Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development.

The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds

Boron-doped nanocrystalline diamond electrodes for neural interfaces:in vivo biocompatibility evaluation

Boron-doped nanocrystalline diamond (BDD) electrodes have recently attracted attention as materials for neural electrodes due to their superior physical and electrochemical properties, however their biocompatibility remains largely unexplored. In this work, we aim to investigate the in vivo biocompatibility of BDD electrodes in relation to conventional titanium nitride (TiN) electrodes using a rat subcutaneous implantation model. High quality BDD films were synthesized on electrodes intended for use as an implantable neurostimulation device. After implantation for 2 and 4 weeks, tissue sections adjacent to the electrodes were obtained for histological analysis. Both types of implants were contained in a thin fibrous encapsulation layer, the thickness of which decreased with time. Although the level of neovascularization around the implants was similar, BDD electrodes elicited significantly thinner fibrous capsules and a milder inflammatory reaction at both time points. These results suggest that BDD films may constitute an appropriate material to support stable performance of implantable neural electrodes over time

Ultrasound-Activated Piezoelectric Nanoparticles Inhibit Proliferation of Breast Cancer Cells

Abstract A nanotechnology-based approach for the inhibition of breast cancer cell proliferation is proposed. The innovative solution consists in a platform based on biocompatible piezoelectric nanoparticles able to target and remotely stimulate HER2-positive breast cancer cells. The anti-proliferative effects of the ultrasound-driven piezoelectric nanoparticle-assisted stimulation significantly reduced the proliferation by inducing the cell cycle arrest. Similarly to a low-intensity alternating electric field, chronic piezoelectric stimulation resulted able to inhibit cancer cell proliferation by upregulating the expression of the gene encoding Kir3.2 inward rectifier potassium channels, by interfering on Ca2+ homeostasis, and by affecting the organization of mitotic spindles during mitosis. The proposed platform, even if specific for HER2-positive cells, shows huge potential and versatility for the treatment of different type of cancers

Hypergravity As a Tool for Cell Stimulation: Implications in Biomedicine

Gravity deeply influences numerous biological events in living organisms. Variations in gravity values induce adaptive reactions that have been shown to play important roles, for instance in cell survival, growth, and spatial organization. In this paper, we summarize effects of gravity values higher than that one experienced by cells and tissues on Earth, i.e., hypergravity, with particular attention to the nervous and the musculoskeletal systems. Besides the biological consequences that hypergravity induces in the living matter, we will discuss the possibility of exploiting this augmented force in tissue engineering and regenerative medicine, and thus hypergravity significance as a new therapeutic approach both in vitro and in vivo

Design, assembly and test of a bioreactor for the electrical stimulation of neuronal cells

Millions of people have suffered injury to their nervous system, which by its limited self- healing capacity, represents life-long complications, associated with loss of motor and sensory function. Though limited, this capacity is being extensively explored, and has been shown to increase through the use of electrical stimulation (ES). Therefore, this work was oriented towards the development of a setup for ES of neuronal cells, allowing the assessment of its potential in promoting neuronal regeneration. An ES chamber was designed using the CAD software Fusion 360TM and produced by machining of a poly(methyl methacrylate) (PMMA) block. A fixation system for con- ductive scaffolds was included, using stainless steel electrodes, which fits the description for a direct coupling method found in the literature. Connection to a power supply or a function generator is possible, allowing for delivery of both direct current (DC) and alternating current (AC) to cells. In a different design, electrical insulation of the media was attempted, defectively. The nature of this work supported the need for incorporating conductive polymers (CPs) in the scaffolds used for neuronal differentiation of cells in the stimulation chamber and so, poly(lactic acid) (PLA) aligned electruspun fibers were produced and coated with poly(3,4-ethylenedioxythiophene) (PEDOT) using vapor-phase polymerization (VPP). In this process, the polymerization takes place through the reaction of Iron(III) p-toluenesul– fonate/Fe(III)Tosylate (FeTos) included in the scaffolds with 3,4-ethylenedioxythiophene (EDOT) on vapor phase. This fibers did not exhibit cytotoxicity and electrical character- ization was attempted, using the bioreactor as a 2-point probe. Film casting using the same polymeric solutions failed, as an increase in the ratio of PLA to FeTos did not reduce film brittleness. In vitro assays were conducted both with and without stimulation. SH-SY5Y extended neurites after only 2 days of exposure to retinoic acid (RA). Cells maintained some level of differentiation and neurite directionality with time, when cultured in the produced fibers. Importantly, an electrical field of 500 mV/ cm was applied 1 h/day, for 9 days, without significant improvements on neuronal differentiation.Milhões de pessoas já sofreram lesões no seu sistema nervoso, o que, dada a sua capaci- dade limitada de regeneração, origina complicações a longo prazo, associadas à perda de função motora e sensorial. Embora limitada, esta capacidade tem sido amplamente explo- rada, e já se provou poder ser melhorada, com recurso a estimulação elétrica. Assim, este trabalho focou-se no desenvolvimento de um sistema para estimulação elétrica de células neuronais, permitindo avaliar o seu potencial para promover regeneração neuronal. Desenhou-se um sistema para estimulação neuronal recorrendo ao software Fusion 360TM e fabricou-se o mesmo por maquinagem de um bloco de PMMA. A montagem inclui um sistema para fixação de scaffolds condutores, usando elétrodos de aço inoxidável, correspondendo a um sistema de acoplamento direto, segundo a literatura. É possível estabelecer contactos elétricos com uma fonte de tensão ou um gerador de funções, o que permite fornecer às células correntes diretas e alternadas. Houve uma tentativa, sem sucesso, para um novo design que permitisse isolamento elétrico do meio. A natureza deste trabalho justificou a incorporação de polímeros condutores nos scaffolds usados para diferenciação neuronal de células no sistema de estimulação desen- volvido. Assim, foram eletrofiadas fibras alinhadas de PLA e, mais tarde, revestidas por PEDOT recorrendo a VPP. Neste processo, a polimerização ocorreu pela reação do FeTos, incluído nos scaffolds, com EDOT em fase de vapor. Foram feitas tentativas de produção de filmes, usando as mesmas soluções poliméricas, contudo verificou-se que o aumento da razão PLA:FeTos não reduziu a sua fragilidade. Foram realizados testes in vitro com e sem estimulação. As células SH-SY5Y estende- ram neurites, com apenas dois dias de exposição a meio contendo ácido retinóico. Quando semeadas nas fibras produzidas, estas células mantiveram um nível moderado de diferen- ciação neuronal ao longo do tempo, alinhando as suas extensões na direção das fibras. É de salientar que a exposição das células a um campo de 500 mV/ cm aplicado 1 h/dia, por 9 dias, não resultou num aumento significativo de diferenciação neuronal

Polydopamine nanoparticles as an organic and biodegradable multitasking tool for neuroprotection and remote neuronal stimulation

Oxidative stress represents a common issue in most neurological diseases, causing severe impairments of neuronal cell physiological activity that ultimately lead to neuron loss of function and cellular death. In this work, lipid-coated polydopamine nanoparticles (L-PDNPs) are proposed both as antioxidant and neuroprotective agents, and as well as a photo-thermal conversion platform able to stimulate neuronal activity. L-PDNPs showed the ability to counteract reactive oxygen species (ROS) accumulation in differentiated SH-SY5Y, prevented mitochondrial ROS-induced dysfunctions, and stimulated neurite outgrowth. Moreover, for the first time in the literature, the photo-thermal conversion capacity of L-PDNPs was used to increase the intracellular temperature of neuron-like cells through near-infrared (NIR) laser stimulation, and this phenomenon was thoroughly investigated using a fluorescent temperature-sensitive dye and modeled from a mathematical point of view. It was also demonstrated that the increment in temperature caused by the NIR stimulation of L-PDNPs was able to produce a Ca2+ influx in differentiated SH-SY5Y, being, to the best of our knowledge, the first example of organic nanostructures used in such an approach. This work could pave the way to new and exciting applications of polydopamine-based and of other NIR-responsive antioxidant nanomaterials in neuronal research

Hybrid Magnetic Nanovectors Promote Selective Glioblastoma Cell Death through a Combined Effect of Lysosomal Membrane Permeabilization and Chemotherapy

Glioblastoma multiforme is the most aggressive brain tumor, due to its high invasiveness and genetic heterogeneity. Moreover, the blood-brain barrier prevents many drugs from reaching a therapeutic concentration at the tumor site, and most of the chemotherapeutics lack in specificity toward cancer cells, accumulating in both healthy and diseased tissues, with severe side effects. Here, we present in vitro investigations on lipid-based nanovectors encapsulating a drug, nutlin-3a, and superparamagnetic iron oxide nanoparticles, to combine the proapoptotic action of the drug and the hyperthermia mediated by superparamagnetic iron oxide nanoparticles stimulated with an alternating magnetic field. The nanovectors are functionalized with the peptide angiopep-2 to induce receptor-mediated transcytosis through the blood-brain barrier and to target a receptor overexpressed by glioma cells. The glioblastoma multiforme targeting efficiency and the blood-brain barrier crossing abilities were tested through in vitro fluidic models, where different human cell lines were placed to mimic the tumor microenvironment. These nanovectors successfully cross the blood-brain barrier model, maintaining their targeting abilities for glioblastoma multiforme with minimal interaction with healthy cells. Moreover, we showed that nanovector-assisted hyperthermia induces a lysosomal membrane permeabilization that not only initiates a caspase-dependent apoptotic pathway, but also enhances the anticancer efficacy of the drug