Leonardo Ricotti

Enhancement of Neurite Outgrowth in Neuronal-Like Cells following Boron Nitride Nanotube-Mediated Stimulation

In this paper, we propose an absolutely innovative technique for the electrical stimulation of cells, based on piezoelectric nanoparticles. Ultrasounds are used to impart mechanical stress to boron nitride nanotubes incubated with neuronal-like PC12 cells. By virtue of their piezoelectric properties, these nanotubes can polarize and convey electrical stimuli to the cells. PC12 stimulated with the present method exhibit neurite sprout 30% greater than the control cultures after 9 days of treatment.

Investigation of interactions between poly-l-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation

Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription – polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

Symbiotic robot organisms: REPLICATOR and SYMBRION projects

Cooperation and competition among stand - alone swarm agents can increase the collective fitness of the whole system. An interesting form of collective system is demonstrated by some bacteria and fungi, which can build symbiotic organisms. Symbiotic communities can enable new functional capabilities which allow all members to survive better in their environment. In this article we show an overview of two large European projects dealing with new collective robotic systems which utilize principles derived from natural symbiosis. The paper provides also an overview of typical hardware, software and methodological challenges arose along these projects, as well as some prototypes and on-going experiments available on this stage.

Microwrinkled Conducting Polymer Interface for Anisotropic Multicellular Alignment

Surfaces with controlled micro and nanoscale topographical cues are useful as smart scaffolds and biointerfaces for cell culture. Recently, use of thin-film and surface wrinkling is emerging as a rapid unconventional method for preparing topographically patterned surfaces, especially suited for the production of smart patterns over large area surfaces. On the other hand, there is an increasing interest in employing conducting polymers as soft, biocompatible, conductive biointerfaces or as parts of bioelectronic devices. A novel convenient and versatile method is presented for producing anisotropic topographical cues at the micro- and nanoscale on conducting polymer surfaces. Micro and nanowrinkles were formed during the heat-shrinking process of a thermo-retractable polystyrene substrate. Surface wrinkling was due to the mismatch between the mechanical properties of a conducting polymer ultrathin film and the substrate. Various geometries of wrinkled structures were prepared, demonstrating the tunability of topography depending on the thickness of the conductive film. A method for patterning the conductive properties of the wrinkled substrates was also presented. Such surfaces acted as smart scaffolds for the functional alignment of cells, envisioning their electrical stimulation. Cell adhesion and proliferation were evaluated, comparing different topographies, and a preferential anisotropic alignment of C2C12 murine skeletal muscle cells along wrinkles was demonstrated. The observed trends were also confirmed concerning the formation of aligned myotubes in C2C12 differentiation stage. Furthermore, improved results in terms of aligned and mature myotube formation were obtained by co-culturing C2C12 cells with a fibroblasts feeder layer. The combination of living cells and tunable conductive nanowrinkles will represent a unique tool for the development of innovative biomedical devices.

Boron Nitride Nanotube-Mediated Stimulation of Cell Co-Culture on Micro-Engineered Hydrogels

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young’s modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds

This study aims at investigating the behavior in terms of the proliferation and skeletal muscle differentiation capability of two myoblastic cell lines, C2C12 and H9c2, on both isotropic and anisotropic electrospun nanofibrous poly(hydroxybutyrate) (PHB) scaffolds, as well as on PHB films and polystyrene controls. After a careful characterization of the matrices in terms of surface morphology, surface roughness and mechanical properties, the proliferation rate and the capability of the two cell lines to form skeletal myotubes were evaluated. Genetic analyses were also performed in order to assess the differentiation level of the cells on the different substrates. We demonstrated that the aligned nanofibrous mesh decreases the proliferation activity and provides a higher differentiative stimulus. We also clarified how the nanofibrous substrate influences myotube formation, and quantified a series of myotube-related parameters for both C2C12 and H9c2 cells.

Bio-hybrid muscle cell-based actuators

Actuation is an essential function of any artificial or living machine, allowing its movement and its interaction with the surrounding environment. Living muscles have evolved over millions of years within animals as nature’s premier living generators of force, work and power, showing unique characteristics in comparison with standard artificial actuators. Current actuation technologies actually represent a real bottleneck in many robotics and ICT applications, including the bio-inspired ones. Main limitations involve inertia and backdrivability, stiffness control and power consumption. The development of novel actuators able to better mimic or even to overcome living muscle performances would open new horizons in robotics and ICT technologies: these components would allow the raise of a new generation of machines, with life-like movements and outstanding performances. An innovative solution to achieve this goal is represented by the merging between artificial and living entities, towards the realization of bio-hybrid devices. The aim of the present article is to describe the scientific and technological efforts made by researchers in the last two decades to achieve cell- or tissue-based actuators, with the dream of matching or outperforming natural muscles and to efficiently power micro- and mini-devices. The main challenges connected to the development of a cell-based actuator are highlighted and the most recent solutions to this scientific/technological problem are depicted, reporting advantages and drawbacks of each single approach. Future perspectives are also described, envisioning bio-hybrid actuators as key components of a new generation of machines able to show life-like movements and behaviors.

Effects of barium titanate nanoparticles on proliferation and differentiation of rat mesenchymal stem cells

Nanomaterials hold great promise in the manipulation and treatments of mesenchymal stem cells, since they allow the modulation of their properties and differentiation. However, systematic studies have to be carried out in order to assess their potential toxicological effects. The present study reports on biocompatibility evaluation of glycol-chitosan coated barium titanate nanoparticles (BTNPs) on rat mesenchymal stem cells (MSCs). BTNPs are a class of ceramic systems which possess interesting features for biological applications thanks to their peculiar dielectric and piezoelectric properties. Viability was evaluated up to 5 days of incubation (concentrations in the range 0-100 μg/ml) both quantitatively and qualitatively with specific assays. Interactions cells/nanoparticles were further investigated with analysis of the cytoskeleton conformation, with SEM and TEM imaging, and with AFM analysis. Finally, differentiation in adipocytes and osteocytes was achieved in the presence of high doses of BTNPs, thus highlighting the safety of these nanostructures towards mesenchymal stem cells.

MEMS Sensor Technologies for Human Centred Applications in Healthcare, Physical Activities, Safety and Environmental Sensing: A Review on Research Activities in Italy

Over the past few decades the increased level of public awareness concerning healthcare, physical activities, safety and environmental sensing has created an emerging need for smart sensor technologies and monitoring devices able to sense, classify, and provide feedbacks to users’ health status and physical activities, as well as to evaluate environmental and safety conditions in a pervasive, accurate and reliable fashion. Monitoring and precisely quantifying users’ physical activity with inertial measurement unit-based devices, for instance, has also proven to be important in health management of patients affected by chronic diseases, e.g., Parkinson’s disease, many of which are becoming highly prevalent in Italy and in the Western world. This review paper will focus on MEMS sensor technologies developed in Italy in the last three years describing research achievements for healthcare and physical activity, safety and environmental sensing, in addition to smart systems integration. Innovative and smart integrated solutions for sensing devices, pursued and implemented in Italian research centres, will be highlighted, together with specific applications of such technologies. Finally, the paper will depict the future perspective of sensor technologies and corresponding exploitation opportunities, again with a specific focus on Italy.

Evolutionary robotics: The next-generation-platform for on-line and on-board artificial evolution

In this paper we present the development of a new self-reconfigurable robotic platform for performing on-line and on-board evolutionary experiments. The designed platform can work as an autonomous swarm robot and can undergo collective morphogenesis to actuate in different morphogenetic structures. The platform includes a dedicated power management, rich sensor mechanisms for on-board fitness measurement as well as very powerful distributed computational system to run learning and evolutionary algorithms. The whole development is performed within several large European projects and is open-hardware and open-software.