Search

Nanostructured and bulk silicon carbide ($\mathrm{Si}\mathrm{C}$) materials are relevant for electronics, nano- and micromechanical systems, and biosensing applications. $\mathrm{Si}\mathrm{C}$ has recently emerged as an alternative platform for nanophotonics and quantum applications due to its intra-band-gap point defects, emitting from the visible to the near-infrared, which are ideal for photoluminescent probes. Here, we use a single-molecule localization microscope to study the photoluminescence (PL) properties of $\mathrm{Si}\mathrm{C}$ point defects in bulk, quantum dots, and nanoparticles of different sizes in the 3C and $4H\text{\ensuremath{-}}\mathrm{Si}\mathrm{C}$ polytypes using intra-band-gap excitation. We study the PL dynamics by using different excitation wavelengths, and we use the point-defect PL intermittency to achieve superresolved images, with a resolution of 20 nm and a minimum distance between emitters of 40 nm. We observe that, while 561 nm is an ideal excitation wavelength to obtain a sufficient blinking behavior, 638 nm is mostly quenching the PL. We further incubate $4H\text{\ensuremath{-}}\mathrm{Si}\mathrm{C}$ nanoparticles with MCF10A cells in vitro and observe superresolved images of the nanoparticles in cells by combining 561 and 638 nm excitation. This approach is very promising for the application of $\mathrm{Si}\mathrm{C}$ fluorescent nanocrystals and their quantum dots, hosting a combination of intra-band-gap color centers and surface defects, for quantum nanophotonics, magnetic sensing, and biomedical imaging, paving the way for single-particle tracking combined with spin sensing within a cellular environment using near-infrared emission.

Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid supports can improve cell adhesion, spreading and differentiation compared to traditional flat, stiff 2D cell culture substrates like Tissue Culture Polystyrene (TCPS). This is due to the fibrous, nanotopographic nature of the amyloid fibril networks and the fact that they closely mimic the mechanical properties and architecture of the extracellular matrix (ECM). However, precise cell responses are strongly dependent on the nanostructure of the network at the cell culture interface, thus accurate characterization of the immobilized network is important. Due to its exquisite lateral resolution and simple sample preparation techniques, Atomic Force Microscopy (AFM) is an ideal technique to characterize the fibril network morphology. Thus, here we describe a detailed protocol, for the characterization of amyloid fibril networks by tapping mode AFM.

Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale structures in composite materials, such as protein assemblies for use in consumer films or gels. As a recently developed force spectroscopy technique, bimodal force spectroscopy relates frequency shifts in cantilevers simultaneously excited at multiple frequencies to the elastic properties of the contacted material, yet its utility for quantitative characterization of biopolymer assemblies has been limited. In this study, a linear correlation between experimental frequency shift and Young's modulus of polymer films was used to calibrate bimodal force spectroscopy and quantify Young's modulus of two protein nanostructures: β-lactoglobulin fibrils and zein nanoparticles. Cross-sectional Young's modulus of protein fibrils was determined to be 1.6 GPa while the modulus of zein nanoparticles was determined as 854 MPa. Parallel measurement of β-lactoglobulin fibril by a competing pulsed-force technique found a higher cross-sectional Young's modulus, highlighting the importance of comparative calibration against known standards in both pulsed and bimodal force spectroscopies. These findings demonstrate a successful procedure for measuring mechanical properties of individual protein assemblies with potential use in biological or packaging applications using bimodal force spectroscopy.

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or β-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis.

Designing surfaces with antifouling and antimicrobial properties has an important route to solve problems, such as infections and fouling, in healthcare and industrial applications. Recently, there has been considerable interest in developing surfaces with polymers because of their distinct properties, such as their length scale, their processability, low cost, tunable properties, and diverse functionalities. This article reviews the polymer systems developed as antifouling–antimicrobial surface coatings, termed as biopassive and bioactive polymers respectively. Many different types of bioactive and biopassive polymeric systems and their limitations are presented.

The aim of this work was to immobilize short melanocyte-stimulating hormone (MSH) anti-inflammatory peptide sequences onto a model surface using resorcinarenes, which are known to attach to a wide variety of hydrophilic materials. The first stage comprised the biological evaluation of a synthetic MSH peptide attached to different linking tethers (poly(ethylene glycol), PEG 350, octanoic acid or cholesterol) for the ability to inhibit inflammatory signaling. Findings showed that a glycinelysine-proline-D-valine sequence inhibited inflammatory signaling most effectively when attached to a PEG 350 tether. This molecule was selected for a second stage which comprised of synthesizing the MSH peptide attached to resorcinarene groups via a PEG tether and immobilizing it onto glass coverslips. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF–SIMS) indicated the presence of a surface attached peptide. The ability of the immobilized peptide to inhibit inflammatory signaling was then determined by culturing RN22 Schwann neuronal cells and human dermal fibroblast cells on functional surfaces and measuring NF-kB/p65 inflammatory transcription factor activation. Significant inhibition of inflammatory signaling was observed in cells cultured on functional surfaces. In conclusion, we therefore describe the development of a new approach enabling the rapid immobilization of short biologically active peptides. This work has potential for generating a ‘‘dip-and-dry’’ approach for altering the surface properties of biomaterial and medical devices. Many implantable medical devices can initiate an acute inflammatory response which in turn is detrimental to the function of the device. This can be triggered either by the initial implantation injury or by the biomaterial itself. Examples of this include coronary restenosis after stent implantation, the ‘stress cracking’ of pacemakers and the failure of peripheral nerve guides. Inflammation can be treated by steroids or non-steroidal anti-inflammatories (NSAIDs). However, these compounds frequently lack sensitivity or a specific targeted site of action, leading to side effects. Therefore the development of more specific and localized forms of treatment is important for controlling inflammation associated with biomaterials. Previous work has investigated modifying the material surface chemistry or immobilizing non-bio-fouling molecules, such as PEG. However, these techniques are not always appropriate because they do not specifically target inflammatory signaling. An alternative approach is through the exploitation of naturally occurring peptides to reduce inflammation. One potential peptide is a-MSH which has multiple roles in the body including the control of inflammation. An analogue of a-MSH, NleDPhe-a-MSH, has already been shown to increase allograft survival when injected into rats. Previous research has shown that the minimal sequence required for anti-inflammatory biology is a tripeptide at the carboxyl terminal consisting of lysine-proline-valine (KPV). This tripeptide mimics the anti-inflammatory effect of the full-length peptide, but is more specific as it does not exhibit melanotropic activity or stimulate dopa oxidase activity. This peptide has the ability to inhibit nuclear factor-kB (NF-kB) activation when stimulated by a wide range of proinflammatory agents, reducing the production of proinflammatory cytokines under its control. KPV can stimulate the direct production of the anti-inflammatory cytokine interleukin-10 (IL-10) and inhibit inflammation in vivo. Additionally, the peptide has been demonstrated to possess antimicrobial properties in vitro. The short tripeptide sequence makes it amenable for straightforward laboratory synthesis and this, in combination with its pharmacological potency, makes it an attractive candidate for immobilization on to medical device surfaces for creating a localized anti-inflammatory effect. a-MSH and its analogues have previously been immobilized using polyelectrolyte films and also as an analogue of the tripeptide (GKP-D-V), where it was attached to polystyrene beads. Both techniques demonstrated that the peptide retained anti-inflammatory properties when immobilized in vitro. Prostheses of titanium beads coated with polyelectrolyte multilayer films functionalized with a-MSH also retained the ability to inhibit inflammation when implanted into rats. The D-Val substitution in KP-D-V leads to greater anti-inflammatory properties than the KPV peptide and the parent molecule, a-MSH, probably due to reduced degradation. The present work describes a new methodology for immobilization of the tripeptide analogue GKP-D-V using resorcinarene group molecules. Resorcinarenes can be immobilized through hydrogen bonding of the hydroxylated bowl to hydrophilic materials through simple immersion of the material into a solution of the resorcinarene compound. This forms a new surface on that material, the properties of which then depend on the nature of the group covalently attached to the resorcinarene. Previous research has investigated the use of attaching alkyl chains and

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range ( 0.5–4.7 lm), and mean distance between peaks (RSm) gradually varying from 214 lm to 33 lm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra 1.53 lm/RSm 79 lm in Dex-deprived OI medium, and Ra 0.93 lm/RSm 135 lm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone., This work was supported by the Foundation for Science and Technology for the PhD Grant of A.B. Faia-Torres (SFRH/BD/36476/2007). We are also grateful to the Swiss National Science Foundation (SNSF) Grant P300P3_154664 for providing funds to MC.

The success of synthetic bone implants requires good interface between the material and the host tissue. To study the biological relevance of fi bronectin (FN) density on the osteogenic commitment of human bone marrow mesenchymal stem cells (hBMMSCs), human FN was adsorbed in a linear density gradient on the surface of PCL. The evolution of the osteogenic markers alkaline phosphatase and collagen 1 alpha 1 was monitored by immunohistochemistry, and the cytoskeletal organization and the cell-derived FN were assessed. The functional analysis of the gradient revealed that the lower FN-density elicited stronger osteogenic expression and higher cytoskeleton spreading, hallmarks of the stem cell commitment to the osteoblastic lineage. The identifi cation of the optimal FN density regime for the osteogenic commitment of hBM-MSCs presents a simple and versatile strategy to signifi cantly enhance the surface properties of polycaprolactone as a paradigm for other synthetic polymers intended for bone-related applications., This work was supported by the Foundation for Science and Technology for the PhD grant of A.B. Faia-Torres (SFRH/BD/36476/2007). We are also grateful to the Swiss National Science foundation (SNSF) grant P300P3_154664 for providing funds to MC.

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data., PLoS ONE, 6 (12), ISSN:1932-6203

Background Increasing evidence shows that the cancer microenvironment affects both tumorigenesis and the response of cancer to drug treatment. Therefore in vitro models that selectively reflect characteristics of the in vivo environment are greatly needed. Current methods allow us to screen the effect of extrinsic parameters such as matrix composition and to model the complex and three-dimensional (3D) cancer environment. However, 3D models that reflect characteristics of the in vivo environment are typically too complex and do not allow the separation of discrete extrinsic parameters. Methodology/Principal Findings In this study we used a poly(ethylene glycol) (PEG) hydrogel-based microwell array to model breast cancer cell behavior in multilayer cell clusters that allows a rigorous control of the environment. The innovative array fabrication enables different matrix proteins to be integrated into the bottom surface of microwells. Thereby, extrinsic parameters including dimensionality, type of matrix coating and the extent of cell-cell adhesion could be independently studied. Our results suggest that cell to matrix interactions and increased cell-cell adhesion, at high cell density, induce independent effects on the response to Taxol in multilayer breast cancer cell clusters. In addition, comparing the levels of apoptosis and proliferation revealed that drug resistance mediated by cell-cell adhesion can be related to altered cell cycle regulation. Conversely, the matrix-dependent response to Taxol did not correlate with proliferation changes suggesting that cell death inhibition may be responsible for this effect. Conclusions/Significance The application of the PEG hydrogel platform provided novel insight into the independent role of extrinsic parameters controlling drug response. The presented platform may not only become a useful tool for basic research related to the role of the cancer microenvironment but could also serve as a complementary platform for in vitro drug development. ISSN:1932-6203

We report on the optically detected magnetic resonance of nitrogen-vacancy centres in blinking nanodiamonds to measure magnetic fields from magnetically-labeled MCF10A cells. Magnetic fields imaging is reconstructed with a localization microscope down to 25 nm.