Publications

The full list of publications of the Lab is maintained by the Enlighten: Publications service of the University of Glasgow, including deposited author copies and full references. Below you can find a curated collection of the most recent ones. Please click on the title to see the abstracts and get the reference to the publisher's website.

Recent selected publications

• 2023

• Sylvain Bonnefond, Antoine Reynaud, Julie Cazareth, Sophie Abélanet, Massimo Vassalli, Frédéric Brau, Gian Luca Lippi

  ; Nanoscatterer-Assisted Fluorescence Amplification Technique; Nanomaterials (2023) doi: 10.3390/nano13212875

  Weak fluorescence signals, which are important in research and applications, are often masked by the background. Different amplification techniques are actively investigated. Here, a broadband, geometry-independent and flexible feedback scheme based on the random scattering of dielectric nanoparticles allows the amplification of a fluorescence signal by partial trapping of the radiation within the sample volume. Amplification of up to a factor of 40 is experimentally demonstrated in ultrapure water with dispersed TiO2 nanoparticles (30 to 50 nm in diameter) and fluorescein dye at 200 μmol concentration (pumped with 5 ns long, 3 mJ laser pulses at 490 nm). The measurements show a measurable reduction in linewidth at the emission peak, indicating that feedback-induced stimulated emission contributes to the large gain observed.

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• Dipankar Ghosh, Libby J Marshall, Giuseppe Ciccone, Wanli Liu, Adam Squires, Annela Seddon, Massimo Vassalli, Dave J Adams

  ; Fine‐Tuning Supramolecular Assemblies by Controlling Micellar Aggregates; Marcomolecular Materials and Engineering (2023) doi: https://doi.org/10.1002/mame.202300082

  Supramolecular assembly can be used to fabricate complex functional materials by organizing simple building blocks. However, it is difficult to control the hierarchical assembly across multiple length scales. The correlation of a supramolecular gel network and a pre-gelling aggregate will help to understand how a molecular-level assembly is translated into a higher order. Here, a functional dipeptide 2NapFF is used that can assemble in different micellar structures at high pH by varying the counterion. Replacing the counterions with a divalent calcium salt results in a cross-linked gel network, or an interesting analog “gel noodles.” The physical properties of the gel noodles can be varied by choosing specific micellar assemblies as the pre-gel. The mechanical rigidity of the gel networks is compared by nanoindentation and tensile testing, and the pattern to the structures of the micelles observed by small-angle X-ray scattering is correlated. The supramolecular assembly can be fine-tuned by using different micelles as the pre-gel without affecting the inherent gel-state properties.

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• Federica Viti, Roberto De Giorgio, Isabella Ceccherini, Arti Ahluwalia, Maria M. Alves, Chiara Baldo, Giannina Baldussi, Elena Bonora, Osvaldo Borrelli, Luigi Dall’Oglio, Paolo De Coppi, Carlotta De Filippo, Pascal de Santa Barbara, Antonella Diamanti, Carlo Di Lorenzo, Ruggero Di Maulo, Antonio Galeone, Paolo Gandullia, Sohaib K. Hashmi, Florence Lacaille, Laurence Lancon, Salvatore Leone, Maxime M. Mahé, Maria Judit Molnar, Alessandro Palmitelli, Silvia Perin, Alessio Pini Prato, Nikhil Thapar, Massimo Vassalli, Robert O. Heuckeroth

  ; Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting; Digestive Diseases and Sciences (2023) doi: 10.1007/s10620-023-08066-1

  Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype–phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first ‘European Forum on Visceral Myopathy’. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

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• Martina Zambito, Federica Viti, Alessia G. Bosio, Isabella Ceccherini, Tullio Florio, Massimo Vassalli

  ; The Impact of Experimental Conditions on Cell Mechanics as Measured with Nanoindentation; Nanomaterials (2023) doi: 10.3390/nano13071190

  The evaluation of cell elasticity is becoming increasingly significant, since it is now known that it impacts physiological mechanisms, such as stem cell differentiation and embryogenesis, as well as pathological processes, such as cancer invasiveness and endothelial senescence. However, the results of single-cell mechanical measurements vary considerably, not only due to systematic instrumental errors but also due to the dynamic and non-homogenous nature of the sample. In this work, relying on Chiaro nanoindenter (Optics11Life), we characterized in depth the nanoindentation experimental procedure, in order to highlight whether and how experimental conditions could affect measurements of living cell stiffness. We demonstrated that the procedure can be quite insensitive to technical replicates and that several biological conditions, such as cell confluency, starvation and passage, significantly impact the results. Experiments should be designed to maximally avoid inhomogeneous scenarios to avoid divergences in the measured phenotype.

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• Libby J. Marshall, Matthew Wallace, Najet Mahmoudi, Giuseppe Ciccone, Claire Wilson, Massimo Vassalli, Dave J. Adams

  ; Hierarchical Composite Self-Sorted Supramolecular Gel Noodles; Advanced Materials (2023) doi: 10.1002/adma.202211277

  Multicomponent supramolecular systems can be used to achieve different properties and new behaviors compared to their corresponding single component systems. Here, we use a two-component system, showing that a non-gelling component modifies the assembly of the gelling component, allowing access to co-assembled structures that cannot be formed from the gelling component alone. We characterize the systems across multiple length scales, from the molecular level by NMR and CD spectroscopy, to the microstructure level by SANS and finally to the material level using nanoindentation and rheology. By exploiting the enhanced mechanical properties achieved through addition of the second component, we formed multicomponent noodles with superior mechanical properties to those formed by the single component system. Furthermore, the non-gelling component can be triggered to crystallize within the multicomponent noodles, allowing us to prepare new types of hierarchical composite noodles.

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• Ewan A Ross, Lesley-Anne Turner, Hannah Donnelly, Anwer Saeed, Monica P Tsimbouri, Karl V Burgess, Gavin Blackburn, Vineetha Jayawarna, Yinbo Xiao, Mariana AG Oliva, Jennifer Willis, Jaspreet Bansal, Paul Reynolds, Julia A Wells, Joanne Mountford, Massimo Vassalli, Nikolaj Gadegaard, Richard OC Oreffo, Manuel Salmeron-Sanchez, Matthew J Dalby

  ; Nanotopography reveals metabolites that maintain the immunomodulatory phenotype of mesenchymal stromal cells; Nature Communications (2023) doi: 10.1038/s41467-023-36293-7

  Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.

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• Federica Viti, Francesca Micaela Pramotton, Michela Martufi, Raffaella Magrassi, Nicoletta Pedemonte, Mario Nizzari, Francesca Cella Zanacchi, Benedetta De Michele, Manuela Alampi, Martina Zambito, Giuseppe Santamaria, Adriana Bajetto, Sabah Sardar, Valeria Tomati, Paolo Gandullia, Costanza Giampietro, Tullio Florio, Francesco Beltrame, Massimo Vassalli, Isabella Ceccherini

  ; Patient's dermal fibroblasts as disease markers for visceral myopathy; Biomaterials Advances (2023) doi: 10.1016/j.bioadv.2023.213355

  Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

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• Carolin Grandy, Fabian Port, Jonas Pfeil, Mariana AG Oliva, Massimo Vassalli, Kay-Eberhard Gottschalk

  ; Cell shape and tension alter focal adhesion structure; Biomaterials Advances (2023) doi: 10.1016/j.bioadv.2022.213277

  Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.

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• 2022

• J. Mejía Morales, P. Glynne-Jones, M. Vassalli, G. L. Lippi

  ; Acoustofluidic interferometric device for rapid single-cell physical phenotyping; European Biophysics Journal (2022) doi: 10.1007/s00249-021-01585-7

  High-throughput single-cell analysis based on physical properties (such as morphology or mechanics) is emerging as a powerful tool to inform clinical research, with a great potential for translation towards diagnosis. Here we present a novel microfluidic approach adopting acoustic waves to manipulate and mechanically stimulate single cells, and interferometry to track changes in the morphology and measure size, deformability, and refractive index of non-adherent cells. The method is based on the integration within the acoustofluidic channel of a low-finesse Fabry–Perot resonator, providing very high sensitivity and a speed potentially suitable to obtain the high-throughput necessary to handle the variability stemming from the biological diversity of single cells. The proposed approach is applied to a set of different samples: reference polystyrene beads, algae and yeast. The results demonstrate the capability of the acoustofluidic interferometric device to detect and quantify optomechanical properties of single cells with a throughput suitable to address label-free single-cell clinical analysis.

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• J.Mouro, P.Paoletti, M.Sartore, M.Vassalli, B.Tiribilli

  ; Photothermal Self-Excitation of a Phase-Controlled Microcantilever for Viscosity or Viscoelasticity Sensing; Sensors (2022) doi: 10.3390/s22218421

  This work presents a feedback closed-loop platform to be used for viscosity or viscoelasticity sensing of Newtonian or non-Newtonian fluids. The system consists of a photothermally excited microcantilever working in a digital Phase-Locked Loop, in which the phase between the excitation signal to the cantilever and the reference demodulating signals is chosen and imposed in the loop. General analytical models to describe the frequency and amplitude of oscillation of the cantilever immersed in viscous and viscoelastic fluids are derived and validated against experiments. In particular, the sensitivity of the sensor to variations of viscosity of Newtonian fluids, or to variations of elastic/viscous modulus of non-Newtonian fluids, are studied. Interestingly, it is demonstrated the possibility of controlling the sensitivity of the system to variations of these parameters by choosing the appropriate imposed phase in the loop. A working point with maximum sensitivity can be used for real-time detection of small changes of rheological parameters with low-noise and fast-transient response. Conversely, a working point with zero sensitivity to variations of rheological parameters can be potentially used to decouple the effect of simultaneous external factors acting on the resonator.

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• Giulio Capponi, Martina Zambito, Igor Neri, Francesco Cottone, Maurizio Mattarelli, Massimo Vassalli, Silvia Caponi, Tullio Florio

  ; Cellular Mechanosensitivity: Validation of an Adaptable 3D-Printed Device for Microindentation; Nanomaterials (2022) doi: 10.3390/nano12152691

  Mechanotransduction refers to the cellular ability to sense mechanical stimuli from the surrounding environment and convert them into biochemical signals that regulate cellular physiology and homeostasis. Mechanosensitive ion channels (MSCs), especially ones of Piezo family (Piezo1 and Piezo2), play a crucial role in mechanotransduction. These transmembrane proteins directly react to mechanical cues by triggering the onset of an ionic current. The relevance of this mechanism in driving physiology and pathology is emerging, and there is a growing need for the identification of an affordable and reliable assay to measure it. Setting up a mechanosensitivity assay requires exerting a mechanical stimulus on single cells while observing the downstream effects of channels opening. We propose an open-hardware approach to stimulate single adherent cells through controlled microindentation, using a 3D-printed actuation platform. We validated the device by measuring the mechanosensitivity of a neural mice cell line where the expression level and activity of Piezo1 were genetically and pharmacologically manipulated. Moreover, this extremely versatile device could be integrated with different read-out technologies, offering a new tool to improve the understanding of mechanotransduction in living cells.

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• Luigi Sforna, Antonio Michelucci, Francesco Morena, Chiara Argentati, Fabio Franciolini, Massimo Vassalli, Sabata Martino, Luigi Catacuzzeno

  ; Piezo1 controls cell volume and migration by modulating swelling-activated chloride current through Ca2+ influx; J. Cellular Physiology (2022) doi: 10.1002/jcp.30656

  Regulatory volume decrease (RVD), a homeostatic process responsible for the re-establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling-activated Cl− current (ICl,swell), which can be modulated by cytoplasmic Ca2+. Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca2+-permeable channel Piezo1. We hypothesized that, by controlling cytoplasmic Ca2+ and in turn ICl,swell, Piezo1 is involved in the fine regulation of RVD and cell migration. We compared RVD and ICl,swell in wild-type (WT) HEK293T cells, which express endogenous levels of Piezo1, and in cells overexpressing (OVER) or knockout (KO) for Piezo1. Compared to WT, RVD was markedly increased in OVER, while virtually absent in KO cells. Consistently, ICl,swell amplitude was highest in OVER and lowest in KO cells, with WT cells displaying an intermediate level, suggesting a Ca2+-dependent modulation of the current by Piezo1 channels. Indeed, in the absence of external Ca2+, ICl,swell in both WT and OVER cells, as well as the RVD probed in OVER cells, were significantly lower than in the presence of Ca2+ and no longer different compared to KO cells. However, the Piezo-mediated Ca2+ influx was ineffective in enhancing ICl,swell in the absence of releasable Ca2+ from intracellular stores. The different expression levels of Piezo1 affected also cell migration which was strongly enhanced in OVER, while reduced in KO cells, as compared to WT. Taken together, our data indicate that Piezo1 controls RVD and migration in HEK293T cells by modulating ICl,swell through Ca2+ influx.

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• Francesca Baldini, Mohamad Khalil, Alice Bartolozzi, Massimo Vassalli, Agostino Di Ciaula, Piero Portincasa, Laura Vergani

  ; Relationship between Liver Stiffness and Steatosis in Obesity Conditions: In Vivo and In Vitro Studies; Biomolecules (2022) doi: 10.3390/biom12050733

  Obesity is a major risk factor for metabolic dysfunction such as non-alcoholic fatty liver disease (NAFLD). The NAFLD spectrum ranges from simple steatosis, to steatohepatitis, fibrosis, and cirrhosis. The aim of this study is to characterize the grade of steatosis being associated with overnutrition and obesity, both at the level of single hepatocyte and whole liver, and to correlate it with the hepatocyte/liver stiffness and dysfunction. For the in vivo study, 60 subjects were enrolled and grouped based on the stage of liver steatosis/fibrosis according to biochemical analyses, liver ultrasonography (USG) and acoustic radiation force impulse shear wave elastography (ARFI-SWE). For single hepatocyte analyses we employed in vitro models of moderate and severe steatosis on which to assess the single cell biomechanics by Single Cell Force Spectroscopy (SCFS) and Quantitative Phase Microscopy (QPM). Results show that in vivo liver stiffness depends mainly on the extent of fat accumulation and not on fibrosis. These results parallel the in vitro observations showing that hepatocyte stiffness and dysfunction increase with increasing fat accumulation and lipid droplet enlargement. Our findings indicate that the extent of steatosis markedly affects the biomechanical properties of both liver and single hepatocytes thus proving insights about the role of modulation of liver/hepatocyte elasticity as a physical mechanism transducing the obesity-dependent excess of plasmatic lipids towards liver steatosis and dysfunction.

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• Aldo Ferrari, Massimo Vassalli, Silvia Caponi

  ; Nanoengineering for Mechanobiology “N4M-20”; European Biophysics Journal (2022) doi: 10.1007/s00249-022-01596-y

  It will soon be 3 years since the “Nanoengineering for Mechanobiology” (N4M) young and growing community of mechanobiology enthusiasts last met on the Ligurian coast, in the sunny village of Camogli. It was March 2019, as we gathered for the 5th edition of the symposium, N4M-19 (Ferrari et al. 2019), the yearly rendezvous open to material technology, engineering, exact and life sciences. Back then, we had no idea of the kabbalah reserved to those numbers, which was shortly to be revealed. [...]

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• Giuseppe Ciccone, Mariana Azevedo Gonzalez Oliva, Nelda Antonovaite, Ines Lüchtefeld, Manuel Salmeron-Sanchez, Massimo Vassalli

  ; Experimental and Data Analysis Workflow for Soft Matter Nanoindentation ; Journal of Visualized Experiments (JoVE) (2022) doi: 10.3791/63401

  Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (μm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells. In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

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• 2021

• Bin Yang, Marina Lledos, Riaz Akhtar, Giuseppe Ciccone, Long Jiang, Emanuele Russo, Sunil Rajput, Chunyu Jin, Maria Galini Faidra Angelerou, Thomas Arnold, Jonathan Rawle, Massimo Vassalli, Maria Marlow, Dave J. Adams, Mischa Zelzer

  ; Surface-controlled spatially heterogeneous physical properties of a supramolecular gel with homogeneous chemical composition; Chemical Science (2021) doi: 10.1039/D1SC04671C

  Controlling supramolecular self-assembly across multiple length scales to prepare gels with localised properties is challenging. Most strategies concentrate on fabricating gels with heterogeneous components, where localised properties are generated by the stimuli-responsive component. Here, as an alternative approach, we use a spiropyran-modified surface that can be patterned with light. We show that light-induced differences in surface chemistry can direct the bulk assembly of a low molecular weight gelator, 2-NapAV, meaning that mechanical gel properties can be controlled by the surface on which the gel is grown. Using grazing incidence X-ray diffraction and grazing incidence small angle X-ray scattering, we demonstrate that the origin of the different gel properties relates to differences in the architectures of the gels. This provides a new method to prepare a single domain (i.e., chemically homogeneous) hydrogel with locally controlled (i.e., mechanically heterogeneous) properties.

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• Silvia Caponi, Alessandra Passeri, Giulio Capponi, Daniele Fioretto, Massimo Vassalli, Maurizio Mattarelli

  ; Non-contact elastography methods in mechanobiology: a point of view; Eur Biophys J (2021) doi: 10.1007/s00249-021-01567-9

  In recent decades, mechanobiology has emerged as a novel perspective in the context of basic biomedical research. It is now widely recognized that living cells respond not only to chemical stimuli (for example drugs), but they are also able to decipher mechanical cues, such as the rigidity of the underlying matrix or the presence of shear forces. Probing the viscoelastic properties of cells and their local microenvironment with sub-micrometer resolution is required to study this complex interplay and dig deeper into the mechanobiology of single cells. Current approaches to measure mechanical properties of adherent cells mainly rely on the exploitation of miniaturized indenters, to poke single cells while measuring the corresponding deformation. This method provides a neat implementation of the everyday approach to measure mechanical properties of a material, but it typically results in a very low throughput and invasive experimental protocol, poorly translatable towards three-dimensional living tissues and biological constructs. To overcome the main limitations of nanoindentation experiments, a radical paradigm change is foreseen, adopting next generation contact-less methods to measure mechanical properties of biological samples with sub-cell resolution. Here we briefly introduce the field of single cell mechanical characterization, and we concentrate on a promising high resolution optical elastography technique, Brillouin spectroscopy. This non-contact technique is rapidly emerging as a potential breakthrough innovation in biomechanics, but the application to single cells is still in its infancy.

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• Daniel McDowall, Matthew Walker, Massimo Vassalli, Marco Cantini, Nikul Khunti, Charlotte J. C. Edwards-Gayle, Nathan Cowieson and Dave Adams

  ; Controlling the formation and alignment of low molecular weight gel ‘noodles’; Chemical Communications (2021) doi: 10.1039/D1CC03378F

  We show how to control the formation and alignment of gel ‘noodles’. Nanostructure alignment can be achieved reproducibly by extensional deformation as the filaments form. Using a spinning technique, very long and highly aligned filaments can be made. The Young’s moduli of the gel noodles are similar to that of a bulk gel. By using two syringe pumps in a concentric flow setup, we show that a filament-in-filament morphology can be created.

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• Daniele Obino, Massimo Vassalli, Alberto Franceschi, Andrea Alessandrini, Paolo Facci, Federica Viti

  ; An Overview on Microfluidic Systems for Nucleic Acids Extraction from Human Raw Samples; Sensors (2021) doi: 10.3390/s21093058

  Nucleic acid (NA) extraction is a basic step for genetic analysis, from scientific research to diagnostic and forensic applications. It aims at preparing samples for its application with biomolecular technologies such as isothermal and non-isothermal amplification, hybridization, electrophoresis, Sanger sequencing and next-generation sequencing. Multiple steps are involved in NA collection from raw samples, including cell separation from the rest of the specimen, cell lysis, NA isolation and release. Typically, this process needs molecular biology facilities, specialized instrumentation and labor-intensive operations. Microfluidic devices have been developed to analyze NA samples with high efficacy and sensitivity. In this context, the integration within the chip of the sample preparation phase is crucial to leverage the promise of portable, fast, user-friendly and economic point-of-care solutions. This review presents an overview of existing lab-on-a-chip (LOC) solutions designed to provide automated NA extraction from human raw biological fluids, such as whole blood, excreta (urine and feces), saliva. It mainly focuses on LOC implementation aspects, aiming to describe a detailed panorama of strategies implemented for different human raw sample preparations.

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• Oana Dobre, Mariana A. G. Oliva, Giuseppe Ciccone, Sara Trujillo, Aleixandre Rodrigo‐Navarro, Douglas Cormac Venters, Virginia Llopis‐Hernandez, Massimo Vassalli, Cristina Gonzalez‐Garcia, Matthew J. Dalby, Manuel Salmeron‐Sanchez

  ; A Hydrogel Platform that Incorporates Laminin Isoforms for Efficient Presentation of Growth Factors – Neural Growth and Osteogenesis; Advanced Functional Materials (2021) doi: 10.1002/adfm.202010225

  Laminins (LMs) are important structural proteins of the extracellular matrix (ECM). The abundance of every LM isoform is tissue‐dependent, suggesting that LM has tissue‐specific roles. LM binds growth factors (GFs), which are powerful cytokines widely used in tissue engineering due to their ability to control stem cell differentiation. Currently, the most commonly used ECM mimetic material in vitro is Matrigel, a matrix of undefined composition containing LM and various GFs, but subjected to batch variability and lacking control of physicochemical properties. Inspired by Matrigel, a new and completely defined hydrogel platform based on hybrid LM‐poly(ethylene glycol) (PEG) hydrogels with controllable stiffness (1–25 kPa) and degradability is proposed. Different LM isoforms are used to bind and efficiently display GFs (here, bone morphogenetic protein (BMP‐2) and beta‐nerve growth factor (β‐NGF)), enabling their solid‐phase presentation at ultralow doses to specifically target a range of tissues. The potential of this platform to trigger stem cell differentiation toward osteogenic lineages and stimulate neural cells growth in 3D, is demonstrated. These hydrogels enable 3D, synthetic, defined composition, and reproducible cell culture microenvironments reflecting the complexity of the native ECM, where GFs in combination with LM isoforms yield the full diversity of cellular processes.

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• Tom Hodgkinson, P. Monica Tsimbouri, Virginia Llopis-Hernandez, Paul Campsie, David Scurr, Peter G. Childs, David Phillips, Sam Donnelly, Julia A. Wells, Fergal J. O’Brien, Manuel Salmeron-Sanchez, Karl Burgess, Morgan Alexander, Massimo Vassalli, Richard O. C. Oreffo, Stuart Reid, David J. France, Matthew J. Dalby

  ; The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells; Science Advances (2021) doi: 10.1126/sciadv.abb7921

  Bioactive metabolites have wide-ranging biological activities and are a potential source of future research and therapeutic tools. Here, we use nanovibrational stimulation to induce osteogenic differentiation of mesenchymal stem cells, in the absence of off-target, nonosteogenic differentiation. We show that this differentiation method, which does not rely on the addition of exogenous growth factors to culture media, provides an artifact-free approach to identifying bioactive metabolites that specifically and potently induce osteogenesis. We first identify a highly specific metabolite, cholesterol sulfate, an endogenous steroid. Next, a screen of other small molecules with a similar steroid scaffold identified fludrocortisone acetate with both specific and highly potent osteogenic-inducing activity. Further, we implicate cytoskeletal contractility as a measure of osteogenic potency and cell stiffness as a measure of specificity. These findings demonstrate that physical principles can be used to identify bioactive metabolites and then enable optimization of metabolite potency can be optimized by examining structure-function relationships.

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• Oropesa-Nuñez, R., Mescola, A., Vassalli, M. and Canale, C.

  ; Impact of Experimental Parameters on Cell–Cell Force Spectroscopy Signature; Sensors (2021) doi: 10.3390/s21041069

  Atomic force microscopy is an extremely versatile technique, featuring atomic-scale imaging resolution, and also offering the possibility to probe interaction forces down to few pN. Recently, this technique has been specialized to study the interaction between single living cells, one on the substrate, and a second being adhered on the cantilever. Cell–cell force spectroscopy offers a unique tool to investigate in fine detail intra-cellular interactions, and it holds great promise to elucidate elusive phenomena in physiology and pathology. Here we present a systematic study of the effect of the main measurement parameters on cell–cell curves, showing the importance of controlling the experimental conditions. Moreover, a simple theoretical interpretation is proposed, based on the number of contacts formed between the two interacting cells. The results show that single cell–cell force spectroscopy experiments carry a wealth of information that can be exploited to understand the inner dynamics of the interaction of living cells at the molecular level.

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• M. Vassalli

  ; Meet the editor series - Massimo Vassalli; Biophysical Reviews (2021) doi: 10.1007/s12551-021-00786-w

  It is my pleasure to write a few words to introduce myself to the readers of Biophysical Reviews as part of the ‘meet the editors’ series.

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• Morales, J. M., Hammarström, B., Lippi, G. L., Vassalli, M. and Glynne-Jones, P.

  ; Acoustofluidic phase microscopy in a tilted segmentation-free configuration; Biomicrofluidics (2021) doi: 10.1063/5.0036585

  A low-cost device for registration-free quantitative phase microscopy (QPM) based on the transport of intensity equation of cells in continuous flow is presented. The method uses acoustic focusing to align cells into a single plane where all cells move at a constant speed. The acoustic focusing plane is tilted with respect to the microscope’s focal plane in order to obtain cell images at multiple focal positions. As the cells are displaced at constant speed, phase maps can be generated without the need to segment and register individual objects. The proposed inclined geometry allows for the acquisition of a vertical stack without the need for any moving part, and it enables a cost-effective and robust implementation of QPM. The suitability of the solution for biological imaging is tested on blood samples, demonstrating the ability to recover the phase map of single red blood cells flowing through the microchip.

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• Gavazzo, P., Viti, F., Donnelly, H., Oliva, M. A. G., Salmeron-Sanchez, M. , Dalby, M. J. and Vassalli, M.

  ; Biophysical phenotyping of mesenchymal stem cells along the osteogenic differentiation pathway; Cell Biology and Toxicology (2021) doi: 10.1007/s10565-020-09569-7

  Mesenchymal stem cells represent an important resource, for bone regenerative medicine and therapeutic applications. This review focuses on new advancements and biophysical tools which exploit different physical and chemical markers of mesenchymal stem cell populations, to finely characterize phenotype changes along their osteogenic differentiation process. Special attention is paid to recently developed label-free methods, which allow monitoring cell populations with minimal invasiveness. Among them, quantitative phase imaging, suitable for single-cell morphometric analysis, and nanoindentation, functional to cellular biomechanics investigation. Moreover, the pool of ion channels expressed in cells during differentiation is discussed, with particular interest for calcium homoeostasis. Altogether, a biophysical perspective of osteogenesis is proposed, offering a valuable tool for the assessment of the cell stage, but also suggesting potential physiological links between apparently independent phenomena.

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• 2020

• Ridone, P., Pandzic, E., Vassalli, M. , Cox, C. D., Macmillan, A., Gottlieb, P. A. and Martinac, B.

  ; Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters; Journal of General Physiology (2020) doi: 10.1085/jgp.201912515

  The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP’s response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure–response curve, increased channel latency in response to mechanical stimuli, and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM superresolution imaging revealed that, at the nanoscale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels.

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• Hammarström, B., Vassalli, M. and Glynne-Jones, P.

  ; Acoustic focussing for sedimentation-free high-throughput imaging of microalgae; Journal of Applied Phycology (2020) doi: 10.1007/s10811-019-01907-5

  Microalgae play a key role in aquatic ecology, and methods providing species determination and enumeration can provide critical information about—for instance—harmful algae blooms (HABs) or spreading of invasive species. A crucial step in current methods is the use of sedimentation. This provides the enrichment needed to achieve statistical counts of sometimes rare species within reasonable timeframes, but it comes with the drawback of aggregating the sample. This is a real challenge for computer-aided identification as particle aggregates can often be erroneously classified. In this paper, we propose an alternative method based on flow-through imaging aided by acoustic-focussing, as this provides better input-data for automated counting-methods while simultaneously removing the need for manual sample preparation. We demonstrate that by acoustically focussing microalgae and other particulates in a fast-flowing water sample, it is possible to analyse up to 8 mL sample per minute with sufficient image quality to discriminate the invasive species Ostreopsis ovata from other particulates in samples taken directly from the Mediterranean. We also showcase the ability to achieve sharp images in flow-through at magnifications up to × 50.

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• Gambardella, C., Piazza, V., Vassalli, M. , Sbrana, F., Lavorano, S., Garaventa, F. and Faimali, M.

  ; Microplastics ingestion in the ephyra stage of Aurelia sp. triggers acute and behavioral responses.; Ecotoxicology and Environmental Safety (2020) doi: 10.1016/j.ecoenv.2019.109983

  For the first time, we report a correspondence between microplastics (MP) ingestion and ecotoxicological effects in gelatinous zooplankton (Cnidarian jellyfish). The ephyra stage of the jellyfish Aurelia sp. was exposed to both environmental and high concentrations of fluorescent 1–4 μm polyethylene MP (0.01–10 mg/L). After 24 and 48 h, MP accumulation, acute (Immobility) and behavioral (Frequency pulsation) endpoints were investigated. MP were detected by confocal and tomographic investigations on gelatinous body and mouth, either attached on the surface or ingested. This interaction was responsible for impairing ephyrae survival and behavior at all tested concentrations after 24 h. Acute and behavioral effects were also related to mechanical disturbance, caused by MP, triggering a loss of radial symmetry. Contaminated ephyrae exposed to clean seawater showed full recovery after 72 h highlighting the organisms without the microspheres, attached on body jellyfish surface around the mouth and lappets. In conclusion, short-term exposure to MP affects ephyrae jellyfish health, impairing both their survival and behavior. Polyethylene MP temporarily affect both Immobility and Frequency of pulsation of Aurelia sp. jellyfish. This study provides a first step towards understanding and clarifying the potential impacts of MP contamination in gelatinous zooplankton.

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• Bartolozzi, A., Viti, F., De Stefano, S., Sbrana, F., Petecchia, L., Gavazzo, P. and Vassalli, M.

  ; Development of label-free biophysical markers in osteogenic maturation; Journal of the Mechanical Behavior of Biomedical Materials (2020) doi: 10.1016/j.jmbbm.2019.103581

  The spatial and temporal changes of morphological and mechanical properties of living cells reflect complex functionally-associated processes. Monitoring these modifications could provide a direct information on the cellular functional state. Here we present an integrated biophysical approach to the quantification of the morphological and mechanical phenotype of single cells along a maturation pathway. Specifically, quantitative phase microscopy and single cell biomechanical testing were applied to the characterization of the maturation of human foetal osteoblasts, demonstrating the ability to identify effective label-free biomarkers along this fundamental biological process.

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• Mattarelli, M., Vassalli, M. and Caponi, S.

  ; Relevant length scales in Brillouin imaging of biomaterials: the interplay between phonons propagation and light focalization; ACS Photonics (2020) doi: 10.1021/acsphotonics.0c00801

  Recent advances in photonics technologies pushed optical microscopy toward new horizons in materials characterization. In this framework, Brillouin microscopy emerged as an innovative method to provide images of materials with mechanical contrast without any physical contact, but exploiting the light-matter interaction. Brillouin imaging holds great promise: to allow mechanical analysis inside soft and heterogeneous materials, addressing the pivotal role played by viscoelastic properties in the physiology and pathology of living tissues and cells. Nevertheless, extending the approach of Brillouin imaging to characterize elastic heterogeneities of micro- and nanostructured samples is especially challenging, and it poses a critical question about the actual spatial resolution reachable in the mechanical characterization. We focus this critical review on the key quantities that define the spatial resolution in the Brillouin scattering process, and we highlight that not only the optical focalization of the light, but also the acoustic excitations present in the material influence the information collected from a sample by Brillouin imaging. Referring to the body of knowledge gained in the field of material science, we review new results and recently obtained progresses in the more unexplored context of life science. In future developments, a comprehensive strategy to tackle both the acoustic and the optical aspects of the measurement will be required to maximize the efficacy of the technique.

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• Wich Orapiriyakul, Monica P. Tsimbouri, Peter Childs, Paul Campsie, Julia Wells, Marc A. Fernandez-Yague, Karl Burgess, K. Elizabeth Tanner, Manlio Tassieri, Dominic Meek, Massimo Vassalli, Manus J. P. Biggs, Manuel Salmeron-Sanchez, Richard O. C. Oreffo, Stuart Reid, and Matthew J. Dalby

  ; Nanovibrational stimulation of mesenchymal stem cells induces therapeutic reactive oxygen species and inflammation for 3D bone tissue engineering; ACS Nano (2020) doi: 10.1021/acsnano.0c03130

  There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs’ responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells’ mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

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• Ciccone, G., Dobre, O. , Gibson, G. M. , Rey, J. M., Gonzalez-Garcia, C., Vassalli, M. , Salmeron-Sanchez, M. and Tassieri, M.

  ; What caging force cells feel in 3D hydrogels: a rheological perspective; Advanced Healthcare Materials (2020) doi: 10.1002/adhm.202000517

  It has been established that the mechanical properties of hydrogels control the fate of (stem) cells. However, despite its importance, a one‐to‐one correspondence between gels' stiffness and cell behavior is still missing from literature. In this work, the viscoelastic properties of poly(ethylene‐glycol) (PEG)‐based hydrogels are investigated by means of rheological measurements performed at different length scales. The outcomes of this work reveal that PEG‐based hydrogels show significant stiffening when subjected to a compressional deformation, implying that conventional bulk rheology measurements may overestimate the stiffness of hydrogels by up to an order of magnitude. It is hypothesized that this apparent stiffening is caused by an induced “tensional state” of the gel network, due to the application of a compressional normal force during sample loading. Moreover, it is shown that the actual stiffness of the hydrogels is instead accurately determined by means of both passive‐video‐particle‐tracking (PVPT) microrheology and nanoindentation measurements, which are inherently performed at the cell's length scale and in absence of any externally applied force in the case of PVPT. These results underpin a methodology for measuring hydrogels' linear viscoelastic properties that are representative of the mechanical constraints perceived by cells in 3D hydrogel cultures.

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• Lüchtefeld, I., Bartolozzi, A., Mejía Morales, J., Dobre, O. , Basso, M., Zambelli, T. and Vassalli, M.

  ; Elasticity spectra as a tool to investigate actin cortex mechanics; Journal of Nanobiotechnology (2020) doi: 10.1186/s12951-020-00706-2

  Background

  The mechanical properties of single living cells have proven to be a powerful marker of the cell physiological state. The use of nanoindentation-based single cell force spectroscopy provided a wealth of information on the elasticity of cells, which is still largely to be exploited. The simplest model to describe cell mechanics is to treat them as a homogeneous elastic material and describe it in terms of the Young’s modulus. Beside its simplicity, this approach proved to be extremely informative, allowing to assess the potential of this physical indicator towards high throughput phenotyping in diagnostic and prognostic applications.

  Results

  Here we propose an extension of this analysis to explicitly account for the properties of the actin cortex. We present a method, the Elasticity Spectra, to calculate the apparent stiffness of the cell as a function of the indentation depth and we suggest a simple phenomenological approach to measure the thickness and stiffness of the actin cortex, in addition to the standard Young’s modulus.

  Conclusions

  The Elasticity Spectra approach is tested and validated on a set of cells treated with cytoskeleton-affecting drugs, showing the potential to extend the current representation of cell mechanics, without introducing a detailed and complex description of the intracellular structure.

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• 2019

• Pietro Ridone, Massimo Vassalli, Boris Martinac

  ; Piezo1 mechanosensitive channels: what are they and why are they important; Biophysical Reviews (2019) doi: 10.1007/s12551-019-00584-5

  Mechanosensitive (MS) ion channels are integral membrane proteins which play a crucial role in fast signaling during mechanosensory transduction processes in living cells. They are ubiquitous and old in the evolutionary sense, given their presence in cells from all three kingdoms of life found on Earth, including bacterial, archaeal, and eukaryotic organisms. As molecular transducers of mechanical force, MS channels are activated by mechanical stimuli exerted on cellular membranes, upon which they rapidly and efficiently convert these stimuli into electrical, osmotic, and/or chemical intracellular signals. Most of what we know about the gating mechanisms of MS channels comes from the work carried out on bacterial channels. However, recent progress resulting from identification and structural information of eukaryotic K2P-type TREK and TRAAK as well as Piezo1 and Piezo2 MS channels has greatly contributed to our understanding of the common biophysical principles underlying the gating mechanism and evolutionary origins of these fascinating membrane proteins. Using Piezo1 channels as an example, we briefly describe in this review what we have learned about their biophysics, physiological functions, and potential roles in “mechanopathologies.”

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• Bruno Tiribilli, Michele Basso, Franco Quercioli, Massimo Vassalli

  ; Optical refraction with a toy robot; Physics Education (2019) doi: 10.1088/1361-6552/ab3e25

  A mechanical model of light propagation helps to show that optical refraction is related to the speed of light and how it changes from one medium to another. A tricycle toy robot is used to realize the model. Left and right wheels independently change their speed (high or low) in response to the local color (white or black, respectively) of the ground pattern detected by the corresponding optical sensor. Because of this simple rule the robot path deviates when passing, for example, from a clear to a dark area, in this way mimicking the refraction of a beam of light at an air–glass interface. Black silhouettes, representing optical components, are positioned on a clear mat along the robot trip. The robot trajectories reproduce phenomena such as refraction and total internal reflection showing a perfect analogy to light path described by geometrical optics in a sort of live ray-tracing.

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• Margheri, G., Tiribilli, B., Trigari, S. and Vassalli, M.

  ; Switchable lensed linear micro axicon in plasmonic structures for all optical light processing; IEEE Photonics Technology Letters (2019) doi: 10.1109/LPT.2019.2934583

  We propose a simple route to build up Fresnel bi-mirror microaxicons based on the localized absorption of an astigmatic pump laser beam by a plasmonic metal foil. The corresponding local overheating produces two main effects: the thermal swelling of the glass and the local increase of its refractive index. As a result, a two-mirrors axicon-like element coupled to a gradient index microlens is produced. We modelled the optothermal formation of this microelement by Finite Element Modeling and tested the predicted temperature raise by Scanning Thermal Microscopy (SThM). We calculated and measured the far field distribution of an impinging probe beam that passes through the lensed microaxicon, finding in both cases a noticeable change in the far field pattern when the pumping light is ON evidencing at the same time the good modulation capability of this opto-thermal element. A similar, even if smaller, effect was also found when the probe light impinges from the air side, where no gradient index region is present. This configuration allowed us to measure and confirm the theoretically expected angular deflection and, indirectly, the high value of the vertical thermomechanical deformation induced by the tight astigmatic focusing of the pump beam.

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• Baldini, F., Bartolozzi, A., Ardito, M., Voci, A., Portincasa, P., Vassalli, M. and Vergani, L.

  ; Biomechanics of cultured hepatic cells during different steatogenic hits; Journal of the Mechanical Behavior of Biomedical Materials (2019) doi: 10.1016/j.jmbbm.2019.05.036

  Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease often associated with overnutrition. Number and morphometry of lipid droplets (LDs) define micro vs macrovesicular steatosis, influence the morphology and function of hepatocytes and possibly their stiffness. The link between grade and features of steatosis and biomechanical properties of single hepatocytes requires deeper investigations. In vitro NAFLD models with distinct steatosis conditions were set by exposing FaO hepatoma cells to single or combined fructose (Fru), fatty acids (FA), and tumor necrosis factor (TNF)α. Single Cell Force Spectroscopy and Quantitative Phase Microscopy quantified the single cell stiffness and a series of morphometric parameters; the mRNA expression of genes involved in lipid metabolism was quantified by real-time PCR. In our models, LD size and number increased with Fru and FA as single agents, and more with combined Fru/FA (macrovesicular steatosis), while FA/TNFα combination increased LD number with a reduction in their size (microvesicular steatosis). We found that the changes in LD size and number influenced cell stiffness and morphometry as follows: (i) single cell elasticity increased in macrovesicular steatosis (maximally with combined Fru/FA); (ii) FA-induced steatosis resulted in cells thinner and larger, whereas combined FA/TNFα shrunk the hepatocytes. Taken together the data on hepatocyte biomechanics show that, in addition to extent of lipid accumulation, cell stiffness is mainly influenced by LD size, while cell morphometry directly relates to LD number. Our findings suggest that a novel mechanobiology perspective might provide future contributions in NAFLD research.

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• Ferrari, A., Capitanio, M., Vassalli, M. and Martinac, B.

  ; Science by the sea: how nanoengineering met mechanobiology in Camogli; Biophysical Reviews (2019) doi: 10.1007/s12551-019-00598-z

  [...] We realized that technology developers, i.e. the engineers, and their adopters, the biologists and medical doctors, had very little occasions to meet and discuss at international congresses. Each category attended specialized meetings, where different scientific languages are spoken, further marking the divide. Based on that dinner chat, Massimo had the brilliant idea of creating a venue where researchers in nanoengineering with interest in biological applications and biologists open to innovative technologies could meet, discuss and develop new ideas. [...]

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• Alfieri, R., Vassalli, M. and Viti, F.

  ; Flow-induced mechanotransduction in skeletal cells; Biophysical Reviews (2019) doi: 10.1007/s12551-019-00596-1

  Human body is subject to many and variegated mechanical stimuli, actuated in different ranges of force, frequency, and duration. The process through which cells “feel” forces and convert them into biochemical cascades is called mechanotransduction. In this review, the effects of fluid shear stress on bone cells will be presented. After an introduction to present the major players in bone system, we describe the mechanoreceptors in bone tissue that can feel and process fluid flow. In the second part of the review, we present an overview of the biological processes and biochemical cascades initiated by fluid shear stress in bone cells.

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• 2018

• Vassalli, M; Penna, A; Sbrana, F; Casabianca, S; Gjeci, N; Capellacci, S; Asnaghi, V; Ottaviani, E; Giussani, V; Pugliese, L; Jauzeine, C; Lemée, R; Hachani, MA; Turki, S; Açaf, L; Abboud-Abi Saab, M; Fricke, A; Mangialajo, L; Bertolotto, R; Totti, C; Accoroni, S; Berdalet, E; Vila, M; Chiantore, MC

  ; Intercalibration of counting methods for Ostreopsis spp. blooms in the Mediterranean Sea.; Ecological Indicators (2018) doi: 10.1016/j.ecolind.2017.07.063

  This paper describes the adoption and validation of two innovative methods for the automated count of Ostreopsis spp. concentration in sea water: a molecular assay based on RT-qPCR and an opto-electronic device implementing automatic recognition algorithms. The proposed approaches were tested on samples coming from different locations along the Mediterranean Sea and compared with the standard counting method based on microscopy observation by a taxonomy expert. The results demonstrate the effectiveness of both automatic approaches which provide a valuable tool, mostly cost and time effective, for the establishment of wide pan-Mediterranean monitoring strategies of Ostreopsis spp. blooms. Moreover, the two automatic methods demonstrated the ability to discriminate for the presence of a different but similar species, O. fattorussoi, for which new species-specific qPCR primers were developed.

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• Bloise, N; Petecchia, L; Ceccarelli, G; Fassina, L; Usai, C; Bertoglio, F; Balli, M; Vassalli, M; De Angelis, GC; Gavazzo, P; Imbriani, M; Visai L

  ; The effect of pulsed electromagnetic field exposure on osteoinduction of human mesenchymal stem cells cultured on nano-TiO2 surfaces.; Plos One (2018) doi: 10.1371/journal.pone.0199046

  Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with TiO2 nanostructured surface might be a great option in bone tissue engineering applications.

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• Vitale, RM; Rispoli, V; Desiderio, D; Sgammato, R; Thellung, S; Canale, C; Vassalli, M; Carbone, M; Ciavatta, ML; Mollo, E; Felicità, V; Arcone, R; Capoggiani, MG; Masullo, M; Florio, T; Amodeo P

  ; In silico identification and experimental validation of novel anti-Alzheimer’s multitargeted ligands from a marine source featuring a “2-aminoimidazole plus aromatic group” scaffold.; ACS Chemical Neuroscience (2018) doi: 10.1021/acschemneuro.7b00416

  Multitargeting or polypharmacological approaches, looking for single chemical entities retaining the ability to bind two or more molecular targets, are a potentially powerful strategy to fight complex, multifactorial pathologies. Unfortunately, the search for multiligand agents is challenging because only a small subset of molecules contained in molecular databases are bioactive and even fewer are active on a preselected set of multiple targets. However, collections of natural compounds feature a significantly higher fraction of bioactive molecules than synthetic ones. In this view, we searched our library of 1175 natural compounds from marine sources for molecules including a 2-aminoimidazole+aromatic group motif, found in known compounds active on single relevant targets for Alzheimer’s disease (AD). This identified two molecules, a pseudozoanthoxanthin (1) and a bromo-pyrrole alkaloid (2), which were predicted by a computational approach to possess interesting multitarget profiles on AD target proteins. Biochemical assays experimentally confirmed their biological activities. The two compounds inhibit acetylcholinesterase, butyrylcholinesterase, and β-secretase enzymes in high- to sub-micromolar range. They are also able to prevent and revert β-amyloid (Aβ) aggregation of both Aβ1–40 and Aβ1–42 peptides, with 1 being more active than 2. Preliminary in vivo studies suggest that compound 1 is able to restore cholinergic cortico-hippocampal functional connectivity.

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• Cacace, T., Bianco, V., Paturzo, M., Memmolo, P., Vassalli, M. , Fraldi, M., Mensitieri, G. and Ferraro, P.

  ; Retrieving acoustic energy densities and local pressure amplitudes in microfluidics by holographic time-lapse imaging.; Lab on a Chip (2018) doi: 10.1039/C8LC00149A

  The development of techniques able to characterize and map the pressure field is crucial for the widespread use of acoustofluidic devices in biotechnology and lab-on-a-chip platforms. In fact, acoustofluidic devices are powerful tools for driving precise manipulation of microparticles and cells in microfluidics in non-contact modality. Here, we report a full and accurate characterization of the movement of particles subjected to acoustophoresis in a microfluidic environment by holographic imaging. The particle displacement along the direction of the ultrasound wave propagation, coinciding with the optical axis, is observed and investigated. Two resonance frequencies are explored, varying for each the amplitude of the applied signal. The trajectories of individual tracers, accomplished by holographic measurements, are fitted with the theoretical model thus allowing the retrieval of the acoustic energy densities and pressure amplitudes through full holographic analysis. The absence of prior calibration, being independent of the object shape and the possibility of implementing automatic analysis make the use of holography very appealing for applications in devices for biotechnologies.

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• 2017

• Petecchia, L., Usai, C., Vassalli, M. and Gavazzo, P.

  ; Biophysical characterization of nanostructured TiO2 as a good substrate for hBM-MSC adhesion, growth and differentiation.; Experimental Cell Research (2017) doi: 10.1016/j.yexcr.2017.06.008

  Mesenchymal stem cells from human bone marrow (hBM-MSC) are widely utilized for clinical applications involving bone healing. In this context, their use has been often optimized in association to variously designed titanium substrates, being this material of great use in orthopaedic implants. According to recent findings, the ability of hBM-MSC to differentiate towards a specific lineage is not only driven by biochemical signals, but physical stimuli, such as rigidity or roughness of the substrate, can also support a commitment towards osteogenic differentiation. Moreover, the presence of features with defined dimensional scales, in particular nanometer-size, also proved to elicit specific biological effects. Here we evaluated the effectiveness of a nano-patterned titanium surface in sustaining hBM-MSC adhesion, growth and differentiation by means of a panel of biophysical tools: morphometry, electrophysiology, intracellular calcium measurements and immunocytochemistry. The results substantiate the idea that this micro-textured titanium dioxide is a good surface for growth and differentiation of hBM-MSC and it exhibits a stimulating action mainly in the initial period of differentiation. Moreover, the basal concentration of free cytosolic Calcium [Ca2+]i is confirmed to be a good hallmark of the hBM-MSC maturation stage. The study could provide relevant hints to help improving the biocompatibility and osteointegration potential of clinical titanium implants.

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• Sbrana, F., Landini, E., Gjeci, N., Viti, F., Ottoviani, E. and Vassalli, M.

  ; OvMeter: an automated 3D-integrated opto-electronic system for Ostreopsis cf. ovata bloom monitoring.; Journal of Applied Phycology (2017) doi: 10.1007/s10811-017-1069-7

  Over the last decade, toxic events along the Mediterranean coast associated with exceptional harmful blooms of the dinoflagellate Ostreopsis cf. ovata have increased in frequency and distribution, causing not only the death of marine organisms and human health problems, but also economic loss on the tourism and aquaculture industries. In order to reduce the burden of routine algal counting, an innovative automated, low-cost, opto-electronic system called OvMeter was developed. It is able to speed up the monitoring process and therefore it enables early warning of incipient harmful algal blooms. An ad-hoc software tool provides automated cell recognition, counting and real-time calculation of the final algal concentration. The core of dinoflagellate recognition relies on a localization step which takes advantage of the synergistic exploitation of 2D bright-field and quantitative phase microscopy images, and a classification phase performed by a machine learning algorithm based on Boosted Trees approach. The architectural design of the OvMeter device is presented here, together with a performance evaluation on sea samples.

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• Cacace, T., Paturzo, M., Memmolo, P., Vassalli, M. , Ferraro, P., Fraldi, M. and Mensitieri, G.

  ; Digital holography as 3D tracking tool for assessing acoustophoretic particle manipulation.; Optics Express (2017) doi: 10.1364/OE.25.017746

  The integration of digital holography (DH) imaging and the acoustic manipulation of micro-particles in a microfluidic environment is investigated. The ability of DH to provide efficient 3D tracking of particles inside a microfluidic channel is exploited to measure the position of multiple objects moving under the effect of stationary ultrasound pressure fields. The axial displacement provides a direct verification of the numerically computed positions of the standing wave’s node, while the particles’ transversal movement highlights the presence of nodes in the planar direction. Moreover, DH is used to follow the aggregation dynamics of trapped spheres in such nodes by using aggregation rate metrics.

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• Palazzolo, G., Moroni, M., Soloperto, A., Aletti, G., Naldi, G., Vassalli, M. , Nieus, T. and Difato, F.

  ; Fast wide-volume functional imaging of engineered in vitro brain tissues.; Scientific Reports (2017) doi: 10.1038/s41598-017-08979-8

  The need for in vitro models that mimic the human brain to replace animal testing and allow high-throughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor the functional states of 3D networks. Herein, we propose an experimental model based on 3D neuronal networks in an alginate hydrogel, a tunable wide-volume imaging approach, and an efficient denoising algorithm to resolve, down to single cell resolution, the 3D activity of hundreds of neurons expressing the calcium sensor GCaMP6s. Furthermore, we implemented a 3D co-culture system mimicking the contiguous interfaces of distinct brain tissues such as the cortical-hippocampal interface. The analysis of the network activity of single and layered neuronal co-cultures revealed cell-type-specific activities and an organization of neuronal subpopulations that changed in the two culture configurations. Overall, our experimental platform represents a simple, powerful and cost-effective platform for developing and monitoring living 3D layered brain tissue on chip structures with high resolution and high throughput.

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• Petecchia, L., Viti, F., Sbrana, F., Vassalli, M. and Gavazzo, P.

  ; A biophysical approach to quantify skeletal stem cells trans-differentiation as a model for the study of osteoporosis. ; Biophysical Chemistry (2017) doi: 10.1016/j.bpc.2017.05.011

  The stroma of human bone marrow contains a population of skeletal stem cells (hBM-MSC) which are common ancestors, among the others, of osteoblasts and adipocytes. It has been proposed that the imbalance between hBM-MSC osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. The possibility to reproduce this mechanism in vitro has been demonstrated, providing a good model to disclose the details of the complex bone-fat generation homeostasis. Nevertheless, the lack of a simple approach to quantitatively assess the actual stage of a cellular population hindered the adoption of this in vitro model. In this work, the direct differentiation of hBM-MSCs towards a single (osteo or adipo) lineage was characterized using quantitative biophysical and biological approaches, together with the parallel process of trans-differentiation from one lineage to the other. The results confirm that the original plasticity of hBM-MSCs is maintained along the initial stages of the differentiation, showing that in vitro conversion of pre-osteoblasts into adipocytes and, vice versa, of pre-adipocytes into osteoblasts is extremely efficient, comparable with the direct differentiation. Moreover, a method based on digital holography is proposed, providing a quantitative indication of the phenotype stage along differentiation.

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• Gavazzo, P, Petecchia, L, Facci, P, Vassalli, M, Viti, F

  ; Controlled single-cell cyclic compression and transcription analysis: a pilot study; Biophysical Chemistry (2017) doi: 10.1016/j.bpc.2017.07.010

  An innovative platform for the study of the molecular mechanisms at the basis of mechanotransduction has been implemented, developing an experimental approach capable of providing controlled dynamic compression stimuli and retrieving the biomolecular response with single-cell sensitivity. The system provides the ability to perform compression-release cycles on single cells with controlled forces in the nN range and a user-defined repetition rate. Experimental procedures to perform qPCR from a small set of single cells were finely tuned. The experimental platform was tested in the context of bone (cell line hFOB 1.19), a physiological environment highly subjected to mechanical stimuli. Target genes were identified in the literature, based on their involvement in the osteogenesis process or in the bone response to mechanical stimuli. qPCR analysis shows an increase in expression of the chosen targets, and confirms the effectiveness of the presented approach for studying living single cells response to dynamic compression.

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• S Alloisio, P Garbati, F Viti, S Dante, R Barbieri, G Arnaldi, A Petrelli, A Gigoni, P Giannoni, R Quarto, M Nobile, M Vassalli, A Pagano

  ; Generation of a functional human neural network by NDM29 overexpression in neuroblastoma cancer cells.; Molecular Neurobiology (2017) doi: 10.1007/s12035-016-0161-3

  Recent advances in life sciences suggest that human and rodent cell responses to stimuli might differ significantly. In this context, the results achieved in neurotoxicology and biomedical research practices using neural networks obtained from mouse or rat primary culture of neurons would benefit of the parallel evaluation of the same parameters using fully differentiated neurons with a human genetic background, thus emphasizing the current need of neuronal cells with human origin. In this work, we developed a human functionally active neural network derived by human neuroblastoma cancer cells genetically engineered to overexpress NDM29, a non-coding RNA whose increased synthesis causes the differentiation toward a neuronal phenotype. These cells are here analyzed accurately showing functional and morphological traits of neurons such as the expression of neuron-specific proteins and the possibility to generate the expected neuronal current traces and action potentials. Their morphometrical analysis is carried out by quantitative phase microscopy showing soma and axon sizes compatible with those of functional neurons. The ability of these cells to connect autonomously forming physical junctions recapitulates that of hippocampal neurons, as resulting by connect-ability test. Lastly, these cells self-organize in neural networks able to produce spontaneous firing, in which spikes can be clustered in bursts. Altogether, these results show that the neural network obtained by NDM29-dependent differentiation of neuroblastoma cells is a suitable tool for biomedical research practices.

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