Live cells can form multifunctional and environmentally responsive multiscale assemblies of living and non-living components. We recently reported the results of a unique approach to introduce supplementary properties, fluorescence in particular, into fibrillar proteins produced by live fibroblasts and extruded into the ECM. In this work, we demonstrate that the physiological secretion of fluorescent nanostructured microfibers upon the spontaneous uptake of the appropriate fluorophore extends to living cells derived by different tissue contexts. We also show that live cells seeded on fluorescent microfibers have a different fate in terms of the cellular morphology, cytoskeleton rearrangement and viability. These results suggest that the microfibers, which are biocompatible and biodegradable, can be used as multiscale biomaterials to direct the cell behaviour. This journal is

Oil spills at sea are a severe global environmental issue. Smart materials with controllable wettability are of global challenging interest in oil-water related applications. Nature offers a versatile platform of remarkable hierarchical structures with a chemical component, which provides bioinspired solutions for solving many challenges. In this study, an approach to achieve robust superhydrophobic/oleophobic property on flexible polydimethylsiloxane (PDMS) surfaces which mimics the hierarchical morphology of the natural lotus leaf surface is shown. The structure is prepared by hydrothermal assembly of zinc oxide nanorods onto the microstructured surface, which results in an underwater superoleophobic surface with an oil contact angle up to 153° which can effectively prevent the surface from being polluted by oils. Our results are significant in terms of their importance to academic research and industrial applications and may lead to an innovative impact in the science field.

The exploitation of cell-instructive scaffolds with uniform physical/chemical surfaces and controlled stiffness will be greatly useful in tissue engineering applications to resemble the extracellular matrix (ECM) or topographical appearance of native tissues. We herein describe a versatile and straightforward method to assemble a polydimethylsiloxane (PDMS)-composite structure in which a uniformly laminin-coated membrane is placed on top of a micropatterned substrate that applies a stiffness gradient. This 'double-sheet' structure provides soft or stiff microdomains that guide the self-patterning of different cell types [e.g. chronic myeloid leukemia (KU812), cervix carcinoma (HeLa), NIH 3T3 and BJ], thereby stimulating their cytoskeletal remodeling. More interestingly, we used these uniform PDMS surfaces with patterned rigidity for obtaining co-cultures of tumor blood cells (KU812) and adherent fibroblasts (NIH 3T3) with spatially-controlled distribution. Thus, beyond single-cell stiffening and mechanosensing, these surfaces should also be used as simple and feasible co-culture systems for mimicking and dissecting the bidirectional interactions between blood cells and specific stromal elements of their in vivo microenvironment.

Bio-nanomaterials offer promise in the field of tissue engineering. Specifically, environmental cues such as the material chemistry, topography and rigidity of the surface to which cells adhere to, can alter and dictate cell shape, proliferation, migration, and gene expression. How deeply each factor (topographical, chemical and mechanical) drives cell response remains incompletely understood. To illustrate cell sensitivities to different factors, we herein present ZnO nanorods (ZnO-Nrds) coated on glass and polydimethylsiloxane (PDMS) substrates and analyzed cell viability and proliferation. The work presented here shows a clear response of various cell lines (mouse embryonic fibroblasts 3T3, human cervix carcinoma HeLa and human osteoblast-like cells MG63) to the rigidity of the underlying surface. The chemical counterpart, given by the presence of ZnO-Nrds, strongly reduced the cell viability of all cell lines. However, the substrate underlying the ZnO coating impacted cell spreading and viability. The substrates exhibited a better ability to neglect cell attachment and proliferation with the ZnO coating and pro-apoptoticity specifically with the PDMS as the underlying substrate which exhibited a "softer" environment with respect to a glass substrate. The results also revealed that the few cells that adhered to the ZnO-Nrds on PDMS and glass showed a rounded morphology. On the basis of these observations, we can correlate common features of phenomenological cell response to chemotactic and durotactic cues. The work presented herein reinforces the response of cells to changes in substrate rigidity. These observations provide a foundation for a potentially promising approach to decrease cell adhesion and thus as an optimal substrate for different applications such as prosthesis design, tissue engineering, anti-bio fouling materials and diagnostics.

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250nm at 2:1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability.

In order to induce bone regeneration several natural and synthetic materials have been proposed. However, single-phase scaffolds present some insurmountable disadvantages such as poor mechanical strength or brittleness and too low or too high degradation rate. In order to overcome these drawbacks, composite systems can be an interesting and promising option. In the present work a novel hybrid porous scaffold for bone tissue engineering is proposed. Chitosan/Forsterite (Ch/FS) composite scaffolds were prepared by freeze-drying method using a chitosan/forsterite ratio of 90/10. The FS nanopowder (Mg2SiO4) is synthesized using a simple sol-gel based method. The FS composition was checked by XRD analysis. The macrostructure of the Ch/FS scaffolds were analyzed by SEM, the FS distribution within the chitosan matrix observed by EDS, the mechanical strength measured by compression test in PBS and the biocompatibility of the composite on human osteosarcoma cell line (MG-63) verified by MTT assay after 48 hours. The porosity appears interconnected and with a pore size ranging from 1 to 100 ?m. The FS is overall distributed within the chitosan matrix. The compression strength of composite scaffolds increased with respect to the pure chitosan scaffolds of more than two times (from 0.8 to 1.9 KPa) and the composites did not show any toxicity effect on human osteosarcoma cells. © (2014) Trans Tech Publications, Switzerland.

There is an exponential growing attention in developing new micro/nano-delivery systems for controlled delivery of bioactive agents, to control cellular functions modulation in a more effective manner. In this study, the effect of dexamethasone encapsulated in calcium carbonate microcubes on proliferation and osteogenic activity of human osteoblasts in vitro was investigated. Dexamethasone-loaded calcium carbonate microcubes are suggested for supportive controlled release and improving osteogenic activity of low dose of dexamethasone. The calcium carbonate microcubes have a cubic structure of about 2 µm and represent a good performance in molecular delivery systems for bone tissue engineering thanks to their potential to mimic the composition, structure, and properties of native bone. The presence of polyelectrolyte multilayers that covered the calcium carbonate microcubes allowed to modulate the release kinetics of active dexamethasone over time. Our dexamethasone-loaded calcium carbonate microcubes provide a simple and easy way to allow controlled release of dexamethasone or other active agents in scaffolds for bone tissue engineering applications.

The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts.MATERIALS & METHODS:Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested.RESULTS & DISCUSSION:Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro.CONCLUSION:This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

A platform is described for the first time for the facile synthesis of oligo- and polythiophene-S-oxides and the corresponding -S,S-dioxides in short times, mild conditions, high yields. Employing ultrasound assistance, brominated thiophenes are selectively mono- or dioxygenated at room temperature. These building blocks are then combined with metalated thiophenes via microwave-assisted cross-coupling reactions through a "Lego-like" strategy to afford unprecedented oligo/polythiophene-S-oxides and mixed -S-oxides/-S,S-dioxides. It is demonstrated that depending on the number, type, and sequence alternation of nonoxygenated, monooxygenated, and dioxygenated thiophene units a very wide property-function tuning can be achieved spanning from frontier orbital energies and energy gaps, to charge transport characteristics and supramolecular H-bonding interactions with specific proteins inside live cells.

Understanding the mechanism of cell migration and interaction with the microenvironment is not only of critical significance to the function and biology of cells, but also has extreme relevance and impact on physiological processes and diseases such as morphogenesis, wound healing, neuron guidance, and cancer metastasis. External guidance factors such as topography and physical cues of the microenvironment promote directional migration and can target specific changes in cell motility and signalling mechanisms. Recent studies have shown that cells can directionally respond to applied electric fields (EFs), in both in vitro and in vivo settings, a phenomenon called electrotaxis. However, the exact cellular mechanisms for sensing electrical signals are still not fully well understood, and it is thus far unknown how cells recognize and respond to electric fields, although some studies have suggested that electro-migration of some cell surface receptors and ion channels in cells could be involved. Applied electric fields may have a potential clinical role in guiding cell migration and present a more precise manageability to change the magnitude and direction of the electric field than most other guidance cues such as chemical cues. Here we present a review of recent studies used for studying electrotaxis to point out similarities, identify points of disagreement, and stimulate new directions for investigation. Insights into the mechanisms by which applied EFs direct cell migration, morphological change and development will enable current and future therapeutic applications to be optimized

A structurally correlated series of cell-permeant thiophene fluorophores, characterized by intense green or red fluorescence inside live mouse embryonic fibroblasts, was developed. The fluorophores displayed rapid internalization, excellent retention inside the cells, and high optical stability in the cytosolic environment and did not alter cell viability and reproducibility. Depending on the molecular structure, they experienced distinct fate inside the cells: from bright and lasting staining of the cytoplasm to selective tagging of a small set of globular proteins. This journal is © The Royal Society of Chemistry 2014.

In our search for thiophene fluorophores that can overcome the limits of currently available organic dyes in live-cell staining, we synthesized biocompatible dithienothiophene-S,S-dioxide derivatives (DTTOs) that were spontaneously taken up by live mouse embryonic fibroblasts and HeLa cells. Upon treatment with DTTOs, the cells secreted nanostructured fluorescent fibrils, while cell viability remained unaltered. Comparison with the behavior of other cell-permeant, newly synthesized thiophene fluorophores showed that the formation of fluorescent fibrils was peculiar to DTTO dyes. Laser scanning confocal microscopy of the fluorescent fibrils showed that most of them were characterized by helical supramolecular organization. Electrophoretic analysis and theoretical calculations suggested that the DTTOs were selectively recognized by the HyPro component of procollagen polypeptide chains and incorporated through the formation of multiple H-bondings.

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro-patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi-nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer-by-layer (LbL) polyelectrolyte multilayer deposition was combined with a micro-molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self-assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4-styrene-sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly-L-arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)(5) -coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586-596. 2012 Wiley Periodicals, Inc. Copyright 2012 Wiley Periodicals, Inc.

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. We developed a novel cell culture technique that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering and more micro-patterning techniques and surface engineering have been tested by switching differentiating myotubes from growth medium (GM) to differentiative media (DM). One of the goals of tissue engineering is to develop tools allowing in vitro construction and mimicking of the final tissue architectures. The fabrication of polyelectrolyte multilayers (PEMs) may represent a promising approach for recreating physiological nanometer-sized cell environments in vitro. In this study we describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation of parallel arrays of mature C2C12 myotubes continuously maintained in GM for 7 days. The structure consists of a 'double-sheet' PDMS structure that provides 'compliant' or 'stiff' microdomains to guide the cell self-patterning, coupled to layer-by-layer (LbL) self-assembled multilayers of biocompatible polyelectrolytes that promote C2C12 myoblasts alignment and differentiation. Our findings have relevance to the interpretation of in vitro data as well as to the study of cellular interactions with biomaterials.

Messenger RNA (mRNA) provides a promising alternative to plasmid DNA as a genetic material for delivery in non-viral gene therapy strategies. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions. Here, mRNA-protamine complex encapsulated poly(?-caprolactone) (PCL) nanoparticles (NPs) are proposed for the intracellular delivery of mRNA molecules. The nanoparticles with a size of about 247 nm in diameter have a core-shell structure with an mRNA-containing inner core surrounded by PCL layers, providing high stability and stealth properties to the nanoparticles. The partial neutralization of the negatively charged mRNA molecules with the cationic protamine allows one to modulate the release kinetics in a pH-dependent manner. At pH 7.4, mimicking the conditions found in the systemic circulation, only 25% of the mRNA is released after 48 hours post incubation, whereas at pH 5.0, recreating the cell endosomal environment, about 60% of the mRNA molecules are released within the same time window post incubation. These NPs show no cytotoxicity to NIH 3T3 fibroblasts, HeLa cells and MG63 osteoblasts up to 8 days of incubation. Given the stability, preferential release behavior, and well-known biocompatibility properties of PCL nanostructures, our non-viral PCL nanoparticles are a promising system that simultaneously resolved the two major problems of mRNA introduction and the instability, opening the door to various new therapeutic strategies using mRNA. This journal is

We have recently reported initial results concerning an original approach to introduce additional properties into fibrillar proteins produced by live fibroblasts and extruded into the ECM. The key to such an approach were biocompatible fluorescent and semiconducting synthetic molecules which penetrated spontaneously the cells and were progressively encompassed via non-bonding interactions during the self-assembly process of the proteins, without altering cell viability and reproducibility. In this paper we demonstrate that the intracellular secretion of fluorescent microfibers can be generalized to living primary and immortalized human/mouse fibroblasts. By means of real-time single-cell confocal microscopy we show that the fluorescent microfibers, most of which display helical morphology, are generated by intracellular coding of the synthetic molecules. We also describe co-localization experiments on the fluorescent microfibers isolated from the cell milieu demonstrating that they are mainly made of type-I collagen. Finally, we report experimental data indicating that the embedded synthetic molecules cause the proteins not only to be fluorescent but also capable of electrical conductivity.

The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of beta-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3 beta (glycogen synthase kinase 3 beta). Herein, we show that GSK3 beta is constitutively Y-216 phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3 beta activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3 beta complex formation. Specifically, GSK3 beta activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the beta-catenin, cyclinD1, C-EBP alpha, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib. (Blood. 2012;119(10):2335-2345)

Osteosarcomas are highly malignant tumors, which develop rapid growth and local infiltration, inducing metastases that spread primarily in the lung. Treatment of these tumors is mainly based on pre- and post-operative chemotherapy and surgery of the primary tumor. Surgical resection though, generates bone defects. Reparation of these weaknesses presents formidable challenges to orthopedic surgery. Medicine regenerative grafts that act as both tumor therapy with constant local drug delivery and tissue regeneration may provide a new prospect to address this need. These implants can provide sustained drug release at the cancer area, decreasing systemic second effects such as inflammation, and a filling of the resected tissues with regenerative biomaterials. In this study microporous poly-?-caprolactone (PCL) scaffolds have been developed for sustained local release of anti-inflammatory drug dexamethasone (DXM), used as drug model, in cancer medicine regenerative field. The microporous PCL matrix of the scaffolds supported the attachment, proliferation and osteogenic differentiation of osteoblast-like cells, while the polyelectrolyte multilayers, anchored to the inner pore surfaces, sustained locally DXM release. These microporous scaffolds demonstrate the ability to deliver DXM as a localized tumor therapy and to promote proliferation and differentiation of osteoblast-like cells in vitro.

Unconventional nanopatterning methods are emerging as powerful tools for the development of controlled shapes and ordered morphology of nanostructured materials with novel properties and tailorable functions. Here, we report a simple yet straightforward and efficient approach for patterning through unconventional dewetting that involves surface tension driven process. Using this innovative approach, we have successfully demonstrated to be able to prepare surface micro-patterns over large areas deposited through Eu3+:TiO2 nanoparticles providing rational control over the local nucleation of nanoparticles. Remarkably, these features could be addressed by polar or apolar solvents, suggesting potential applications in bottom-up nanodevices. This paper represents the first such attempt to create an inorganic materials non-lithographic template for the directed deposition of Eu3+:TiO2 or related metal oxides. The technique, which is driven by the unique chemical properties and geometrical layout of the underlying patterned micrometer-sized templates, enables the construction of micro- and nano-structuration of dispersed inorganic functional materials suitable for electrooptical and photonic applications.

Rationale & aim: Imatinib mesylate (IM), a selective tyrosine kinase inhibitor of the oncoprotein BCR-ABL,is the 'gold standard' for patients with chronic myeloid leukemia (CML) but the drug does not eliminateCML stem cells, leading to disease relapse on drug discontinuation. At present, much effort is focused ondelivery carriers that can increase the intracellular retention and antileukemic impact of IM. We previouslyvalidated IM-loaded polyelectrolyte microcapsules as effective purging agents to eradicate BCR-ABL+ cellsfrom CML patient autografts. The aim is to develop controlled release carriers that can increase theintracellular retention and functionality of IM in leukemia cells. Materials & methods: Herein, novelpolyelectrolyte complexes were used as model carriers for IM in a CML cell line (KU812) and CD34+ cellsfreshly isolated from patients. Results & discussion: Polyelectrolyte complexes promoted a long-actingBCR-ABL kinase inactivation that was necessary to promote apoptosis at approximately twofold lowerintracellular IM dose compared with the microscale formulation polyelectrolyte microcapsules.Conclusion: IM-loaded polyelectrolyte complexes can be used as more efficient delivery devices forovercoming drug resistance of BCR-ABL+ leukemic cells.

We investigated the uptake and release of labeled antibodies from pH-sensitive hydrogel microparticles (i.e. microgels) by means of fluorescence analysis of labeled biological samples. The poly(methacrylic acid) (PMAA) hydrogel is a carbon-based network having carboxylic groups on the surface that dissociate according to their acid-base equilibrium. The ability of the PMAA microgel to encapsulate and release anti-CD4 and anti-CD8 monoclonal antibodies (MAbs), differing for the isotype and labeled with highly photostable fluorophore, was studied in solution by photoluminescence spectroscopy. The experimental results indicated that the uptake and release of the tested antibodies were controlled by pH. Furthermore, confocal microscopy analysis in the solid state revealed that the distribution of the labeled antibodies either on the surface or in the core of the microgel matrix was related to the specific properties of these MAbs. (C) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.