Selective cell cycle arrest in glioblastoma cell lines by quantum molecular resonance alone or in combination with temozolomide.

BACKGROUND: Glioblastoma is the most aggressive form of brain cancer, characterised by high proliferation rates and cell invasiveness. Despite advances in surgery and radio-chemotherapy, patients continue to have poor prognoses, with a survival rate of 14-15 months. Thus, new therapeutic strategies are needed. Non-ionising electromagnetic fields represent an emerging option given the potential advantages of safety, low toxicity and the possibility to be combined with other therapies. METHODS: Here, the anticancer activity of quantum molecular resonance (QMR) was investigated. For this purpose, three glioblastoma cell lines were tested, and the QMR effect was evaluated on cancer cell proliferation rate and aggressiveness. To clarify the QMR mechanism of action, the proteomic asset after stimulation was delineated. Mesenchymal stromal cells and astrocytes were used as healthy controls. RESULTS: QMR affected cancer cell proliferation, inducing a significant arrest of cell cycle progression and reducing cancer tumorigenicity. These parameters were not altered in healthy control cells. Proteomic analysis suggested that QMR acts not only on DNA replication but also on the machinery involved in the mitotic spindle assembly and chromosome segregation. Moreover, in a combined therapy assessment, QMR significantly enhanced temozolomide efficacy. CONCLUSIONS: QMR technology appears to be a promising tool for glioblastoma treatment.

Management and Rehabilitative Treatment in Osteoarthritis with a Novel Physical Therapy Approach: A Randomized Control Study.

Knee osteoarthritis (KOA) is a chronic degenerative disease characterized by progressive joint damage leading to significant disability. Although rehabilitative treatment methods for KOA have been widely implemented, the optimal integrated instrumental physical therapy approach remains unclear. Therefore, this study aimed to analyze the effect of Quantum Molecular Resonance (QMR) on pain reduction as the primary outcome and the functional improvement in activity daily living (ADL) as a secondary outcome. The study was designed as a double-blind, randomized, controlled trial in an outpatient setting. Fifty-four (N = 54) patients were enrolled and then randomized into three groups according to a simple randomization list: Group 1 (intensive protocol, N = 22), Group 2 (extensive protocol, N = 21), and a Sham group (N = 11). Patients were evaluated over time with scales assessing pain and function. Treatment was performed with the QMR model electro-medical device, which generates alternating electric currents characterized by high frequency (4-64 MHz). The results showed that QMR had a positive effect with respect to the Sham group in terms of pain and function (p < 0.01), and intensive treatment was more effective than the extensive treatment in terms of "speed of response" to the treatment (p < 0.05). In conclusion, QMR in KOA could be effective in slowing the progression of clinical symptoms and improving patients' pain and functionality and thus quality of life. Future studies will be necessary to investigate further treatment algorithms and therapeutic associations with rehabilitative exercise.

Nanomechanics controls neuronal precursors adhesion and differentiation.

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine.

Quantum Molecular Resonance Inhibits NLRP3 Inflammasome/Nitrosative Stress and Promotes M1 to M2 Macrophage Polarization: Potential Therapeutic Effect in Osteoarthritis Model In Vitro.

This study aimed to investigate the anti-inflammatory effects of Quantum Molecular Resonance (QMR) technology in an in vitro model of osteoarthritis-related inflammation. The study used THP-1-derived macrophages stimulated with lipopolysaccharide and hyaluronic acid fragments to induce the expression of inflammatory cytokines and nitrosative stress. QMR treatment inhibited COX-2 and iNOS protein expression and activity and reduced NF-κB activity. Furthermore, QMR treatment led to a significant reduction in peroxynitrite levels, reactive nitrogen species that can form during inflammatory conditions, and restored tyrosine nitration values to those similar to sham-exposed control cells. We also investigated the effect of QMR treatment on inflammasome activation and macrophage polarization in THP-1-derived macrophages. Results showed that QMR treatment significantly decreased NLRP3 and activated caspase-1 protein expression levels and downregulated IL-18 and IL-1ß protein expression and secretion. Finally, our findings indicate that QMR treatment induces a switch in macrophage polarization from the M1 phenotype to the M2 phenotype.

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized "self-developing" esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

Acceleration of neuronal precursors differentiation induced by substrate nanotopography.

Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES-derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ∼80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation.

Nanoelectrode Arrays Fabricated by Thermal Nanoimprint Lithography for Biosensing Application.

Electrochemical sensors are devices capable of detecting molecules and biomolecules in solutions and determining the concentration through direct electrical measurements. These systems can be miniaturized to a size less than 1 µm through the creation of small-size arrays of nanoelectrodes (NEA), offering advantages in terms of increased sensitivity and compactness. In this work, we present the fabrication of an electrochemical platform based on an array of nanoelectrodes (NEA) and its possible use for the detection of antigens of interest. NEAs were fabricated by forming arrays of nanoholes on a thin film of polycarbonate (PC) deposited on boron-doped diamond (BDD) macroelectrodes by thermal nanoimprint lithography (TNIL), which demonstrated to be a highly reliable and reproducible process. As proof of principle, gliadin protein fragments were physisorbed on the polycarbonate surface of NEAs and detected by immuno-indirect assay using a secondary antibody labelled with horseradish peroxidase (HRP). This method allows a successful detection of gliadin, in the range of concentration of 0.5-10 µg/mL, by cyclic voltammetry taking advantage from the properties of NEAs to strongly suppress the capacitive background signal. We demonstrate that the characteristics of the TNIL technology in the fabrication of high-resolution nanostructures together with their low-cost production, may allow to scale up the production of NEAs-based electrochemical sensing platform to monitor biochemical molecules for both food and biomedical applications.

Successful muscle regeneration by a homologous microperforated scaffold seeded with autologous mesenchymal stromal cells in a porcine esophageal substitution model.

BACKGROUND: Since the esophagus has no redundancy, congenital and acquired esophageal diseases often require esophageal substitution, with complicated surgery and intestinal or gastric transposition. Peri-and-post-operative complications are frequent, with major problems related to the food transit and reflux. During the last years tissue engineering products became an interesting therapeutic alternative for esophageal replacement, since they could mimic the organ structure and potentially help to restore the native functions and physiology. The use of acellular matrices pre-seeded with cells showed promising results for esophageal replacement approaches, but cell homing and adhesion to the scaffold remain an important issue and were investigated. METHODS: A porcine esophageal substitute constituted of a decellularized scaffold seeded with autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) was developed. In order to improve cell seeding and distribution throughout the scaffolds, they were micro-perforated by Quantum Molecular Resonance (QMR) technology (Telea Electronic Engineering). RESULTS: The treatment created a microporous network and cells were able to colonize both outer and inner layers of the scaffolds. Non seeded (NSS) and BM-MSCs seeded scaffolds (SS) were implanted on the thoracic esophagus of 4 and 8 pigs respectively, substituting only the muscle layer in a mucosal sparing technique. After 3 months from surgery, we observed an esophageal substenosis in 2/4 NSS pigs and in 6/8 SS pigs and a non-practicable stricture in 1/4 NSS pigs and 2/8 SS pigs. All the animals exhibited a normal weight increase, except one case in the SS group. Actin and desmin staining of the post-implant scaffolds evidenced the regeneration of a muscular layer from one anastomosis to another in the SS group but not in the NSS one. CONCLUSIONS: A muscle esophageal substitute starting from a porcine scaffold was developed and it was fully repopulated by BM-MSCs after seeding. The substitute was able to recapitulate in shape and function the original esophageal muscle layer.

Carbon Nanotubes, Directly Grown on Supporting Surfaces, Improve Neuronal Activity in Hippocampal Neuronal Networks.

Carbon nanotube (CNT)-modified surfaces unequivocally demonstrate their biocompatibility and ability to boost the electrical activity of neuronal cells cultured on them. Reasons for this effect are still under debate. However, the intimate contact at the membrane level between these thready nanostructures and cells, in combination with their unique electrical properties, seems to play an important role. The entire existing literature exploiting the effect of CNTs on modulating cellular behavior deals with cell cultures grown on purified multiwalled carbon nanotubes (MWNTs) deposited on a supporting surface via drop-casting or mechanical entrapment. Here, for the first time, it is demonstrated that CNTs directly grown on a supporting silicon surface by a chemical vapor deposition (CVD)-assisted technique have the same effect. It is shown that primary neuronal cells developed above a carpet of CVD CNTs form a healthy and functional network. The resulting neuronal network shows increased electrical activity when compared to a similar network developed on a control glass surface. The low cost and high versatility of the here presented CVD-based synthesis process, together with the possibility to create on supporting substrate patterns of any arbitrary shape of CNTs, open up new opportunities for brain-machine interfaces or neuroprosthetic devices.

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells.

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4-64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling.

Site-Control of InAs/GaAs Quantum Dots with Indium-Assisted Deoxidation.

Site-controlled epitaxial growth of InAs quantum dots on GaAs substrates patterned with periodic nanohole arrays relies on the deterministic nucleation of dots into the holes. In the ideal situation, each hole should be occupied exactly by one single dot, with no nucleation onto planar areas. However, the single-dot occupancy per hole is often made difficult by the fact that lithographically-defined holes are generally much larger than the dots, thus providing several nucleation sites per hole. In addition, deposition of a thin GaAs buffer before the dots tends to further widen the holes in the [110] direction. We have explored a method of native surface oxide removal by using indium beams, which effectively prevents hole elongation along [110] and greatly helps single-dot occupancy per hole. Furthermore, as compared to Ga-assisted deoxidation, In-assisted deoxidation is efficient in completely removing surface contaminants, and any excess In can be easily re-desorbed thermally, thus leaving a clean, smooth GaAs surface. Low temperature photoluminescence showed that inhomogeneous broadening is substantially reduced for QDs grown on In-deoxidized patterns, with respect to planar self-assembled dots.

Continuous adsorption in highly ordered porous matrices made by nanolithography.

The exposed surface area of porous materials is usually determined by measuring the mass of adsorbed gas as a function of vapour pressure. Here we report a comprehensive study of adsorption in systems with closed bottom, not interconnected pores exhibiting different degrees of disorder, produced with methods encompassing nanolithography and dry and wet etching. Detailed adsorption studies of these matrices show hysteresis loops, as found always in pores having sizes of tens to hundreds of nanometres. The observed variations in the loop shape are associated with changes in the pore morphology. In regular pores formed by vertical and smooth walls, continuous adsorption is found for the first time in agreement with thermodynamic considerations valid for ideal pores. This suggests that irregularities in the walls and pore openings are the key factors behind the hysteresis phenomenon. Interestingly, pores having rough walls but a pyramidal shape also do not show any hysteresis.

The order of condensation in capillary grooves.

We consider capillary condensation in a deep groove of width L. The transition occurs at a pressure p(co)(L) described, for large widths, by the Kelvin equation p(sat) - p(co)(L) = 2σ cosθ/L, where θ is the contact angle at the side walls and σ is the surface tension. The order of the transition is determined by the contact angle of the capped end θcap; it is continuous if the liquid completely wets the cap, and first-order otherwise. When the transition is first-order, corner menisci at the bottom of the capillary lead to a pronounced metastability, determined by a complementary Kelvin equation Δp(L) = 2σ sinθcap/L. On approaching the wetting temperature of the capillary cap, the corner menisci merge and a single meniscus unbinds from the bottom of the groove. Finite-size scaling shifts, crossover behaviour and critical singularities are determined at mean-field level and beyond. Numerical and experimental results showing the continuous nature of condensation for θcap = 0 and the influence of corner menisci on adsorption isotherms are presented.

Novel hybrid organic-inorganic spin-on resist for electron- or photon-based nanolithography with outstanding resistance to dry etching.

A new spin-on alumina-based resist exhibits excellent performance in terms of both achievable lateral resolution and etch resistance in fluorine-based non-cryo-cooled dry etching processes. The resist has selectivity greater than 100:1 with respect to the underlying silicon during the etching process, patternability with various lithographic tools (UV, X-rays, electron beam, and nanoimprint lithography), and positive and negative tone behavior depending only on the developer chemistry.

Can hippocampal neurites and growth cones climb over obstacles?

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.