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      Surface Microstructures on Planar Substrates and Textile Fibers Guide Neurite Outgrowth: A Scaffold Solution to Push Limits of Critical Nerve Defect Regeneration?

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          Abstract

          The treatment of critical size peripheral nerve defects represents one of the most serious problems in neurosurgery. If the gap size exceeds a certain limit, healing can't be achieved. Connection mismatching may further reduce the clinical success. The present study investigates how far specific surface structures support neurite outgrowth and by that may represent one possibility to push distance limits that can be bridged. For this purpose, growth cone displacement of fluorescent embryonic chicken spinal cord neurons was monitored using time-lapse video. In a first series of experiments, parallel patterns of polyimide ridges of different geometry were created on planar silicon oxide surfaces. These channel-like structures were evaluated with and without amorphous hydrogenated carbon (a-C:H) coating. In a next step, structured and unstructured textile fibers were investigated. All planar surface materials (polyimide, silicon oxide and a-C:H) proved to be biocompatible, i.e. had no adverse effect on nerve cultures and supported neurite outgrowth. Mean growth cone migration velocity measured on 5 minute base was marginally affected by surface structuring. However, surface structure variability, i.e. ridge height, width and inter-ridge spacing, significantly enhanced the resulting net velocity by guiding the growth cone movement. Ridge height and inter-ridge distance affected the frequency of neurites crossing over ridges. Of the evaluated dimensions ridge height, width, and inter-ridge distance of respectively 3, 10, and 10 µm maximally supported net axon growth. Comparable artificial grooves, fabricated onto the surface of PET fibers by using an excimer laser, showed similar positive effects. Our data may help to further optimize surface characteristics of artificial nerve conduits and bioelectronic interfaces.

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          Most cited references43

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          The establishment of polarity by hippocampal neurons in culture.

          By the end of the first week in culture, hippocampal neurons have established a single axon and several dendrites. These 2 classes of processes differ in their morphology, in their molecular composition, and in their synaptic polarity (Bartlett and Banker, 1984a, b; Caceres et al., 1984). We examined the events during the first week in culture that lead to the establishment of this characteristic form. Hippocampal cells were obtained from 18 d fetal rats, plated onto polylysine-treated coverslips, and maintained in a serum-free medium. The development of individual cells was followed by sequential photography at daily intervals until both axons and dendrites had been established; identification of the processes was confirmed by immunostaining for MAP2, a dendritic marker. Time-lapse video recording was used to follow the early stages of process formation. Hippocampal neurons acquired their characteristic form by a stereotyped sequence of developmental events. The cells first established several, apparently identical, short processes. After several hours, one of the short processes began to grow very rapidly; it became the axon. The remaining processes began to elongate a few days later and grew at a much slower rate. They became the cell's dendrites. Neurons that arose following mitosis in culture underwent this same sequence of developmental events. In a few instances, 2 processes from a cell exhibited the rapid growth typical of axons, but only one maintained this growth; the other retracted and became a dendrite. Axons branched primarily by the formation of collaterals, not by bifurcation of growth cones. As judged by light microscopy, processes are not specified as axons or dendrites when they arise. The first manifestation of neuronal polarity is the acquisition of axonal characteristics by one of the initial processes; subsequently the remaining processes become dendrites.
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            Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend.

            Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-epsilon-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.
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              The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps.

              Peripheral nerve regeneration across long nerve gaps is clinically challenging. Autografts, the standard of therapy, are limited by availability and other complications. Here, using rigorous anatomical and functional measures, we report that aligned polymer fiber-based constructs present topographical cues that facilitate the regeneration of peripheral nerves across long nerve gaps. Significantly, aligned but not randomly oriented fibers elicit regeneration, establishing that topographical cues can influence endogenous nerve repair mechanisms in the absence of exogenous growth promoting proteins. Axons regenerated across a 17 mm nerve gap, reinnervated muscles, and reformed neuromuscular junctions. Electrophysiological and behavioral analyses revealed that aligned but not randomly oriented constructs facilitated both sensory and motor nerve regeneration, significantly improved functional outcomes. Additionally, a quantitative comparison of DRG outgrowth in vitro and nerve regeneration in vivo on aligned and randomly oriented fiber films clearly demonstrated the significant role of sub-micron scale topographical cues in stimulating endogenous nerve repair mechanisms.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                12 December 2012
                : 7
                : 12
                : e50714
                Affiliations
                [1 ]MaTisMed, Materials-Biology Interactions Lab, EMPA Materials Science and Technology, St. Gallen, Switzerland
                [2 ]Technische Universität München, Zoology, Freising-Weihenstephan, Germany
                [3 ]National Center for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
                [4 ]Department of Biological Sciences, Wichita State University, Wichita, United States of America
                [5 ]Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory for Microsystems 2, Lausanne, Switzerland
                Rutgers University, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SW TO AB. Performed the experiments: SW UT MW. Analyzed the data: SW TO AB. Contributed reagents/materials/analysis tools: TL LY AP. Wrote the paper: SW AB. Critical reading of the manuscript: TL. Development of methodology to structure fibers: LY AP. Method development to structure plane surfaces: TL.

                Article
                PONE-D-12-20294
                10.1371/journal.pone.0050714
                3520951
                23251379
                037bda2c-b510-4997-9ab2-58b0ee2d2fad
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 July 2012
                : 23 October 2012
                Page count
                Pages: 10
                Funding
                The study was supported by the EMPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Developmental Biology
                Morphogenesis
                Regeneration
                Neuroscience
                Cellular Neuroscience
                Neuronal Morphology
                Developmental Neuroscience
                Axon Guidance
                Materials Science
                Biomaterials
                Material by Structure
                Polymers
                Microstructure

                Uncategorized
                Uncategorized

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