Electrospinning Technique

Electrospinning is a widely used technique to pro-duce fibers in the nanoscale. In 1934, Formhals was the first to publish a work concerning the electrospinning process. He described it as a method to produce thin polymer filaments by exploiting electrostatic repulsions between sur-face charges. He also gave a rigorous description of the apparatus he used to create the polymer filaments. As previously mentioned, however, the increased popularity in the use of electrospin-ning for producing nanofibers for tissue engineering and other applications was sparked by Reneker in the early 1990s [61, 62]. Prior to Reneker’s work, the effect of fiber diameter on the performance and manufacturing of more complex fibrous structures was known, but the practical generation and implementation of nanoscale fibers were limited [101]. During the early 1990s, the technique would also assume its current appellation, becoming a portmanteau of the words electrostatic and spinning [60]. This technique remains viable primarily because of its simplicity.

The electrospinning process involves apply-ing a voltage, typically 1–30 kV, to charge a poly-mer solution (or melt) loaded into a syringe (Figure 7.1). This high applied voltage causes the polymer solution to be sufficiently charged, and the induced charge distributes evenly through the surface. At this point, the solution experi-ences electrostatic repulsion forces from the sur-face charges and Coulombic forces exerted by the external electric field [60]. When the electro-static repulsion forces combined with the Coulombic force are sufficient to overcome the surface tension of the solution, a stream erupts from the deformed droplet, known as a Taylor cone, at the end of the nozzle. If the cohesion is sufficient, then the stream is elongated and even-tually deposited onto a grounded collector plate.

If cohesion or chain entanglement is not suffi-cient, then electrospraying or droplet formation usually occurs. Jet elongation happens during

the stream’s travel toward the collector. During its flight, the jet undergoes a stretching and whipping process to draw the fiber into an ultrafine long filament. The solvent simultane-ously evaporates and the fibers are deposited on the grounded collector, thereby creating a non-woven, randomly aligned fibrous mat [60].

The parameters chosen during electrospin-ning greatly influence the collected fibers. These parameters are typically divided into three cat-egories: polymer parameters, polymer solution parameters, and parameters of the apparatus.

The type of polymer used and its physical prop-erties greatly affect the nanofibers. These proper-ties include the molecular weight, the molecular weight distribution, and the branching of the polymer [12]. Solution properties found to have an integral role in fiber formation include viscos-ity, polymer concentration, conductivviscos-ity, and surface tension. Important apparatus parame-ters are applied flow rate, voltage, distance from syringe needle tip to collector, type of collector and whether it is static or dynamic, the type of needle used, and the ambient conditions during electrospinning [61, 94–96].

One of the most studied dependent properties of the fibers is the fiber diameter. Several research-ers have attempted to sum up the effects of the many independent variables of electrospinning

on fiber diameter into a succinct mathematical model. Rutledge et al. [102] developed a mathe-matical model that related surface tension γ, static relative permittivity ǫ, flow rate Q, current carried by the fiber I, and the ratio of initial jet length to the nozzle diameter χ to fiber diameter d as follows:

This equation was derived by fitting an exponen-tial model to empirical data. According to this equation, increasing the current-carrying capa-bility of fibers by adding more conductive mate-rials to the polymer solution will significantly reduce fiber diameter [62]. It is also possible to reduce fiber diameter by manipulating other independent variables such as a reduction in either the flow rate Q or the nozzle diameter γ.

Another model relates fiber diameter to the molecular weight of the polymer and the con-centration of the polymer in the spinning solu-tion. It also uses the dimensionless parameter called the Berry number B. The Berry number is the product of the intrinsic viscosity η and poly-mer concentration C, i.e.,

(7.1) d=

γ ǫ

Q² I²

2 π (2lnχ−3)

¹/3

.

(7.2) B=ηC.

FIGURE 7.1 Schematic of an electrospinning setup.

7.2 FABRICATION OF 3D BIOSCAFFOLDS 173 The Berry number has four distinct domains.

According to the value assigned to B of a poly-mer, a researcher can determine the likelihood the polymer will produce nanofibers [62].

Region I, where B < 1, is representative of a very dilute polymer solution with limited chain entanglement. This results in only poly-mer droplets being formed. In Region II, 1 < B < 3, the fiber diameter increases within the range of 100–500 nm as B increases. This region is indicative of molecular entanglement that is just sufficient for fiber formation. Although fiber formation is observed, there is still some droplet formation as a result of polymer relax-ation and surface tension. In Region III, 3 < B < 4, the fiber diameter increases rapidly with B and is in the range of 1,700–2,800 nm. The rapid increase in fiber diameter is attributed to the intensive molecular entanglement resulting in an increase in polymer viscosity. The conse-quence of increased polymer viscosity also means that a higher electric field is required to produce fibers. Finally, in Region IV, where B > 4 and there is significant chain entangle-ment within and among chains fiber diameter is more dependent on the applied voltage/

electric field and other process parameters than it is on B [62].

The two quantitative models for calculating fiber diameter are a sampling of what research-ers have produced thus far, with more complex examples existing. They serve as a suitable start-ing point for researchers, suggeststart-ing parameter boundaries to achieve desired fibers. There are also numerous other variations that can be made to electrospinning parameters to engineer favorable fibers.

7.2.2.1.1 Electrospinning for uniaxially aligned nanofibers

Aligning nanofibrous arrays is useful for a vari-ety of purposes. When fibers are used to impart additional mechanical integrity as a composite component, control of the alignment dictates the degree of structural support the fibers provide. In

the areas of tissue engineering and regenerative medicine, aligned fibers can serve as a physical guide for cellular growth, influence cell adhesion, and modulate cellular patterns found in native tissue [103]. They are particularly useful when employed to regenerate tissues that require direc-tional recruitment and assembly of cells, for example, in neural tissue engineering. Cooper et al. [103] demonstrated the benefits of aligned chitosan-PCL fibers over films and randomly ori-ented fibers of the same materials in promoting the attachment and proliferation of Schwann cells, which are important cells of the peripheral nervous system. The aligned fibers induced cellular responses as a result of topographical and chemical cues for the modulation of neurite outgrowth [104].

One way to fabricate uniaxially aligned nanofibers or parallel arrays of fibers is to use a rapidly rotating drum or cylinder as the collec-tor. The rotating collector forces fibers to align in a perpendicular orientation to the axis of rota-tion of the drum. This method, however, only produces partially aligned fibers [62]. To improve alignment, researchers modified the drum by adding a sharp edge.

Another method to fabricate aligned fibers is to use a pair of split electrodes [60]. Two conduc-tive strips separated by a gap of up to several centimeters allow for the synthesis of aligned nanofibers in the gap. Researchers believe that the insulating gap alters the electrostatic forces acting on the fibers in the gap. As a result, elec-trostatic forces act in opposing directions and fibers are stretched, aligning themselves perpen-dicular to the edge of the gap. The electrostatic repulsion between deposited fibers can further enhance the alignment of the collected fibers.

This technique can also be used to readily stack the aligned fibers into films or mats for practical applications [105].

The overarching concept governing the previ-ously described methods to fabricate aligned fib-ers is the ability to control the electric field. Some researchers have modeled the dominant role of

the electric field in controlling the trajectory of the fiber and ultimately its collection. Manipula-tion of the field, either through the applied volt-age or by the type of collector used, provides the proper fiber geometry for the application.

7.2.2.1.2 Blend and composite nanofibers

As with other fabrication techniques, composites and blend systems can be created to enhance the biocompatibility and structural properties of nanofibrous scaffolds. Blend nanofibers incorpo-rate two or more polymers into a single solution that can be electrospun. Figure 7.2 shows one example, developed by Cooper et al. [106], of polyblend nanofibers of PCL-chitosan for the application of skeletal muscle tissue reconstruc-tion. The combinations of polymeric materials provide increased structural support, enhanced biocompatibility, and desired degradative prop-erties. The material choice is critically important in engineering fiber scaffolds with suitable phys-icochemical properties. Synthetic polymers are widely used because of their uniformity (physical

and chemical properties) and because they are consistently reproduced. Some synthetic poly-mers commonly used in tissue engineering appli-cations are PCL and poly-lactide-co-glycolide acid (PLGA). For nanofibrous scaffold produc-tion, these polymers are particularly suited because they easily form nanostructures from electrospinning, and like many other polymers, they degrade via hydrolysis of their ester linkage.

However, this degradation often occurs at an unfavorably slow rate. To rectify this issue, poly-mers such as PCL, with good mechanical integrity but inherent hydrophobicity, can be altered with natural or more hydrophilic synthetic polymers to produce structurally sound and biocompatible scaffolds. Several research reports have been pub-lished showing the ability of PCL and a naturally occurring polymer to form blended copolymeric nanofibrous structures [35, 106–110].

The resulting fiber morphology appeared to be smooth, and fibers had a mean diameter of approximately 100 nm, a result researchers attrib-ute to the solvent system used. Contact angle

FIGURE 7.2 Electrospun composite nanofibers for a skeletal muscle tissue reconstruction application. Confocal micros-copy images showing immunocytochemistry analysis of actin (left column, green) and myosin heavy chain (MHC) (middle column, red) expressed by muscle cells grown on chitosan-PCL randomly oriented and aligned nanofibrous scaffolds after culture in fusion media for five days. The merged images with nuclei stained with DAPI (blue) are shown in the right column. SEM images showing the morphology of chitosan-PCL nanofibers. Scale bars represent 40 μm for cell/nanofiber structures and 20 μm for SEM images. Reprinted with permission from Ref. 106; copyright 2012 Royal Society of Chemistry.

(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this book.)

7.2 FABRICATION OF 3D BIOSCAFFOLDS 175 measurements were used to determine the

rela-tive hydrophilicity of the scaffolds. The blend scaffold of chitosan and PCL was shown to exhibit a significant decrease in hydrophobicity and degradation rates compared to the pure PCL fiber scaffold. The blend fibers were also opti-mally mixed to produce the most bioactive struc-tures, resulting in a larger percentage of chitosan being used to sustain cells more favorably.

Studies have also been conducted demon-strating that optimizing biocompatibility does not cause severe losses of mechanical integrity [111]. The natural polymer chitosan was again combined with the synthetic polymer PCL. The ultimate goal was to produce a synergistic blend of the two polymers that had the mechanical integrity of PCL and the biocompatibility and bioactivity of chitosan.

Composite nanofibers are a product of ceram-ics or metals added to polymers to increase fiber mechanical properties and enhance bioactivity.

Hydroxyapatite or some other form of calcium phosphate or bioglass is a commonly used ceramic in bioscaffolds because of its chemical similarity to natural mineral components. The increased osteoconductivity, the ability to sup-port bone formation, obtained by adding ceram-ics aids in the formation of natural bone.

Although it is simple to add the mineral compo-nent to the polymer solution to be electrospun, this typically results in the mineral being embed-ded or encapsulated in the fibers, rendering it ineffective. To remedy this problem, several researchers have deposited the mineral on the fibers as a post-fabrication treatment. Incubation in simulated body fluid allows the pores and fiber surfaces to get sufficiently covered in min-erals; this process can take several hours to weeks. To shorten the time it takes to mineralize a scaffold, electrodeposition technology was used to coat the fibers, accomplishing minerali-zation in less than an hour. The resulting fibers had the same morphology compared to simu-lated body fluid (SBF) incubated fibers as well as similar mechanical strength [43].

7.2.2.1.3 Centrifugal electrospinning

Centrifugal electrospinning is another modified electrospinning technique that produces highly aligned nanofibers. This process involves load-ing a spinneret and nozzle onto a circular disk that is attached to a rotating axle. A metallic cylindrical shell is then placed around the disk and grounded to serve as the collector. Centrifu-gal action on the polymer solution provides a uniform distribution of stress, which stretches the polymer into a long fiber if the solution viscosity is ample. This process also allows lower-molecular-weight polymer solutions to be electrospun.

Polymer solution viscosity is a result of the friction between polymer chains. The frictional forces are dependent on the speed of the centri-fuge: as the speed increases, the frictional force increases and consequently the solution viscos-ity increases. The electrocentrifugal technique produces fibers that are better aligned compared to electrospinning, due to its ability to reduce the bending stability of the polymer jet. These fibers, however, exhibit only marginal improve-ments to mechanical properties [112].

7.2.2.1.4 Coaxial electrospinning

Coaxial or core-shell electrospinning is another common modification to the traditional electro-spinning technique to obtain multifunctional nanofibrous scaffolds. The fibers can be spun from many different polymers and polymeric combinations, and a variety of material (syn-thetic or natural) can be placed in the core, all aimed at efficient tissue formation. Most often, biomolecules, which easily lose their bioactivity in the harsh solvents used to dissolve polymers, are encapsulated as the core. The polymer shell surrounding the biomolecule core has the prop-erty of tunable degradability, depending on its composition, which allows these biochemical agents to be released over a favorable time period. The ability to optimize the temporal release of the molecules promotes more effica-cious behavior of the biomolecules [113, 114].

The most common core molecules are sensitive biomolecules like the growth factors bone mor-phogenic protein (BMP) and fiberblast growth factor (FGF), which elicit favorable responses from cells. The favorable responses of cells in the presence of growth factors include enhanced growth, cell proliferation, and cell differentia-tion, all of which contribute to tissue growth. It is necessary that molecules like growth factors be introduced into the wound or defective area in a sustained manner to ensure favorable cel-lular function throughout the wound-healing or tissue-formation process [113, 114].

The polymer shell, in addition to protecting the encapsulated biomolecules and moderating biomolecular release, can function to make cells and the implant interact favorably. This behavior is normally achieved through simple chemical modifications [115] to the polymer shell’s sur-face. Finally, the core-shell method is applicable for encasing fibers that are mechanically strong or have some other benefit but are not benign to the cells at the site of implantation [115].

In addition to encasing biomolecules and other polymers, the core-shell method can be used to alter the physical properties of electro-spun nanofibers in an optimal way [114]. The dynamic evaporation of solvent from the elec-trospun fluid jets presents an opportunity for manipulation of the resulting collected nanofib-ers. As previously explained, electrospinning has an instability region where the fluid poly-mer jets are whipped and bent, stretched and elongated. This is a result of the interactions of the applied electrostatic force, the solution’s intrinsic viscoelasticity, and its surface tension.

As the fluid jets become increasingly sticky due to solvent molecule transfer and evaporation, the electrical forces gradually lose their influ-ence on the fluid jets because electrons can only easily interact with fluids. As a result, the elec-trical drawing process stops when the entire jet, or sometimes the surface of the jet, solidifies.

The use of surfactant solutions as the shell or sheath fluid helps remedy this problem. As a

result of using surfactant solutions, the col-lected fibers have reduced diameters and are smoother compared to fibers spun with no sheath solution [114].

To achieve the core-shell geometry, the outer solution consisting of a solution or a solvent is loaded into a syringe and placed into a pump apparatus. The core solution is then loaded into another syringe and placed into a pump. A metallic capillary that has an inner and outer diameter is attached to the syringes using some type of polymer piping. The piping from the inner diameter is connected to the core solution, and the piping to the outer diameter is con-nected to the sheath solution. Like traditional electrospinning, a voltage is applied to the metal-lic capillary, then a jet forms, and eventually fib-ers are deposited on a grounded collector [114].

7.2.2.1.5 Melt electrospinning

Although electrospun nanofibers from polymer solutions have been successful in producing fib-ers in the submicron region, the potential clini-cal use of these nanofibers could be limited due to the use of harsh solvents and incomplete dry-ing [116]. Therefore, a need for solvent-free pro-cessing exists. Zhmayev et al. showed that by using gas-assisted polymer melt electrospin-ning, it is possible to achieve fibers with sub-micrometer diameters [116]. Air drag in the electrospinning setup, as well as heating pro-vided by the air stream, aid in thinning the fiber [116]. This presents a viable alternative to solu-tions of synthetic polymers such as polylactic acid (PLA). However, this technique would not suffice for some natural polymers because heat-ing could cause structural damage [117].

7.2.2.2 Thermally Induced Phase Separation