Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.     

Evaluating neuronal and glial growth on electrospun polarized matrices: bridging the gap in percussive spinal cord injuries

One of the many obstacles to spinal cord repair following trauma is the formation of a cyst that impedes axonal regeneration. Accordingly, we examined the potential use of electrospinning to engineer an implantable polarized matrix for axonal guidance. Polydioxanone, a resorbable material, was electrospun to fabricate matrices possessing either aligned or randomly oriented fibers. To assess the extent to which fiber alignment influences directional neuritic outgrowth, rat dorsal root ganglia (DRGs) were cultured on these matrices for 10 days. Using confocal microscopy, neurites displayed a directional growth that mimicked the fiber alignment of the underlying matrix. Because these matrices are generated from a material that degrades with time, we next determined whether a glial substrate might provide a more stable interface between the resorbable matrix and the outgrowing axons. Astrocytes seeded onto either aligned or random matrices displayed a directional growth pattern similar to that of the underlying matrix. Moreover, these glia-seeded matrices, once co-cultured with DRGs, conferred the matrix alignment to and enhanced outgrowth exuberance of the extending neurites. These experiments demonstrate the potential for electrospinning to generate an aligned matrix that influences both the directionality and growth dynamics of DRG neurites.     

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的：探讨应用改进静电纺丝技术一次成型制备三维（3D）取向聚乳酸与聚羟基乙酸共聚物（PLGA）纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法：应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组（每组12只）,A组：非取向PLGA神经导管组（阴性对照）;B组：取向PLGA神经导管组,C组：自体神经移植组（阳性对照）,于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果：神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异（P〈0.05）,均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论：无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的:探讨应用改进静电纺丝技术一次成型制备三维(3D)取向聚乳酸与聚羟基乙酸共聚物(PLGA)纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法:应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组(每组12只),A组:非取向PLGA神经导管组(阴性对照);B组:取向PLGA神经导管组,C组:自体神经移植组(阳性对照),于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果:神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异(P0.05),均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论:无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

Electrospinning of collagen nanofibers

Electrospinning is a fabrication process that uses an electric field to control the deposition of polymer fibers onto a target substrate. This electrostatic processing strategy can be used to fabricate fibrous polymer mats composed of fiber diameters ranging from several microns down to 100 nm or less. In this study, we describe how electrospinning can be adapted to produce tissue-engineering scaffolds composed of collagen nanofibers. Optimizing conditions for calfskin type I collagen produced a matrix composed of 100 nm fibers that exhibited the 67 nm banding pattern that is characteristic of native collagen. The structural properties of electrospun collagen varied with the tissue of origin (type I from skin vs type I from placenta), the isotype (type I vs type III), and the concentration of the collagen solution used to spin the fibers. Electrospinning is a rapid and efficient process that can be used to selectively deposit polymers in a random fashion or along a predetermined and defined axis. Toward that end, our experiments demonstrate that it is possible to tailor subtle mechanical properties into a matrix by controlling fiber orientation. The inherent properties of the electrospinning process make it possible to fabricate complex, and seamless, three-dimensional shapes. Electrospun collagen promotes cell growth and the penetration of cells into the engineered matrix. The structural, material, and biological properties of electrospun collagen suggest that this material may represent a nearly ideal tissue engineering scaffold.     

Patterning of polymer nanofiber meshes by electrospinning for biomedical applications

The end-product of the electrospinning process is typically a randomly aligned fiber mesh or membrane. This is a result of the electric field generated between the drop of polymer solution at the needle and the collector. The developed electric field causes the stretching of the fibers and their random deposition. By judicious selection of the collector architecture, it is thus possible to develop other morphologies on the nanofiber meshes. The aim of this work is to prepare fiber meshes using various patterned collectors with specific dimensions and designs and to evaluate how those patterns can affect the properties of the meshes relevant to biomedical applications. This study aims at verifying whether it is possible to control the architecture of the fiber meshes by tailoring the geometry of the collector. Three different metallic collector topographies are used to test this hypothesis. Electrospun nonwoven patterned meshes of polyethylene oxide (PEO) and poly( -caprolactone) (PCL) were successfully prepared. Those fiber meshes were analyzed by scanning electron microscopy (SEM). Both mechanical properties of the meshes and cell contacting experiments were performed to test the effect of the produced patterns over the properties of the meshes relevant for biomedical applications. The present study will evaluate cell adhesion sensitivity to the patterns generated and the effect of those patterns on the tensile properties of the fiber meshes.     

Effect of Process Parameters on the Microstructural Characteristics of Electrospun Poly(Vinyl Alcohol) Fiber Mats

Fiber mats with average fiber diameter ranging between 80 and 250 nm of polyvinyl alcohol (PVA)/water solution having a concentration of 4 wt.% have been prepared by electrospinning method. The influence of applied voltage, flow rate, and needle-to-collector distance on the fiber morphology and diameters has been studied. Scanning electron microscopy and atomic force microscopy are used to characterize the fibers. It has been observed that bead-free fibers of 4 wt.% PVA can be obtained at lower voltages (9 kV). Also, density and the deposition area of the fiber mats showed a clear dependence on the applied voltage, flow rate, and collector distance.     

Carbon nanotube reinforced Bombyx mori silk nanofibers by the electrospinning process

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.     

Direct in vitro electrospinning with polymer melts

The electrospinning of polymer melts can offer an advantage over solution electrospinning, in the development of layered tissue constructs for tissue engineering. Melt electrospinning does not require a solvent, of which many are cytotoxic in nature, and the use of nonwater soluble polymers allows the collection of fibers on water or onto cells. In this article, melt electrospinning of a blend of PEO-block-PCL with PCL was performed with in vitro cultured fibroblasts as the collection target. The significant parameters governing electrospinning polymer melts were determined before electrospinning directly onto fibroblasts. In general, a high electric field resulted in the most homogeneous and smallest fibers, although it is important that an optimal pump rate to the spinneret needs to be determined for different configurations. Many parameters governing melt electrospinning differ to those reported for solution electrospinning: the pump rate was a magnitude lower and the viscosity a magnitude higher than successful parameters for solution electrospinning. Cell vitality was maintained throughout the electrospinning process. Six days after electrospinning, fibroblasts adhered to the electrospun fibers and appeared to detach from the underlying flat substrate. The morphology of the fibroblasts changed from spread and flat, to long and spindle-shaped as adherence onto the fiber progressed. Therefore, an important step for producing layer-on-layer tissue constructs of cells and polymers in view of scaffold construction for tissue engineering was successfully demonstrated. The process of using cultured cells as the collection target was termed "direct in vitro electrospinning".     

A method for studying the structure of uniaxially aligned biopolymers using solid state 15N-nmr: Application to Bombyx mori silk fibroin fibers

Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction. In the present work, a new method is developed in which solid state ¹⁵N-nmr spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield the orientations of specific molecular bonds. Analytical expressions are derived that utilize spectral features read from 15N chemical shift anisotropy line shapes to calculate a discrete number of possible orientations for a specific site. The ¹⁵N-¹H dipolar interaction is employed to further narrow the number of unique orientations possible for a given site. With this method, a neighborhood of possible orientations is quickly determined, and full line-shape simulations within this region of allowed space can be performed to refine the limits of orientation. This technique demonstrates the use of a single type of isotopic label to determine the orientation of a specific molecular group such as a peptide plane within a protein. Results from the application of this method to the Bombyx mori silk fibroin protein provide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies. © 1993 John Wiley & Sons, Inc.     

Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering

Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1+/-0.4MPa, compared to highly anisotropic scaffolds whose modulus was 11.6+/-3.1MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.     

Self‐Actuating Electrocaloric Cooling Fibers

Solid‐state cooling fibers comprising an electrocaloric polymer, poly(vinylidene fluoride‐trifluoroethylene‐chlorofluoroethylene) terpolymer, spray‐coated on a conductive fiber core electrode, and a coaxially coated single‐walled carbon nanotubes outer electrode are reported. The fiber coolers can be less than 160 µm thin and more than 8 cm long. Measured cooling ΔT of the EC fibers is 0.7 °C at an electric field of 100 V µm^?1 applied between the electrodes. The fiber coolers are flexible; 2000 cycles of repeated bending to a 2.5 mm curvature radius do not significantly degrade the cooling ΔT. Self‐actuated bending of the fibers is observed during the EC operation, which allows the EC fibers to move heat from one location to another without any additional driving mechanisms such as electromagnetic motors, pumps, or electrostatic actuation that are commonly used in conventional coolers. The self‐actuating EC fibers represent the first ever active cooler in a thin fiber form factor.     

Improved prediction of the collagen fiber architecture in the aortic heart valve

Living tissues show an adaptive response to mechanical loading by changing their internal structure and morphology. Understanding this response is essential for successful tissue engineering of load-bearing structures, such as the aortic valve. In this study, mechanically induced remodeling of the collagen architecture in the aortic valve was investigated. It was hypothesized that, in uniaxially loaded regions, the fibers aligned with the tensile principal stretch direction. For biaxial loading conditions, on the other hand, it was assumed that the collagen fibers aligned with directions situated between the principal stretch directions. This hypothesis has already been applied successfully to study collagen remodeling in arteries. The predicted fiber architecture represented a branching network and resembled the macroscopically visible collagen bundles in the native leaflet. In addition, the complex biaxial mechanical behavior of the native valve could be simulated qualitatively with the predicted fiber directions. The results of the present model might be used to gain further insight into the response of tissue engineered constructs during mechanical conditioning.     

1H-NMR studies of the intracellular water of skeletal muscle fibers under various physiological conditions.

Intracellular water of frog skeletal muscle fibers has been studied under various physiological conditions by use of the 1H-nuclear magnetic resonance (NMR) technique. 1H-NMR spectra of muscle fibers had single peaks derived from the intracellular water. The spectra changed in a characteristic fashion when the fiber axis was aligned at various angles relative to the magnetic field of the NMR magnet. Further, the relaxation rates of the 1H-NMR spectra changed depending on the water content of muscle fibers, and in association with contraction and rigor formation of muscle fibers. The obtained results indicate that the intracellular water of muscle fibers is structured and aligned along the myofilaments, and further that the state of the intracellular water changes with physiological conditions.     

Collagen fiber alignment does not explain mechanical anisotropy in fibroblast populated collagen gels

Many load-bearing soft tissues exhibit mechanical anisotropy. In order to understand the behavior of natural tissues and to create tissue engineered replacements, quantitative relationships must be developed between the tissue structures and their mechanical behavior. We used a novel collagen gel system to test the hypothesis that collagen fiber alignment is the primary mechanism for the mechanical anisotropy we have reported in structurally anisotropic gels. Loading constraints applied during culture were used to control the structural organization of the collagen fibers of fibroblast populated collagen gels. Gels constrained uniaxially during culture developed fiber alignment and a high degree of mechanical anisotropy, while gels constrained biaxially remained isotropic with randomly distributed collagen fibers. We hypothesized that the mechanical anisotropy that developed in these gels was due primarily to collagen fiber orientation. We tested this hypothesis using two mathematical models that incorporated measured collagen fiber orientations: a structural continuum model that assumes affine fiber kinematics and a network model that allows for nonaffine fiber kinematics. Collagen fiber mechanical properties were determined by fitting biaxial mechanical test data from isotropic collagen gels. The fiber properties of each isotropic gel were then used to predict the biaxial mechanical behavior of paired anisotropic gels. Both models accurately described the isotropic collagen gel behavior. However, the structural continuum model dramatically underestimated the level of mechanical anisotropy in aligned collagen gels despite incorporation of measured fiber orientations; when estimated remodeling-induced changes in collagen fiber length were included, the continuum model slightly overestimated mechanical anisotropy. The network model provided the closest match to experimental data from aligned collagen gels, but still did not fully explain the observed mechanics. Two different modeling approaches showed that the level of collagen fiber alignment in our uniaxially constrained gels cannot explain the high degree of mechanical anisotropy observed in these gels. Our modeling results suggest that remodeling-induced redistribution of collagen fiber lengths, nonaffine fiber kinematics, or some combination of these effects must also be considered in order to explain the dramatic mechanical anisotropy observed in this collagen gel model system.     

Complementary Effects of Two Growth Factors in Multifunctionalized Silk Nanofibers for Nerve Reconstruction

With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the ‘bands of Bungner’ found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.     

Morphology of the core fibrous layer of the cetacean tail fluke

The cetacean tail fluke blades are not supported by any vertebral elements. Instead, the majority of the blades are composed of a densely packed collagenous fiber matrix known as the core layer. Fluke blades from six species of odontocete cetaceans were examined to compare the morphology and orientation of fibers at different locations along the spanwise and chordwise fluke blade axes. The general fiber morphology was consistent with a three‐dimensional structure comprised of two‐dimensional sheets of fibers aligned tightly in a laminated configuration along the spanwise axis. The laminated configuration of the fluke blades helps to maintain spanwise rigidity while allowing partial flexibility during swimming. When viewing the chordwise sectional face at the leading edge and mid‐chord regions, fibers displayed a crossing pattern. This configuration relates to bending and structural support of the fluke blade. The trailing edge core was found to have parallel fibers arranged more dorso‐ventrally. The fiber morphology of the fluke blades was dorso‐ventrally symmetrical and similar in all species except the pygmy sperm whale (Kogia breviceps), which was found to have additional core layer fiber bundles running along the span of the fluke blade. These additional fibers may increase stiffness of the structure by resisting tension along their long spanwise axis.     

Manipulation of microparticles for construction of array patterns by negative dielectrophoresis using multilayered array and grid electrodes

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative‐dielectrophoresis (n‐DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non‐uniform electric fields. The MLAG electrode was mounted to a target substrate in a face‐to‐face configuration with a spacer. When an AC voltage (4.60 V_rms and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 µm diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays. Biotechnol. Bioeng. 2009; 104: 709–718 © 2009 Wiley Periodicals, Inc.     

The influence of fiber orientation on the equilibrium properties of neutral and charged biphasic tissues

Constitutive models facilitate investigation into load bearing mechanisms of biological tissues and may aid attempts to engineer tissue replacements. In soft tissue models, a commonly made assumption is that collagen fibers can only bear tensile loads. Previous computational studies have demonstrated that radially aligned fibers stiffen a material in unconfined compression most by limiting lateral expansion while vertically aligned fibers buckle under the compressive loads. In this short communication, we show that in conjunction with swelling, these intuitive statements can be violated at small strains. Under such conditions, a tissue with fibers aligned parallel to the direction of load initially provides the greatest resistance to compression. The results are further put into the context of a Benninghoff architecture for articular cartilage. The predictions of this computational study demonstrate the effects of varying fiber orientations and an initial tare strain on the apparent material parameters obtained from unconfined compression tests of charged tissues.     

Electrospinning: A fascinating fiber fabrication technique

With the emergence of nanotechnology, researchers become more interested in studying the unique properties of nanoscale materials. Electrospinning, an electrostatic fiber fabrication technique has evinced more interest and attention in recent years due to its versatility and potential for applications in diverse fields. The notable applications include in tissue engineering, biosensors, filtration, wound dressings, drug delivery, and enzyme immobilization. The nanoscale fibers are generated by the application of strong electric field on polymer solution or melt. The non-wovens nanofibrous mats produced by this technique mimics extracellular matrix components much closely as compared to the conventional techniques. The sub-micron range spun fibers produced by this process, offer various advantages like high surface area to volume ratio, tunable porosity and the ability to manipulate nanofiber composition in order to get desired properties and function. Over the years, more than 200 polymers have been electropun for various applications and the number is still increasing gradually with time. With these in perspectives, we aim to present in this review, an overview of the electrospinning technique with its promising advantages and potential applications. We have discussed the electrospinning theory, spinnable polymers, parameters (solution and processing), which significantly affect the fiber morphology, solvent properties and melt electrospinning (alternative to solution electrospinning). Finally, we have focused on varied applications of electrospun fibers in different fields and concluded with the future prospects of this efficient technology.