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Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography
Abstract
Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, due to the random architecture of these artificial networks. Important groundwork resulted therefore in the development of methods to confine neuronal adhesion at specific locations to match
predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of
the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning.
Here we report the development and characterization of a novel approach that; by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere non-adhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the
integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on
mechanotransductive processes, and can be used in their original state without any coating requirements.
These micropatterned substrates were validated using: i) a neuron-like PC12 cell line; ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated; in case of the hippocampal neurons up to the formation of mature, functionally active and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional inter-dot communication between these subgroups of cells was observed.
... Recently, we demonstrated the fabrication of complex micropatterns of nanostructured zirconia films able to control cell adhesion and their spatial distribution [32,33]. Zirconia nanostructured substrates have been used in a variety of experimental studies [32,34-39] to provide cellular microenvironments with controllable and reproducible biomimetic nanotopographies to unravel their impact on the mechanisms underlying mechanotransduction and to guide and control cellular behaviour [32,33]. ...
... Recently, we demonstrated the fabrication of complex micropatterns of nanostructured zirconia films able to control cell adhesion and their spatial distribution [32,33]. Zirconia nanostructured substrates have been used in a variety of experimental studies [32,34-39] to provide cellular microenvironments with controllable and reproducible biomimetic nanotopographies to unravel their impact on the mechanisms underlying mechanotransduction and to guide and control cellular behaviour [32,33]. Furthermore, these nanotopographical surfaces have been tested and validated as micropatterned substrates on a neuronal-like cell line (PC12), CA3-CA1 primary hippocampal neurons and astrocytes. ...
... Furthermore, these nanotopographical surfaces have been tested and validated as micropatterned substrates on a neuronal-like cell line (PC12), CA3-CA1 primary hippocampal neurons and astrocytes. For the neuronal cells, it has been shown that they grow and differentiate in a confined manner in these nanostructured zirconia micropatterns [32,33]. For astrocytes, so far the effective confinement of astrocytes in a nanostructured zirconia pattern has also been demonstrated in Ref. [33]. ...
Astrocytes’ organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation.
... The droplets are dried after being evaporated and precipitated. Thermolysis reactors decompose the dried particles into microporous particles that are sintered into Tissue Eng Regen Med Improved Hummer's method GO/CS/DA [20,65] Hydrolysis method TiO 2 /PEGylated CS [20,66] Electrospraying method GO/alginate microgels [67] rGO/alginate microgels IBAD/IME method TiN microelectrode arrays [68][69][70][71][72][73][74][75] Ionic modification and ultrasonic treatment MWCNTs/PCL scaffold [76,77] Covalent attachment Mesoporous silica/PAMAM [76] Coating method Au/immobilized silica spheres [4,9,76,78,79] BP nanosheets/GelMA nanocomposite BP/natural polymers ...
... Recent studies reveal that intracortical microelectrodes (IME) can be utilized to enhance and mimic biological tissue, especially nervous tissue and have been extensively studied. Nano-architectural approaches examine the surface of the implants and several approaches have been discussed by Kim et al. to improve IME biocompatibility and improve the quality and longevity of the implant [68][69][70][71]. A possible substitute to the titanium nitride (TiN) deposition approach has been invented by Ryynänen et al. ...
Tissue engineering is a research domain that deals with the growth of various kinds of tissues with the help of synthetic composites. With the culmination of nanotechnology and bioengineering, tissue engineering has emerged as an exciting domain. Recent literature describes its various applications in biomedical and biological sciences, such as facilitating the growth of tissue and organs, gene delivery, biosensor-based detection, etc. It deals with the development of biomimetics to repair, restore, maintain and amplify or strengthen several biological functions at the level of tissue and organs. Herein, the synthesis of nanocomposites based on polymers, along with their classification as conductive hydrogels and bioscaffolds, is comprehensively discussed. Furthermore, their implementation in numerous tissue engineering and regenerative medicine applications is also described. The limitations of tissue engineering are also discussed here. The present review highlights and summarizes the latest progress in the tissue engineering domain directed at functionalized nanomaterials.
... We believe that our model also supports the idea that the vesicular diffusive motions in a crowded environment, and the downstream RRP filling dynamics, are fundamental key factors regulating the efficacy of synaptic transmission. Future routes of investigation will include extending the range of predictions of our model also to more complex processes such as short-and long-term synaptic plasticity, but also testing if different synaptic network topologies can change the non-linear features of neurotransmission (Schulte et al., 2018). The vast toolkit of methods now available to measure and manipulate synaptic transmission will make it feasible to achieve such aims in the near future . ...
Neurons in the central nervous system communicate with each other by activating billions of tiny synaptic boutons distributed along their fine axons. These presynaptic varicosities are very crowded environments, comprising hundreds of synaptic vesicles. Only a fraction of these vesicles can be recruited in a single release episode, either spontaneous or evoked by action potentials. Since the seminal work by Fatt and Katz, spontaneous release has been modelled as a memoryless process. Nevertheless, at central synapses, experimental evidence indicates more complex features, including non‐exponential distributions of release intervals and power‐law behaviour in their rate. To describe these features, we developed a probabilistic model of spontaneous release based on Brownian motion of synaptic vesicles in the presynaptic environment. To account for different diffusion regimes, we based our simulations on fractional Brownian motion. We show that this model can predict both deviation from the Poisson hypothesis and power‐law features in experimental quantal release series, thus suggesting that the vesicular motion by diffusion could per se explain the emergence of these properties. We demonstrate the efficacy of our modelling approach using electrophysiological recordings at single synaptic boutons and ultrastructural data. When this approach was used to simulate evoked responses, we found that the replenishment of the readily releasable pool driven by Brownian motion of vesicles can reproduce the characteristic binomial release distributions seen experimentally. We believe that our modelling approach supports the idea that vesicle diffusion and readily releasable pool dynamics are crucial factors for the physiological functioning of neuronal communication. image
Key points
We developed a new probabilistic model of spontaneous and evoked vesicle fusion based on simple biophysical assumptions, including the motion of vesicles before they dock to the release site.
We provide closed‐form equations for the interval distribution of spontaneous releases in the special case of Brownian diffusion of vesicles, showing that a power‐law heavy tail is generated.
Fractional Brownian motion (fBm) was exploited to simulate anomalous vesicle diffusion, including directed and non‐directed motion, by varying the Hurst exponent.
We show that our model predicts non‐linear features observed in experimental spontaneous quantal release series as well as ultrastructural data of synaptic vesicles spatial distribution.
Evoked exocytosis based on a diffusion‐replenished readily releasable pool might explain the emergence of power‐law behaviour in neuronal activity.
... These changes might artificially be mimicked by inducing the mobilization and/or recruitment of vesicles to the presynaptic AZ, or by inserting/removing voltage-dependent calcium channels (VDCCs), although the effectiveness of such manipulations in modulating synaptic strength should still be established. More futuristic avenues relate to the de novo formation of synaptic contacts and axonal growth guidance, which would allow to reproduce and investigate realistic neural circuits in vitro with the help of novel techniques for neuronal culture patterning (Schulte et al., 2016(Schulte et al., , 2018. ...
Chemical synapses are tiny and overcrowded environments, deeply embedded inside brain tissue and enriched with thousands of protein species. Many efforts have been devoted to developing custom approaches for evaluating and modifying synaptic activity. Most of these methods are based on the engineering of one or more synaptic protein scaffolds used to target active moieties to the synaptic compartment or to manipulate synaptic functioning. In this review, we summarize the most recent methodological advances and provide a description of the involved proteins as well as the operation principle. Furthermore, we highlight their advantages and limitations in relation to studies of synaptic transmission in vitro and in vivo. Concerning the labeling methods, the most important challenge is how to extend the available approaches to the in vivo setting. On the other hand, for those methods that allow manipulation of synaptic function, this limit has been overcome using optogenetic approaches, that can be more easily applied to the living brain. Finally, future applications of these methods to neuroscience, as well as new potential routes for development, are discussed.
... PC12 and PC12-27 neuron-like cell lines, derived from rat pheochromocytoma, were grown on glass poly-L-lysine coated coverslips or plastic Petri dishes in a CO 2 incubator (5% CO 2 , 37 • C; Heraeus Instruments GmbH, Hanau, Germany) in DMEM medium (Dulbecco's Modified Eagle Medium, Gibco™) supplemented with 10% (v/v) horse serum, 5% (v/v) FCS (Fetal Calf Serum, Gibco™), 2 mM GlutaMax (Gibco™) and antibiotics (Penicillin 100 U/ml and Streptomycin 100 mg/ml, Gibco™) (Schulte et al., 2018). After reaching 90% of confluence, cells were lysed and diluted for validation analyses. ...
Background:
Mitochondria and their dynamics fuel most cellular processes both in physiological and pathological conditions. In the central nervous system, mitochondria sustain synaptic transmission and plasticity via multiple mechanisms which include their redistribution and/or expansion to higher energy demanding sites, sustaining activity changes and promoting morphological circuit adaptations.
New method:
To be able to evaluate changes in mitochondrial number and protein phenotype, we propose a novel methodological approach where the simultaneous analysis of both mitochondrial DNA and protein content is performed on each individual microsample, avoiding non-homogeneous loss of material.
Results:
We validated this method on neuronal-like cells and tissue samples and obtained estimates for the mitochondrial/genomic DNA ratio as well as for the abundance of protein counterparts. When the mitochondrial content per cell was evaluated in different brain areas, our results matched the known regional variation in aerobic-anaerobic metabolism. When long-term potentiation (LTP) was induced on hippocampal neurons, we detected increases in the abundance of mitochondria that correlated with the degree of synaptic enhancement.
Conclusions:
Our approach can be effectively used to study the mitochondrial content andits changes in different brain cells and tissues.
... In EB formation, these two features were further shown to increase the cell packing density [37], form tighter junctions, and modulate ECM networks [36,37]. Previous studies also established that microwell device's 3D topography and surface coating could affect cell behaviors (e.g., proliferation, differentiation) [17,41,79,80] and tissue morphogenesis (e.g., surface wrinkling) [10,81]. Furthermore, the provided deep and highly-networked (as discussed in section 3.1) microwells were previously found to enhance cell aggregation, nutrient exchange, and physiological functions of spheroids [21,38]. ...
The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices (ECM) for cell micro-environmental modulation. Matrigel embedding is problematic for long-term culture and clinical applications due to high inconsistency and other limitations. In this study, we developed a novel microwell culture platform based on 3D printing. This platform, without using Matrigel or external signaling molecules (i.e., SMAD and Wnt inhibitors), successfully generated matured human cerebral organoids with robust formation of high-level features (i.e., wrinkling/folding, lumens, neuronal layers). The formation and timing were comparable or superior to the current Matrigel methods, yet with improved consistency. The effect of microwell geometries (curvature and resolution) and coating materials (i.e., mPEG, Lipidure, BSA) was studied, showing that mPEG outperformed all other coating materials, while curved-bottom microwells outperformed flat-bottom ones. In addition, high-resolution printing outperformed low-resolution printing by creating faithful, isotropically-shaped microwells. The trend of these effects was consistent across all developmental characteristics, including EB formation efficiency and sphericity, organoid size, wrinkling index, lumen size and thickness, and neuronal layer thickness. Overall, the microwell device that was mPEG-coated, high-resolution printed, and bottom curved demonstrated the highest efficacy in promoting organoid development. This platform provided a promising strategy for generating uniform and mature human cerebral organoids as an alternative to Matrigel/ECM-embedding methods.
... According to previous results 1-3 , the b parameter of the cluster-assembled ZrO2 thin films grown on flat substrates is b = 0.368 ± 0.001 on silicon, or b = 0.31 ± 0.09 on glass coated with a monomolecular PAcrAm-g-(PMOXA, NH2, Si) layer 4 . These values are compatible with the prediction of the ballistic deposition growth model (b = 0.32 -0.25), which assumes that clusters impinge with a direction perpendicular to the plane of the substrate, and they do not diffuse significantly upon landing [5][6][7] . ...
Mechanosensing, the ability of cells to perceive and interpret the microenvironmental biophysical cues (such as the nanotopography), impacts strongly on cellular behaviour through mechanotransductive processes and signalling. These events are predominantly mediated by integrins, the principal cellular adhesion receptors located at the cell/extracellular matrix (ECM) interface. Because of the typical piconewton force range and nanometre length scale of mechanotransductive interactions, achieving a detailed understanding of the spatiotemporal dynamics occurring at the cell/microenvironment interface is challenging; sophisticated interdisciplinary methodologies are required. Moreover, an accurate control over the nanotopographical features of the microenvironment is essential, in order to systematically investigate and precisely assess the influence of the different nanotopographical motifs on the mechanotransductive process. In this framework, we were able to study and quantify the impact of microenvironmental nanotopography on early cellular adhesion events by means of adhesion force spectroscopy based on innovative colloidal probes mimicking the nanotopography of natural ECMs. These probes provided the opportunity to detect nanotopography-specific modulations of the molecular clutch force loading dynamics and integrin clustering at the level of single binding events, in the critical time window of nascent adhesion formation. Following this approach, we found that the nanotopographical features are responsible for an excessive force loading in single adhesion sites after 20 – 60 s of interaction, causing a drop in the number of adhesion sites. However, by manganese treatment we demonstrated that the availability of activated integrins is a critical regulatory factor for these nanotopography-dependent dynamics.
... Real tissues exhibit definite architectural shapes (pits, folds and branching points) that may create "hot spots" of tensional stress due to localized cell distortion. Important groundwork towards the regeneration of neural tissue also resulted from the development of methods to confine neuronal adhesion at specific locations to match predefined network topologies and connectivity that they in their niche environments (Brusatin et al. 2018, Schulte et al. 2018. The phenomenon in which the cells retain information from historical stimuli, the so-called "time-retention effect", is another fact to take into consideration at the time to modulate stem cell differentiation through a biophysical stimulus. ...
... In fact, even isotropic and disordered nanotopographies (made by quite different methods and materials, e.g. silica nanobeads, carbon nanotubes, silicon nanowires, assembled zirconia nanoclusters, and nanorough glass surfaces generated by reactive-ion etching or platinum-coated polystyrene nanopattern) have been reported to promote neuritogenesis, neuronal differentiation, and neural network maturation (Migliorini et al. 2011;Kang et al. 2012;Fabbro et al. 2012;Bugnicourt et al. 2014;Schulte et al. 2016c, b;Chen et al. 2018;Baek et al. 2018;Schulte et al. 2018). Nanotopographies with a suitable dimensionality have the ability to modulate integrin adhesion complexes (IAC) in a way that impacts on neuronal cell decision making, programming, and fate (Yang et al. 2013(Yang et al. , 2014Schulte et al. 2016c, b;Maffioli et al. 2017;Chen et al. 2018;Baek et al. 2018). ...
Although many details remain still elusive, it became increasingly evident in recent years that mechanosensing of microenvironmental biophysical cues and subsequent mechanotransduction are strongly involved in the regulation of neuronal cell development and functioning. This review gives an overview about the current understanding of brain and neuronal cell mechanobiology and how it impacts on neurogenesis, neuronal migration, differentiation, and maturation. We will focus particularly on the events in the cell/microenvironment interface and the decisive extracellular matrix (ECM) parameters (i.e. rigidity and nanometric spatial organisation of adhesion sites) that modulate integrin adhesion complex-based mechanosensing and mechanotransductive signalling. It will also be outlined how biomaterial approaches mimicking essential ECM features help to understand these processes and how they can be used to control and guide neuronal cell behaviour by providing appropriate biophysical cues. In addition, principal biophysical methods will be highlighted that have been crucial for the study of neuronal mechanobiology.
... In fact, even isotropic and disordered nanotopographies (made by quite different methods and materials, such as e.g. silica nanobeads, carbon nanotubes, silicon nanowires, assembled zirconia nanoclusters, nanorough glass surfaces generated by reactiveion etching or platinum-coated polystyrene nanopattern) have been reported to promote neuritogenesis, neuronal differentiation and neural network maturation (Migliorini et al., 2011), (Kang et al., 2012), (Fabbro et al., 2012), (Bugnicourt et al., 2014), (Schulte et al., 2016c), (Schulte et al., 2016b), , (Baek et al., 2018), (Schulte et al., 2018). Nanotopographies with a suitable dimensionality have the ability to modulate integrin adhesion complexes (IAC) in a way that impacts on neuronal cell decision making, programming and fate (Yang et al., 2013), (Yang et al., 2014), (Schulte et al., 2016c), (Schulte et al., 2016b), (Maffioli et al., 2017), , (Baek et al., 2018). ...
Although many details remain still elusive, it became increasingly evident in recent years that mechanosensing of microenvironmental biophysical cues and subsequent mechanotransduction are strongly involved in the regulation of neuronal cell development and functioning. This review gives an overview about the current understanding of brain and neuronal cell mechanobiology and how it impacts on neurogenesis, neuronal migration, differentiation, and maturation. Therein; we are focussing particularly on the events in the cell/microenvironment interface and the decisive extracellular matrix (ECM) parameters (i.e. rigidity and nanometric spatial organisation of adhesion sites) that modulate integrin adhesion complex-based mechanosensing and mechanotransductive signalling. It will also be outlined how biomaterial approaches mimicking essential ECM features help to understand these processes and how they can be used to control and guide neuronal cell behaviour by providing appropriate biophysical cues. In addition, principal biophysical methods will be highlighted that have been crucial for the study of neuronal mechanobiology.
Nanostructured zirconia and gold films (ns-Au/ZrO x ) have been demonstrated as devices characterized by non-linear and hysteretic electrical behavior, with short-term memory and potentiation/depression activity. Here we investigate the conduction mechanisms regulating the non-linear behavior of the nanostructured bilayer Au/ZrO x films. In particular, we investigated the hysteretic I-V curves following the Chua’s approach to memristive systems and separately modelling ion migration and electron transport in the films. The conduction mechanisms exhibited by the bilayered nanostructured system are strongly influenced by the nanogranular morphology that dynamically changes because of electrical stimuli; structural rearrangements are particularly promoted by intense local electric fields and high mobility along bottlenecks and edges in the microstructure. Electron transport is accounted for the Schottky barrier at the electrode interfaces and Poole-Frenkel effect in the bulk nanogranular material, according to a dynamic reorganization of the cluster-assembled network. A model for Poole-Frenkel effect is here discussed to include saturation of the Coulombic traps in the high applied field regime; the proposed model has been validated with experimental voltage ramps with varying sweep-velocity and at different temperatures (from 300 to 200 K), as also by a power exponent parameter analysis.
First-line treatments for post-traumatic stress disorder (PTSD) encompass a wide range of pharmacotherapies and psychotherapies. However, many patients fail to respond to such interventions, highlighting the need for novel approaches. Due to its ability to modulate cortical activity, non-invasive brain stimulation (NIBS) could represent a valuable therapeutic tool. Therefore, the aim of this systematic review is to summarize and discuss the existing evidence on the ameliorative effects of NIBS on PTSD and comorbid anxiety and depressive symptoms. Our goal is also to debate the effectiveness of an integrated approach characterized by the combination of NIBS and psychotherapy. This search was conducted following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines in the PubMed, PsycINFO, PsycARTICLES, PSYINDEX, MEDLINE, and ERIC databases. Overall, 31 studies met the eligibility criteria, yielding a total of 26 clinical trials employing transcranial magnetic stimulation (TMS) and 5 making use of transcranial direct-current stimulation (tDCS). From these studies, it emerged that NIBS consistently reduced overall PTSD symptoms’ severity as well as comorbid anxiety and depressive symptoms. Moreover, we speculate that combining NIBS with prolonged exposure or cognitive processing therapy might represent a promising therapeutic approach for consistently ameliorating subjects’ clinical conditions.
The neuroscience field has increased enormously over the last decades, achieving the possible real application of neuronal cultures not only for reproducing neural architectures resembling in vivo tissues, but also for the development of functional devices. In this context, surface patterning for cell confinement is crucial, and new active materials together with new protocols for preparing substrates suitable for confining cells, guiding their processes in the desired configuration are extremely appreciated. Here, TiO2 sol-gel derived films were selected as proof-of-concept materials to grow neurons in suitable confined configurations, taking advantage of the biocompatible properties of modified TiO2 substrates. TiO2 sol-gel derived films were made compatible with the growth of neurons thanks to a stable and controlled poly-lysine coating, obtained by silanization chemistry and streptavidin-biotin interactions. Moreover, a spotting protocol, here described and optimized, allowed the simple preparation of arrays of neurons, where cell adhesion was guided in specific areas and the neurites development driven in the desired arrangement. The resulting arrays were successfully tested for the growth and differentiation of neurons, demonstrating the possible adhesion of cells in specific areas of the film, therefore paving the way to applications such as the direct growth of excitable cells nearby electrodes of devices, with an evident enhancement of cell-electrodes communication.
The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.
The ability to control adhesion and the spatial organization of cells over nanoscale surfaces is essential in tissue engineering, regenerative medicine, the growth of organoids and spheroids as an in-vitro-model of human development and disease. Nonetheless, despite the several different works that have explored the influence of nanotopography on cell adhesion and clustering, little is known about how the forces arising from membrane conformational change developing during cell adaptation to a nanorough surface, and the cell-cell adhesion forces, interact to guide cell assembly. Here, starting from the works of Decuzzi and Ferrari, who examined how the energy of a cell varies while adhering to a nanoscale surface, and of Armstrong and collaborators, who developed a continuous model of cell-cell adhesion and morphogenesis, we provide a description of how nanotopography can modulate cellular clustering. In simulations where the parameters of the model were varied over large intervals, we found that nanoroughness may induce cell aggregation from a homogenous, uniform state, also for weak cell-cell adhesion. Results of the model are relevant in bio-engineering and biomedical nanotechnology, and may be of interest for those involved in the design and fabrication of biomaterials and scaffolds for tissue formation and repair.
Nanomedicine, a scientific branch of nanotechnology that operates on the same scale as biology, offers the possibility of influencing the healing process from inside of the body by manipulating the matter at cellular or molecular levels. Throughout this book, current healing approaches based on this revolutionary new technology are summarized from a scientific assessment. The aim of the authors is to give, through select examples, a deep insight to nanotechnology status and the great progress that its rigorous application will bring to human health. The authors' commitment is to broaden the vision of health professionals who will eventually be the future users of this knowledge.
Mechanosensing, the ability of cells to perceive and interpret the microenvironmental biophysical cues (such as the nanotopography), impacts strongly on cellular behaviour through mechanotransductive processes and signalling. These events are predominantly mediated by integrins, the principal cellular adhesion receptors located at the cell/extracellular matrix (ECM) interface.
Because of the typical picoNewton force range and nanometre length scale of mechanotransductive interactions, achieving a detailed understanding of the spatiotemporal dynamics occurring at the cell/microenvironment interface is challenging, and requires sophisticated interdisciplinary methodologies. Moreover, an accurate control over the nanotopographic features of the microenvironment is essential, in order to systematically investigate and precisely assess the influence of the different nanotopographic motifs on the mechanotransductive process.
In this framework, we were able to study and quantify the impact of microenvironmental nanotopography on early cellular adhesion events by means of adhesion force spectroscopy based on innovative colloidal probes mimicking the nanotopography of natural ECMs.
These probes provided the unique opportunity to detect nanotopography-specific modulations of the molecular force loading dynamics and integrin clustering at the level of single binding events, in the critical time window of nascent adhesion formation. Following this approach, we found that the availability of activated integrins is a critical regulatory factor for these nanotopography-dependent dynamics.
Intracortical microelectrodes (IME) are neural devices that initially were designed to function as neuroscience tools to enable researchers to understand the nervous system. Over the years, technology that aids interfacing with the nervous system has allowed the ability to treat patients with a wide range of neurological injuries and diseases. Despite the substantial success that has been demonstrated using IME in neural interface applications, these implants eventually fail due to loss of quality recording signals. Recent strategies to improve interfacing with the nervous system have been inspired by methods that mimic the native tissue. This review focusses on one strategy in particular, nano-architecture, a term we introduce that encompasses the approach of roughening the surface of the implant. Various nano-architecture approaches have been hypothesized to improve the biocompatibility of IMEs, enhance the recording quality, and increase the longevity of the implant. This review will begin by introducing IME technology and discuss the challenges facing the clinical deployment of IME technology. The biological inspiration of nano-architecture approaches will be explained as well as leading fabrication methods used to create nano-architecture and their limitations. A review of the effects of nano-architecture surfaces on neural cells will be examined, depicting the various cellular responses to these modified surfaces in both in vitro and pre-clinical models. The proposed mechanism elucidating the ability of nano-architectures to influence cellular phenotype will be considered. Finally, the frontiers of next generation nano-architecture IMEs will be identified, with perspective given on the future impact of this interfacing approach.
Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals-cell shape and cytoskeletal contractile force-instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.
Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be “interpreted” by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD) allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calcium signaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive mechanotransductive signaling hubs participating in neuronal differentiation were dissected.
Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin-ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking 'adaptor' protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin-ECM bonds-the molecular clutches-respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.
Ideally, elucidating the role of specific brain circuits in animal behavior would require the ability to measure activity at all involved synapses, possibly with unrestricted field of view, thus even at those boutons deeply located into the brain. Here, we introduce and validate an efficient scheme reporting synaptic vesicle cycling in vivo. This is based on SynaptoZip, a genetically encoded molecule deploying in the vesicular lumen a bait moiety designed to capture upon exocytosis a labeled alien peptide, Synbond. The resulting signal is cumulative and stores the number of cycling events occurring at individual synapses. Since this functional signal is enduring and measurable both online and ex post, SynaptoZip provides a unique method for the analysis of the history of synaptic activity in regions several millimeters below the brain surface. We show its broad applicability by reporting stimulus-evoked and spontaneous circuit activity in wide cortical fields, in anesthetized and freely moving animals.
Cell membrane roughness has been proposed as a sensitive feature to reflect cellular physiological conditions. In order to know whether membrane roughness is associated with the substrate properties, we employed the non-interferometric wide-field optical profilometry (NIWOP) technique to measure the membrane roughness of living mouse embryonic fibroblasts with different conditions of the culture substrate. By controlling the surface density of fibronectin (FN) coated on the substrate, we found that cells exhibited higher membrane roughness as the FN density increased in company with larger focal adhesion (FA) sizes. The examination of membrane roughness was also confirmed with atomic force microscopy. Using reagents altering actin or microtubule cytoskeletons, we provided evidence that the dynamics of actin filaments rather than that of microtubules plays a crucial role for the regulation of membrane roughness. By changing the substrate rigidity, we further demonstrated that the cells seeded on compliant gels exhibited significantly lower membrane roughness and smaller FAs than the cells on rigid substrate. Taken together, our data suggest that the magnitude of membrane roughness is modulated by way of actin dynamics in cells responding to substrate properties.
The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting. We also discuss the implementation of these technologies in compound identification, screening, and development, ranging from disease modeling to assessment of efficacy and safety profiles.
The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signalling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterised by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological in vivo ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation.
Background
Thanks to mechanotransductive components cells are competent to perceive nanoscale topographical features of their environment and to convert the immanent information into corresponding physiological responses. Due to its complex configuration, unraveling the role of the extracellular matrix is particularly challenging. Cell substrates with simplified topographical cues, fabricated by top-down micro- and nanofabrication approaches, have been useful in order to identify basic principles. However, the underlying molecular mechanisms of this conversion remain only partially understood.
Results
Here we present the results of a broad, systematic and quantitative approach aimed at understanding how the surface nanoscale information is converted into cell response providing a profound causal link between mechanotransductive events, proceeding from the cell/nanostructure interface to the nucleus. We produced nanostructured ZrO2 substrates with disordered yet controlled topographic features by the bottom-up technique supersonic cluster beam deposition, i.e. the assembling of zirconia nanoparticles from the gas phase on a flat substrate through a supersonic expansion. We used PC12 cells, a well-established model in the context of neuronal differentiation. We found that the cell/nanotopography interaction enforces a nanoscopic architecture of the adhesion regions that affects the focal adhesion dynamics and the cytoskeletal organization, which thereby modulates the general biomechanical properties by decreasing the rigidity of the cell. The mechanotransduction impacts furthermore on transcription factors relevant for neuronal differentiation (e.g. CREB), and eventually the protein expression profile. Detailed proteomic data validated the observed differentiation. In particular, the abundance of proteins that are involved in adhesome and/or cytoskeletal organization is striking, and their up- or downregulation is in line with their demonstrated functions in neuronal differentiation processes.
Conclusion
Our work provides a deep insight into the molecular mechanotransductive mechanisms that realize the conversion of the nanoscale topographical information of SCBD-fabricated surfaces into cellular responses, in this case neuronal differentiation. The results lay a profound cell biological foundation indicating the strong potential of these surfaces in promoting neuronal differentiation events which could be exploited for the development of prospective research and/or biomedical applications. These applications could be e.g. tools to study mechanotransductive processes, improved neural interfaces and circuits, or cell culture devices supporting neurogenic processes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12951-016-0171-3) contains supplementary material, which is available to authorized users.
Surface-active polymers that display nonfouling properties and carry binding groups that can adsorb onto different substrates are highly desirable. We present a postmodification protocol of an active-ester-containing polymer that allows the creation of such a versatile platform. Poly(pentafluorophenyl acrylate) has been postmodified with a fixed grafting ratio of a nonfouling function (mPEG) and various combinations of functional groups, such as amine, silane and catechol, which can provide strong affinity to two model substrates: SiO2 and TiO2 . Adsorption, stability and resistance to nonspecific protein adsorption of the polymer films were studied. A polymer was obtained that maintained its surface functionality under a variety of harsh conditions. EG surface-density calculations show that this strategy generates a denser packing when both negatively and positively charged groups are present within the backbone, and readily allows the fabrication of a broad combinatorial matrix.
Technologies for microfluidics and biological microelectromechanical systems have been rapidly progressing over the past decade, enabling the development of unique microplatforms for in vitro human central nervous system (CNS) and related disease models. Most fundamental techniques include manipulation of axons, synapses, and neuronal networks, and different culture conditions are possible, such as compartmental, co-culturing, and 3D. Various CNS disease models, such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), epilepsy, N-methyl-D-aspartate receptor (NMDAR) encephalitis, migraine, diffuse axonal injury, and neuronal migration disorders, have been successfully established on microplatforms. In this review, we summarize fundamental technologies and current existing CNS disease models on microplatforms. We also discuss possible future directions, including application of these methods to pathological studies, drug screening, and personalized medicine, with 3D and personalized disease models that could generate more realistic CNS disease models.
Complex network topologies represent the necessary substrate to support complex brain functions. In this work, we reviewed in vitro neuronal networks coupled to Micro-Electrode Arrays (MEAs) as biological substrate. Networks of dissociated neurons developing in vitro and coupled to MEAs, represent a valid experimental model for studying the mechanisms governing the formation, organization and conservation of neuronal cell assemblies. In this review, we present some examples of the use of statistical Cluster Coefficients and Small World indices to infer topological rules underlying the dynamics exhibited by homogeneous and engineered neuronal networks.
Neuronal growth cones are exquisite sensory-motor machines capable of transducing features contacted in their local extracellular environment into guided process extension during development. Extensive research has shown that chemical ligands activate cell surface receptors on growth cones leading to intracellular signals that direct cytoskeletal changes. However, the environment also provides mechanical support for growth cone adhesion and traction forces that stabilize leading edge protrusions. Interestingly, recent work suggests that both the mechanical properties of the environment and mechanical forces generated within growth cones influence axon guidance. In this review we discuss novel molecular mechanisms involved in growth cone force production and detection, and speculate how these processes may be necessary for the development of proper neuronal morphogenesis.
Fullerene-like MoS2 and WS2 nanoparticles can be used as building blocks for the fabrication of fluid and solid lubricants. Metal dichalcogenide films have a very low friction coefficient in vacuum, therefore they have mostly been used as solid lubricants in space and vacuum applications. Unfortunately, their use is significantly hampered by the fact that in the presence of humidity, oxygen and moisture, the low-friction properties of these materials rapidly degrade due to oxidation. The use of closed-cage MoS2 and WS2 nanoparticles may eliminate this problem, although the fabrication of lubricant thin films starting from dichalcogenide nanoparticles is, to date, a difficult task. Here we demonstrate the use of supersonic cluster beam deposition for the coating of complex mechanical elements (angular contact ball bearings) with nanostructured MoS2 and WS2 thin films. We report structural and tribological characterization of the coatings in view of the optimization of tribological performances for aerospace applications.
Micropatterning strategies, which enable control over cell and tissue architecture in vitro, have emerged as powerful platforms for modelling tissue microenvironments at different scales and complexities. Here, we provide an overview of popular micropatterning techniques, along with detailed descriptions, to guide new users through the decision making process of which micropatterning procedure to use, and how to best obtain desired tissue patterns. Example techniques and the types of biological observations that can be made are provided from the literature. A focus is placed on microcontact printing to obtain co-cultures of patterned, confluent sheets, and the challenges associated with optimizing this protocol. Many issues associated with microcontact printing, however, are relevant to all micropatterning methodologies. Finally, we briefly discuss challenges in addressing key limitations associated with current micropatterning technologies.
Patterned gold-polydimethylsiloxane (Au-PDMS) nanocomposites
were fabricated by supersonic cluster beam implantation (SCBI) of
neutral gold nanoparticles in PDMS through stencil masks. The influence
of nanoparticle dose on the surface roughness and morphology of the
micropatterned regions of the nanocomposite was characterized.
Nanoparticle implantation causes the swelling of PDMS without affecting
substantially the lateral resolution of the patterns. In order to have
an insight on the mechanism and the influence of nanoparticle
implantation on the polymeric matrix, large-scale molecular dynamics
simulations of the implantation process have been performed. The
simulations show that even a single cluster impact on PDMS substrate
strongly affects the polymer local temperature and density. Our results
show that SCBI is a promising methodology for the efficient fabrication
of nanocomposite microstructures on polymers with interesting
morphological, structural and functional properties.
Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.
To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.
Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.
The quantitative determination of key adherent cell culture characteristics such as confluency, morphology and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post-hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (< 1s per 1208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
In this Historical Perspective, we ask what information is needed beyond connectivity diagrams to understand the function of nervous systems. Informed by invertebrate circuits whose connectivities are known, we highlight the importance of neuronal dynamics and neuromodulation, and the existence of parallel circuits. The vertebrate retina has these features in common with invertebrate circuits, suggesting that they are general across animals. Comparisons across these systems suggest approaches to study the functional organization of large circuits based on existing knowledge of small circuits.
In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency E was >75% during lengthy in chip culture, with ∼85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.
Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs), or an extracellular matrix such as Matrigel™. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel™ ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with 3D cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications.
Gas phase nanoparticle production, manipulation and deposition is of primary importance for the synthesis of nanostructured materials and for the development of industrial processes based on nanotechnology. In this review we present and discuss this approach, introducing cluster sources, nanoparticle formation and growth mechanisms and the use of aerodynamic focusing methods that are coupled with supersonic expansions to obtain high intensity cluster beams with a control on nanoparticle mass and spatial distribution. The implication of this technique for the synthesis of nanostructured materials is also presented and applications are highlighted.
The interplay between anatomical connectivity and dynamics in neural networks plays a key role in the functional properties of the brain and in the associated connectivity changes induced by neural diseases. However, a detailed experimental investigation of this interplay at both cellular and population scales in the living brain is limited by accessibility. Alternatively, to investigate the basic operational principles with morphological, electrophysiological and computational methods, the activity emerging from large in vitro networks of primary neurons organized with imposed topologies can be studied. Here, we validated the use of a new bio-printing approach, which effectively maintains the topology of hippocampal cultures in vitro and investigated, by patch-clamp and MEA electrophysiology, the emerging functional properties of these grid-confined networks. In spite of differences in the organization of physical connectivity, our bio-patterned grid networks retained the key properties of synaptic transmission, short-term plasticity and overall network activity with respect to random networks. Interestingly, the imposed grid topology resulted in a reinforcement of functional connections along orthogonal directions, shorter connectivity links and a greatly increased spiking probability in response to focal stimulation. These results clearly demonstrate that reliable functional studies can nowadays be performed on large neuronal networks in the presence of sustained changes in the physical network connectivity.
Neuron-glia cultures serve as a valuable model system for exploring the bio-molecular activity of single cells. Since neurons in culture can be conveniently recorded with great fidelity from many sites simultaneously, it has long been suggested that uniform cultured neurons may also be used to investigate network-level mechanisms pertinent to information processing, activity propagation, memory, and learning. But how much of the functionality of neural circuits can be retained in vitro remains an open question. Recent studies utilizing patterned networks suggest that they provide a most useful platform to address fundamental questions in neuroscience. Here we review recent efforts in the realm of patterned networks' activity investigations. We give a brief overview of the patterning methods and experimental approaches commonly employed in the field, and summarize the main results reported in the literature. The general picture that emerges from these reports indicates that patterned networks with uniform connectivity do not exhibit unique activity patterns. Rather, their activity is very similar to that of unpatterned uniform networks. However, by breaking the connectivity homogeneity, using a modular architecture, it is possible to introduce pronounced topology-related gating and delay effects. These findings suggest that patterned cultured networks may serve as a new platform for studying the role of modularity in neuronal circuits.
This book brings together two of the most exciting and widely studied subjects in modern physics: namely fractals and surfaces. To the community interested in the study of surfaces and interfaces, it brings the concept of fractals. To the community interested in the exciting field of fractals and their application, it demonstrates how these concepts may be used in the study of surfaces. The authors cover, in simple terms, the various methods and theories developed over the past ten years to study surface growth. They describe how one can use fractal concepts successfully to describe and predict the morphology resulting from various growth processes. Consequently, this book will appeal to physicists working in condensed matter physics and statistical mechanics, with an interest in fractals and their application. The first chapter of this important new text is available on the Cambridge Worldwide Web server: http://www.cup.cam.ac.uk/onlinepubs/Textbooks/textbookstop.html
Human neural stem cells (hNSCs) can alter their fate choice in response to the biophysical cues provided during development. In particular, it has been reported that the differentiation of neural stem cells (NSCs) is enhanced by anisotropic contact, which facilitates focal adhesion (FA) formation and cytoskeletal organization. However, biomolecular mechanism how the cells process the biophysical cues from these anisotropic geometries to their fate commitment is still poorly understood due to the limited availability of geometrical diversities (contact width above 50 nm) applicable to cell studies. Here, we firstly demonstrate that biomolecular mechanism for enhanced neurogenesis on anisotropic nanostructure critically depended on resolution of contact feature. We observed a totally different cellular response to anisotropic geometries by firstly utilizing a high-resolution nanogroove (HRN) structure with extremely narrow contact width (15 nm). The width scale is sufficiently low to suppress the integrin clustering and enable us to elucidate how the contact area influences the neurogenesis of hNSCs at aligned state. Both HRN and control nanogroove pattern (CN) with the contact width of 1 μm induced the spontaneous topographic alignment of hNSCs. However, intriguingly, the focal adhesion (FA) formation and cytoskeletal reorganization were substantially limited on HRN although the cells on CN showed enhanced FA formation as compared with flat surfaces. In particular, the hNSCs on HRN surface exhibited strikingly lower fraction of nuclear yes-associated protein (YAP) than on CN surface, which was turned out to be regulated by Rho GTPase in the same way the cells sense the mechanical property of environment. Considering previously reported role of YAP on neurogenesis, our finding newly substantiates that YAP and Rho GTPase also can be transducers of hNSCs to process topographical alternation to fate decision. Furthermore, this study with unprecedented high-resolution nanostructure suggests a novel geometry sensing model where the functional crosstalk between YAP signaling and Rho GTPase integrally regulate the fate commitment of hNSCs.
The formation of correct synaptic structures and neuronal connections is paramount for normal brain development and a functioning adult brain. The integrin family of cell adhesion receptors and their ligands play essential roles in the control of several processes regulating neuronal connectivity - including neurite outgrowth, the formation and maintenance of synapses, and synaptic plasticity - that are affected in neurodevelopmental disorders, such as autism spectrum disorders (ASDs) and schizophrenia. Many ASD- and schizophrenia-associated genes are linked to alterations in the genetic code of integrins and associated signalling pathways. In non-neuronal cells, crosstalk between integrin-mediated adhesions and the actin cytoskeleton, and the regulation of integrin activity (affinity for extracellular ligands) are widely studied in healthy and pathological settings. In contrast, the roles of integrin-linked pathways in the central nervous system remains less well defined. In this Review, we will provide an overview of the known pathways that are regulated by integrin-ECM interaction in developing neurons and in adult brain.We will also describe recent advances in the identification of mechanisms that regulate integrin activity in neurons, and highlight the interesting emerging links between integrins and neurodevelopment.
The formation of correct synaptic structures and neuronal connections is paramount for normal brain development and a functioning adult brain. The integrin family of cell adhesion receptors and their ligands play essential roles in the control of several processes regulating neuronal connectivity - including neurite outgrowth, the formation and maintenance of synapses, and synaptic plasticity - that are affected in neurodevelopmental disorders, such as autism spectrum disorders (ASDs) and schizophrenia. Many ASD- and schizophrenia-associated genes are linked to alterations in the genetic code of integrins and associated signalling pathways. In non-neuronal cells, crosstalk between integrin-mediated adhesions and the actin cytoskeleton, and the regulation of integrin activity (affinity for extracellular ligands) are widely studied in healthy and pathological settings. In contrast, the roles of integrin-linked pathways in the central nervous system remains less well defined. In this Review, we will provide an overview of the known pathways that are regulated by integrin-ECM interaction in developing neurons and in adult brain. We will also describe recent advances in the identification of mechanisms that regulate integrin activity in neurons, and highlight the interesting emerging links between integrins and neurodevelopment.
Nanostructured films obtained by assembling preformed atomic clusters are of strategic importance for a wide variety of applications. The deposition of clusters produced in the gas phase onto a substrate offers the possibility to control and engineer the structural and functional properties of the cluster-assembled films. To date, the microscopic mechanisms underlying the growth and structuring of cluster-assembled films are poorly understood, and, in particular, the transition from the submonolayer to the thin-film regime is experimentally unexplored. Here we report the systematic characterization by atomic force microscopy of the evolution of the structural properties of cluster-assembled films deposited by supersonic cluster beam deposition. As a paradigm of nanostructured systems, we focus our attention on cluster-assembled zirconia films, investigating the influence of the building block dimensions on the growth mechanisms and roughening of the thin films, following the growth process from the early stages of the submonolayer to the thin-film regime. Our results demonstrate that the growth dynamics in the submonolayer regime determines different morphological properties of the cluster-assembled thin film. The evolution of the roughness with the number of deposited clusters reproduces the growth exponent of the ballistic deposition in the 2+1 model from the submonolayer to the thin-film regime.
Integrin-mediated adhesions between cells and the extracellular matrix are fundamental for cell function, and one of their main roles is to sense and respond to mechanical force. Here we discuss the different mechanisms that can confer mechanosensitivity to adhesions. We first address molecular mechanisms mediated by force-induced changes in molecular properties, such as binding dynamics or protein conformation. Then, we discuss recent evidence on how these mechanisms are integrated with cellular and extracellular parameters such as myosin and actin activity, membrane tension, and ECM properties, endowing cells with an exquisite ability to both detect and respond to physical and mechanical cues from their environment.
Regulation of human pluripotent stem cell (hPSC) behaviors has been mainly studied through exploration of biochemical factors. However, current directed differentiation protocols for hPSCs that completely rely on biochemical factors remain suboptimal. It has recently become evident that coexisting biophysical signals in the stem cell microenvironment, including nanotopographic cues, can provide potent regulatory signals to mediate adult stem cell behaviors, including self-renewal and differentiation. Herein, we utilized a recently developed, large-scale nanofabrication technique based on reactive-ion etching (RIE) to generate random nanoscale structures on glass surfaces with high precision and reproducibility. We report here that hPSCs are sensitive to nanotopographic cues and such nanotopographic sensitivity can be leveraged for improving directed neuronal differentiation of hPSCs. We demonstrate early neuroepithelial conversion and motor neuron (MN) progenitor differentiation of hPSCs can be promoted using nanoengineered topographic substrates. We further explore how hPSCs sense substrate nanotopography and relay this biophysical signal through a regulatory signaling network involving cell adhesion, actomyosin cytoskeleton, and Hippo/YAP signaling to mediate neuroepithelial induction of hPSCs. Our study provides an efficient method for large-scale production of MNs from hPSCs, useful for regenerative medicine and cell-based therapies.
Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer’s ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.
The development of smart prosthetics, scaffolds, and biomaterials for tissue engineering and organ-on-a-chip devices heavily depends on the understanding and control of biotic/abiotic interfaces. In recent years, the nanometer scale emerged as the predominant dimension for processes impacting on protein adsorption and cellular responses on surfaces. In this context, the extracellular matrix (ECM) can be seen as the prototype for an intricate natural structure assembled by nanoscale building blocks forming highly variable nanoscale configurations, dictating cellular behavior and fate. How exactly the ECM nanotopography influences mechanotransduction, that is, the cellular capacity to convert information received from the ECM into appropriate responses, remains partially understood due to the complexity of the involved biological structures, limiting also the attempts to artificially reproduce the nanoscale complexity of the ECM. In this Account, we describe and discuss our strategies for the development of an efficient and large-scale bottom-up approach to fabricate surfaces with multiscale controlled disorder as substrates to study quantitatively the effect of nanoscale topography on biological entities. Our method is based on the use of supersonic cluster beam deposition (SCBD) to assemble, on a substrate, neutral clusters produced in the gas phase and accelerated by a supersonic expansion. The assembling of clusters in the ballistic deposition regime follows simple scaling laws, allowing the quantitative control of surface roughness and asperity layout over large areas. Due to their biocompatibility, we focused on transition metal oxide nanostructured surfaces assembled by titania and zirconia clusters. We demonstrated the engineering of structural and functional properties of the cluster-assembled surfaces with high relevance for interactions at the biotic/abiotic interface. We observed that isoelectric point and wettability, crucial parameters for the adhesion of biological entities on surfaces, are strongly influenced and controlled by the nanoscale roughness. By developing a high-throughput method (protein surface interaction microarray, PSIM), we characterized quantitatively the capacity of the nanostructured surfaces to adsorb proteins, showing that with increasing roughness the adsorption rises beyond what could be expected by the increase in specific area, paralleled by an almost linear decrease in protein binding affinity. We also determined that the spatial layout of the surface asperities effectively perceived by the cells mimics at the nanoscale the topographical ECM characteristics. The interaction with these features consequently regulates parameters significant for cell adhesion and mechanotransductive signaling, such as integrin clustering, focal adhesion maturation, and the correlated cellular mechanobiology, eventually impacting the cellular program and differentiation, as we specifically showed for neuronal cells.
Integrins are a large family of extracellular matrix (ECM) receptors. In the developing and adult brain, many integrins are present at high levels at synapses. The tetrapartite structure of synapses - which comprises presynaptic and postsynaptic neurons, the ECM and glial processes - places synaptic integrins in an excellent position to sense dynamic changes in the synaptic environment and use this information to coordinate further changes in synapse structure and function that will shape neural circuit properties. Recent developments in our understanding of the cellular and physiological roles of integrins, which range from control of neural process outgrowth and synapse formation to regulation of synaptic plasticity and memory, enable us to attempt a synthesis of synaptic integrin function.
Nanostructured zirconium dioxide (zirconia) films are very promising for catalysis and biotechnological applications: a precise control of the interfacial properties of the material at different length scales and, in particular, at the nanoscale, is therefore necessary. Here, we present the characterization of cluster-assembled zirconia films produced by supersonic cluster beam deposition possessing cubic structure at room temperature and controlled nanoscale morphology. We characterized the effect of thermal annealing in reducing and oxidizing conditions on the crystalline structure, grain dimensions, and topography. We highlight the mechanisms of film growth and phase transitions, which determine the observed interfacial morphological properties and their resilience against thermal treatments.
A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.
Cell function depends on tissue rigidity, which cells probe by applying and transmitting forces to their extracellular matrix, and then transducing them into biochemical signals. Here we show that in response to matrix rigidity and density, force transmission and transduction are explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. We demonstrate that force transmission is regulated by a dynamic clutch mechanism, which unveils its fundamental biphasic force/rigidity relationship on talin depletion. Force transduction is triggered by talin unfolding above a stiffness threshold. Below this threshold, integrins unbind and release force before talin can unfold. Above the threshold, talin unfolds and binds to vinculin, leading to adhesion growth and YAP nuclear translocation. Matrix density, myosin contractility, integrin ligation and talin mechanical stability differently and nonlinearly regulate both force transmission and the transduction threshold. In all cases, coupling of talin unfolding dynamics to a theoretical clutch model quantitatively predicts cell response.
Communication, the effective delivery of information, is fundamental to life across all scales and species. Nervous systems (by necessity) may be most specifically adapted among biological tissues for high rate and complexity of information transmitted, and thus, the properties of neural tissue and principles of its organization into circuits may illuminate capabilities and limitations of biological communication. Here, we consider recent developments in tools for studying neural circuits with particular attention to defining neuronal cell types by input and output information streams-i.e., by how they communicate. Complementing approaches that define cell types by virtue of genetic promoter/enhancer properties, this communication-based approach to defining cell types operationally by input/output (I/O) relationships links structure and function, resolves difficulties associated with single-genetic-feature definitions, leverages technology for observing and testing significance of precisely these I/O relationships in intact brains, and maps onto processes through which behavior may be adapted during development, experience, and evolution.
The fundamental mechanisms of complex neural computation remain largely unknown, especially in respect to the characteristics of distinct neural circuits within the mammalian brain. The bottom-up approach of building well-defined neural networks with controlled topology has immense promise for improved reproducibility and increased target selectivity and response of drug action, along with hopes to unravel the relationships between functional connectivity and its imprinted physiological and pathological functions. In this review, we summarize the different approaches available for engineering neural networks treated analogously to a mathematical graph consisting of cell bodies and axons as nodes and edges, respectively. After discussing the advances and limitations of the current techniques in terms of cell placement to the nodes and guiding the growth of axons to connect them, the basic properties of patterned networks are analyzed in respect to cell survival and activity dynamics, and compared to that of in vivo and random in vitro cultures. Besides the fundamental scientific interest and relevance to drug and toxicology tests, we also visualize the possible applications of such engineered networks. The review concludes by comparing the possibilities and limitations of the different methods for realizing in vitro engineered neural networks in 2D.
Biophysical signals act as potent regulators of stem cell function, lineage commitment, and epigenetic status. In recent years, synthetic biomaterials have been used to study a wide range of outside-in signaling events, and it is now well appreciated that material cues modulate the epigenome. Here, we review the role of extracellular signals in guiding stem cell behavior via epigenetic regulation, and we stress the role of physicochemical material properties as an often-overlooked modulator of intracellular signaling. We also highlight promising new research tools for ongoing interrogation of the stem cell-material interface.
Neurons and neural stem cells are sensitive to their mechanical and topographical environment, and cell-substrate binding contributes to this sensitivity to activate signaling pathways for basic cell functions. Many transmembrane proteins transmit signals into and out of the cell including integrins, growth factor receptors, G-protein coupled receptors, cadherins, cell adhesion molecules, and ion channels. Specifically, integrins are one of the main transmembrane proteins that transmit force across the cell membrane between a cell and its extracellular matrix, making them critical in the study of cell-material interactions. This review focuses on mechanotransduction, defined as the conversion of force a cell generates through cell-substrate bonds to a chemical signal, of neural cells. The chemical signals relay information via pathways through the cellular cytoplasm to the nucleus, where signaling events can affect gene expression. Pathways and the cellular response initiated by substrate binding are explored to better understand their effect on neural cells mechanotransduction. As the results of mechanotransduction affects cell adhesion, cell shape, and differentiation, knowledge regarding neural mechanotransduction is critical for most regenerative strategies in tissue engineering, where novel environments are developed to improve conduit design for central and peripheral nervous system repair in vivo.
Great emphasis is placed on the development of integrated approaches for the
synthesis and the characterization of ad hoc nanostructured platforms, to be
used as templates with controlled morphology and chemical properties for the
investigation of specific phenomena of great relevance for technological
applications in interdisciplinary fields such as biotechnology, medicine and
advanced materials. Here we discuss the crucial role and the advantages of thin
film deposition strategies based on cluster-assembling from supersonic cluster
beams. We select cluster-assembled nanostructured titania (ns-TiO2) as a case
study to demonstrate that accurate control over morphological parameters can be
routinely achieved, and consequently over several relevant interfacial
properties and phenomena, like surface charging in a liquid electrolyte, and
proteins and nanoparticles adsorption.
This review is an attempt to shed light on the wealth of the close association between micro/
nanotechnologies and neurobiology. With this aim, we have identified four specific areas in the field of neuroscience that fruitfully exploit the conceptual approaches of physics together with the engineering tools originally developed for microelectronics. Each of these areas are developed in this review within a dedicated section. The first section ‘‘Isolating environmental parameters’’ illustrates how fabricating specific microenvironments allows one to isolate the cell response to specific physiological stimuli, either chemical or topographical. In a complementary way, the section ‘‘Revealing the properties of brain cells’’ shows how the design of rather artificial in vitro culture conditions can give precious insights into the
field of neurobiology. The acquired knowledge concerning the response of isolated neurons to external physical cues may inspire methodologies for mastering the architecture, polarity and connectivity of in vitro neuronal assemblies. These aspects are developed in the section ‘‘Controlling neuronal architectures’’. Finally, a section entitled ‘‘Instrumentation of neuronal networks’’ reports some salient examples of recent innovative achievements in the field of neuron–electronic interfacing, illustrating how the issue of neuronal contact with micro-structured environments supports the development of advanced multiplexed electrophysiology methods. We will finally conclude this review by introducing some medical applications originating from the contribution of micro/nanotechnologies to the field of neuroscience. Our take-home message is that configuring the cellular microenvironment using microengineering tools has a strong positive impact in neuroscience at different levels, from the most fundamentals of cellular neurobiology to medical applications.
http://authors.elsevier.com/a/1P-o1cBkcAEMc
Neurons are sensitive to topographical cues provided either by in vivo or in vitro environments on the micrometric scale. We have explored the role of randomly distributed silicon nano-pillars on primary hippocampal neurite elongation and axonal differentiation. We observed that neurons adhere on the upper part of nano-pillars with a typical distance between adhesion points of about 500nm. These neurons produce less neurites, elongate faster, and differentiate an axon earlier than those grown on flat silicon surfaces. Moreover, when confronted to a differential surface topography, neurons specify an axon preferentially onto nano-pillars. As a whole, these results highlight the influence of the physical environment in many aspects of neuronal growth.
Integrin-mediated focal adhesions (FAs) are large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix and take part in adhesion-mediated signaling. These adhesions are highly complex and diverse at the molecular level; thus, assigning particular structural or signaling functions to specific components is highly challenging. Here, we combined functional, structural and biophysical approaches to assess the role of a major FA component, namely, integrin-linked kinase (ILK), in adhesion formation. We show here that ILK plays a key role in the formation of focal complexes, early forms of integrin adhesions, and confirm its involvement in the assembly of fibronectin-bound fibrillar adhesions. Examination of ILK-null fibroblasts by cryo-electron tomography pointed to major structural changes in their FAs, manifested by disarray of the associated actin filaments and an increase in the packing density of FA-related particles (FARPs). Interestingly, adhesion of the mutant cells to the substrate required a higher ligand density than control cells. These data indicate that ILK has a key role in integrin adhesion assembly and sub-structure, and in the regulation of the FA-associated cytoskeleton.
Biological cells are well known to respond to a multitude of chemical signals. In the nervous system, chemical signaling has been shown to be crucially involved in development, normal functioning, and disorders of neurons and glial cells. However, there are an increasing number of studies showing that these cells also respond to mechanical cues. Here, we summarize current knowledge about the mechanical properties of nervous tissue and its building blocks, review recent progress in methodology and understanding of cellular mechanosensitivity in the nervous system, and provide an outlook on the implications of neuromechanics for future developments in biomedical engineering to aid overcoming some of the most devastating and currently incurableCNSpathologies such as spinal cord injuries and multiple sclerosis. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 15 is July 11, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Intense and collimated supersonic cluster beams have been produced by exploiting inertial focusing effects. To this goal we have developed and tested a novel focusing nozzle (focuser). Using this device with a pulsed microplasma cluster source we have obtained cluster beams with a divergence of 10 mrad and average densities of 3×1010 atoms/cm3 (2×1012 atoms/cm3 pulsed) corresponding to deposition rates of 2 nm/s at 300 mm distance from the source nozzle. With a focusing nozzle cluster thermal relaxation and mass distribution in a supersonic expansion can be controlled. We have measured the cluster transverse velocities, with extremely high precision, by characterizing the cluster beam deposition on a substrate by an atomic force microscope. Besides the relevance for the understanding of relaxation processes in expanding jets, the inertial focusing of clusters has several important consequences for the synthesis of nanostructured films with controlled structure and for all the experimental techniques requiring intense and collimated cluster beams. Due to its simplicity the focusing nozzle presented here can be used with a wide variety of cluster sources. © 2001 American Institute of Physics.
The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.
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