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Objective. The goal of this work is to develop and characterize an innovative experimental framework to design interconnected (i.e. modular) heterogeneous (cortical-hippocampal) neuronal cultures with a three-dimensional (3D) connectivity and to record their electrophysiological activity using micro-electrode arrays (MEAs). Approach. A two-compartment polymeric mask for the segregation of different neuronal populations (cortex and hippocampus) was coupled to the MEA surface. Glass microbeads were used as a scaffold to mimic the 3D brain micro-architecture. Main results. We built a fully functional heterogeneous 3D neuronal network. From an electrophysiological point of view, we found that the heterogeneity induces a global increase of the activity rate, while the 3D connectivity modulates the duration and the organization of the bursting activity. Significance. In vivo, studies of network dynamics and interactions between neuronal populations are often time-consuming, low-throughput, complex, and suffer from reproducibility. On the other hand, most of the commonly used in vitro brain models are too simplified and thus far from the in vivo situation. The achieved results demonstrate the feasibility to build a more realistic and controllable experimental in vitro model of interconnected brain regions on-a-chip whose applications may have impacts on the study of neurological disorders that impair the connectivity between brain areas (e.g. Parkinson disease).

The increase of different types of cell cultures, which can be used for the in vitro studies of physiological and/or pathological processes, has introduced the need to improve culture techniques through the use of materials and culture media that promote growth, recreating a cellular micro-environment that can be asserted in in vivo condition. The standard methods for the functionalization of supports used for cell cultures are based on the use of synthetic or natural biopolymers, which generally have high costs, such as poly-lysine and polyornithine. The aim of this work is to demonstrate the alternative use of the polysaccharide chitosan as adhesion factor and structural component for 2D/3D neuronal cultures. Thanks to its versatility, it could be easily functionalized for the fabrication of personalized of in vitro models

Wireless and non-invasive stimulation of neural system, especially at the central level, is considered a critical issue not only for the treatment of a variety of pathological conditions, such as epilepsy, chronic pain, and obsessive-compulsive disorders, but also for reducing the debilitating motor symptoms of movement disorders such as Parkinson’s disease, dystonia, and essential tremor. In this chapter, the potential of piezoelectric nanostructured materials for remote non-invasive neural stimulation is presented.

Evaluation and understanding the effect of drug delivery in in vitro systems is fundamental in drug discovery. We present an assay based on real-time electrical impedance spectroscopy (EIS) measurements that can be used to follow the internalisation and cytotoxic effect of a matrix metalloproteinase (MMP)–sensitive liposome formulation loaded with oxaliplatin (OxPt) on colorectal cancer cells. The EIS response identified two different cellular processes: (i) a negative peak in the cell index (CI) within the first 5 h, due to onset of liposome endocytosis, followed by (ii) a subsequent CI increase, due to the reattachment of cells until the onset of cytotoxicity with a decrease in CI. Free OxPt or OxPt-loaded Stealth liposomes did not show this two-stage EIS response; the latter can be due to the fact that Stealth cannot be cleaved by MMPs and thus is not taken up by the cells. Real-time bright-field imaging supported the EIS data, showing variations in cell adherence and cell morphology after exposure to the different liposome formulations. A drastic decrease in cell coverage as well as rounding up of cells during the first 5 h of exposure to OxPt-loaded (MMP)-sensitive liposome formulation is reflected by the first negative EIS response, which indicates the onset of liposome endocytosis. [Figure not available: see fulltext.]

Neuro-electronic interfaces play a fundamental role for studying brain functions and cellular mechanisms of information processing both in vitro and in vivo. Here we present a new concept in which electronic devices are integrated with models of neuronal networks of human origin together with nanotools to characterize the (patho)physiology of such in vitro complex systems. Large-scale CMOS micro-transducer arrays, coupled to 2D and 3D networks are presented together with new techniques for non-invasively modulate the cell activity. Specifically, piezo-electric nano particles and gold nano rods are used to excite-inhibit the neural activity that is recorded by the micro-transducer arrays. Concept, technology, and preliminary results are presented toward the implementation of a bio-hybrid brain-on-a-chip.

The brain is the most complex organ of our body. Such a complexity spans from the single-cell morphology up to the intricate connections that hundreds of thousands of neurons establish to create dense neuronal networks. All these components are involved in the genesis of the rich patterns of electrophysiological activity that characterize the brain. Over the years, researchers coming from different disciplines developed in vitro simplified experimental models to investigate in a more controllable and observable way how neuronal ensembles generate peculiar firing rhythms, code external stimulations, or respond to chemical drugs. Nowadays, such in vitro models are named brain-on-a-chip pointing out the relevance of the technological counterpart as artificial tool to interact with the brain: multi-electrode arrays are well-used devices to record and stimulate large-scale developing neuronal networks originated from dissociated cultures, brain slices, up to brain organoids. In this review, we will discuss the state of the art of the brain-on-a-chip, highlighting which structural and biological features a realistic in vitro brain should embed (and how to achieve them). In particular, we identified two topological features, namely modular and three-dimensional connectivity, and a biological one (heterogeneity) that takes into account the huge number of neuronal types existing in the brain. At the end of this travel, we will show how 'far' we are from the goal and how interconnected-brain-regions-on-a-chip is the most appropriate wording to indicate the current state of the art.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

[EN] Herein we investigate the feasibility of detecting photo-induced surface stress changes using the deflection response of cantilevers. For this purpose, silicon microcantilevers have been functionalised with spiropyran photochromic molecules, using both a monolayer and a polymeric brushes approach. Uponultraviolet light irradiation, the spiropyran unit is converted to the merocyanine form due to the photo-induced cleavage of the Cspiro-O bond. The two forms of the molecule have dramatically different charge,polarity and molecular conformations. This makes spiropyrans an ideal system to study the correlation between photo-induced molecular changes and corresponding changes in surface stress. Our investigations include monitoring the changes in static cantilever deflection, and consequently, surface stress of the spiropyran functionalised cantilevers on exposure to ultraviolet light. Cantilever deflection data reveals that ultraviolet induced conformational changes in the spiropyran moiety cause a change incompressive surface stress and this varies with the type of functionalisation method implemented. The change in surface stress response from the spiropyran polymer brushes functionalised cantilevers gives an average surface stress change of 98 Nm−1(n = 24) while the spiropyran monolayer coated cantilevers have an average surface stress change of about 446 Nm−1(n = 8) upon irradiation with UV light. This project was supported by the Dublin Institute of Technology Research Support Programme with the IPS grant scheme. The project was also supported by Science Foundation Ireland underINSIGHT award SFI/12/RC/2289. F.B.L. acknowledges the Ramón y Cajal Programme (Ministerio de Economía y Competitividad) and to Marian M. De Pancorbo for letting him to use her laboratory facilities at UPV/EHU. C.G. thanks Rob Cain for technical assistance with the Veriscan3000 system.

Cardiomyocytes (CM) placed on microelectrode array (MEA) were simultaneously probed with cantilever from atomic force microscope (AFM) system. This electric / nanomechanical combination in real time recorded beating force of the CMs cluster and the triggering electric events. Such "organ-on-a-chip" represents a tool for drug development and disease modeling. The human pluripotent stem cells included the WT embryonic line CCTL14 and the induced dystrophin deficient line reprogrammed from fibroblasts of a patient affected by Duchenne Muscular Dystrophy (DMD, complete loss of dystrophin expression). Both were differentiated to CMs and employed with the AFM/MEA platform for diseased CMs’ drug response testing and DMD characterization. The dependence of cardiac parameters on extracellular Ca2+ was studied. The differential evaluation explained the observed effects despite variability of biological samples. The β-adrenergic stimulation (isoproterenol) and antagonist trials (verapamil) addressed ionotropic and chronotropic cell line-dependent features. For the first time, a distinctive beating-force relation for DMD CMs was measured on the 3D cardiac in vitro model.

Articular cartilage is a tissue of fundamental importance for the mechanics of joints, since it provides a smooth and lubricated surface for the proper transfer of loads. From a mechanical point of view, this tissue is an anisotropic poroviscoelastic material: its characteristics at the macroscopic level depend on the complex microscopic architecture. With the ability to probe the local microscopic features, dynamic nanoindentation test is a powerful tool to investigate cartilage mechanics. In this work we focus on a length scale where the time dependent behaviour is regulated by poroelasticity more than viscoelasticity and we aim to understand the effect of the anisotropic permeability on the mechanics of the superficial layer of the articular cartilage. In a previous work, a finite element model for the dynamic nanoindentation test has been presented. In this work, we improve the model by considering the presence of an anisotropic permeability tensor that depends on the collagen fibers distribution. Our sensitivity analysis highlights that the permeability decreases with increasing indentation, thus making the tissue stiffer than the case of isotropic permeability, when solicited at the same frequency. With this improved model, a revised identification of the mechanical and physical parameters for articular cartilage is provided. To this purpose the model was used to simulate experimental data from tests performed on bovine tissue, giving a better estimation of the anisotropy in the elastic properties. A relation between the identified macroscopic anisotropic permeability properties and the microscopic rearrangement of the fiber/matrix structure during indentation is also provided.

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

The ability to sense mechanical stimuli and elaborate a response to them is a fundamental process in all organisms, driving crucial mechanisms ranging from cell volume regulation up to organ development or regeneration. Nevertheless, only in few cases the underlying molecular players are known. In particular, mammals possess a large variety of mechanoreceptors, providing highly specialized functions in sensory cells, but also several housekeeping molecular systems are involved in the complex mechanism of mechanotransduction. Recently, a new class of almost ubiquitous membrane channels has been identified in mammalians, namely piezo1 and piezo2, that is thought to play a crucial role in the mechanobiology of mammals. This review focuses on recent findings on these novel channels, and highlights open biophysical questions that largely remain to be addressed.

Studies in vitro have demonstrated that β3-adrenergic receptors (β3-ARs) regulate protein metabolism in skeletal muscle by promoting protein synthesis and inhibiting protein degradation. In this study, we evaluated whether activation of β3-ARs by the selective agonist CL316,243 modifies the functional and structural properties of skeletal muscles of healthy mice. Daily injections of CL316,243 for 15 days resulted in a significant improvement in muscle force production, assessed by grip strength and weight tests and an increased myofiber cross-sectional area, indicative of muscle hypertrophy. In addition, atomic force microscopy revealed a significant effect of CL316,243 on the transversal stiffness of isolated muscle fibers. Interestingly, the expression level of mammalian target of rapamycin (mTOR) downstream targets and neuronal nitric oxide synthase (NOS) was also found to be enhanced in tibialis anterior and soleus muscles of CL316,243 treated mice, in accordance with previous data linking β3-ARs to mTOR and NOS signaling pathways. In conclusion, our data suggest that CL316,243 systemic administration might be a novel therapeutic strategy worthy of further investigations in conditions of muscle wasting and weakness associated with aging and muscular diseases.

Graphene-derived materials, such as graphene oxide (GO), have been widely explored for biomedical and biological applications, including cancer research. Despite some recent works proving that GO inhibits the migration and invasion of different cancer cells, so far most of these in vitro studies have been conducted using GO sheets dispersed in solution or as a planar film. On the contrary, little is known about cellular activities, such as cell viability, adhesion, and spreading, when cancer cells interface with GO functionalized hydrogel-based surfaces, biomechanically and structurally more similar to the tumor environment. Here, we evaluate the interactions of human breast cancer cells (MDA-MB-231) with alginate (Alg)/GO hydrogel-based substrates, and compare them with a cancer cell line from human osteosarcoma (HOS) and healthy murine fibroblasts (3T3). We observed that GO addition selectively inhibits malignant breast cancer cell adhesion efficiency and spreading area, while promotes HOS and 3T3 adhesive processes. Furthermore, we did not observe the same results over Alg substrates with GO nanosheets dispersed in the medium, without embedment into the Alg. This suggests that cancer (MDA-MB-231 and HOS) and healthy (3T3) cell adhesion efficacy does not depend on the cellular tumoral nature and it is driven by the topographical cues provided by the GO-based substrates, whose physical-mechanical characteristics better mimic those of the cell native tissue. We envision that this study can provide a rational for future design and use of graphene-based nanomaterials for cancer research by deepening the knowledge of graphene-cancer cell specific interactions.

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 μm2 areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun-exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure-the former even more than the latter-are thicker and present a larger D-period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.

In this paper an open-loop actuation strategy based on a Prandtl-Ishlinskii hysteresis model is applied to a commercial atomic force microscope, equipped with an open-loop piezoelectric tube scanner. Estimates of the scanner vertical displacement, necessary to train the model, are obtained by exploiting the interference pattern generated by the laser beam used in the instrument to sense the cantilever deflection. Experimental results show that the undesired hysteresis effects are drastically reduced.

The availability of 3D biomimetic in vitro neuronal networks of mammalian neurons represents a pivotal step for the development of brain-on-a-chip experimental models to study neuronal (dys)functions and particularly neuronal connectivity. The use of hydrogel-based scaffolds for 3D cell cultures has been extensively studied in the last years. However, limited work on biomimetic 3D neuronal cultures has been carried out to date. In this respect, here we investigated the use of a widely popular polysaccharide, chitosan (CHI), for the fabrication of a microbead based 3D scaffold to be coupled to primary neuronal cells. CHI microbeads were characterized by optical and atomic force microscopies. The cell/scaffold interaction was deeply characterized by transmission electron microscopy and by immunocytochemistry using confocal microscopy. Finally, a preliminary electrophysiological characterization by micro-electrode arrays was carried out.

Objective We aim to develop a novel non-invasive or minimally invasive method for neural stimulation to be applied in the study and treatment of brain (dys)functions and neurological disorders. Approach We investigate the electrophysiological response of in vitro neuronal networks when subjected to low-intensity pulsed acoustic stimulation, mediated by piezoelectric nanoparticles adsorbed on the neuronal membrane. Main results We show that the presence of piezoelectric barium titanate nanoparticles induces, in a reproducible way, an increase in network activity when excited by stationary ultrasound waves in the MHz regime. Such a response can be fully recovered when switching the ultrasound pulse off, depending on the generated pressure field amplitude, whilst it is insensitive to the duration of the ultrasound pulse in the range 0.5 s-1.5 s. We demonstrate that the presence of piezoelectric nanoparticles is necessary, and when applying the same acoustic stimulation to neuronal cultures without nanoparticles or with non-piezoelectric nanoparticles with the same size distribution, no network response is observed. Significance We believe that our results open up an extremely interesting approach when coupled with suitable functionalization strategies of the nanoparticles in order to address specific neurons and/or brain areas and applied in vivo, thus enabling remote, non-invasive, and highly selective modulation of the activity of neuronal subpopulations of the central nervous system of mammalians.

Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivationand electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in bothapplications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring.

Nanostructured polymeric capsules are regarded as highly promising systems with different potential applications ranging from drug delivery, biosensing and artificial cells. To fully exploit this potential, it is required to produce bio-activated stable and biocompatible capsules. To this purpose, in present work we proposed the combination of the layer-by-layer self assembly method with bacterial S-layer technology to fabricate stable and biocompatible polymeric capsules having a well defined arrangement of functional groups allowing the covalent attachment of antibody molecules. Hollow microcapsules were obtained by the layer-by-layer self assembly of oppositely charged polyelectrolytes onto colloidal particles, followed by removal of the cores at acidic pH. S-layers were crystallized onto the shell of the obtained capsules. Quartz crystal microbalance was used to characterize the crystallization process onto planar surfaces. S-layer containing capsules were investigated by atomic force microscopy. Immunoenzymatic tests were performed to assess the effective modification of the S-layer with antibody molecules both on planar surfaces and on hollow capsules. Fluorescent microscopy was employed to visualize the presence of the antibody molecules onto the capsule shell and immunological tests used to assess the bioactivity of the immobilized antibodies. Finally, the in vitro cytotoxicity of fabricated S-layer containing capsules was studied. The obtained results demonstrated the possibility to fabricate bio-activated S-layer containing capsules with improved features in terms of biocompatibility.

As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.

Cantilever transducers, which are recognized as a promising platform for the next generation of chemical and biological sensors, are currently under extensive research. Here we report composite porous silicon-crystalline silicon microcantilevers made of SOI wafers, where the porous silicon surface provides an excellent interface for immobilization of the biosensing layer. We start with crystalline silicon cantilevers fabricated from SOI wafers and we establish a surface layer of porous silicon on the cantilevers by vapor phase stain etching. The processed porous silicon does not introduce significant static bending of the cantilevers indicating that it does not introduce significant residual stress. It does provide an excellent biocompatible material for immobilization of a wide variety of chemical and biological materials, resulting in enhanced sensitivity as demonstrated on the covalently immobilized antibody binding its complementary antigen.

Nuclear aggregates of polyamines (NAPs) are supramolecular compounds generated by the self-assembly of protonated nuclear polyamines (spermine, spermidine and putrescine) and phosphate ions. In the presence of genomic DNA, the hierarchical process of self-structuring ultimately produces nanotube-like polymers that envelop the double helix. Because of their modular nature and their aggregation-disaggregation dynamics, NAPs confer plasticity and flexibility to DNA. Through the disposition of charges, NAPs also enable a bidirectional stream of information between the genome and interacting moieties. High mobility group (HMG) B1 is a non-histone chromosomal protein that binds to DNA and that influences multiple nuclear processes. Because genomic DNA binds to either NAPs or HMGB1 protein, we explored the ability of in vitro self-assembled NAPs ( iv NAPs) to mediate the DNA-HMGB1 interaction. To this end, we structured DNA-NAPs-HMGB1 and DNA-HMGB1-NAPs ternary complexes in vitro through opportune sequential incubations. Mobility shift electrophoresis and atomic force microscopy showed that the DNA- iv NAPs-HGMB1 complex had conformational assets supposedly more suitable those of the DNA-HGMB1- iv NAPs to comply with the physiological and functional requirements of DNA. Our findings indicated that iv NAPs act as mediators of the DNA-HMGB1 interaction.

Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150–4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150–200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.

Micro-cantilevers can be utilized as sensors primarily due to their low stiffness/high flexibility and high resonance frequency. The cantilever system is therefore very sensitive to small changes of properties that can be detected either as change of deflection, or of resonance frequency. The development of small forces in the near-surface layers results in significant property changes of micro-cantilevers allowing their use for the detection of very small effects, e.g. associated with the adsorption of biological or chemical substances on the cantilever surfaces. While many experiments providing proof of principle deformation have been reported in the literature, quantitative analysis of the cantilever transduction effect requires precise characterization of the device deflection in response to known forces, i.e. detector calibration. The present paper addresses two aspects of this issue: property evaluation of micro-cantilever components, particularly the coating layer, and the use of analytical and numerical models of cantilever deformation using the concept of eigenstrain, a term that is used in residual stress theory to describe inelastic deformation. The analytical model proposed here accounts for the presence of surface tension and thermal mismatch effects, and predicts the resulting cantilever curvature. The numerical model presented is developed within the framework of finite element analysis, and allows the prediction of effects of cantilever attachment to the chip on complete cantilever deflection. Comparisons of model predictions with the experimental data on cantilever deflection due to changes in temperature obtained using optical interferometry show good agreement. © 2007 Elsevier B.V. All rights reserved.

Here we investigate the feasibility of detecting the photo-reversible inter-conversion between the two thermo-dynamically stable states of the photochromic molecule spiropyran, using silicon microcantilever sensors. The merging of microcantilever technology and spiropyran photochromic brushes for the first time, offers a good route for the pairing of a sensitive transducer with a sensing surface which can be optically controlled. Photo-induced switching between the spiropyran isomers has been tracked through changes in surface stress of the cantilever that can be detected by monitoring the cantilever deflection.

The most important aspect of biosensor development with high sensitivity is the oriented immobilization of antibodies on the solid substrate. Human IgG was immobilized on gold-coated silicon employing protein A, protein G and neutravidin immobilization procedures. The amount of human IgG immobilized was analyzed by 3, 3', 4, 4'- tetramethyl benzidine (TMB) substrate assay and was maximum when protein A immobilization procedure was followed. Human IgG coated biosensing surface was regenerated by treatment with glycine-HCl buffer (50 mM, pH 2.2). Atomic force microscopy was used to analyze the distribution of biomolecules immobilized on gold-coated silicon. Our study indicates that human IgG molecules were uniformly bound to gold-coated silicon by protein A immobilization procedure.

We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.

In this paper, a quantitative interpretation for atomic force microscopy-based dynamic nanoindentation (AFM-DN) tests on the superficial layers of bovine articular cartilage (AC) is provided. The relevant constitutive parameters of the tissue are estimated by fitting experimental results with a finite element model in the frequency domain. Such model comprises a poroelastic stress–strain relationship for a fibril reinforced tissue constitution, assuming a continuous distribution of the collagen network orientations. The identification procedure was first validated using a simplified transversely isotropic constitutive relationship; then, the experimental data were manually fitted by using the continuous distribution fibril model. Tissue permeability is derived from the maximum value of the phase shift between the input harmonic loading and the harmonic tissue response. Tissue parameters related to the stiffness are obtained from the frequency response of the experimental storage modulus and phase shift. With this procedure, an axial to transverse stiffness ratio (anisotropy ratio) of about 0.15 is estimated.

Dysregulated mechano-activated calcium (Ca2+) and reactive oxygen species (ROS) signaling pathways underscore a growing number of diseases; however, a lack of mechanistic detail has limited the discovery of therapeutic targets. The cytoskeleton has garnered recent interest as it integrates and focuses mechanical stress on mechano-activated enzymes, ion channels, and proteins. In striated muscle, we recently discovered the microtubule network as the critical cytoskeletal element which activates X-ROS, a novel pathway in which the mechanical stress of stretch/contraction drives NADPH Oxidase 2 (NoX2) ROS production. In healthy muscle, X-ROS sensitizes the activation of ligand or stretch-activated Ca2+ channels and pharmacologic modulation of MT density ‘tunes’ X-ROS. In disparate models of muscular dystrophy, we find elevated MT network density drives excess X-ROS and Ca2+ signaling dysfunction. Acute pharmacologic ablation of MT network structure in diseased muscle revealed the MT network as a target with translational potential, however MT structure is critical to normal cellular function and significant MT ablation may limit this approach. We identified elevated levels of α-tubulin de-tyrosination - a post-translational modification (PTM) associated with increased mechanical properties of the MT network - in muscle exhibiting both enhanced MT density and X-ROS. In dystrophic mdx muscle, the pharmacologic reduction of α-tubulin de-tyrosination had no effect on the overall density or structure of the MT cytoskeleton, yet significantly reduced X-ROS and Ca2+ dysregulation in vitro and limited contraction-induced muscle injury in vivo. Measures of sarcomere and sub-sarcolemmal cytoskeletal mechanics revealed that a reduction in α-tubulin de-tyrosination decreased the mechanical stiffness of the MT cytoskeletal network. We conclude that α-tubulin de-tyrosination is a PTM that regulates the mechanosensitivity of striated muscle and propose that pharmacological targeting of this PTM will have broad therapeutic potential for the muscular dystrophies.

How can microelectromechanical systems (MEMS) experts support molecular biologists in studying DNA hybridization? Cantilever-based devices are an example of how a ‘simple’ sensor can be tailored by microfabrication techniques and used to achieve an unprecedented performance. We review fascinating experiments, which use different mechanical transduction principles for detecting and analyzing small quantities of materials. The principles of these experiments allow biologists to study surface biochemistry on a nano-scale and offer new, exciting opportunities in developing microscopic biomedical analysis systems with unique characteristics. Cantilever sensors rely on relatively well known and simple transduction principles, and have attracted the interest of many researchers. This is, at least in part, because of the merging of silicon microfabrication techniques and surface functionalization biochemistry, together with the development of multi-cantilever sensing methods offering new opportunities in physical and (bio)chemical sensing.

Interparticle and surface forces are of great importance in many fields of pure and applied science. We present an apparatus to measure the normal and friction forces acting between a particle (radius of 0.5–20 μm) and another solid surface. The apparatus is based on the principle of an atomic force microscope. For quantitative friction measurements we propose a method to determine the lateral spring constants of atomic force microscope cantilevers with attached spherical particles.

In this work we investigate the influence of the use of YSZ and CeO2/YSZ as buffers in the growth of SrBi2Ta2O9 (SBT). We show that by using YSZ only the a-axis oriented Pyrochlore phase could be obtained. On the other hand a CeO2/YSZ double-buffer layer gave a c-axis oriented SBT with no amorphous SiO2 inter- diffusion layer. From the piezoelectric response. nano-meter scale ferroelectric domains could be clearly identified in polycrystalline SBT thin films. In spite that SBT is an anisotropic material with an in- plane easy axis for polarization, we observe in our films a clear ferroelectric behavior of the Capacitance versus Voltage characteristics.

The bending of microfabricated silicon nitride cantilevers was used to determine surface stress changes at solid–liquid interfaces. The radius of curvature of the bent cantilever is directly proportional to changes in the differential surface stress between its opposite sides. To demonstrate the possibilities and limitations of the technique, cantilevers coated on both sides with gold and densely packed monolayers of different thiols were put in a constant flow of aqueous electrolyte solution and the deflection was measured using a optical lever technique. Changes in the surface stress for the different thiol monolayers due to specific proton adsorption are presented. Possible applications and improvements of this technique are discussed.