Multiaxial filament winding of biopolymer microfibers with a collagen resin binder for orthobiologic medical device biomanufacturing.

INTRO: Multiaxial filament winding is an additive manufacturing technique used extensively in large industrial and military manufacturing yet unexplored for biomedical uses. This study adapts filament winding to biomanufacture scalable, strong, three-dimensional microfiber (3DMF) medical device implants for potential orthopedic applications. Methods: Polylactide, polydioxanone, or nanocellulose microfiber filaments were wound through a collagen "resin" bath to create organized, stable orthobiologic implants, which are sized for common ligament (e.g., anterior cruciate ligament) and tendon (e.g., rotator cuff) injuries and can be manufactured at industrial scale using a small footprint, economical, high-output benchtop system. Ethylene oxide or electron beam sterilized 3DMF samples were analyzed by scanning electron microscopy (SEM), underwent ASTM1635-based degradation testing, tensile testing, ISO 10993-based cytocompatibility, and biocompatibility testing, quantified for human platelet-rich plasma (PRP) absorption kinetics, and examined for adhesion of bioceramics and lyophilized collagen after coating. Results: 3DMF implants had consistent fiber size and high alignment by SEM. Negligible mass and strength loss were noted over 4 months in culture. 3DMF implants initially exceeded 1,000 N hydrated tensile strength and retained over 70% strength through 4 months in culture, significantly stronger than conventionally produced implants made by fused fiber deposition 3D printing. 3DMF implants absorbed over 3x their weight in PRP within 5 minutes, were cytocompatible and biocompatible, and could readily bind tricalcium phosphate and calcium carbonate coatings discretely on implant ends for further orthobiologic material functionalization. The additive manufacturing process further enabled engineering implants with suture-shuttling passages for facile arthroscopic surgical delivery. Conclusion and Clinical Significance: This accessible, facile, economical, and rapid microfiber manufacturing platform presents a new method to engineer high-strength, flexible, low-cost, bio-based implants for orthopedic and extended medical device applications. .

Gene electrotransfer of FGF2 enhances collagen scaffold biocompatibility.

Tendon injuries are a common athletic injury that have been increasing in prevalence. While there are current clinical treatments for tendon injuries, they have relatively long recovery times and often do not restore native function of the tendon. In the current study, gene electrotransfer (GET) parameters for delivery to the skin were optimized with monophasic and biphasic pulses with reporter and effector genes towards optimizing underlying tendon healing. Tissue twitching and damage, as well as gene expression and distribution were evaluated. Bioprinted collagen scaffolds, mimicking healthy tendon structure were then implanted subcutaneously for biocompatibility and angiogenesis analyses when combined with GET to accelerate healing. GET of human fibroblast FGF2 significantly increased angiogenesis and biocompatibility of the bioprinted implants when compared to implant only sites. The combination of bioprinted collagen fibers and angiogenic GET therapy may lead to better graft biocompatibility in tendon repair.

Reduction of plasmid vector backbone length enhances reporter gene expression.

Gene therapy has a wide range of applications for various types of pathologies. Viral methods of gene delivery provide high levels of gene expression but have various safety concerns. Non-viral methods are largely known to provide lower levels of expression. We aim to address this issue by using plasmid DNA with smaller backbones to increase gene expression levels when delivered using non-viral methods. In this study we compare gene expression levels between two vectors with firefly luciferase encoding gene insert using liposome complexes and gene electrotransfer as delivery methods. A 2-fold reduction in plasmid vector backbone size, disproportionately enhanced gene expression levels more than 10-fold in rat tenocytes in vitro, and rat myocardium in vivo, while improvements in delivery to the skin were more moderate.

Assembled Cell-Decorated Collagen (AC-DC) Fiber Bioprinted Implants with Musculoskeletal Tissue Properties Promote Functional Recovery in Volumetric Muscle Loss.

Musculoskeletal tissue injuries, including volumetric muscle loss (VML), are commonplace and often lead to permanent disability and deformation. Addressing this healthcare need, an advanced biomanufacturing platform, assembled cell-decorated collagen (AC-DC) bioprinting, is invented to rapidly and reproducibly create living biomaterial implants, using clinically relevant cells and strong, microfluidic wet-extruded collagen microfibers. Quantitative analysis shows that the directionality and distribution of cells throughout AC-DC implants mimic native musculoskeletal tissue. AC-DC bioprinted implants further approximate or exceed the strength and stiffness of human musculoskeletal tissue and exceed collagen hydrogel tensile properties by orders of magnitude. In vivo, AC-DC implants are assessed in a critically sized muscle injury in the hindlimb, with limb torque generation potential measured over 12 weeks. Both acellular and cellular implants promote functional recovery compared to the unrepaired group, with AC-DC implants containing therapeutic muscle progenitor cells promoting the highest degree of recovery. Histological analysis and automated image processing of explanted muscle cross-sections reveal increased total muscle fiber count, median muscle fiber size, and increased cellularization for injuries repaired with cellularized implants. These studies introduce an advanced bioprinting method for generating musculoskeletal tissue analogs with near-native biological and biomechanical properties with the potential to repair myriad challenging musculoskeletal injuries.

Cardioporation enhances myocardial gene expression in rat heart.

Damage from myocardial infarction (MI) and subsequent heart failure are serious public health concerns. Current clinical treatments and therapies to treat MI damage largely do not address the regeneration of cardiomyocytes. In a previous study, we established that it is possible to promote regeneration of cardiac muscle with vascular endothelial growth factor B gene delivery directly to the ischemic myocardium. In the current study we aim to optimize cardioporation parameters to increase expression efficiency by varying electrode configuration, applied voltage, pulse length, and plasmid vector size. By using a surface monopolar electrode, optimized pulsing conditions and reducing vector size, we were able to prevent ventricular fibrillation, increase survival, reduce tissue damage, and significantly increase gene expression levels.

Monopolar gene electrotransfer enhances plasmid DNA delivery to skin.

A novel monopolar electroporation system and methodologies were developed for in vivo electroporation intended for potential clinical applications such as gene therapy. We hypothesized that an asymmetric anode/cathode electrode applicator geometry could produce favorable electric fields for electroporation, without the typical drawback associated with traditional needle and parallel plate geometries. Three monopolar electrode applicator prototypes were built and tested for gene delivery of reporter genes to the skin in a guinea pig model. Gene expression was evaluated in terms of kinetics over time and expression distribution within the treatment site. Different pulsing parameters, including pulse amplitude, pulse duration, and pulse number were evaluated. Monopolar gene electrotransfer significantly enhanced gene expression compared to controls over the course of 21 days. Gene expression distribution was observed throughout the full thickness of the epidermis, as well as notable expression in the deeper layers of the skin, including the dermis, and the underlying striated muscle without any damage at the treatment site, which is a substantial improvement over previously reported expression confined to the epidermis only. Expression distribution observed is consistent with the electric field distribution model, indicating that our novel electrode geometry results in targeted electroporation and gene transfer. This is important, as it may facilitate translation of many electroporation-based clinical therapies including gene therapies, IRE, and ECT.

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's 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.

Biomanufacturing organized collagen-based microfibers as a Tissue ENgineered Device (TEND) for tendon regeneration.

Approximately 800, 000 surgical repairs are performed annually in the U.S. for debilitating injuries to ligaments and tendons of the foot, ankle, knee, wrist, elbow and shoulder, presenting a significant healthcare burden. To overcome current treatment shortcomings and advance the treatment of tendon and ligament injuries, we have developed a novel electrospun Tissue ENgineered Device (TEND), comprised of type I collagen and poly(D,L-lactide) (PDLLA) solubilized in a benign solvent, dimethyl sulfoxide (DMSO). TEND fiber alignment, diameter and porosity were engineered to enhance cell infiltration leading to promote tissue integration and functional remodeling while providing biomechanical stability. TEND rapidly adsorbs blood and platelet-rich-plasma (PRP), and gradually releases growth factors over two weeks. TEND further supported cellular alignment and upregulation of tenogenic genes from clinically relevant human stem cells within three days of culture. TEND implanted in a rabbit Achilles tendon injury model showed new in situ tissue generation, maturation, and remodeling of dense, regularly oriented connective tissue in vivo. In all, TEND's organized microfibers, biological fluid and cell compatibility, strength and biocompatiblility make significant progress towards clinically translating electrospun collagen-based medical devices for improving the clinical outcomes of tendon injuries.

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.

Coalesced thermal and electrotransfer mediated delivery of plasmid DNA to the skin.

Efficient gene delivery and expression in the skin can be a promising minimally invasive technique for therapeutic clinical applications for immunotherapy, vaccinations, wound healing, cancer, and peripheral artery disease. One of the challenges for efficient gene electrotransfer (GET) to skin in vivo is confinement of expression to the epithelium. Another challenge involves tissue damage. Optimizing gene expression profiles, while minimizing tissue damage are necessary for therapeutic applications. Previously, we established that heating pretreatment to 43 °C enhances GET in vitro. We observed a similar trend in vivo, with an IR-pretreatment for skin heating prior to GET. Currently, we tested a range of GET conditions in vivo in guinea pigs with and without preheating the skin to ~43 °C. IR-laser heating and conduction heating were tested in conjunction with GET. In vivo electrotransfer to the skin by moderately elevating tissue temperature can lead to enhanced gene expression, as well as achieve gene transfer in epidermal, dermal, hypodermal and muscle tissue layers.

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.

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 'pneumatospinning,' 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.

VEGF-B electrotransfer mediated gene therapy induces cardiomyogenesis in a rat model of cardiac ischemia.

Atherosclerosis induced myocardial infarction (MI) continues to be a major public health concern. Regenerative therapies that restore cardiac muscle cells are largely absent. The rate of cardiomyogenesis in adults is insufficient to compensate for MI damage. In this study, we explored the capacity of a gene therapy approach to promote cardiomyogenesis. We hypothesized that VEGF-B, critical during fetal heart development, could promote cardiomyogenesis in adult ischemic hearts. Gene electrotransfer (GET), a physical method of in vivo gene delivery, was adapted to the rat model of MI. Favorable pulsing parameters were then used for delivery of pVEGF-B and compared to a sham control in terms of infarct size, cardiomyocyte proliferation and presence of new cardiomyocytes. Ki67 immunoreactivity was used for proliferation analysis. Newly synthetized DNA was labeled with BrdU to identify new cells post-infarction. Cardiac troponin co-localization indicated proliferating and new cardiomyocytes histologically. Eight weeks post-treatment, GET pVEGF-B treated hearts had significantly smaller infarcts than the sham control group (p < 0.04). Proliferating and new cardiomyocytes were only present in the GET of pVEGF-B group, and absent in the controls. In summary, GET pVEGF-B promoted cardiomyogenesis post-MI, demonstrating for the first time direct evidence of myocardial regeneration post-infarction.

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

INTRODUCTION: Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. METHODS AND RESULTS: Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. CONCLUSION: Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. STATEMENT OF SIGNIFICANCE: This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

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.

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.

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 < 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 < 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.

Recellularized human dermis for testing gene electrotransfer ex vivo.

Gene electrotransfer (GET) is a proven and valuable tool for in vivo gene delivery to a variety of tissues such as skin, cardiac muscle, skeletal muscle, and tumors, with controllable gene delivery and expression levels. Optimizing gene expression is a challenging hurdle in preclinical studies, particularly for skin indications, due to differences in electrical conductivity of animal compared to human dermis. Therefore, the goal of this study was to develop an ex vivo model for GET using recellularized human dermis to more closely mimic human skin. Decellularized human dermis (DermACELL(®)) was cultured with human dermal fibroblasts and keratinocytes for 4 weeks. After one week of fibroblast culture, fibroblasts infiltrated and dispersed throughout the dermis. Air-liquid interface culture led to epithelial cell proliferation, stratification and terminal differentiation with distinct basal, spinous, granular and cornified strata. Firefly luciferase expression kinetics were evaluated after GET of recellularized constructs for testing gene delivery parameters to skin in vitro. Elevated luciferase expression persisted up to a week following GET compared to controls without electrotransfer. In summary, recellularized dermis structurally and functionally resembled native human skin in tissue histological organization and homeostasis, proving an effective 3D human skin model for preclinical gene delivery studies.

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's 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 (p<0.03), 40 Volts (p<0.05), and 90 Volts (p<0.05), but not with 60 Volts (p<0.09) while maintaining a pulse width of 20 milliseconds. The group with gene electro transfer of plasmid encoding vascular endothelial growth factor had increased perfusion in the area at risk compared to control groups. Troponin and creatine kinase increased across all groups, suggesting equivalent ischemia in all groups prior to treatment. Echocardiography was used to assess ejection fraction, cardiac output, stroke volume, left ventricular end diastolic volume, and left ventricular end systolic volume. No statistically significant differences in these parameters were detected during a 2-week time period. However, directional trends of these variables were interesting and offer valuable information about the feasibility of gene electro transfer of vascular endothelial growth factor in the ischemic heart. The results demonstrate that gene electro transfer can be applied safely and can increase perfusion in an ischemic area. Additional study is needed to evaluate potential efficacy.

Enhanced chemoresistance of squamous carcinoma cells grown in 3D cryogenic electrospun scaffolds.

It is critically important to study head and neck squamous cell carcinoma tumorigenic mechanisms in order to gain a better understanding of tumor development, progression, and treatment. Unfortunately, a representative three-dimensional (3D) model for these evaluations has yet to be developed. The purpose of this study was to replicate tumor extracellular matrix (ECM) morphology utilizing electrospinning technology. First, the tumor ECM was evaluated by decellularizing tumor samples and analyzing the fibrous structure of the ECM by scanning electron microscopy. Cryogenic electrospun silk scaffolds were then fabricated to mimic the tumor ECM, and were found to be similar in fiber orientation and fiber dimensions to the native tumor ECM. Tumor cells were cultured on these ECM mimicking scaffolds and compared to an in vivo model of the same derivative human tumor in terms of proliferation and differentiation. The tumor cells in the 3D model show similar phenotypes to those found in vivo, contrasting to the same cells grown in two-dimensional (2D) culture. The sensitivity of the tumor cells to paclitaxel was compared between 2D culture and 3D culture. The results indicate that increased drug concentrations, orders of magnitude higher than the IC90 for 2D culture, had minimal effects on HN12 cell viability in the 3D model. In conclusion, an in vitro tumor model has been developed that will allow for a better understanding of tumor biology and aid chemotherapeutic drug development and accurate evaluation of drug efficacy.