Michelle K. Leach

A culture system to study oligodendrocyte myelination processes using engineered nanofibers

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.

Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers

Neuritogenesis, neuronal polarity formation, and maturation of axons and dendrites are strongly influenced by both biochemical and topographical extracellular components. The aim of this study was to elucidate the effects of polylactic acid electrospun fiber topography on primary motor neuron development, because regeneration of motor axons is extremely limited in the central nervous system and could potentially benefit from the implementation of a synthetic scaffold to encourage regrowth. In this analysis, we found that both aligned and randomly oriented submicron fibers significantly accelerated the processes of neuritogenesis and polarity formation of individual cultured motor neurons compared to flat polymer films and glass controls, likely due to restricted lamellipodia formation observed on fibers. In contrast, dendritic maturation and soma spreading were inhibited on fiber substrates after 2 days in vitro. This study is the first to examine the effects of electrospun fiber topography on motor neuron neuritogenesis and polarity formation. Aligned nanofibers were shown to affect the directionality and timing of motor neuron development, providing further evidence for the effective use of electrospun scaffolds in neural regeneration applications.

Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(L-lactic acid)

Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(L-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 +/- 18 microm. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices.

A facile approach for the fabrication of core–shell PEDOT nanofiber mats with superior mechanical properties and biocompatibility

The development of modern biomedical nanotechnology requires conductive polymeric nanofibers with excellent mechanical and biocompatible properties to meet the needs of practical applications in complex biological systems. In the study, we developed a novel facile method to fabricate poly(3,4-ethylenedioxythiophene) (PEDOT) nanofiber mats by electrospinning combined with in situ interfacial polymerization. The PEDOT nanofiber mats displayed superior mechanical properties (tensile strength: 8.7 ± 0.4 MPa; Youngs modulus: 28.4 ± 3.3 MPa) and flexibility, which can almost be restored to its original shape even after serious twisting and crimping. Especially, from the results of the cellular morphology and proliferation of human cancer stem cells (hCSCs) cultured on the PEDOT nanofiber mats for 3 days, evidence was provided that the PEDOT nanofiber mats have similar biocompatibility to tissue culture plates (TCPs). Combined with an outstanding electrical conductivity of 7.8 ± 0.4 S cm-1, these excellent mechanical and biocompatible properties make the PEDOT nanofiber mats promising candidates in biotechnology applications, such as electroactive substrates/scaffolds for tissue engineering, drug delivery, cell culture, and implanted electrodes.

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Synthesis, copolymerization and peptide-modification of carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) for neural electrode interfaces☆

BACKGROUND Conjugated polymers have been developed as effective materials for interfacing prosthetic device electrodes with neural tissue. Recent focus has been on the development of conjugated polymers that contain biological components in order to improve the tissue response upon implantation of these electrodes. METHODS Carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) monomer was synthesized in order to covalently bind peptides to the surface of conjugated polymer films. EDOTacid was copolymerized with EDOT monomer to form stable, electrically conductive copolymer films referred to as PEDOT-PEDOTacid. The peptide GGGGRGDS was bound to PEDOT-PEDOTacid to create peptide functionalized PEDOT films. RESULTS The PEDOT-PEDOTacid-peptide films increased the adhesion of primary rat motor neurons between 3 and 9 times higher than controls, thus demonstrating that the peptide maintained its biological activity. CONCLUSIONS The EDOT-acid monomer can be used to create functionalized PEDOT-PEDOTacid copolymer films that can have controlled bioactivity. GENERAL SIGNIFICANCE PEDOT-PEDOTacid-peptide films have the potential to control the behavior of neurons and vastly improve the performance of implanted electrodes. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.

Fabrication and characterization of a novel fluffy polypyrrole fibrous scaffold designed for 3D cell culture

Three dimensional (3D) cell culture in functional scaffolds to mimic the cell natural growth state is important for the construction of cell based implants in vitro for tissue engineering applications. Herein, we report a novel fluffy polypyrrole (PPy) fibrous scaffold (fluffy-PPy scaffold) fabricated by means of an improved electrospinning process combined with in situ surface polymerization, in which PPy hollow fibers are discrete from one another with deep interconnected pores of ∼100 μm. This unique spatial structure permits the easy entry of cells into the fluffy-PPy scaffold with no extra help to achieve complicated 3D cell culture methodologies. The cell proliferation and morphology of cardiomyocytes (as a model cell) cultured in the fluffy-PPy scaffold were tested over a 3 day culture period. Evidence was provided that cardiomyocytes entered into the interior of the fluffy-PPy scaffold and formed stable cell-fiber constructs, and the rate of cell proliferation was higher than that on a traditional electrospun PPy fibrous mesh (mesh-PPy scaffold) and tissue culture plates (TCP). These results demonstrate that the fluffy-PPy scaffold not only achieved 3D cell culture, but also resulted in increased cell proliferation. Therefore, we suggest that the fluffy-PPy scaffold may be an appropriate choice as a functional scaffold capable of supporting 3D cell culture in the field of cardiac tissue engineering.

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

The influence of type-I collagen-coated PLLA aligned nanofibers on growth of blood outgrowth endothelial cells

Nanofibrous scaffolds have been applied widely in tissue engineering to simulate the nanostructure of natural extracellular matrix (ECM) and promote cell bioactivity. The aim of this study was to design a biocompatible nanofibrous scaffold for blood outgrowth endothelial cells (BOECs) and investigate the interaction between the topography of the nanofibrous scaffold and cell growth. Poly(L-lactic acid) (PLLA) random and aligned nanofibers with a uniform diameter distribution were fabricated by electrospinning. NH(3) plasma etching was used to create a hydrophilic surface on the nanofibers to improve type-I collagen adsorption; the conditions of the NH(3) plasma etching were optimized by XPS and water contact angle analysis. Cell attachment, proliferation, viability, phenotype and morphology of BOECs cultured on type-I collagen-coated PLLA film (col-Film), random fibers (col-RFs) and aligned fibers (col-AFs) were detected over a 7 day culture period. The results showed that collagen-coated PLLA nanofibers improved cell attachment and proliferation; col-AFs induced the directional growth of cells along the aligned nanofibers and enhanced endothelialization. We suggest that col-AFs may be a potential implantable scaffold for vascular tissue engineering.

Nanoporous fibers of type-I collagen coated poly(L-lactic acid) for enhancing primary hepatocyte growth and function

Advanced scaffold materials are required for liver tissue engineering, to improve primary hepatocyte activity and hepatic function in vitro. The nanotopography of the scaffold material plays an important role in the regulation of cell growth and function. Therefore, in the current study, we developed a novel scaffold composed of type-I collagen coated nanoporous poly(l-lactic acid) (PLLA) fibers (nPFs) to provide a nanotopography with a combination of fibrous and porous features for the culture of primary hepatocytes. The interaction between the nanotopography and the hepatocytes was described by testing cell morphology, retention, activity and hepatic function over a 15 day culture period. Primary hepatocytes cultured on the nPFs formed large-area stable immobilized monolayers after 3 days of culture, and displayed excellent cell bioactivity with higher levels of liver-specific function maintenance, in terms of albumin secretion, urea synthesis, and CYP1A and UGT enzymatic activity, than those cultured on type-I collagen coated non-porous PLLA fibers (Fs). These results indicate that the combined fibrous and porous nanotopography of nPFs has a superior promoting effect on primary hepatocyte culture compared to the non-porous fibrous nanotopography of Fs. The nPFs may be a suitable material for liver tissue engineering research and potential therapeutic applications, such as in bioartificial liver devices, and as a substrate for primary hepatocyte culture.