Aligned fibrous PVDF-TrFE scaffolds with Schwann cells support neurite extension and myelination in vitro.
ABSTRACT: OBJECTIVE:Polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), which is a piezoelectric, biocompatible polymer, holds promise as a scaffold in combination with Schwann cells (SCs) for spinal cord repair. Piezoelectric materials can generate electrical activity in response to mechanical deformation, which could potentially stimulate spinal cord axon regeneration. Our goal in this study was to investigate PVDF-TrFE scaffolds consisting of aligned fibers in supporting SC growth and SC-supported neurite extension and myelination in vitro. APPROACH:Aligned fibers of PVDF-TrFE were fabricated using the electrospinning technique. SCs and dorsal root ganglion (DRG) explants were co-cultured to evaluate SC-supported neurite extension and myelination on PVDF-TrFE scaffolds. MAIN RESULTS:PVDF-TrFE scaffolds supported SC growth and neurite extension, which was further enhanced by coating the scaffolds with Matrigel. SCs were oriented and neurites extended along the length of the aligned fibers. SCs in co-culture with DRGs on PVDF-TrFE scaffolds promoted longer neurite extension as compared to scaffolds without SCs. In addition to promoting neurite extension, SCs also formed myelin around DRG neurites on PVDF-TrFE scaffolds. SIGNIFICANCE:This study demonstrated PVDF-TrFE scaffolds containing aligned fibers supported SC-neurite extension and myelination. The combination of SCs and PVDF-TrFE scaffolds may be a promising tissue engineering strategy for spinal cord repair.
Project description:The path created by aligned Schwann cells (SCs) after nerve injury underlies peripheral nerve regeneration. We developed geometric bioinspired substrates to extract key information needed for axon guidance by deconstructing the topographical cues presented by SCs. We have previously reported materials that directly replicate SC topography with micro- and nanoscale resolution, but a detailed explanation of the means of directed axon extension on SC topography has not yet been described. Here, using neurite tracing and time-lapse microscopy, we analyzed the SC features that influence axon guidance. Novel poly(dimethylsiloxane) materials, fabricated via photolithography, incorporated bioinspired topographical components with the shapes and sizes of aligned SCs, namely somas and processes, where the lengths of the processes were varied but the soma geometry and dimensions were kept constant. Rat dorsal root ganglia neurites aligned to all materials presenting bioinspired topography after 5days in culture and aligned to bioinspired materials presenting soma and process features after only 17h in culture. The key findings of this study were: neurite response to underlying bioinspired topographical features was time dependent, with neurites aligned most strongly to materials presenting combinations of soma and process features at 5days, with higher than average density of either process or soma features, but at 17h they aligned more strongly to materials presenting average densities of soma and process features and to materials presenting process features only. These studies elucidate the influence of SC topography on axon guidance in a time-dependent setting and have implications for the optimization of nerve regeneration strategies.
Project description:Developing robust in vitro models of in vivo environments has the potential to reduce costs and bring new therapies from the bench top to the clinic more efficiently. This study aimed to develop a biomaterial platform capable of modeling isotropic-to-anisotropic cellular transitions observed in vivo, specifically focusing on changes in cellular organization following spinal cord injury. In order to accomplish this goal, nebulized solvent patterning of aligned, electrospun poly-l-lactic acid (PLLA) fiber substrates was developed. This method produced a clear topographic transitional boundary between aligned PLLA fibers and an isotropic PLLA film region. Astrocytes were then seeded on these scaffolds, and a shift between oriented and non-oriented astrocytes was created at the anisotropic-to-isotropic fiber/film transition (AFFT) boundary. Orientation of chondroitin sulfate proteoglycans (CSPGs) and fibronectin produced by these astrocytes was analyzed, and it was found that astrocytes growing on the aligned fibers produced aligned arrays of CSPGs and fibronectin, while astrocytes growing on the isotropic film region produced randomly-oriented CSPG and fibronectin arrays. Neurite extension from rat dissociated dorsal root ganglia (DRG) was studied on astrocytes cultured on anisotropic, aligned fibers, isotropic films, or from fibers to films. It was found that neurite extension was oriented and longer on PLLA fibers compared to PLLA films. When dissociated DRG were cultured on the astrocytes near the AFFT boundary, neurites showed directed orientation that was lost upon growth into the isotropic film region. The AFFT boundary also restricted neurite extension, limiting the extension of neurites once they grew from the fibers and into the isotropic film region. This study reveals the importance of anisotropic-to-isotropic transitions restricting neurite outgrowth by itself. Furthermore, we present this scaffold as an alternative culture system to analyze neurite response to cellular boundaries created following spinal cord injury and suggest its usefulness to study cellular responses to any aligned-to-unorganized cellular boundaries seen in vivo.
Project description:Researchers are investigating the use of biomaterials with aligned guidance cues, like those provided by aligned electrospun fibers, to facilitate axonal growth across critical-length peripheral nerve defects. To enhance the regenerative outcomes further, these aligned fibers can be designed to provide local, sustained release of therapeutics. The drug fingolimod improved peripheral nerve regeneration in preclinical rodent models by stimulating a pro-regenerative Schwann cell phenotype and axonal growth. However, the systemic delivery of fingolimod for nerve repair can lead to adverse effects, so it is necessary to develop a means of providing sustained delivery of fingolimod local to the injury. Here we created aligned fingolimod-releasing electrospun fibers that provide directional guidance cues in combination with the local, sustained release of fingolimod to enhance neurite outgrowth and stimulate a pro-regenerative Schwann cell phenotype. Electrospun fiber scaffolds were created by blending fingolimod into poly(lactic-co-glycolic acid) (PLGA) at a w/w% (drug/polymer) of 0.0004, 0.02, or 0.04%. We examined the effectiveness of these scaffolds to stimulate neurite extension in vitro by measuring neurite outgrowth from whole and dissociated dorsal root ganglia (DRG). Subsequently, we characterized Schwann cell migration and gene expression in vitro. The results show that drug-loaded PLGA fibers released fingolimod for 28 days, which is the longest reported release of fingolimod from electrospun fibers. Furthermore, the 0.02% fingolimod-loaded fibers enhanced neurite outgrowth from whole and dissociated DRG neurons, increased Schwann cell migration, and reduced the Schwann cell expression of promyelinating factors. The in vitro findings show the potential of the aligned fingolimod-releasing electrospun fibers to enhance peripheral nerve regeneration and serve as a basis for future in vivo studies.
Project description:Magnetic electrospun fibers are of interest for minimally invasive biomaterial applications that also strive to provide cell guidance. Magnetic electrospun fibers can be injected and then magnetically positioned in situ, and the aligned fiber scaffolds provide consistent topographical guidance to cells. In this study, magnetically responsive aligned poly-l-lactic acid electrospun fiber scaffolds were developed and tested for neural applications. Incorporating oleic acid-coated iron oxide nanoparticles significantly increased neurite outgrowth, reduced the fiber alignment, and increased the surface nanotopography of the electrospun fibers. After verifying neuron viability on two-dimensional scaffolds, the system was tested as an injectable three-dimensional scaffold. Small conduits of aligned magnetic fibers were easily injected in a collagen or fibrinogen hydrogel solution and repositioned using an external magnetic field. The aligned magnetic fibers provided internal directional guidance to neurites within a three-dimensional collagen or fibrin model hydrogel, supplemented with Matrigel. Neurites growing from dorsal root ganglion explants extended 1.4-3× farther on the aligned fibers compared with neurites extending in the hydrogel alone. Overall, these results show that magnetic electrospun fiber scaffolds can be injected and manipulated with a magnetic field in situ to provide directional guidance to neurons inside an injectable hydrogel. Most importantly, this injectable guidance system increased both neurite alignment and neurite length within the hydrogel scaffold.
Project description:RalA and RalB are small GTPases that are involved in cell migration and membrane dynamics. We used transgenic mice in which one or both GTPases were genetically ablated to investigate the role of RalGTPases in the Schwann cell (SC) response to nerve injury and repair. RalGTPases were dispensable for SC function in the naive uninjured state. Ablation of both RalA and RalB (but not individually) in SCs resulted in impaired axon remyelination and target reinnervation following nerve injury, which resulted in slowed recovery of motor function. Ral GTPases were localized to the leading lamellipodia in SCs and were required for the formation and extension of both axial and radial processes of SCs. These effects were dependent on interaction with the exocyst complex and impacted on the rate of SC migration and myelination. Our results show that RalGTPases are required for efficient nerve repair by regulating SC process formation, migration, and myelination, therefore uncovering a novel role for these GTPases.
Project description:Development of the peripheral nervous system requires radial axonal sorting by Schwann cells (SCs). To accomplish sorting, SCs must both proliferate and undergo morphogenetic changes such as process extension. Signaling studies reveal pathways that control either proliferation or morphogenesis, and laminin is essential for SC proliferation. However, it is not clear whether laminin is also required for SC morphogenesis. By using a novel time-lapse live-cell-imaging technique, we demonstrated that laminins are required for SCs to form a bipolar shape as well as for process extension. These morphological deficits are accompanied by alterations in signaling pathways. Phosphorylation of Schwannomin at serine 518 and activation of Rho GTPase Cdc42 and Rac1 were all significantly decreased in SCs lacking laminins. Inhibiting Rac1 and/or Cdc42 activities in cultured SCs attenuated laminin-induced myelination, whereas forced activation of Rac1 and/or Cdc42 in vivo improved sorting and hypomyelinating phenotypes in SCs lacking laminins. These findings indicate that laminins play a pivotal role in regulating SC cytoskeletal signaling. Coupled with previous results demonstrating that laminin is critical for SC proliferation, this work identifies laminin signaling as a central regulator coordinating the processes of proliferation and morphogenesis in radial axonal sorting.
Project description:This study was undertaken to examine the bioactivity, specificity, and reversibility of lithium's action on the growth, survival, proliferation, and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cyclic adenosine monophosphate (cAMP) analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin, and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific, and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact, and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation, and myelination while maintaining the integrity of pre-existing myelinated fibers.
Project description:Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm(-1) can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.
Project description:The myelination of axons in peripheral nerves requires precisely coordinated proliferation and differentiation of Schwann cells (SCs). We found that the activity of the mechanistic target of rapamycin complex 1 (mTORC1), a key signaling hub for the regulation of cellular growth and proliferation, is progressively extinguished as SCs differentiate during nerve development. To study the effects of different levels of sustained mTORC1 hyperactivity in the SC lineage, we disrupted negative regulators of mTORC1, including TSC2 or TSC1, in developing SCs of mutant mice. Surprisingly, the phenotypes ranged from arrested myelination in nerve development to focal hypermyelination in adulthood, depending on the level and timing of mTORC1 hyperactivity. For example, mice lacking TSC2 in developing SCs displayed hyperproliferation of undifferentiated SCs incompatible with normal myelination. However, these defects and myelination could be rescued by pharmacological mTORC1 inhibition. The subsequent reconstitution of SC mTORC1 hyperactivity in adult animals resulted in focal hypermyelination. Together our data suggest a model in which high mTORC1 activity promotes proliferation of immature SCs and antagonizes SC differentiation during nerve development. Down-regulation of mTORC1 activity is required for terminal SC differentiation and subsequent initiation of myelination. In distinction to this developmental role, excessive SC mTORC1 activity stimulates myelin growth, even overgrowth, in adulthood. Thus, our work delineates two distinct functions of mTORC1 in the SC lineage essential for proper nerve development and myelination. Moreover, our studies show that SCs retain their plasticity to myelinate and remodel myelin via mTORC1 throughout life.
Project description:Schwann cells (SCs) promote axonal integrity independently of myelination by poorly understood mechanisms. Current models suggest that SC metabolism is critical for this support function and that SC metabolic deficits may lead to axonal demise. The LKB1-AMP-activated protein kinase (AMPK) kinase pathway targets several downstream effectors, including mammalian target of rapamycin (mTOR), and is a key metabolic regulator implicated in metabolic diseases. We found through molecular, structural and behavioral characterization of SC-specific mutant mice that LKB1 activity is central to axon stability, whereas AMPK and mTOR in SCs are largely dispensable. The degeneration of axons in LKB1 mutants was most dramatic in unmyelinated small sensory fibers, whereas motor axons were comparatively spared. LKB1 deletion in SCs led to abnormalities in nerve energy and lipid homeostasis and to increased lactate release. The latter acts in a compensatory manner to support distressed axons. LKB1 signaling is essential for SC-mediated axon support, a function that may be dysregulated in diabetic neuropathy.