We report a straightforward method for creating large-area, microscale resolution patterns of functional amines on self-assembled monolayers by the photoinduced local acidification of a flat elastomeric stamp enriched with photoacid. The limited diffusivity of the photoactivated merocyanine acid in poly(dimethylsiloxane) (PDMS) enabled to confine efficient deprotection of N-tert-butyloxycarbonyl amino group (N-Boc) to line widths below 10 μm. The experimental setup is very simple and is built around the conventional HD-DVD optical pickup. The method allows cost-efficient, maskless, large-area chemical patterning while avoiding potentially cytotoxic photochemical reaction products. The activation of the embedded photoacid occurs within the stamp upon illumination with the laser beam and the process is fully reversible. Preliminary positive results highlight the possibility of repeatable use of the same stamp for the creation of different patterns.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

We developed an experimentally simple method for localized N-tert-butyloxycarbonyl amino group (N-Boc) deprotection on surface applicable to meso-scale on-demand patterning for further synthetic modifications. The chemical patterning is accomplished on heated self-assembled monolayers using micro-drop equipment and water as the sole chemical.

There is a growing need for novel in vitro corneal models to replace animal-based ex vivo tests in drug permeability studies. In this study, we demonstrated a corneal mimetic that models the stromal and epithelial compartments of the human cornea. Human corneal epithelial cells (HCE-T) were grown on top of a self-supporting porcine collagen-based hydrogel. Cross-sections of the multi-layers were characterized by histological staining and immunocytochemistry of zonula oc-cludens-1 protein (ZO-1) and occludin. Furthermore, water content and bssic elastic properties of the synthetized collagen type I-based hydrogels were measured. The apparent permeability coefficient (Papp) values of a representative set of ophthalmic drugs were measured and correlated to rabbit cornea Papp values found in the literature. A multilayered structure of HCE-T cells and the expression of ZO-1 and occludin in the full thickness of the multilayer were observed. The hydrogel-based corneal model exhibited an excellent correlation to rabbit corneal permeability (r = 0.96), whereas the insert-grown HCE-T multilayer was more permeable and the correlation to the rabbit corneal permeability was lower (r = 0.89). The hydrogel-based human corneal model predicts the rabbit corneal permeability more reliably in comparison to HCE-T cells grown in inserts. This in vitro human corneal model can be successfully employed for drug permeability tests whilst avoiding ethical issues and reducing costs.

To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

By conducting a systematic study of model lipid membranes using the atomic force microscopy (AFM) indentation, we demonstrate the importance of an experimental protocol on the determination of their mechanical parameters. We refine the experimental approach by analyzing the influence of the contact mechanics models used to process the data, substrate preparation, and indenter geometry. We show that both bending rigidity and area compressibility can be determined from a single AFM indentation measurement.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell–cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Recombinant human collagen type I hydrogels as superior cell carriers for corneal epithelial stem cells

Self-supporting, shapeable hydrogels that consist of self-assembling synthetic peptides mimic the structural blocks of the extracellular matrix (ECM). Although they have been developed for regenera ...

Increasing the imaging rate of atomic force microscopy (AFM) without impairing of the imaging quality is a challenging task, since the increase in the scanning speed leads to a number of artifacts related to the limited mechanical bandwidth of the AFM components. One of these artifacts is the loss of contact between the probe tip and the sample. We propose to apply an additional nonlinear force on the upper surface of a cantilever, which will help to keep the tip and surface in contact. In practice, this force can be produced by the precisely regulated airflow. Such an improvement affects the AFM system dynamics, which were evaluated using a mathematical model that is presented in this paper. The model defines the relationships between the additional nonlinear force, the pressure of the applied air stream, and the initial air gap between the upper surface of the cantilever and the end of the air duct. It was found that the nonlinear force created by the stream of compressed air (aerodynamic force) prevents the contact loss caused by the high scanning speed or the higher surface roughness, thus maintaining stable contact between the probe and the surface. This improvement allows us to effectively increase the scanning speed by at least 10 times using a soft (spring constant of 0.2 N/m) cantilever by applying the air pressure of 40 Pa. If a stiff cantilever (spring constant of 40 N/m) is used, the potential of vertical deviation improvement is twice is large. This method is suitable for use with different types of AFM sensors and it can be implemented practically without essential changes in AFM sensor design.

Abstract A new strategy towards tubular hydrogen‐bonded polymers based on the self‐assembly of isocytosine tautomers in orthogonal directions is proposed and experimentally verified, including by 1H fast magic‐angle spinning (MAS) solid‐state NMR. The molecular tubes obtained possess large internal diameter and tailor‐made outer functionalities rendering them potential candidates for a number of applications.

The design and synthesis of new stimuli-responsive hydrogen-bonding monomers that display a diversity of self-assembly pathways is of central importance in supramolecular chemistry. Here we describe the aggregation properties of a simple, intrinsically C 2 -symmetric enantiopure bicyclic cavity compound bearing a terminally unsubstituted ureidopyrimidinone fragment fused with a pyrrole moiety in different solvents and in the absence and presence of C 60 and C 70 guests. The tetrameric cyclic aggregate is selectively obtained in chlorinated solvents, where only part of the available hydrogen bonding sites are utilized, whereas in toluene or upon addition of C 70 guests, further aggregation into tubular supramolecular polymers is achieved. The open-end cyclic assemblies rearrange into a closed-shell capsule upon introduction of C 60 with an accompanied symmetry breaking of the monomer. Our study demonstrates that a C 60 switch can be used to simultaneously control the topology and occupancy of tubular assemblies resulting from the aggregation of small monomers.

A non-raster scanning technique for atomic force microscopy (AFM) imaging which combines rotational and translational motion is presented. The use of rotational motion for the fast scan axis allows us to significantly increase the scanning speed while imaging a large area (diameter > 30 μm). An image reconstruction algorithm and the factors influencing the resolution of the technique are discussed. The experimental results show the potential of the rotational scanning technique for high-throughput large area AFM investigation.

We report simple and low-cost setup based on a modified DVD optical pickup unit for detection and photothermal actuation of microcantilever oscillations. Small rectangular cantilevers (length 7 width 3 mu m and thickness 100 nm) were successfully actuated and detected in both air and liquid environments. Applications of such a setup for microcantilever-based biochemical and physical sensors and in atomic force microscopy are envisaged. (C) 2016 Elsevier B.V. All rights reserved.

There are three areas in which conventional AFM has limitations: (i) low imaging rate, (ii) probe-sample force interaction, and (iii) the planar nature of the sample. We are developing two high-speed force microscopy techniques. One high-speed AFM (HS AFM) technique is a DC method in which the tip is in continuous contact with the specimen. This routinely allows video-rate imaging (30 frames per second, fps) and has achieved imaging at over 1000 fps, i.e., 100,000 times faster than conventional AFM. Damage to specimens resulting from this high-speed contact-mode imaging is surprisingly less than would be caused at normal speeds. The behavior of the cantilever and tip at these high velocities has been investigated and superlubricity is a key component in the success of this technique. The other high-speed force microscope is a non-contact method based on shear-force microscopy (ShFM). In this HS ShFM, a vertically-mounted laterally-oscillating probe detects the sample surface at about 1 nm from it as a result of the change in the mechanical properties of the water confined between the probe tip and the sample. With this technique, very low normal forces are applied to the specimen. Conventional AFMs require planar samples because the probe scans in a plane. It is as if the tip is only 'seeing' the sample from above. We have overcome this limitation be steering the tip of a nanorod in a three dimensional scan with six degrees of freedom using holographically generate optical traps such that it is possible to scan around a sample.

A study was carried out on the effect of the reaction temperature in atomic layer deposition on the structural and piezoelectric properties of thin ZnO films (5–50 nm) by scanning electron microscopy, X-ray diffraction, piezoresponse force microscopy, and surface acoustic wave (SAW) spectroscopy. The piezoresponse was shown to be mainly a function of ZnO grain size and the monocrystalline structure perpendicular to the substrate plane. The results demonstrate promise for the use of textured ZnO films as sensitive coatings for acoustic gas sensors in instruments based on the SAW transducer amplification-frequency response.

The mechanical characterization of biomolecular motors requires force sensors with sub-piconewton resolution. The coupling of a nanoscale motor to this type of microscale sensors introduces structural deformations in the motor according to the thermally activated degrees of freedom of the sensor. At present, no simple solution is available to reduce these effects. Here, we exploit the advantages of micro-fabricated cantilevers to produce a force sensor with essentially one degree of freedom and a spring constant of 0.03 pN nm − 1 for the study of the molecular motor protein kinesin-1. During processive runs, the cantilever constrains the movement of the cargo binding domain of kinesin in a straight line, parallel to the microtubule track, and excludes specific reaction coordinates such as cargo rotation. In these conditions, we measured a step size of 8.0 ± 0.4 nm and a maximal unloaded velocity of 820 ± 80 nm s − 1 at saturated adenosine triphosphate (ATP) concentration. We concluded that the motor does not need to rotate its tail as it moves through consecutive stepping cycles.

Atomic force microscopy (AFM) has been very successful in measuring forces perpendicular to the sample plane. Here, we present the advantages of turning the AFM cantilever 90° in order for it to be perpendicular to the sample. This rotation leads naturally to the detection of in-plane forces with some extra advantages with respect to the AFM orientation. In particular, the use of extremely small (1μm wide) and soft (k≅10(-5)N/m) micro-fabricated cantilevers is demonstrated by recording their thermal power spectral density in ambient conditions and in liquid. These measurements lead to the complete characterisation of the sensors in terms of their stiffness and resonant frequency. Future applications, which will benefit from the use of this force microscopy technique, are also described.