Bio-inspired microneedle design for efficient drug/vaccine coating.

Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport. To realize these MNs, two-photon polymerization (TPP) technique was used. Liquid coating capabilities of structured and non-structured MNs were compared. Moreover, both in-vivo and ex-vivo skin tests were performed to prove that MNs pierce the skin. We show that the arrays of MNs can be accurately replicated using a micro-moulding technique. We believe this design will be beneficial for the process of drug/vaccine loading onto the needles' surfaces, by making it more efficient and by reducing the drug/vaccine wastage during MN coating process.

Nanopatterning of Bionic Materials.

The nanopatterning of bionic materials, performed by means of laser processes that utilize pulsed laser sources with short and ultrashort pulse durations, is a rapidly growing field [...].

Robustness of antiadhesion between nanofibers and surfaces covered with nanoripples of varying spatial period.

Since nanofibers have a high surface-to-volume ratio, van der Waals forces render them attracted to virtually any surface. The high ratio provides significant advantages for applications in drug delivery, wound healing, tissue regeneration, and filtration. Cribellate spiders integrate thousands of nanofibers into their capture threads as an adhesive to immobilize their prey. These spiders have antiadhesive nanoripples on the calamistrum, a comb-like structure on their hindmost legs, and are thus an ideal model for investigating how nanofiber adhesion can be reduced. We found that these nanoripples had similar spacing in the cribellate species Uloborus plumipes, Amaurobius similis, and Menneus superciliosus, independent of phylogenetic relation and size. Ripple spacing on other body parts (i.e., cuticle, claws, and spinnerets), however, was less homogeneous. To investigate whether a specific distance between the ripples determines antiadhesion, we fabricated nanorippled foils by nanosecond UV laser processing. We varied the spatial periods of the nanoripples in the range ~203-613 nm. Using two different pulse numbers resulted in ripples of different heights. The antiadhesion was measured for all surfaces, showing that the effect is robust against alterations across the whole range of spatial periods tested. Motivated by these results, we fabricated irregular surface nanoripples with spacing in the range ~130-480 nm, which showed the same antiadhesive behavior. The tested surfaces may be useful in tools for handling nanofibers such as spoolers for single nanofibers, conveyor belts for producing endless nanofiber nonwoven, and cylindrical tools for fabricating tubular nanofiber nonwoven. Engineered fibers such as carbon nanotubes represent a further candidate application area.

Laser-processed antiadhesive bionic combs for handling nanofibers inspired by nanostructures on the legs of cribellate spiders.

Nanofibers are drawing the attention of engineers and scientists because their large surface-to-volume ratio is favorable for applications in medicine, filter technology, textile industry, lithium-air batteries, and optical sensors. However, when transferring nanofibers to a technical product in the form of a random network of fibers, referred to as nonwoven fabric, the stickiness of the freshly produced and thus fragile nanofiber nonwoven remains a problem. This is mainly because nanofibers strongly adhere to any surface because of van der Waals forces. In nature, there are animals that are actually able to efficiently produce, process, and handle nanofibers, namely cribellate spiders. For that, the spiders use the calamistrum, a comb-like structure of modified setae on the metatarsus of the hindmost (fourth) legs, to which the 10-30 nm thick silk nanofibers do not stick due to a special fingerprint-like surface nanostructure. In this work, we present a theoretical model of the interaction of linear nanofibers with a sinusoidally corrugated surface. This model allows for a prediction of the adhesive interaction and, thus, the design of a suitable surface structure to prevent sticking of an artificially nonwoven of nanofibers. According to the theoretical prediction, a technical analogon of the nanoripples was produced by ultrashort pulse laser processing on different technically relevant metal surfaces in the form of so-called laser-induced periodic surface structures (LIPSS). Subsequently, by means of a newly established peel-off test, the adhesion of an electrospun polyamide fiber-based nonwoven was quantified on such LIPSS-covered aluminium alloy, steel, and titanium alloy samples, as well as on polished (flat) control samples as reference and, additionally, on samples with randomly rough surfaces. The latter revealed that the adhesion of electrospun nanofiber nonwoven is significantly lowered on the nanostructured surfaces compared with the polished surfaces.

Ambient Climate Influences Anti-Adhesion between Biomimetic Structured Foil and Nanofibers.

Due to their uniquely high surface-to-volume ratio, nanofibers are a desired material for various technical applications. However, this surface-to-volume ratio also makes processing difficult as van der Waals forces cause nanofibers to adhere to virtually any surface. The cribellate spider Uloborus plumipes represents a biomimetic paragon for this problem: these spiders integrate thousands of nanofibers into their adhesive capture threads. A comb on their hindmost legs, termed calamistrum, enables the spiders to process the nanofibers without adhering to them. This anti-adhesion is due to a rippled nanotopography on the calamistrum. Via laser-induced periodic surface structures (LIPSS), these nanostructures can be recreated on artificial surfaces, mimicking the non-stickiness of the calamistrum. In order to advance the technical implementation of these biomimetic structured foils, we investigated how climatic conditions influence the anti-adhesive performance of our surfaces. Although anti-adhesion worked well at low and high humidity, technical implementations should nevertheless be air-conditioned to regulate temperature: we observed no pronounced anti-adhesive effect at temperatures above 30 °C. This alteration between anti-adhesion and adhesion could be deployed as a temperature-sensitive switch, allowing to swap between sticking and not sticking to nanofibers. This would make handling even easier.

An Improved Transwell Design for Microelectrode Ion-Flux Measurements.

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures.

Femtosecond Laser-Processing of Pre-Anodized Ti-Based Bone Implants for Cell-Repellent Functionalization.

Microstructures and nanostructures can be used to reduce the adhesion of the cells on the auxiliary material. Therefore, the aim of our work was to fabricate laser-induced hierarchical microstructures and nanostructures by femtosecond laser-treatment (wavelength 1040 nm, pulse length 350 fs, repetition rates in the kHz range) to reduce the cell adhesion. Additionally, surface chemistry modification by optimized electrochemical anodization was used to further reduce the cell adhesion. For testing, flat plates and bone screws made of Ti-6Al-4V were used. Bone-forming cells (human osteoblasts from the cell line SAOS-2) were grown on the bone implants and additional test samples for two to three weeks. After the growth period, the cells were characterized by scanning electron microscopy (SEM). While earlier experiments with fibroblasts had shown that femtosecond laser-processing followed by electrochemical anodization had a significant impact on cell adhesion reduction, for osteoblasts the same conditions resulted in an activation of the cells with increased production of extracellular matrix material. Significant reduction of cell adhesion for osteoblasts was only obtained at pre-anodized surfaces. It could be demonstrated that this functionalization by means of femtosecond laser-processing can result in bone screws that hinder the adhesion of osteoblasts.

Spatial Period of Laser-Induced Surface Nanoripples on PET Determines Escherichia coli Repellence.

Bacterial adhesion and biofilm formation on surfaces are associated with persistent microbial contamination, biofouling, and the emergence of resistance, thus, calling for new strategies to impede bacterial surface colonization. Using ns-UV laser treatment (wavelength 248 nm and a pulse duration of 20 ns), laser-induced periodic surface structures (LIPSS) featuring different sub-micrometric periods ranging from ~210 to ~610 nm were processed on commercial poly(ethylene terephthalate) (PET) foils. Bacterial adhesion tests revealed that these nanorippled surfaces exhibit a repellence for E. coli that decisively depends on the spatial periods of the LIPSS with the strongest reduction (~91%) in cell adhesion observed for LIPSS periods of 214 nm. Although chemical and structural analyses indicated a moderate laser-induced surface oxidation, a significant influence on the bacterial adhesion was ruled out. Scanning electron microscopy and additional biofilm studies using a pili-deficient E. coli TG1 strain revealed the role of extracellular appendages in the bacterial repellence observed here.

Microgrinding of lensed fibers by means of a scanning-probe microscope setup.

We describe a precision grinding procedure that allows treating a previously etched fiber tip to conform to predefined shapes, including hemispherical and axial conical (axicon) lenses. The grinding method is based on mechanical polishing with the fiber tip moving in a translational mode inside a conical polishing surface. The grinding procedure is performed in a homemade scanning probe microscope equipped with a shear-force sensor based on a piezoelectric tuning fork as well as with capacitor position sensors. The scanning probe microscope is operated either as atomic force microscope for topographic characterization of the tip shape and the polishing surface or as a scanning near-field microscope for measurement of the light focusing properties of the ground microlenses.

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene.

Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200-430 nm and a depth of 30-100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.

The external scent efferent system of selected European true bugs (Heteroptera): a biomimetic inspiration for passive, unidirectional fluid transport.

In this work, we present structured capillaries that were inspired by the microstructures of the external scent efferent system as found in different European true bug species (Pentatomidae and Cydnidae). These make use of small, orientated structures in order to facilitate fluid movement towards desired areas where defensive substances are evaporated. Gland channels and microstructures were investigated by means of scanning electron microscopy and abstracted into three-dimensional models. We used these models to create scent channel replicas from different technical substrates (steel and polymers) by means of laser ablation, laser structuring and casting. Video analysis of conducted fluid-flow experiments showed that bug-inspired, artificial scent fluid channels can indeed transport different fluids (water solutions and oils/lubricants) passively in one direction (velocities of up to 1 mm s-1), while halting the fluid movement in the opposite direction. At the end of this contribution, we present a physical theory that explains the observed fluid transport and sets the rules for performance optimization in future work.

Homogeneous application of imaging criteria in a multicenter trial supported by investigator training: A report from the WAKE-UP study.

BACKGROUND AND PURPOSE: WAKE-UP is a randomized, placebo-controlled trial of thrombolysis in stroke with unknown time of symptom onset using magnetic resonance imaging criteria to determine patients' eligibility. As it is a multicenter trial, homogeneous interpretation of criteria is an important contributor to the trial's success. We describe the investigator image training as well as results of the quality control done by the central image reading board (CIRB). METHODS: Investigators at local centers were given an imaging manual and passed a software-based image training prior to being allowed to judge images in the trial. Throughout the trial, the CIRB gave feedback to recruiting centers in cases of disagreement regarding a patient's randomization. We evaluated the investigators performance in the image training and analyzed results of this quality control from the first 1069 screened patients. Additionally, we obtained feedback from investigators regarding their experiences with the trial. RESULTS: Four-hundred-and-sixty physicians from eight European countries took part in the image training, of whom 436 (95%) successfully completed it. In the trial, agreement rates between the local investigators and members of the CIRB were high for the presence of an acute ischemic lesion (94%, κ = 0.87) as well as for the judgment of infarct extent (93%, κ = 0.87). Agreement for the criterion of DWI-FLAIR mismatch was 74%, κ = 0.60. The majority of investigators reported that the DWI-FLAIR mismatch was the hardest imaging criterion to evaluate. Ninety-one percent of investigators who responded to our survey stated that the image training specifically increased their confidence when assessing the DWI-FLAIR mismatch. CONCLUSIONS: Despite its multicenter design, the WAKE-UP study has demonstrated a high level of homogeneity amongst raters in interpreting the various imaging criteria for patient randomization, including the novel criterion of DWI-FLAIR mismatch. Systematic image training increased the confidence of investigators in applying imaging criteria.

Laser-induced optical breakdown spectroscopy of polymer materials based on evaluation of molecular emission bands.

Laser-induced breakdown spectroscopy (LIBS) for composition analysis of polymer materials results in optical spectra containing atomic and ionic emission lines as well as molecular emission bands. In the present work, the molecular bands are analyzed to obtain spectroscopic information about the plasma state in an effort to quantify the content of different elements in the polymers. Polyethylene (PE) and a rubber material from tire production are investigated employing 157nmF2 laser and 532nm Nd:YAG laser ablation in nitrogen and argon gas background or in air. The optical detection reaches from ultraviolet (UV) over the visible (VIS) to the near infrared (NIR) spectral range. In the UV/VIS range, intense molecular emissions, C2 Swan and CN violet bands, are measured with an Echelle spectrometer equipped with an intensified CCD camera. The measured molecular emission spectra can be fitted by vibrational-rotational transitions by open access programs and data sets with good agreement between measured and fitted spectra. The fits allow determining vibrational-rotational temperatures. A comparison to electronic temperatures Te derived earlier from atomic carbon vacuum-UV (VUV) emission lines show differences, which can be related to different locations of the atomic and molecular species in the expanding plasma plume. In the NIR spectral region, we also observe the CN red bands with a conventional CDD Czerny Turner spectrometer. The emission of the three strong atomic sulfur lines between 920 and 925nm is overlapped by these bands. Fitting of the CN red bands allows a separation of both spectral contributions. This makes a quantitative evaluation of sulfur contents in the start material in the order of 1wt% feasible.

Adaptive camouflage: what can be learned from the wetting behaviour of the tropical flat bugs Dysodius lunatus and Dysodiusmagnus.

The neotropical flat bug species Dysodius lunatus and Dysodius magnus show a fascinating camouflage principle, as their appearance renders the animal hardly visible on the bark of trees. However, when getting wet due to rain, bark changes its colour and gets darker. In order to keep the camouflage effect, it seems that some Dysodius species benefit from their ability to hold a water film on their cuticle and therefore change their optical properties when also wetted by water. This camouflage behaviour requires the insect to have a hydrophilic surface and passive surface structures which facilitate the liquid spreading. Here we show morphological and chemical characterisations of the surface, especially the cuticular waxes of D. magnus Scanning electron microscopy revealed that the animal is covered with pillar-like microstructures which, in combination with a surprising chemical hydrophilicity of the cuticle waxes, render the bug almost superhydrophilic: water spreads immediately across the surface. We could theoretically model this behaviour assuming the effect of hemi-wicking (a state in which a droplet sits on a rough surface, partwise imbibing the structure around).  Additionally the principle was abstracted and a laser-patterned polymer surface, mimicking the structure and contact angle of Dysodius wax, shows exactly the behaviour of the natural role model - immediate spreading of water and the formation of a thin continuous water film changing optical properties of the surface.

Simple and versatile methods for the fabrication of arrays of live mammalian cells.

Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.

Incorporation of a lauric acid-conjugated GRGDS peptide directly into the matrix of a poly(carbonate-urea)urethane polymer for use in cardiovascular bypass graft applications.

Gly-Arg-Gly-Asp-Ser (GRGDS) was modified by conjugation to lauric acid (LA) to facilitate incorporation into the matrix of a poly(carbonate-urea)urethane (PCU) used in vascular bypass grafts. GRGDS and LA-GRGDS were synthesized using solid phase Fmoc chemistry and characterized by high performance liquid chromatography and Fourier transform infrared spectroscopy. LA-GRGDS was passively coated and incorporated as nanoparticle dispersion on the PCU films. Biocompatibility of the modified surfaces was investigated. Endothelial cells seeded on LA-GRGDS coated and incorporated PCU showed after 48 h and 72 h a significant (p < 0.05) increase in metabolism compared with unmodified PCU. The platelet adhesion and hemolysis studies showed that the modification of PCU had no adverse effect. In conclusion, LA-conjugated RGD derivatives, such as LA-GRGDS, that permit solubility into solvents used in solvent casting methodologies should have wide applicability in polymer development for use in coronary, vascular, and dialysis bypass grafts, and furthermore scaffolds utilized for tissue regeneration and tissue engineering.

Cell microarrays on photochemically modified polytetrafluoroethylene.

We studied the adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVEC) and human embryonic kidney cells (HEK) on modified spots at polytetrafluoroethylene (PTFE) surfaces. The viability of the cells was assessed using an aqueous non-radioactive cell proliferation assay. Round spots with a diameter of 100 microm were modified by exposure to the ultraviolet (UV) light of a Xe(2)(*)-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere employing a contact mask. The spots were arranged in a quadratic pattern with 300 microm center-to-center spot distances. With optimized degree of modification, the cells adhered to the modified spots with a high degree of selectivity (70-90%). The adhered cells on the spots proliferated. This resulted in a significant increase in the number of adhering HUVECS or HEK cells after seeding and in the formation of confluent cell clusters after 3-4 days. With higher start seeding density, these clusters were not only confined to the modified spots but extended several micrometer to the neighborhood. The high potential of the cell microarrays for gene analysis in living cells was demonstrated with HEK cells transfected by yellow fluorescent protein (YFP).

Enhanced Ca2+Entry and Tyrosine Phosphorylation Mediate Nanostructure-Induced Endothelial Proliferation.

Nanostructured substrates have been recognized to initiate transcriptional programs promoting cell proliferation. Specifically ß-catenin has been identified as transcriptional regulator, activated by adhesion to nanostructures. We set out to identify processes responsible for nanostructure-induced endothelial ß-catenin signaling. Transmission electron microscopy (TEM) of cell contacts to differently sized polyethylene terephthalate (PET) surface structures (ripples with 250 to 300 nm and walls with 1.5 µm periodicity) revealed different patterns of cell-substrate interactions. Cell adhesion to ripples occurred exclusively on ripple peaks, while cells were attached to walls continuously. The Src kinase inhibitor PP2 was active only in cells grown on ripples, while the Abl inhibitors dasatinib and imatinib suppressed ß-catenin translocation on both structures. Moreover, Gd3+ sensitive Ca2+ entry was observed in response to mechanical stimulation or Ca2+ store depletion exclusively in cells grown on ripples. Both PP2 and Gd3+ suppressed ß-catenin nuclear translocation along with proliferation in cells grown on ripples but not on walls. Our results suggest that adhesion of endothelial cells to ripple structured PET induces highly specific, interface topology-dependent changes in cellular signalling, characterized by promotion of Gd3+ -sensitive Ca2+ entry and Src/Abl activation. We propose that these signaling events are crucially involved in nanostructure-induced promotion of cell proliferation.

Nanopatterned polymer substrates promote endothelial proliferation by initiation of ß-catenin transcriptional signaling.

Control of endothelial phenotype involves a variety of signaling pathways and transcriptional regulators, including the junctional protein ß-catenin. This multifunctional signaling molecule is part of adhesion contacts in the endothelium and is able to translocate into the nucleus to activate genetic programs and control proliferation and the fate of the cells. We investigated the influence of laser-generated nanopatterns on polymeric cell culture substrates on endothelial tissue architecture, proliferation and ß-catenin signaling. For our experiments human microvascular endothelial cells or CD34(+) endothelial progenitor cells, isolated from human adipose tissue, were cultured on polyethylene terephthalate (PET) substrates with oriented nanostructures with lateral periodicities of 1.5 µm and 300 nm, respectively. The surface topography and chemistry of the PET substrates were characterized by electron microscopy, atomic force microscopy, water contact angle measurement and X-ray photoelectron spectroscopy. Analysis of cell phenotype markers as well as ß-catenin signaling revealed that short-term culture of endothelial cells on nanostructured substrates generates a proliferative cell phenotype associated with nuclear accumulation of ß-catenin and activation of specific ß-catenin target genes. The effects of the nanostructures were not directly correlated with nanostructure-induced alignment of cells and were also clearly distinguishable from the effects of altered PET surface chemistry due to photomodification. In summary, we present a novel mechanism of surface topology-dependent control of transcriptional programs in mature endothelium and endothelial progenitor cells.