Direct Neurite Outgrowth Research Articles

Smart Polymeric 3D Microscaffolds Hosting Spheroids for Neuronal Research via Quantum Metrology.

Toward the aim of reducing animal testing, innovative in vitro models are required. Here, this study proposes a novel smart polymeric microscaffold to establish an advanced 3D model of dopaminergic neurons. These scaffolds are fabricated with Ormocomp via Two-Photon Polymerization. The scaffolds are further enhanced by functionalization with fluorescent nanodiamonds (FNDs), which can serve as quantum nanosensors for both magnetic and temperature sensing.The material biocompatibility is tested using two different cell lines, SH-SY5Y and A431, with cell viability over 98%. A total of 69% of the FNDs are grafted on the structure compared to those that remained on the glass surface. Cells are tested with the scaffolds in several microenvironments, and the final assembly required for 3D quantum metrology experiments achieved 91% biocompatibility. Subsequently, embryoid bodies containing dopaminergic neurons, the cell type affected by Parkinson's disease (PD), are integrated with FND-functionalized scaffolds. This 3D model is successfully established, demonstrated by strong interactions between dopaminergic neurons and the scaffold, with the directional growth of neurites along the 3D scaffold. Ultimately, this study have developed a 3D platform that enables the readout of signaling in a model that holds great potential for future PD research.

Long-range directional growth of neurites induced by magnetic forces.

The ability to control the growth and orientation of neurites over long distances has significant implications for regenerative therapies and the development of physiologically relevant brain tissue models. In this study, the forces generated on magnetic nanoparticles internalised within intracellular endosomes are used to direct the orientation of neuronal outgrowth in cell cultures. Following differentiation, neurite orientation was observed after 3 days application of magnetic forces to human neuroblastoma (SH-SY5Y) cells, and after 4 days application to rat cortical primary neurons. The direction of neurite outgrowth was quantified using a 2D Fourier transform analysis, showing agreement with the derived magnetic force vectors. Orientation control was found to be effective over areas >1cm2 using modest forces of ∼10 fN per endosome, apparently limited only by the local confluence of the cells. A bioinformatics analysis of protein expression in cells exposed to magnetic forces revealed changes to cell signaling and metabolic pathways resulting in enhanced carbohydrate metabolism, as well as the perturbation of processes related to cellular organisation and proliferation. Additionally, in cell culture regions where the measured force vectors converged, large (∼100 µm) SH-SY5Y neuroclusters loaded with nanoparticles were found, connected by unusually thick linear neurite fibres. This could suggest a magnetically driven enhancement of neurocluster growth, with the clusters themselves contributing to the local forces that direct outgrowth. Such structures, which have not been previously observed, could provide new insights into the development and possible enhancement of neural circuitry. STATEMENT OF SIGNIFICANCE: A magnetic force approach for directing outgrowth in neuronal cells over macroscopic areas is successfully demonstrated. Cells were incubated with magnetic nanoparticles which were sequestered into intracellular compartments. Permanent magnet arrays created local intracellular magnetic force vectors mediated via the internalized nanoparticles, which were found to precisely guide neurite orientation. Analysis of cellular protein expression suggested the mechanism for directed growth involved specific cell signaling and metabolic pathways. In addition, highly unusual straight and thick neural fibers were observed that connected large 'magnetic' spherical cell clusters. The results reported will advance nanotechnology and cell therapy for neuro-regeneration where magnetic forces could help to reconnect damaged neurons, or even build artificial neuronal architectures.

Open-End Control of Neurite Outgrowth Lengths with Steep Bending Confinement Microchannel Patterns for Miswiring-Free Neuronal Network Formation.

Wiring technology to control the length and direction of neurite outgrowth and to connect them is one of the most crucial development issues for forming single-cell-based neuronal networks. However, with current neurite wiring technology, it has been difficult to stop neurite extension at a specific length and connect it to other neurites without causing miswiring due to over-extension. Here, we examined a novel method of wiring neurites without miswiring by controlling the length of neurites in open-ended bending microchannel arrays connected beyond the maximum bending angle of neurite outgrowth. First, we determined the maximum bending angle of neurite elongation to pass through the bending point of a bending microfluidic channel; the maximum angle (the critical angle) was 90°. Next, we confirmed the control of neurite outgrowth length in open-ended microchannels connected at 120°, an angle beyond the maximum bending angle. The neurites stopped when elongated to the bend point, and no further elongation was observed. Finally, we observed that in bending microchannel arrays connected at an angle of 120°, two neurite outgrowths stopped and contacted each other without crossing over the bend point. The results show that the steep bending connection pattern is a robust open-end neurite wiring technique that prevents over-extension and miswiring.

Placental growth factor promotes neural invasion and predicts disease prognosis in resectable pancreatic cancer

BackgroundSurgery represents the only curative treatment option for pancreatic ductal adenocarcinoma (PDAC), but recurrence in more than 85% of patients limits the success of curative-intent tumor resection. Neural invasion (NI), particularly the spread of tumor cells along nerves into extratumoral regions of the pancreas, constitutes a well-recognized risk factor for recurrence. Hence, monitoring and therapeutic targeting of NI offer the potential to stratify recurrence risk and improve recurrence-free survival. Based on the evolutionary conserved dual function of axon and vessel guidance molecules, we hypothesize that the proangiogenic vessel guidance factor placental growth factor (PlGF) fosters NI. To test this hypothesis, we correlated PlGF with NI in PDAC patient samples and functionally assessed its role for the interaction of tumor cells with nerves.MethodsSerum levels of PlGF and its soluble receptor sFlt1, and expression of PlGF mRNA transcripts in tumor tissues were determined by ELISA or qPCR in a retrospective discovery and a prospective validation cohort. Free circulating PlGF was calculated from the ratio PlGF/sFlt1. Incidence and extent of NI were quantified based on histomorphometric measurements and separately assessed for intratumoral and extratumoral nerves. PlGF function on reciprocal chemoattraction and directed neurite outgrowth was evaluated in co-cultures of PDAC cells with primary dorsal-root-ganglia neurons or Schwann cells using blocking anti-PlGF antibodies.ResultsElevated circulating levels of free PlGF correlated with NI and shorter overall survival in patients with PDAC qualifying for curative-intent surgery. Furthermore, high tissue PlGF mRNA transcript levels in patients undergoing curative-intent surgery correlated with a higher incidence and greater extent of NI spreading to tumor-distant extratumoral nerves. In turn, more abundant extratumoral NI predicted shorter disease-free and overall survival. Experimentally, PlGF facilitated directional and dynamic changes in neurite outgrowth of primary dorsal-root-ganglia neurons upon exposure to PDAC derived guidance and growth factors and supported mutual chemoattraction of tumor cells with neurons and Schwann cells.ConclusionOur translational results highlight PlGF as an axon guidance factor, which fosters neurite outgrowth and attracts tumor cells towards nerves. Hence, PlGF represents a promising circulating biomarker of NI and potential therapeutic target to improve the clinical outcome for patients with resectable PDAC.Graphical

Synergy of Nanotopography and Electrical Conductivity of PEDOT/PSS for Enhanced Neuronal Development.

Biomaterials able to promote neuronal development and neurite outgrowth are highly desired in neural tissue engineering for the repair of damaged or disrupted neural tissue and restoring the axonal connection. For this purpose, the use of either electroactive or micro- and nanostructured materials has been separately investigated. Here, the use of a nanomodulated conductive poly(3,4-ethylendioxithiophene) poly(styrenesulfonate) (PEDOT/PSS) substrate that exhibits instructive topographical and electrical cues at the same time was investigated for the first time. In particular, thin films featuring grooves with sizes comparable with those of neuronal neurites (NanoPEDOT) were fabricated by electrochemical polymerization of PEDOT/PSS on a nanomodulated polycarbonate template. The ability of NanoPEDOT to support neuronal development and direct neurite outgrowth was demonstrated by assessing cell viability and proliferation, expression of neuronal markers, average neurite length, and direction of neuroblastoma N2A cells induced to differentiate on this novel support. In addition to the beneficial effect of the nanogrooved topography, a 30% increase was shown in the average length of neurites when differentiating cells were subjected to an electrical stimulation of a few microamperes for 6 h. The results reported here suggest a favorable effect on the neuronal development of the synergistic combination of nanotopography and electrical stimulation, supporting the use of NanoPEDOT in neural tissue engineering to promote physical and functional reconnection of impaired neural networks.

Biocompatible neuronal stimulant-embedded poly(γ-benzyl-L-glutamate) peptide-based scaffolds promote differentiation, growth and functional maturation of human induced pluripotent stem cell-derived retinal ganglion cells

BackgroundTo culture human induced pluripotent stem cells(hiPSCs)- derived retinal ganglion cells (RGCs) with appropriate structure and functionality is critical for cell therapy of optic nerve degeneration. In this study, we aimed to assess the effectiveness of three biocompatible scaffolds -poly(γ-benzyl-L-glutamate) (PBG)-based three-dimensional fibrous scaffolds in promoting the structural and functional maturation of hiPSC-derived RGCs. MethodsThe study investigated three scaffolds (1) PBG, (2) PBG with glutamic acid (PBGA20), and (3) PBGA20 with sodium salt (PBGA20-Na). The biocompatibility was evaluated in vivo with C57BL/6 mice. Neurite outgrowth was assessed using immunostaining for axon markers, while fluorescence-based Ca2+ imaging and patch-clamp technique were employed to measure neuronal calcium signaling and electrophysiological activity, respectively. Significant findingsAll three PBG-based scaffolds exhibited excellent biocompatibility in vivo and effectively supported the directional outgrowth of neurites from hiPSC-derived RGCs while the PBGA20-Na group demonstrated the most favorable outcomes. The presence of glutamic acid in PBGA20 and PBGA20-Na scaffolds led to an increase in calcium signaling within hiPSC-derived RGCs. HiPSC-derived RGCs cultured on PBGA20 and PBGA20-Na scaffolds exhibited an earlier onset of electrophysiological activities with lower thresholds. These promising results provide a foundation for potential sheet transplantation of hiPSC-derived RGCs as cell therapy for optic nerve regeneration.

Electrohydrodynamic Printing of Microfibrous Architectures with Cell‐Scale Spacing for Improved Cellular Migration and Neurite Outgrowth (Small 19/2023)

Microfibrous Architectures In article number 2207331, Jiankang He and co-workers propose an electrohydrodynamic printing strategy for the fabrication of orderly parallel microfibers with cell-scale spacing. The resultant microfibrous architectures with cell-scale spacing can accelerate the directional outgrowth of neurites and collective migration of Schwann cells to form highly-aligned and densely-organized neurite constructs.

Bionic opto-responsive fiber for directing neurite growth

Mounting evidence indicates that the nervous system plays a central function in pathogenesis. To study the association of the nervous system in diseases, tools that allow delicate control of neural behavior are crucial. Wireless electrical modulation of neural cells via opto-stimulation is an emerging approach to trigger and control neural behavior. Furthermore, to imitate the highly organized natural neural network, neurite outgrowth needs to be guided to achieve directionality. This work successfully combines bionic fibers with nano-groove topography and evident opto-response to guide neurite outgrowth. With excellent biocompatibility, the bionic opto-responsive fibers spatiotemporally controlled and guided PC12 neurite outgrowth. Transient photocurrent measurement revealed that this effect was attributed to anodic faradaic photocurrents. This bionic opto-responsive fiber has great potential to act as a tool to control and guide neurite outgrowth to study nervous system/disease interaction.

Directional control of neurite outgrowth: emerging technologies for Parkinson's disease using magnetic nanoparticles and magnetic field gradients.

A challenge in current stem cell therapies for Parkinson's disease (PD) is controlling neuronal outgrowth from the substantia nigra towards the targeted area where connectivity is required in the striatum. Here we present progress towards controlling directional neurite extensions through the application of iron-oxide magnetic nanoparticles (MNPs) labelled neuronal cells combined with a magnetic array generating large spatially variant field gradients (greater than 20 T m-1). We investigated the viability of this approach in both two-dimensional and organotypic brain slice models and validated the observed changes in neurite directionality using mathematical models. Results showed that MNP-labelled cells exhibited a shift in directional neurite outgrowth when cultured in a magnetic field gradient, which broadly agreed with mathematical modelling of the magnetic force gradients and predicted MNP force direction. We translated our approach to an ex vivo rat brain slice where we observed directional neurite outgrowth of transplanted MNP-labelled cells from the substantia nigra towards the striatum. The improved directionality highlights the viability of this approach as a remote-control methodology for the control and manipulation of cellular growth for regenerative medicine applications. This study presents a new tool to overcome challenges faced in the development of new therapies for PD.

Bridging the electrode–neuron gap: finite element modeling of in vitro neurotrophin gradients to optimize neuroelectronic interfaces in the inner ear

Although cochlear implant (CI) technology has allowed for the partial restoration of hearing over the last few decades, persistent challenges (e.g., poor performance in noisy environments and limited ability to decode intonation and music) remain. The “electrode–neuron gap” is inherent to these challenges and poses the most significant obstacle to advancing past the current plateau in CI performance. We propose the development of a “neuro-regenerative nexus”—a biological interface that doubly preserves native spiral ganglion neurons (SGNs) while precisely directing the growth of neurites arising from transplanted human pluripotent stem cell (hPSC)-derived otic neuronal progenitors (ONPs) toward the native SGN population. We hypothesized that the Polyhedrin Delivery System (PODS®-recombinant human brain-derived neurotrophic factor [rhBDNF]) could stably provide the adequate BDNF concentration gradient to hPSC-derived late-stage ONPs to facilitate otic neuronal differentiation and directional neurite outgrowth. To test this hypothesis, a finite element model (FEM) was constructed to simulate BDNF concentration profiles generated by PODS®-rhBDNF based on initial concentration and culture device geometry. For biological validation of the FEM, cell culture experiments assessing survival, differentiation, neurite growth direction, and synaptic connections were conducted using a multi-chamber microfluidic device. We were able to successfully generate the optimal BDNF concentration gradient to enable survival, neuronal differentiation toward SGNs, directed neurite extension of hPSC-derived SGNs, and synaptogenesis between two hPSC-derived SGN populations. This proof-of-concept study provides a step toward the next generation of CI technology. Statement of significanceOur study demonstrates that the generation of in vitro neurotrophin concentration gradients facilitates survival, neuronal differentiation toward auditory neurons, and directed neurite extension of human pluripotent stem cell-derived auditory neurons. These findings are indispensable to designing a bioactive cochlear implant, in which stem cell-derived neurons are integrated into a cochlear implant electrode strip, as the strategy will confer directional neurite growth from the transplanted cells in the inner ear. This study is the first to present the concept of a “neuro-regenerative nexus” congruent with a bioactive cochlear implant to eliminate the electrode-neuron gap—the most significant barrier to next-generation cochlear implant technology.

A Novel in vitro Model Delineating Hair Cell Regeneration and Neural Reinnervation in Adult Mouse Cochlea.

The study of an adult mammalian auditory system, such as regeneration, has been hampered by the lack of an in vitro system in which hypotheses can be tested efficiently. This is primarily due to the fact that the adult inner ear is encased in the toughest bone of the body, whereas its removal leads to the death of the sensory epithelium in culture. We hypothesized that we could take advantage of the integral cochlear structure to maintain the overall inner ear architecture and improve sensory epithelium survival in culture. We showed that by culturing adult mouse cochlea with the (surrounding) bone intact, the supporting cells (SCs) survived and almost all hair cells (HCs) degenerated. To evaluate the utility of the explant culture system, we demonstrated that the overexpression of Atoh1, an HC fate-determining factor, is sufficient to induce transdifferentiation of adult SCs to HC-like cells (HCLCs). Transdifferentiation-derived HCLCs resemble developmentally young HCs and are able to attract adult ganglion neurites. Furthermore, using a damage model, we showed that degenerated adult ganglions respond to regenerated HCLCs by directional neurite outgrowth that leads to HCLC-neuron contacts, strongly supporting the intrinsic properties of the HCLCs in establishing HCLC-neuron connections. The adult whole cochlear explant culture is suitable for diverse studies of the adult inner ear including regeneration, HC-neuron pathways, and inner ear drug screening.

Bionic Opto-Responsive Fiber for Directing Neurite Growth

Bionic Opto-Responsive Fiber for Directing Neurite Growth

Interaction of micropatterned topographical and biochemical cues to direct neurite growth from spiral ganglion neurons

Functional outcomes with neural prosthetic devices, such as cochlear implants, are limited in part due to physical separation between the stimulating elements and the neurons they stimulate. One strategy to close this gap aims to precisely guide neurite regeneration to position the neurites in closer proximity to electrode arrays. Here, we explore the ability of micropatterned biochemical and topographic guidance cues, singly and in combination, to direct the growth of spiral ganglion neuron (SGN) neurites, the neurons targeted by cochlear implants. Photopolymerization of methacrylate monomers was used to form unidirectional topographical features of ridges and grooves in addition to multidirectional patterns with 90o angle turns. Microcontact printing was also used to create similar uni- and multi-directional patterns of peptides on polymer surfaces. Biochemical cues included peptides that facilitate (laminin, LN) or repel (EphA4-Fc) neurite growth. On flat surfaces, SGN neurites preferentially grew on LN-coated stripes and avoided EphA4-Fc-coated stripes. LN or EphA4-Fc was selectively adsorbed onto the ridges or grooves to test the neurite response to a combination of topographical and biochemical cues. Coating the ridges with EphA4-Fc and grooves with LN lead to enhanced SGN alignment to topographical patterns. Conversely, EphA4-Fc coating on the grooves or LN coating on the ridges tended to disrupt alignment to topographical patterns. SGN neurites respond to combinations of topographical and biochemical cues and surface patterning that leverages both cues enhance guided neurite growth.

Neurite Extension and Orientation of Spiral Ganglion Neurons Can Be Directed by Superparamagnetic Iron Oxide Nanoparticles in a Magnetic Field.

IntroductionNeuroregeneration is a major challenge in neuroscience for treating degenerative diseases and for repairing injured nerves. Numerous studies have shown the importance of physical stimulation for neuronal growth and development, and here we report an approach for the physical guidance of neuron orientation and neurite growth using superparamagnetic iron oxide (SPIO) nanoparticles and magnetic fields (MFs).MethodsSPIO nanoparticles were synthesized by classic chemical co-precipitation methods and then characterized by transmission electron microscope, dynamic light scattering, and vibrating sample magnetometer. The cytotoxicity of the prepared SPIO nanoparticles and MF was determined using CCK-8 assay and LIVE/DEAD assay. The immunofluorescence images were captured by a laser scanning confocal microscopy. Cell migration was evaluated using the wound healing assay.ResultsThe prepared SPIO nanoparticles showed a narrow size distribution, low cytotoxicity, and superparamagnetism. SPIO nanoparticles coated with poly-L-lysine could be internalized by spiral ganglion neurons (SGNs) and showed no cytotoxicity at concentrations less than 300 µg/mL. The neurite extension of SGNs was promoted after internalizing SPIO nanoparticles with or without an external MF, and this might be due to the promotion of growth cone development. It was also confirmed that SPIO can regulate cell migration and can direct neurite outgrowth in SGNs preferentially along the direction imposed by an external MF.ConclusionOur results provide a fundamental understanding of the regulation of cell behaviors under physical cues and suggest alternative treatments for sensorineural hearing loss caused by the degeneration of SGNs.

Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation.

Neuron differentiation includes formation and outgrowth of neurites that differentiate into axons or dendrites. Directed neurite outgrowth is controlled by growth cones that protrude and retract actin-rich structures to sense environmental cues. These cues control local actin filament dynamics, steer growth cones toward attractants and away from repellents, and navigate neurites through the developing brain. Rodent hippocampal neurons are widely used to study the mechanisms underlying neuron differentiation. Genetic manipulation of isolated neurons including gene inactivation or reporter gene expression can be achieved by classical transfections methods, but these methods are restricted to neurons cultured for several days, after neurite formation or outgrowth. Instead, electroporation allows gene manipulation before seeding. However, reporter gene expression usually takes up to 24 h, and time course of gene inactivation depends on the half live of the targeted mRNA and gene product. Hence, these methods do not allow to study early aspects of neuron differentiation. In the present study, we provide a detailed protocol in which we combined electroporation-based gene manipulation of mouse hippocampal neurons before initial seeding with a replating step after 2 d in vitro (DIV) that resets neurons into an undifferentiated stage. By categorizing neurons according to their differentiation stage, thorough morphometric analyses, live imaging of actin dynamics in growth cones as well as guidance cue-mediated growth cone morphologic changes, we demonstrate that differentiation and function of replated neurons did not differ from non-replated neurons. In summary, we provide a protocol that allows to thoroughly characterize differentiation of mouse primary hippocampal neurons.

A directional 3D neurite outgrowth model for studying motor axon biology and disease

We report a method to generate a 3D motor neuron model with segregated and directed axonal outgrowth. iPSC-derived motor neurons are cultured in extracellular matrix gel in a microfluidic platform. Neurons extend their axons into an adjacent layer of gel, whereas dendrites and soma remain predominantly in the somal compartment, as verified by immunofluorescent staining. Axonal outgrowth could be precisely quantified and was shown to respond to the chemotherapeutic drug vincristine in a highly reproducible dose-dependent manner. The model was shown susceptible to excitotoxicity upon exposure with excess glutamate and showed formation of stress granules upon excess glutamate or sodium arsenite exposure, mimicking processes common in motor neuron diseases. Importantly, outgrowing axons could be attracted and repelled through a gradient of axonal guidance cues, such as semaphorins. The platform comprises 40 chips arranged underneath a microtiter plate providing both throughput and compatibility to standard laboratory equipment. The model will thus prove ideal for studying axonal biology and disease, drug discovery and regenerative medicine.

Decellularized optic nerve functional scaffold transplant facilitates directional axon regeneration and remyelination in the injured white matter of the rat spinal cord

Axon regeneration and remyelination of the damaged region is the most common repair strategy for spinal cord injury. However, achieving good outcome remains difficult. Our previous study showed that porcine decellularized optic nerve better mimics the extracellular matrix of the embryonic porcine optic nerve and promotes the directional growth of dorsal root ganglion neurites. However, it has not been reported whether this material promotes axonal regeneration in vivo. In the present study, a porcine decellularized optic nerve was seeded with neurotrophin-3-overexpressing Schwann cells. This functional scaffold promoted the directional growth and remyelination of regenerating axons. In vitro, the porcine decellularized optic nerve contained many straight, longitudinal channels with a uniform distribution, and microscopic pores were present in the channel wall. The spatial micro topological structure and extracellular matrix were conducive to the adhesion, survival and migration of neural stem cells. The scaffold promoted the directional growth of dorsal root ganglion neurites, and showed strong potential for myelin regeneration. Furthermore, we transplanted the porcine decellularized optic nerve containing neurotrophin-3-overexpressing Schwann cells in a rat model of T10 spinal cord defect in vivo. Four weeks later, the regenerating axons grew straight, the myelin sheath in the injured/transplanted area recovered its structure, and simultaneously, the number of inflammatory cells and the expression of chondroitin sulfate proteoglycans were reduced. Together, these findings suggest that porcine decellularized optic nerve loaded with Schwann cells overexpressing neurotrophin-3 promotes the directional growth of regenerating spinal cord axons as well as myelin regeneration. All procedures involving animals were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Sun Yat-sen University (approval No. SYSU-IACUC-2019-B034) on February 28, 2019.

Synergistic effect of CNTF and GDNF on directed neurite growth in chick embryo dorsal root ganglia.

It is often critical to improve the limited regenerative capacity of the peripheral nerves and direct neural growth towards specific targets, such as surgically implanted bioengineered constructs. One approach to accomplish this goal is to use extrinsic neurotrophic factors. The candidate factors first need to be identified and characterized in in vitro tests for their ability to direct the neurite growth. Here, we present a simple guidance assay that allows to assess the chemotactic effect of signaling molecules on the growth of neuronal processes from dorsal root ganglia (DRG) using only standard tissue culture materials. We used this technique to quantitatively determine the combined and individual effects of the ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth. We demonstrated that these two neurotrophic factors, when applied in a 1:1 combination, but not individually, induced directed growth of neuronal processes towards the source of the gradient. This chemotactic effect persists without significant changes over a wide (10-fold) concentration range. Moreover, we demonstrated that other, more general growth parameters that do not evaluate growth in a specific direction (such as, neurite length and trajectory) were differentially affected by the concentration of the CNTF/GNDF mixture. Furthermore, GDNF, when applied individually, did not have any chemotactic effect, but caused significant neurite elongation and an increase in the number of neurites per ganglion.

Self-Folding 3D Silk Biomaterial Rolls to Facilitate Axon and Bone Regeneration.

Biomaterial scaffold designs are needed for self-organizing features related to tissue formation while also simplifying the fabrication processes involved. Toward this goal, silk protein-based self-folding scaffolds to support 3D cell culture, while providing directional guidance and promotion of cell growth and differentiation, are reported. A simple and robust one-step self-folding approach is developed using bilayers consisting of a hydrogel and silk film in aqueous solution. The 3D silk rolls, with patterns transferred from the initially prepared 2D films, guide the directional outgrowth of neurites and also promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The osteogenic outcomes are further supported by enhanced biomechanical performance. By utilizing this self-folding method, cocultures of neurons and hMSCs are achieved by patterning cells on silk films and then converting these materials into a 3D format with rolling, mimicking aspects of the structure of osteons and providing physiologically relevant structures to promote bone regeneration. These results demonstrate the utility of self-folded silk rolls as efficient scaffold systems for tissue regeneration, while exploiting relatively simple 2D designs programmed to form more complex 3D structures.

Magnetic Organization of Neural Networks via Micro‐Patterned Devices

AbstractGuiding neuronal migration and outgrowth has great importance for therapeutic applications and for bioelectronics interfaces. Many efforts have been devoted to the development of tools to form predesigned structured neuronal networks. Here, a unique approach to localize cell bodies and direct neurite outgrowth is described based on local magnetic manipulations. Inspired by spintronic devices, a multi‐layer deposition process is developed to generate nanometric‐thick films with perpendicular magnetization that provide stable attraction forces toward the entire magnetic pads. PC12 cells, a common neuronal model, are transformed to magnetic units by incubation with superparamagnetic nanoparticles, which are then plated and differentiated atop the substrates. The vast majority of MNPs‐loaded cells adhere to the magnetic pads, showing high affinity to the magnetic patterns in correlation with numerical simulations of the magnetic force strength. Additionally, neuronal growth analysis shows that the magnetic substrate is effective in directing the extending neurites, which tend to remain atop the magnetic pads, and even follow complex patterns such as hexagons. This suggests that the MNPs diffuse into the neuronal processes throughout network formation. The ability to remotely control neuronal motility together with network design via smart magnetic materials opens possibilities for new neuronal interfaces and implantable therapeutic devices.