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Abstract Tissue nanotransfection (TNT) topically delivers Etv2, Foxc2, and Fli1 (EFF) plasmids increasing vasculogenic fibroblasts (VF) and promoting vascularization in ischemic murine skin. Human dermal fibroblasts respond to EFF nanoelectroporation with elevated expression of endothelial genes in vitro, which is linked to increased ten-eleven translocase 1/2/3 (TET) expression. Single cell RNA sequencing dependent validation of VF induction reveals a TET-dependent transcript signature. TNTEFF also induces TET expression in vivo, and fibroblast-specific EFF overexpression leads to VF-transition, with TET-activation correlating with higher 5-hydroxymethylcytosine (5-hmC) levels in VF. VF emergence requires TET-dependent demethylation of endothelial genes in vivo, enhancing VF abundance and restoring perfusion in diabetic ischemic limbs. TNTEFF improves perfusion and wound closure in diabetic mice, while increasing VF in cultured human skin explants. Suppressed in diabetes, TET1/2/3 play a critical role in TNT-mediated VF formation which supports de novo blood vessel development to rescue diabetic ischemic tissue.

Abstract Sweating and heat buildup at the skin-liner interface is a major challenge for persons with limb loss. Liners made of heat-non-conducting materials may cause sweating of the residual limb and may result in liners slipping off the skin surface especially on a warm day or during high activity, causing skin breakdown and affecting limb health. To address this, we evaluated the efficacy of the vented liner-socket system (VS, Össur) compared to Seal-In silicone liner and non-vented socket (nVS, Össur) in reducing relative humidity (RH) during increased sweat. Nine individuals with limb loss using nVS were randomized to VS or nVS and asked for activity in a 20-min treadmill walk. RH was significantly attenuated (p = 0.0002) and perceived sweating, as reported by prosthesis users, improved (p = 0.028) with VS, patient-reported comprehensive lower limb amputee socket survey (CLASS) outcomes to determine the suspension, stability, and comfort were not significantly different between VS and nVS. There are limited rigorous scientific studies that clearly provide evidence-based guidelines to the prosthetist in the selection of liners from numerous available options. The present study is innovative in clearly establishing objective measures for assessing humidity and temperatures at the skin-liner interface while performing activity. As shown by the measured data and perceived sweat scores provided by the subjects based on their daily experience, this study provided clear evidence establishing relative humidity at the skin-liner interface is reduced with the use of a vented liner-socket system when compared to a similar non-vented system.

Of all the numerous nanosized extracellular vesicles released by a cell, the endosomal-originated exosomes are increasingly recognized as potential therapeutics, owing to their inherent stability, low immunogenicity, and targeted delivery capabilities. This review critically evaluates the transformative potential of exosome-based modalities across pharmaceutical and precision medicine landscapes. Because of their precise targeted biomolecular cargo delivery, exosomes are posited as ideal candidates in drug delivery, enhancing regenerative medicine strategies, and advancing diagnostic technologies. Despite the significant market growth projections of exosome therapy, its utilization is encumbered by substantial scientific and regulatory challenges. These include the lack of universally accepted protocols for exosome isolation and the complexities associated with navigating the regulatory environment, particularly the guidelines set forth by the U.S. Food and Drug Administration (FDA). This review presents a comprehensive overview of current research trajectories aimed at addressing these impediments and discusses prospective advancements that could substantiate the clinical translation of exosomal therapies. By providing a comprehensive analysis of both the capabilities and hurdles inherent to exosome therapeutic applications, this article aims to inform and direct future research paradigms, thereby fostering the integration of exosomal systems into mainstream clinical practice.

The α-tocotrienol (TCT) form of natural vitamin E is more potent than the better known α-tocopherol against stroke. Angiographic studies of canine stroke have revealed beneficial cerebrovascular effects of TCT. This work seeks to understand the molecular basis of such effect. In mice, TCT supplementation improved perfusion at the stroke-affected site by inducing miR-1224. miRNA profiling of a laser-capture-microdissected stroke-affected brain site identified miR-1224 as the only vascular miR induced. Lentiviral knockdown of miR-1224 significantly blunted the otherwise beneficial effects of TCT on stroke outcomes. Studies on primary brain microvascular endothelial cells revealed direct angiogenic properties of miR-1224. In mice not treated with TCT, advance stereotaxic delivery of an miR-1224 mimic to the stroke site markedly improved stroke outcomes. Mechanistic studies identified Serpine1 as a target of miR-1224. Downregulation of Serpine1 augmented the angiogenic response of the miR-1224 mimic in the brain endothelial cells. The inhibition of Serpine1, by dietary TCT and pharmacologically, increased cerebrovascular blood flow at the stroke-affected site and protected against stroke. This work assigns Serpine1, otherwise known to be of critical significance in stroke, a cerebrovascular function that worsens stroke outcomes. miR-1224-dependent inhibition of Serpine1 can be achieved by dietary TCT as well as by the small-molecule inhibitor TM5441.

Here, the authors report on the discovery of physiological vasculogenic fibroblasts capable of forming functional blood vessels. In vivo tissue reprogramming triggered by topical tissue nanotransfection (TNT) of a single anti-miR-200b oligonucleotide achieved therapeutic tissue vascularization.

Abstract This work rests on our non-viral tissue nanotransfection (TNT) platform to deliver MyoD (TNTMyoD) to injured tissue in vivo. TNTMyoD was performed on skin and successfully induced expression of myogenic factors. TNTMyoD was then used as a therapy 7 days following volumetric muscle loss (VML) of rat tibialis anterior and rescued muscle function. TNTMyoD is promising as VML intervention.

Summary: Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n = 3), gingiva (HGG, n = 13), and mature skin (MS, n = 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.

Tissue nanotransfection (TNT), a cutting-edge technique of in vivo gene therapy, has gained substantial attention in various applications ranging from in vivo tissue reprogramming in regenerative medicine, and wound healing to cancer treatment. This technique harnesses the advancements in the semiconductor processes, facilitating the integration of conventional transdermal gene delivery methods—nanoelectroporation and microneedle technologies. TNT silicon chips have demonstrated considerable promise in reprogramming fibroblast cells of skin in vivo into vascular or neural cells in preclinical studies to assist in the recovery of injured limbs and damaged brain tissue. More recently, the application of TNT chips has been extended to the area of exosomes, which are vital for intracellular communication to track their functionality during the wound healing process. In this review, we provide an in-depth examination of the design, fabrication, and applications of TNT silicon chips, alongside a critical analysis of the electroporation-based gene transfer mechanisms. Additionally, the review discussed the existing limitations and challenges in the current technique, which may project future trajectories in the landscape of gene therapy. Through this exploration, the review aims to shed light on the prospects of TNT in the broader context of gene therapy and tissue regeneration.

Abstract Antibiotic‐resistant infections caused by bacterial pathogens pose a serious threat to public health, hampering wound healing and causing significant morbidities worldwide. A biomedical fiber‐device that functions as a drugless antiseptic is introduced as a solution to this problem. Through stitching, piercing, or topical application to the wound, this fiber slows down the proliferation of pathogenic bacteria, thereby reducing the risks associated with inflammation and inhibiting infections. The fiber's bacterial proliferation inhibition function is based on the galvanic effect, which disturbs bacterial quorum sensing. Detailed herein are the fiber design optimization, scalable fabrication approach, electrical function characterization, and antiseptic function verification in cultures of typical wound pathogens. Such a fiber—mechanically and environmentally resilient, insensitive to harsh storage conditions with nominally infinite shelf‐life, resulting from machining rather than pharmacochemical fabrication— provides a cost‐effective and widely available alternative to current antibiotic treatments of physical injury.

Abstract Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5 V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Porcine respiratory coronavirus AR310 particles (105) were placed in direct contact with the fabric for 1 or 5 min. Following one minute of contact, zeta potential of the porcine coronavirus was significantly lowered indicating destabilization of its electrokinetic properties. Size-distribution plot showed appearance of aggregation of the virus. Testing of the cytopathic effects of the virus showed eradication of infectivity as quantitatively assessed by PI-calcein and MTT cell viability tests. This work provides the rationale to consider the studied electroceutical fabric, or other materials with comparable property, as material of choice for the development of PPE in the fight against COVID-19.

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5′-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.

Background and Aims. The cellular mechanism of liver injury related to arsenic toxicity is ill defined. It is thought that oxidative stress and mitochondrial dysfunction may play some role in arsenic-induced liver damage. In this study, we evaluated subcellular events within the primary cultured mouse hepatocytes when exposed to inorganic arsenic. Methods. Primary cultured mouse hepatocytes were treated with 10 μM arsenic for different time periods. Reactive oxygen species (ROS) formation, functional changes of the lysosome and mitochondria, and mode of hepatocytes death were studied by laser confocal microscopy, fluorescence spectroscopy, and flow cytometry. Expression of proapoptotic member of the BCL-2 family of genes BAX and antiapoptotic BCL-2 mRNA expression were studied by real-time PCR. Cytochrome c expression was studied by Western blotting. Results. Fluorescence spectroscopy as well as flow cytometric analysis revealed that arsenic-induced formation of ROS was time dependent. Confocal microscopy showed initiation of ROS formation from periphery of the hepatocytes at 30 min of arsenic exposure that progressed to central part of the hepatocytes at 3 h of arsenic exposure. The ROS formation was found to be NADPH oxidase (NOX) dependent. This low level of intracellular ROS induced lysosomal membrane permeabilization (LMP) and subsequently released cathepsin B to the cytosol. The LMP further increased intracellular ROS which in turn triggered induction of mitochondrial permeability transition (MPT). Pretreatment of hepatocytes with LMP inhibitor bafilomycin A (BafA) significantly decreased, and LMP inducer chloroquine (ChQ) significantly increased the production of ROS suggesting that LMP preceded enhanced ROS generation in response to arsenic. MPT was accompanied with increase in BAX : BCL2 mRNA ratio resulting in upregulation of caspase 3 and increased hepatocyte apoptosis. Conclusion. Although arsenic-related oxidative liver injury is well established, neither the site of origin of ROS nor the early sequence of events in arsenic toxicity due to ROS is known. We believe that our study provides evidences elucidating the early sequence of events that culminates in the death of the mouse hepatocytes during arsenic exposure.

Summary: Pseudomonas aeruginosa biofilms represent a major threat to health care. Rugose small colony variants (RSCV) of P. aeruginosa, isolated from chronic infections, display hyperbiofilm phenotype. RSCV biofilms are highly resistant to antibiotics and host defenses. This work shows that RSCV biofilm aggregates consist of two distinct bacterial subpopulations that are uniquely organized displaying contrasting physiological characteristics. Compared with that of PAO1, the extracellular polymeric substance of RSCV PAO1ΔwspF biofilms presented unique ultrastructural characteristics. Unlike PAO1, PAO1ΔwspF released fragmented extracellular DNA (eDNA) from live cells. Fragmented eDNA, thus released, was responsible for resistance of PAO1ΔwspF biofilm to disruption by DNaseI. When added to PAO1, such fragmented eDNA enhanced biofilm formation. Disruption of PAO1ΔwspF biofilm was achieved by aurine tricarboxylic acid, an inhibitor of DNA-protein interaction. This work provides critical novel insights into the contrasting structural and functional characteristics of a hyperbiofilm-forming clinical bacterial variant relative to its own wild-type strain. : Microbiology; Microbiofilms Subject Areas: Microbiology, Microbiofilms

Non-invasive, repeated interrogation of the same wound is necessary to understand the tissue repair continuum. In this work, we sought to test the significance of non-invasive high-frequency high-resolution ultrasound technology for such interrogation. High-frequency high-resolution ultrasound imaging was employed to investigate wound healing under fetal and adult conditions. Quantitative tissue cellularity and elastic strain was obtained for visualization of unresolved inflammation using Vevo strain software. Hemodynamic properties of the blood flow in the artery supplying the wound-site were studied using color Doppler flow imaging. Non-invasive monitoring of fetal and adult wound healing provided unprecedented biomechanical and functional insight. Fetal wounds showed highly accelerated closure with transient perturbation of wound tissue cellularity. Fetal hemodynamics was unique in that sharp fall in arterial pulse pressure (APP) which was rapidly restored within 48h post-wounding. In adults, APP transiently increased post-wounding before returning to the pre-wounding levels by d10 post-wounding. The pattern of change in the elasticity of wound-edge tissue of diabetics was strikingly different. Severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of the non-diabetic group. Wound bed of adult diabetic mice (db/db) showed persistent hypercellularity compared to littermate controls (db/+) indicative of prolonged inflammation. Normal skin strain of db/+ and db/db were asynchronous. In db/db, severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of non-diabetics. This study showcases a versatile clinically relevant imaging platform suitable for real-time analyses of functional wound healing.

At the site of injury, macrophages exit their characteristic phenotype undergoing direct conversion to fibroblasts. Keratinocyte-derived miR-21, packaged in extracellular vesicles, enables such plasticity which accounts for the vast majority of all fibroblasts in the granulation tissue.