Biocompatible SU-8-Based Microprobes for Recording Neural Spike Signals From Regenerated Peripheral Nerve Fibers

A biocompatible neural microprobe constructed using well-established SU-8 microfabrication techniques is described that was designed to record fiber spike signals from regenerated axons within peripheral nerves. These microprobes features bipolar longitudinal gold electrodes recessed below the surface within “grooves” designed to guide the growth of regenerating axons along the length of the grooves and limit the number of fibers that come in contact with the longitudinal electrodes. In addition, screening microprobe toxicity using cultures of human skin fibroblasts, the biocompatibility of these SU-8 microprobes for neural interface applications, in particular, was specifically verified using primary cultures of two sensitive cell types found in peripheral nerves: purified Schwann cells and explanted dorsal root ganglion (DRG) neurons and their fibers. The SU-8 microprobes were surgically implanted into transected rat Sciatic nerves within a unique peripheral nerve regeneration tube. Long-term fiber spike signals were recorded with these SU-8 microprobes in 13 chronically implanted rats for periods from 4 to 51 weeks without any signs of tissue damage or inflammatory reaction.      

Covalent binding of placental derived proteins to silk fibroin improves schwann cell adhesion and proliferation

Schwann cells play a key role in peripheral nerve regeneration. Failure in sufficient formation of Büngner bands due to impaired Schwann cell proliferation has significant effects on the functional outcome after regeneration. Therefore, the growth substrate for Schwann cells should be considered with highest priority in any peripheral nerve tissue engineering approach. Due to its excellent biocompatibility silk fibroin has most recently attracted considerable interest as a biomaterial for use as conduit material in peripheral nerve regeneration. In this study we established a protocol to covalently bind collagen and laminin, which have been isolated from human placenta, to silk fibroin utilizing carbodiimide chemistry. Altered adhesion, viability and proliferation of Schwann cells were evaluated. A cell adhesion assay revealed that the functionalization with both, laminin or collagen, significantly improved Schwann cell adhesion to silk fibroin. Moreover laminin drastically accelerated adhesion. Schwann cell proliferation and viability assessed with BrdU and MTT assay, respectively, were significantly increased in the laminin-functionalized groups. The results suggest beneficial effects of laminin on both, cell adhesion as well as proliferative behaviour of Schwann cells. To conclude, the covalent tailoring of silk fibroin drastically enhances its properties as a cell substratum for Schwann cells, which might help to overcome current hurdles bridging long distance gaps in peripheral nerve injuries with the use of silk-based nerve guidance conduits.     

Regeneration of peripheral nerves by bioabsorbable polymer tubes with fibrin gel

We developed two types of nerve conduits, straight tubes, and bellows tubes, for peripheral nerve regeneration with bioabsorbable polymer membranes. Mechanical properties of these straight and bellows tubes were analyzed. 30 straight tubes and 30 bellows tubes were implanted to a nerve defect made in a rat sciatic nerve and the nerve regeneration in the tube was investigated. A half of these tubes were utilized alone and the others were filled with fibrin gel made from coagulated plasma and implanted. Bellows tubes were superior in mechanical characteristics to straight tubes and the inner cavities of the bellows tubes were suitably maintained after implantation. At 4 weeks after operations, remyelinations were observed in the regenerated tissue at the location of the middle parts of the tubes filled with fibrin gel whereas no remyelinations in the tubes without fibrin gel. The use of fibrin gel as filling materials within the tubes significantly enhanced the nerve regeneration. The fibrin gel might be soft nanomaterials significantly enhance the regeneration of the peripheral nerves. We concluded that the developed bioabsorbable bellows tubes filled with fibrin gel were effective for peripheral nerve regeneration.     

Novel crosslinked alginate/hyaluronic acid hydrogels for nerve tissue engineering

Artificial tissue engineering scaffolds can potentially provide support and guidance for the regrowth of severed axons following nerve injury. In this study, a hybrid biomaterial composed of alginate and hyaluronic acid (HA) was synthesized and characterized in terms of its suitability for covalent modification, biocompatibility for living Schwann cells and feasibility to construct three dimensional (3D) scaffolds. Carbodiimide mediated amide formation for the purpose of covalent crosslinking of the HA was carried out in the presence of calcium ions that ionically crosslink alginate. Amide formation was found to be dependent on the concentrations of carbodiimide and calcium chloride. The double-crosslinked composite hydrogels display biocompatibility that is comparable to simple HA hydrogels, allowing for Schwann cell survival and growth. No significant difference was found between composite hydrogels made from different ratios of alginate and HA. A 3D BioPlotter^TM rapid prototyping system was used to fabricate 3D scaffolds. The result indicated that combining HA with alginate facilitated the fabrication process and that 3D scaffolds with porous inner structure can be fabricated from the composite hydrogels, but not from HA alone. This information provides a basis for continuing in vitro and in vivo tests of the suitability of alginate/HA hydrogel as a biomaterial to create living cell scaffolds to support nerve regeneration.     

Synthetic collagen fibers coated with a synthetic peptide containing the YIGSR sequence of laminin to promote peripheral nerve regeneration in vivo

The usefulness of collagen fibers and the YIGSR sequence (Tyr-lle-Gly-Ser-Arg) of laminin for nerve regeneration were examined in vivo. Type I collagen gel (G-group), Type I collagen fibers (F-group), Type I collagen fibers coated with laminin (L-group) or the YIGSR sequence (Y-group) were packed into silicone tubes, 15 mm long, and transplanted to the sciatic nerves of Wistar rats. Empty silicone tubes were used as the control. The animals were sacrificed 8 weeks after transplantation. Bridging of the nerve was confirmed in the F-(7/12), Y-(7/10) and L-group (6/10), but no bridging was observed in any of the animals of the G- and control group. Nerve regeneration among the space of collagen fibers was observed, and it was suggested that fibroblasts infiltrated the gap in the substance of the degenerated collagen fibers were followed by Schwann cells on the basis of immunocytochemistry. The number of myelinated axons per regenerated tissue in the tube (density), and total area of myelinated axons per measured regenerated tissue in the tube (% axon area) in each the L- and Y-group were significantly higher than that in the F-group (P < 0.05). These results suggest the possibility of obtaining adequate nerve regeneration with new artificial materials only. © 1999 Kluwer Academic Publishers     

Schwann cell-seeded scaffold with longitudinally oriented micro-channels for reconstruction of sciatic nerve in rats

To provide a more permissive environment for axonal regeneration, Schwann cells (SCs) were introduced into a collagen-chitosan scaffold with longitudinally oriented micro-channels (L-CCH). The SC-seeded scaffold was then used for reconstruction of a 15-mm-long sciatic nerve defect in rats. The axonal regeneration and functional recovery were examined by a combination of walking track analysis, electrophysiological assessment, Fluoro-Gold retrograde tracing, as well as morphometric analyses to both regenerated axons and target muscles. The findings showed that SCs adhered and migrated into the L-CCH scaffold and displayed a longitudinal arrangement in vitro. Axonal regeneration as well as functional recovery was in the similar range between SCs-seeded scaffold and autograft groups, which were superior to those in L-CCH scaffold alone group. These indicate that the SCs-seeded L-CCH scaffold, which resembles the microstructure as well as the permissive environment of native peripheral nerves, holds great promise in nerve regeneration therapies.     

Novel use of biodegradable casein conduits for guided peripheral nerve regeneration

Recent advances in nerve repair technology have focused on finding more biocompatible, non-toxic materials to imitate natural peripheral nerve components. In this study, casein protein cross-linked with naturally occurring genipin (genipin-cross-linked casein (GCC)) was used for the first time to make a biodegradable conduit for peripheral nerve repair. The GCC conduit was dark blue in appearance with a concentric and round lumen. Water uptake, contact angle and mechanical tests indicated that the conduit had a high stability in water and did not collapse and cramped with a sufficiently high level of mechanical properties. Cytotoxic testing and terminal deoxynucleotidyl transferase dUTP nick-end labelling assay showed that the GCC was non-toxic and non-apoptotic, which could maintain the survival and outgrowth of Schwann cells. Non-invasive real-time nuclear factor-κB bioluminescence imaging accompanied by histochemical assessment showed that the GCC was highly biocompatible after subcutaneous implantation in transgenic mice. Effectiveness of the GCC conduit as a guidance channel was examined as it was used to repair a 10 mm gap in the rat sciatic nerve. Electrophysiology, labelling of calcitonin gene-related peptide in the lumbar spinal cord, and histology analysis all showed a rapid morphological and functional recovery for the disrupted nerves. Therefore, we conclude that the GCC can offer great nerve regeneration characteristics and can be a promising material for the successful repair of peripheral nerve defects.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

Bridging defects in nerve continuity: influence of variations in synthetic fiber composition

Synthetic filaments introduced into a silicone tube may help to enhance axonal growth over extended defects in nerve continuity [1]. Here we test the influence of number (0, 3, 7 or 15), size (diameter 150 or 250 m) and material of filaments (polyamide or catgut) enclosed in such tubes (inner diameter 1.98 mm) on axonal growth across a 10 mm defect in rat sciatic nerve. The morphology of the tube content was analyzed four weeks post-surgery. The area of the formed tissue matrix inside the tube showed no difference between the groups. Myelinated axons were observed in the formed tissue matrix inbetween and peripheral to the filaments, however, separated from the filaments by concentric cell layers. The number of myelinated axons was less in the tubes with 15 filaments, most pronounced when catgut filaments were used. In most cases, except in tubes with 15 catgut filaments, fibers had grown into the distal nerve segment (pinch reflex test/light microscopy). We conclude that an intrinsic framework consisting of a limited number of synthetic filaments inside an extrinsic framework (silicone tube) does not disturb nerve regeneration. The formed tissue matrix was neither influenced by the presence or the numbers (if less than or equal to seven filaments), type of filaments nor the size of the filaments indicating the importance of the inserted nerve segments. © 1999 Kluwer Academic Publishers     

Synthesis,characterization r）oly [LA-co-（GIc-alt-Lys）] for and biological evaluation of nerve regeneration scaffold

A novel nerve repairing material poly [LA-co.（GIc-alt-Lys）] （PLGL） was synthesized. The viability and growth of Schwann cells （SCs） co-cultured With poly （D, L- lactic acid） （PDLLA） films （control group） and PLGL films were evaluated by MTT assay and SEM observation. Then, contact angle measurement, histological assessment and enzyme-linked immunosorbent assay （ELISA） testing on inflammatory-related cyto- kines such as IL-10 and TGF-β1 were performed. The results showed that, compared with PDLLA, PLGL films possesses better hydrophilicity, biocompatibility, degradation property and less inflammatory reaction. The present study indicated that PLGL scaffolds would meet the requirements of artificial nerve scaffold and have a potential application in the fields of nerve regeneration.     

Impact of Scaffold Micro and Macro Architecture on Schwann Cell Proliferation under Dynamic Conditions in a Rotating Wall Vessel Bioreactor

Over the last decade tissue engineering has emerged as a powerful alternative to regenerate lost tissues owing to trauma or tumor. Evidence shows that Schwann cell containing scaffolds have improved performance in vivo as compared to scaffolds that depend on cellularization post implantation. However, owing to limited supply of cells from the patients themselves, several approaches have been taken to enhance cell proliferation rates to produce complete and uniform cellularization of scaffolds. The most common approach is the application of a bioreactor to enhance cell proliferation rate and therefore reduce the time needed to obtain sufficiently significant number of glial cells, prior to implantation.In this study, we show the application of a rotating wall bioreactor system for studying Schwann cell proliferation on nanofibrous spiral shaped scaffolds, prepared by solvent casting and salt leaching techniques. The scaffolds were fabricated from polycaprolactone (PCL), which has ideal mechanical properties and upon degradation does not produce acidic byproducts. The spiral scaffolds were coated with aligned or random nanofibers, produced by electrospinning, to provide a substrate that mimics the native extracellular matrix and the essential contact guidance cues.At the 4 day time point, an enhanced rate of cell proliferation was observed on the open structured nanofibrous spiral scaffolds in a rotating wall bioreactor, as compared to static culture conditions. However, the cell proliferation rate on the other contemporary scaffolds architectures such as the tubular and cylindrical scaffolds show reduced cell proliferation in the bioreactor as compared to static conditions, at the same time point. Moreover, the rotating wall bioreactor does not alter the orientation or the phenotype of the Schwann cells on the aligned nanofiber containing scaffolds, wherein, the cells remain aligned along the length of the scaffolds. Therefore, these open structured spiral scaffolds pre-cultured with Schwann cells, in bioreactors could potentially shorten the time needed for grafts for peripheral nerve regeneration.     

Microhardness evaluations of the bone growing into porous implants

Small cylinders of a new composite porous material consisting of a dense alumina core coated with two layers of beads of the same material, bonded to each other and to the underlying surface by a high-temperature melting glass have been implanted in the proximal femurs of rabbits. The explants were carried out 1, 4, 6, 8, and 18 weeks after surgery. The bone fragment containing the implant was embedded in methyl methacrylate without performing decalcification, and morphological observations were carried out. These showed that four weeks after surgery it is already possible to observe the development of bone spicules in the implant porosities. Along with these studies, microhardness measurements were carried out by using a microhardness tester connected to an image analyser. The mineralized tissue in close contact with the implant showed, one month after surgery, a compression strength similar to that of healthy bone.     

The synthesis and characterization of a novel biodegradable and electroactive polyphosphazene for nerve regeneration

Conductive polymers have been of great interest to the biopharmaceutical industry because of their cell adhesion and proliferation. In this paper, a novel electrically-conductive and biodegradable polyphosphazene polymer containing parent aniline pentamer (PAP) and glycine ethyl ester (GEE) as side chains was synthesized through a nucleophilic substitution reaction for its potential application in nerve regeneration. The electrical conductivity of the polymer was ~ 2 × 10^? 5 S/cm in the semiconducting region upon preliminarily protonic-doped experiment. Degradation studies carried out in phosphate-buffered saline at 37 °C showed a mass loss of ~ 50% after 70 days. In vitro cytotoxicity to the RSC96 Schwann cells was evaluated using the cell viability assay. The polymer exhibited no cytotoxicity, indicating that such a polyphosphazene polymer has potential as scaffold material in tissue engineering for peripheral nerve regeneration or other biomedical devices that require electroactivity.     

Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels

Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.     

Rapid continuous 3D printing of customizable peripheral nerve guidance conduits

Engineered nerve guidance conduits (NGCs) have been demonstrated for repairing peripheral nerve injuries. However, there remains a need for an advanced biofabrication system to build NGCs with complex architectures, tunable material properties, and customizable geometrical control. Here, a rapid continuous 3D-printing platform was developed to print customizable NGCs with unprecedented resolution, speed, flexibility, and scalability. A variety of NGC designs varying in complexity and size were created including a life-size biomimetic branched human facial NGC. In vivo implantation of NGCs with microchannels into complete sciatic nerve transections of mouse models demonstrated the effective directional guidance of regenerating sciatic nerves via branching into the microchannels and extending toward the distal end of the injury site. Histological staining and immunostaining further confirmed the progressive directional nerve regeneration and branching behavior across the entire NGC length. Observational and functional tests, including the von Frey threshold test and thermal test, showed promising recovery of motor function and sensation in the ipsilateral limbs grafted with the 3D-printed NGCs.     

Spatiotemporally controlled and multifactor involved assay of neuronal compartment regeneration after chemical injury in an integrated microfluidics

Studies on the degeneration and regeneration of neurons as individual compartments of axons or somata can provide critical information for the clinical therapy of nervous system diseases. A controllable in vitro platform for multiple purposes is key to such studies. In the present study, we describe an integrated microfluidic device designed for achieving localized stimulation to neuronal axons or somata. We observed neuronal compartment degeneration after localized chemical stimulation and regeneration under the accessorial function of an interesting compound treatment or coculture with desired cells in controllable chambers. In a spatiotemporally controlled manner, this device was used to investigate hippocampal neuronal soma and axon degeneration after acrylamide stimulation, as well as subsequent regeneration after treatment with the monosialoganglioside GM1 or with cocultured glial cells (astrocytes or Schwann cells). To gain insight into the molecular mechanisms that mediate neuronal injury and regeneration, as well as to investigate whether acrylamide stimulation to neurons induces changes in Ca(2+) concentrations, the related neuronal genes and real-time Ca(2+) signal in neurons were also analyzed. The results showed that neuronal axons were more resistant to acrylamide injury than neuronal somata. Under localized stimulation, axons had self-destruct programs different from somata, and somatic injury caused the secondary response of axon collapse. This study provides a foundation for future in-depth analyses of spatiotemporally controlled and multifactor neuronal compartment regeneration after various injuries. The microfluidic device is also useful in evaluating potential therapeutic strategies to treat chemical injuries involving the central nervous system.     

Aligned collagen–GAG matrix as a 3D substrate for Schwann cell migration and dendrimer-based gene delivery

The development of artificial off-the-shelf conduits that facilitate effective nerve regeneration and recovery after repair of traumatic nerve injury gaps is of fundamental importance. Collagen–glycosaminoglycan (GAG) matrix mimicking Schwann cell (SC) basal lamina has been proposed as a suitable and biologically rational substrate for nerve regeneration. In the present study, we have focused on the permissiveness of this matrix type for SC migration and repopulation, as these events play an essential role in nerve remodeling. We have also demonstrated that SCs cultured within collagen–GAG matrix are compatible with non-viral dendrimer-based gene delivery, that may allow conditioning of matrix-embedded cells for future gene therapy applications.     

Fabrication and characterization of <Emphasis Type="Italic">Antheraea pernyi</Emphasis> silk fibroin-blended P(LLA-CL) nanofibrous scaffolds for peripheral nerve tissue engineering

Electrospun nanofibers have gained widespreading interest for tissue engineering application. In the present study, ApF/P(LLA-CL) nanofibrous scaffolds were fabricated via electrospinning. The feasibility of the material as tissue engineering nerve scaffold was investigated in vitro. The average diameter increased with decreasing the blend ratio of ApF to P(LLA-CL). Characterization of ¹³C NMR and FTIR clarified that there is no obvious chemical bond reaction between ApF and P(LLA-CL). The tensile strength and elongation at break increased with the content increase of P(LLA-CL). The surface hydrophilic property of nanofibrous scaffolds enhanced with the increased content of ApF. Cell viability studies with Schwann cells demonstrated that ApF/P(LLA-CL) blended nanofibrous scaffolds significantly promoted cell growth as compare to P(LLA-CL), especially when the weight ratio of ApF to P(LLA-CL) was 25:75. The present work provides a basis for further studies of this novel nanofibrous material (ApF/P(LLA-CL)) in peripheral nerve tissue repair or regeneration.     

Preparation and characterisation of zein/polyphenol nanofibres for nerve tissue regeneration

Bridging strategies are required to repair peripheral nerve injuries that result in gaps >5–8 mm. Limitations such as donor‐site morbidity and size mismatches with receptor sites for autografts, together with immunological problems associated with allografts and xenografts, have created an increased interest in the field of manufactured nerve guide conduits. In this study, zein, a plant protein‐based polymer, was electrospun to prepare nanofibrous mats. An important challenge with zein mats is the rapid change from fibre to film under aqueous conditions. Tannic acid (TA), which is a polyphenol, was selected to prepare a blend of zein/TA with different weight ratios to investigate its effect on the wetting resistance of nanofibres. The electrospun mats were characterised and evaluated by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM). Also, degradation and mechanical properties of the mats were studied. Results showed that TA had a significant effect on the resistance to film formation in nanofibres. Moreover, the degradation and elongation at break of mats were increased with increase in TA concentration. For the investigation of the peripheral nerve regeneration potential, Schwann cells were selected for cytotoxicity evaluation by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide assay and cell morphology by SEM. Schwann cells had good biocompatibility with zein/TA blends (%) of 90/10 and 80/20.Inspec keywords: polymer fibres, biomedical materials, electrospinning, cellular biophysics, tissue engineering, proteins, molecular biophysics, neurophysiology, nanofibres, injuries, nanomedicine, toxicology, scanning electron microscopy, nanofabrication, polymer blends, polymer films, wetting, Fourier transform infrared spectra, elongationOther keywords: SEM, Schwann cells, nerve tissue regeneration, peripheral nerve injuries, donor‐site morbidity, size mismatches, receptor sites, immunological problems, allografts, xenografts, manufactured nerve guide conduits, plant protein‐based polymer, nanofibrous mats, zein mats, aqueous conditions, tannic acid, wetting resistance, electrospun mats, scanning electron microscopy, film formation, TA concentration, peripheral nerve regeneration potential, cell morphology, weight ratios, zein‐polyphenol nanofibres, electrospinning, zein‐TA blends, Fourier transform infrared spectroscopy, mechanical properties, elongation‐at‐break, cytotoxicity evaluation, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide assay, biocompatibility     

The effects of implanted ionomeric and acrylic bone cements on peripheral nerve function

The effects of two experimental ionomeric and one commercial acrylic bone cement and set ionomeric microimplant bone substitute (lonogran®) on peripheral nerve conduction, 1 and 3 weeks after implantation, have been compared. In 44 experiments the rat saphenous nerve was exposed midway between the ankle and thigh and bone cement placed into a pocket created in the connective tissue adjacent to the nerve. In terminal experiments, 1 and 3 weeks later, stimulating electrodes were placed on the saphenous nerve at the ankle, and the amplitude and conduction velocity of the compound action potential (CAP) evoked was recorded through another pair of electrodes positioned on the nerve proximal to the implant, in the thigh. One week after placing an ionomeric bone cement (HVA or V-4), no neural activity could be recorded. Three weeks, after HVA implantation apparently normal CAPs were recorded indicating a recovery from a temporary nerve conduction block, but 3 weeks after V-4 implantation only small CAPs were recorded and these could be attributed to axonal regeneration. After implantation of acrylic bone cement, small CAPs were recorded after 1 week, and within 3 weeks nerve conduction appeared to have completely recovered. Three weeks after placing set ionomeric microimplant particles the amplitude and conduction velocity of the CAP was similar to the controls.