Surface Tension and Neuronal Sorting in Magnetically Engineered Brain-Like Tissue.

Engineered 3D brain-like models have advanced the understanding of neurological mechanisms and disease, yet their mechanical signature, while fundamental for brain function, remains understudied. The surface tension for instance controls brain development and is a marker of cell-cell interactions. Here, 3D magnetic brain-like tissue spheroids composed of intermixed primary glial and neuronal cells at different ratios are engineered. Remarkably, the two cell types self-assemble into a functional tissue, with the sorting of the neuronal cells toward the periphery of the spheroids, whereas the glial cells constitute the core. The magnetic fingerprint of the spheroids then allows their deformation when placed under a magnetic field gradient, at a force equivalent to a 70 g increased gravity at the spheroid level. The tissue surface tension and elasticity can be directly inferred from the resulting deformation, revealing a transitional dependence on the glia/neuron ratio, with the surface tension of neuronal tissue being much lower. The results suggest an underlying mechanical contribution to the exclusion of the neurons toward the outer spheroid region, and depict the glia/neuron organization as a sophisticated mechanism that should in turn influence tissue development and homeostasis relevant in the neuroengineering field.

Modulated nanowire scaffold for highly efficient differentiation of mesenchymal stem cells.

BACKGROUND: Nanotopographical cues play a critical role as drivers of mesenchymal stem cell differentiation. Nanowire scaffolds, in this regard, provide unique and adaptable nanostructured surfaces with focal points for adhesion and with elastic properties determined by nanowire stiffness. RESULTS: We show that a scaffold of nanowires, which are remotely actuated by a magnetic field, mechanically stimulates mesenchymal stem cells. Osteopontin, a marker of osteogenesis onset, was expressed after cells were cultured for 1 week on top of the scaffold. Applying a magnetic field significantly boosted differentiation due to mechanical stimulation of the cells by the active deflection of the nanowire tips. The onset of differentiation was reduced to 2 days of culture based on the upregulation of several osteogenesis markers. Moreover, this was observed in the absence of any external differentiation factors. CONCLUSIONS: The magneto-mechanically modulated nanosurface enhanced the osteogenic differentiation capabilities of mesenchymal stem cells, and it provides a customizable tool for stem cell research and tissue engineering.

Transient cell stiffening triggered by magnetic nanoparticle exposure.

BACKGROUND: The interactions between nanoparticles and the biological environment have long been studied, with toxicological assays being the most common experimental route. In parallel, recent growing evidence has brought into light the important role that cell mechanics play in numerous cell biological processes. However, despite the prevalence of nanotechnology applications in biology, and in particular the increased use of magnetic nanoparticles for cell therapy and imaging, the impact of nanoparticles on the cells' mechanical properties remains poorly understood. RESULTS: Here, we used a parallel plate rheometer to measure the impact of magnetic nanoparticles on the viscoelastic modulus G*(f) of individual cells. We show how the active uptake of nanoparticles translates into cell stiffening in a short time scale (< 30 min), at the single cell level. The cell stiffening effect is however less marked at the cell population level, when the cells are pre-labeled under a longer incubation time (2 h) with nanoparticles. 24 h later, the stiffening effect is no more present. Imaging of the nanoparticle uptake reveals almost immediate (within minutes) nanoparticle aggregation at the cell membrane, triggering early endocytosis, whereas nanoparticles are almost all confined in late or lysosomal endosomes after 2 h of uptake. Remarkably, this correlates well with the imaging of the actin cytoskeleton, with actin bundling being highly prevalent at early time points into the exposure to the nanoparticles, an effect that renormalizes after longer periods. CONCLUSIONS: Overall, this work evidences that magnetic nanoparticle internalization, coupled to cytoskeleton remodeling, contributes to a change in the cell mechanical properties within minutes of their initial contact, leading to an increase in cell rigidity. This effect appears to be transient, reduced after hours and disappearing 24 h after the internalization has taken place.

3D Printed Microneedle Array for Electroporation.

In-vitro transfection of cells by electroporation is a widely used approach in cell biology and medicine. The transfection method is highly dependent on the cell culture's electrical resistance, which is strongly determined by differences in the membranes, but also on the morphology of the electrodes. Microneedle (MN)-based electrodes have been used to concentrate the electrical field during electroporation, and therefore maximize its effect on cell membrane permeability. So far, the methods used for the fabrication of MN electrodes have been relatively limited with respect to the needle design. In this work, we provide a method to fabricate MNs using 3D printing, which is a technology that provides a high degree of flexibility with respect to geometry and dimensions. Pyramidal-shaped MN designs were fabricated and tested on HCT116 cancer cells. Customization of the tips of the pyramids permits tailoring of the electrical field in the vicinity of the cell membranes. The fabricated device enables low-voltage (2 V) electroporation, eliminating the need for the use of specialized chemical buffers. The results show the potential of this method, which can be exploited and optimized for many different applications, and offer a very accessible approach for in-vitro electroporation and cell studies. The MNs can be customized to create complex structures, for example, for a multi-culture cell environment.

Magnetic molding of tumor spheroids: emerging model for cancer screening.

Three-dimensional tissue culture, and particularly spheroid models, have recently been recognized as highly relevant in drug screening, toxicity assessment and tissue engineering due to their superior complexity and heterogeneity akin to thein vivomicroenvironment. However, limitations in size control, shape reproducibility and long maturation times hinder their full applicability. Here, we report a spheroid formation technique based on the magnetic aggregation of cells with internalized magnetic nanoparticles. The method yields magnetic spheroids with high sphericity and allows fine-tuning the final spheroid diameter. Moreover, cohesive spheroids can be obtained in less than 24 h. We show the proof of concept of the method using the CT26 murine colon carcinoma cell line and how different cell proliferation and invasion potentials can be attained by varying the spheroid size. Additionally, we show how the spheroid maturation impacts cell invasion and doxorubicin penetrability, highlighting the importance of this parameter in drug screening and therapeutic applications. Finally, we demonstrate the capability of the method to allow the measurement of the surface tension of spheroids, a relevant output parameter in the context of cancer cell invasion and metastasis. The method can accommodate other cell lines able to be magnetically labeled, as we demonstrate using the U-87 MG human glioblastoma cell line, and shows promise in the therapeutic screening at early time points of tissue formation, as well as in studies of drug and nanoparticle tumor penetration.

3D Magnetic Alignment of Cardiac Cells in Hydrogels.

Tissue engineering aims to repair or replace deficient tissue by delivering constructs that mimic the native in vivo structure. One challenge in cardiac tissue engineering approaches is to achieve intrinsic cardiac organization, particularly the alignment of cardiomyocytes. Here, we propose a strategy for 3D manipulation and alignment of cardiomyocytes by combining magnetism and a hydrogel. The advantage of using magnetic forces is that they act remotely on the cells when these are endowed with magnetization via the internalization of magnetic nanoparticles. The magnetic actuation then allows obtaining, almost instantaneously and before gel transition, an aligned biomimetic cardiac tissue construct. Gel transition enables us to keep the cellular pattern once the magnetic field was removed. This cardiac tissue engineering approach was tested with both H9c2 cell line and primary cardiomyocytes, and with both a synthetic hydrogel and a natural one, Pluronic F-127 and fibrin, respectively. Key parameters of the anisotropic tissue formation were assessed. Hydrogel rheology is provided, and the impact of cell density and magnetic labeling on cell-cell alignment is assessed. Immunofluorescence confirms the presence of several cardiac markers upon chaining, demonstrating the functionality of the tissue-like cell alignment obtained via magnetic actuation.

Iron-Based Core-Shell Nanowires for Combinatorial Drug Delivery and Photothermal and Magnetic Therapy.

Combining different therapies into a single nanomaterial platform is a promising approach for achieving more efficient, less invasive, and personalized treatments. Here, we report on the development of such a platform by utilizing nanowires with an iron core and iron oxide shell as drug carriers and exploiting their optical and magnetic properties. The iron core has a large magnetization, which provides the foundation for low-power magnetic manipulation and magnetomechanical treatment. The iron oxide shell enables functionalization with doxorubicin through a pH-sensitive linker, providing selective intracellular drug delivery. Combined, the core-shell nanostructure features an enhanced light-matter interaction in the near-infrared region, resulting in a high photothermal conversion efficiency of >80% for effective photothermal treatment. Applied to cancer cells, the collective effect of the three modalities results in an extremely efficient treatment with nearly complete cell death (∼90%). In combination with the possibility of guidance and detection, this platform provides powerful tools for the development of advanced treatments.

Inductively actuated micro needles for on-demand intracellular delivery.

Methods that provide controlled influx of molecules into cells are of critical importance for uncovering cellular mechanisms, drug development and synthetic biology. However, reliable intracellular delivery without adversely affecting the cells is a major challenge. We developed a platform for on-demand intracellular delivery applications, with which cell membrane penetration is achieved by inductive heating of micro needles. The micro needles of around 1 µm in diameter and 5 µm in length are made of gold using a silicon-based micro fabrication process that provides flexibility with respect to the needles' dimensions, pitch, shell thickness and the covered area. Experiments with HCT 116 colon cancer cells showed a high biocompatibility of the gold needle platform. Transmission electron microscopy of the cell-needle interface revealed folding of the cell membrane around the needle without penetration, preventing any delivery, which was confirmed using the EthD-1 fluorescent dye. The application of an alternating magnetic field, however, resulted in the delivery of EthD-1 by localized heating of the micro needles. Fluorescence quantification showed that intracellular delivery, with as high as 75% efficiency, is achieved for specific treatment times between 1 and 5 minutes. Overexposure of the cells to the heated micro needles, i.e. longer magnetic field application, leads to an increase in cell death, which can be exploited for cleaning the platform. This method allows to perform intracellular deliver by remotely activating the micro needles via a magnetic field, and it is controlled by the application time, making it a versatile and easy to use method. The wireless actuation could also be an attractive feature for in-vivo delivery and implantable devices.

Scalable High-Affinity Stabilization of Magnetic Iron Oxide Nanostructures by a Biocompatible Antifouling Homopolymer.

Iron oxide nanostructures have been widely developed for biomedical applications because of their magnetic properties and biocompatibility. In clinical applications, stabilization of these nanostructures against aggregation and nonspecific interactions is typically achieved using weakly anchored polysaccharides, with better-defined and more strongly anchored synthetic polymers not commercially adopted because of their complexity of synthesis and use. Here, we show for the first time stabilization and biocompatibilization of iron oxide nanoparticles by a synthetic homopolymer with strong surface anchoring and a history of clinical use in other applications, poly(2-methacryloyloxyethyl phosphorylcholine) [poly(MPC)]. For the commercially important case of spherical particles, binding of poly(MPC) to iron oxide surfaces and highly effective individualization of magnetite nanoparticles (20 nm) are demonstrated. Next-generation high-aspect-ratio nanowires (both magnetite/maghemite and core-shell iron/iron oxide) are, furthermore, stabilized by poly(MPC) coating, with the nanowire cytotoxicity at large concentrations significantly reduced. The synthesis approach exploited to incorporate functionality into the poly(MPC) chain is demonstrated by random copolymerization with an alkyne-containing monomer for click chemistry. Taking these results together, poly(MPC) homopolymers and random copolymers offer a significant improvement over current iron oxide nanoformulations, combining straightforward synthesis, strong surface anchoring, and well-defined molecular weight.

Mesenchymal stem cells cultured on magnetic nanowire substrates.

Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 µm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing hMSCs differentiation.

Highly Efficient Thermoresponsive Nanocomposite for Controlled Release Applications.

Highly efficient magnetic release from nanocomposite microparticles is shown, which are made of Poly (N-isopropylacrylamide) hydrogel with embedded iron nanowires. A simple microfluidic technique was adopted to fabricate the microparticles with a high control of the nanowire concentration and in a relatively short time compared to chemical synthesis methods. The thermoresponsive microparticles were used for the remotely triggered release of Rhodamine (B). With a magnetic field of only 1 mT and 20 kHz a drug release of 6.5% and 70% was achieved in the continuous and pulsatile modes, respectively. Those release values are similar to the ones commonly obtained using superparamagnetic beads but accomplished with a magnetic field of five orders of magnitude lower power. The high efficiency is a result of the high remanent magnetization of the nanowires, which produce a large torque when exposed to a magnetic field. This causes the nanowires to vibrate, resulting in friction losses and heating. For comparison, microparticles with superparamagnetic beads were also fabricated and tested; while those worked at 73 mT and 600 kHz, no release was observed at the low field conditions. Cytotoxicity assays showed similar and high cell viability for microparticles with nanowires and beads.

Tunable magnetic nanowires for biomedical and harsh environment applications.

We have synthesized nanowires with an iron core and an iron oxide (magnetite) shell by a facile low-cost fabrication process. The magnetic properties of the nanowires can be tuned by changing shell thicknesses to yield remarkable new properties and multi-functionality. A multi-domain state at remanence can be obtained, which is an attractive feature for biomedical applications, where a low remanence is desirable. The nanowires can also be encoded with different remanence values. Notably, the oxidation process of single-crystal iron nanowires halts at a shell thickness of 10 nm. The oxide shell of these nanowires acts as a passivation layer, retaining the magnetic properties of the iron core even during high-temperature operations. This property renders these core-shell nanowires attractive materials for application to harsh environments. A cell viability study reveals a high degree of biocompatibility of the core-shell nanowires.

Cytotoxicity and intracellular dissolution of nickel nanowires.

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis, and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage, and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Cytotoxic effects of nickel nanowires in human fibroblasts.

The increasing interest in the use of magnetic nanostructures for biomedical applications necessitates rigorous studies to be carried out in order to determine their potential toxicity. This work attempts to elucidate the cytotoxic effects of nickel nanowires (NWs) in human fibroblasts WI-38 by a colorimetric assay (MTT) under two different parameters: NW concentration and exposure time. This was complemented with TEM and confocal images to assess the NWs internalization and to identify any changes in the cell morphology. Ni NWs were fabricated by electrodeposition using porous alumina templates. Energy dispersive X-ray analysis, scanning electron microscopy and transmission electron microscopy imaging were used for NW characterization. The results showed decreased cell metabolic activity for incubation times longer than 24 h and no negative effects for exposure times shorter than that. The cytotoxicity effects for human fibroblasts were then compared with those reported for HCT 116 cells, and the findings point out that it is relevant to consider the cellular size. In addition, the present study compares the toxic effects of equivalent amounts of nickel in the form of its salt to those of NWs and shows that the NWs are more toxic than the salts. Internalized NWs were found in vesicles inside of the cells where their presence induced inflammation of the endoplasmic reticulum.

Enteritis Regional (Enfermedad de Crohn) / Regional enteritis (ChronÂ's disease.)

Enteritis Regional o Enfermedad de Crohn, forma parte de las enfermedades inflamatorias intestinales como Colitis Ulcerativa, el Crohn, enfermedades inflamatorias bacterianas y virales, diverticulosis, colitis por irradiación y enterocolitis por fármacos. Las que más se relacionan entre si son: Colitis ulcerativas y la enfermedad de Crohn. Etiología desconocida. El tubo digestivo no puede diferenciar los antígenos extraños de los propios. Se cree que hay influencia genética e inmunológica, aparece entre los 20-40 años. La primera lesión es una diminuta úlcera aftoide sobre una base linfoide, aparece por brotes, en forma lineal y de carácter segmentaria. La enfermedad de Crohn incluye cualquier segmento de la boca del ano, 20% infesta el Colon, otras veces al gleo distal y colon derecho, 20% se observa a veces en el intestino delgado, 10% estómago duodeno. Posible etiología: Defecto de la regulación de la disminución de fenómenos inmunológicos y deficiencias en la producción de alotipos o anticuerpos citoplasmáticos de leutróficos. Diagnóstico: Tomografía abdominal computarizada, tomografía perineal o ultrasonografía rectal, inyección de leucocitos marcados con indio o con tectenio, aspiración guiada por US.