Regenerative Peripheral Nerve Interface for Prostheses Control: Electrode Comparison.

BACKGROUND: This study compared epimysial patch electrodes with intramuscular hook electrodes using monopolar and bipolar recording configurations. The purpose was to determine which strategy transduced muscle signals with better fidelity for control of myoelectric prostheses. METHODS: One of the two electrode styles, patch (n = 4) or hook (n = 6) was applied to the left extensor digitorum longus muscle in rats. Electrodes were evaluated at the time of placement and at monthly intervals for 4 months. Evaluations consisted of evoked electromyography signals from stimulation pulses applied to the peroneal and tibial nerves in both monopolar and bipolar recording configurations. RESULTS: Compared with hook electrodes, patch electrodes recorded larger signals of interest and minimized muscle tissue injury. A bipolar electrode configuration significantly reduced signal noise when compared with a monopolar configuration. CONCLUSION: Epimysial patch electrodes outperform intramuscular hook electrodes during chronic skeletal muscle implantation.

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.

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.

Highly Aligned Poly(3,4-ethylene dioxythiophene) (PEDOT) Nano- and Microscale Fibers and Tubes.

This study reports a facile method for the fabrication of aligned Poly(3,4-ethylene dioxythiophene) (PEDOT) fibers and tubes based on electrospinning and oxidative chemical polymerization. Discrete PEDOT nano- and microfibers and nano- and microtubes are difficult to fabricate quickly and reproducibly. We employed poly(lactide-co-glycolide) (PLGA) polymers that were loaded with polymerizable 3,4-ethylene dioxythiophene (EDOT) monomer to create aligned nanofiber assemblies using a rotating glass mandrel during electrospinning. The EDOT monomer/PLGA polymer blends were then polymerized by exposure to an oxidative catalyst (FeCl3). PEDOT was polymerized by continuously dripping a FeCl3 solution onto the glass rod during electrospinning. The resulting PEDOT fibers were conductive, aligned and discrete. Fiber bundles could be easily produced in lengths of several centimeters. The PEDOT sheath/PLGA core fibers were immersed in chloroform to remove the PLGA and any residual EDOT resulting in hollow PEDOT tubes. This approach made it possible to easily generate large areas of aligned PEDOT fibers/tubes. The structure and properties of the aligned assemblies were measured using optical microscopy, electron microscopy, Raman spectroscopy, thermal gravimetric analysis, and DC conductivity measurements. We also demonstrated that the aligned PEDOT sheath/PLGA core fiber assemblies could be used in supporting and directing the extension of dorsal root ganglia (DRG) neurons in vitro.

Combining topographical and genetic cues to promote neuronal fate specification in stem cells.

There is little remedy for the devastating effects resulting from neuronal loss caused by neural injury or neurodegenerative disease. Reconstruction of damaged neural circuitry with stem cell-derived neurons is a promising approach to repair these defects, but controlling differentiation and guiding synaptic integration with existing neurons remain significant unmet challenges. Biomaterial surfaces can present nanoscale topographical cues that influence neuronal differentiation and process outgrowth. By combining these scaffolds with additional molecular biology strategies, synergistic control over cell fate can be achieved. Here, we review recent progress in promoting neuronal fate using techniques at the interface of biomaterial science and genetic engineering. New data demonstrates that combining nanofiber topography with an induced genetic program enhances neuritogenesis in a synergistic fashion. We propose combining patterned biomaterial surface cues with prescribed genetic programs to achieve neuronal cell fates with the desired sublineage specification, neurochemical profile, targeted integration, and electrophysiological properties.

Mechanical tension applied to substrate films specifies location of neuritogenesis and promotes major neurite growth at the expense of minor neurite development.

One obstacle in neural repair is facilitating axon growth long enough to reach denervated targets. Recent studies show that axonal growth is accelerated by applying tension to bundles of neurites, and additional studies show that mechanical tension is critical to all neurite growth. However, no studies yet describe how individual neurons respond to tensile forces applied to cell bodies and neurites simultaneously; neither do any test motor neurons, a phenotype critical to neural repair. Here we examine the growth of dissociated motor neurons on stretchable substrates. E15 spinal motor neurons were cultured on poly-lactide-co-glycolide films stretched at 4.8, 9.6, or 14.3 mm day(-1). Morphological analysis revealed that substrate stretching has profound effects on developing motor neurons. Stretching increases major neurite length; it also forces neuritogenesis to occur nearest poles of the cell closest to the sources of tension. Stretching also reduces the number of neurites per neuron. These data show that substrate stretching affects neuronal morphology by specifying locations on the cell where neuritogenesis occurs and favoring major neurite growth at the expense of minor neurites. These results serve as a building block for development of new techniques to control and improve the growth of neurons for nerve repair purposes.

Electrically stimulated signals from a long-term Regenerative Peripheral Nerve Interface.

Despite modern technological advances, the most widely available prostheses provide little functional recovery beyond basic grasping. Although sophisticated upper extremity prostheses are available, optimal prosthetic interfaces which give patients high-fidelity control of these artificial limbs are limited. We have developed a novel Regenerative Peripheral Nerve Interface (RPNI), which consists of a unit of free muscle that has been neurotized by a transected peripheral nerve. In conjunction with a biocompatible electrode on the muscle surface, the RPNI facilitates signal transduction from a residual peripheral nerve to a neuroprosthetic limb. The purpose of this study was to explore signal quality and reliability in an RPNI following an extended period of implantation. Following a 14-month maturation period, electromyographic signal generation was evaluated via electrical stimulation of the innervating nerve. The long-term RPNI was viable and healthy, as demonstrated by evoked compound muscle action potentials as well as histological tissue analysis. Signals exceeding 4 mV were successfully acquired and amplitudes were consistent across multiple repetitions of applied stimuli. There were no evident signs of muscle denervation, significant scar tissue, or muscle necrosis. This study provides further evidence that after a maturation period exceeding 1 year, reliable and consistent signals can still be acquired from an RPNI.

Quantification of muscle-derived signal interference during monopolar needle electromyography of a peripheral nerve interface in the rat hind limb.

High-fidelity signal acquisition is critical for the fundamental control of a neuroprosthesis. Our group has developed a bio-artificial interface consisting of a muscle graft neurotized by a severed nerve in a rat hind limb model. This regenerative peripheral nerve interface (RPNI) permits nerve signal transmission, amplification, and detection via in situ electromyography (EMG). Our study examined the magnitude of signal interference from simultaneously contracting muscles adjacent to our muscle of interest. In eighteen F344 rats, the extensor digitorum longus (EDL) muscle was used to fabricate simulated RPNI constructs of various sizes in which the neurovascular pedicle was preserved, obviating the need for reinnervation or revascularization. After 3 weeks of recovery, in situ EMG testing was performed using electrical stimulation of the common peroneal nerve. A recording needle was placed in the EDL muscle with a reference/ground electrode in the contralateral toe webspace, comprising a monopolar recording configuration. The superficial peroneal nerve was transected to further isolate stimulation of the anterior compartment. Recordings from the EDL were performed before and after excision of the tibialis anterior (TA) and extensor hallucis longus (EHL) muscles. After TA/EHL excision, EDL compound muscle action potential (CMAP) peak-to-peak amplitudes were significantly lower by an average of 7.4±5.6(SD) mV, or 32±18%, (paired t(17)=-5.7, p<;0.0001). A significant positive linear correlation was seen between CMAP amplitude and EDL mass both before TA/EHL excision (r=0.68, n=18, p<;0.01) and after TA/EHL excision (r=0.79, n=18, p<;0.0001). EDL mass did not correlate with differences in CMAP amplitude or area caused by TA/EHL excision. Monopolar needle EMG recordings from the EDL muscle are significantly, but predictively, contaminated by concomitant muscular contractions in the anterior compartment of the rat hind limb. Further investigation of strategies to reduce this signal interference, including electrode choice or configuration, use of bioelectrical insulators, and filtering methods, is warranted to promote high-fidelity signal acquisition for prosthetic control.

Growth of primary motor neurons on horizontally aligned carbon nanotube thin films and striped patterns.

OBJECTIVE: Carbon nanotubes (CNTs) are attractive for use in peripheral nerve interfaces because of their unique combination of strength, flexibility, electrical conductivity and nanoscale surface texture. Here we investigated the growth of motor neurons on thin films of horizontally aligned CNTs (HACNTs). APPROACH: We cultured primary embryonic rat motor neurons on HACNTs and performed statistical analysis of the length and orientation of neurites. We next presented motor neurons with substrates of alternating stripes of HACNTs and SiO2. MAIN RESULTS: The neurons survived on HACNT substrates for up to eight days, which was the full duration of our experiments. Statistical analysis of the length and orientation of neurites indicated that the longest neurites on HACNTs tended to align with the CNT direction, although the average neurite length was similar between HACNTs and glass control substrates. We observed that when motor neurons were presented with alternating stripes of HACNTs and SiO2, the proportion of neurons on HACNTs increases over time, suggesting that neurons selectively migrate toward and adhere to the HACNT surface. SIGNIFICANCE: The behavior of motor neurons on CNTs has not been previously investigated, and we show that aligned CNTs could provide a viable interface material to motor neurons. Combined with emerging techniques to build complex hierarchical structures of CNTs, our results suggest that organised CNTs could be incorporated into nerve grafts that use physical and electrical cues to guide regenerating axons.

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.

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; Young's 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.

Critical variables in the alignment of electrospun PLLA nanofibers.

Electrospun polymer nanofibers show promise as components of scaffolds for tissue engineering because of their ability to orient regenerating cells. Our research focuses on aligned electrospun fiber scaffolds for nerve regeneration. Critical to this are highly aligned fibers, which are frequently difficult to manufacture reproducibly. Here we show that three variables: the distance between the spinneret tip and collector, the addition of DMF to the solvent, and placement of an aluminum sheet on the spinneret together greatly improve the alignment of electrospun poly-L-lactide (PLLA) nanofibers. We identified the most important variable as tip-to-collector distance. Nanofiber alignment was maximal at 30cm compared to shorter distances. DMF:chloroform (1:9) improved nanofiber uniformity and was integral to maintaining a uniform stream over the 30cm tip-to-collector distance. Other ratios caused splattering of the solution or flattening or beading of the fibers and non-uniform fiber diameter. The aluminum sheet helped to stabilize the electric field and improve fiber alignment provided that it was placed at 1cm behind the tip, while other distances destabilized the stream and worsened alignment. This study demonstrates that control of these variables produces dramatic improvement in reproducibly obtaining high alignment and uniform morphology of electrospun PLLA nanofibers.

Erythropoietin-loaded oligochitosan nanoparticles for treatment of periventricular leukomalacia.

In this study, a single intraperitoneal injection of erythropoietin (EPO) loaded oligochitosan nanoparticles (epo-NPs) (average diameter 266 nm) was investigated as a treatment for periventricular leukomalacia (PVL). Nanoparticles were fabricated using a gelation technology process. PVL rats models were prepared to examine the therapeutic efficacy of epo-NPs and analyze the mechanism by which epo-NPs protect white matter. The metabolization of epo-NPs in the liver was also investigated. The pathology and behavioral data show that this single injection of a low quantity of epo-NPs had an excellent therapeutic effect on the rat model of PVL. The EPO release curve in phosphate buffered saline solution was a good fit with the zero-order kinetics distribution and was maintained at around 25% in 48 h. In vivo experiments demonstrated that 50 IU/kg epo-NPs had the same effect as a 5000 IU/kg direct injection of free EPO. Nanoparticles prolonged the time course of EPO metabolization in the liver and the stable release of EPO from the nanoparticles kept the plasma concentration of EPO at around 100 IU/ml during the 8-12h post-injection. Therefore, we suggest that oligochitosan based nanoparticles are an effective vehicle for drug delivery.

A novel fluffy conductive polypyrrole nano-layer coated PLLA fibrous scaffold for nerve tissue engineering.

In this study, a novel three-dimensional fluffy PPy conductive fibrous scaffold (3D-cFSs) was fabricated by electrospinning technique combined with situ surface polymerization. Chemical compositions, morphology were characterized by fourier transform infrared (FTIR) and scanning electron microscopy (SEM). The results showed that the average diameter of PPy coated PLLA fibers in the 3D-cFSs was 2.086 microm, the thickness of PPy nano-layer was -45 nm. These PPy coated PLLA fibers were in discrete state, the size of interconnected pores in the 3D-cFSs was from 50 microm to 100 microm, this unique structure ensured that cells can entry into internal of 3D-cFSs smoothly without any other extra help to achieve three-dimensional cell culture (3D-culture). Rat pheochromocytoma 12 (PC12) cells (as model cell) were cultured in the 3D-cFSs to evaluate its potential application for nerve tissue engineering. The interaction between cell and scaffold was test by detecting the cell proliferation, viability, and morphology. After 3 days culture, the number of PC12 in 3D-cFSs were much higher than that on the conductive fibrous meshes (cFMs) and well developed cell-fibers constructs were observed from fluorescence image and SEM of PC12 in the central of 3D-cFSs. These results showed that the 3D-cFSs provided cell 3D-culture, and improved cell growth. Therefore, we suggest that the 3D-cFSs maybe a suitable scaffold for the nerve tissue engineering as cells substrate to apply electrical stimulation.

Electrospun fibers and tissue engineering.

Electrospinning is an exciting technique attracting more and more attention as a potential solution to the current challenges in the field of tissue engineering. This technique can be used to produce fibrous scaffolds with excellent biocompatibility and biodegradability, as well as suitable micro-/nanostructure to induce desired cellular activities and to guide tissue regeneration. In order to develop electrospun fibrous scaffolds for these applications, different biocompatible materials including natural polymers, synthetic polymers and inorganic substances and preparations have been used to fabricate electrospun fibers with different structures and morphologies. This review briefly describes the development of the technique, focusing on several typically electrospun materials, surface modification of electrospun fibers, and current applications in tissue engineering.

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.

Stages of neuronal morphological development in vitro--an automated assay.

Following plating in vitro, neurons pass through a series of morphological stages as they adhere and mature. These morphological stage transitions can be monitored as a function of time to evaluate the relative health and development of neuronal cultures under different conditions. While morphological development is usually quite obvious to the experienced eye, it can often be difficult to quantify in a meaningful way. Morphology quantification typically relies on manual image measurement and can therefore be tedious, time consuming and prone to human error. Here we report the successful development of an automated process using the commercially available image analysis program MetaMorph(®) to analyze the morphology and quantify the growth of embryonic spinal motor neurons in vitro. Our process relied on the Neurite Outgrowth and Cell Scoring modules included in MetaMorph(®) and on analyzing the exported data with an algorithm written in MATLAB(®). We first adopted a series of stages of motor neuron development in vitro. Neurons were classified into these stages directly from the available output of MetaMorph(®) using the algorithm written in MATLAB(®). We validated the results of the automated analysis against a manual analysis of the same images and found no statistically significant difference between the two methods. When properly configured, automated image analysis with MetaMorph(®) is a rapid and reliable alternative to manual measurement and has the potential to accelerate the research process.

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.

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.

Electrospun chitosan nanofibers for hepatocyte culture.

In this study, we developed a method to obtain high surface area nanofiber meshes composed of chitosan of a number of molecular weights. This method required decreasing the viscosity and surface tension of the chitosan solution as well as optimization of the electrospinning parameters such as applied voltage and environmental humidity. These chitosan nanofiber meshes were developed as a culture substrate for hepatocytes. The fibers exhibited a uniform diameter distribution (average diameter: 112 nm) and FTIR results indicate that the chemical structure of chitosan is stable during the electrospinning process. The attachment, morphology and activity of hepatocytes cultured on the chitosan nanofiber meshes were tested. The results showed that the chitosan nanofibers are biocompatible with hepatocytes and that these chitosan nanofiber meshes could be useful tissue culture substrates for various applications, including bioartificial liver-assist devices and tissue engineering for liver regeneration.