Metal Ion Doping of Alginate-Based Surface Coatings Induces Adipogenesis of Stem Cells.

Metal ions are important effectors of protein and cell functions. Here, polyelectrolyte multilayers (PEMs) made of chitosan (Chi) and alginate (Alg) were doped with different metal ions (Ca2+, Co2+, Cu2+, and Fe3+), which can form bonds with their functional groups. Ca2+ and Fe3+ ions can be deposited in PEM at higher quantities resulting in more positive ζ potentials and also higher water contact angles in the case of Fe3+. An interesting finding was that the exposure of PEM to metal ions decreases the elastic modulus of PEM. Fourier transformed infrared (FTIR) spectroscopy of multilayers provides evidence of interaction of metal ions with the carboxylic groups of Alg but not for hydroxyl and amino groups. The observed changes in wetting and surface potential are partly related to the increased adhesion and proliferation of multipotent C3H10T1/2 fibroblasts in contrast to plain nonadhesive [Chi/Alg] multilayers. Specifically, PEMs doped with Cu2+ and Fe3+ ions greatly promote cell attachment and adipogenic differentiation, which indicates that changes in not only surface properties but also the bioactivity of metal ions play an important role. In conclusion, metal ion-doped multilayer coatings made of alginate and chitosan can promote the differentiation of multipotent cells on implants without the use of other morphogens like growth factors.

Engineering of Stable Cross-Linked Multilayers Based on Thermo-Responsive PNIPAM-Grafted-Chitosan/Heparin to Tailor Their Physiochemical Properties and Biocompatibility.

The thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) is ubiquitously applied in controlled drug release and tissue engineering. However, the lack of bioactivity of PNIPAM restricts its use in cell-containing systems being a thermo-responsive adhesive substratum with no regulating effect on cell growth and differentiation. In this study, integrating PNIPAM with chitosan into PNIPAM-grafted-chitosan (PNIPAM-Chi) allows a layer-by-layer assembly with bioactive heparin to fabricate PNIPAM-modified polyelectrolyte multilayers (PNIPAM-PEMs). Grafting PNIPAM chains of either 2 (LMW) or 10 kDa (HMW) on the chitosan backbone influences the cloud point (CP) temperature in the range from 31 to 33 °C. PNIPAM-Chi with either a higher molecular weight or a higher degree of substitution of PNIPAM chains exhibiting a significant increase in diameter above CP as ensured by dynamic light scattering is selected to fabricate PEM with heparin as a polyanion at pH 4. Little difference of layer growth is detected between the chosen PNIPAM-Chi used as polycations by surface plasmon resonance, while multilayers formed with HMW-0.02 are more hydrated and show striking swelling-and-shrinking abilities when studied with quartz crystal microbalance with dissipation monitoring. Subsequently, the multilayers are covalently cross-linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to strengthen the stability of the systems under physiological conditions. Ellipsometry results confirm the layer integrity after exposure to the physiological buffer at pH 7.4 compared to those without cross-linking. Moreover, significantly higher adhesion and more spreading of C3H10T1/2 multipotent embryonic mouse fibroblasts on cross-linked PEMs, particularly with heparin terminal layers, are observed owing to the bioactivity of heparin. The slightly more hydrophobic surfaces of cross-linked PNIPAM-PEMs at 37 °C also increase cell attachment and growth. Thus, layer-by-layer constructed PNIPAM-PEM with cross-linking represents an interesting cell culture system that can be potentially employed for thermally uploading and controlled release of growth factors that further promotes tissue regeneration.

Extracellular matrix-inspired surface coatings functionalized with dexamethasone-loaded liposomes to induce osteo- and chondrogenic differentiation of multipotent stem cells.

Biomimetic surface coatings can be combined with conventional implants to mimic the extracellular matrix (ECM) of the surrounding tissue to make them more biocompatible. Layer-by-layer technique (LbL) can be used for making surface coatings by alternating adsorption of polyanions and polycations from aqueous solutions without need of chemical reactions. Here, polyelectrolyte multilayer (PEM) systems is made of hyaluronic acid (HA) as polyanion and Collagen I (Col) as polycation to mimic the ECM of connective tissue. The PEM are combined with dexamethasone (Dex)-loaded liposomes to achieve a local delivery and protection of this drug for stimulation of osteo- and chondrogenic differentiation of multipotent stem cells. The liposomes possess a positive surface charge that is required for immobilization on the PEM. The surface properties of PEM system show a positive zeta potential after liposome adsorption and a decrease in wettability, both promoting cell adhesion and spreading of C3H10T1/2 multipotent embryonic mouse fibroblasts. Differentiation of C3H10T1/2 was more prominent on the PEM system with embedded Dex-loaded liposomes compared to the basal PEM system and the use of free Dex-loaded liposomes in the supernatant. This was evident by immunohistochemical staining and an upregulation of the expression of genes, which play a key role in osteogenesis (RunX2, ALP, Osteocalcin (OCN)) and chondrogenesis (Sox9, aggrecan (ACAN), collagen type II), determined by quantitative Real-time polymerase chain reaction (qRT-PCR) after 21 days. These findings indicate that the designed liposome-loaded PEM system have high potential for use as drug delivery systems for implant coatings that can induce bone and cartilage differentiation needed for example in osteochondral implants.

Effect of metal ions on the physical properties of multilayers from hyaluronan and chitosan, and the adhesion, growth and adipogenic differentiation of multipotent mouse fibroblasts.

Polyelectrolyte multilayers (PEMs) consisting of the polysaccharides hyaluronic acid (HA) as the polyanion and chitosan (Chi) as the polycation were prepared with layer-by-layer technique (LbL). The [Chi/HA]5 multilayers were exposed to solutions of metal ions (Ca2+, Co2+, Cu2+ and Fe3+). Binding of metal ions to [Chi/HA]5 multilayers by the formation of complexes with functional groups of polysaccharides modulates their physical properties and the bioactivity of PEMs with regard to the adhesion and function of multipotent murine C3H10T1/2 embryonic fibroblasts. Characterization of multilayer formation and surface properties using different analytical methods demonstrates changes in the wetting, surface potential and mechanical properties of multilayers depending on the concentration and type of metal ion. Most interestingly, it is observed that Fe3+ metal ions greatly promote adhesion and spreading of C3H10T1/2 cells on the low adhesive [Chi/HA]5 PEM system. The application of intermediate concentrations of Cu2+, Ca2+ and Co2+ as well as low concentrations of Fe3+ to PEMs also results in increased cell spreading. Moreover, it can be shown that complex formation of PEMs with Cu2+ and Fe3+ ions leads to increased metabolic activity in cells after 24 h and induces cell differentiation towards adipocytes in the absence of any additional adipogenic media supplements. Overall, complex formation of [Chi/HA]5 PEM with metal ions like Cu2+ and Fe3+ represents an interesting and cheap alternative to the use of growth factors for making cell-adhesive coatings and guiding stem cell differentiation on implants and scaffolds to regenerate connective-type of tissues.

Atomic Layer Deposition of Cobalt Phosphide for Efficient Water Splitting.

Transition-metal phosphides (TMP) prepared by atomic layer deposition (ALD) are reported for the first time. Ultrathin Co-P films were deposited by using PH3 plasma as the phosphorus source and an extra H2 plasma step to remove excess P in the growing films. The optimized ALD process proceeded by self-limited layer-by-layer growth, and the deposited Co-P films were highly pure and smooth. The Co-P films deposited via ALD exhibited better electrochemical and photoelectrochemical hydrogen evolution reaction (HER) activities than similar Co-P films prepared by the traditional post-phosphorization method. Moreover, the deposition of ultrathin Co-P films on periodic trenches was demonstrated, which highlights the broad and promising potential application of this ALD process for a conformal coating of TMP films on complex three-dimensional (3D) architectures.

Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

Stimuli-Responsive Multilayers Based on Thiolated Polysaccharides That Affect Fibroblast Cell Adhesion.

Control of the biomaterial properties through stimuli-responsive polymeric platforms has become an essential technique in recent biomedical applications. A multilayer system of thiolated chitosan (t-Chi) and thiolated chondroitin sulfate (t-CS), consisting of five double layers ([t-Chi/t-CS]5), was fabricated here by applying a layer-by-layer coating strategy. To represent a novel class of chemically tunable nanostructures, the ability to cross-link pendant thiol groups was tested by a rise from pH 4 during layer formation to pH 9.3 and a more powerful chemical stimulus by using chloramine-T (ChT). Following both treatments, the resulting multilayers showed stimuli-dependent behavior, as demonstrated by their content of free thiols, wettability, surface charge, elastic modulus, roughness, topography, thickness, and binding of fibronectin. Studies with human dermal fibroblasts further demonstrated the favorable potential of the ChT-responsive multilayers as a cell-adhesive surface compared to pH-induced cross-linking. Because the [t-Chi/t-CS]5 multilayer system is responsive to stimuli such as the pH and redox environment, multilayer systems with disulfide bond formation may help to tailor their interaction with cells, film degradation, and controlled release of bioactive substances like growth factors in a stimuli-responsive manner useful in future wound healing and tissue engineering applications.

Introduction of Laser Interference Lithography to Make Nanopatterned Surfaces for Fundamental Studies on Stem Cell Response.

The extracellular matrix (ECM) is a nanostructured environment that provides chemical, mechanical, and topographical stimuli for various cellular functions. Here, we introduce the application of laser interference lithography (LIL) to generate hexagonally arranged gold nanostructures of three different dimensions on silicon to study the effect of feature dimensions on human adipose-derived stem cells (hADSC) in terms of adhesion, growth, and differentiation. Self-assembled monolayers (SAM) were used to passivate the background silicon surface with a long-chain polyethylene glycol (PEG), whereas the gold nanostructures were activated with mercaptoundecanoic acid (MUDA) to direct protein adsorption and cell adhesive structures to them, only. It was possible to show that the size and distance of the nanostructures affected the spreading of hADSC with a decrease of cell size with the increase of feature dimensions, which corresponded also to the expression of focal adhesions and presence of the small GTPase RhoA. Effects of these early events, related to outside-in signal transduction, were visible by an enhanced cell growth on smaller feature dimensions and distinct effects on cell differentiation. Because of the precise control of chemical and topographical cues, the presented system offers great potential to study effects of material topography on stem cell behavior, which may pave the way for applications in tailoring surfaces of implants and tissue engineering scaffolds.

Inverse spin Hall effect in a complex ferromagnetic oxide heterostructure.

We present spin pumping and inverse spin Hall effect (ISHE) in an epitaxial complex oxide heterostructure. Ferromagnetic La0.7Sr0.3MnO3 (LSMO) is used as a source of spin pumping while the spin sink exhibiting the ISHE consists of SrRuO3 (SRO). SRO is a ferromagnetic oxide with metallic conductivity, however, with a Curie temperature (TC) of 155 K, thus well below room temperature. This choice allows to perform the experiment above and below TC of the SRO and to demonstrate that SRO not only shows an ISHE of a magnitude comparable to Pt (though with opposite sign) in its non magnetic state but also exhibits a finite ISHE even 50 K below TC.

Influence of the doping level on the porosity of silicon nanowires prepared by metal-assisted chemical etching.

A systematic method to control the porosity of silicon nanowires is presented. This method is based on metal-assisted chemical etching (MACE) and takes advantage of an HF/H2O2 etching solution and a silver catalyst in the form of a thin patterned film deposited on a doped silicon wafer. It is found that the porosity of the etched nanowires can be controlled by the doping level of the wafer. For low doping concentrations, the wires are primarily crystalline and surrounded by only a very thin layer of porous silicon (pSi) layer, while for highly doped silicon, they are porous in their entire volume. We performed a series of controlled experiments to conclude that there exists a well-defined critical doping concentration separating the crystalline and porous regimes. Furthermore, transmission electron microscopy investigations showed that the pSi has also a crystalline morphology on a length scale smaller than the pore size, determined from positron annihilation lifetime spectroscopy to be mesoscopic. Based on the experimental evidence, we devise a theoretical model of the pSi formation during MACE and apply it for better control of the nanowire morphology.

Improved stability and cell response by intrinsic cross-linking of multilayers from collagen I and oxidized glycosaminoglycans.

Stability of surface coatings against environmental stress, such as pH, high ionic strength, mechanical forces, and so forth, is crucial for biomedical application of implants. Here, a novel extracellular-matrix-like polyelectrolyte multilayer (PEM) system composed of collagen I (Col I) and oxidized glycosaminoglycans (oGAGs) was stabilized by intrinsic cross-linking due to formation of imine bonds between aldehydes of oxidized chondroitin sulfate (oCS) or hyaluronan (oHA) and amino groups of Col I. It was also found that Col I contributed significantly more to overall mass in CS-Col I than in HA-Col I multilayer systems and fibrillized particularly in the presence of native and oxidized CS. Adhesion and proliferation studies with murine C3H10T1/2 embryonic fibroblasts demonstrated that covalent cross-linking of oGAG with Col I had no adverse effects on cell behavior. By contrast, it was found that cell size and polarization was more pronounced on oGAG-based multilayer systems, which corresponded also to the higher stiffness of cross-linked multilayers as observed by studies with quartz crystal microbalance (QCM). Overall, PEMs prepared from oGAG and Col I give rise to stable PEM constructs due to intrinsic cross-linking that may be useful for making bioactive coatings of implants and tissue engineering scaffolds.

Nanoscaled surface patterns influence adhesion and growth of human dermal fibroblasts.

In general, there is a need for passivation of nanopatterned biomaterial surfaces if cells are intended to interact only with a feature of interest. For this reason self-assembled monolayers (SAM), varying in chain length, are used; they are highly effective in preventing protein adsorption or cell adhesion. In addition, a simple and cost-effective technique to design nanopatterns of various sizes and distances, the so-called nanosphere lithography (NSL), is discussed, which allows the control of cell adhesion and growth depending on the feature dimensions. Combining both techniques results in highly selective nanostructured surfaces, showing that single proteins selectively adsorb on activated nanopatterns. Additionally, adhesion and growth of normal human dermal fibroblasts (NHDF) is strongly affected by the nanostructure dimensions, and it is proven that fibronectin (FN) matrix formation of these cells is influenced, too. Moreover, the FN fibrils are linked to the hexagonally close-packed nanopatterns. As a result, the system presented here can be applied in tissue engineering and implant design due to the fact that the nanopattern dimensions give rise to further modifications and allow the introduction of chemical heterogeneity to guide stem cell differentiation in the future.

Ag-mediated charge transport during metal-assisted chemical etching of silicon nanowires.

The charge transport mechanism during metal-assisted chemical etching of Si nanowires with contiguous metal films has been investigated. The experiments give a better insight how the charges and reaction products can penetrate to the etching front. The formation of a layer of porous Si between the metal film and the bulk Si is a prerequisite for the etching process. The electronic holes (positive charges) necessary for the etching of porous Si are generated at the surface of the metal in contact with the oxidative agent. Because of the insulating character of the thin walls of the porous Si, the transport of the electronic holes through this layer is not possible. Instead, it is found that the transport of electronic holes proceeds primarily by means of the Ag/Ag(+) redox pair circulating in the electrolyte and diffusing through the etched pores in the Si. The charge transport occurs without the ionic contribution at the positions where the metal is in direct contact with the Si. Here, an electropolishing process takes place, leading to an extensive removal of the Si and sinking in of the film into the Si substrate.

Enantiopure chiral poly(glycerol methacrylate) self-assembled monolayers knock down protein adsorption and cell adhesion.

Chirality plays a fundamental role not only in biological systems, but also in synthetic materials intended for bio-applications. Self-assembled monolayers (SAMs) are prepared on gold surfaces through a "grafting to" method from racemic or enantiopure chiral poly(glycerol methacrylate)s (PGMA(rac), PGMA(R), and PGMA(S)), having a thiol endgroup. Such SAMs constitute a chemically and structurally well-defined model substrate for studying protein adsorption and cell adhesion as a function of the polymer chirality. Surface plasmon resonance measurements reveal that PGMA SAMs greatly reduce the adsorption of bovine serum albumin (BSA) compared to bare gold surfaces. Interestingly, enantiopure SAMs based on PGMA(R) or PGMA(S) show a significantly larger reduction in BSA adsorption than PGMA(rac)-covered surfaces. Studies with the monocytic cell line THP-1 show a similar relationship between enantiopurity of PGMA SAMs and the extent of cell adhesion. Ellipsometry and Raman spectroscopy measurements indicate that SAMs formed by PGMA(rac) have a higher grafting density compared to SAMs of PGMA(R) and PGMA(S). This seems to be due to the ability of PGMA(rac) to form more intermolecular hydrogen bonds among polymer chains compared to the enantiopure PGMAs. Circular dichroism spectroscopy provide evidence that enantiopure polymers adopt a chiral ordered conformation, most likely helical, in aqueous solutions. It is concluded that a higher water content of SAMs formed by enantiopure PGMA(S) and PGMA(R) SAMs arises from the macromolecular chiral conformation adopted by their enantiopure PGMA chains, and it is the decisive reason for the reduced BSA adsorption and cell adhesion as compared to PGMA(rac) SAMs.

Sub-20 nm Si/Ge superlattice nanowires by metal-assisted etching.

An effective and low-cost method to fabricate hexagonally patterned, vertically aligned Si/Ge superlattice nanowires with diameters below 20 nm is presented. By combining the growth of Si/Ge superlattices by molecular beam epitaxy, prepatterning the substrate by anodic aluminum oxide masks, and finally metal-assisted chemical wet etching, this method generates highly ordered hexagonally patterned nanowires. This technique allows the fabrication of nanowires with a high area density of 10(10) wires/cm(2), including the control of their diameter and length.

Silicon nanocolumns on nanosphere lithography templated substrates: effects of sphere size and substrate temperature.

Glancing angle ion beam sputter deposition was used to grow regular arrays of Si nanocolumns with a nominal height of 650 nm at room temperature on polystyrene nanospheres with sphere diameters between 260 nm and 3550 nm, and at elevated temperatures on SiO2 nanospheres with a sphere diameter of 360 nm. Top view and cross sectional scanning electron microscopy reveals that the Si nanocolumns resemble cylinder-like structures, terminated by a hemispherical cap. Diameter, height and inter-column-spacing are found to depend linearly on the nanosphere diameter, thus giving the possibility to grow arrays of vertical Si columns with distinct porosities. For the growth at elevated temperatures, it was found that while on non-patterned substrates diffusion effects lead to broadening and finally merging of initially separated nanocolumns, on nanosphere patterned substrates this broadening effect is only moderate. No merging of columns is observable in this case, but a decrease of the column height due to a temperature-driven inter-column densification.

Ordered arrays of silicon nanowires produced by nanosphere lithography and molecular beam epitaxy.

Because of their importance in fundamental research and possible applications in nanotechnology and nanoelectronics, semiconductor nanowires have attracted much interest. In addition to the growth itself, the control of the size and location is an essential problem. Here we show the growth of ordered arrays of vertically aligned silicon nanowires by molecular beam epitaxy using prepatterned arrays of gold droplets on Si(111) substrates. The ordered arrays of gold particles were produced by nanosphere lithography.