Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features.

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.

Decellularized extracellular matrix-based scaffold and hypoxic priming: A promising combination to improve the phenotype of degenerate intervertebral disc cells.

AIMS: The main cause of low back pain is the intervertebral disc (IVD) degeneration. Designing an effective disc regeneration strategy still remains a major challenge, especially for the lack of effective self-healing capacity. Understanding the properties of IVD cells in the degenerate microenvironment could help to develop in situ regeneration strategies. The objective of the present study was to investigate the ability of degenerate cells to respond to conditions they experience physiologically in their niche in vivo, namely the presence of the hypoxic environment and trophic factors. MAIN METHODS: Degenerate cells from IVD of patients operated for herniated disc were exposed to hypoxic priming and decellularized Wharton's jelly matrix (DWJM) as scaffold and trophic factors source for 48 h in culture. Cell response was evaluated in terms of cell viability, proliferation, cytoskeletal organization, migratory ability and expression of discogenic transcription factors (SOX9, TRPS1), hypoxia-inducible factor 1α (HIF-1α) and longevity transcription factor FOXO3a. The recruitment of HIF-1α at FOXO3a and SOX9 gene promoters was analyzed by Chromatin immunoprecipitation. KEY FINDINGS: Degenerate IVD cells were able to re-acquire the discogenic phenotype, and to re-adapt to hypoxia after exposure to hypoxic priming and DWJM. We demonstrated for the first time that HIF-1α is specifically recruited at the promoter of SOX9 and FOXO3a which are crucial for IVD homeostasis and repair. SIGNIFICANCE: These results open new avenues to engineer IVD by demonstrating that appropriate stimuli are able to dampen the degenerated IVD cell phenotype and to promote anabolic activity in cells which are constitutively characterized by poor reparative capacity.

A rhabdomyosarcoma hydrogel model to unveil cell-extracellular matrix interactions.

Three-dimensional (3D) culture systems have progressively attracted attention given their potential to overcome limitations of classical 2D in vitro systems. Among different supports for 3D cell culture, hydrogels (HGs) offer important advantages such as tunable mechanical and biological properties. Here, a biocompatible hyaluronic acid-polyethylene glycol HG was developed to explore the pro-migratory behavior of alveolar rhabdomyosarcoma (ARMS) cells. Proteomic analysis of ARMS xenografts unveiled the composition of the extracellular matrix (ECM) elucidating the most representative proteins. In parallel, HGs were obtained by the combination of a thiol-containing hyaluronic acid derivative and different polyethylene glycol (PEG) dimaleimide polymers. The selection of the optimal HG for ARMS cell growth was made based on degradation time, swelling, and cell distribution. Rheology measures and mechanical properties were assessed in the presence or absence of ECM proteins (collagen type I and fibronectin), as well as viability tests and cell distribution analysis. The role of ITGA5, the receptor of fibronectin, in determining ARMS cell migration was validated in vitro upon ITGA5 silencing. In vivo, cell dissemination and the capacity for engrafting were validated after injecting ARMS cell populations enriched for the level of ITGA5 in zebrafish embryos. To study the interactions with ARMS-specific ECM proteins (HG + P), the key players from the Rho and heat-shock pathways were investigated by reverse phase protein array (RPPA). Our data suggest that the developed 3D ARMS model is useful for identifying potential physical hallmarks that allow cancer cells to resist therapy, escape from the immune-system and increase dissemination.

Plasmon Couplings from Subsystem Time-Dependent Density Functional Theory.

Many applications in plasmonics are related to the coupling between metallic nanoparticles (MNPs) or between an emitter and a MNP. The theoretical analysis of such a coupling is thus of fundamental importance to analyze the plasmonic behavior and to design new systems. While classical methods neglect quantum and spill-out effects, time-dependent density functional theory (TD-DFT) considers all of them and with Kohn-Sham orbitals delocalized over the whole system. Thus, within TD-DFT, no definite separation of the subsystems (the single MNP or the emitter) and their couplings is directly available. This important feature is obtained here using the subsystem formulation of TD-DFT, which has been originally developed in the context of weakly interacting organic molecules. In subsystem TD-DFT, interacting MNPs are treated independently, thus allowing us to compute the plasmon couplings directly from the subsystem TD-DFT transition densities. We show that subsystem TD-DFT, as well as a simplified version of it in which kinetic contributions are neglected, can reproduce the reference TD-DFT calculations for gap distances greater than about 6 Å or even smaller in the case of hybrid plasmonic systems (i.e., molecules interacting with MNPs). We also show that the subsystem TD-DFT can be also used as a tool to analyze the impact of charge-transfer effects.

Extracellular Matrix From Decellularized Wharton's Jelly Improves the Behavior of Cells From Degenerated Intervertebral Disc.

Regenerative therapies for intervertebral disc (IVD) injuries are currently a major challenge that is addressed in different ways by scientists working in this field. Extracellular matrix (ECM) deriving from decellularized non-autologous tissues has been established as a biomaterial with remarkable regenerative capacity and its potential as a therapeutic agent is rising. In the present study, we investigated the potential of decellularized Wharton's jelly matrix (DWJM) from human umbilical cord to act as an ECM-based scaffold for IVD cell culturing. An efficient detergent-enzymatic treatment (DET) was used to produce DWJM maintaining its native microarchitecture. Afterward, immunofluorescence, biochemical assays and electron microscopy analysis showed that DWJM was able to produce sizeable 3D cell aggregates, when combined with human mesenchymal stromal cells isolated from WJ (MSCs) and IVD cells. These latter cells are characterized by the loss of their chondrocyte-like phenotype since they have been isolated from degenerated IVD and in vitro expanded to further de-differentiate. While the effect exerted by DWJM on MSCs was essentially the induction of proliferation, conversely, on IVD cells the DWJM promoted cell differentiation toward a discogenic phenotype. Notably, for the first time, the ability of DWJM to improve the degenerated phenotype of human IVD cells was demonstrated, showing that the mere presence of the matrix maintained the viability of the cells, and positively affected the expression of critical regulators of IVD homeostasis, such as SOX2, SOX9, and TRPS1 transcription factors at specific culture time. Our data are in line with the hypothesis that the strengthening of cell properties in terms of viability and expression of specific proteins at precise times represents an important condition in the perspective of guiding the recovery of cellular functionality and triggering regenerative potential. Currently, there are no definitive surgical or pharmacological treatments for IVD degeneration (IDD) able to restore the disc structure and function. Therefore, the potential of DWJM to revert degenerated IVD cells could be exploited in the next future an ECM-based intradiscal injectable therapeutic.

Rhabdomyosarcoma Cells Produce Their Own Extracellular Matrix With Minimal Involvement of Cancer-Associated Fibroblasts: A Preliminary Study.

BACKGROUND: The interplay between neoplastic cells and surrounding extracellular matrix (ECM) is one of the determinant elements for cancer growth. The remodeling of the ECM by cancer-associated fibroblasts (CAFs) shapes tumor microenvironment by depositing and digesting ECM proteins, hence promoting tumor growth and invasion. While for epithelial tumors CAFs are well characterized, little is known about the stroma composition of mesenchymal cancers, such as in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma during childhood and adolescence. The aim of this work is to identify the importance of CAFs in specifying RMS microenvironment and the role of these stromal cells in RMS growth. METHODS: We assessed in two dimensional (2D) and three dimensional (3D) systems the attraction between RMS cells and fibroblasts using epithelial colon cancer cell line as control. CAFs were studied in a xenogeneic mouse model of both tumor types and characterized in terms of fibroblast activation protein (FAP), mouse PDGFR expression, metalloproteases activation, and ECM gene and protein expression profiling. RESULTS: In 2D model, the rate of interaction between stromal and malignant cells was significantly lower in RMS with respect to colon cancer. Particularly, in 3D system, RMS spheroids tended to dismantle the compact aggregate when grown on the layer of stromal cells. In vivo, despite the well-formed tumor mass, murine CAFs were found in low percentage in RMS xenogeneic samples. CONCLUSIONS: Our findings support the evidence that, differently from epithelial cancers, RMS cells are directly involved in their own ECM remodeling, and less dependent on CAFs support for cancer cell growth.

Alveolar Rhabdomyosarcoma Decellularization.

In cancer research, it is an urgent need in the obtainment of a simple and reproducible model that mimics in all the complexity the pathological microenvironment. Specifically, the will to improve the overall survival of young patients affected by rhabdomyosarcoma compels researchers to develop new models resembling the multifaceted environment of the pathology to deeply study the disease under novel and different aspects. To this end, we developed a decellularization protocol for alveolar rhabdomyosarcoma (ARMS) able to maintain the three-dimensional structure. The attained extracellular matrix (ECM) can be used as 3D in vitro model suitable to both recapitulate the in vivo cancer microenvironment, and also for drug testing. Here, we first describe a detergent-enzymatic method and then we analyze the decellularization efficiency and the scaffold proteins.

Exciton-Plasmon Coupling Enhancement via Metal Oxidation.

In this paper, we report on the effect of metal oxidation on strong coupling interactions between silver nanostructures and a J-aggregated cyanine dye. We show that metal oxidation can sensibly affect the plexcitonic system, inducing a change in the coupling strength. In particular, we demonstrate that the presence of oxide prevents the appearance of Rabi splitting in the extinction spectra for thick spacers. In contrast, below a threshold percentage, the oxide layer results in an higher coupling strength between the plasmon and the Frenkel exciton. Contrary to common belief, a thin oxide layer seems thus to act, under certain conditions, as a coupling mediator between an emitter and a localized surface plasmon excited in a metallic nanostructure. This suggests that metal oxidation can be exploited as a means to enhance light-matter interactions in strong coupling applications.

Distributed feedback imprinted electrospun fiber lasers.

Imprinted, distributed feedback lasers are demonstrated on individual, active electrospun polymer nanofibers. In addition to advantages related to miniaturization, optical confinement and grating nanopatterning lead to a significant threshold reduction compared to conventional thin-film lasers. The possibility of imprinting arbitrary photonic crystal geometries on electrospun lasing nanofibers opens new opportunities for realizing optical circuits and chips.

Metallic-like stoichiometric copper sulfide nanocrystals: phase- and shape-selective synthesis, near-infrared surface plasmon resonance properties, and their modeling.

In the realm of semiconductor nanomaterials, a crystal lattice heavily doped with cation/anion vacancies or ionized atomic impurities is considered to be a general prerequisite to accommodating excess free carriers that can support localized surface plasmon resonance (LSPR). Here, we demonstrate a surfactant-assisted nonaqueous route to anisotropic copper sulfide nanocrystals, selectively trapped in the covellite phase, which can exhibit intense, size-tunable LSPR at near-infrared wavelengths despite their stoichiometric, undoped structure. Experimental extinction spectra are satisfactorily reproduced by theoretical calculations performed by the discrete dipole approximation method within the framework of the Drude-Sommerfeld model. The LSPR response of the nanocrystals and its geometry dependence are interpreted as arising from the inherent metallic-like character of covellite, allowed by a significant density of lattice-constitutional valence-band free holes. As a consequence of the unique electronic properties of the nanocrystals and of their monodispersity, coherent excitation of symmetric radial breathing modes is observed for the first time in transient absorption experiments at LSPR wavelengths.

Strong coupling between a dipole emitter and localized plasmons: enhancement by sharp silver tips.

In this work sharp silver nanotips are analyzed and proposed as useful plasmonic tools to reduce the threshold for the onset of strong coupling in the electromagnetic interaction of a point-like emitter with localized surface plasmons. If compared to similarly-sized spherical nanoparticles, conically-shaped nanoparticles turn out to be extremely useful to reduce the oscillator strength requirements for the emitting dipole, a reduction of the threshold by one sixth being obtained in a double cone configuration. Moreover the transition to the strong coupling regime is analyzed for several cone apertures, revealing a nonmonotonic behavior with the appearance of an optimal cone geometry. The emitted-light spectrum is obtained from the computation of the perturbative decay rate and photonic Lamb shift in the classical framework of the Discrete Dipole Approximation. This combined classical-quantum electrodynamics treatment is useful for the theoretical investigation on nonperturbative light-matter interactions involving complex shaped nanoparticles or aggregates.

Active role of oxide layers on the polarization of plasmonic nanostructures.

In this paper a theoretical study of polarization properties of a silver nanosphere touching a homogeneous silver substrate and covered by oxide layers of increasing thickness, is reported. Oxide layers are often deposited on metallic nanostructures in metal-enhanced fluorescence (MEF) or surface-enhanced raman scattering experiments to avoid nonradiative energy transfer from emitters to the metal, and to increase the nanoparticles stability against thermal processes and laser exposure. Not much has been said on the effect of the oxide on the field enhancement of such kind of plasmonic systems. This work aims at filling this gap by shedding light on the effects of the oxide coverage on the near and far field behavior: numerical simulations performed in the framework of the discrete dipole approximation show the presence of new resonances in the absorption spectra and, of major importance for MEF applications, a strong enhancement of the near field around the nanosphere.

Pure white hybrid light-emitting device with color rendering index higher than 90.

The realization of white-light sources with a combination of high color rendering index (CRI), which is the average of the first eight rendering indices, and the deep-red color rendering R9 is an important challenge in the field of solid-state lighting. Herein, we report on a pure white hybrid light-emitting device combining a deep-blue emission from a polymer with blue, green, and red emissions from ternary CdSe/ZnS quantum dots. By carefully designing the device structure and tuning the ratio of QDs with different sizes, high CRI of 94 and R9 of 92 at 525 cd/m(2) were achieved.

Enhanced fluorescence by metal nanospheres on metal substrates.

We investigate the metal enhanced fluorescence by silver nanospheres on a thin silver substrate. Experimental measurements for core/shell colloidal nanocrystals embedded in a polymer matrix show a fluorescence enhancement factor of about 9. We apply the discrete dipole approximation method to describe the local-field enhancement factor (LFEF). We find that the observed fluorescence enhancement is related to the broad LFEF profile induced by the substrate.

In the rat maximal dentate activation model of partial complex epilepsy, the anticonvulsant activity of levetiracetam is modulated by nitric oxide-active drugs.

The effects of nitric oxide-active drugs on the anticonvulsant action of the antiepileptic drug levetiracetam in an experimental model of partial complex seizures named maximal dentate gyrus activation were studied in rats. Levetiracetam was given alone or in combination with 7-nitroindazole, a preferential inhibitor of neuronal nitric oxide synthase, or with L: -arginine, the precursor of nitric oxide synthesis. The maximal dentate activation parameters were the time of latency and the durations of maximal dentate activation and afterdischarge responses. The administration of levetiracetam showed an anticonvulsant effect that was increased when given in combination with 7-nitroindazole. The co-administration of levetiracetam and L: -arginine, which is pro-convulsant, did not significantly modify all the parameters. The present results indicate that the acute administration of levetiracetam, at the lower effective dose, exerts an efficacious inhibitory effect on the severity of maximal dentate activation seizures. Levetiracetam-induced antiepileptic effect is significantly increased by the simultaneous inhibition of neuronal nitric oxide synthase.

Nitric oxide- and cGMP-active compounds affect the discharge of substantia nigra pars reticulata neurons: in vivo evidences in the rat.

The nitric oxide (NO)-active drugs influence on the bioelectric activity of neurons of the pars reticulata of the substantia nigra was studied in urethane-anesthetized rats. A first group of animals was treated with 7-nitro-indazole (7-NI), a preferential inhibitor of neuronal NO synthase. In a second group of rats, electrophysiological recordings were coupled with microiontophoretic administration of Nomega-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor), 3-morpholino-sydnonimin-hydrocloride (SIN-1, a NO donor) and 8-Br-cGMP (a cell-permeable analogue of cGMP, the main second-messenger of NO neurotransmission). 7-NI and L-NAME caused a statistically significant decrease in the firing rate of most of the responsive cells, while application of SIN-1 and 8-Br-CGMP induced statistically significant excitatory effects. The results suggest a NO mediated excitatory modulation of the SNr neurons activity with a possible involvement of the cGMP pathway.

Intensity of GABA-evoked responses is modified by nitric oxide-active compounds in the subthalamic nucleus of the rat: a microiontophoretic study.

We have previously described modulatory effects of nitric oxide (NO)-active drugs on subthalamic nucleus (STN) neurons. In this study, the effects of microiontophoretically applied NO-active compounds on GABA-evoked responses were investigated in subthalamic neurons extracellularly recorded from anesthetized rats: 45 of 62 cells were excited by S-nitroso-glutathione (SNOG), an NO donor, whereas 28 of 43 neurons were inhibited by Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. Nearly all neurons responding to SNOG and/or L-NAME showed significant inhibitory responses to the administration of iontophoretic GABA. In these cells, the changes induced by NO-active drugs in the magnitude of GABA-evoked responses were used as indicators of NO modulation. In fact, when an NO-active drug was co-iontophoresed with GABA, significant changes in GABA-induced responses were observed: generally, decreased magnitudes of GABA-evoked responses were observed during continuous SNOG ejection, whereas the administration of L-NAME enhanced GABA responses. In contrast, glutamate-evoked responses were enhanced by SNOG and dampened by L-NAME co-iontophoresis. Furthermore, the iontophoretic administration of bicuculline (a GABA(A) receptor antagonist) completely abolished the GABA-evoked inhibitory responses and reduced the magnitude of both the SNOG- and L-NAME-induced effects. The results suggest that the NO-mediated modulation of subthalamic neurons could also be a result of an interaction between NO and GABA(A) neurotransmission. Increased NOS activity has been shown in the hyperactive STN neurons of parkinsonian patients; on the basis of our observations about the influence of NO-active drugs on the baseline and GABA-evoked activity of subthalamic cells, such hyperactivity suggests the involvement of increased NO levels and reduced sensitivity to GABA.