatory cytokines. Angiogenesis within the burn area was also improved in MAPC cell treated mice. However, no significant effect of MAPC cell treatment was observed on extracellular matrix production. Treatment of burns with MAPC cells improved burn injury repair with reduced time to healing, decreased inflammation and increased angiogenesis. These findings demonstrate the promising effects of MAPC cells on burn injury repair and suggest MAPC cells as a candidate source for clinical cell therapies.