Exploring Electrospun Scaffold Innovations in Cardiovascular Therapy: A Review of Electrospinning in Cardiovascular Disease.

Cardiovascular disease (CVD) remains the leading cause of mortality worldwide. In particular, patients who suffer from ischemic heart disease (IHD) that is not amenable to surgical or percutaneous revascularization techniques have limited treatment options. Furthermore, after revascularization is successfully implemented, there are a number of pathophysiological changes to the myocardium, including but not limited to ischemia-reperfusion injury, necrosis, altered inflammation, tissue remodeling, and dyskinetic wall motion. Electrospinning, a nanofiber scaffold fabrication technique, has recently emerged as an attractive option as a potential therapeutic platform for the treatment of cardiovascular disease. Electrospun scaffolds made of biocompatible materials have the ability to mimic the native extracellular matrix and are compatible with drug delivery. These inherent properties, combined with ease of customization and a low cost of production, have made electrospun scaffolds an active area of research for the treatment of cardiovascular disease. In this review, we aim to discuss the current state of electrospinning from the fundamentals of scaffold creation to the current role of electrospun materials as both bioengineered extracellular matrices and drug delivery vehicles in the treatment of CVD, with a special emphasis on the potential clinical applications in myocardial ischemia.

Delivery of a mitochondria-targeted antioxidant from biocompatible, polymeric nanofibrous scaffolds.

Cardiovascular disease has been associated with increased levels of reactive oxygen species (ROS). Recently, we have shown that a critical balance between cytosolic ROS and mitochondrial ROS is crucial in cardiovascular health and that modulation of mitochondrial ROS helps prevent detrimental effects of cytosolic ROS on endothelial cells (EC) in transgenic animals. Here, we report the development of a controlled delivery system for a mitochondria-targeted antioxidant, JP4-039, from an electrospun scaffold made of FDA-approved biocompatible polymeric nanofibers. We demonstrate that the active antioxidant moiety was preserved in released JP4-039 for over 72 h using electron paramagnetic resonance. We also show that both the initial burst release of the drug within the first 20 min and the ensuing slow and sustained release that occurred over the next 24 h improved tube formation in human coronary artery ECs (HCAEC) in vitro. Taken together, these findings suggest that electrospinning methods can be used to upload mitochondrial antioxidant (JP4-039) onto a biocompatible nanofibrous PLGA scaffold, and the uploaded drug (JP4-039) retains nitroxide antioxidant properties upon release from the scaffold, which in turn can reduce mitochondrial ROS and improve EC function in vitro.

Novel Honokiol-eluting PLGA-based scaffold effectively restricts the growth of renal cancer cells.

Renal Cell Carcinoma (RCC) often becomes resistant to targeted therapies, and in addition, dose-dependent toxicities limit the effectiveness of therapeutic agents. Therefore, identifying novel drug delivery approaches to achieve optimal dosing of therapeutic agents can be beneficial in managing toxicities and to attain optimal therapeutic effects. Previously, we have demonstrated that Honokiol, a natural compound with potent anti-tumorigenic and anti-inflammatory effects, can induce cancer cell apoptosis and inhibit the growth of renal tumors in vivo. In cancer treatment, implant-based drug delivery systems can be used for gradual and sustained delivery of therapeutic agents like Honokiol to minimize systemic toxicity. Electrospun polymeric fibrous scaffolds are ideal candidates to be used as drug implants due to their favorable morphological properties such as high surface to volume ratio, flexibility and ease of fabrication. In this study, we fabricated Honokiol-loaded Poly(lactide-co-glycolide) (PLGA) electrospun scaffolds; and evaluated their structural characterization and biological activity. Proton nuclear magnetic resonance data proved the existence of Honokiol in the drug loaded polymeric scaffolds. The release kinetics showed that only 24% of the loaded Honokiol were released in 24hr, suggesting that sustained delivery of Honokiol is feasible. We calculated the cumulative concentration of the Honokiol released from the scaffold in 24hr; and the extent of renal cancer cell apoptosis induced with the released Honokiol is similar to an equivalent concentration of direct application of Honokiol. Also, Honokiol-loaded scaffolds placed directly in renal cell culture inhibited renal cancer cell proliferation and migration. Together, we demonstrate that Honokiol delivered through electrospun PLGA-based scaffolds is effective in inhibiting the growth of renal cancer cells; and our data necessitates further in vivo studies to explore the potential of sustained release of therapeutic agents-loaded electrospun scaffolds in the treatment of RCC and other cancer types.

A Decision Tree Based Methodology for Evaluating Creativity in Engineering Design.

Multiple metrics have been proposed to measure the creativity of products, yet there is still a need for effective, reliable methods to assess the originality of new product designs. In the present article we introduce a method to assess the originality of concepts that are produced during idea generation activities within engineering design. This originality scoring method uses a decision tree that is centered around distinguishing design innovations at the system level. We describe the history and the development of our originality scoring method, and provide evidence of its reliability and validity. A full protocol is provided, including training procedures for coders and multiple examples of coded concepts that received different originality scores. We summarize data from over 500 concepts for garbage collection systems that were scored by Kershaw et al. (2015). We then show how the originality scoring method can be applied to a different design problem. Our originality scoring method, the Decision Tree for Originality Assessment in Design (DTOAD), has been a useful tool to identify differences in originality between various cohorts of Mechanical Engineering students. The DTOAD reveals cross-sectional differences in creativity between beginning and advanced students, and shows longitudinal growth in creativity from the beginning to the end of the undergraduate career, thus showing how creativity can be influenced by the curriculum. The DTOAD can be applied to concepts produced using different ideation procedures, including concepts produced both with and without a baseline example product, and concepts produced when individuals are primed to think of different users for their designs. Finally, we show how our the DTOAD compares to other measurements of creativity, such as novelty, fixation, and remoteness of association.

pH-responsive drug release from functionalized electrospun poly(caprolactone) scaffolds under simulated in vivo environment.

The difference in the tumor environment from the normal healthy tissue can be therapeutically exploited to develop new strategies for controlled and site-specific drug delivery. In the present study, a continuous flow system is designed to represent the in vivo environment of a tumor tissue and drug release is studied at different pH that represents normal tissue pH, tumor tissue pH, and stomach pH. The results obtained from these experiments were translated to a human embryonic kidney cell culture system and the effect of drug released from these functionalized PCL scaffolds on cell viability was studied. A significant decrease in cell viability was observed with the doxorubicin hydrochloride concentration that would be released at acidic pH, either present as a result of tumor extracellular environment or could be achieved via fabrication of a composite scaffold with a polyvinyl alcohol hydrogel containing acid. In the end, a study using zebrafish as an animal model is also undertaken in order to study the drug release from the scaffolds in vivo.

Functionalization of electrospun poly(caprolactone) fibers for pH-controlled delivery of doxorubicin hydrochloride.

Functionalized electrospun polymer fibers are a promising candidate for controlled delivery of chemotherapeutic drugs to improve the therapeutic efficacy and to reduce the potential toxic effects by delivering the drug at a rate governed by the physiological need of the site of action. In this study, poly(caprolactone) (PCL) fibers were fabricated by electrospinning, followed by hydrolyzation to introduce functional groups on the fiber surface. Characterization studies were performed on these functionalized fibers using X-ray photoelectron spectroscopy, scanning electron microscopy, and Toluidine Blue O dye assay. The pH-sensitivity of the functional groups on the fiber surface and doxorubicin hydrochloride was utilized to bind the drug electrostatically to these functionalized PCL fibers. The effect of pH on drug loading and release kinetics was investigated. Results indicate successful electrostatic binding of the drug to functionalized electrospun fibers and a high drug payload. The drug delivery response can be modulated by introduction of suitable stimuli (pH).

Effect of stiffness of micron/sub-micron electrospun fibers in cell seeding.

The objective of this study was to develop a predictive model for cell seeding depth in electrospun scaffold as a function of fiber stiffness. Electrospun scaffolds (micron and submicron) and 3T3 fibroblasts are used as scaffold-cell systems under vacuum seeding conditions. Atomic force microscopy is used to determine the Young's modulus (E) as a function of fiber diameter. A higher E value led to a lower depth of cell seeding (closer to the surface) indicating that nanofibrous scaffolds offer higher resistance to cell movement compared to microfibrous scaffold. An energy balance model was developed to predict cell seeding depth as a function of E for various vacuum pressures. Experimental data was used in the model to extract unknown parameters to predict cell seeding depth as a function of vacuum pressure for different stiffness scaffolds.

Hydrolyzed Poly(acrylonitrile) Electrospun Ion-Exchange Fibers.

A potential ion-exchange material was developed from poly(acrylonitrile) fibers that were prepared by electrospinning followed by alkaline hydrolysis (to convert the nitrile group to the carboxylate functional group). Characterization studies performed on this material using X-ray photoelectron spectroscopy, scanning electron microscopy, Fourier-Transform infra-red spectroscopy, and ion chromatography confirmed the presence of ion-exchange functional group (carboxylate). Optimum hydrolysis conditions resulted in an ion-exchange capacity of 2.39 meq/g. Ion-exchange fibers were used in a packed-bed column to selectively remove heavy-metal cation from the background of a benign, competing cation at a much higher concentration. The material can be efficiently regenerated and used for multiple cycles of exhaustion and regeneration.

Fibronectin adsorption on functionalized electrospun polycaprolactone scaffolds: experimental and molecular dynamics studies.

Designing scaffolds to modulate protein adsorption is a key to building advanced scaffolds for tissue regeneration. Protein adsorption to tissue engineering scaffolds is critical in early cell attachment, survival, and eventual proliferation. The goal of this study is to examine the effect of functionalization on fibronectin adsorption to electrospun polycaprolactone (PCL) scaffolds through experimentation using fluorescently labeled fibronectin and to couple this experimental data with analysis of interaction energies obtained through molecular dynamics (MD) simulations to develop a better understanding of the adsorption process. This study is the first to analyze and compare experimental and MD simulation results of fibronectin adsorption on functionalized electrospun PCL scaffolds. Electrospun nanofiber PCL scaffolds were treated with either 1 N NaOH (hydrolyzed) or 46% hexamethylenediamine (HMD) (aminated) solution to be compared with untreated (control) scaffolds. We found that aminated PCL scaffolds experimentally adsorbed more fibronectin than control scaffolds, whereas hydrolyzed scaffolds showed decreased adsorption. MD simulations carried out with NVT ensemble at a temperature of 310 K indicated a higher work of adhesion for both functionalized scaffolds over control. Also, the simulations revealed different conformations of fibronectin on each scaffold type after adsorption, with the arginine-glycine-aspartic acid sequence appearing most accessible on the aminated scaffolds. This suggests that functionalization affects not only the quantity of protein that will adsorb on a scaffold but how it attaches as well, which could affect subsequent cell attachment.

Osmotic damage as a predictor of motility loss during convective desiccation of bovine sperm.

Current state-of-the art technologies are lagging in the application of desiccation storage to mammalian cells using nonreducing sugars. For bovine sperm, motility is irreversibly lost before reaching a sufficiently low moisture content necessary for preservation. It is hypothesized that much of the damage during drying is related to the osmotic stress encountered due to increased osmolarity of the extracellular environment. To test this hypothesis, we subjected sperm to liquid hyperosmotic environments for varying time-periods and measured their motility. We then extracted parameters for two models for motility loss based on these experiments: a first-order rate injury model (Fast or Slow) and a multi-modal (MM) injury model. The MM injury model incorporated an additional function accounting for damage induced by a time-independent osmotic change. Based on these models, we predicted sperm motility loss measured from natural and forced convective desiccation experiments. The MM injury model was able to closely bracket motility loss for desiccation as an osmotic change event with time-independent and time-dependent components. While the mechanistic basis of osmotic damage requires further exploration, the model can serve as a bracketing tool for predicting motility loss during desiccation based on excipients designed to minimize osmotic damage.

Altering surface characteristics of polypropylene mesh via sodium hydroxide treatment.

Incisional hernias represent a serious and common complication following laparotomy. The use of synthetic (e.g. polypropylene) meshes to aid repair of these hernias has considerably reduced recurrence rates. While polypropylene is biocompatible and has a long successful clinical history in treating hernias and preventing reherniation, this material may suffer some limitations, particularly in challenging patients at risk of wound failure due to, for example, an exaggerated inflammation reaction, delayed wound healing, and infection. Surface modification of the polypropylene mesh without sacrificing its mechanical properties, critical for hernia repair, represents one way to begin to address these clinical complications. Our hypothesis is treatment of a proprietary polypropylene mesh with sodium hydroxide (NaOH) will increase in vitro NIH/3T3 cell attachment, predictive of earlier and improved cell colonization and tissue integration of polypropylene materials. Our goal is to achieve this altered surface functionality via enhanced removal of chemicals/oils used during material synthesis without compromising the mechanical properties of the mesh. We found that NaOH treatment does not appear to compromise the mechanical strength of the material, despite roughly a 10% decrease in fiber diameter. The treatment increases in vitro NIH/3T3 cell attachment within the first 72 h and this effect is sustained up to 7 days in vitro. This research demonstrates that sodium hydroxide treatment is an efficient way to modify the surface of polypropylene hernia meshes without losing the mechanical integrity of the material. This simple procedure could also allow the attachment of a variety of biomolecules to the polypropylene mesh that may aid in reducing the complications associated with polypropylene meshes today.

A study of the effect of sorbitol on osmotic tolerance during partial desiccation of bovine sperm.

The goal of the study was to improve the partial desiccation survival of bovine sperm by decreasing the dehydration induced osmotic injury. The protective role of sorbitol, a polyol, was investigated by (i) studying the osmotic behavior of sperm in hypertonic Tyrode's buffer in the presence of sorbitol and trehalose, (ii) studying the effect of sorbitol and trehalose on sperm motility following partial dehydration. The osmotic behavior studies included the assessment of motility and volumetric responses in the presence of the additives. For the drying experiments, motility was assayed after drying the samples to different end water content followed by immediate rehydration. Compared to the effect of "intracellular+extracellular" trehalose alone, results showed a much improved motility in the presence of sorbitol and trehalose. While the drying results suggest an enhanced osmotolerance in the presence of sorbitol, the study of motility under hypertonic conditions combined with the sperm volume excursion experiments suggest that sorbitol imparts the enhancement by permeating into the cell cytoplasm.

Thermal therapy in urologic systems: a comparison of arrhenius and thermal isoeffective dose models in predicting hyperthermic injury.

The Arrhenius and thermal isoeffective dose (TID) models are the two most commonly used models for predicting hyperthermic injury. The TID model is essentially derived from the Arrhenius model, but due to a variety of assumptions and simplifications now leads to different predictions, particularly at temperatures higher than 50 degrees C. In the present study, the two models are compared and their appropriateness tested for predicting hyperthermic injury in both the traditional hyperthermia (usually, 43-50 degrees C) and thermal surgery (or thermal therapy/thermal ablation, usually, >50 degrees C) regime. The kinetic parameters of thermal injury in both models were obtained from the literature (or literature data), tabulated, and analyzed for various prostate and kidney systems. It was found that the kinetic parameters vary widely, and were particularly dependent on the cell or tissue type, injury assay used, and the time when the injury assessment was performed. In order to compare the capability of the two models for thermal injury prediction, thermal thresholds for complete killing (i.e., 99% cell or tissue injury) were predicted using the models in two important urologic systems, viz., the benign prostatic hyperplasia tissue and the normal porcine kidney tissue. The predictions of the two models matched well at temperatures below 50 degrees C. At higher temperatures, however, the thermal thresholds predicted using the TID model with a constant R value of 0.5, the value commonly used in the traditional hyperthermia literature, are much lower than those predicted using the Arrhenius model. This suggests that traditional use of the TID model (i.e., R=0.5) is inappropriate for predicting hyperthermic injury in the thermal surgery regime (>50 degrees C). Finally, the time-temperature relationships for complete killing (i.e., 99% injury) were calculated and analyzed using the Arrhenius model for the various prostate and kidney systems.

Controlled vacuum seeding as a means of generating uniform cellular distribution in electrospun polycaprolactone (PCL) scaffolds.

A major challenge encountered in using electrospun scaffolds for tissue engineering is the non-uniform cellular distribution in the scaffold with increasing depth under normal passive seeding conditions. Because of the small surface pores, typically few microns in diameter, cells tend to congregate and proliferate on the surface much faster compared to penetrating the scaffold interior. In order to overcome this problem, we used a vacuum seeding technique on polycaprolactone electrospun scaffolds while using NIH 3T3 fibroblasts as the model cell system. This serves as a precursor to the bilayer skin model where the fibroblasts would be residing at an intermediate layer and the keratinocytes would be on the top. Vacuum seeding was used in this study to enhance fibroblasts seeding and proliferation at different depths. Our results show that the kinetics of cell attachment and proliferation were a function of varying vacuum pressure as well as fiber diameter. Cell attachment reached a maxima somewhere between 2-8 in. Hg vacuum pressure and fell for lower vacuum pressures presumably because of cell loss through the filtration process. Cell proliferation and collagen secretion over five days indicated that vacuum pressure did not affect cellular function adversely. We also compared the combined impact of scaffold architecture (400 nm versus 1100 nm average diameter fiber scaffolds) and vacuum pressure. At a given pressure, more cells were retained in the 400 nm scaffolds compared to 1100 nm scaffolds. In addition, the cell intensity profile shows cell intensity peak shift from the top to the inner layers of the scaffold by lowering the vacuum pressure from 0 in. Hg to 20 in. Hg. For a given vacuum pressure the cells were seeded deeper within the 1100 nm scaffold. The results indicate that cells can be seeded in electrospun scaffolds at various depths in a controlled manner using a simple vacuum seeding technique. The depth of seeding is a function of pressure and scaffold fiber diameter.

Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment.

In order to develop scaffolds for tissue regeneration applications, it is important to develop an understanding of the kinetics of cell attachment as a function of scaffold geometry. In the present study, we investigated how the specific surface area of electrospun scaffolds affected cell attachment and spreading. Number of cells attached to the scaffold was measured by the relative intensity of a metabolic dye (MTS) and cell spreading was analysed for individual cells by measuring the area of projected F-actin cytoskeleton. We varied the fibre diameter to obtain a specific surface area distribution in the range 2.24-18.79 microm(-1). In addition, we had one case where the scaffolds had beads in them and therefore had non-uniform fibres. For each of these different geometries, we varied the cell-seeding density (0.5-1 x 10(5)) and the serum concentration (0-12%) over the first 8 h in an electrospun polycaprolactone NIH 3T3 fibroblast system. Cells on beaded scaffolds showed the lowest attachment and almost no F-actin spreading in all experiments indicating uniform fibre diameter is essential for electrospun scaffolds. For the uniform fibre scaffolds, cell attachment was a function of scaffold specific surface area (SSA) (18.79-2.24 microm(-1)) and followed two distinct trends: when scaffold SSA was < 7.13 microm(-1), cell adhesion rate remained largely unchanged; however, for SSA > 7.13 microm(-1) there was a significant increase in cellular attachment rate with increasing SSA. This indicated that nanofibrous scaffolds increased cellular adhesion compared to microfibrous scaffolds. This phenomenon is true for serum concentrations of 7.5% and higher. For 5% and lower serum concentration, cell attachment is low and higher SSA fails to make a significant improvement in cell attachment. When cell attachment was investigated at a single-cell level by measuring the projected actin area, a similar trend was noted where the effect of higher SSA led to higher projected area for cells at 8 h. These results indicate that uniform electrospun scaffolds with SSA provide a faster cell attachment compared to lower SSA and beaded scaffolds. These results indicate that continuous electrospun nanofibrous scaffolds may be a good substrate for rapid tissue regeneration.

Desiccation tolerance in bovine sperm: a study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator.

Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal.

Developing a predictive tool for reactive oxygen species damage during bovine sperm storage at ambient temperature.

Preservation of sperm at ambient storage temperature (25°C) provides a simplified alternative for short-term storage applications. The goals of the study were (i) to improve the storage ability of bovine sperm in liquid suspension by scavenging the reactive oxygen species (ROS) with the help of antioxidants and chelators, (ii) to develop a predictive tool to determine sperm motility as a function of storage temperature. Two types of antioxidants (catalase and superoxide dismutase, SOD) and 2 chelators (Desferal and ethylenediaminetetraacetic acid, EDTA) were used. Different concentrations of the supplements were added in egg yolk Tris (EYT) extender and stored at 4°C, room temperature (25°C), and 37°C, respectively. Motility and membrane integrity (MI) were assessed on days 1, 4, 7, 10, and 14 for samples stored at 4°C and 25°C. Results did not suggest the beneficial effect of SOD and EDTA. Catalase caused a slight improvement in motility at 4°C storage and its effectiveness was found to increase at higher storage temperature. The presence of Desferal resulted in a much higher increase in motility and membrane integrity at both 4°C and 25°C. Similarly, the fitting of Arrhenius damage model to the motility data provided a useful tool to predict motility degradation time as a function of storage temperature.

Role of fiber diameter in adhesion and proliferation of NIH 3T3 fibroblast on electrospun polycaprolactone scaffolds.

The goal of the current study was to find the quantitative relationship between electrospun polycaprolactone scaffold fiber diameter and NIH 3T3 fibroblast adhesion and growth kinetics. By varying 3 important process parameters--solution concentration, voltage, and collector screen distance--different average fiber diameters ranging from 117 to 1,647 nm were obtained. Although 117 nm represented the lowest possible fiber diameter obtainable, these fibers had beads in them. An increase in fiber diameter to 428 nm led to uniform fibers without any beads. Fiber distribution pattern was a single mode for all the scaffolds except at the largest-diameter end. The diameter distribution changed from single to bimodal at 1,647 nm, suggesting some instability in the process. It was found that cell adhesion and growth kinetics are significantly affected as a function of fiber diameter. Beaded scaffolds offered the lowest cell adhesion and minimal growth kinetics despite having the lowest average fiber diameter. When uniform fibers were formed and the average fiber was in the nanofiber range (428-1051 nm), cell adhesion and growth kinetics decreased as a function of increasing fiber diameter. Cell adhesion kinetics remained invariant when the average fiber diameter was in the micron range (1,647 nm), whereas cell-growth kinetics were slightly greater than with 900 nm scaffolds. We propose that the uniformness of fibers and the average fiber diameter may play an important role in modulating cellular attachment and proliferation in electrospun tissue engineering scaffolds.

A flexible approach to the preparation of polymer scaffolds for tissue engineering.

Porous polyester thermoset xerogels have been produced via sol-gel chemistry as a first step in the development of sol-gel derived tissue engineering scaffolds templated by replica molding and/or salt leaching. The pore structure of these untemplated thermosets is tunable and can be altered independent of or in tandem with alterations in composition. Cytocompatibility studies on these xerogels imply the effects of both pore size and materials chemistry, with fully aliphatic polyesters with large pore structures allowing the growth of mammalian cells. To the best of our knowledge, this represents the first report examining the preparation and potential of sol-gel derived porous polymer xerogels as tissue engineering scaffolds.