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Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ 1 ) followed by a slower one (τ 2 ). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ 1 with respect to τ 2 . Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca 2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca 2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity., (© 2024. The Author(s).)

Actin-binding filamin C (FLNC) is expressed in cardiomyocytes, where it localizes to Z-discs, sarcolemma, and intercalated discs. Although FLNC truncation variants ( FLNCtv ) are an established cause of arrhythmias and heart failure, changes in biomechanical properties of cardiomyocytes are mostly unknown. Thus, we investigated the mechanical properties of human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) carrying FLNCtv . CRISPR/Cas9 genome-edited homozygous FLNC KO-/- hiPSC-CMs and heterozygous knock-out FLNC KO+/- hiPSC-CMs were analyzed and compared to wild-type FLNC (FLNC WT ) hiPSC-CMs. Atomic force microscopy (AFM) was used to perform micro-indentation to evaluate passive and dynamic mechanical properties. A qualitative analysis of the beating traces showed gene dosage-dependent-manner "irregular" peak profiles in FLNC KO+/- and FLNC KO-/- hiPSC-CMs. Two Young's moduli were calculated: E1, reflecting the compression of the plasma membrane and actin cortex, and E2, including the whole cell with a cytoskeleton and nucleus. Both E1 and E2 showed decreased stiffness in mutant FLNC KO+/- and FLNC KO-/- iPSC-CMs compared to that in FLNC WT . The cell adhesion force and work of adhesion were assessed using the retraction curve of the SCFS. Mutant FLNC iPSC-CMs showed gene dosage-dependent decreases in the work of adhesion and adhesion forces from the heterozygous FLNC KO+/- to the FLNC KO-/- model compared to FLNC WT , suggesting damaged cytoskeleton and membrane structures. Finally, we investigated the effect of crenolanib on the mechanical properties of hiPSC-CMs. Crenolanib is an inhibitor of the Platelet-Derived Growth Factor Receptor α (PDGFRA) pathway which is upregulated in FLNCtv hiPSC-CMs. Crenolanib was able to partially rescue the stiffness of FLNC KO-/- hiPSC-CMs compared to control, supporting its potential therapeutic role.

The production of glass foams obtained by recycling post-consumer glass and textile industry processing waste is presented. The mechanical, thermal and acoustic properties were characterized as a function of process temperature and time. The results showed that it is possible to produce glass foams with thermal and acoustic insulation properties from a mixture consisting of 96.5% of glass waste, 1% of textile waste and 2.5% of manganese dioxide, processed at temperatures between 800 and 900 °C for a time between 30 and 90 min. The samples had density in the range of 200-300 kg m -3 , porosity of 87-92%, thermal conductivity of 85-105 mW m -1 K -1 , noise-reducing factors of 0.15-0.40 and compressive strength of 1.2-3.0 MPa. Although their insulation performance was not as outstanding as that of polymer foams, these materials can emerge as competitive candidates for applications requiring non-flammability and high-temperature load bearing capacity in combination with low weight, mechanical strength, and thermal and acoustic insulation properties. The use of secondary raw materials (which accounted for 97.5% by weight of the synthetic blend) limits the energy required compared to that needed for the extraction, transportation and processing of primary raw materials, making these foams attractive also in terms of environmental footprint.

Given the clinical effect of progeria syndrome, understanding the cell mechanical behavior of this pathology could benefit the patient's treatment. Progeria patients show a point mutation in the lamin A/C gene (LMNA), which could change the cell's biomechanical properties. This paper reports a mechano-dynamic analysis of a progeria mutation (c.1824 C > T, p.Gly608Gly) in neonatal rat ventricular myocytes (NRVMs) using cell indentation by atomic force microscopy to measure alterations in beating force, frequency, and contractile amplitude of selected cells within cell clusters. Furthermore, we examined the beating rate variability using a time-domain method that produces a Poincaré plot because beat-to-beat changes can shed light on the causes of arrhythmias. Our data have been further related to our cell phenotype findings, using immunofluorescence and calcium transient analysis, showing that mutant NRVMs display changes in both beating force and frequency. These changes were associated with a decreased gap junction localization (Connexin 43) in the mutant NRVMs even in the presence of a stable cytoskeletal structure (microtubules and actin filaments) when compared with controls (wild type and non-treated cells). These data emphasize the kindred between nucleoskeleton (LMNA), cytoskeleton, and the sarcolemmal structures in NRVM with the progeria Gly608Gly mutation, prompting future mechanistic and therapeutic investigations.

Fibrotic tissues share many common features with neoplasms where there is an increased stiffness of the extracellular matrix (ECM). In this review, we present recent discoveries related to the role of the mechanosensitive ion channel Piezo1 in several diseases, especially in regulating tumor progression, and how this can be compared with cardiac mechanobiology. Based on recent findings, Piezo1 could be upregulated in cardiac fibroblasts as a consequence of the mechanical stress and pro-inflammatory stimuli that occurs after myocardial injury, and its increased activity could be responsible for a positive feedback loop that leads to fibrosis progression. The increased Piezo1-mediated calcium flow may play an important role in cytoskeleton reorganization since it induces actin stress fibers formation, a well-known characteristic of fibroblast transdifferentiation into the activated myofibroblast. Moreover, Piezo1 activity stimulates ECM and cytokines production, which in turn promotes the phenoconversion of adjacent fibroblasts into new myofibroblasts, enhancing the invasive character. Thus, by assuming the Piezo1 involvement in the activation of intrinsic fibroblasts, recruitment of new myofibroblasts, and uncontrolled excessive ECM production, a new approach to blocking the fibrotic progression can be predicted. Therefore, targeted therapies against Piezo1 could also be beneficial for cardiac fibrosis.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell-substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell's ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disorder characterized by progressive replacement of cardiomyocytes by fibrofatty tissue, ventricular dilatation, cardiac dysfunction, arrhythmias, and sudden cardiac death. Interest in molecular biomechanics for these disorders is constantly growing. Atomic force microscopy (AFM) is a well-established technic to study the mechanobiology of biological samples under physiological and pathological conditions at the cellular scale. However, a review which described all the different data that can be obtained using the AFM (cell elasticity, adhesion behavior, viscoelasticity, beating force, and frequency) is still missing. In this review, we will discuss several techniques that highlight the potential of AFM to be used as a tool for assessing the biomechanics involved in ACM. Indeed, analysis of genetically mutated cells with AFM reveal abnormalities of the cytoskeleton, cell membrane structures, and defects of contractility. The higher the Young's modulus, the stiffer the cell, and it is well known that abnormal tissue stiffness is symptomatic of a range of diseases. The cell beating force and frequency provide information during the depolarization and repolarization phases, complementary to cell electrophysiology (calcium imaging, MEA, patch clamp). In addition, original data is also presented to emphasize the unique potential of AFM as a tool to assess fibrosis in cardiac tissue.

Single-cell adhesion measured with atomic force microscopy (AFM) offers outstanding time and force resolution and allows the investigation of many important phenomena with unmatched precision. However, this technique suffers from serious practical limitations that hinder its effective application to a broader set of situations. Here we propose a different strategy based on the fabrication of large cantilevers and on the culture of the cells directly on them. Cantilevers are fabricated by standard micromachining, with an active area of 300 × 300 µm. A wedged structure is created so that the cantilever surface lies parallel to the substrate when mounted on an AFM system, so that the adhesion measurement probes the whole surface area at the same time. Thanks to the large area, cells can be seeded and grown on the cantilevers the day before the experiment, and let recover to optimal condition for the experiment. We used Human Embryonic Kidney cells, HEK 293A, to demonstrate the measurement of adhesion forces of up to 100 cells in parallel, and obtain a straightforward measurement of the average single cell adhesion energy. Our approach can improve significantly the cell-cell and cell-substrate adhesion statistics, reduce the experiment time and allow the investigation of the adhesion properties of cells that do not grow well in solution or on low adherent substrates, or that develop their characteristic features only after several hours or days of culture on a solid and adherent substrate., (© 2021. European Biophysical Societies' Association.)

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene ( LMNA ), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA -cardiomyopathy.

Purpose of Review: The giant protein titin forms the "elastic" filament of the sarcomere, essential for the mechanical compliance of the heart muscle. Titin serves a biological spring, and therefore structural modifications of titin affect function of the myocardium and are associated with heart failure and cardiomyopathy., Recent Findings: In this review, we discuss the current understanding of titin's biophysical properties and how modifications contribute to cardiac function and heart failure. In addition, we review the most recent data on the clinical impact and phenotype heterogeneity of TTN truncating variants, including diseases involving striated muscles, and prospects for future therapies. Because of the giant structure of the titin protein and the complexity of its function, titin's role in health and disease is not yet completely understood. Future research efforts need to focus on novel therapeutic approaches able to modulate titin transcriptional and post-translational modification., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Danon disease is a severe X-linked disorder caused by deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Clinical manifestations are phenotypically diverse and consist of hypertrophic and dilated cardiomyopathies, skeletal myopathy, retinopathy, and intellectual dysfunction. Here, we investigated the metabolic landscape of Danon disease by applying a multi-omics approach and combined structural and functional readouts provided by Raman and atomic force microscopy. Using these tools, Danon patient-derived cardiac tissue, primary fibroblasts, and human induced pluripotent stem cells differentiated into cardiomyocytes (hiPSC-CMs) were analyzed. Metabolic profiling indicated LAMP-2 deficiency promoted a switch toward glycolysis accompanied by rerouting of tryptophan metabolism. Cardiomyocytes' energetic balance and NAD+/NADH ratio appeared to be maintained despite mitochondrial aging. In turn, metabolic adaption was accompanied by a senescence-associated signature. Similarly, Danon fibroblasts appeared more stress prone and less biomechanically compliant. Overall, shaping of both morphology and metabolism contributed to the loss of cardiac biomechanical competence that characterizes the clinical progression of Danon disease., Competing Interests: The authors declare no conflict of interest.

Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (F unfold ). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se . Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling., (Copyright © 2020 Lanzicher, Zhou, Saripalli, Keschrumrus, Smith, Mayans, Sbaizero and Granzier.)

Given the clinical effect of laminopathies, understanding lamin mechanical properties will benefit the treatment of heart failure. Here we report a mechano-dynamic study of LMNA mutations in neonatal rat ventricular myocytes (NRVM) using single cell spectroscopy with Atomic Force Microscopy (AFM) and measured changes in beating force, frequency and contractile amplitude of selected mutant-expressing cells within cell clusters. Furthermore, since beat-to-beat variations can provide clues on the origin of arrhythmias, we analyzed the beating rate variability using a time-domain method which provides a Poincaré plot. Data were further correlated to cell phenotypes. Immunofluorescence and calcium imaging analysis showed that mutant lamin changed NRVMs beating force and frequency. Additionally, we noted an altered microtubule network organization with shorter filament length, and defective hemichannel membrane localization (Connexin 43). These data highlight the interconnection between nucleoskeleton, cytoskeleton and sarcolemmal structures, and the transcellular consequences of mutant lamin protein in the pathogenesis of the cardiac laminopathies ., (© 2020 The Author(s).)

Background: Laminopathies are genetic diseases caused by mutations in the nuclear lamina., Objective: Given the clinical impact of laminopathies, understanding mechanical properties of cells bearing lamin mutations will lead to advancement in the treatment of heart failure., Methods: Atomic force microscopy (AFM) was used to analyze the viscoelastic behavior of neonatal rat ventricular myocyte cells expressing three human lamin A/C gene (LMNA) mutations., Results: Cell storage modulus was characterized, by two plateaus, one in the low frequency range, a second one at higher frequencies. The loss modulus instead showed a "bell" shape with a relaxation toward fluid properties at lower frequencies. Mutations shifted the relaxation to higher frequencies, rendering the networks more solid-like. This increase of stiffness with mutations (solid like behavior) was at frequencies around 1 Hz, close to the human heart rate., Conclusions: These features resulted from a combination of the properties of cytoskeleton filaments and their temporary cross-linker. Our results substantiate that cross-linked filaments contribute, for the most part, to the mechanical strength of the cytoskeleton of the cell studied and the relaxation time is determined by the dissociation dynamics of the cross-linking proteins. The severity of biomechanical defects due to these LMNA mutations correlated with the severity of the clinical phenotype.

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte-myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1 Pkp2-shRNA ) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1 Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1 Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 ( Itga1 ) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, sudden cardiac death and heart transplant. DCM is inherited in approximately 50% of cases, in which the most frequent genetic defects are truncation variants of the titin gene ( TTN tv). TTN encodes titin, which is the largest protein in the body and is an essential component of the sarcomere. Titin serves as a biological spring, spanning half of the sarcomere and connecting the Z-disk to the M-line, with scaffold and signaling functions. Truncations of titin are believed to lead to either haploinsufficiency and loss-of-function, or to a "poison peptide" effect. However, other titin mechanisms are postulated to influence cardiac function including post-translational modifications, in particular changes in titin phosphorylation that alters the stiffness of the protein, and diversity of alternative splicing that generates different titin isoforms. In this article, we review the role of TTN mutations in development of DCM, how differential expression of titin isoforms relate to DCM pathophysiology, and discuss how post-translational modifications of titin can affect cardiomyocyte function. Current research efforts aim to elucidate the contribution of titin to myofibril assembly, stability, and signal transduction, and how mutant titin leads to cardiac dysfunction and human disease. Future research will need to translate this knowledge toward novel therapeutic approaches that can modulate titin transcriptional and post-translational defects to treat DCM and heart failure., Highlights: - Titin (TTN) truncation variants are the most frequent cause of dilated cardiomyopathy, one of the main causes of heart failure and heart transplant. Titin is a giant protein, and the mechanisms causing the disease are both complex and still incompletely understood.- This review discusses the role of titin in myocardial function and in disease. In particular, we discuss TTN gene structure, the complexity of genotype-phenotype correlation in human disease, the physiology of TTN and the role of post-translation modification.- Additional studies will be required to clarify whether missense variants are associated with cardiac disease. While initial studies suggested a role of non-synonymous variants in arrhythmogenic cardiomyopathy, confirmatory investigations have been hampered by the complexity of the protein structure and function., (Copyright © 2019 Tharp, Haywood, Sbaizero, Taylor and Mestroni.)

Purpose of Review: The purpose of this review is to provide an update on lamin A/C (LMNA)-related cardiomyopathy and discuss the current recommendations and progress in the management of this disease. LMNA-related cardiomyopathy, an inherited autosomal dominant disease, is one of the most common causes of dilated cardiomyopathy and is characterized by steady progression toward heart failure and high risks of arrhythmias and sudden cardiac death., Recent Findings: We discuss recent advances in the understanding of the molecular mechanisms of the disease including altered cell biomechanics, which may represent novel therapeutic targets to advance the current management approach, which relies on standard heart failure recommendations. Future therapeutic approaches include repurposed molecularly directed drugs, siRNA-based gene silencing, and genome editing. LMNA-related cardiomyopathy is the focus of active in vitro and in vivo research, which is expected to generate novel biomarkers and identify new therapeutic targets. LMNA-related cardiomyopathy trials are currently underway.

Heart failure is a disease of epidemic proportion and a leading cause of mortality in the world. Because cardiac myocytes are terminally differentiated cells with minimal intrinsic ability to self-regenerate, cardiac tissue engineering has emerged as one of the most realistic therapeutic strategies for cardiac repair. We have previously proven the ability of carbon nanotube scaffolds to promote cardiomyocyte proliferation, maturation, and long-term survival. Here, we tested if three-dimensional scaffolds of carbon nanotube-based composites can also promote cardiomyocyte growth, electrophysiological maturation, and formation of functional syncytia. To this purpose, we developed an elastomeric scaffold that consists of a microporous and self-standing material made of polydimethylsiloxane (PDMS) containing micrometric cavities, and integrated multiwall carbon nanotubes (MWCNTs) into the scaffold. We combined microscopy, cell biology, and calcium imaging to investigate whether neonatal rat ventricular myocytes (NRVMs) cultured on the 3D-PDMS+MWCNT acquire a more viable and mature phenotype compared to control. We found that when cultured in the 3D-PDMS+MWCNTs, NRVMs showed improved viability ( p < 0.005 at day 3) and more defined and mature sarcomeric phenotype compared to 3D-PDMS control. These modifications were associated with an increase of connexin-43 gene expression, gap junction areas ( p < 0.005 at day 3), and a more mature electrophysiological phenotype of syncytia and calcium transients. Finally, 3D-PDMS+MWCNT boosted NRVMs proliferation ( p < 0.005 at day 3) while hindering cardiac fibroblasts proliferation compared to control PDMS. Thus, 3D-PDMS+MWCNT has the ability to promote viability, proliferation and functional maturation of cardiac myocytes. These properties are essential in cardiac tissue engineering and offer novel perspectives in the development of innovative therapies for cardiac repair.

Morphology together with the capability to respond to surrounding stimuli are key elements governing the spatial interaction of living cells with the environment. In this respect, biomechanical stimulation can trigger significant physiological cascades that can potentially modulate toxicity. Deoxynivalenol (DON, vomitoxin) is one of the most prevalent mycotoxins produced by Fusarium spp. and it was used to explore the delicate interaction between biomechanical stimulation and cytotoxicity in A431 cells. In fact, in addition of being a food contaminant, DON is a relevant toxin for several organ systems. The combination between biomechanical stimulation and the mycotoxin revealed how DON can impair crucial functions affecting cellular morphology, tubulin and lysosomes at concentrations even below those known to be cytotoxic in routine toxicity studies. Sub-toxic concentrations of DON (0.1-1 μM) impaired the capability of A431 cells to respond to a biomechanical stimulation that normally sustains trophic effects in these cells. Moreover, the effects of DON (0.1-10 μM) were partially modulated by the application of uniaxial stretching (0.5 Hz, 24 h, 15% deformation). Ultimately, proteomic analysis revealed the potential of DON to alter several proteins necessary for cell adhesion and cytoskeletal modulation suggesting a molecular link between biomechanics and the cytotoxic potential of the mycotoxin.

Aims: Given the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM), and molecular modeling was used to analyse the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte model., Methods and Results: The severity of biomechanical defects due to the three LMNA mutations correlated with the severity of the clinical phenotype. AFM and molecular modeling identified distinctive biomechanical and structural changes, with increasing severity from E161K to N195K and D192G, respectively. Additionally, the biomechanical defects were rescued with a p38 MAPK inhibitor., Conclusions: AFM and molecular modeling were able to quantify distinct biomechanical and structural defects in LMNA mutations E161K, D192G, and N195K and correlate the defects with clinical phenotypic severity. Improvements in cellular biomechanical phenotype was demonstrated and may represent a mechanism of action for p38 MAPK inhibition therapy that is now being used in human clinical trials to treat laminopathies.

In a tissue continuously challenged by mechanical stresses, such as the skin or the heart, cells perceive information about their microenvironment through several adhesive protein complexes and activate cell-signaling events to maintain tissue cohesion. Consequently, alteration of cell adhesion components leads to aberrant assembly of the associated cytoplasmic scaffolding and signaling pathways, which may reflect changes to the tissue physiology and mechanical resistance. Desmoplakin is an essential component of the cell-cell junction, anchoring the desmosomal protein complex to the intermediate filaments (IFs). Inherited mutations in desmoplakin are associated with both heart and skin disease (cardiocutaneous syndrome). In this study, we investigated the mechanical properties of human keratinocytes harboring a cardiocutaneous-associated homozygous C-terminal truncation in desmoplakin (JD-1) compared to a control keratinocyte line (K1). Using Single Cell Force Spectroscopy (SCFS) AFM-based measurements, JD-1 keratinocytes displayed an overall alteration in morphology, elasticity, adhesion capabilities and viscoelastic properties, highlighting the profound interconnection between the adhesome pathways and the IF scaffold., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cardiovascular diseases (CVDs) are the number one cause of death globally, therefore interest in studying aetiology, hallmarks, progress and therapies for these disorders is constantly growing. Over the last decades, the introduction and development of atomic force microscopy (AFM) technique allowed the study of biological samples at the micro- and nanoscopic level, hence revealing noteworthy details and paving the way for investigations on physiological and pathological conditions at cellular scale. The present work is aimed to collect and review the literature on cardiomyocytes (CMs) studied by AFM, in order to emphasise the numerous potentialities of this approach and provide a platform for researchers in the field of cardiovascular diseases. Original data are also presented to highlight the application of AFM to characterise the viscoelastic properties of CMs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

It is widely accepted that the pathological state of cells is characterized by a modification of mechanical properties, affecting cellular shape and viscoelasticity as well as adhesion behaviour and motility. Thus, assessing these parameters could represent an interesting tool to monitor disease development and progression, but also the effects of drug treatments. Since biomechanical properties of cells are strongly related to cytoskeletal architecture, in this work we extensively studied the effects of selective impairments of actin microfilaments and microtubules on HeLa cells through force-deformation curves and stress relaxation tests with atomic force microscopy. Confocal microscopy was also used to display the effects of the used drugs on the cytoskeletal structure. In synergy with the aforementioned methods, stress relaxation data were used to assess the storage and loss moduli, as a complementary way to describe the influence of cytoskeletal components on cellular viscoelasticity. Our results indicate that F-actin and microtubules play a complementary role in the cell stiffness and viscoelasticity, and both are fundamental for the adhesion properties. Our data support also the application of biomechanics as a tool to study diseases and their treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lamins are type V intermediate filaments that collectively form a meshwork underneath the inner nuclear membrane, called nuclear lamina. Furthermore, they are also present in the nucleoplasm. Lamins are experiencing a growing interest, since a wide range of diseases are induced by mutations in the gene coding for A-type lamins, globally known as laminopathies. Moreover, it has been demonstrated that lamins are involved in other pathological conditions, like cancer. The role of lamins has been studied from several perspectives, exploiting different techniques and procedures. This multidisciplinary approach has contributed to resolving the unique features of lamins and has provided a thorough insight in their role in living organisms. Yet, there are still many unanswered questions, which constantly generate research in the field. The present work is aimed to review some interesting experimental techniques performed so far to study lamins. Scientists can take advantage of this collection for their novel investigations, being aware of the already pursued and consolidated methodologies. Hopefully, advances in these research directions will provide insights to achieve better diagnostic procedures and effective therapeutic options., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The ability of the adult heart to regenerate cardiomyocytes (CMs) lost after injury is limited, generating interest in developing efficient cell-based transplantation therapies. Rigid carbon nanotubes (CNTs) scaffolds have been used to improve CMs viability, proliferation, and maturation, but they require undesirable invasive surgeries for implantation. To overcome this limitation, we developed an injectable reverse thermal gel (RTG) functionalized with CNTs (RTG-CNT) that transitions from a solution at room temperature to a three-dimensional (3D) gel-based matrix shortly after reaching body temperature. Here we show experimental evidence that this 3D RTG-CNT system supports long-term CMs survival, promotes CMs alignment and proliferation, and improves CMs function when compared with traditional two-dimensional gelatin controls and 3D plain RTG system without CNTs. Therefore, our injectable RTG-CNT system could potentially be used as a minimally invasive tool for cardiac tissue engineering efforts.

In the last decades, atomic force microscopy (AFM) underwent a rapid and stunning development, especially for studying mechanical properties of biological samples. The numerous discoveries relying to this approach, have increased the credit of AFM as a versatile tool, and potentially eligible as a diagnostic equipment. Meanwhile, it has become strikingly evident that lamins are involved on the onset and development of certain diseases, including cancer, Hutchinson-Gilford progeria syndrome, cardiovascular pathologies, and muscular dystrophy. A new category of pathologies has been defined, the laminopathies, which are caused by mutations in the gene encoding for A-type lamins. As the majority of medical issues, lamins, and all their related aspects can be considered as a quite complex problem. Indeed, there are many facets to explore, and this definitely requires a multidisciplinary approach. One of the most intriguing aspects concerning lamins is their remarkable contribute to cells mechanics. Over the years, this has led to the speculation of the so-called "structural hypothesis", which attempts to elucidate the etiology and some features of the laminopathies. Among the various techniques tried to figure out the role of lamins in the cells mechanics, the AFM has been already successfully applied, proving its versatility. Therefore, the present work aims both to highlight the qualities of AFM and to review the most relevant knowledge about lamins, in order to promote the study of the latter, taking advantage from the former. Microsc. Res. Tech. 80:97-108, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

An easy, low-cost and fast wet processing-based method named ASB-SANS (Auxiliary Solvent-Based Sublimation-Aided NanoStructuring) has been used to fabricate poly(l-lactic acid) (PLLA) highly ordered and hierarchically organized 2D fibrillar patterns, with fiber widths between 40 and 500 nm and lengths exceeding tens of microns. A clear contact guidance effect of these nanofibrillar scaffolds with respect to HeLa and NIH-3T3 cells growth has been observed, on top of an overall good viability. For NIH-3T3 pronounced elongation of the cells was observed, as well as a remarkable ability of the patterns to guide the extension of pseudopodia. Moreover, SEM imaging revealed filopodia stemming from both sides of the pseudopodia and aligned with the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. These results validate ASB-SANS as a technique capable to provide biocompatible 2D nanofibrillar patterns suitable for studying phenomena of contact guidance (and, more in general, the behavior of cells onto nanofibrous scaffolds), at very low costs and in an extremely easy way, accessible to virtually any laboratory., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Single-step and three-step irradiated and annealed ultra-high molecular weight polyethylene (UHMWPE) hip liners have been studied by means of Raman spectroscopy (RS) and Fourier transform infrared spectroscopy (FT-IR), in order to clarify the microstructural modifications induced by in vitro oxidative degradation and wear. These spectroscopic techniques enabled us to measure profiles of oxidation index (OI), crystalline (αc), amorphous (αa), and third phase (αb) fractions along the subsurface of the acetabular cups as a function of in vitro oxidation time or after standard testing in hip simulator. Microtomed sections of the liners after accelerated aging (ASTM F2003-02) showed that oxidation profiles developed differently during the first two weeks, while all samples aged longer than 2 weeks revealed OI increasing with lower rates. The initial oxidation of the single-step-annealed material was higher than the one retrieved from the 3-step-annealed material and showed a peak of OI located at a depth of ~1mm below the exposed surface. The profiles of αc, calculated from the same sample cross-sections, followed trends similar to the respective OI profiles, which enabled a phenomenological (but quantitative) correlation between oxidation and crystallization processes to be obtained. Wear simulation under edge loading conditions was conducted on series of four samples of the above two types of irradiated and annealed materials, and for two different liner thicknesses (5.9 and 7.9 mm). The wear rates calculated at the end of the test were very low for all samples (max 2.08 mm(3)/mc for the thinner liners of the single-step irradiated and annealed material). The results indicated that there was a statistically significant increase in both wear rate and volume loss only for the thinner single-step irradiated and annealed liners. Surface analyses by Raman spectroscopy revealed distinct gradients of crystallinity, amorphous, and third phase fractions along the in-depth axis. In both types of UHMWPE materials, the worn area showed an increase of crystallinity at the expense of the third phase. Differences in crystallinity profiles observed at the wear zone of liners with different thicknesses were correlated to the higher contact stress experienced by thinner liners., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKIIδ and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (∼30%) passive force reduction caused by CaMKIIδ and a much smaller (∼6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKIIδ can alter the passive force of myocytes in the heart (although CaMKIIδ appears to be more potent) during physiological and pathophysiological states., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Atomic force microscopy (AFM) cell loading/unloading curves were used to provide comprehensive insights into biomechanical behavior of cardiomyocytes carrying the lamin A/C (LMNA) D192G mutation known to cause defective nuclear wall, myopathy and severe cardiomyopathy. Our results suggested that the LMNA D192G mutation increased maximum nuclear deformation load, nuclear stiffness and fragility as compared to controls. Furthermore, there seems to be a connection between this lamin nuclear mutation and cell adhesion behavior since LMNA D192G cardiomyocytes displayed loss of AFM probe-to-cell membrane adhesion. We believe that this loss of adhesion involves the cytoskeletal architecture since our microscopic analyses highlighted that mutant LMNA may also lead to a morphological alteration in the cytoskeleton. Furthermore, chemical disruption of the actin cytoskeleton by cytochalasin D in control cardiomyocytes mirrored the alterations in the mechanical properties seen in mutant cells, suggesting a defect in the connection between the nucleoskeleton, cytoskeleton and cell adhesion molecules in cells expressing the mutant protein. These data add to our understanding of potential mechanisms responsible for this fatal cardiomyopathy, and show that the biomechanical effects of mutant lamin extend beyond nuclear mechanics to include interference of whole-cell biomechanical properties.

Cells and tissues in our body are continuously subjected to mechanical stress. Mechanical stimuli, such as tensile and contractile forces, and shear stress, elicit cellular responses, including gene and protein alterations that determine key behaviors, including proliferation, differentiation, migration, and adhesion. Several tools and techniques have been developed to study these mechanobiological phenomena, including micro-electro-mechanical systems (MEMS). MEMS provide a platform for nano-to-microscale mechanical stimulation of biological samples and quantitative analysis of their biomechanical responses. However, current devices are limited in their capability to perform single cell micromechanical stimulations as well as correlating their structural phenotype by imaging techniques simultaneously. In this study, a biocompatible and optically transparent MEMS for single cell mechanobiological studies is reported. A silicon nitride microfabricated device is designed to perform uniaxial tensile deformation of single cells and tissue. Optical transparency and open architecture of the device allows coupling of the MEMS to structural and biophysical assays, including optical microscopy techniques and atomic force microscopy (AFM). We demonstrate the design, fabrication, testing, biocompatibility and multimodal imaging with optical and AFM techniques, providing a proof-of-concept for a multimodal MEMS. The integrated multimodal system would allow simultaneous controlled mechanical stimulation of single cells and correlate cellular response.

Atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS) and Poisson statistic were used to analyze the detachment work recorded during the removal of gold-covered microspheres from cardiac fibroblasts. The effect of Cytochalasin D, a disruptor of the actin cytoskeleton, on cell adhesion was also tested. The adhesion work was assessed using a Poisson analysis also derived from single-cell force spectroscopy retracting curves. The use of Poisson analysis to get adhesion work from AFM curves is quite a novel method, and in this case, proved to be effective to study the short-range and long-range contributions to the adhesion work. This method avoids the difficult identification of minor peaks in the AFM retracting curves by creating what can be considered an average adhesion work. Even though the effect of actin depolymerisation is well documented, its use revealed that control cardiac fibroblasts (CT) exhibit a work of adhesion at least 5 times higher than that of the Cytochalasin treated cells. However, our results indicate that in both cells short-range and long-range contributions to the adhesion work are nearly equal and the same heterogeneity index describes both cells. Therefore, we infer that the different adhesion behaviors might be explained by the presence of fewer membrane adhesion molecules available at the AFM tip-cell interface under circumstances where the actin cytoskeleton has been disrupted., (Copyright © 2014. Published by Elsevier B.V.)

Previous investigations suggested that lamin A/C gene (LMNA) mutations, which cause a variety of human diseases including muscular dystrophies and cardiomyopathies, alter the nuclear mechanical properties. We hypothesized that biomechanical changes may extend beyond the nucleus.

AFM was used to collect the whole force-deformation cell curves. They provide both the elasticity and adhesion behavior of mouse primary cardiac fibroblasts. To confirm the hypothesis that a link exists between the membrane receptors and the cytoskeletal filaments causing therefore changing in both elasticity and adhesion behavior, actin-destabilizing Cytochalsin D was administrated to the fibroblasts. From immunofluorescence observation and AFM loading/unloading curves, cytoskeletal reorganization as well as a change in the elasticity and adhesion was indeed observed. Elasticity of control fibroblasts is three times higher than that for fibroblasts treated with 0.5 μM Cytochalasin. Moreover, AFM loading-unloading curves clearly show the different mechanical behavior of the two different cells analyzed: (i) for control cells the AFM cantilever rises during the dwell time while cells with Cytochalasin fail to show such an active resistance; (ii) the maximum force to deform control cells is quite higher and as far as adhesion is concern (iii) the maximum separation force, detachment area and the detachment process time are much larger for control compared to the Cytochalasin treated cells. Therefore, alterations in the cytoskeleton suggest that a link must exist between the membrane receptors and the cytoskeletal filaments beneath the cellular surface and inhibition of actin polymerization has effects on the whole cell mechanical behavior as well as adhesion., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Palytoxins (PLTXs) are known seafood contaminants and their entrance into the food chain raises concern about possible effects on human health. The increasing number of analogs being identified in edible marine organisms complicates the estimation of the real hazard associated with the presence of PLTX-like compounds. So far, 42-OH-PLTX is one of the few congeners available, and the study of its toxicity represents an important step toward a better comprehension of the mechanism of action of this family of compounds. From this perspective, the aim of this work was to investigate the in vivo and in vitro effect of 42-OH-PLTX on skeletal muscle, one of the most sensitive targets for PLTXs. Our results demonstrate that 42-OH-PLTX causes damage at the skeletal muscle level with a cytotoxic potency similar to that of PLTX. 42-OH-PLTX induces cytotoxicity and cell swelling in a Na(+)-dependent manner similar to the parent compound. However, the limited Ca(2+)-dependence of the toxic insult induced by 42-OH-PLTX suggests a specific mechanism of action for this analog. Our results also suggest an impaired response to the physiological agonist acetylcholine and altered cell elasticity., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Mechanical properties of single cells are of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this respect, atomic force microscopy (AFM) has become a powerful tool for imaging and assessing mechanical properties of biological samples. However, while these tests are typically carried out on chemically fixed cells, the most important data is that on living cells. The present study applies AFM technique to assess the Young's modulus of two cell lines: mouse embryonic fibroblasts (NIH/3T3) and human epithelial cancer cells (SW-13). Both living cells and those fixed with paraformaldehyde were investigated. This analysis quantifies the difference between Young's modulus for these two conditions and provides a coefficient to relate them. Knowing the relation between Young's modulus of living and fixed cells, allows carrying out and comparing data obtained during steady-state measurements on fixed cells that are more frequently available in the clinical and research settings and simpler to maintain and probe., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Palytoxin (PLTX) is one of the most toxic seafood contaminants ever isolated. Reports of human food-borne poisoning ascribed to PLTX suggest skeletal muscle as a primary target site. Primary cultures of mouse skeletal muscle cells were used to study the relationship between Ca(2+) response triggered by PLTX and the development of myotoxic insult. Ca(2+) imaging experiments revealed that PLTX causes a transitory intracellular Ca(2+) response (transient phase) followed by a slower and more sustained Ca(2+) increase (long-lasting phase). The transient phase is due to Ca(2+) release from intracellular stores and entry through voltage-dependent channels and the Na(+)/Ca(2+) exchanger (reverse mode). The long-lasting phase is due to a massive and prolonged Ca(2+) influx from the extracellular compartment. Sulforhodamine B assay revealed that the long-lasting phase is the one responsible for the toxicity in skeletal muscle cells. Our data analyzed, for the first time, pathways of PLTX-induced Ca(2+) entry and their correlation with PLTX-induced toxicity in skeletal muscle cells. The cellular morphology changes induced by PLTX and the sensitivity to gadolinium suggest a role for stretch-activated channels.

The investigation of single cells is a topic in continuous evolution. The complexity of the cellular matrix, the huge variety of cells, the interaction of one cell with the other are all factors that must be taken into consideration in the study of the cellular structure and mechanics. In this project, we developed different types of bioMEMS for cell's stretching, both transparent devices based on silicon nitride and non-transparent silicon based. While the use of silicon devices is limited to reflection microscopes, transparent bioMEMS can be used with transmission and reflection microscopes but can also be easily coupled with other tools such as patch clamp analyzers or atomic force microscope. This improvement will open brand new possibilities in the biological investigation field. We used these two BioMEMS to stretch a single cell in a controlled way and, as a first investigation, we focused on its morphology. We noticed that during a controlled stretch, cells react to the applied deformation. A hysteretic behavior on the ratio between area and perimeter has been highlighted.

Purpose: The aims of this study were to compare the crystal chemical properties of some commercial mineral trioxide aggregate (MTA) and Portland cements (PC) and to propose a new white MTA product., Methods: The samples (four MTA and two PC types) were analyzed by 1) optical microscopy; 2) laser granulometry; 3) X-ray diffraction and fluorescence; 4) scanning electron microscopy (SEM) and electron probe microanalysis (EPM) (wavelength-dispersive)., Results: MTA and PC specimens yielded similar characteristics in their clinker component. The MTA-Angelus specimens displayed a composition overlapping the classical clinker composition (wt%) i.e. 25 silica, nine alumina and 66 lime. However, the bismite, present in large amounts (~15-19 wt%) in all MTA products, contained considerable and diffused heavy (toxic) metals as Pb and Mo, other than Bi. In the MTA clinkers the formation of Portlandite, at water-clinker interface, is favored by the smaller grain size of the MTA particles. However, this may also favor the diffusion of the toxic elements linked to Bi., Significance: In terms of bulk physico-chemical properties, the MTA products can be easily substituted by a fine-grained Portland clinker by adding a non-toxic radio-opaque component, for example, Ba-carbonate.

Micromechanical systems are increasingly being used as tools in biological applications, since their characteristic dimensions permit to operate at the same length scale of the structures under investigation. Here, we present a methodology for the design, fabrication and operation of a tool for the assessment of mechanical properties of single cells. In particular, we describe a microsystems platform to study bio-mechanical response of single living cells to in-plane biaxial stretching. The proposed device employs a new linkage design in order to obtain the displacement of the quadrants of a sliced circular plate in mutually-orthogonal directions using just one linear actuator. With this linkage geometry, the whole device has only one degree of freedom. This results in a very predictable and reliable mechanical behaviour, thereby allowing use a simple and easily available control electronics. Results of this study have relevance for the design of a powerful yet simple BioMEMS platform for the characterization of living cells as in-plane bi-axial loading simulated the conditions experienced by cells in vivo more realistically than a uniaxial stretching.

Amongst the processes that have been implemented in microfluidic devices, electrophoretic transport of charged molecules, along microfluidic channels, is one of the most commonly found. However, less work has been done about continuous, pressure gradient driven flow systems where an electric field is applied orthogonally with respect to the microchannel walls. The perspective applications of this technique, include continuous flow separation and concentration of analyte molecules, and the kinetic control of surface reactions. In order to dimensioning and optimizing such a device, a mathematical model has been formulated and analyzed both with numeric and analytic methods. The given solutions let the designer of microfluidic devices able to estimate the concentration profiles along the microchannel length, as a function of the main system parameters. As a practical example of application which could be of great interest in biotechnology applications, the results relative to the simulation of the electrostatic induced cross flow of single strand DNA oligonucleotides of about 20 bases has been reported.