Project description:Although evidence has shown that very small electric currents produce a beneficial therapeutic result for wounds, non-invasive EMF therapy has consisted mostly of anecdotal clinical reports with very few well controlled laboratory mechanistic studies. In this study, we evaluated the effects and potential mechanisms of a non-invasive EMF device on skin wound repair. In vitro analyses with human skin keratinocyte cultures demonstrated that the non-invasive EMF has a very strong effect on accelerating keratinocyte migration and a relatively weaker effect on promoting keratinocyte proliferation. The positive effects of the non-invasive EMF on cell migration and proliferation seem keratinocyte specific without such effects seen on dermal fibroblasts. cDNA microarray and RT-PCR performed revealed increased expression of CRK7 and HOXC8 genes in treated keratinocytes. This study suggests that a non-invasive electric magnetic field accelerates wound reepithelialization through a mechanism of promoting keratinocyte migration and proliferation, possibly due to upregulation of CRK7 and HOXC8 genes. Experiment Overall Design: Non-invasive EMF: Experiment Overall Design: In this study, we used the Field Therapy Accelerator (FTA; Advatech, Miami, Florida) device to produce a magnetic field which in turn induces a non-invasive electric current in the targetâs surroundings. All human and animal studies have been approved by the authors' Institutional Review Board. The FTA device generates a continuous series of direct current (DC)-like pulses of voltage followed for a very short time interval by a negative voltage spike. The induced EMF comprises of a series of concentric circles centered on the axis of the coil. The EMF vectors are at each point perpendicular to the surface of the circle and in the plane of that circle, that plane being parallel to the face of the coil. Experiment Overall Design: In vitro Studies with Cell Cultures: Experiment Overall Design: Cells and Cell Cultures: Normal human keratinocytes were maintained in growth medium of EpiLife (Cascade Biologics, Portland, Oregon) with human keratinocyte growth supplement at concentration of 0.2% v/v of bovine pituitary extract, 5 ug/ml bovine insulin, 0.18 ug/ml hydrocortisone, 5 ug/ml bovine transferrin, 0.2 ng/ml human epidermal growth factor (Cascade Biologics, Portland, Oregon), plus antibiotics of 100 U/ml penicillin G and 100 ug/ml streptomycin at 37ÂºC and 5% CO2. Media were changed every 24 hours. Experiment Overall Design: cDNA Microarray and RT-PCR Analysis: Experiment Overall Design: Cell cultures and migration assay: Normal human epidermal keratinocytes were maintained in 35-mm cell culture dishes and in EpiLife at 37ÂºC and 5% CO2. Keratinocytes were maintained in growth media and grown to confluency. At time 0, a cross-shaped wound gap or cell-free zone was made among confluent monolayer cells in the center of the culture dish, and detached cells were washed off with PBS and then replaced with growth medium. Non-invasive EMF treatment: Group 1 was treated for 1 hour immediately after wounding, at a frequency of 2080 cycles/second, electric field strength of 20mV/cm, and duty cycle of 90%. Group 2 was the control group, and received no treatment but was placed in the same hood at temperatures of 31Â±2ÂºC for the same period of time as their treatment counterparts. Each group was performed in triplicates. Experiment Overall Design: cDNA Microarray: RNA was isolated from cells using an RNeasy Mini RNA Isolation Kit (Qiagen Sciences, Germantown, Maryland), and converted to double stranded cDNA using a cDNA RT Kit (Applied Biosystems, Foster City, California). An in vitro transcription reaction was subsequently performed to produce biotin-labeled complementary RNA (cRNA) from the cDNA. Gene expression profiles were assessed using the Affymetrix Human Genome HU133A 2.0 GeneChip array, containing 22,277 sequenced human genes. GeneChip arrays were scanned using a GeneArray Scanner (Hewlett-Packard, Santa Clara, Calif). Hybridization data were analyzed using MAS 5.

Project description:In this project we aimed at deciphering modulation in genes expression induced by external pulsed electric fields applied in reversible electroporation-based treatments. Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel extracellular matrix, which was the point of interest in our to be published study. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute, a tissue model rich in endogeneous extracellular matrix such as collagens, was used to demonstrate the effects of microsecond and millisecond pulsed electric fields applied respectively in electrochemotherapy (ECT) treatment and gene electrotransfer (GET) strategy. Our analyses, focused on matrisome genes and extracellular matrix remodeling, underpin that pulsed electric fields, a technology already approved for clinical use combined with anti-cancer agents, are particularly promising to provide local and effective treatment of abnormal extracellular matrix. Part of this dataset was used to describe how pulsed electric field on its own (with no addition of external drugs) induce extracellular matrix (ECM) remodeling at human dermal scale, by focusing at genes related to matrisome subset. In this manuscript to be published, electrochemotherapy (ECT) parameters were named SP for "short pulses" and gene electrotransfer (GET) parameters were named LP for "long pulses". W demonstrated that these both types of electric parameters inducing reversible electroporation of the cells whitin the dermal tissue substitute induced 1) a rapid modulation (4h after electrostimulation) of mRNA’s genes composing the matrisome, particularly a down-regulation of pro-collagens and ECM maturation’s enzymes such as transglutaminase TG2 and LOX-like; 2) a transient decrease in pro-collagens production and hydroxyproline tissue content within a week after electrostimulation; 3) a long-lasting ROS-dependent over-activation of MMPs for at least 48h and 4) a down-regulation at both mRNA and protein level of pro-fibrotic TGF-β.

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