Nerve Growth Factor Gradient Research Articles

Fabrication of 3D Scaffolds Displaying Biochemical Gradients along Longitudinally Oriented Microchannels for Neural Tissue Engineering.

Biochemical and physical guidance cues are both pivotal for axonal guidance and nerve regeneration. However, fabrication of a platform that can integrate biochemical gradients and topographical guidance cues remains challenging, especially in a three-dimensional (3D) scaffold that closely mimics in vivo axonal outgrowth conditions and ready to be used for in vivo nerve repair. In this study, a new method was introduced to construct 3D scaffolds displaying continuous biochemical gradients along longitudinally oriented microchannels by combining the modified 3D printing and directional freezing techniques. Fluorescence analysis and ELISA results demonstrated that a continuous biochemical gradient was formed, and scanning electron microscopy revealed a longitudinally oriented microstructure. Dorsal root ganglia explants seeded on the longitudinal sections of the newly developed scaffold (scaffold with nerve growth factor gradient along oriented microstructure, G-NGF + OS) showed that 81.3 ± 4.5% of neurites oriented within ±10°, 0.3 ± 0.1 of guidance ratio, and 1.5-fold of the average length of neurites on the high-nerve growth factor (NGF) concentration side compared to that on the low-NGF concentration side, which were significantly higher than those in the other groups. In addition, the G-NGF + OS scaffold was used to repair a 15 mm sciatic nerve defect in rats. Immunofluorescence staining, Fluoro-Gold retrograde tracing, and transmission electron microscopy results confirmed that the G-NGF + OS scaffold enhanced nerve regeneration to the distal target and promoted myelination of regenerated axons. More importantly, the sciatic functional index and the von Frey test demonstrated that the G-NGF + OS scaffold accelerated sensory and motor functional recovery. These results provide new insights into the importance of combining topographical guidance cues with bioactive molecule gradient cues for neural tissue engineering. The 3D scaffold displaying biochemical gradients along longitudinally oriented microchannels represents a promising platform for the development of advanced devices for severe nervous system injuries.

Aligned PCL Fiber Conduits Immobilized with Nerve Growth Factor Gradients Enhance and Direct Sciatic Nerve Regeneration

AbstractTopographical guidance and chemotaxis are crucial factors for peripheral nerve regeneration. This study describes the preparation of highly aligned poly(ε‐caprolactone) (PCL) fiber conduits coated with a concentration gradient of nerve growth factor (NGF) (A/G‐PCL) using a newly designed electrospinning receiving device. The A/G‐PCL conduits are confirmed in vitro to enhance and attract the neurite longitudinal growth of dorsal root ganglion (DRG) neurons toward their high‐concentration gradient side. In vivo, the A/G‐PCL conduits are observed to direct a longitudinal stronger attraction of axons and migration of Schwann cells in 15 mm rat sciatic nerve defects. At 12 weeks, rats transplanted with A/G‐PCL conduits show satisfactory morphological and functional improvements in g‐ratio, total number, and area of myelinated nerve fibers as well as the sciatic function index, compound muscle action potentials, and muscle wet weight ratio as compared to aligned PCL fibers conduits with uniform NGF (A/U‐PCL). The performance of A/G‐PCL is similar to that of autografts. Moreover, mRNA‐seq and RT‐PCR results reveal that Rap1, MAPK, and cell adhesion molecules signaling pathways are closely associated with axon chemotactic response and attraction. Altogether, by combining structural guidance with axon chemotaxis, the NGF‐gradient/aligned PCL fiber conduits represent a promising approach for peripheral nerve defect repair.

Chemotactic responses of growing neurites to precisely controlled gradients of nerve growth factor

Chemotaxis plays a key role in many biological systems. In particular in the context of the developing nervous system, growing neurites can respond in vitro to shallow gradients of chemotropic molecules such as nerve growth factor (NGF). However, in such studies the gradient parameters are often not well controlled. Here we present a dataset of ~3500 images of early postnatal rat dorsal root ganglion (DRG) explants growing in 40 different precisely controlled combinations of absolute concentration and gradient steepness of NGF. Each image has been segmented into neurite and explant-body regions. We provide computer code for exploration and quantification of the data, including a Fourier analysis of the outer contour of neurite growth, which allows quantities such as outgrowth and guidance as a function of concentration and gradient steepness to be easily extracted. This is the most comprehensive quantitative dataset of chemotactic responses yet available for any biological system, which we hope will be useful for exploring the biological mechanisms governing chemotaxis.

Multi-tubule conduit-filler constructs loaded with gradient-distributed growth factors for neural tissue engineering applications.

Chitosan/silk fibroin/glycerophosphate gels were loaded with nerve growth factor (NGF) and further processed into multi-tubule fillers. NGF was loaded into the fillers in such a way so that a NGF gradient was established longitudinally along the filler length. A type of poly(lactide-co-glycolide)(PLGA)/chitosan(CH) porous conduit was fabricated using a pre-crosslinking method. The filler was fully filled into the lumen of conduits to build multi-tubule conduit-filler constructs that are intended for long-gap peripheral nerve repair. In vitro degradation in a lysozyme-contained medium revealed that constructs had degradation-tolerant features and the optimized multi-tubule filler was capable of maintaining its multi-tubules unblocked for around 10-week. After being degraded for various periods up to 8 weeks, the optimal conduit-filler constructs showed confirmative ability to retain their compressive load, deformation recovery and tensile strength at about 80N/m, 75% and 15N/cm2 in wet state, respectively. The constructs were able to administer NGF release and to maintain persistent NGF gradients longitudinally distributed inside the PLGA/CH conduit for about 6 weeks or even longer. The PC12 cell neurite extension assay confirmed that the presently developed multi-tubule conduit-filler constructs were reliable for effectively preserving the bioactivity of released NGF.

Establishment of nerve growth factor gradients on aligned chitosan-polylactide /alginate fibers for neural tissue engineering applications

Nerve conduits containing aligned fibrous fillers with gradiently distributed signal molecules are essential for long-gap nerve repair. This study was to develop an approach for establishing nerve growth factor (NGF) gradients onto the aligned chitosan-polylactide (CH-PLA) fibers. CH-PLA containing 37wt% of PLA was spun into fibers using a wet-spinning technique. CH-PLA fibers showed much higher wet-state tensile strength, enhanced degradation tolerance and significantly lower swelling degree in comparison to chitosan fibers. The CH-PLA fibers with diameters from 40 to 60μm were selected and segmentally coated in bundles using NGF-contained alginate solutions to establish NGF gradients lengthwise along fibers. The diameter of resulting NGF-loaded CH-PLA/alginate fibers was well controlled within a range between 60 and 120μm. Calcium ion crosslinked alginate coating layers on fibers showed abilities to administer the sustainable NGF release in a gradient distribution manner for at least 5 weeks. NGF-induced neurite outgrowth of PC12 cells confirmed that bioactivity of NGF released from fibers was well retained.

Enhanced peripheral nerve regeneration through asymmetrically porous nerve guide conduit with nerve growth factor gradient.

In this study, we fabricated a nerve guide conduit (NGC) with nerve growth factor (NGF) gradient along the longitudinal direction by rolling a porous polycaprolactone membrane with NGF concentration gradient. The NGF immobilized on the membrane was continuously released for up to 35 days, and the released amount of the NGF from the membrane gradually increased from the proximal to distal NGF ends, which may allow a neurotrophic factor gradient in the tubular NGC for a sufficient period. From the in vitro cell culture experiment, it was observed that the PC12 cells sense the NGF concentration gradient on the membrane for the cell proliferation and differentiation. From the in vivo animal experiment using a long gap (20 mm) sciatic nerve defect model of rats, the NGC with NGF concentration gradient allowed more rapid nerve regeneration through the NGC than the NGC itself and NGC immobilized with uniformly distributed NGF. The NGC with NGF concentration gradient seems to be a promising strategy for the peripheral nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 52-64, 2018.

Sensitivity and Robustness in an Axon Guidance Signaling System

Correct functioning of the brain requires the orderly wiring of billions of cells during development. A major mechanism that mediates this is the guidance of axons to their targets by extracellular chemical gradients. Although well studied, the computational rules by which even simple guidance decisions are made are not yet understood at a biophysical level. The task of a growing axon to estimate a direction signal is immense, as unavoidable biological noise corrupts the measurement at all levels of processing. Here, we show how the modulation of growth by a balance of positive and negative feedback can explain the remarkable chemotactic sensitivity observed in vitro. We perform a detailed analysis of the experimental data of ref. [1] which characterizes the neurite growth of ∼2500 rat dorsal root ganglia explants in very shallow gradients of nerve growth factor. Constrained by these data, we construct a model chemical signaling system for growth and guidance in the developing brain. In the model, amplification of the gradient signal occurs via paracrine signalling between cell bodies within the ganglion, while robustness is conferred by the dynamics of receptor trafficking. The model gives a unified and quantitative account of experimentally observed behavior, and yields testable predictions with implications for understanding brain development and repair after injury.[1] Mortimer, D. et al (2009) A Bayesian model predicts the response of axons to molecular gradients. Proc. Natl. Acad. Sci. U.S.A, 106(25), 10296-10301.

Axon Guidance Studies Using a Microfluidics-Based Chemotropic Gradient Generator.

Microfluidics can be used to generate flow-driven gradients of chemotropic guidance cues with precisely controlled steepnesses for indefinite lengths of time. Neuronal cells grown in the presence of these gradients can be studied for their response to the effects exerted by the cues. Here we describe a polydimethylsiloxane (PDMS) microfluidics chamber capable of producing linear gradients of soluble factors, stable for at least 18 h, suitable for axon guidance studies. Using this device we demonstrate turning of superior cervical ganglion axons by gradients of nerve growth factor (NGF). The chamber produces robust gradients, is inexpensive to mass produce, can be mounted on a tissue culture dish or glass coverslip for long term time-lapse microscopy imaging, and is suitable for immunostaining.

Experimental and computational models of neurite extension at a choice point in response to controlled diffusive gradients

Ojective. Axons are guided toward desired targets through a series of choice points that they navigate by sensing cues in the cellular environment. A better understanding of how microenvironmental factors influence neurite growth during development can inform strategies to address nerve injury. Therefore, there is a need for biomimetic models to systematically investigate the influence of guidance cues at such choice points. Approach. We ran an adapted in silico biased turning axon growth model under the influence of nerve growth factor (NGF) and compared the results to corresponding in vitro experiments. We examined if growth simulations were predictive of neurite population behavior at a choice point. We used a biphasic micropatterned hydrogel system consisting of an outer cell restrictive mold that enclosed a bifurcated cell permissive region and placed a well near a bifurcating end to allow proteins to diffuse and form a gradient. Experimental diffusion profiles in these constructs were used to validate a diffusion computational model that utilized experimentally measured diffusion coefficients in hydrogels. The computational diffusion model was then used to establish defined soluble gradients within the permissive region of the hydrogels and maintain the profiles in physiological ranges for an extended period of time. Computational diffusion profiles informed the neurite growth model, which was compared with neurite growth experiments in the bifurcating hydrogel constructs. Main results. Results indicated that when applied to the constrained choice point geometry, the biased turning model predicted experimental behavior closely. Results for both simulated and in vitro neurite growth studies showed a significant chemoattractive response toward the bifurcated end containing an NGF gradient compared to the control, though some neurites were found in the end with no NGF gradient. Significance. The integrated model of neurite growth we describe will allow comparison of experimental studies against growth cone guidance computational models applied to axon pathfinding at choice points.

Coiled polymeric growth factor gradients for multi-luminal neural chemotaxis

In the injured adult nervous system, re-establishment of growth-promoting molecular gradients is known to entice and guide nerve repair. However, incorporation of three-dimensional chemotactic gradients in nerve repair scaffolds, particularly in those with multi-luminal architectures, remains extremely challenging. We developed a method that establishes highly tunable three-dimensional molecular gradients in multi-luminal nerve guides by anchoring growth-factor releasing coiled polymeric fibers onto the walls of collagen-filled hydrogel microchannels. Differential pitch in the coiling of neurotrophin-eluting fibers generated sustained chemotactic gradients that appropriately induced the differentiation of Pheochromocytoma (PC12) cells into neural-like cells along an increasing concentration of nerve growth factor (NGF). Computer modeling estimated the stability of the molecular gradient within the luminal collagen, which we confirmed by observing the significant effects of neurotrophin gradients on axonal growth from dorsal root ganglia (DRG). Neurons growing in microchannels exposed to a NGF gradient showed a 60% increase in axonal length compared to those treated with a linear growth factor concentration. In addition, a two-fold increment in the linearity of axonal growth within the microchannels was observed and confirmed by a significant reduction in the turning angle ratios of individual axons. These data demonstrate the ability of growth factor-loaded polymeric coiled fibers to establish three-dimensional chemotactic gradients to promote and direct nerve regeneration in the nervous system and provides a unique platform for molecularly guided tissue repair.

Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding.

Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding; however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration.

Microfluidic gradients reveal enhanced neurite outgrowth but impaired guidance within 3D matrices with high integrin ligand densities.

The density of integrin-binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell-adhesive ligands on neurite chemoattraction is not well understood. A platform is presented here that combines gradient-generating microfluidic devices with 3D protein-engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia-derived spheroids. Spheroids are encapsulated in elastin-like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade-off between neurite extension and neurite guidance is reminiscent of the well-known trade-off between adhesive forces at the leading and trailing edges of a migrating cell, implying that a similar matrix-mediated balance of forces regulates neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels.

Sensory axon guidance with semaphorin 6A and nerve growth factor in a biomimetic choice point model

The direct effect of guidance cues on developing and regenerating axons in vivo is not fully understood, as the process involves a multiplicity of attractive and repulsive signals, presented both as soluble and membrane-bound ligands. A better understanding of axon guidance is critical to functional recovery following injury to the nervous system through improved outgrowth and mapping of damaged nerves. Due to their implications as inhibitors to central nervous system regeneration, we investigated the repulsive properties of semaphorin 6A and ephrin-B3 on E15 rat dorsal root ganglion explants, as well as possible interactions with soluble gradients of chemoattractive nerve growth factor (NGF). We employed a 3D biomimetic in vitro choice point model, which enabled the simple and rapid preparation of patterned gel growth matrices with quantifiable presentation of guidance cues in a specifiable manner that resembles the in vivo presentation of soluble and/or immobilized ligands. Neurites demonstrated an inhibitory response to immobilized Sema6A by lumbosacral dorsal root ganglion explants, while no such repulsion was observed for immobilized ephrin-B3 by explants at any spinal level. Interestingly, Sema6A inhibition could be partially attenuated in a concentration-dependent manner through the simultaneous presentation of soluble NGF gradients. The in vitro model described herein represents a versatile and valuable investigative tool in the quest for understanding developmental processes and improving regeneration following nervous system injury.

21am2-E2 PC12Dパターニングのための誘電泳動法を用いたBio-MEMSデバイスの開発

Because neurons do not spontaneously regenerate their axons, the method of promoting re-combination of a nerve axon is studied by many researchers. The growth direction of nerve axon is drawn by nerve growth factor (NGF). In previous studies, it was demonstrated that axon extension direction is controlled by the concentration gradient of NGF. NGF is mainly secreted from glial cells and neurons. Position information between nerve cell-nerve cell is expected to improve directional control of Axon and extension velocity. Therefore, in this study, we propose a development of Bio-MEMS device that can trap the PC12D by using dielectrophoresis to examine the interaction effect between the position of nerve cell and the expansion direction of nerve axon and the velocity of axon extension.

The effects of gradients of nerve growth factor immobilized PCLA scaffolds on neurite outgrowth in vitro and peripheral nerve regeneration in rats

Introducing concentration gradients of nerve growth factor (NGF) into conduits for repairing of peripheral nerve injury is crucial for nerve regeneration and guidance. Herein, combining differential adsorption of NGF/silk fibroin (SF) coating, the gradient of NGF-immobilized membranes (G-Ms) and nanofibrous nerve conduits (G-nNCs) were successfully fabricated. The efficacy of NGF gradients was confirmed by a quantitative comparison of dorsal root ganglia (DRG) neurite outgrowth on the G-Ms or uniform NGF-immobilized membranes (U-Ms). Significantly, the neurite turning ratio was 0.48 ± 0.11 for G-M group, but it was close to zero for U-M group. The neurite length of DRGs in the middle of the G-Ms was significantly longer than that of U-M group, even though the average NGF concentration was approximated. Furthermore, 12 weeks after implantation in rats with a 14 mm gap of sciatic nerve injury, G-nNCs achieved satisfying outcomes of nerve regeneration associated with morphological and functional improvements, which was superior to that of the uniform NGF-immobilized nNCs (U-nNCs). Sciatic function index (SFI), compound muscle action potentials (CMAPs), total number of myelinated nerve fibers, thickness of myelin sheath were similar for the G-nNCs and autografts, with the G-nNCs having a higher density of axons than the autografts. Our results demonstrated the significant role of introducing NGF gradients into scaffolds in promoting nerve regeneration.

A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen

During nervous system development growing axons are often guided by diffusible chemical gradients. An important contribution to our understanding of the mechanisms involved in this process has been made by in vitro assays. However, an inexpensive and simple assay which allows the establishment of stable and reproducible gradients in a 3D collagen environment has been lacking. Here we present a simple two-compartment diffusion chamber for this purpose. We show that gradient steepnesses of up to 2% are achieved within 1h post setup, and a gradient persists for at least 2 days. We demonstrate the assay by showing robust chemoattraction of dorsal root ganglion neurites by gradients of nerve growth factor (NGF), and chemorepulsion of olfactory bulb neurites by gradients of Slit2.

Matrix Metalloproteinase-8 Is Involved in Dermal Nerve Growth: Implications for Possible Application to Pruritus from In Vitro Models

Cutaneous nerve density is related to abnormal itch perception in dermatoses, such as atopic dermatitis and xerosis. However, the mechanisms underlying the elongation of dermal nerve fibers within the interstitial collagen (CoL) matrix are poorly understood. In this study, a culture system of rat dorsal root ganglion neurons consisting of type I CoL and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. Nerve fibers penetrating into type I CoL gel were observed in the presence of the NGF concentration gradient. Levels of matrix metalloproteinase-8 (MMP-8) mRNA and protein were increased in the cultured neurons and the conditioned medium, respectively. The nerve fiber penetration was dose dependently inhibited by MMP-8 blockers. Moreover, MMP-8 immunoreactivity was partially localized at growth cones in NGF-responsive nerve fibers. Semaphorin 3A stimulation also showed the opposite effects on these NGF-dependent events. Intriguingly, MMP-8 expression was upregulated by type I and III CoLs, which are substrates for this enzyme. These results suggested that MMP-8 is involved in sensory nerve growth within the interstitial CoL matrix through modulation by the axonal guidance molecules and/or extracellular matrix components. These findings provide insight into the development of pruritus involving skin nerve density.

Cyclic nucleotide-dependent switching of mammalian axon guidance depends on gradient steepness

Correct wiring of the nervous system during development requires axons to respond appropriately to gradients of attractive and repulsive guidance cues. However, the steepness and concentration of these gradients vary in vivo, for instance, with distance from the target. Understanding how these changing conditions affect the navigation strategies used by developing axons is important for understanding how they are guided over long distances. Previous work has shown that cyclic nucleotide levels determine whether axons are attracted or repelled by steep gradients of the same guidance cue, but it is unknown whether this is also true for shallow gradients. We therefore investigated the guidance responses of rat superior cervical ganglion (SCG) axons in both steep and shallow gradients of nerve growth factor (NGF). In steep gradients we found that cyclic nucleotide-dependent switching occurred, consistent with previous reports. Surprisingly however, we found that in shallow NGF gradients, cyclic nucleotide-dependent switching did not occur. These results suggest that there may be substantial differences in the way axons respond to gradient-based guidance cues depending on where they are within the gradient.

Nanofibrous scaffold with incorporated protein gradient for directing neurite outgrowth.

Concentration gradient of diffusible bioactive chemicals assumes many important roles in regulating cellular behavior. Among the many factors influencing functional recovery after nerve injury, such as topographical and biochemical signals, concentration gradients of neurotrophic factors provide chemotactic cues for neurite outgrowth and targeted renervation. In this study, a concentration gradient of nerve growth factor (NGF, 0-250μg/ml) was incorporated throughout the thickness of poly(ε-caprolactone)-poly(ethylene glycol) coaxial electrospun nanofibrous scaffolds (∼700μm thick with ∼800nm average fiber diameter). The existence of the protein gradient upon protein release was demonstrated using a customized under-agarose-PC12 neurite outgrowth assay. When exposed to scaffolds endowed with NGF concentration gradient (NGF-CG), a significant difference in the percentage of cells bearing neurite outgrowth was observed (7.1 ± 1.9% vs. 0.8 ± 0.3% for cells exposed to high vs. low concentration surface, respectively; p < 0.05). In contrast, no significant difference was observed when cells were exposed to scaffolds that encapsulated a fixed concentration of NGF. Direct culture of PC12 cells on the substrates demonstrated the cytocompatibility and the effect of diffusible NGF gradient on neurite outgrowth. A significant difference in the percentage of cells with neurite extensions was observed when PC12 cells were seeded on NGF-CG scaffolds (21.2 ± 3.6% vs. 10.4 ± 1.3% on high vs. low concentration surface, respectively; p < 0.05). Furthermore, Z-stack confocal microscopy tracking of neurite extensions revealed the chemotatic guidance effect of NGF concentration gradient. Directed and enhanced neurite penetration into the scaffolds towards increasing NGF concentration was observed. In vitro release study indicated that the encapsulated NGF was released in a sustained manner for at least 30days (80.4 ± 3.6% released). Taken together, this study demonstrates the feasibility of incorporating concentration gradient of diffusible bioactive chemicals in nanofibrous scaffolds via the coaxial electrospinning technique.

The Response of Dorsal Root Ganglion Axons to Nerve Growth Factor Gradients Depends on Spinal Level

Directed sensory axon regeneration has the potential to promote functional recovery after peripheral nerve injury. Using a novel guidance assay to generate precisely controllable nerve growth factor gradients, we show for the first time that the guidance and outgrowth response of rat dorsal root ganglion neurons to identical nerve growth factor gradients depends on the rostrocaudal origin of the dorsal root ganglion explant. These findings have implications for the study of peripheral nerve regeneration in response to exogenous neurotrophins such as nerve growth factor, and provide new insight into the clinical potential of nerve growth factor in the treatment of nerve injury.