Q admitted that they had to put out disinformation at times for operational reasons.
Electrospun Silk Biomaterial Scaffolds for Regenerative Medicine
Electrospun Nanofibers for Regenerative Medicine**
This article reviews recent progress in applying electrospun nanofibers to the emerging field of regenerative medicine. We begin with a brief introduction to electrospinning and nanofibers, with a focus on issues related to the selection of materials, incorporation of bioactive molecules, degradation characteristics, control of mechanical properties, and facilitation of cell infiltration. We then discuss a number of approaches to fabrication of scaffolds from electrospun nanofibers, including techniques for controlling the alignment of nanofibers and for producing scaffolds with complex architectures. We also highlight applications of the nanofiber-based scaffolds in four areas of regenerative medicine that involve nerves, dural tissues, tendons, and the tendon-to-bone insertion site. We conclude this review with perspectives on challenges and future directions for design, fabrication, and utilization of scaffolds based on electrospun nanofibers.
Many of the tissues in the human body do not have the capacity to regenerate, so damage to these tissues is irreversible. In addition to the poor ability to heal, injuries to tissues such as nerve, tendon, cartilage, and myocardium also result in significant pain and disability. Even with surgical intervention, return of function is often limited and the healing response is scar-mediated rather than regenerative. Patients suffering from organ trauma, disease, or congenital abnormality must rely on organ transplantation to regain function. In spite of its enormous success clinically, this approach is plagued by post-surgical immune reactions and a severe limitation in the number of available donors, leaving thousands of patients on waiting lists. In the United States, 18 people die each day before a suitable organ donor is found. To address these and other issues related to tissue damage and organ transplantation, regenerative medicine has emerged as an interdisciplinary research field that incorporates biology, materials science, and engineering to develop functional substitutes that are safe and readily available for patients with damaged tissues or organs. In regenerative medicine, elements of scaffold design, cellular control, and signaling are integrated to enhance healing or replace an injured tissue or organ.
One of the major challenges in regenerative medicine is to design and fabricate a suitable scaffold. In order to achieve the desirable functionality of the tissue or organ to be replaced, the scaffold needs to be carefully engineered to elicit specific responses from local cells and organ systems. In one approach, a donor organ is decellularized and the remaining extracellular matrix (ECM) is used as a scaffold.  The scaffold is then seeded with patient-specific cells to create a functional substitute for implantation. Although this new strategy can mitigate the immune response commonly seen with the conventional transplantation approaches by using patient-specific cells, the availability of organs that can be used for decellularization remains a stringent limitation. This limitation has inspired biomedical engineers to construct tissues and organs in the laboratory using synthetically derived scaffolds. To this end, cells are cultured on different scaffolds and then a whole organ is assembled from multiple cell-scaffold constructs.
Historically, cells were grown and studied as monolayers on tissue culture plates. In recent years, advances in biomaterial synthesis and microfabrication have made it possible to pattern cells into complex, three-dimensional structures by using appropriate scaffolds as the templates. With an ever-growing understanding of the intricate interactions between cells and their microenvironments in tissues, more attention is now given to the fabrication of scaffolds capable of recapitulating key features of the ECM that control the migration, proliferation, and differentiation of cells. The ECM is often composed of interwoven protein fibers such as fibrillar collagen and elastin, with diameters ranging from tens to hundreds of nanometers. This matrix also contains nanoscale adhesion proteins that serve as specific binding sites for cell adhesion. Signaling to cells from the ECM occurs by direct interactions between ligands on the ECM and cell receptors, the sequestration of growth factors by the ECM, spatial cues, and mechanical force transduction. As such, the microenvironment provided by the ECM can control the behavior and fate of a cell. Many techniques have been developed for fabricating fibrous scaffolds to be used as ECM substitutes; electrospinning has recently emerged as one of the most successful techniques, owning to its ability to generate fibers similar to the fibrous structures of native ECM.
Electrospinning is a remarkably simple, robust, and versatile technique capable of generating fibers with diameters down to the nanoscale. A non-woven mat of electrospun nanofibers possesses high porosity and spatial interconnectivity well-suited for nutrient and waste transport and cell communication. A scaffold based on electrospun nanofibers also has a large specific surface area for loading of bioactive molecules to facilitate efficient and selective cellular responses. Electrospinning has been applied to more than 100 different types of polymers. Naturally occurring matrix proteins including collagen, elastin, and fibrinogen and synthetic polymers such as poly(ε-caprolactone) (PCL) and poly(lactic-co-glycolic) acid (PLGA) can all be prepared as nanofibers by electrospinning. Since different tissues have distinct criteria for scaffold functionality, having a wide range of materials to choose from allows one to articulate the compositions and other properties of electrospun nanofibers to meet different demands. In addition, many tissues, including the sciatic nerve, heart, tendon, and blood vessel, have unique anisotropic structures and architectures (Fig. 1) that cannot be recapitulated by scaffolds fabricated using conventional methods. Electrospinning, in contrast, can be easily used to generate assemblies of aligned nanofibers to mimic the anisotropy of these tissues. To this end, scaffolds based on electrospun nanofibers with various alignments have shown superior capacity in shaping cell morphology, guiding cell migration, and affecting cell differentiation when compared to other types of scaffolds both in vitroand in vivo.
The aim of this article is to provide a review of recent work using electrospun nanofibers as scaffolds for regenerative medicine. We emphasize how the alignment of electrospun nanofibers can be controlled to present the right structural cues for the manipulation of cell attachment, proliferation, and migration in vitro and in vivo. We also highlight the applications of nanofiber-based scaffolds in four different areas that involve nerves, dural tissues, tendons, and the tendon-to-bone insertion site.
2. Electrospinning of nanofibers
2.1 Setup and principle
Four major components are required for electrospinning (Fig. 2, center): a spinneret (e.g., a hypodermic needle with blunt-tip), a syringe pump for ejecting the polymer solution at a controlled rate, a direct current (DC) power supply up to 30 kV, and a grounded collector (e.g., a piece of aluminum foil). When the polymer solution emerges from a spinneret, it initially forms a droplet due to the confinement of surface tension. If a high voltage is applied to the spinneret, charges of the same sign will be built on the surface of the droplet. Once the repulsion among the charges is sufficiently strong to overcome the surface tension, a Taylor cone will be formed, followed by a liquid jet directed towards the grounded collector. The jet will experience both solvent evaporation and whipping instability before it reaches the collector. As a result of stretching by electrostatic repulsion and whipping, the liquid jet will be continuously reduced in size until it has been solidified or deposited on the collector. By adjusting experimental parameters such as the concentration of polymer solution, the voltage, and the distance between spinneret and collector, fibers with uniform diameters can be routinely produced.
2.2 Materials consideration
Electrospinning has already been successfully applied to generate nanofibers from more than 100 different types of synthetic and natural polymers. Synthetic polymers are relatively less expensive and more convenient to work with than natural polymers. For scaffold fabrication, the most commonly used synthetic polymers include PCL, PLGA, poly(ethylene oxide) (PEO), and poly(L-lactic) acid (PLA). Although these polymers are biocompatible and biodegradable, they may cause significant inflammation and foreign body reaction when implanted in vivo. Natural polymers are therefore more desirable to avoid complications from severe immune reaction. The most abundant natural polymers are type I and type III collagens; together they account for almost one third of the proteins in the human body. Nanofibers electrospun from collagens will swell when exposed to the moisture in air and tend to lose their fibrous morphology in a short period of time. Cross-linking is thus required to maintain the fibrous morphology after electrospinning. The toxicity associated with some of the cross-linkers may compromise the usefulness of such nanofibers in vivo. Additionally, the mechanical strength of collagen nanofibers are typically very weak. To improve stability and mechanical strength, collagens are often mixed with other polymers and then electrospun into fibers. For example, Boyce and co-workers have demonstrated electrospinning with a blend of collagen and PCL. Tensile testing indicates that even inclusion of PCL at a low concentration of 10% could significantly improve the stability and stiffness of the nanofibers.
2.3 Incorporation of bioactive molecules
Bioactive molecules released from a scaffold at a controlled rate can be used to stimulate the proliferation and differentiation of seeded cells during in vitro culture, thereby encouraging tissue regeneration after implantation in vivo. Many different types of bioactive molecules have been incorporated into scaffolds of electrospun nanofibers, including growth factors. Growth factors are endogenous proteins capable of binding to cell receptors and directing cellular activities. The biggest challenge in incorporating a growth factor into a scaffold is how to preserve its bioactivity. Several factors in an electrospinning process can lead to deactivation of a growth factor: the high voltage applied, the high density of charges built on the nanofiber, and the involvement of an organic solvent. The last problem can be potentially solved by adding a hydrophilic component such as PEG or hydroxyapatite (HAp), which can bind to the growth factor and protect it from deactivation by an organic solvent during electrospinning. The acidic degradation products of some synthetic polymers (e.g., PLGA) is another potential source of deactivation for the growth factor. A porogen such as PEG can be added into the scaffold to facilitate the diffusion of degradation products and thereby maintain the local pH. However, this approach is limited by its destructive effect on the integrity of the nanofibers.
Successful delivery also depends on the release profile of bioactive molecules incorporated into the scaffold. There are currently four methods for incorporating bioactive molecules into a scaffold:physical adsorption, covalent attachment, electrospinning with a co-axial spinneret (Fig. 2, top left), and addition of bioactive molecules to the electrospinning solution (Fig. 2, top right). Different methods of incorporation typically result in distinct release profiles. For physical adsorption, growth factors are attached to the scaffold mainly through electrostatic interactions. Although this is the simplest way to incorporate growth factors with relatively high bioactivity, the release is typically rapid. One study reported a burst release during the first 5 days, with complete release within 20 days. The release profiles for scaffolds prepared by electrospinning with a co-axial spinneret or from a solution mixed with bioactive molecules are similar to each other: there is an initial burst release followed by a sustained, first order release. The initial burst release can be attributed to the migration of the growth factors during the drying process, which tends to concentrate a certain fraction of the growth factor molecules near the surface of the fibers. After burst release, the release mode is primarily driven by diffusion and polymer degradation. Jeong and co-workers developed a method to program the release of the bioactive molecules. They first encapsulated the bioactive molecules in cross-linked polymer particles, which were then blended with a polymer solution and electrospun through a single nozzle. The polymer particles must be cross-linked so they were just swollen in the electrospinning solution instead of being dissolved. By adjusting the physical and chemical properties of the particles, programmed release was achieved. For covalent attachment, burst release is avoided since the release is typically controlled by enzymatic cleavage. However, covalent attachment cannot be employed to routinely load electrospun nanofibers with bioactive molecules due to the technical complexity required for fabrication.
2.4 Degradation characteristics
Once implanted in the body, the scaffold should undergo degradation at a rate matching that of tissue regeneration. For individual nanofibers, the degradation profile is mainly determined by the polymer itself as hydrolysis of the polymer backbone is believed to be the prevailing mechanism. Most of the synthetic polymers are semi-crystalline, implying that their chains fold into crystalline regions in addition to amorphous regions. In the absence of an enzyme, water penetrates the surface of a nanofiber and preferentially attacks the amorphous regions first, converting the long polymer chains into shorter and eventually water-soluble species. Since the crystalline regions are still intact, the nanofiber does not fall apart. As hydrolysis continues, the nanofiber eventually starts to disintegrate and disappear. In the presence of an enzyme, the nanofiber can be digested by the enzyme, resulting in a rapid loss of mass.
When assembled into a scaffold, the structure of the scaffold also plays a very important role in determining the degradation profile of nanofibers. When compared with a thin film cast from the same polymer, a scaffold made of electrospun nanofibers has a higher porosity and therefore the degradation product will be able to diffuse away more quickly. Otherwise, the accumulation of acidic degradation products will act as a catalyst to make the degradation process faster. As a result, a scaffold based on electrospun nanofibers would require a longer time to degrade than a bulk film of the same mass due to the difference in porosity.  Some researchers have also attributed the slow degradation rates of nanofiber scaffolds to the increase in chain orientation and thus higher crystallinity, as the strong electric field involved in an electrospinning process tended to align the polymer chains parallel to the field.
The porosity of a nanofiber-based scaffold is a key factor in controlling the degradation profile. A number of methods have been developed for manipulating the porosity of a nanofiber scaffold, including those based on variation of the size of nanofibers, salt leaching, cryogenic electrospinning, and removal of a sacrificial component. These methods will be discussed in Section 2.6, as cell infiltration is also affected by the porosity.
2.5 Mechanical properties
The mechanical properties of a nanofiber-based scaffold depend on a number of parameters, including the composition, molecular structure, and size of individual nanofibers, as well as the alignment and density of the nanofibers. For example, scaffolds made of PLGA nanofibers can be ten times stiffer than scaffolds made of PCL nanofibers. Ramakrishna and co-workers have found that the rotating speed of a mandrel was a dominant parameter in inducing a highly ordered molecular structure in an electrospun PLLA fiber, which consequently led to higher tensile modulus and strength. Leong and co-workers reported an increase in both strength and stiffness as the fiber diameter was reduced from ~5 μm to ~200 nm.Encapsulation of different drugs may also exert different impacts on the mechanical properties of a single nanofiber. An increase in mechanical strength was reported when 10–20 wt% retinoic acid was encapsulated whereas an opposite trend was observed when 10–20 wt% bovine serum albumin (BSA) was added.
Alignment of nanofibers results in significant stiffening in the direction of alignment and increased scaffold anisotropy. This is an important feature to mimic when engineering anisotropic load-bearing tissues such as tendons, annulus fibrosis, and myocardium. Mauck and co-workers recapitulated the complex tissue organization and mechanical properties of the annulus fibrosus with anisotropic, nanofibrous laminates seeded with mesenchymal stem cells. The scaffolds approached the mechanical properties of native tissues after 10 weeks of culture. Modification of the surface of the fibers with precipitated bioapatite can also lead to dramatic increases in scaffold stiffness.
The mechanical properties of scaffolds can have strong impacts on cell proliferation and stem cell differentiation. Discher and co-workers demonstrated that the commitment of stem cells to a particular phenotype was highly dependent on the stiffness of substrate. The most compliant surfaces were neurogenic while the stiffest matrices were osteogenic. Ingber and co-workers demonstrated that cell phenotypes could be affected by cellular adhesion to the ECM and the mechanical tension in cytoskeleton. In general, rigid substrates tend to promote cell spreading by resisting cell tension. Rigid substrates supporting higher levels of isometric tension in the cell allow spreading and growth of cells such as fibroblasts and endothelial cells. Flexible substrates that cannot withstand stretching will result in retracting, rounding up, and the down regulation of genes associated with proliferation. Controlling the mechanical properties of both bulk and individual nanofibers will help optimize scaffolds for tissue regeneration by recapitulating the properties of the tissue being replaced and by providing the appropriate cues for the seeded cells.
2.6 Cell infiltration
The porosity of a nanofiber scaffold can directly affect the infiltration of cells. Although many research groups have focused on the development of fibers with reduced diameters to increase the specific surface area for loading of bioactive molecules, it has been shown that the scaffolds consisting of thinner fibers tend to have a lower porosity due to a denser packing of the fibers. One technique for increasing the porosity of a scaffold is based on salt leaching. The setup for this technique is identical to that of electrospinning with a co-axial spinneret (Fig. 2, top left). The nanofibers produced using this technique had a core-sheath structure with the polymer as the core and crystals of the salt in the sheath. The high voltage was able to stretch the jet of polymer solution into a nanofiber while the salt crystals were formed and attached to the surface. The salt crystals were then dissolved in water to generate a mat with high porosity, which could facilitate cell infiltration up to 4 mm in depth. Other approaches include selective removal of sacrificial fibers in a scaffold prepared using a dual spinneret system. To this end, Mauck and co-workers co-electrospun PCL and PEO (a water-soluble polymer) from two separate spinnerets to form a dual-polymer scaffold (Fig. 2, bottom left). The PEO fibers were dissolved gradually in the cell culture medium and cells were found to be present throughout the entire scaffold.[64c] Although these two methods could considerably improve cell infiltration, they also led to compromised structural integrity and macroscopic delamination. Recently, Jun and co-workers developed a semi-spherical bowl with small needles randomly distributed on the inner surface to fabricate cotton-ball-like, uncompressed scaffolds of electrospun nanofibers (Fig. 2, bottom right). This type of scaffold has very high porosity and encourages inward cell migration. The major limitation of this type of scaffold is the difficulty of transferring it to other substrates without destroying its hierarchal structure.
Although many efforts have been devoted to increasing the infiltration of cells into a nanofibrous constructs, a robust and transferrable scaffold has yet to be developed and remains a major goal of future work.
3. Controlling the alignment of nanofibers
In many applications, it is desirable to have a scaffold made of aligned nanofibers, as the anisotropy in topography and structure can greatly affect not only the mechanical properties but also cell adhesion, proliferation, and alignment. Aligned fibrous scaffolds may thus be useful in replicating the ECM for a specific tissue type such as tendon, where collagen fibrils are aligned parallel to each other. To this end, Ouyang and co-workers studied human tendon stem/progenitor cells (hTSPCs) cultured on scaffolds made of aligned or random PLLA nanofibers. Tendon-specific genes were up-regulated in hTSPCs cultured on the aligned nanofibers compared to those on the random fibers. Another example is cardiac tissue, where the ventricular myocardium is composed of perpendicularly interwoven collagen stripes. This unique anisotropy in cardiac tissue gives rise to its special directionally dependent electrical and mechanical properties. Entcheva and co-workers showed that primary cardiomyocytes cultured on a scaffold of aligned PLLA nanofibers developed mature contractile machinery (sarcomeres). Excitability of the engineered constructs was confirmed by optical imaging of electrical activity using voltage-sensitive dyes.
Besides mimicking the ECM, the alignment of electrospun nanofibers in a scaffold can also guide the migration and extension of cells. For example, aligned electrospun nanofibers have been used to guide the neuronal growth from neural stem cells (NSCs). Ramakrishna and coworkers have shown that axons of up to 100 μm were formed on aligned fibers, which could be attributed to the enhanced contact guidance.We have also demonstrated that electrospun nanofibers with a uniaxial alignment could induce the differentiation of mouse embryonic stem cells (ESCs) into neural lineages with less possibility of scar tissue formation. The alignment associated with the fibers can also accelerate the rate of wound closure. This is because the alignment confines the migratory route of the cells to a certain direction so that a shorter time will be needed for cells cultured on aligned nanofibers to cover the same area comparing with those cultured on random nanofibers. As a demonstration, we have recently shown that dural fibroblasts could cover the entire scaffold with a radial alignment at a faster speed relative to a scaffold made of random nanofibers.
A number of methods have been developed for controlling the alignment of electrospun nanofibers. These methods can be categorized into three major categories depending on the type of forces involved (Fig. 3): mechanical, electrostatic, and magnetic. These methods are described in the subsequent sections.
3.1 Alignment caused by mechanical forces
In order to align nanofibers using mechanical forces, a metallic rotating mandrel is often used as the collector (Fig. 3A). When the electrospun nanofibers are collected by a rotating mandrel, the rotating speed of the mandrel determines the degree of alignment of the non-woven mat. Bowlin and co-workers showed that random collagen fibers were collected at linear velocity lower than 0.16 m s−1, while significant alignment was observed at linear velocities higher than 1.4 m s−1.[25a] Whereas a higher speed can result in better alignment, a linear velocity higher than 45 m s−1 will encourage formation of necks in the nanofibers. At very high rotation speeds, the velocity of the electrospinning jet may be slower than the linear velocity of the mandrel; this may create stress residues and thus necking in the nanofibers. In order to obtain aligned, uniform nanofibers, it is essential to rotate the mandrel at an appropriate and well-controlled speed. 
The alignment of nanofibers collected by conventional rotating mandrels can sometimes be affected by the mandrel width, as the two ends of the collector can act as electrodes too and exert electrostatic forces on the fibers, influencing their alignment. Yarin and co-workers modified the design of a drum by replacing it with a tapered, wheel-like disk. A typical disk is significantly thinner than a commonly used mandrel and the fibers are mostly collected on the sharp edge. The authors also simulated the electrostatic field of the rotating disk and revealed that the field strength increased dramatically near the edge of the disk, which in turn forced the charged fibers to continuously wound on the edge. The major drawback of a rotating disc is its small collecting area, which limits its application for experiments where fabrication of large scaffolds is desirable. Another alternative collector is to use a drum made of nonconductive materials as the collector. Fann and co-workers attempted to collect aligned fibers on a plastic mandrel that included a sharp metallic pin in the interior. By moving the position of the pin, aligned fibers were obtained. The major disadvantage of this design is that the thickness of the scaffold is limited. The plastic mandrel cannot quickly dissipate the charges on the deposited fibers, and the accumulated charges thus repel the incoming fibers, preventing further deposition. One approach to reduce the accumulation of residual charges is to use an alternating current (AC) power supply instead of the typical DC source. Tepper and co-workers showed that nanofibers of PEO collected on the rotating mandrel showed greater alignment when using an AC rather than DC power supply. The electrospinning jet from an AC system consists of short segments of alternating polarity, which would neutralize each other, thus minimizing charge accumulation and leading to improved fiber alignment. One has to be extremely cautious whenever an AC power supply is used as it may generate a high current between the spinneret and the collector.
3.2 Alignment caused by electrostatic forces
Since electrostatic charges are distributed along the electrospinning jet, an external electric field can be used to manipulate and control the alignment of nanofibers. We have developed a collector consisting of two pieces of conductive substrates which are separated by a void gap to collect uniaxially aligned nanofibers across the gap (Fig. 3B). Nanofibers descending from the spinneret will experience two types of electrostatic forces. The splitting of electric field with electric field lines pointing towards the two electrodes produces the first force. This force will pull the fiber towards the electrodes and further induce opposite charges on the surfaces of the electrodes when the fiber travels to their vicinity. This gives rise to a second force, which stretches the nanofiber across the gap to render it perpendicular to the edges of the electrodes. These two types of electrostatic forces work together to produce a uniaxially aligned array of nanofibers. If the fibers spanning across the void gap discharge very slowly and repel each other, the extent of alignment will be improved with deposition time.
One of the remarkable features associated with the gap technique is that it is convenient to transfer the aligned fibers onto other solid substrates for further applications. Single fibers collected across the gap can be easily picked up and tested without transferring to other substrates. As single fibers are very sensitive to stress and strain, avoiding transferring the single fibers is highly desirable in mechanical testing assays.In particular, it has been established that uniaxially aligned fibers could be directly deposited on an insulating substrate on which the pair-wise electrodes can be patterned. This variant is commonly used to fabricate multilayered constructs by controlling the scheme or the configuration for applying high voltage. The detailed explanation will be provided in Section 4.1.
In addition to uniaxial alignment, it is sometimes desirable to align the fibers into other patterns. For example, scaffolds made of radially aligned nanofibers may improve wound healing by providing contact guidance for cell migration. In this case, radially aligned nanofibers will encourage cells to migrate from the peripheral healthy tissue towards the central, injured site. Radial alignment can be achieved using a ring collector with a point electrode in the center. In order to ensure that all the fibers pass through the central point electrode, the needle electrode should be slightly higher than its peripheral ring collector (Fig. 3C). Since cells tend to migrate along the fibers, cells seeded around the periphery of a radially aligned scaffold would follow the nanofibers and migrate inward to cover the whole scaffold at a speed faster than if they were seeded on a scaffold made of random nanofibers.
Another useful type of alignment can be found in scaffolds that contain arrays of microwells. These scaffolds can be fabricated using an array of metallic beads as the collector (Fig. 3D). Whereas fibers deposited on the beads were randomly oriented, those deposited across the gaps between adjacent beads were uniaxially aligned. The resultant non-woven mat had concave microwells at the positions corresponding to the beads. The size of the microwells and the distance between adjacent microwells can both be tailored to accommodate different applications. Dorsal root ganglia (DRG) cultured in the microwells extended neurites to adjacent microwells and formed neural networks on the entire scaffold.This type of scaffold containing aligned nanofibers and patterned microwells may be a useful platform for research related to cell-cell communication and cell microarray assays.
3.3 Alignment caused by magnetic forces
Alignment of electrospun nanofibers can also be achieved by applying an external magnetic field (Fig. 3E). Jiang and co-workers demonstrated that magnetized poly(vinyl alcohol) (PVA) fibers could be stretched into parallel fibers over large areas (more than 5 × 5 cm2) using a magnetic field. The polymer solution was magnetized by adding a small amount (less than 0.5 wt%) of magnetic nanoparticles. The solution was electrospun into nanofibers in the presence of a magnetic field generated by two parallel-positioned permanent magnets. The magnetic field stretched the fibers across the gap to form a uniaxially aligned array as they landed onto the magnets. Nanofibers of PVA were also electrospun without magnetic nanoparticles in a magnetic field, and they were randomly distributed without any alignment. The authors concluded that the magnetic field could assist alignment of electrospun nanofibers, but only when electrospinning a magnetizable solution in a magnetic field. Yang and co-workers also demonstrated that by increasing the flow rate of the electrospun jet, the morphology of resultant nanofibers would change from uniaxially aligned to wavy. In this case, both PLGA and poly(vinyl pyrrolidone) (PVP) were electrospun at different flow rates in a magnetic field. At a flow rate of 0.5 mL h−1, the PLGA and PVP fibers were straight and uniaxially aligned, while at a flow rate of 3.0 ml h−1, the fibers became wavy in morphology without losing the overall alignment. The authors also concluded that the generation of aligned polymer fibers (both straight and wavy) by magnetic field-assisted electrospinning was independent of the solution and solvent used and did not require the solution to be magnetically active. Although both research groups obtained well aligned nanofibers, their interpretation of the phenomenon was contradictory to each other. Further efforts are needed to investigate the mechanism of alignment for nanofibers electrospun in a magnetic field.
4. Nanofiber scaffolds with complex architectures
Nonwoven mats of electrospun nanofibers can be further processed into scaffolds with complex architectures such as stacked arrays and tubular conduits. Stacked arrays of electrospun nanofibers are made of multiple layers of fibers that are sequentially deposited on top of one another. The main motivation for developing this type of scaffold is to mimic some natural tissue with respect to its biochemical, structural, and mechanical properties, as well as to enable the formation of multilayered tissues. Tubular conduits made of electrospun nanofibers are usually used for applications in vascular or neural tissue engineering as they resemble the hollow structures of these tissues (see Fig. 1). Here we only focus on the fabrication methods for these scaffolds with complex architectures.
4.1 Stacked arrays of nanofibers
Uniaxially aligned nanofibers can be stacked into multilayered films with a controllable, hierarchically porous structure. When a pair of electrodes separated by a void gap is used as the collector, the uniaxially aligned nanofibers can be sequentially transferred onto a substrate. By rotating the substrate between the deposition of different layers, it is fairly straightforward to obtain a multilayered film with the nanofibers in each layer oriented along a different direction. Alternatively, the void gap can be replaced by a highly insulating substrate such as quartz or polystyrene. By patterning the collector into an array of electrodes on an insulating substrate, the deposition of nanofibers can easily be directed to generate a multilayered film (Fig. 4A). In one study, this was achieved by alternating the scheme for applying the high voltage. The nanofibers in each layer were uniaxially aligned, with their long axes rotated by 60 degrees between adjacent layers (Fig. 4B). We have applied this type of multilayered scaffold to neural tissue engineering and showed that neurites derived from embryonic stem cells (ESCs) followed the pattern of the underlying scaffold, including at the intersection of two perpendicular fibers. Wang and co-workers also developed scaffolds using a layer-by-layer approach. They first deposited a layer of random fibers mat on a grounded electrode, which was immersed in cell culture medium. Cells were then seeded onto the first layer of the scaffold, after which the second layer of fibers was directly electrospun on top of the cells (Fig. 4C). By repeating these two steps, scaffolds with a multilayered architecture and embedded with cells were constructed. Figure 4D shows the cross-sectional fluorescent image of the cell-fiber construct, with the fibers in green and cell nucleus in blue. A major advantage of this approach is that the composition of the fibers can be easily tailored. Different growth factors or drugs can be encapsulated in different layers, and the topography of each layer can also be altered.
4.2 Conduits assembled from nanofibers
Conduits comprised of random or circumferentially aligned fibers can be readily fabricated by depositing nanofibers on a rotating mandrel.[70b] However, conduits with nanofibers aligned parallel to the long axis of the tube are sometimes more desirable as cells could then be guided to migrate along the tube. To address this issue, Ramakrishna and co-workers developed a method for fabricating a tube consisting of diagonally aligned electrospun fibers through a combination of electrostatic and mechanical forces. A knife-edged auxiliary electrode was charged with a polarity opposite to the spinneret and placed at a 45-angle relative to the long axis of the rotating Teflon tube, creating an electrostatic field that promoted a diagonal alignment for the nanofibers deposited on the collector. By rotating the Teflon tube, they obtained a tubular conduit with uniform thickness and superior mechanical strength. Chang and co-workers also developed an electrostatic method for fabricating multiple, interconnected conduits composed of electrospun fibers (Fig. 4E). In this case, nanofibers were deposited on collectors with different designs or structures. Upon removal of the collectors, nanofiber conduits with desired configurations were obtained. Alternatively, a tubular conduit can also be fabricated by simply rolling up a nonwoven mat of electrospun nanofibers and securing the edges through the use of solvent, glue, or heating. This technique allows one to use multiple, different layers, for example, a mesh of aligned fibers in the inner layer and a nonwoven mat of random fibers in the outer layer (Fig. 4F). Since a mat made of random fibers has a larger porosity comparing to the aligned counterpart, the multilayered conduits would support better nutrient transport and cell outgrowth without compromising contact guidance. Recently, a biomimetic scaffold was fabricated by rolling electrospun nanofiber matrices in a concentric manner with an open central cavity to replicate bone marrow cavity as well as the lamellar structure of bone. In this case, a rectangular strip of electrospun nanofibers was rolled around a 1-mm thick Teflon rod to produce the concentric structure. The compressive modulus of the scaffold was found to be in the mid-range of human trabecular bone. The porous structure also encouraged osteoblast infiltration and ECM secretion by mimicking the native structure of the bone.
5. Applications in regenerative medicine
5.1 Nerve injury repair
The recovery of injuries to both peripheral and central nervous systems can greatly benefit from the neural tissue engineering strategy that uses a scaffold or conduit to facilitate the re-growth of nerves. The most severe injury to a peripheral nervous system (PNS) is the complete transection of the nerve fiber. After the injury, protease activity increases at the site of injury, giving rise to a series of degradation events at the distal ends of the injured nerve fiber. In the central nervous system (CNS), the initial neurological damage provokes a series of cellular and biochemical responses, resulting in a secondary injury. The secondary injury prohibits nerve regeneration and causes more cell death, creating a cavity at the injury site and glial scar around the lesion. Since the environment at the injured site discourages the elongation and re-innervation of axonal re-growth, CNS nerve repairs are more challenging than PNS nerve repairs.
The extension of a regenerating axon requires positive cues to be built within the scaffold. The growth cone at the tip of the axon cylindrically extends into the ECM searching for cues and retracts when inhibitory molecules are encountered or if no positive cues are found. It then transduces the guidance cues into intracellular signals for neurite extension and orientation. Conventional hydrogel scaffolds are isotropic, and hence they cannot provide any directional cues. Microchannels, microridges, microgrooves, and stripes can provide topographical cues to direct neurite extension, but the dimension of these microstructures are on the same scale as the diameter of axons or cells and thus they are unable to guide sub-cellular events. Electrospun nanofibers, on the other hand, are more physiologically relevant to the fibrous structures of native ECM of neural tissues and can thus interact intimately with the growth cones to provide contact guidance for directed neurite extension.
Many research groups have demonstrated that uniaxially aligned nanofibers can not only provide directional contact guidance, but also encourage longer axonal protrusion to bridge the severe nerve defect. Ramakrishna and co-workers have demonstrated that neurites sprouting from neonatal mouse cerebellum C17.2 NSCs ran parallel to the aligned PLLA fibers and axons of up to 100 μm were formed due to the enhanced contact guidance. We also showed that the average length of the neurites projected from chicken primary DRG cultured on aligned nanofibers was ~1,100 μm, whereas this value was ~800 μm on random nanofibers. Figure 5, A and B, shows a projection of the neurites from DRG cultured on aligned and random nanofibers, respectively. The neurites were stained with anti-neurofilament 200, which recognize an epitope present on neurofilaments 200 kDa. The anisotropy of a scaffold made of aligned nanofibers also encouraged the differentiation of ESCs towards a neural lineage. CE3 mouse ESCs were induced to become neural progenitor cells by adding retinoic acid to embryoid body (EB) cultures, and after for 4 days the EBs were seeded onto electrospun nanofibers and allowed to differentiate. An increase in the proportion of neurons and neural precursor cells and a decrease in the proportion of astrocytes was found for aligned nanofibers compared to random nanofibers. Neural guidance conduits (NGCs) with variations in both fiber organization and composition can be easily constructed by rolling and sealing non-woven mats of nanofibers. Leong and co-workers demonstrated that NGCs composed of axially aligned fibers were able to improve peripheral nerve regeneration across a 15-mm nerve defect compared with NGCs consisting of random or circumferentially aligned fibers. Bellamkonda and co-workers examined the repair of a 17-mm nerve gap using a NGC fabricated by stacking 10–12 layers of electrospun nanofiber mats within two halves of a longitudinally split polysulfone tube. The results demonstrated that the aligned rather than the random construct successfully promoted regeneration of axons across the 17-mm nerve gap, re-innervated muscles, and formed new neuromuscular junctions.We also compared the healing effects of conduits base on silicone tubes and conduits of electrospun nanofibers over 10-mm defects in the sciatic nerves of rats. Figure 5, C and D, shows the results 8 weeks post operation. The conduits based on electrospun nanofibers were composed of two layers, with aligned nanofibers as the inner layer and random nanofibers as the outer layer. Toluidine blue staining of cross sections of the regenerated nerves indicated that the two types of conduits were comparable to each other in terms of regenerative capacity. However, 3D isometric reconstruction images revealed much more uniform nerve regeneration in the conduit made of nanofibers, especially in the middle zone (see the insets). These results indicated that the aligned nanofibers on the inner surface of the conduit could facilitate the regeneration of nerve by providing guidance to neurite extension, while the porosity in wall was advantages for the transport of nutrients and metabolic waste. This type of conduit is promising for applications in peripheral nerve repair.
The large surface area of a scaffold of electrospun nanofibers may provide superior cues for the differentiation of NSCs by accommodating a higher concentration of neurotrophic factors. For example, aminolysation of the surface of PCL electrospun fibers with ethylene diamine improved the adhesion and migration of adult rat brain derived NSCs into the scaffold. Fiber diameter is a significant factor in controlling the differentiation of NSCs. Reduction in diameter led to increased cell proliferation, spreading, and differentiation. A lower degree of cell aggregation was also reported for scaffolds made of nanofibers with smaller diameters. Electrical stimulation has been recognized as another contributing factor for neurite sprouting. To this end, we fabricated a scaffold of conductive core-sheath nanofibers through a combination of electrospinning and aqueous polymerization. Specifically, electrospun nanofibers of PCL or PLLA were employed as templates to generate uniform sheaths of conductive polypyrrole (PPy) via in situpolymerization. Figure 5E shows a TEM image of hollow PPy tubes obtained by dissolving the core polymer. Electrical stimulation, when applied through the scaffolds of conductive core-sheath nanofibers, was found to further increase the maximum length of neurites for aligned samples by 47% relative to the controls with no electrical stimulation. Figure 5F shows the synergetic effect of electrical stimulation and topographic guiding in promoting the uniaxial extension of neurites from DRG over long distances.
Although many efforts have been devoted to the field of nerve repair, challenges still exist. For example, the development of double-layered NGCs (with random nanofibers as the outer layer and the aligned nanofibers as the inner layer) has achieved some promising results, but the central cavity in the NGCs fails to provide contact guidance for neuronal outgrowth and thus will probably lead to a mismatch between proximal and distal ends. An NGC that provides 3D contact guidance throughout the whole conduit has yet to be developed.
5.2 Dura mater repair
The dura mater is the outermost layer of the three meninges that surround the brain and the spinal cord. Its major function is to help retain the cerebrospinal fluid (CSF). Traumatic injury or surgical operation often leaves a defect in the dura mater, which needs to be covered by a dural substitute to prevent CSF leakage and promote the regeneration of dura tissue. Materials that have been used as dural substitutes include native autografts, xenografts, and synthetic polymers. Whereas native autografts such as fascia lata work well as dural substitutes due to their low immunogenicity, their availability and the morbidity associated with the explanted sites severely limits their use. Various xenologous collagen grafts, including bovine and ovine pericardium, have been developed as alternatives, but with limited success due to the risk of disease transmission and immunogenicity from animal-derived materials. Other concerns such as low-tensile strength and rapid bioresorption also plague the use of xenologous collagen grafts. Scaffolds based on synthetic, biodegradable polymers have gained popularity in recent years because of their low cost, zero risk of disease transmission, and good mechanical properties. In particular, the high rate of cell infiltration and the ability to provide directional contact guidance have made nonwoven mats of electrospun nanofibers a new class of promising dural substitutes.
The regeneration rate of the injury site is a key factor in evaluating the efficacy of a dural substitute. Several prior studies have shown that cells cultured on scaffolds of uniaxially aligned nanofibers tend to migrate along the nanofibers. Based on this observation, we developed nanofibers with radial alignment to specifically target dura mater repair and other applications involving wound closure. In a typical dural defect, the injured site is still surrounded by healthy, intact tissue. By accelerating the migration of dura fibroblasts from the periphery, it is possible to achieve fast closure for the dural defect. We have demonstrated that dural fibroblasts were able to cover the entire surface of a scaffold made of radially aligned fibers within 4 days, while a void still existed on a control based on random nanofibers, indicating a faster migration rate for the cells on radially aligned nanofibers (Fig. 6, A and B). An enlarged view of the center regions are shown Figure 6, C and D, where a void of cells can be clearly seen on the random scaffold.
Synthetic materials possess a range of advantages over collagen matrices: they are cost effective, mechanically strong, and less prone to diseases transfer. The ability to generate radially aligned topography distinguishes electrospinning from other techniques in the fabrication of dural substitutes. A current limitation of electrospun dural substitutes is that they are not suitable for small dural defects where on-lay transplantation (without suturing) is needed. Further research should include efforts to alter the surface chemistry of the electrospun nanofibers so that they can be used for both on-lay and in-lay purposes.
5.3 Tendon/ligament repair
Tendon (connecting muscle and bone) and ligament (connecting bone to bone) tissues are compositionally, structurally, and mechanically similar. Both tissues are loaded primarily in one direction and their ECM (mostly type I collagen) has a uniaxially aligned structure, leading to highly anisotropic mechanical properties. Tendon/ligament has a low propensity for regeneration due to its high ECM density, collagen organization, and low vascularity. The scar-mediated healing response of tendon/ligament and its inability to regenerate has led to the investigation of tissue engineering approaches to replace the damaged or diseased tissue.
Braided and knitted fabrics have been used as scaffolds for tendon/ligament repair. The major drawbacks of these constructs are the poor performance in mass transport, cell attachment, and cell infiltration.Scaffolds based on electrospun nanofibers have started to gain popularity in the field of tendon/ligament repair not only because of the high porosity and high cell infiltration rate, but also due to the ease of generating uniaxial alignment to mimic the anisotropic structure of native tissues. Fibroblasts and BMSCs are often cultured on aligned nanofibers to generate a cell-seeded scaffold. Ouyang and co-workers found that, when seeded on aligned PLLA fibers, human tendon progenitor cells (hTSPCs) oriented themselves along the direction of the fibers and expressed higher level of tendon specific genes than those seeded on random fibers. Figure 7, A and B, shows alkaline phosphatase (ALP) stating of hTSPCs cultured on scaffolds consisting of aligned and random fibers, respectively, in normal osteogenic induction media for 2 weeks. Figure 7, C and D, shows alizarin red S staining of hTSPCs cultured on aligned and random scaffolds under the same conditions. These results indicated the pluripotent nature of the hTSPCs. Figure7, E and F, shows Masson’s trichrome staining of collagen fibers formed on the fibrous scaffolds after 6 weeks in vivo. Whereas aligned fibers induced the formation of aligned collagen fibrils similar to the native system, random fibers resulted in the formation of haphazard collagen fibrils. Shin and co-workers investigated the influence of nanofiber alignment and the direction of mechanical strain stimulation on ECM production of human anterior cruciate ligament fibroblast (HLF). The HLFs on the aligned nanofibers were spindle-shaped and oriented in the direction of the nanofibers. Significantly more collagen was synthesized on aligned fibers than on random fibers. After culture at 5% uniaxial strain for 24 h at a frequency of 12 cycles per min, the HLFs produced more collagen on the longitudinally stretched scaffolds than on those stretched in the transverse direction. Similar results were obtained by Lu and co-workers, who cultured human rotator cuff fibroblasts on scaffolds of aligned and random PLGA nanofibers. Distinct integrin expression profiles on these two scaffolds were observed, with higher expression of integrin on aligned nanofibers. Quantitative analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber alignment and that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically relevant mechanical properties were maintained.
In addition to fiber alignment, the crimped nature of collagen fibrils in tendon/ligament is an important feature that should be considered when preparing nanofiber scaffolds for application in tendon/ligament repair. Fiber crimp leads to relatively high extension under low loads, providing the characteristic non-linear mechanical behavior of tendon and ligaments and possibly shielding cells from high shear stresses. Electrospun nanofibers could be induced to crimp upon removal from a mandrel that rotates at a very high speed (Fig. 7D). The crimped morphology could be retained for at least 4 weeks in PBS at 37 °C (Fig. 7E). The crimping effect was determined to be a result of the residual stresses resident in the fibers during the fiber alignment process. The same group also produced crimp in electrospun nanofibers by using a temperature higher than the glass-transition temperature of the polymer. The resultant crimped fibers exhibited a long toe region in their stress-strain curve, reproducing a characteristic of the collagen fibrils in native tendon/ligament.
The ease of producing uniaxial alignment represents a major advantage for electrospinning in fabricating scaffolds for tendon/ligament repair. Growing interest has been focused on generating crimped structures with tension bearing capacity similar to that of native tendon/ligament. While the shape of the stress-strain curve for a scaffold made of crimped, electrospun nanofibers resembles that of native tendon/ligament, the magnitude of the curve for these constructs is much smaller than that of native tissue. The insufficient mechanical properties for the demanding mechanical physiological conditions of tendons and ligaments may lead to premature failure of the healing tendon/ligament. Future work should focus on improving the mechanical properties of aligned and crimped nanofiber scaffolds.
5.4 Tendon-to-bone insertion site repair
The tendon-to-bone insertion (the enthesis) can generally be characterized as either fibrous or fibrocartilagious. At a fibrous insertion, a tendon attaches to the bone at an acute angle through collagen fibers that extend directly to the bone. In contrast, a fibrocartilagious insertion is characterized by a functionally graded transitional zone of tendon, followed by uncalcified fibrocartilage, mineralized fibrocartilage, and bone. The transitional zone exhibits a gradual change in mineral content, spatial organization, cell type, and signaling molecules. While the tendon tissue is made up of densely packed and well-aligned collagen fibrils, the bone tissue is made up of less oriented and highly mineralized collagen fibrils. No sharp boundary exists between the tendon and bone; rather, a functionally graded architecture connects the very different tissues, mitigating stress concentrations and enabling the transmission of forces.
Tendon-to-bone insertion repair is a well-known clinical challenge. For example, surgical repair of the injured rotator cuff usually involves suturing the torn tendon to the humeral head. Although the tendon is re-attached to its anatomic footprint, the functionally graded transitional tissue is not regenerated and the repair often fails. In order to improve outcomes after tendon-to-bone repair, tissue engineers seek to develop a scaffold that recapitulates the native, graded structure of fibrocartilagious tendon-to-bone insertion. Such a scaffold may facilitate the surgical repair and provide functional recovery of a robust attachment between the repaired tendon and bone. Two different methods have been demonstrated for fabricating scaffolds to be used for tendon-to-bone repair. In the first method, stratified scaffolds were fabricated by co-culture of multiple types of cells. The expectation is that the interactions among different cell types would eventually result in cell-mediated development of a functional insertion. In the second method, stem cells are seeded onto a graded scaffold with variation in surface chemistry such as the concentration of bioactive molecules or mechanical properties. The argument is that the stem cells will respond to the different local stimuli, differentiating into various types of cells and thus generating a functional insertion. The fabrication of a scaffold with a graded coating of mineral on electrospun nanofibers falls into the second approach.
The potential use of electrospun nanofiber scaffolds in addressing tendon-to-bone insertion repair has been investigated by several groups.[111b,113] In one study, we gradually added 10 times concentrated simulated body fluid (10SBF) into a glass vial containing a piece of the electrospun nanofiber scaffold (Fig. 8A). A mineral gradient was formed along the length of the scaffold due to the difference in immersion time along the vertical direction. Significantly, the gradient in mineral content resulted in a gradient in stiffness for the scaffold and thus the MC3T3 mouse preosteoblasts were found to preferentially attach to the end higher in mineralization.[111b] Figure 8, B and C, show SEM images of the scaffold. The fluorescence micrographs in Figure 8, D and E, indicate that the MC3T3 cells had a low affinity for the unmineralized end of the scaffold and a high affinity for the highly mineralized end of the scaffold.
We also fabricated scaffolds with an “aligned-to-random” transition for the electrospun PLGA nanofibers that could mimic the structural organization of collagen fibers at the tendon-to-bone insertion site (Fig. 8F). Figure 8G shows an SEM image taken from the boundary between the aligned and random nanofibers. As shown in Figure 8H, tendon fibroblasts cultured on such a scaffold exhibited highly organized and haphazardly oriented morphologies, on the aligned and random portions of the scaffold, respectively.[113a]
Gradation in surface-attached bioactive molecules can also be achieved by adding a solution gradually. For example, Chen and co-workers fabricated a poly(methyl glutarimide) scaffold with graded fibronectin by placing the scaffold of electrospun nanofibers vertically in a container, into which a fibronectin solution was added in a controlled manner.[113b] They also observed a positive correlation between the population of NIH/3T3 fibroblasts and the concentration of fibronectin. A technique called twin-screw extrusion/electrospinning was utilized by Kalyon and co-workers to fabricated scaffolds of electrospun PCL nanofibers with gradation in β-tricalcium phosphate (β-TCP) concentration along the thickness direction.[113c, d] The technique involved the use of a twin screw extruder with fully intermeshing and rotating screws integrated with a multichannel spinneret, which was connected to a high-voltage power supply. Injection ports on the side surface of the barrel enabled the introduction of β-TCP continuously. The screws were rotated to allow for mixing. The feeding rate for the β-TCP solution was gradually increased over time. The resultant scaffold had a gradient in β-TCP concentration along the thickness direction. The graded scaffold was then seeded and cultured with MC3T3. After 4 weeks, the tissue construct showed continuous gradation in ECM, including both collagen synthesis and mineralization.[113d]
These studies demonstrate that electrospinning is a promising approach to the fabrication of graded scaffolds with physical and chemical properties similar to the natural ECM of the tendon-to-bone insertion. However, this technique is still in the early stages of development. Most of the studies to date have focused on the establishment of new fabrication systems to produce the desired gradients, whereas the differentiation of stem cells such as bone mesenchymal stem cells (BMSCs) on such scaffolds has not yet been systematically investigated. In vivo studies should also be carried out to assess the efficacy of such graded scaffolds in tendon-to-bone insertion repair.
6. Concluding remarks
Electrospinning is a remarkably simple and versatile technique. This technique can generate nanofibers on a large scale while also allowing one to tailor many aspects of the resulting scaffold: i) the fiber diameter can easily be controlled by varying the concentration of the solution, ejection rate, applied voltage, and the distance between the spinneret and the collector; ii) the nanofibers can be readily fabricated with a hollow or core-sheath structure by altering the configuration of the spinneret; iii) the nanofibers can be made highly porous by inducing a phase separation between the polymer and the solvent; iv) the nanofibers can be aligned into a rich variety of structures by using specially designed collectors; and v) the nanofibers can be stacked and/or folded to form complex structures or architectures. Electrospinning has been successfully applied to a wide range of biomaterials, including natural proteins (e.g., collagen and elastin) and synthetic polymers such as PLGA, PCL, PLA, and PGA that have been approved by Federal Drug Administration (FDA) as biocompatible and biodegradable materials for clinical use. Its capabilities have also been extended to include inorganic materials such as hydroxyapatite (HA), bioactive glasses, and their composites with polymers. Because of the high porosity and large surface area, a scaffold derived from electrospun nanofibers can mimic the hierarchical structure of ECM that is critical for cell attachment and spreading as well as nutrient/waste transportation. If necessary, the nanofibers can be further functionalized via encapsulation or attachment of bioactive species such as ECM proteins, enzymes, DNAs, and growth factors to better control the proliferation and differentiation of cells seeded on the scaffolds. These attributes make electrospun nanofibers well-suited as scaffolds for regenerative medicine.
Whereas impressive progress has been made in applying electrospun nanofibers as scaffolds for regenerative medicine, challenges still exist. Low cell infiltration rates remain one of the most significant hurdles that must be cleared before this technology can be applied to most in vivo systems. Although the porosity of a nanofiber-based scaffold can be enlarged to facilitate cell infiltration by incorporating a sacrificial component, the mechanical properties and integrity of the scaffold are often compromised. Moreover, due to the abundance of nutrients at the surface of the scaffold, it is likely that cells will prefer to stay on the surface without migrating into the bulk. Direct electrospinning of live cells has emerged as a novel technique to address this challenge. To this end, Jayasinghe and co-workers used a coaxial electrospinning method to enable the flow of highly concentrated cellular suspension in the inner capillary while the outer needle accommodated the flow of a poly(dimethylsiloxane) (PDMS) medium. A large population of the cells remained viable post-electrospinning for a relatively long period of time, as assessed by flow cytometry.
Another technical challenge for the application of electrospun nanofiber scaffolds is that the diameter of the fibers cannot be easily reduced to a scale below 100 nm, which is the upper limit of native ECM fibers. In particular, the nanoscale size is difficult to achieve for the natural and synthetic polymers commonly used for tissue engineering applications. To better mimic the ECM, it is desirable to produce fibers with diameters thinner than 100 nm, preferably in the range of 10–50 nm. When the size of electrospun fibers is reduced, an additional issue will arise in that the porosity of the scaffold will decrease accordingly. There is a strong need to develop a method capable of generating fibers with diameters identical to that of native ECM fibers while maintaining a high porosity and thus high rates for cell infiltration and mass transport. As a better understanding of the electrospinning process is achieved through theoretical modeling, it is expected that all these technical problems will be solved in the near future.
**This work was supported in part by an NIH Director’s Pioneer Award (DP1 OD000798), a grant from the NIH (1R01 AR060820-10), a musculoskeletal core center grant from the NIH (1P30 AR057235-01), and startup funds from Washington University in St. Louis. We thank Yu Zhang for his help with the preparation of this manuscript.
Wenying Liu, Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130 (USA)
Prof. Stavros Thomopoulos, Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110 (USA). Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130 (USA)
Prof. Younan Xia, Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130 (USA)
Electrospun Nanofibers: New Concepts, Materials, and Applications.
Electrospun nanofibers: solving global issues
Porous structures with their high surface areas have found applications in many different areas. Nanofibers, with their large surface-to-volume ratio, have the potential for use in various applications where high porosity is desirable. A porous structure made out of nanofibers is a dynamic system where the pore size and shape can change, unlike conventional rigid porous structures. Nanofibers can also be linked to form a rigid structure if required. Perhaps the most versatile process for producing nanofibers with relatively high productivity is electrospinning. Porous, nanofiber meshes made by electrospinning have been identified for use in numerous applications (Fig. 1).
There are several methods of producing nanofibers, from high-volume production methods such as melt fibrillation1, island-in-sea2, and gas jet3 techniques, to highly precise methods like nanolithography4, 5 and self-assembly6, 7, 8, 9. However, their usefulness is limited by combinations of restricted material ranges, possible fiber assembly, cost, and production rate. Here, electrospinning has an advantage with its comparative low cost and relatively high production rate. Micron size yarns consisting of nanofibers can be produced at a rate of 70 m/min, and different assemblies can be formed.
Almost any soluble polymer with sufficiently high molecular weight can be electrospun. Nanofibers made of natural polymers, polymer blends, nanoparticle- or drug-impregnated polymers, and ceramic precursors have been successfully demonstrated. Different fiber morphologies have also been shown, such as beaded, ribbon, porous, and core-shell fibers10 (Fig. 2).
As with conventional fiber-spinning process, the solution typically passes through a spinneret during electrospinning, although there are a few exceptions. But instead of using air or mechanical devices to create the extrusion force, a high voltage is applied such that the particles within the solution are charged, thereby creating a repulsive force. At a critical voltage, the repulsive force overcomes the surface tension of the solution and a jet erupts from the tip of the spinneret. Unlike conventional spinning, the jet is only stable near the tip of the spinneret, after which the jet is subject to bending instability11. As the charged jet accelerates toward regions of lower potential, the solvent evaporates while the entanglements of the polymer chains prevent the jet from breaking up. This results in fiber formation. Generally, a grounded plate is used to collect the fibers (Fig. 3).
Electrospinning was first patented in the US in 190212; however, the process was largely forgotten until the 1990s. With interest in the field of nanoscience and nanotechnology, researchers began new investigations of nanofiber production using electrospinning13. This effort has grown, with over 200 universities (Fig. 4) and research institutes worldwide studying various aspects of the electrospinning process and the fibers it produces. The number of patents for processes and applications based on electrospinning has also grown in recent years (Fig. 5). Startups such as eSpin Technologies, NanoTechnics, and KATO Tech are just some of the companies seeking to reap the unique advantages offered by electrospinning, while companies such as Donaldson Company and Freudenberg have been using electrospun fibers in their air filtration products for the last two decades.
The ability to form porous fibers through electrospinning means that the surface area of the fiber mesh can be increased tremendously. Phase separation is proposed as the main mechanism behind the formation of porous fibers. When more volatile solvents are used, solvent-rich regions begin to form during electrospinning that transform into pores14. Another method of producing porous nanofibers is the spinning of a blend of two different polymers. One of the polymers is removed after fiber formation by dissolution in a solvent in which the other polymer is insoluble15.
Since stretching of the solution arises from repulsive charges, the electrospinning jet path is very chaotic and only nonwoven meshes are produced using a typical setup. Nevertheless, more ordered assemblies that allow the porosity of the mesh to be controlled have been produced through clever manipulation of the setup and solution composition. Several methods have been developed that yield aligned fibers with various degrees of order16, 17, 18, 19 and fiber directions20, 21 for two- and three-dimensional assemblies22, 23, 24, 25, 26 (Fig. 6). Such assemblies are usually achieved through control of the electric field between the tip of the spinneret and the collector, use of a dynamic collector such as a rotating mandrel, or a combination of both. Li et al.20 used a pair of parallel conducting electrodes to create an electric field such that the electrospun fibers are preferentially aligned across the gap in between the electrodes. Boland et al.16 used a rotating drum at a speed of 1000 rpm to collect aligned fibers. To fabricate a tubular scaffold, electrospun fibers can be deposited on a rotating tube and the deposited fiber layer subsequently extracted from the tube. Fiber alignment can be controlled using auxiliary electrodes to create an electric field profile that influences the flight of the electrospinning jet (Fig. 7).
With such versatility, electrospun fibers are being explored for use in many different applications. Currently, most tests use nonwoven fiber meshes made out of smooth fibers. Ceramic nanofibers derived from nonwoven electrospun fiber meshes have opened up new areas of opportunities. Besides nonwoven meshes, testing of other fibrous assemblies for potential applications has been limited. Nevertheless, the versatility of electrospun fibers can be seen in the established results and on-going research in major areas like healthcare, biotechnology and environmental engineering, defense and security, and energy storage and generation.
Current medical practice is based almost entirely on treatment regimes. However, it is envisaged that medicine in the future will be based heavily on early detection and prevention before disease manifestation. Together with nanotechnology, new treatment modalities will emerge that will significantly reduce medical costs.
With recent developments in electrospinning, both synthetic and natural polymers can be produced as nanofibers with diameters ranging from tens to hundreds of nanometers with controlled morphology and function. The potential of these electrospun nanofibers in human healthcare applications is promising, for example in tissue/organ repair and regeneration, as vectors to deliver drugs and therapeutics, as biocompatible and biodegradable medical implant devices, in medical diagnostics and instrumentation, as protective fabrics against environmental and infectious agents in hospitals and general surroundings, and in cosmetic and dental applications.
Tissue/organ repair and regeneration are new avenues for potential treatment, circumventing the need for donor tissues and organs in transplantation and reconstructive surgery. In this approach, a scaffold is usually required that can be fabricated from either natural or synthetic polymers by many processing techniques including electrospinning and phase separation.
The biocompatibility of the scaffold is usually tested ex vivo by culturing organ-specific cells on the scaffold and monitoring cell growth and proliferation. An animal model is used to study the biocompatibility of the scaffold in a biological system before the scaffold is introduced into patients for tissue-regeneration applications.
Nanofiber scaffolds are well suited to tissue engineering as the scaffold can be fabricated and shaped to fill anatomical defects; its architecture can be designed to provide the mechanical properties necessary to support cell growth, proliferation, differentiation, and motility; and it can be engineered to provide growth factors, drugs, therapeutics, and genes to stimulate tissue regeneration. An inherent property of nanofibers is that they mimic the extracellular matrices (ECM) of tissues and organs. The ECM is a complex composite of fibrous proteins such as collagen and fibronectin, glycoproteins, proteoglycans, soluble proteins such as growth factors, and other bioactive molecules that support cell adhesion and growth. Studies of cell-nanofiber interactions have shown that cells adhere and proliferate well when cultured on polymer nanofibers27, 28, 29.
One of our aims is to fabricate electrospun polymer nanofiber scaffolds for engineering blood vessels, nerves, skin, and bone. We have demonstrated that human coronary artery smooth muscle cells cultured on synthetic nanofibrous scaffolds of the copolymer poly(L-lactic acid)/poly(ɛ-caprolactone), or PLLA/PCL, show normal morphology and good proliferation. The cells organize along the aligned nanofibers in a directional manner typified by the orientation of the cytoskeletal protein α-actin (Fig. 8), suggesting that nanofiber orientation can impart a functional development on the cells30.
On collagen-modified nanofibers, human coronary artery endothelial cells exhibit cobble-stone morphology (Fig. 9a), typical of endothelial cells cultured on a polystyrene surface with comparable adhesion and proliferation rates31. On aligned PLLA nanofibers, c17.2 neural cells adhere, elongate along the fibers, and neurites extend along the direction of the aligned fibers (Fig. 9b)32. Human dermal fibroblasts have been demonstrated to grow better on collagen nanofibrous scaffolds than polystyrene tissue culture surfaces (Fig. 9c)33.
A recent study carried out with human coronary endothelial cells cultured on nanofibrous scaffolds34 indicates that nanofiber scaffolds positively promote cell-matrix and cell-cell interactions, with the cells having a normal phenotypic shape and gene expression. This can be attributed to the ECM-like properties of the nanofiber scaffolds that mimic the natural tissue environment.
Further research is required to elucidate the influence of nanofibers on the biochemical pathways and cellular signaling mechanisms that regulate cell morphology, growth, proliferation, differentiation, motility, and genotype. Insight into how natural ECM components secreted by cells replace the biodegradable polymeric scaffolds is also needed. This complete understanding of cell-nanofiber scaffold interactions will pave the way for successful engineering of various tissues and organs, such as vascular grafts, nerve, skin and bone regeneration, cornea transplants, skeletal and cardiac muscle engineering, gastrointestinal and renal/urinary replacement therapy, and even stem cell expansion and differentiation to specific cells types and organ regeneration.
In the pharmaceutical and cosmetic industry, nanofibers are promising tools for controlled delivery of drugs, therapeutics, molecular medicines, and body-care supplements. For example, DNA covalently attached to a patterned carbon nanofiber array and inserted into cells by centrifuging the cells onto the array, does not affect cell viability and the gene encoded by the inserted DNA is expressed35. This could pave the way for the development of a ‘smart’ polymeric drug delivery system.
In another system36, 37, a drug-bound, pH-responsive polymer is targeted to diseased cells through cell receptor binding of a ligand. It is subsequently endocytosed into the endosomal compartment of the cells. In the low pH environment of the endosome, the polymer backbone separates from the drug, destabilizes the endosomal membrane, and releases the drug into the cytoplasmic compartment of the cells. This system of drug delivery can also be used to deliver therapeutics, silencing RNA, antisense oligonucleotides, and vaccines to specific cell types, targeting specific compartments and organelles.
The core-shell nanofibers developed in our laboratory can also be used to encapsulate drugs and therapeutics for drug delivery applications. Release kinetic studies of core-shell nanofibers with fluorescein-isothiocyanate-conjugated bovine serum albumin (FITC-BSA) encapsulated in the core show a gradual release of FITC-BSA when cultured with human dermal fibroblasts (HDFs), instead of a burst release profile without the cells (Fig. 10). However, a faster release of FITC-BSA is observed when the nanofibers are cultured with cells. This is likely to be the result of a higher polymer degradation rate in the presence of degradative enzymes secreted by the cells, which could be desirable for applications like wound dressing, where an initially higher but sustained release of antibiotics is preferred. This release profile is crucial for regulating cell growth if the nanofiber scaffolds for tissue engineering applications are to encapsulate bioactive molecules or allow slow passive delivery if the nanofibers are used for drug delivery applications. Biological or chemical ligands can be conjugated onto the nanofibers for cell-specific targeting, or as biosensors responsive to physiological changes to mediate controlled delivery of insulin in diabetes patients. In cosmetics, nanofiber masks can be impregnated with skin-revitalizing factors for skin health and renewal.
Nanofibers can also be fabricated from shape-memory materials38. These can be implanted into the body using a laparoscope (a long, slender medical instrument for examining the interior of an organ or to perform minor surgery), minimizing complex surgical procedures. The fibers then change shape in response to the increased temperature. Such nanofibers have potential as vascular stents, bone void fillers39, hernia repairs, and general tissue repairs40. Because of the large surface-to-volume ratio, nanofibers can also be used in diagnostics for large-scale disease and genetic screening and even as filters in medical instruments for membrane-impermeable biomolecules, bacteria, and viral particles.
Biotechnology and environmental engineering applications
High porosity, interconnectivity, microscale interstitial space, and a large surface-to-volume ratio mean that nonwoven electrospun nanofiber meshes are an excellent material for membrane preparation, especially in biotechnology and environmental engineering applications (Fig. 11). Ligand molecules, biomacromolecules, or even cells can be attached or hybridized with the nanofiber membrane for applications in protein purification and waste water treatment (affinity membranes), enzymatic catalysis or synthesis (membrane bioreactors), and, in the future, chemical analysis and diagnostics (biosensors).
Electrospun nanofibers can form an effective size exclusion membrane for particulate removal from wastewater. Particle removal from air by a nanofiber membrane has been studied by Gibson et al.41. The nanofiber membrane shows an extremely effective removal (∼100% rejection) of airborne particles with diameters between 1 μm and 5 μm by both physical trapping and adsorption. For particle removal from aqueous solution, our recent results show that electrospun membranes can successfully remove particles 3-10 μm in size (>95 % rejection) without a significant drop in flux performance42. No particles were found trapped in the membrane, so the membrane could be effectively recovered upon cleaning. This opens up new avenues of application of electrospun membranes for the pretreatment of water prior to reverse osmosis.
In our laboratory, nanofiber membranes are also being tested as affinity (or adsorptive) membranes. Affinity membranes are a broad class of membranes that selectively capture specific target molecules (or ligates) by immobilizing a specific capturing agent (or ligand) onto the membrane surface. In biotechnology, affinity membranes have applications in protein (such as IgG) purification and toxin (such as endotoxin) removal from bioproducts. In the environmental industry, affinity membranes have applications in organic waste removal and heavy metal removal in water treatment.
To be used as affinity membranes, electrospun nanofibers must be surface functionalized with ligands. In most cases, the ligand molecules should be covalently attached on the membrane to prevent leaching of the ligands. Cellulose nanofiber membranes have been surface functionalized with cibacron blue for the purification of albumin43. Cellulose nanofiber membranes functionalized with protein A/G (a recombinant 50 449 Da protein from Pierce Biotechnology that has an increased ability to bind IgG molecules) shows a high ability to capture IgG molecules with a capacity of ∼134 μg/cm2, which is higher than that of the commercialized membrane (∼80 μg/cm2).
Water pollution is now becoming a critical global issue. One important class of inorganic pollutant of great physiological significance is heavy metals, e.g. Hg, Pb, Cu, and Cd. The distribution of these metals in the environment is mainly attributed to the release of metal-containing wastewaters from industries. For example, copper smelters may release high quantities of Cd, one of the most mobile and toxic among the trace elements, into nearby waterways44. It is impossible to eliminate some classes of environmental contaminants completely, such as metals, by conventional water purification methods. Affinity membranes will play a critical role in wastewater treatment to remove (or recycle) heavy metals ions in the future. Polymer nanofibers functionalized with a ceramic nanomaterial, such as hydrated alumina/alumina hydroxide and iron oxides, could be suitable materials for fabrication of affinity membranes for water industry applications. The polymer nanofiber membrane acts as a carrier of the reactive nanomaterial that can attract toxic heavy metal ions, such as As, Cr, and Pb, by adsorption/chemisorption and electrostatic attraction mechanisms.
Compared with heavy metal pollutants, overall water quality is much more sensitive to organic pollutants. Although such organics are usually no more than 1% of the pollution in a river, they tend to use up its dissolved oxygen, making the water unable to sustain life. While the transformations and pathways of metals in the environment have been studied to some extent, much less information is available on most commercial organic products because of their complex structures. Again, affinity membranes provide an alternative approach for removing organic molecules from wastewater. For example, β-cyclodextrin is a cyclic oligosaccharide comprising of seven glucose units. It has a stereo-specific toroidal structure with a hydrophobic interior and hydrophilic exterior that can capture hydrophobic organic molecules from water by forming an inclusion complex. β-cyclodextrin has been introduced into a poly(methyl methacrylate) nanofiber membrane using a physical mixing method to develop an affinity membrane for organic waste removal45.
Electrospun nanofibers have also received great attention for sensor applications because of their unique high surface area. This is one of the most desirable properties for improving the sensitivity of conductometric sensors because a larger surface area will absorb more of a gas analyte and change the sensor’s conductivity more significantly. Nanofibers functionalized with a semiconductor oxide such as MoO3, SnO2, or TiO2 show an electrical resistance that is sensitive to harmful chemical gases like ammonia and nitroxide46. Single polypyrrole nanofibers containing avidin were studied as biosensors for detecting biotin-labeled biomolecules such as DNA. Specific binding of the biomolecules to the nanofibers changes the electrical resistance of a single nanofiber47. A fluorescent polymer, poly(acrylic acid)-poly(pyrene methanol), or PAA-PM, was used as a sensing material for the detection of organic and inorganic waste. The fluorescence is quenched by adsorbed metal ions Fe3+ or Hg2+ or 2,4-dinitrotoluene (DNT) on the nanofiber surfaces48. In our laboratory, nylon-6 nanofiber was functionalized with biotinylated glucose oxidase to develop a novel biosensor for testing glucose concentration49.
Defense and security applications
Military, firefighter, law enforcement, and medical personnel require high-level protection when dealing with chemical and biological threats (which include chemicals like nerve agents, mustard gas, blood agents such as cyanides, and biological toxins such as bacterial spores, viruses, and rickettsiae) in many environments ranging from combat to urban, agricultural, and industrial. Current protective clothing is based on full barrier protection such as hazardous materials (HAZMAT) suits, or permeable adsorptive protective overgarments such as those used by the US military. The obvious limitations of these suits are weight and moisture retention, which prevent the user from donning them for long periods.
Nanostructures with their small size, large surface area50, and light weight will improve, by orders of magnitude, our capability to:
- Detect chemical and biological warfare agents with sensitivity and selectivity;
- Protect through filtration and destructive decomposition of harmful toxins; and
- Provide site-specific in vivo prophylaxis.
Polymer nanofibers are considered as excellent membrane materials for this purpose owing to their light weight, high surface area, and breathable (porous) nature51. The high sensitivity of nanofibers toward warfare agents makes them excellent candidates as sensing interfaces for chemical and biological toxins in concentration levels of parts per billion52. Governments across the world are investing in strengthening the protection levels offered to soldiers in the battlefield53. Various methods of modifying nanofiber surfaces to enhance their capture and decontamination capability of warfare agents are currently under investigation. One protection method is through chemical surface modification and attachment of reactive groups such as oximes, cyclodextrins, and chloramines54, 55 that bind and detoxify warfare agents.
In association with the Defense Science and Technology Agency (DSTA) in Singapore, our laboratory is working on functionalizing nanofibers to be used in facemasks for chemical and biowarfare defense (Fig. 12). The facemask consists of two main components: a high-efficiency particulate air (HEPA) filtering layer and an activated charcoal bed that adsorbs harmful gases and contaminants.
Nanofiber membranes may be used to replace the activated charcoal in adsorbing toxins from the atmosphere. Active reagents can be embedded into the nanofiber membrane by chemical functionalization, post-spinning modification, or through using nanoparticle polymer composites (Fig. 13). Preliminary tests using chemical warfare simulators such as paraoxon and dimethyl methyl phosphonate on the functionalized fibers show evidence of decontamination. Metal nanoparticles (Ag, MgO, Ni, Ti, etc.), which have proven abilities in decomposing warfare agents, can also be embedded in the nanofibers.
There are many avenues for future research in nanofibers from the defense perspective. As well as serving protection and decontamination functions, nanofiber membranes will also have to provide the durability, washability, resistance to intrusion of all liquids, and tear strength required of battledress fabrics.
Energy generation applications
Natural energy resources such as crude oil, coal, natural gas, and uranium are a necessity for everyday life. Rapid economic growth in Asia and the subsequent increase in demand for energy mean that the rate of oil production is no longer adequate. This is evident in the soaring price of crude oil, which has reached over $60 per barrel56. Large volumes of carbon dioxide emitted by the burning of fossil fuels is also the main culprit in climate change. Thus, there is an urgent need to identify new sources of energy that are environmentally friendly and able to replace current supplies. Polymer batteries, fuel cells, photovoltaic cells, wind power generators, and geothermal power generators are some possible alternatives.
Given their high porosity and inherent large total surface area, electrospun nanofiber membranes are being considered for polymer batteries57, 58, 59, photovoltaic cells60, 61, 62, 63, and polymer electrolyte membrane fuel cells (PEMFCs).
Polymer batteries have been developed for PC notebooks and cell phones to replace conventional, bulky lithium batteries. The components of polymer batteries are a carbon anode, a lithium cobalt oxide cathode, and a polymer gel electrolyte. When a battery is subjected to charging, Li+ ions are confined in the carbon anode. On discharging, the Li+ions move to cathode. Noteworthy properties of polymer batteries are less electrolyte leakage, high dimension flexibility, and high energy density per weight. However, there is still a need to improve energy density per weight of polymer batteries to increase their market share. Choi et al.57 and Kim et al.59 have assembled a new type of polymer battery using poly(vinylidene fluoride), or PVDF, nanofiber membranes (Fig. 14). The porous structure of the PVDF nanofiber membrane favors high uptake (350 wt.%) of lithium electrolyte so that electrolyte leakage is reduced. These factors make it possible to hold a large quantity of lithium electrolyte in thinner battery packs. The large surface area of the nanofibrous network also enhances ion conductivity, thus polymer batteries comprising nanofiber membranes may improve energy density per weight as compared with conventional polymer batteries.
Most conventional photovoltaic cells use single-crystalline, polycrystalline, or amorphous Si. It is well known that a single-crystal Si cell can achieve an energy translation efficiency of ∼20%, and this value is higher than other types of solar cells. However, the biggest shortcoming for single-crystal Si solar cells is their high manufacturing cost. There is also a need for a large surface area to obtain sufficient electrical output.
As an alternative, Grätzel and colleagues64 have developed dye-sensitized solar cells. The principle here is that sensitizing dye molecules coated onto TiO2 nanoparticles absorb photons and transfer excited electrons through the conduction band of TiO2 to the cathode. A nanotopographic TiO2 layer works as the electrode and enhances the total surface area to achieve a high electrical output. Dye-sensitized solar cells are less costly to manufacture than Si-based solar cells, but there are issues that need to be addressed, including reducing electrolyte leakage and improving the energy conversion efficiency (generally ∼4–10%). With respect to electrolyte leakage, an alternative solution is to use a viscous polymer gel electrolyte. However, it is difficult to infuse a viscous gel into a conventional TiO2nanotopographic layer. Song et al.61, 62, 63 have solved this problem by using TiO2nanofiber membranes fabricated by electrospinning in combination with sol-gel processes (Fig. 15). The viscous polymer gel electrolyte can easily penetrate into the porous nanofiber membrane. Their assembled TiO2 nanofiber dye-sensitized solar cells are able to achieve an energy conversion efficiency of 6.2%63.
Electricity generation in PEMFCs is through the chemical reaction of hydrogen at the anode and oxygen at the cathode (Fig. 16). Protons are transmitted through an electrolyte membrane that contains distilled water, while electrons are transmitted from the anode to the cathode. The key properties of electrolyte membranes are high proton conductivity and shielding of electron transport. As the membrane needs to hold distilled water for proton conductivity, water retention of the membrane is also important. Nafion® (DuPont), a perfluorosulfonic acid polymer film, has been widely used so far. However, Nafion membranes are expensive at up to $800/kg. For the same membrane area, electrospun Nafion fiber membranes require less material than conventional Nafion fuel cell membranes, thereby reducing cost. Porous nanofiber membranes are also able to hold distilled water, thus enhancing proton conductivity. Therefore, such nanofiber membranes have the potential to be used in PEMFCs.
Given the versatility of electrospinning for generating highly porous nanofiber meshes made out of different materials, it is no surprise that it has found possible uses in different fields ranging from healthcare, biotechnology, and environmental engineering to defense and security, and energy generation. Electrospinning may be able to produce microengineered scaffolds for tissue engineering. Improved wound dressings could be made out of nanofiber meshes impregnated with drugs. Membranes for water treatment or use in biotechnology could be made of electrospun fibers. Nanofiber clothing and filters could deal more effectively with chemical and biological threats. In the future, we may no longer be dependent on crude oil thanks to more efficient conversion of other energy sources to electricity. With the ability to mass-produce nanofibers, electrospinning may well be one of the most significant nanotechnologies of this century.
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Chemtrails And Aerosol Fibers
Rain On Texas Town
Weather Modification Patents
- 1891 – 0462795 – method of producing rain-fall
- 1914 – 1103490 – rain maker (balloon images)
- 1917 – 1225521 – protection from poisonous gas in warfare
- 1920 – 1338343 – process and apparatus for the production of intense artificial clouds, fogs, or mists
- 1924 – 1512783 – composition for dispelling fogs
- 1927 – 1619183 – process of producing smoke clouds from moving aircraft
- 1928 – 1665267 – process of producting artificial fogs
- 1932 – 1892132 – atomizing attachment for airplane engine exhausts
- 1933 – 1928963 – electrical system and method (for spraying chemtrails)
- 1934 – 1957075 – airplane spray equipment
- 1936 – 2045865 – skywriting apparatus
- 1936 – 2052626 – method of dispelling fog (mit)
- 1937 – 2068987 – process of dissipating fog
- 1939 – 2160900 – method for vapor clearing
- 1941 – 2232728 – method and composition for dispelling vapors
- 1941 – 2257360 – desensitized pentaerythritol tetranitrate explosive
- 1946 – 2395827 – airplane spray unit (us. dept. of agriculture)
- 1946 – 2409201 – smoke-producing mixture
- 1949 – 2476171 – smoke screen generator
- 1949 – 2480967 – aerial discharge device
- 1950 – 2527230 – method of crystal formation and precipitation
- 1951 – 2550324 – process for controlling weather
- 1951 – 2570867 – method of crystal formation and precipitation (general electric)
- 1952 – 2582678 – material disseminating apparatus for airplanes
- 1952 – 2591988 – production of tio2 pigments (dupont)
- 1952 – 2614083 – metal chloride screening smoke mixture
- 1953 – 2633455 – smoke generator
- 1954 – 2688069 – steam generator
- 1955 – 2721495 – method and apparatus for detecting minute crystal forming particles suspended in a gaseous atmosphere (general electric)
- 1956 – 2730402 – controllable dispersal device
- 1957 – 2801322 – decomposition chamber for monopropellant fuel
- 1958 – 2835530 – process for the condensation of atmospheric humidity and dissolution of fog
- 1959 – 2881335 – generation of electrical fields (haarp – for re-charging clouds!)
- 1959 – 2903188 – control of tropical cyclone formation
- 1959 – 2908442 – method for dispersing natural atmospheric fogs and clouds
- 1960 – 2962450 – fog dispelling composition (see references)
- 1960 – 2963975 – cloud seeding carbon dioxide bullet
- 1961 – 2986360 – aerial insecticide dusting device
- 1962 – 3044911 – propellant system
- 1962 – 3056556 – method of artificially influencing the weather
- 1964 – 3120459 – composite incendiary powder containing metal coated oxidizing salts
- 1964 – 3126155 – silver iodide cloud seeding generator (main commercial ingrediant)
- 1964 – 3127107 – generation of ice-nucleating crystals
- 1964 – 3131131 – electrostatic mixing in microbial conversions
- 1965 – 3174150 – self-focusing antenna system (haarp)
- 1966 – 3257801 – pyrotechnic composition comprising solid oxidizer, boron and aluminum additive and binder
- 1966 – 3234357 – electrically heated smoke producing device
- 1966 – 3274035 – metallic composition for production of hydroscopic smoke
- 1967 – 3300721 – means for communication through a layer of ionized gases (haarp)
- 1967 – 3313487 – cloud seeding apparatus
- 1967 – 3338476 – heating device for use with aerosol containers
- 1968 – 3410489 – automatically adjustable airfoil spray system with pump
- 1969 – 3429507 – rainmaker
- 1969 – 3430533 – aircraft dispensor pod having self-sealing ejection tubes
- 1969 – 3432208 – fluidized particle dispenser (us air force)
- 1969 – 3437502 – titanium dioxide pigment coated with silica and aluminum (dupont)
- 1969 – 3441214 – method and apparatus for seeding clouds
- 1969 – 3445844 – trapped electromagnetic radiation communications system (haarp – raytheon)
- 1969 – 3456880 – method of producing precipitation from the atmosphere
- 1970 – 3517512 – apparatus for suppressing contrails
- 1970 – 3518670 – artificial ion cloud
- 1970 – 3531310 – production of improved metal oxide pigment
- 1970 – 3534906 – control of atmospheric particles (dow chemical co)
- 1970 – 3545677 – method of cloud seeding
- 1971 – 3564253 – system and method for irradiation of planet surface areas (space solar mirrors)
- 1971 – 3587966 – freezing nucleation
- 1971 – 3601312 – methods of increasing the likelihood of precipatation by the artificial introduction of sea water vapor into the atmosphere winward of an air lift region
- 1971 – 3608810 – methods of treating atmospheric conditions
- 1971 – 3608820 – treatment of atmospheric conditions by intermittent dispensing of materials therein
- 1971 – 3613992 – weather modification method
- 1971 – 3630950 – combustible compositions for generating aerosols, particularly suitable for cloud modification and weather control and aerosolization process
- 1972 – 3647710 – method for dispersing fog with phosphate salt compositions
- 1972 – 3653383 – algin sponge and process therefor
- 1972 – 3659785 – weather modification utilizing microencapsulated material (gross)
- 1972 – 3666176 – solar temperature inversion device
- 1972 – 3677840 – pyrotechnics comprising oxide of silver for weather modification use
- 1972 – 3690552 – fog dispersal
- 1973 – 3722183 – device for clearing impurities from the atmosphere
- 1973 – 3769107 – pyrotechnic composition for generating lead based smoke (are you serious?)
- 1974 – 3784099 – air pollution control method
- 1974 – 3785557 – cloud seeding system
- 1974 – 3795626 – weather modification process
- 1974 – 3808595 – chaff dispensing system
- 1974 – 3813875 – rocket having barium release system to create ion clouds in the upper atmopsphere (nasa)
- 1974 – 3835059 – methods of generating ice nuclei smoke particles for weather modification and apparatus therefore
- 1974 – 3835293 – electrical heating aparatus for generating super heated vapors
- 1975 – 3877642 – freezing nucleant
- 1975 – 3882393 – communications system utilizing modulation of the characteristic polarization of the ionosphere (haarp – us navy)
- 1975 – 3896993 – process for local modification of fog and clouds for triggering their precipitation and for hindering the development of hail producing clouds
- 1975 – 3899144 – powder contrail generation
- 1975 – 3902934 – gas generating compositions
- 1976 – 3940059 – method of fog dispersion
- 1976 – 3940060 – vortex ring generator
- 1976 – 3990987 – smoke generator
- 1976 – 3992628 – countermeasure system for laser radiation
- 1976 – 3994437 – broadcast dissemination of trace quantities of biologically active chemicals
- 1977 – re29142 – combustible compositions for generating aerosols, particularly suitable for cloud modification and weather control and aerosolization process
- 1977 – 4035726 – method of controlling and/or improving high-latitude and other communications or radio wave surveillance systems by partial control of radio wave et al (haarp – cornell univ.)
- 1977 – 4042196 – method and apparatus for triggering a substantial change in earth characteristics and measuring earth changes
- 1978 – 4096005 – pyrotechnic cloud seeding composition
- 1978 – 4111911 – method of hardening liquid resols
- 1978 – 4129252 – method and apparatus for production of seeding materials
- 1979 – 4141274 – weather modification automatic cartridge dispenser
- 1979 – 4167008 – fluid bed chaff dispenser
- 1981 – 4269637 – high-performance mhd solid gas generator
- 1982 – 4347284 – white cover sheet material capable of reflecting ultraviolet rays
- 1982 – 4362271 – procedure for the artificial modification of atmospheric precipitation as well as compounds with a dimethyl sulfoxide base for use in carrying out said procedure
- 1983 – 4402480 – atmosphere modification satellite (space)
- 1983 – 4412654 – laminar microjet atomizer and method of aerial spraying of liquids
- 1983 – 4415265 – method and apparatus for aerosol particle absorption spectroscopy
- 1983 – 4373391 – relative humidity sensitive material
- 1984 – 4470544 – method of and means for weather modification
- 1984 – 4475927 – bipolar fog abatement system
- 1986 – 4600147 – liquid propane generator for cloud seeding apparatus
- 1987 – 4633714 – aerosol particle charge and size analyzer
- 1987 – 4643355 – method and apparatus for modification of climatic conditions
- 1987 – 4653690 – method of producing cumulus clouds
- 1987 – 4684063 – particulates generation and removal
- 1987 – 4686605 – method and apparatus for altering a region in the earth’s atmosphere, ionosphere, and/or magnetosphere (haarp – raythen)
- 1987 – 4704942 – charged aerosol (anti-airborne biological warfare agent)
- 1987 – 4712155 – method and apparatus for creating an artificial electron cyclotron heating region of plasma (haarp – raytheon)
- 1988 – 4744919 – method of dispersing particulate aerosol tracer
- 1988 – 4766725 – method of suppressing formation of contrails and solution therefor
- 1989 – 4829838 – method and apparatus for the measurement of the size of particles entrained in a gas
- 1989 – 4836086 – apparatus and method for the mixing and diffusion of warm and cold air for dissolving fog
- 1989 – 4873928 – nuclear-sized explosions without radiation (haarp – raytheon)
- 1990 – 4948257 – laser optical measuring device and method for stabilizing fringe pattern spacing
- 1990 – 4948050 – liquid atomizing apparatus for aerial spraying
- 1990 – 4959559 – (united states) electromagnetic or other directed energy pulse launcher
- 1991 – 4999637 – creation of artificial ionization clouds above the earth (haarp – raytheon)
- 1991 – 5003186 – stratospheric welsbach seeding for reduction of global warming
- 1991 – 5005355 – method of suppressing formation of contrails and solution therefor
- 1991 – 5038664 – method for producing a shell of relativistic particles at an altitude above the earths surface (haarp – raytheon)
- 1991 – 5041760 – method and apparatus for generating and utilizing a compound plasma configuration
- 1991 – 5041834 – artificial ionospheric mirror composed of a plasma layer which can be tilted (haarp – raytheon)
- 1991 – 5056357 – acoustic method for measuring properties of a mobile medium
- 1991 – 5059909 – determination of particle size and electrical charge
- 1992 – 5104069 – apparatus and method for ejecting matter from an aircraft
- 1992 – 5110502 – method of suppressing formation of contrails and solution therefor
- 1992 – 5148173 – millimeter wave screening cloud and method
- 1992 – 5156802 – inspection of fuel particles with acoustics
- 1992 – 5174498 – cloud seeding
- 1993 – 5245290 – device for determining the size and charge of colloidal particles by measuring electroacoustic effect
- 1994 – 5286979 – process for absorbing ultraviolet radiation using dispersed melanin (wow!)
- 1994 – 5296910 – method and apparatus for particle analysis
- 1994 – 5327222 – displacement information detecting apparatus
- 1994 – 5357865 – method of cloud seeding
- 1994 – 5360162 – method and composition for precipitation of atmospheric water
- 1995 – 5383024 – optical wet steam monitor
- 1995 – 5425413 – method to hinder the formation and to break-up overhead atmospheric inversions, enhance ground level air circulation and improve urban air quality
- 1995 – 5434667 – characterization of particles by modulated dynamic light scattering
- 1995 – 5441200 – tropical cyclone disruption (hurricanes!)
- 1996 – 5486900 – measuring device for amount of charge of toner and image forming apparatus having the measuring device
- 1996 – 5556029 – method of hydrometeor dissipation (clouds)
- 1997 – 5628455 – method and apparatus for modification of supercooled fog
- 1997 – 5631414 – method and device for remote diagnostics of ocean-atmosphere system state
- 1997 – 5639441 – methods for fine particle formation
- 1998 – 5762298 – use of artificial satellites in earth orbits adaptively to modify the effect that solar radiation would otherwise have on earth’s weather (space)
- 1999 – 5912396 – system and method for remediation of selected atmospheric conditions (solar powered blimp to ionize clouds)
- 1999 – 5922976 – method of measuring aerosol particles using automated mobility-classified aerosol detector
- 1999 – 5949001 – method for aerodynamic particle size analysis
- 1999 – 5984239 – weather modification by artificial satellites (space)
- 2000 – 6025402 – chemical composition for effectuating a reduction of visibility obscuration, and a detoxifixation of fumes and chemical fogs in spaces of fire origin
- 2000 – 6030506 – preparation of independently generated highly reactive chemical species
- 2000 – 6034073 – solvent detergent emulsions having antiviral activity
- 2000 – 6045089 – solar-powered airplane
- 2000 – 6056203 – method and apparatus for modifying supercooled clouds
- 2000 – 6110590 – synthetically spun silk nanofibers and a process for making the same
- 2001 – 6263744 – automated mobility-classified-aerosol detector
- 2001 – 6281972 – method and apparatus for measuring particle-size distribution
- 2001 – 6315213 – method of modifying the weather
- 2002 – 6382526 – process and apparatus for the production of nanofibers
- 2002 – 6408704 – aerodynamic particle size analysis method and apparatus
- 2002 – 6412416 – propellant-based aerosol generation devices and method (us army)
- 2003 – 6520425 – process and apparatus for the production of nanofibers
- 2003 – 6539812 – system for measuring the flow-rate of a gas by means of ultrasound
- 2003 – 6553849 – electrodynamic particle size analyzer
- 2003 – 6569393 – method and device for cleaning the atmosphere
- 2011 – 7965488 – methods of removing aerosols from the atmosphere (ionizing antennas)
- 1991 – Stratospheric Welsbach seeding for reduction of global warming
- 1999 – Process for sequestering into the ocean the atmospheric greenhouse gas carbon dioxide by means of supplementing the ocean with ammonia or salts thereof
- 1999 – 6200530 – Water-bouyant particulate materials containing micronutrients for phytoplankton
- 2000 – WO/2000/010691A1 – METHOD AND APPARATUS FOR EXTRACTING AND SEQUESTERING CARBON DIOXIDE
- 2001 – Method of sequestering carbon dioxide with spiral fertilization
- 2001 – US20010022952 – METHOD AND APPARATUS FOR EXTRACTING AND SEQUESTERING CARBON DIOXIDE
- 2009 – US20090177569 – WATER ALTERATION STRUCTURE RISK MANAGEMENT OR ECOLOGICAL ALTERATION MANAGEMENT SYSTEMS AND METHODS
BOWERS, JEFFREY A. (KIRKLAND, WA, US), CALDEIRA, KENNETH G. (CAMPBELL, CA, US), CHAN, ALISTAIR K. (STILLWATER, MN, US), GATES III, WILLIAM H. (REDMOND, WA, US), HYDE, RODERICK A. (REDMOND, WA, US), ISHIKAWA, MURIEL Y. (LIVERMORE, CA, US), KARE, JORDIN T. (SEATTLE, WA, US), LATHAM, JOHN (BOULDER, CO, US), MYHRVOLD, NATHAN P. (MEDINA, WA, US), SALTER, STEPHEN H. (EDINBURGH, GB), TEGREENE, CLARENCE T. (BELLEVUE, WA, US), WOOD JR., LOWELL L. (BELLEVUE, WA, US)
- 2005 – 6890497 – METHOD FOR EXTRACTING AND SEQUESTERING CARBON DIOXIDE
- 2009 – WO/2009/086559A1 – WATER ALTERATION STRUCTURE AND SYSTEM
- 2009 – US20090173386 – WATER ALTERATION STRUCTURE APPLICATIONS AND METHODS
- 2009 – US20090173404 – WATER ALTERATION STRUCTURE AND SYSTEM
- 2009 – US20090173801 – WATER ALTERATION STRUCTURE AND SYSTEM HAVING BELOW SURFACE VALVES OR WAVE REFLECTORS
- 2009 – US20090175685 – WATER ALTERATION STRUCTURE MOVEMENT METHOD AND SYSTEM
- 2009 – US20090202413 – CARBON SEQUESTRATION AND PRODUCTION OF HYDROGEN AND HYDRIDE
- 2010 – 7655193 – APPARATUS FOR EXTRACTING AND SEQUESTERING CARBON DIOXIDE
KEN CALDEIRA, GREGORY RAU – THIS APPLICATION IS A DIVISIONAL OF U.S. NON-PROVISIONAL APPLICATION SER. NO. 09/759,781, FILED JAN. 11, 2001, WHICH IS A CONTINUATION-IN-PART OF U.S. APPLICATION SER. NO. 09/314,220, FILED MAY 19, 1999, WHICH CLAIMS THE BENEFIT OF U.S. PROVISIONAL APPLICATION NO. 60⁄096,846, FILED AUG. 18, 1998.
- 2010 – WO/2010/138195A1 – WATER ALTERATION STRUCTURE AND SYSTEM HAVING HEAT TRANSFER CONDUIT
- 2010 – US20100300560 – WATER ALTERATION STRUCTURE AND SYSTEM HAVING HEAT TRANSFER CONDUIT
- 2011 – 8012453 – CARBON SEQUESTRATION AND PRODUCTION OF HYDROGEN AND HYDRIDE
- 2011 – 8048309 – SEAWATER-BASED CARBON DIOXIDE DISPOSAL
A DEVICE SUITABLE FOR RETENTION IN A NASAL PASSAGE OF A RUMINANT, THE DEVICE BEING ARRANGED TO PASS A RUMINANT EXHALATION, THE DEVICE INCLUDING A FIRST STRUCTURE CONFIGURED TO OXIDIZE METHANE GAS IN THE RUMINANT EXHALATION, THE DEVICE BEING FURTHER ARRANGED TO PASS PRODUCTS OF THE OXIDIZED METHANE GAS TOWARD A NASAL PASSAGE EXIT.
- 2012 – WO/2012/064367A2 – TREATMENT OF RUMINANT EXHALATIONS
- 2012 – WO/2012/064367A3 TREATMENT OF RUMINANT EXHALATIONS
- 2012 – US20120115240 – TREATMENT OF RUMINANT EXHALATIONS
- 2012 – EP2435633A1 – WATER ALTERATION STRUCTURE AND SYSTEM HAVING HEAT TRANSFER CONDUIT
- 2012 – 8152091 – PRODUCTION OR DISTRIBUTION OF RADIATIVE FORCING AGENTS
- 2014 – 20140145002 A1 – System for facilitating cloud formation and cloud precipitation – Ken Caldeira, cloud ion cannon
Alert friends in Victoria, Australia, have reported numerous sightings of the same phenomenon in just the past week ( May 1st thru to the 7th, 2017 )
I am observing these fibers regularly here in North Canterbury, and record my sightings on camera.
Patents for electrospun nanofibers in the years 2000, 2002 and 2003 can be found here:
By Wayne K., 7 May 2017
On Tuesday, the 18th of April this year in the sleepy little gold-rush town of Thames, which is at the southwestern end of the Coromandel Peninsula in New Zealand’s North Island, I was taking my mum out for a walk along the foreshore. It was such a perfect day, until I noticed a long white stringy thread sail by in the air.
When we looked up, we saw that the mostly clear blue sky was full of fibers descending. I estimated that some were around 10 meters or more in length, and they were landing everywhere, as well as hanging off power lines and trees.
The threads also had some foamy stuff in clumps attached, like a detergent foam, and a passer-by commented that people were seeing a lot of it up at the Pak’ n Save car park landing on the cars.
I was so shocked at the sight of it, I grabbed my camera after I got home and took some pictures of it, as well as picking up some samples on a stick, since I’d read about this happening elsewhere in the world and they always warned not to touch it if you find it, and some people had become sick after encountering it. It maybe a coincidence, but I did feel ill the night after I touched it. I had picked it off my clothes.
By the late afternoon, the amount of it hanging off some of the power lines was incredible, with some really long ones stretching between power poles and trees.
Somebody I spoke to reckoned it was cobwebs and a common occurrence, but there weren’t any spiders visible, and it seemed a bit thick for cobweb material…it was more like string, as the photos show.
Most of it was gone after it rained, but a few threads were still visible days later in the power lines and trees.
Didn’t see any mention of it in the local paper.
NASA confirms: Sea levels have been FALLING across the planet for two years … lamestream media is SILENT
July 26, 2017 by: Mike Adams
(Natural News) As the global warming narrative unravels under revelations of scientific fraud, data alteration and faked “hockey stick” data models, the fake news media remains suspiciously silent over the fact that NASA now confirms ocean levels have been falling for nearly two years.
On a NASA page intended to spread climate alarmism (https://climate.nasa.gov/vital-signs/sea-level/), NASA’s own data reveal that world-wide ocean levels have been falling for nearly two years, dropping from a variation of roughly 87.5mm to below 85mm.
These data, of course, clearly contradict the false narrative of rapid, never-ending rising ocean levels that flood continents and drown cities — a key element of the climate change “boogeyman” fiction that’s used to scare gullible youth into making Al Gore rich.
Check out the sea level chart for yourself, showing the downward trend across 2016 –2017:
Thanks to Charles of Ruakaka, Northland for alerting us to this photograph which appeared in the LA Times on June the 21st, 2017 in an article titled: ‘Summer and winter solstice celebrations around the globe,’ by Marc Martin.
The article does not mention a word about the elephant in the living room, in spite of the fact that festival goers were there to see the sun rise – and thus presumably were looking at the sky.
In effect, the current global warming software models used by the IPCC and cited by the media wildly over-estimate the warming effects of CO2 emissions. How much do they over-estimate warming? By about 50%.
In other words, the climate change threat has been wildly overstated. The fear mongering of Al Gore and the government-funded science community can truly only be described as a “junk science hoax.”
Climate alarmists suddenly find themselves admitting they were wrong all along
The war on humanity continues unabated in Texas, where over one million acres of land are being carpet-bombed with the aerial spraying of neurotoxic chemicals in an effort to kill mosquitoes. Texas authorities are openly lying to citizens, claiming these poisons are magically not poisons at all when they come into contact with humans. Even more alarmingly, Texas authorities are urging their citizens to use other toxic chemicals on their skin — chemicals that are part of a binary weapon system that causes Alzheimer’s and dementia by incurring permanent brain damage (see the science, below).
“U.S. Air Force C-130 cargo planes began spraying insecticides over three eastern Texas counties over the weekend and will expand to other areas over the next two weeks, officials from the Texas Department of State Health Services (DSHS) said,” reports Reuters. “About 1.85 million acres have been treated as of Tuesday, according to the department.”
In effect, the State of Texas is unleashing weapons of mass destruction against its own citizens, generating trillions of dollars in long-term health care costs that financially benefit the drug makers who sell prescription “treatments” for Alzheimer’s, dementia, cancer and other diseases caused by chemical exposure. The aerial bombardment of high-density population centers, it turns out, is a revenue generating activity for the for-profit “sick care” industry that has infiltrated the entire Texas legislature.
“DSHS is working with counties that have requested assistance with mosquito control to coordinate spraying by two state contractors and federal support through FEMA and the U.S. Air Force Reserve,” reports the official DSHS website. “A total of approximately 4.36 million acres has been sprayed across all areas.”
The odd-looking cloud formations shown during the 6 o’clock news on TV One on Monday, January the 15th by weatherman, Daniel Corbett, were introduced as: “Some really cool-looking clouds…” Let’s hope that one day mainstream weather presenters talk about the weather modification technology that is in play globally. Note what appears to be an obvious aerosol trail in the first image at the level of his head.
In my opinion, the odd formations and aerosol trails, of which these images provide an example of, provide evidence to show that so-called “global warming” and extremes in weather are being manufactured to meet nefarious agendas.
Currently there is a poll on the TVNZ website (see below), asking the public if they believe New Zealand’s record rain is the result of (carbon-dioxide, fossil-fuel induced) “global warming.” Evidence shows that global warming is an engineered hoax that is being promoted as a repairable environmental issue – supposedly repairable if the public pays carbon taxes, among other sacrifices. Will the powers-that-be cease manipulating the weather if we pay carbon taxes? They have too much to gain from controlling the weather, so it seems highly unlikely. Carbon taxes may be used to fund weather modification globally?
No, it is not a negative of an eye above. It appears to show bright-white aerosol material falling in wisps. These stunning photographs were taken by activist, Marian Sutherland at 8.16pm on the 15th of March, 2018 from the Hawarden region.
Hawarden is a small town in Canterbury, which is in New Zealand’s South Island. It is located near Waikari, just off State Highway 7.
A big thank-you to Marian for her continued efforts to expose the manipulation of the atmosphere over Canterbury.
These photos were kindly supplied by Brad of Whangarei and show Air Zealand’s Airbus A320’s, which are used domestically and internationally. Note the relatively untarnished metal surrounding the pipes, suggesting that it is a retrofit and it appears that rivets have been removed in order to install these. Also, note that these pipes are not evident in the image of the A320 taken from the Air New Zealand website at the time of writing.
Also, note that in the last two images shown of an aircraft in flight, which are taken from a YouTube video titled: ‘NOZZLE TRAILS – block the sun – focus on airports/aircraft’, three trails appear to be being emitted adjacent the left engine, although it is not known what model of aircraft this is, nor which airline the plane belongs to.
Doctor warns world about “chemtrail lung,” a new health epidemic causing brain and lung problems across society
Doctor warns world about “chemtrail lung,” a new health epidemic causing brain and lung problems across society
The existence of chemtrails used to be a topic of debate, but they are now being more widely acknowledged by experts like meteorologists to scientists. It’s becoming increasingly difficult to deny that they exist as more and more people are coming down with illnesses related to chemtrails.
When TV host Rachel Reenstra had trouble overcoming a persistent cough, accompanied by aches, pains, and fever, she visited a doctor. After chest x-rays revealed a type of bronchitis, she was given antibiotics, which only seemed to make her feel worse.
Her doctor told her that lots of bacterial infections are going around, and when she asked him where they are coming from, he told her the truth that many doctors wouldn’t dare reveal to their patients: Chemtrails are at the heart of widespread lung problems right now. Surprised by his candor, she asked if she could videotape him talking about the phenomenon.
Information related to false-flag attacks and the symbolic appearance of foot wear in images from around the world. This, according to Ole, is a Freemasonic message that is being sent and more. Ole’s website: https://www.lightonconspiracies.com/
Researcher, Clifford Carnicom: What Are We Breathing? Urgent Research Update Via Lost Arts Radio | Richard Sacks, published on 19 Nov 2017.
Scientists say the last major Alpine Fault earthquake, in 1717, brought long-term disruption across the South Island and “there will be a similar earthquake soon”.
The 1717 quake is estimated to have had a magnitude of around 8.1, according to a special anniversary edition of the New Zealand Journal of Geology and Geophysics, published on Tuesday. That estimate comes from evidence for the ground breaking along 380km of the South Island.
“Land on the south-eastern side of the fault moved up relative to the north-western side in most places and 7–8 metres south in a matter of seconds,” an editorial said.
Dutchsinse: 9/10/2018 — Major Earthquake Activity underway – Spread of M7.0 + M6.0 — New Zealand to Indonesia
NewsHub, 29 8 2018
The “meteor” spotted above the South Island on Monday night may have actually been a flight full of Australians.
Canterbury residents reported seeing the alleged meteor just after 6pm on Monday night – the exact time a Qantas flight was passing over the area on its way to Sydney from Santiago.
The Boeing 747 crossed New Zealand’s east coast above Rangiora at 5:54pm and passed over the West Coast just south of Hokitika at 6:07pm, flying at a height of 12 kilometres. An aircraft at this height would most likely leave a contrail behind it – however, Qantas doubts its aircraft is responsible.
An Air New Zealand Dreamliner flew over Whangarei this morning at about 9.30am on its way to Tokyo and emitted a polluting aerosol trail overhead, which spread out within 15 minutes into an ugly, wispy mass of particulates, some of which were descending faster than others, which people may be breathing in in the not to distant future. The third photo of the trail was taken about 15 minutes after it passed by demonstrating that it was not a condensation trail.
Malcolm Scott: Complaint to Ombudsman Regarding The Withholding of Information About Geoengineering in NZ By Minister for the Environment
Christchurch-based researcher, Malcolm Scott received a reply from Hon David Parker, the Minister for the Environment which claimed that New Zealand is not involved in any program of atmospheric geoengineering. Malcolm has advised the Minister in his reply dated the 18th of June, that his withholding of information may be in breach of the Official Information Act (OIA) and that the matter is being referred to the Ombudsman. See letters below. Thank-you to Malcolm, who will provide us with updates.
Link to the petition referred to in references here: https://www.change.
FBI / 4CHAN ANONYMOUS INSIDER RESURFACES ON MARCH 5TH
On March 5th, the FBI / 4Chan insider again granted an interview to Victurus Libertas.
There wasn’t a whole lot of new intel here, and we will only quote part of it. Clearly, there was disappointment about the arrests not proceeding as quickly:
The People outnumber the Government 300 to 1. It’s a numbers game.
We expected high-level arrests to happen weeks ago, but discovered the bee keepers had interfered at the highest levels.
There is an internal war within our Government, as many know.
The Shadow State is like a vampire facing dawn. They do not go easily into sunlit areas….
THE ACCOUNTS ARE VERY SIMILAR
During the same exact timeframe where the FBI / 4Chan souce was telegraphing mass arrests, the TBR News insider was revealing the same things:
Washington, D.C. February 18, 2017: “One of the most prolific of the anti-Trump mainline news media is the Washington Post.
At one time, the Post was a powerful national influence on the American political scene.
With the advent of the Internet and its presentation of a wide spectrum of genuine news, the Post, and its equal, the New York Times have rapidly lost both influence and paid subscribers.
The Post was bought by Jeff Bezos, a wealthy owner of Amazon and who, after purchasing the Post from the Graham family, got a multi-million dollar contract to work with the CIA.
Between Bezos and Soros, the media has been doing its best to wreck the presidency of Donald Trump.
Why are they interested in so disrupting the American political and social scene?
Trump is a loose cannon in their minds because he is popular with a very large segment of the American public.
The economic oligarchs who basically run the American political institutions do not like Trump because they cannot buy him.
Washington, D.C. March 14, 2017: “”One of the coming releases of highly negative culled email finds is a truly disgusting compendium of top American political, business and social leaders’ downloading and participation in pedophilic pornography.
The viewers, and participants include: Two former Presidents, sixteen Senators, thirty five Congressmen, seventy-seven top bureaucrats, fifty-two bankers and other leading businessmen are topics of this file.
[This is] a file replete with pictures of the perps that would be forbidden on XHamster, the Ukrainian pornographic site.
An advanced posting of this filth has been circulating, via the Deep Internet, to various interested people and when, not if, it goes public, it will put the Podesta papers to shame.
It will also put a number of the nation’s top leaders to even worse shame.
The outraged public might well end up displaying the perverts in trees.
A Washington wind chime!”
Let’s be clear that I have no interest in seeing “highly negative” and “truly disgusting… pictures… of this filth” with my own eyes.
I do, however, very much want to see this group, which is actively attempting to kill as many people as possible by any means necessary, finally be stopped.
These same people absolutely appear to have been behind the worst mass shooting in American history, according to this same FBI insider.
They are willing to do just about anything to try to distract us from their inevitable collapse.
This includes formally stating, through a source obtaining intel through Podesta, that “UFOs are Real.” That just happened on October 11th.
Given everything we have heard, the revelation of warm, habitable caves in Antarctica could be another step in this same direction.
THE NEXT BIG STORY: WAS SETH RICH THE LEAKER OF PEDOGATE EMAILS?
As we saw just a few paragraphs back, the first major FBI insider leaks on 4Chan were on July 2nd, 2016.
This person referred to the FBI having “Clinton Foundation secret records”, and said “There is enough for [Hillary] and the entire government to be brought down.”
This included information on human trafficking — how “many politicians trade girls like cattle”.
Eight days after the FBI insider revealed they had these documents on 4Chan, the DNC staffer who apparently leaked them was murdered.
Seth Rich could well have been the next Edward Snowden, but sadly in this case he did not make it.
He was shot. Nothing of his was stolen. He was brought to the hospital with a good chance of survival. They expected him to make a complete recovery.
Suddenly, the DNC staff came in, with all their own people, kicked out all the medical staff and locked it all down. When they finally emerged, Seth Rich was dead.
Our own insiders have said this was a badly botched job by the Cabal-controlled elements of the FBI. This still has the potential to become a huge, huge story.
THE STORY FIRST BREAKS ON FOX NEWS AS OF MAY 16TH, 2017
As much as I have voiced disgust of Fox News on this site, it does appear that they sided with the Alliance some time around the 2016 election — at least partly.
This is a war, and the management of this network decided not to keep supporting the side that was obviously losing — and cut some kind of deal.
Like it or not, Fox was the first to interview a private investigator hired by Seth Rich’s family named Rod Wheeler.
In this groundbreaking interview, Wheeler said Seth’s murder was an inside job.
This story went wild in the alternative media, and this article by Claire Bernish on May 18th, 2017 is particularly noteworthy if you are looking for more.
If this is true — and apparently the proof already exists — it utterly destroys the idea that these emails came from “the Russians.”
That means this dizzying nightmare of truth journalism being censored by Apple, Facebook, Google, YouTube, Twitter, et cetera as “Fake News” is a big joke.
I have completely stopped all audio work other than Coast to Coast and Jimmy Church, as no one else has adequate protection in place to stop the stealing.
Most of the stuff people try to send me on Facebook says “this attachment could not be loaded.”
According to Corey Goode and other insiders, all of this blatant, in-your-face censorship was intended to stop Pedogate from going any further.
Any time you type in a truth journalist’s name on YouTube and look for keywords like “NEW,” forget it. Unless you can find their real channel, it will not be new at all.
This makeshift lie about “Russian hackers” has been bought by some of the public, but most people see right through it.
They just don’t want to face how ugly the truth really is.
FAMOUS HACKER KIM DOTCOM CONFIRMS SETH RICH WAS WIKILEAKS SOURCE ON FRIDAY
The next stunning development in this story came on Friday, May 19th, when the famous hacker Kim Dotcom confirmed Seth Rich was the Wikileaks source.
This was another event of seismic proportions. Its full ramifications may only just now be coming into view.
His exact wording was, ““If Congress includes #SethRich case into their Russia probe I’ll give written testimony with evidence that Seth Rich was @Wikileaks source:”
— Kim Dotcom (@KimDotcom) May 19, 2017
Then after Sean Hannity publicly asked for more details, Dotcom released the following:
— Kim Dotcom (@KimDotcom) May 20, 2017
DID A VANITY FAIR ARTICLE THREATEN SETH RICH IN JANUARY 2016?
Hollywood locals know that no one gets into the Vanity Fair Oscars party for less than thirty thousand dollars per person, reserving it only for the elite.
I have noted extensive Cabal propaganda emerging from this magazine, and have published examples on this site in the past.
Once again, the Gateway Pundit broke this story of Vanity Fair’s apparent threat as of Thursday, May 25th:
T. A. Frank… wrote a piece in January of 2016 published in Vanity Fair that appears more than sinister to many following the Seth Rich murder investigation.
Frank begins his article as follows:
For the second time in eight years, Hillary Clinton sees the Democratic nomination being pawed by a charming interloper, like a priceless vase grabbed by a panda.
She’d prefer to shoot the panda, but that could mean breaking the vase, and onlookers would object.
[This is] a wordless feel-hope montage that is awfully good, good enough that I can’t help feeling both moved by it and resentful that it works on me.
Maybe shoot the panda.
The piece is creepy in and of itself because of the weird and sadistic references to shooting a panda.
But what really makes the piece creepy is that Seth Rich’s alias on the Internet was panda!
Internet hacker Kim Dot Com confirmed that Seth Rich’s alias on the Internet was panda.
The article goes on to show how the Vanity Fair author T. A. Frank was forced to write a Tweet dismissing the whole thing as a “coincidence.”
Given everything that we now know, that does not seem to be the case.
The threat was put in writing as of January 2016. Seth Rich died that same July.
TWO DAYS LATER: INCREDIBLE ALLIANCE-DRIVEN DISCLOSURE ON HISTORY CHANNEL
Just two days after that dream, which rocked me to my core, Snowden journalist Glenn Greenwald announced a stunning disclosure on History channel.
That same night, Sunday, June 18th, featured the first installment of a four-part series entitled “America’s War on Drugs.”
I will let Greenwald tell you what was going on in his own words:
Starting Sunday night and running through Wednesday, the History Channel is showing a new four-part series called “America’s War on Drugs.”
Not only is it an important contribution to recent American history, it’s also the first time U.S. television has ever told the core truth about one of the most important issues of the past 50 years.
That core truth is: The war on drugs has always been a pointless sham.
For decades the federal government has engaged in a shifting series of alliances of convenience with some of the world’s largest drug cartels.
So while the U.S. incarceration rate has quintupled since President Richard Nixon first declared the war on drugs in 1971, top narcotics dealers have simultaneously enjoyed protection at the highest levels of power in America….
The first episode opens with the voice of Lindsay Moran, a one-time clandestine CIA officer, declaring, “The agency was elbow deep with drug traffickers.”
Then Richard Stratton, a marijuana smuggler turned writer and television producer, explains,
“Most Americans would be utterly shocked if they knew the depth of involvement that the Central Intelligence Agency has had in the international drug trade.”
Next, New York University professor Christian Parenti tells viewers,
“The CIA is from its very beginning collaborating with mafiosas who are involved in the drug trade, because these mafiosas will serve the larger agenda of fighting communism.”
For the next eight hours, the series sprints through history that’s largely the greatest hits of the U.S. government’s partnership with heroin, hallucinogen, and cocaine dealers.
That these greatest hits can fill up most of four two-hour episodes demonstrates how extraordinarily deep and ugly the story is.
This only further underlined the significance of what my dream had just said: Something Very Big is Coming.
Any open, formal, public disclosure of the contents of this History channel documentary would be curtains for the Cabal.
Earlier in February 2017, Pablo Escobar’s son revealed his infamous father was producing and selling drugs for the CIA all along.
What did the mainstream media have to say about this? Take a guess.
JULY 16TH: PENCE REFERENCES THE “FACE OF MARS”
Another strange development, and possible sign of impending disclosure, occurred on July 16th, 2017.
My dream had said that the coming disclosures would validate everything that Alex Jones and I have been revealing throughout our entire careers.
I have been educating people about the Face on Mars ever since I first found out about it in 1993.
On July 16th, Vice President Pence thanked NASA for “making science fiction science fact,” and then made a very enigmatic comment.
Pence’s speech was obviously well-rehearsed, and the wording very carefully chosen.
Although “the face of Mars” could simply be a strangely poetic reference to its surface, the story of the Face on Mars — and nearby pyramids — is also well known.
I have referenced it in The Antarctic Atlantis and other videos as well as featuring it again in The Ascension Mysteries, along with many other anomalies.
WILCOCK HAD BEEN THREATENED, WAS REMAINING SILENT
As all of these amazing stories were taking place, I was experiencing severe threats about coming forward in any way.
Corey Goode, an insider I have a weekly show with on Gaia called Cosmic Disclosure, was being massively attacked by what we came to call the Dark Alliance.
These attacks were highly overblown, exaggerated and at times completely fabricated, having nothing to do with the man I personally knew.
I was already not feeling good about jumping back in. I had not written a single thing on my website since April 29th, 2017.
The threats reached my front door on July 3rd, when a trusted insider conveyed a threat to me.
WHAT WAS COREY GOODE LEAKING?
They are completely in alignment with everything else we have been discussing here, and are worthy of repetition:
Update Summary (Full update to come): I have recently had meetings with a highly placed contact. I met him in the FEMA Mass Casualties response course when I was still in the Texas State Guard.
He stopped talking to me when I began Cosmic Disclosure. He suddenly contacted me again right after the elections had concluded.
He shared a cluster of Intel with me before going dark again… Until now.
I had reported that there were teams of US Special Forces that were conducting “domestic surveillance and investigations” of a Satanic group.
[This is a group] that had infiltrated all aspects of government agencies and the military.
These Special Forces teams have been in place since the middle of the election cycle.
[DW: I have heard about these groups from other insiders as well. There are far more than you might think, and they are prepared to conduct strikes if necessary.
The teams are stationed in plainclothes near the centers of Cabal power — namely media, financial and governmental. And they are ready to go.]
The report states that the conspiracy involves the majority of powerful people within the UN, EU, US, State and local (City) power structures.
These government organizations are completely complicit in what is going on. All the way down to local Post Offices and Police Offices.
I have to tell you, this was a very creepy report.
These military investigators have expressed their shock and anger to their superiors as well as their desire to take them down.
They report that it is so systemic that they are at a loss of how to counter and defeat these people without a military coup.
This would involve US Special Forces and trusted “Military Contractors” that come from Alliance countries (And are already in place in the US).
Some of these Special Forces Investigators have been removed from the investigation because they are HIGHLY suspected as being involved in torturing and killing some of these low level “Pedo-Gate” suspects.
[DW: Although this is very unethical, it is not hard to see how an “alpha male” type could be horrified enough to do this as he learns the truth firsthand.
In facing this level of evil, we have to be very careful that we do not become the embodiment of that which we are fighting.]
The scope of this cult takeover of government is shocking to even the people that think they have seen it all, including myself.
These investigators can’t figure out how the government was infiltrated to such a degree.
THINGS GOT REALLY BAD AT THIS POINT
Four days before I wrote this, on August 11th, 2017, the brakes had mysteriously failed on my car. Thankfully it only occurred just after making it down the mountain.
Bear in mind that my brakes went out BEFORE I had a chance to actually publicize this highly significant new intel.
This alleged attack was apparently part of Operation Snow White, as I came to find out.
This is another person the Cabal would definitely not want to have out there talking if anything like this were to break into mainstream news.
Sen. De Camp’s work had eventually made its way to the front page of the Washington Times in a stunning headline:
If the Pedogate story was about to break loose, DeCamp would have been one of the best witnesses of all for any type of Congressional hearings.
This story also found its way into a stunning documentary called Conspiracy of Silence, which was censored from publication — but a copy was later found.
INCREDIBLE SUPPORT, INCREDIBLE LOSSES
Just four days after my brakes went out, my top insider Pete Peterson had his house seized and everything start to get thrown into dumpsters.
You rallied around him incredibly with a fundraiser that brought him over 50K of critically-needed funds, proving that you had our backs.
The ridiculously fake, contrived hate attacks from the Dark Alliance could do nothing to stop this outpouring of support for an American hero.
Just six days later, on August 21st, our equally valuable insider William Tompkins died from a brain bleed after a tragic fall.
Although Tompkins was 94 years old, this was a stunning and horrible loss. I was about to reconnect with him and had his number sitting on my desk.
This also occurred just hours before the total solar eclipse. His fall may have been “nudged” by some means as described before, at this symbolic time.
Very shortly thereafter, another insider we will call Paul had everything he owned stolen from him. A bullet was left on the countertop in his now-empty home.
I was deeply traumatized by all of these events, the death threats, the property losses and the actual deaths of Marrs, Sen. De Camp and Tompkins.
THE HURRICANES WERE THE NEXT BIG THING
Shortly after all of these events took place, Houston got completely wiped out by a mega-hurricane — followed by Puerto Rico.
The financial losses that will ultimately be caused by these events potentially number in the hundreds of billions.
In both cases, these hurricanes were gigantic, and happened to steer precisely into areas that would cause maximum damage and financial loss.
At the same time, a Hollywood movie entitled Geostorm was about to launch on October 20th, in which satellite technology is used to control the weather.
The satellites end up being overtaken and used to create mega-disasters — hence the name Geostorm.
I did not mention the movie at the time, but I could see that with all we know about HAARP microwave technology, these hurricanes could have been steered.
This is another area where people who are not sufficiently informed could think this is impossible, ridiculous, et cetera.
Simply put, if you concentrate enough energy into a beam, you can create strong heat — which will create a high-pressure zone that steers weather systems.
Corey Goode and other intel sources confirmed this was happening. I alluded to this in Personal and Global Attacks Become Lethal, on September 23, 2017.
Both hurricanes appeared to be a direct attack against the US infrastructure, and therefore against the US aspect of the Alliance, by the Cabal.
Consider that the Alliance may be working through this administration to expose the Cabal.
If so, a collapsed US economy could lead to a public uprising that would ruin any effort to continue this process
FINISHING UP WITH THE 4CHAN INSIDER
As we learned in the previous article exposing the Vegas atrocity, the 4Chan insider revealed that the Alliance is preparing a major, public announcement.
This would forever validate every “conspiracy theory” about the Cabal. The exact nature of what will be disclosed remains mysterious.
However, we have been told it will destroy the credibility of the Federal Reserve, the CIA and the FBI — or at least the Cabal-controlled portions of them.
Furthermore, this insider revealed that the hurricanes threw off the timing of this announcement completely.
He didn’t want to speculate as to whether they had been deliberately steered, but did say the media was able to utterly block any announcement for weeks because of them.
The president was tied up in all sorts of mandatory meetings and public appearances in response to endless criticism from the press.
That finally brings us to the present. The 4Chan insider has continued “singing like a songbird” since our last update.
Now we will conclude this investigation with the latest things he has had to say.
Rohrabacher told OAN in the second part of the exclusive interview (to be aired on Saturday) that “the American people don’t know as much as they should” and would be “outraged if they knew the full measure of the information withheld from them.”
Trump doesn’t have a party. When it’s all said and done, we will all realize that “WE, THE PEOPLE OF THIS GREAT UNION”, ARE THE ONLY F—ING PARTY HE CONSIDERS HIMSELF, TO REPRESENT.I mean, you do realize you’re watching the Dems right now, try to backdoor the inevitable demise of their failing “Russia collusion” shit.
As for the Dems, they all knew they were finished, when AWAN WAS STOPPED AND DETAINED AT THE AIRPORT. It’s over, friends.
When it’s all done, you’ll know who WAS DIRTY, but you’ll also have to learn to accept and appreciate what they risked on Trump, to get out and help.
This should not be a new concept for you. This is not “news”. It’s fact.
Stick with me on this… you’ll want to remember this forever. READ THIS CAREFULLY!1. Lynch and Bill Clinton PURPOSEFULLY PLANNED to have an unironic, questionably shady, private meeting on the tarmac.
3. They leaked it themselves so when they’d awkwardly and eventually DENY rumors that they’d met (also started and circulated by them) in an undisclosed airplane meeting…
Something extraordinary was revealed today. Former high-level officials and scientists with deep black experience who have always remained in the shadows came forward on one platform. These insiders have long-standing connections to government agencies which may have programs investigating unidentifed aerial phenomena (UAP/UFOs). The team includes a 25-year veteran of the CIA’s Directorate of Operations, a Lockheed Martin Program Director for Advanced Systems at “Skunk Works”, and a former deputy Assistant Secretary of Defense for Intelligence.
Today marked the official launch of To The Stars Academy of Arts & Science (TTS/AAS) an innovative Public Benefit Corporation which will advance research into unexplained phenomena and develop related technology. It has established three synergistic divisions: Science, Areospace, and Entertainment. “We believe there are discoveries within our reach that will revolutionize the human experience,” says company President and CEO Tom DeLonge. Please see my previous story,- released yesterday – for background on today’s announcement which was live-streamed and is archived on the company website.
Today’s launch of the TTS/AAS
According to a TTS /AAS statement, its team members who have been “operating under the shadows of top-secrecy for decades” believe that “there is sufficient credible evidence of UAP that proves exotic technologies exist that could revolutionize the human experience.”
This could represent the beginning of a trend towards a new openness on the part of the U.S. government. These people know more than any of us, and they will be releasing important data on UAP in the future. As an investgative reporter with a long standing interest in this subject, who has worked with a few Academy team members in the past, I was shown some of this data at a meeting with them on Oct 4.
Present for that four hour meeting was Luis Eiizondo, who ran an important program at the DOD. Elizondo is a senior career intelligence officer whose experience includes working with the U.S. Army, the Dept of Defense, the National Counterintelligence Executive, and the Director of National Intelligence. He served as the Director for the National Programs Special Management Staff in the Office of the Secretary of Defense. He is also the former Director of Programs to investigate Unidentified Aerial Threats for the Office of the Secretary of Defense.
“Lue will architect partnerships with some very sensitive places, to help protect us and the technology involved, so we may achieve for you the extraordinary,” DeLonge said at today’s event.
Lue [the “nickname” for Luis] had resigned his position at the DOD literally the day before we met. I was able to verify who he was and what his tasks were at the Pentagon. He received the highest commendations from his superiors. I was told that important unclassified data and documentation are expected to be released through the Academy’s on-line Community of Interest (COI) in collaboration with the US government, which will be set up soon.
I asked him if these unidentified objects were considered to be threats. “They did not exhibit overt hostility,” he said. “But something unexplained is always assumed to be a potential threat until we are certain it isn’t. On the bright side, I believe we are closer than ever before in our understanding of how it operates,” he told me.
At the event today, Elizondo explained his work this way: “I am accustomed to being involved in close-hold, nuanced programs involving national security. This includes being a Counterintelligence Special Agent, a case officer, and intelligence practitioner. However, by far the most interesting effort I was involved with was the topic of Advanced Aerial Threats.”
He stated that he ran “a sensitive aerospace threat identification program focusing on unidentified aerial technologies.” His job at TTS/AAS will be “to work as a liaison and interlocutor in the collection of this information for the Science division, allowing us to collect facts for analysis.” How will this be done? One way is through the COI, which will “provide a platform for the detection and triangulation of phenomena events in real time, and allows two-way communication between the public and our team regarding this information.”
As a result of his position managing the DOD program for almost a decade, Lue said “I learned that the phenomena is indeed real.”
In short, less that two weeks after leaving the Pentagon, Luis Elizondo confirmed that UFOs are a real; they exist, and they have been officially documented. Can anyone argue with this fact now, given where this man comes from and what he knows?
A former high ranking DOD official – who only left his position last week – has confirmed that an official UFO Program exists, answering a question so many have debated and speculated about for decades.
It is very important to understand that Lue does not speak for the DOD, since he is no longer employed there; he speaks independently as part of the TTS Academy.
Lue also stated: “We are also planning to provide never before released footage from real US Government systems…not blurry, amateur photos, but real data and real videos.” And even more significant: “We are inviting our Government colleagues and friends in Defense to participate regularly with their own findings.”
Another member of the team, Stephen Justice, an experienced aerospace engineer, explained that he will use scientific disoveries about the travel of UAP through space, demonstrating almost unimaginable technology, to develop technology that can change the world. Steve recently retired as a Program Director for Advanced Systems at Lockheed Martin after a thirty-one year career in the Skunk Works. “How dare we think that the physics we have today is all that there is,” he told me in an interview a few days ago. The opportunity here, as he sees it, is to “stand in the future and look back.””
Steve’s objective is to harvest this advanced technology to build a vehicle that will allow for almost instantaneous travel through land, air, oceans and space, by engineering the fabric of space-time. Such a vehicle could also float, like something from science fiction. Steve says we have had glimpses of the science that could make this possible. It is not outside the realm of possibiity, if the necessary resources are in place.
A TTS Academy concept for a vehicle drawn by Stephen Justice, embodying the technology they seek to understand and develop – a visionary concept for a revolutionary electromagnetic vehicle based on technology observed in unidentified aerial phenomena
TTS Academy member Chris Mellon served as Deputy Assistant Secretary of Defense for Intelligence for two administrations, and early in his career drafted the legislation that established the US Special Operations Command at a time when terrorism was not yet on the radar. He presented an overview of a 2004 incident involving the aircraft carrier USS Nimitz which was detected on two separate radars.
“Two F-18s approach, the four aviators see that the object has no wings or exhaust — it is white, oblong, some 40 ft long and perhaps 12 ft thick”, he said. “One pilot pursues the craft while his wingman stays high. The pilots are astonished to see the object suddenly reorient itself toward the approaching F-18. In a series of discrete tumbling maneuvers that seem to defy the laws of physics, the object takes a position directly behind the approaching F-18.”
The lengthy event occurred in broad daylight off the California coast, and gun camera footage was taken. At one point the object went from hovering at 80,000 feet to dropping at supersonic speeds, and came to a complete stop at 50 feet above the ocean. “More F-18’s are dispatched but with similar results,” Mellon stated. “The secret machine easily evades the F-18s. Dozens of military personnel aboard the various planes and ships involved are privy to these interactions.”
Tom DeLonge’s final message today sums up the position of all involved: “No one person, one government, or one hidden institution should own this information and technology.”
The TTS Academy intends to release game-changing information of the type interested people have been seeking for a long time. “The fact is, we might not necessarily be alone,” Luis Elizondo says. “Now is the time to move forward and try to answer some of life’s deepest mysteries.”
“The [fraudulent] report claimed that the ‘pause’ or ‘slowdown’ in global warming in the period since 1998 – revealed by UN scientists in 2013 – never existed, and that world temperatures had been rising faster than scientists expected. Launched by NOAA with a public relations fanfare, it was splashed across the world’s media, and cited repeatedly by politicians and policy makers.”
“In an exclusive interview, Dr Bates accused the lead author of the paper, Thomas Karl, who was until last year director of the NOAA section that produces climate data – the National Centers for Environmental Information (NCEI) – of ‘insisting on decisions and scientific choices that maximised warming and minimised documentation…in an effort to discredit the notion of a global warming pause, rushed so that he could time publication to influence national and international deliberations on climate policy’.”
“None of the data on which the paper was based was properly ‘archived’ – a mandatory requirement meant to ensure that raw data and the software used to process it is accessible to other scientists, so they can verify NOAA results.”
“But Dr Bates said this increase in temperatures was achieved by dubious means. Its key error was an upwards ‘adjustment’ of readings from fixed and floating buoys, which are generally reliable, to bring them into line with readings from a much more doubtful source – water taken in by ships. This, Dr Bates explained, has long been known to be questionable: ships are themselves sources of heat, readings will vary from ship to ship, and the depth of water intake will vary according to how heavily a ship is laden – so affecting temperature readings.”
“Moreover, the…software [used in the fraudulent study] was afflicted by serious bugs. They caused it to become so ‘unstable’ that every time the raw temperature readings were run through the computer, it gave different results.”
Get it? Fraud all the way along the line. And a cover-up, to make an examination of the fraud-details impossible.
At long last, put official science and its enablers in the dock.
Media headlines, like those in the Washington Post, are all abuzz with reports of 2016 being the hottest year on record.
Why do warming propagandists talk about “hottest years?”
For the same reason they talk about ice; to distract us from the fact their computer models are wrong.
Global warming policy is completely predicated on models that project a much warmer planet than real-world observations record. The globe has simply failed to warm as they expected.
Absent confirmation of their prognostications, the warming campaign needs another narrative. Their go-to tactic is to hype natural events as warming. They also “adjust” data to help them spin their tale.
Facts, however, are stubborn things:
- Satellite measurements show the Earth has not experienced any significant warming since 1998
- Computer models project warming that has not occurred
- 1998 and 2015-2016 were times of naturally occurring El Niños
- If 2016 was “the warmest” year, it was so by insignificant hundredths of a degree
- The margin of error is actually a far larger one-tenth of a degree
- Temperature data adjustments by NASA and NOAA are highly suspect and not supported by satellites
- Ice, by the way, expanded in the South at the same time it retracted in the North, neither a “warming” event
Climate Depot quoted MIT atmospheric scientist Richard Lindzen, who said the following in an interview on Howie Carr’s radio show:
“‘To imply that a rise of temperature of a tenth of a degree is proof that the world is coming to an end — has to take one back to the dark ages.’ He added, ‘As long as you can get people excited as to whether it’s a tenth of a degree warmer or cooler, then you don’t have to think, you can assume everyone who is listening to you is an idiot.’”
“Lindzen continued, ‘The whole point is so crazy because the temperature is always going up or down a little. What is astonishing is that in the last 20 years it hasn’t done much of anything… What they don’t mention is there has been a big El Niño in 2016 and in recent months the temperature has been dropping back into a zero-trend level.’”
If actual temperature measurements confirmed the climate computer models, well, that might be a real story.
Hyping meaningless “adjusted” temperatures to hundredths of a degree during an El Niño event is not.
For nature and people too,
2016 allegedly ‘hottest year’
by immeasurable 1/100 of a degree
While satellites show ‘pause’ continues
By Marc MoranoRead the facts at CFACT’s Climate Depot
At 11.34am approximately, a plane identified as Korean Airlines KAL130 using Flightradar24.com in real time, was seen emitting an aerosol trail which persisted and expanded over time. The photos show the trail being sprayed at 11.34am approx, then the same trail is shown dispersing at about 11.40am and 11.46am.
Flightradar24.com shows that this plane was at an altitude of approximately 26,000 feet at the time.
This is not the first time the Korean Airline has been caught spraying aerosols in this region. Refer:
Northland’s pastures are looking increasingly desiccated and as was the case with droughts here since early-2010, aerosol trails have been an all too common sight in our skies. The following photos were taken this week from Whangarei, Northland and show a typical display of pollutants criss-crossing the once relatively pristine skies.
Inland near Waikari & Hawarden, North Canterbury, South Island, Aotearoa-New Zealand
Chemtrail dregs looking like a dirty oil slick in the sky. Taken looking west towards the hills in the late afternoon.
I DO NOT CONSENT to being sprayed!
Read ” Chemtrails, HAARP, and the Full Spectrum Dominance of Planet Earth “by Elana Freeland.
Detection of ‘abundant’ magnetite particles raises concerns because of suggested links to Alzheimer’s disease
Toxic nanoparticles from air pollution have been discovered in human brains in “abundant” quantities, a newly published study reveals.
The detection of the particles, in brain tissue from 37 people, raises concerns because recent research has suggested links between these magnetite particles and Alzheimer’s disease, while air pollution has been shown to significantly increase the risk of the disease. However, the new work is still a long way from proving that the air pollution particles cause or exacerbate Alzheimer’s.
“This is a discovery finding, and now what should start is a whole new examination of this as a potentially very important environmental risk factor for Alzheimer’s disease,” said Prof Barbara Maher, at Lancaster University, who led the new research. “Now there is a reason to go on and do the epidemiology and the toxicity testing, because these particles are so prolific and people are exposed to them.”