Advances in 3D tissue models for neural engineering: self-assembled versus engineered tissue models.

Neural tissue engineering has emerged as a promising field that aims to create functional neural tissue for therapeutic applications, drug screening, and disease modelling. It is becoming evident in the literature that this goal requires development of three-dimensional (3D) constructs that can mimic the complex microenvironment of native neural tissue, including its biochemical, mechanical, physical, and electrical properties. These 3D models can be broadly classified as self-assembled models, which include spheroids, organoids, and assembloids, and engineered models, such as those based on decellularized or polymeric scaffolds. Self-assembled models offer advantages such as the ability to recapitulate neural development and disease processes in vitro, and the capacity to study the behaviour and interactions of different cell types in a more realistic environment. However, self-assembled constructs have limitations such as lack of standardised protocols, inability to control the cellular microenvironment, difficulty in controlling structural characteristics, reproducibility, scalability, and lengthy developmental timeframes. Integrating biomimetic materials and advanced manufacturing approaches to present cells with relevant biochemical, mechanical, physical, and electrical cues in a controlled tissue architecture requires alternate engineering approaches. Engineered scaffolds, and specifically 3D hydrogel-based constructs, have desirable properties, lower cost, higher reproducibility, long-term stability, and they can be rapidly tailored to mimic the native microenvironment and structure. This review explores 3D models in neural tissue engineering, with a particular focus on analysing the benefits and limitations of self-assembled organoids compared with hydrogel-based engineered 3D models. Moreover, this paper will focus on hydrogel based engineered models and probe their biomaterial components, tuneable properties, and fabrication techniques that allow them to mimic native neural tissue structures and environment. Finally, the current challenges and future research prospects of 3D neural models for both self-assembled and engineered models in neural tissue engineering will be discussed.

Deciphering platinum dissolution in neural stimulation electrodes: Electrochemistry or biology?

Platinum (Pt) is the metal of choice for electrodes in implantable neural prostheses like the cochlear implants, deep brain stimulating devices, and brain-computer interfacing technologies. However, it is well known since the 1970s that Pt dissolution occurs with electrical stimulation. More recent clinical and in vivo studies have shown signs of corrosion in explanted electrode arrays and the presence of Pt-containing particulates in tissue samples. The process of degradation and release of metallic ions and particles can significantly impact on device performance. Moreover, the effects of Pt dissolution products on tissue health and function are still largely unknown. This is due to the highly complex chemistry underlying the dissolution process and the difficulty in decoupling electrical and chemical effects on biological responses. Understanding the mechanisms and effects of Pt dissolution proves challenging as the dissolution process can be influenced by electrical, chemical, physical, and biological factors, all of them highly variable between experimental settings. By evaluating comprehensive findings on Pt dissolution mechanisms reported in the fuel cell field, this review presents a critical analysis of the possible mechanisms that drive Pt dissolution in neural stimulation in vitro and in vivo. Stimulation parameters, such as aggregate charge, charge density, and electrochemical potential can all impact the levels of dissolved Pt. However, chemical factors such as electrolyte types, dissolved gases, and pH can all influence dissolution, confounding the findings of in vitro studies with multiple variables. Biological factors, such as proteins, have been documented to exhibit a mitigating effect on the dissolution process. Other biological factors like cells and fibro-proliferative responses, such as fibrosis and gliosis, impact on electrode properties and are suspected to impact on Pt dissolution. However, the relationship between electrical properties of stimulating electrodes and Pt dissolution remains contentious. Host responses to Pt degradation products are also controversial due to the unknown chemistry of Pt compounds formed and the lack of understanding of Pt distribution in clinical scenarios. The cytotoxicity of Pt produced via electrical stimulation appears similar to Pt-based compounds, including hexachloroplatinates and chemotherapeutic agents like cisplatin. While the levels of Pt produced under clinical and acute stimulation regimes were typically an order of magnitude lower than toxic concentrations observed in vitro, further research is needed to accurately assess the mass balance and type of Pt produced during long-term stimulation and its impact on tissue response. Finally, approaches to mitigating the dissolution process are reviewed. A wide variety of approaches, including stimulation strategies, coating electrode materials, and surface modification techniques to avoid excess charge during stimulation and minimise tissue response, may ultimately support long-term and safe operation of neural stimulating devices.

Growing human-scale scala tympani-likein vitrocell constructs.

Emerging materials and electrode technologies have potential to revolutionise development of higher resolution next-generation, bionic devices. However, barriers associated with the extended timescales, regulatory constraints, and opportunity costs of preclinical and clinical studies, can inhibit such innovation. Development ofin vitromodels that mimic human tissues would provide an enabling platform to overcome many of these barriers in the product development pathway. This research aimed to develop human-scale tissue engineered cochlea models for high throughput evaluation of cochlear implants on the bench. Novel mould-casting techniques and stereolithography three-dimensional (3D) printing approaches to template hydrogels into spiral-shaped structures resembling the scala tympani were compared. While hydrogels are typically exploited to support 3D tissue-like structures, the challenge lies in developing irregular morphologies like the scala tympani, in which the cochlear electrodes are commonly implanted. This study successfully developed human-scale scala tympani-like hydrogel structures that support viable cell adhesion and can accommodate cochlear implants for future device testing.

Improving Deep Brain Stimulation Electrode Performance in vivo Through Use of Conductive Hydrogel Coatings.

Active implantable neurological devices like deep brain stimulators have been used over the past few decades to treat movement disorders such as those in people with Parkinson's disease and more recently, in psychiatric conditions like obsessive compulsive disorder. Electrode-tissue interfaces that support safe and effective targeting of specific brain regions are critical to success of these devices. Development of directional electrodes that activate smaller volumes of brain tissue requires electrodes to operate safely with higher charge densities. Coatings such as conductive hydrogels (CHs) provide lower impedances and higher charge injection limits (CILs) than standard platinum electrodes and support safer application of smaller electrode sizes. The aim of this study was to examine the chronic in vivo performance of a new low swelling CH coating that supports higher safe charge densities than traditional platinum electrodes. A range of hydrogel blends were engineered and their swelling and electrical performance compared. Electrochemical performance and stability of high and low swelling formulations were compared during insertion into a model brain in vitro and the formulation with lower swelling characteristics was chosen for the in vivo study. CH-coated or uncoated Pt electrode arrays were implanted into the brains of 14 rats, and their electrochemical performance was tested weekly for 8 weeks. Tissue response and neural survival was assessed histologically following electrode array removal. CH coating resulted in significantly lower voltage transient impedance, higher CIL, lower electrochemical impedance spectroscopy, and higher charge storage capacity compared to uncoated Pt electrodes in vivo, and this advantage was maintained over the 8-week implantation. There was no significant difference in evoked potential thresholds, signal-to-noise ratio, tissue response or neural survival between CH-coated and uncoated Pt groups. The significant electrochemical advantage and stability of CH coating in the brain supports the suitability of this coating technology for future development of smaller, higher fidelity electrode arrays with higher charge density requirement.

Challenges and solutions for fabrication of three-dimensional cocultures of neural cell-loaded biomimetic constructs.

Fabrication of three-dimensional (3D) constructs to model body tissues and organs can contribute to research into tissue development and models for studying disease, as well as supporting preclinical drug screening in vitro. Furthermore, 3D constructs can also be used for diagnosis and therapy of disease conditions via lab on a chip and microarrays for diagnosis and engineered products for tissue repair, replacement, and regeneration. While cell culture approaches for studying tissue development and disease in two dimensions are long-established, the translation of this knowledge into 3D environments remains a fertile field of research. In this Tutorial, we specifically focus on the application of biosynthetic hydrogels for neural cell encapsulation. The Tutorial briefly covers background on using biosynthetic hydrogels for cell encapsulation, as well as common fabrication techniques. The Methods section focuses on the hydrogel design and characterization, highlighting key elements and tips for more effective approaches. Coencapsulation of different cell types, and the challenges associated with different growth and maintenance requirements, is the main focus of this Tutorial. Much care is needed to blend different cell types, and this Tutorial provides tips and insights that have proven successful for 3D coculture in biosynthetic hydrogels.

Subthreshold Electrical Stimulation for Controlling Protein-Mediated Impedance Increases in Platinum Cochlear Electrode.

OBJECTIVE: This study evaluated subthreshold biphasic stimulation pulses as a strategy to stabilize electrode impedance via control of protein adsorption. Following implantation, cochlear electrodes undergo impedance fluctuations thought to be caused by protein adsorption and/or inflammatory responses. Impedance increases can impact device power consumption, safe charge injection limits, and long-term stability of electrodes. METHODS: Protein-mediated changes in polarization impedance (Zp) were measured by voltage transient responses to biphasic current pulses and electrochemical impedance spectroscopy, with and without protein solutions. Four subthreshold stimulation regimes were studied to assess their effects on protein adsorption and impedance; (1) symmetric charge-balanced pulses delivered continuously, (2) at 10% duty cycle, (3) at 1% duty cycle, and (4) an asymmetric charge balanced pulse delivered continuously with a cathodic phase twice as long as the anodic phase. RESULTS: The Zp of electrodes incubated in protein solutions without stimulation for 2 h increased by between ∼28% and ∼55%. Subthreshold stimulation reduced the rate at which impedance increased following exposure to all protein solutions. Decreases in Zp were dependent on the type of protein solution and the stimulation regime. Subthreshold stimulation pulses were more effective when delivered continuously compared to 1% and 10% duty cycles. CONCLUSION: These results support the potential of subthreshold stimulation pulses to mitigate protein-mediated increase in impedance. SIGNIFICANCE: This research highlights the potential of clinically translatable stimulation pulses to mitigate perilymph protein adsorption on cochlear electrodes, a key phenomenon precursor of the inflammatory response.

Ambient-Temperature Waterborne Polymer/rGO Nanocomposite Films: Effect of rGO Distribution on Electrical Conductivity.

Electrically conductive polymer/rGO (reduced graphene oxide) films based on styrene and n-butyl acrylate are prepared by a variety of aqueous latex based routes involving ambient temperature film formation. Techniques based on miniemulsion polymerization using GO as surfactant and "physical mixing" approaches (i.e., mixing an aqueous polymer latex with an aqueous GO dispersion) are employed, followed by heat treatment of the films to convert GO to rGO. The distribution of GO sheets and the electrical conductivity depend strongly on the preparation method, with electrical conductivities in the range 9 × 10-4 to 3.4 × 102 S/m. Higher electrical conductivities are obtained using physical mixing compared to miniemulsion polymerization, which is attributed to the former providing a higher level of self-alignment of rGO into larger linear domains. The present results illustrate how the distribution of GO sheets within these hybrid materials can to some extent be controlled by judicious choice of preparation method, thereby providing an attractive means of nanoengineering for specific potential applications.

Conductive elastomer composites for fully polymeric, flexible bioelectronics.

Flexible polymeric bioelectronics have the potential to address the limitations of metallic electrode arrays by minimizing the mechanical mismatch at the device-tissue interface for neuroprosthetic applications. This work demonstrates the straightforward fabrication of fully organic electrode arrays based on conductive elastomers (CEs) as a soft, flexible and stretchable electroactive composite material. CEs were designed as hybrids of polyurethane elastomers (PU) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS), with the aim of combining the electrical properties of PEDOT:PSS with the mechanical compliance of elastomers. CE composites were fabricated by solvent casting of PEDOT:PSS dispersed in dissolved PU at different conductive polymer (CP) loadings, from 5 wt% to 25 wt%. The formation of PEDOT:PSS networks within the PU matrix and the resultant composite material properties were examined as a function of CP loading. Increased PEDOT:PSS loading was found to result in a more connected network within the PU matrix, resulting in increased conductivity and charge storage capacity. Increased CP loading was also determined to increase the Young's modulus and reduce the strain at failure. Biological assessment of CE composites showed them to mediate ReNcell VM human neural precursor cell adhesion. The increased stiffness of CE films was also found to promote neurite outgrowth. CE sheets were directly laser micromachined into a functional array and shown to deliver biphasic waveforms with comparable voltage transients to Pt arrays in in vitro testing.

Tissue engineered hydrogels supporting 3D neural networks.

Promoting nerve regeneration requires engineering cellular carriers to physically and biochemically support neuronal growth into a long lasting functional tissue. This study systematically evaluated the capacity of a biosynthetic poly(vinyl alcohol) (PVA) hydrogel to support growth and differentiation of co-encapsulated neurons and glia. A significant challenge is to understand the role of the dynamic degradable hydrogel mechanical properties on expression of relevant cellular morphologies and function. It was hypothesised that a carrier with mechanical properties akin to neural tissue will provide glia with conditions to thrive, and that glia in turn will support neuronal survival and development. PVA co-polymerised with biological macromolecules sericin and gelatin (PVA-SG) and with tailored nerve tissue-like mechanical properties were used to encapsulate Schwann cells (SCs) alone and subsequently a co-culture of SCs and neural-like PC12s. SCs were encapsulated within two PVA-SG gel variants with initial compressive moduli of 16 kPa and 2 kPa, spanning a range of reported mechanical properties for neural tissues. Both hydrogels were shown to support cell viability and expression of extracellular matrix proteins, however, SCs grown within the PVA-SG with a higher initial modulus were observed to present with greater physiologically relevant morphologies and increased expression of extracellular matrix proteins. The higher modulus PVA-SG was subsequently shown to support development of neuronal networks when SCs were co-encapsulated with PC12s. The lower modulus hydrogel was unable to support effective development of neural networks. This study demonstrates the critical link between hydrogel properties and glial cell phenotype on development of functional neural tissues. STATEMENT OF SIGNIFICANCE: Hydrogels as platforms for tissue regeneration must provide encapsulated cellular progenitors with physical and biochemical cues for initial survival and to support ongoing tissue formation as the artificial network degrades. While most research focuses on tailoring scaffold properties to suit neurons, this work aims to support glia SCs as the key cellular component that physically and biochemically supports the neuronal network. The challenge is to modify hydrogel properties to support growth and development of multiple cell types into a neuronal network. Given SCs ability to respond to substrate mechanical properties, the significance of this work lies in understanding the relationship between dynamic hydrogel mechanical properties and glia SCs development as the element that enables formation of mature, differentiated neural networks.

Cytotoxic Activity of a Black Bean (Phaseolus vulgaris L.) Extract and its Flavonoid Fraction in Both In Vitro and In Vivo Models of Lymphoma.

BACKGROUND: Black bean (Phaseolus vulgaris L.) is a very common legume seed in Mexican diet. Flavonoids and crude extracts from different plants have been reported as effective agents for chemoprevention and cytotoxicity in several cancer cell lines. We investigated the effects of black bean hulls extract (BBE) and its flavonoid fraction (FF) on lymphoma cells. METHODS: BBE and FF were characterized by high-performance liquid chromatography. Viability and flow cytometry assays were carried out. Finally, a mouse model was generated to test the in vivo effect of both fractions. RESULTS: Both BBE and FF inhibited cell proliferation in a dose-dependent way. In addition, cells underwent apoptosis, and the cellular population at S-phase increased after exposure to these fractions. Furthermore, mice treated with BBE or FF increased the overall survival by 5 or 6 days, respectively, in comparison with a placebo group (p = 0.056). DISCUSSION: BBE and FF had cytotoxic action by driving OCI-Ly7 cells into apoptosis as well as blocking progression to G2/M phase. In addition, BBE and FF treatments were effective in xenograft models.

Visual Prosthesis: Interfacing Stimulating Electrodes with Retinal Neurons to Restore Vision.

The bypassing of degenerated photoreceptors using retinal neurostimulators is helping the blind to recover functional vision. Researchers are investigating new ways to improve visual percepts elicited by these means as the vision produced by these early devices remain rudimentary. However, several factors are hampering the progression of bionic technologies: the charge injection limits of metallic electrodes, the mechanical mismatch between excitable tissue and the stimulating elements, neural and electric crosstalk, the physical size of the implanted devices, and the inability to selectively activate different types of retinal neurons. Electrochemical and mechanical limitations are being addressed by the application of electromaterials such as conducting polymers, carbon nanotubes and nanocrystalline diamonds, among other biomaterials, to electrical neuromodulation. In addition, the use of synthetic hydrogels and cell-laden biomaterials is promising better interfaces, as it opens a door to establishing synaptic connections between the electrode material and the excitable cells. Finally, new electrostimulation approaches relying on the use of high-frequency stimulation and field overlapping techniques are being developed to better replicate the neural code of the retina. All these elements combined will bring bionic vision beyond its present state and into the realm of a viable, mainstream therapy for vision loss.

Producing 3D neuronal networks in hydrogels for living bionic device interfaces.

Hydrogels hold significant promise for supporting cell based therapies in the field of bioelectrodes. It has been proposed that tissue engineering principles can be used to improve the integration of neural interfacing electrodes. Degradable hydrogels based on poly (vinyl alcohol) functionalised with tyramine (PVA-Tyr) have been shown to support covalent incorporation of non-modified tyrosine rich proteins within synthetic hydrogels. PVA-Tyr crosslinked with such proteins, were explored as a scaffold for supporting development of neural tissue in a three dimensional (3D) environment. In this study a model neural cell line (PC12) and glial accessory cell line, Schwann cell (SC) were encapsulated in PVA-Tyr crosslinked with gelatin and sericin. Specifically, this study aimed to examine the growth and function of SC and PC12 co-cultures when translated from a two dimensional (2D) environment to a 3D environment. PC12 differentiation was successfully promoted in both 2D and 3D at 25 days post-culture. SC encapsulated as a single cell line and in co-culture were able to produce both laminin and collagen-IV which are required to support neuronal development. Neurite outgrowth in the 3D environment was confirmed by immunocytochemical staining. PVA-Tyr/sericin/gelatin hydrogel showed mechanical properties similar to nerve tissue elastic modulus. It is suggested that the mechanical properties of the PVA-Tyr hydrogels with native protein components are providing with a compliant substrate that can be used to support the survival and differentiation of neural networks.

Organic electrode coatings for next-generation neural interfaces.

Traditional neuronal interfaces utilize metallic electrodes which in recent years have reached a plateau in terms of the ability to provide safe stimulation at high resolution or rather with high densities of microelectrodes with improved spatial selectivity. To achieve higher resolution it has become clear that reducing the size of electrodes is required to enable higher electrode counts from the implant device. The limitations of interfacing electrodes including low charge injection limits, mechanical mismatch and foreign body response can be addressed through the use of organic electrode coatings which typically provide a softer, more roughened surface to enable both improved charge transfer and lower mechanical mismatch with neural tissue. Coating electrodes with conductive polymers or carbon nanotubes offers a substantial increase in charge transfer area compared to conventional platinum electrodes. These organic conductors provide safe electrical stimulation of tissue while avoiding undesirable chemical reactions and cell damage. However, the mechanical properties of conductive polymers are not ideal, as they are quite brittle. Hydrogel polymers present a versatile coating option for electrodes as they can be chemically modified to provide a soft and conductive scaffold. However, the in vivo chronic inflammatory response of these conductive hydrogels remains unknown. A more recent approach proposes tissue engineering the electrode interface through the use of encapsulated neurons within hydrogel coatings. This approach may provide a method for activating tissue at the cellular scale, however, several technological challenges must be addressed to demonstrate feasibility of this innovative idea. The review focuses on the various organic coatings which have been investigated to improve neural interface electrodes.