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Extracellular vesicles (EVs) are nanovesicles released by all eukaryotic cells. This work reports the first nanoscale fluorescent visualization of tumor-originating vesicles bearing an angiogenic microRNA (miR)-126 cargo. In a validated experimental model of lethal murine vascular neoplasm, tumor-originating EV delivered its miR-126 cargo to tumor-associated macrophages (TAMs). Such delivery resulted in an angiogenic (LYVE

Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and β-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

Hollow needle array-based tissue nanotransfection (TNT) presents an in vivo transfection approach that directly translocate exogeneous genes to target tissues by using electric pulses. In this work, the gene delivery process of TNT was simulated and experimentally validated. We adopted the asymptotic method and cell-array-based model to investigate the electroporation behaviors of cells within the skin structure. The distribution of nonuniform electric field across the skin results in various electroporation behavior for each cell. Cells underneath the hollow microchannels of the needle exhibited the highest total pore numbers compared to others due to the stronger localized electric field. The percentage of electroporated cells within the skin structure, with pore radius over 10 nm, increases from 25% to 82% as the applied voltage increases from 100 to 150 V/mm. Furthermore, the gene delivery behavior across the skin tissue was investigated through the multilayer-stack-based model. The delivery distance increased nonlinearly as the applied voltage and pulse number increased, which mainly depends on the diffusion characteristics and electric conductivity of each layer. It was also found that the skin is required to be exfoliated prior to the TNT procedure to enhance the delivery depth. This work provides the foundation for transition from the study of murine skin to translation use in large animals and human settings.

Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In

Wireless electroceutical dressing (WED) fabric kills bacteria and disrupts bacterial biofilm. This work tested, comparing with standard of care topical antibiotic ketoconazole, whether the weak electric field generated by WED is effective to manage infection caused by ketoconazole-resistant yeast Candida albicans. WED inhibited Candida albicans biofilm formation and planktonic growth. Unlike ketoconazole, WED inhibited yeast to hyphal transition and downregulated EAP1 curbing cell attachment. In response to WED-dependent down-regulation of biofilm-forming BRG1 and ROB1, BCR1 expression was markedly induced in what seems to be a futile compensatory response. WED induced NRG1 and TUP1, negative regulators of filamentation; it down-regulated EFG1, a positive regulator of hyphal pathway. Consistent with the anti-hyphal properties of WED, the expression of ALS3 and HWP1 were diminished. Ketoconazole failed to reproduce the effects of WED on NRG1, TUP1 and EFG1. WED blunted efflux pump activity; this effect was in direct contrast to that of ketoconazole. WED exposure compromised cellular metabolism. In the presence of ketoconazole, the effect was synergistic. Unlike ketoconazole, WED caused membrane depolarization, changes in cell wall composition and loss of membrane integrity. This work presents first evidence that weak electric field is useful in managing pathogens which are otherwise known to be antibiotic resistant.

Bidirectional cell-cell communication via paracrine mechanisms involving nano-sized extracellular vesicles have emerged as a predominant mechanism of cellular signaling. Unlike other shedding vesicles of similar size, exosomes selectively package their cargo using defined mechanisms within the cells. Recent research on exosome signaling describes a messenger-recipient cell dichotomy. The heterogeneous origin of exosome populations, although previously described, has as-yet been incompletely characterized using this dichotomy and thus does not currently provide a complete understanding of exosome populations. In this work, we outline the fundamentally bidirectional nature of exosomes and replace this dichotomy with a messenger-recipient-effector network formed by repackaging and rerelease events. This network further confounds the determination of messenger cell identity among an already heterogeneous exosome population and has major implications for future clinical application. Redefining the axiom of exosome signaling provides a route for future research to consider a multi-system-based approach and underscores a need for enhanced identification methods. This shift also has implications for the use of exosomes as therapeutic agents. Exosome biogenesis and its manipulation will be crucial for the development of curative endogenous exosomes and their synthetic, exogenously produced counterparts. Directed cargo loading, optimal shell composition, and robust production platforms are just some of the design aspects that need to be considered. As tissue-specific therapeutic agents, exosome design will also need to incorporate repackaging mechanics to prevent off-target effects and increase efficacy. A comprehensive current understanding of exosome biogenesis mechanisms amidst the heterogenous EV population will propel the field towards clinical viability.

We report a minimally invasive, synaptic transistor-based construct to monitor in vivo neuronal activity via a longitudinal study in mice and use depolarization time from measured data to predict the onset of polyneuropathy. The synaptic transistor is a three-terminal device in which ionic coupling between pre- and post-synaptic electrodes provides a framework for sensing low-power (sub μW) and high-bandwidth (0.1–0.5 kHz) ionic currents. A validated first principles-based approach is discussed to demonstrate the significance of this sensing framework and we introduce a metric, referred to as synaptic efficiency to quantify structural and functional properties of the electrodes in sensing. The application of this framework for in vivo neuronal sensing requires a post-synaptic electrode and its reference electrode and the tissue becomes the pre-synaptic signal. The ionic coupling resembles axo-axonic junction and hence we refer to this framework as an ad hoc synaptic junction. We demonstrate that this arrangement can be applied to measure excitability of sciatic nerves due to a stimulation of the footpad in cohorts of m+/db and db/db mice for detecting loss in sensitivity and onset of polyneuropathy. The signal attributes were subsequently integrated with machine learning-based framework to identify the probability of polyneuropathy and to detect the onset of diabetic polyneuropathy.

Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exo(κ-GFP)) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exo(κ-GFP) were also characterized. Unlike skin exosome, wound-edge Exo(κ-GFP) demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by wound-macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exo(κ-GFP) to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exo(κ-GFP) in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNP(κ)) were designed with 94.3% encapsulation efficiency. Application of TLNP(κ/si-hnRNPA2B1) to murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to TLNP(κ/si-control) group. Although no significant difference in wound closure or re-epithelialization was observed, TLNP(κ/si-hnRNPA2B1) treated group showed significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to TLNP(κ/si-control) group. Furthermore, TLNP(κ/si-hnRNPA2B1) treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exo(κ-GFP) are recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties post-injury.

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 0.5V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Respiratory coronavirus particles (105) were placed in direct contact with the fabric for 1 or 5 minutes. Viral particles (2.5-4x104) were recovered from the fabric. Following one minute of contact, zeta potential of the 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.

Nanotechnology research is a rapidly expanding to treat a variety of ailments that a patient may be experiencing. In the scope of diabetes, such research and development include glucose monitoring is one with the use of nanosensors, the management of wounds, and the promotion of wound healing. Nanosensors can simplify the glucose-monitoring process by providing much less noninvasive and increased feedback to the patient about glucose concentrations over traditional glucose monitors. As for the chronic wounds that are often concomitant with diabetes, notably via a pressure ulcer on the foot, nanotechnology seeks to correct this otherwise dysregulated wound-healing process. More specifically, nanotechnology aims to aid the wound-healing process through hemostasis, infection mitigation, control of inflammation, and promotion of cell regeneration and skin remodeling.

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., Graphical Abstract, Highlights • Hyperbiofilm clinical isolate PAO1ΔwspF contain unique cell state and organization • Bacterial cells in PAO1ΔwspF biofilm are morphologically and physiologically unique • PAO1ΔwspF, unlike PAO1 that undergo explosive lysis, release eDNA from live cells • Aurine tricarboxylic acid, not DNAseI as for PAO1, disrupts PAO1ΔwspF biofilm, Microbiology; Microbiofilms

Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue., 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.

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency)1–6. In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing7,8. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection9,10. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods11,12. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

This work rests on our recent report on the successful use of tissue nanotransfection (TNT) delivery of Ascl1, Brn2, and Myt1l (TNT(ABM)) to directly convert skin fibroblasts into electrophysiologically active induced neuronal cells (iN) in vivo. Here we report that in addition to successful neurogenic conversion of cells, TNT(ABM) caused neurotrophic enrichment of the skin stroma. Thus, we asked whether such neurotrophic milieu of the skin can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Topical cutaneous TNT(ABM) caused elevation of endogenous NGF and other co-regulated neurotrophic factors such as Nt3. TNT(ABM) spared loss of cutaneous PGP9.5(+) mature nerve fibers in db/db diabetic mice. This is the first study demonstrating that under conditions of in vivo reprogramming, changes in the tissue microenvironment can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from its predictable path of loss under conditions of diabetes.

OBJECTIVE: Shilajit is a pale-brown to blackish-brown organic mineral substance available from Himalayan rocks. We demonstrated that in type I obese humans, shilajit supplementation significantly upregulated extracellular matrix (ECM)–related genes in the skeletal muscle. Such an effect was highly synergistic with exercise. The present study (clinicaltrials.gov ) aimed to evaluate the effects of shilajit supplementation on skin gene expression profile and microperfusion in healthy adult females. METHODS: The study design comprised six total study visits including a baseline visit (V1) and a final 14-week visit (V6) following oral shilajit supplementation (125 or 250 mg bid). A skin biopsy of the left inner upper arm of each subject was collected at visit 2 and visit 6 for gene expression profiling using Affymetrix Clariom™ D Assay. Skin perfusion was determined by MATLAB processing of dermascopic images. Transcriptome data were normalized and subjected to statistical analysis. The differentially regulated genes were subjected to Ingenuity Pathway Analysis (IPA(®)). The expression of the differentially regulated genes identified by IPA(®) were verified using real-time polymerasechain reaction (RT-PCR). RESULTS: Supplementation with shilajit for 14 weeks was not associated with any reported adverse effect within this period. At a higher dose (250 mg bid), shilajit improved skin perfusion when compared to baseline or the placebo. Pathway analysis identified shilajit-inducible genes relevant to endothelial cell migration, growth of blood vessels, and ECM which were validated by quantitative real-time polymerasechain reaction (RT-PCR) analysis. CONCLUSIONS: This work provides maiden evidence demonstrating that oral shilajit supplementation in adult healthy women induced genes relevant to endothelial cell migration and growth of blood vessels. Shilajit supplementation improved skin microperfusion.

Objective The objective of this work was to causatively link biofilm properties of bacterial infection to specific pathogenic mechanisms in wound healing. Background Staphylococcus aureus is one of the four most prevalent bacterial species identified in chronic wounds. Causatively linking wound pathology to biofilm properties of bacterial infection is challenging. Thus, isogenic mutant stains of S. aureus with varying degree of biofilm formation ability was studied in an established preclinical porcine model of wound biofilm infection. Methods Isogenic mutant strains of S. aureus with varying degree (ΔrexB > USA300 > ΔsarA) of biofilm-forming ability were used to infect full-thickness porcine cutaneous wounds. Results Compared with that of ΔsarA infection, wound biofilm burden was significantly higher in response to ΔrexB or USA300 infection. Biofilm infection caused degradation of cutaneous collagen, specifically collagen 1 (Col1), with ΔrexB being most pathogenic in that regard. Biofilm infection of the wound repressed wound-edge miR-143 causing upregulation of its downstream target gene matrix metalloproteinase-2. Pathogenic rise of collagenolytic matrix metalloproteinase-2 in biofilm-infected wound-edge tissue sharply decreased collagen 1/collagen 3 ratio compromising the biomechanical properties of the repaired skin. Tensile strength of the biofilm infected skin was compromised supporting the notion that healed wounds with a history of biofilm infection are likely to recur. Conclusion This study provides maiden evidence that chronic S. aureus biofilm infection in wounds results in impaired granulation tissue collagen leading to compromised wound tissue biomechanics. Clinically, such compromise in tissue repair is likely to increase wound recidivism.

Peripheral vasculopathies cause severe wound hypoxia inducing the hypoxamiR miR-210. High level of miR-210, persisting in wound-edge tissue as ischemic memory, suppresses oxidative metabolism and inhibits cell proliferation necessary for healing. In wound-edge tissue of chronic wound patients, elevated miR-210 was tightly associated with inhibition of epidermal cell proliferation as evident by lowered Ki67 immunoreactivity. To inhibit miR-210 in murine ischemic wound-edge tissue, we report the formulation of antihypoxamiR functionalized gramicidin lipid nanoparticles (AFGLN). A single intradermal delivery of AFGLN encapsulating LNA-conjugated anti-hypoximiR-210 (AFGLNmiR-210) lowered miR-210 level in the ischemic wound-edge tissue. In repTOP™mitoIRE mice, AFGLNmiR-210 rescued keratinocyte proliferation as visualized by in vivo imaging system (IVIS). 31P NMR studies showed elevated ATP content at the ischemic wound-edge tissue following AFGLNmiR-210 treatment indicating recovering bioenergetics necessary for healing. Consistently, AFGLNmiR-210 improved ischemic wound closure. The nanoparticle based approach reported herein is effective for miR-directed wound therapeutics warranting further translational development.

Wound assessment relies on visual evaluation by physicians. Such assessment is largely subjective and presents the opportunity to explore the use of emergent technologies.Emergent and powerful noninvasive imaging technologies applicable to assess burn and chronic wounds are reviewed.The need to estimate wound depth is critical in both chronic wound and burn injury settings. Harmonic ultrasound technology is powerful to study wound depth. It addresses the limitations of optical imaging with limited depth of penetration. What if a wound appears epithelialized by visual inspection, which shows no discharge yet is covered by repaired skin that lacks barrier function? In this case although the wound is closed as defined by current standards, it remains functionally open, presenting the risk of infection and other postclosure complications. Thus, assessment of skin barrier function is valuable in the context of assessing wound closure. Options for the study of tissue vascularization are many. If noncontact and noninvasive criteria are of importance, laser speckle imaging is powerful. Fluorescence imaging is standard in several clinical settings and is likely to serve the wound clinics well as long as indocyanine green injection is not of concern. A major advantage of harmonic ultrasound imaging of wound depth is that the same system is capable of providing information on blood flow dynamics in arterial perforators.With many productive imaging platforms to choose from, wound care is about to be transformed by technology that would help assess wound severity.

Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal role in the scavenging of apoptotic cells from affected tissue. We have previously reported that apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. In this work, we test the hypothesis that MFG-E8 helps resolve inflammation, supports angiogenesis, and accelerates wound closure. MFG-E8−/− mice displayed impaired efferocytosis associated with exaggerated inflammatory response, poor angiogenesis, and wound closure. Wound macrophage-derived MFG-E8 was recognized as a critical driver of wound angiogenesis. Transplantation of MFG-E8−/− bone marrow to MFG-E8+/+ mice resulted in impaired wound closure and compromised wound vascularization. In contrast, MFG-E8−/− mice that received wild-type bone marrow showed improved wound closure and improved wound vascularization. Hyperglycemia and exposure to advanced glycated end products inactivated MFG-E8, recognizing a key mechanism that complicates diabetic wound healing. Diabetic db/db mice suffered from impaired efferocytosis accompanied with persistent inflammation and slow wound closure. Topical recombinant MFG-E8 induced resolution of wound inflammation, improvements in angiogenesis, and acceleration of closure, upholding the potential of MFG-E8–directed therapeutics in diabetic wound care.

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2 , Ascl1 , and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2 , and that some induced neurons passed through a nestin-positive cell stage. From the Clinical Editor In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

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.

BACKGROUND: Diabetic wounds have become one of the most challenging public health issues of the 21st century, yet there is no effective treatment available. We have previously shown that the diabetic wound healing impairment is associated with increased inflammation and decreased expression of the regulatory microRNA miR-146a. We have conjugated miR-146a to cerium oxide nanoparticles (CNP-miR146a) to target reactive oxygen species (ROS) and inflammation. This study aimed to evaluate the consequences of CNP-miR146a treatment of diabetic wounds. STUDY DESIGN: Eight-millimeter wounds were created on the dorsal skin of Db/Db mice and treated with PBS or differing concentrations of CNP-mir146a (1; 10; 100; or 1,000 ng) at the time of wounding. Rate of wound closure was measured until the wounds were fully healed. At 4 weeks post-healing, a dumbbell-shaped skin sample was collected, with the healed wound in the center, and an Instron 5942 testing unit was used to measure the maximum load and modulus. RESULTS: Our data showed that diabetic wounds treated with PBS or 1 ng CNP-miR146a took 18 days to heal. Treatment with 10, 100, or 1,000 ng of CNP+miR-146a effectively enhanced healing, and wounds were fully closed at day 14 post-wounding. The healed skin from the CNP-miR146a-treated group showed a trend of improved biomechanical properties (increased maximum load and modulus), however it did not reach significance. CONCLUSIONS: We found that a 100-ng dose of CNP-miR146a improved diabetic wound healing and did not impair the biomechanical properties of the skin post-healing. This nanotechnology-based therapy is promising, and future studies are warranted to transfer this therapy to clinical application. (J Am Coll Surg 2019;228:107–115. 2018 by the American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Tissue repair and regeneration rely on the function of miRNA, molecular silencers that enact post-transcriptional gene silencing of coding genes. Disruption of miRNA homeostasis is developmentally lethal, indicating that fetal tissue development is tightly controlled by miRNAs. Multiple critical facets of adult tissue repair are subject to control by miRNAs, as well. Sources of cell pool for tissue repair and regeneration are diverse and provided by processes including cellular dedifferentiation, transdifferentiation, and reprogramming. Each of these processes is regulated by miRNAs. Furthermore, induced pluripotency may be achieved by miRNA-based strategies independent of transcription factor manipulation. The observation that miRNA does not integrate into the genome makes miRNA-based therapeutic strategies translationally valuable. Tools to manipulate cellular and tissue miRNA levels include mimics and inhibitors that may be specifically targeted to cells of interest at the injury site. Here, we discuss the extraordinary importance of miRNAs in tissue repair and regeneration based on emergent reports and rapid advances in miRNA-based therapeutics.

Tissue injury transiently silences miRNA-dependent posttranscriptional gene silencing in its effort to unleash adult tissue repair. Once the wound is closed, miRNA biogenesis is induced averting neoplasia. In this work, we report that Dicer plays an important role in reestablishing the barrier function of the skin post-wounding via a miRNA-dependent mechanism. MicroRNA expression profiling of skin and wound-edge tissue revealed global upregulation of miRNAs following wound closure at day 14 post-wounding with significant induction of Dicer expression. Barrier function of the skin, as measured by trans-epidermal water loss, was compromised in keratinocyte-specific conditional (K14/Lox-Cre) Dicer-ablated mice because of malformed cornified epithelium lacking loricrin expression. Studies on human keratinocytes recognized that loricrin expression was inversely related to the expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). Compared to healthy epidermis, wound-edge keratinocytes from Dicer-ablated skin epidermis revealed elevated p21(Waf1/Cip1) expression. Adenoviral and pharmacological suppression of p21(Waf1/Cip1) in keratinocyte-specific conditional Dicer-ablated mice improved wound healing indicating a role of Dicer in the suppression of p21(Waf1/Cip1). This work upholds p21(Waf1/Cip1) as a druggable target to restore barrier function of skin suffering from loss of Dicer function as would be expected in diabetes and other forms of oxidant insult.

Significance: Angiogenesis is the process by which new blood vessels develop from a pre-existing vascular system. It is required for physiological processes such as developmental biology and wound healing. Angiogenesis also plays a crucial role in pathological conditions such as tumor progression. The underlying importance of angiogenesis necessitates a highly regulated process. Recent Advances: Recent works have demonstrated that the process of angiogenesis is regulated by small noncoding RNA molecules called microRNAs (miRs). These miRs, collectively referred to as angiomiRs, have been reported to have a profound effect on the process of angiogenesis by acting as either pro-angiogenic or anti-angiogenic regulators. Critical Issues: In this review, we will discuss the role of miR-200b as a regulator of angiogenesis. Once the process of angiogenesis is complete, anti-angiogenic miR-200b has been reported to provide necessary braking. Downregulation of miR-200b has been reported across various tumor types, as deregulated angiogenesis is necessary for tumor development. Transient downregulation of miR-200b in wounds drives wound angiogenesis. Future Directions: New insights and understanding of the molecular mechanism of regulation of angiogenesis by miR-200b has opened new avenues of possible therapeutic interventions to treat angiogenesis-related patho-physiological conditions. Antioxid. Redox Signal. 22, 1257–1272.

Lyophilized keratinocyte-targeted nanocarriers (TLN(κ)) loaded with locked nucleic acid (LNA) modified anti-miR were developed for topical application to full thickness burn injury. TLN(κ) were designed to selectively deliver LNA-anti-miR-107 to keratinocytes using the peptide sequence ASKAIQVFLLAG. TLN(κ) employed DOTAP/DODAP combination pH-responsive lipid components to improve endosomal escape. To minimize interference of clearance by non-targeted cells, especially immune cells in the acute wound microenvironment, surface charge was neutralized. Lyophilization was performed to extend the shelf life of the lipid nanoparticles (LNPs). Encapsulation efficiency of anti-miR in lyophilized TLN(κ) was estimated to be 96.54%. Cargo stability of lyophilized TLN(κ) was tested. After 9 days of loading with anti-miR-210, TLN(κ) was effective in lowering abundance of the hypoxamiR miR-210 in keratinocytes challenged with hypoxia. Keratinocyte uptake of DiD-labeled TLN(κ) was selective and exceeded 90% within 4 hr. Topical application of hydrogel-dispersed lyophilized TLN(κ) encapsulating LNA anti-miR-107 twice a week significantly accelerated wound closure and restoration of skin barrier function. TLN(κ/anti-miR-107) application depleted miR-107 and upregulated dicer expression, which accelerated differentiation of keratinocytes. Expression of junctional proteins such as claudin-1, loricrin, filaggrin, ZO-1, and ZO-2 were significantly upregulated following TLN(κ/anti-miR-107) treatment. These LNPs are promising as topical therapeutic agents in the management of burn injury.

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. In vivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

Unlike the epidermis, which regenerates continually, hair follicles anchored in the subcutis periodically regenerate by spontaneous repetitive cycles of growth (anagen), degeneration (catagen), and rest (telogen). The loss of hair follicles in response to injuries or pathologies such as alopecia endangers certain inherent functions of the skin. Thus, it is of interest to understand mechanisms underlying follicular regeneration in adults. In this work, a phytochemical rich in the natural vitamin E tocotrienol (TRF) served as a productive tool to unveil a novel epidermal pathway of hair follicular regeneration. Topical TRF application markedly induced epidermal hair follicle development akin to that during fetal skin development. This was observed in the skin of healthy as well as diabetic mice, which are known to be resistant to anagen hair cycling. TRF suppressed epidermal E-cadherin followed by 4-fold induction of β-catenin and its nuclear translocation. Nuclear β-catenin interacted with Tcf3. Such sequestration of Tcf3 from its otherwise known function to repress pluripotent factors induced the plasticity factors Oct4, Sox9, Klf4, c-Myc, and Nanog. Pharmacological inhibition of β-catenin arrested anagen hair cycling by TRF. This work reports epidermal E-cadherin/β-catenin as a novel pathway capable of inducing developmental folliculogenesis in the adult skin.

Cutaneous microvasculopathy complicates wound healing. Functional assessment of gated individual dermal microvessels is therefore of outstanding interest. Functional performance of laser speckle contrast imaging (LSCI) systems is compromised by motion artefacts. To address such weakness, post-processing of stacked images is reported. We report the first post-processing of binary raw data from a high-resolution LSCI camera. Sharp images of low-flowing microvessels were enabled by introducing inverse variance in conjunction with speckle contrast in Matlab-based program code. Extended moving window averaging enhanced signal-to-noise ratio. Functional quantitative study of blood flow kinetics was performed on single gated microvessels using a free hand tool. Based on detection of flow in low-flow microvessels, a new sharp contrast image was derived. Thus, this work presents the first distinct image with quantitative microperfusion data from gated human foot microvasculature. This versatile platform is applicable to study a wide range of tissue systems including fine vascular network in murine brain without craniotomy as well as that in the murine dorsal skin. Importantly, the algorithm reported herein is hardware agnostic and is capable of post-processing binary raw data from any camera source to improve the sensitivity of functional flow data above and beyond standard limits of the optical system.