Plasma-Activated Air Mediates Plasmid DNA Delivery In Vivo

Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P \u3c 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P \u3c 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets

Vascular Endothelial Growth Factor-A Gene Electrotransfer Promotes Angiogenesis in a Porcine Model of Cardiac Ischemia

This study aimed to assess safety and therapeutic potential of gene electrotransfer (GET) as a method for delivery of plasmid encoding vascular endothelial growth factor A (VEGF-A) to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. GET following plasmid encoding VEGF-A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of the saline vehicle. Animals were monitored for 7 weeks and the hearts were evaluated for angiogenesis, myocardial infarct size and left ventricular contractility. Arteriograms suggest growth of new arteries as early as 2 weeks after treatment in electrotransfer animals. There is a significant reduction of infarct area and left ventricular contractility is improved in GET-treated group compared with controls. There was no significant difference in mortality of animals treated with GET of plasmid encoding VEGF-A from the control groups. Gene delivery of plasmid encoding VEGF-A to ischemic myocardium in a porcine model can be accomplished safely with potential for myocardial repair and regeneration

Pneumatospinning of Collagen Microfibers From Benign Solvents

Introduction. Current collagen fiber manufacturing methods for biomedical applications, such as electrospinning and extrusion, have had limited success in clinical translation, partially due to scalability, cost, and complexity challenges. Here we explore an alternative, simplified and scalable collagen fiber formation method, termed \u27pneumatospinning,\u27 to generate submicron collagen fibers from benign solvents. Methods and results. Clinical grade type I atelocollagen from calf corium was electrospun or pneumatospun as sheets of aligned and isotropic fibrous scaffolds. Following crosslinking with genipin, the collagen scaffolds were stable in media for over a month. Pneumatospun collagen samples were characterized using Fourier-transform infrared spectroscopy, circular dichroism, mechanical testing, and scanning electron microscopy showed consistent fiber size and no deleterious chemical changes to the collagen were detected. Pneumatospun collagen had significantly higher tensile strength relative to electrospun collagen, with both processed from acetic acid. Stem cells cultured on pneumatospun collagen showed robust cell attachment and high cytocompatibility. Using DMSO as a solvent, collagen was further co-pneumatospun with poly(d, l-lactide) to produce a blended microfibrous biomaterial. Conclusions. Collagen microfibers are shown for the first time to be formed using pneumatospinning, which can be collected as anisotropic or isotropic fibrous grafts. Pneumatospun collagen can be made with higher output, lower cost and less complexity relative to electrospinning. As a robust and rapid method of collagen microfiber synthesis, this manufacturing method has many applications in medical device manufacturing, including those benefiting from anisotropic microstructures, such as ligament, tendon and nerve repair, or for applying microfibrous collagen-based coatings to other materials

Mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors

Accumulating data indicates that some cancer treatments can restore anticancer immunosurveillance through the induction of tumor immunogenic cell death (ICD). Nanosecond pulsed electric fields (nsPEF) have been shown to efficiently ablate melanoma tumors. In this study we investigated the mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors. Our data show that in vitro nsPEF (20-200, 200-ns pulses, 7 kV/cm, 2 Hz) caused a rapid dose-dependent cell death which was not accompanied by caspase activation or PARP cleavage. The lack of nsPEF-induced apoptosis was confirmed in vivo in B16F10 tumors. NsPEF also failed to trigger ICD-linked responses such as necroptosis and autophagy. Our results point at necrosis as the primary mechanism of cell death induced by nsPEF in B16F10 cells. We finally compared the antitumor immunity in animals treated with nsPEF (750, 200-ns, 25 kV/cm, 2 Hz) with animals were tumors were surgically removed. Compared to the naïve group where all animals developed tumors, nsPEF and surgery protected 33% (6/18) and 28.6% (4/14) of the animals, respectively. Our data suggest that, under our experimental conditions, the local ablation by nsPEF restored but did not boost the natural antitumor immunity which stays dormant in the tumor-bearing host

Thermal Assisted In Vivo Gene Electrotransfer

Gene electrotransfer is an effective approach for delivering plasmid DNA to a variety of tissues. Delivery of molecules with electric pulses requires control of the electrical parameters to achieve effective delivery. Since discomfort or tissue damage may occur with high applied voltage, the reduction of the applied voltage while achieving the desired expression may be an important improvement. One possible approach is to combine electrotransfer with exogenously applied heat. Previous work performed in vitro demonstrated that increasing temperature before pulsing can enhance gene expression and made it possible to reduce electric fields while maintaining expression levels. In the study reported here, this combination was evaluated in vivo using a novel electrode device designed with an inserted laser for application of heat. The results obtained in this study demonstrated that increased temperature during electrotransfer increased expression or maintained expression with a reduction in applied voltage. With further optimization this approach may provide the basis for both a novel method and a novel instrument that may greatly enhance translation of gene electrotransfer

Mechanisms and Immunogenicity of nsPEF-Induced Cell Death in B16F10 Melanoma Tumors

Accumulating data indicates that some cancer treatments can restore anticancer immunosurveillance through the induction of tumor immunogenic cell death (ICD). Nanosecond pulsed electric fields (nsPEF) have been shown to efficiently ablate melanoma tumors. In this study we investigated the mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors. Our data show that in vitro nsPEF (20-200, 200-ns pulses, 7 kV/cm, 2 Hz) caused a rapid dose-dependent cell death which was not accompanied by caspase activation or PARP cleavage. The lack of nsPEF-induced apoptosis was confirmed in vivo in B16F10 tumors. NsPEF also failed to trigger ICD-linked responses such as necroptosis and autophagy. Our results point at necrosis as the primary mechanism of cell death induced by nsPEF in B16F10 cells. We finally compared the antitumor immunity in animals treated with nsPEF (750, 200-ns, 25 kV/cm, 2 Hz) with animals were tumors were surgically removed. Compared to the naive group where all animals developed tumors, nsPEF and surgery protected 33% (6/18) and 28.6% (4/14) of the animals, respectively. Our data suggest that, under our experimental conditions, the local ablation by nsPEF restored but did not boost the natural antitumor immunity which stays dormant in the tumor-bearing host

Gene Electro Transfer of Plasmid Encoding Vascular Endothelial Growth Factor for Enhanced Expression and Perfusion in the Ischemic Swine Heart

Myocardial ischemia can damage heart muscle and reduce the heart\u27s pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (

Comprehensive Collagen Crosslinking Comparison of Microfluidic Wet-Extruded Microfibers for Bioactive Surgical Suture Development

Collagen microfiber-based constructs have garnered considerable attention for ligament, tendon, and other soft tissue repairs, yet with limited clinical translation due to strength, biocompatibility, scalable manufacturing, and other challenges. Crosslinking collagen fibers improves mechanical properties; however, questions remain regarding optimal crosslinking chemistries, biocompatibility, biodegradation, long-term stability, and potential for biotextile assemble at scale, limiting their clinical usefulness. Here, we assessed over 50 different crosslinking chemistries on microfluidic wet-extruded collagen microfibers made with clinically relevant collagen to optimize collagen fibers as a biotextile yarn for suture or other medical device manufacture. The endogenous collagen crosslinker, glyoxal, provides extraordinary fiber ultimate tensile strength near 300MPa, and Young\u27s modulus of over 3GPa while retaining 50% of the initial load-bearing capacity through 6 months as hydrated. Glyoxal crosslinked collagen fibers further proved cytocompatible and biocompatible per ISO 10993-based testing, and further elicits a predominantly M2 macrophage response. Remarkably these strong collagen fibers are amenable to industrial braiding to form strong collagen fiber sutures. Collagen microfluidic wet extrusion with glyoxal crosslinking thus progress bioengineered, strong, and stable collagen microfibers significantly towards clinical use for potentially promoting efficient healing compared to existing suture materials. STATEMENT OF SIGNIFICANCE: Towards improving clinical outcomes for over 1 million ligament and tendon surgeries performed annually, we report an advanced microfluidic extrusion process for type I collagen microfiber manufacturing for biological suture and other biotextile manufacturing. This manuscript reports the most extensive wet-extruded collagen fiber crosslinking compendium published to date, providing a tremendous recourse to the field. Collagen fibers made with clinical-grade collagen and crosslinked with glyoxal, exhibit tensile strength and stability that surpasses all prior reports. This is the first report demonstrating that glyoxal, a native tissue crosslinker, has the extraordinary ability to produce strong, cytocompatible, and biocompatible collagen microfibers. These collagen microfibers are ideal for advanced research and clinical use as surgical suture or other tissue-engineered medical products for sports medicine, orthopedics, and other surgical indications

Development of an Animal Model for Radiofrequency Ablation of Primary, Virally Induced Hepatocellular Carcinoma in the Woodchuck

To develop a consistent and reproducible method in an animal model for studies of radiofrequency ablation of primary HCC

Direct crystal formation from micronized bone and lactic acid: The writing on the wall for calcium-containing crystal pathogenesis in osteoarthritis?

INTRODUCTION:Pathological calcium-containing crystals accumulating in the joints, synovial fluid, and soft tissues are noted in most elderly patients, yet arthritic crystal formation remains idiopathic. Interestingly, elevated lactic acid and bone erosion are frequently among the comorbidities and clinical features of patients with highest incidence of crystal arthropathies. This work shows that bone particulates (modeling bone erosion) dissolve in lactic acid and directly generate crystals, possibly presenting a mechanism for crystal accumulation in osteoarthritis. METHODS AND RESULTS:Micronized human bone (average particle size of 160 μm x 79 μm) completely dissolved in lactic acid in 48 hours, and in synovial fluid with 500 mMol lactic acid in 5 days, generating birefringent rhomboid and rod-shaped crystals. SEM analysis with energy dispersive x-ray spectroscopy of these crystals showed average dimensions of around 2 μm x 40 μm, which contained oxygen, calcium and phosphorous at 8.64:1.85:1. Raman spectroscopy of the generated crystals further showed 910/cm and 1049/cm peaks, aligning with calcium oxalate monohydrate and calcium pyrophosphate, respectively. CONCLUSIONS:This work shows that lactic acid and micronized mineralized bone together directly generate calcium-containing crystals. These observations may provide insights into the elusive etiology of arthritis with crystal involvement, possibly indicating lactic acid as a clinical target for treatment