Daniel Wesner

Poly(ester-ether)s: II. Properties of electrospun nanofibres from polydioxanone and poly(methyl dioxanone) blends and human fibroblast cellular proliferation†

This article deals with an in-depth study of the thermal, mechanical and degradation behaviours of nanofibres from polydioxanone (PDX) and polydl-3-methyl-1,4-dioxan-2-one (PMeDX) and a comparison with their blend films. Varying ratios of both polymers were blended and electrospun from solution. Electrospun fibres exhibited a melting transition at 109 °C independently of the PMeDX content, which corresponds to the melting of PDX nanofibres. As a result of the drawing process, PMeDX had a reduced plasticizing effect on PDX. In general, it was observed that overall crystallinity of the fibres decreased from 53% to 36% with increasing PMeDX content and this impacted on their mechanical properties. The Youngs moduli decreased as the PMeDX content of the fibres increased. However, an increase in strain at break and peak stress was noted as a result of a decrease in the fibre diameter. AFM images of the electrospun fibres showed an increasing degree of morphological heterogeneity with increasing PMeDX content. Thermal degradation studies showed that electrospun mats were thermally more stable than blend films, as confirmed by a two-fold increase in activation energy. The hydrolytic degradation of the electrospun mats conducted in phosphate buffer solution at 37 °C showed that the degradation followed a surface erosion mechanism as opposed to bulk degradation observed for blend films. Degradation of fibres was found to be mainly dependent on their diameter. On the other hand, the degradation of blend films depended on the overall crystallinity of the blends. Electrospun PDX/PMeDX nanofibrous scaffolds were also subjected to cell viability studies with human dermal fibroblasts, in which they did not show illicit response and demonstrated excellent cell attachment and proliferation.

Poly(ester-ether)s: III. assessment of cell behaviour on nanofibrous scaffolds of PCL, PLLA and PDX blended with amorphous PMeDX

The aim of this paper is to investigate the physico-chemical properties, degradation behaviour and cellular response of electrospun fibre-scaffolds of semi-crystalline PCL, PLLA and PDX blended with amorphous poly(methyl dioxanone) (PMeDX). Electrospun PCL/PMeDX and PLLA/PMeDX blend mats in varying weight ratios of the two components were fabricated and their overall performance was compared with similar composition PDX/PMeDX scaffolds. DSC analysis showed almost no change in crystallization temperature of PCL with increasing PMeDX content and TGA showed a different degradation profile as PMeDX content increased. The appearance of two crystallization peaks for PLLA/PMeDX blends suggested stereocomplex formation. As noted from AFM images, addition of PMeDX caused a change in the width of the lamellae from 14.8 ± 2.9 nm in 100/0 mat to 32.0 ± 11.5 nm in 85/15 mat. Moreover, PCL/PMeDX blend mats show a significant drop in Youngs modulus for 93/7, 90/10 and 85/15 compositions compared to 100/0 and 98/2. On the other hand, no clear trend in mechanical properties was observed for espun PLLA/PMeDX mats with increasing PMeDX content. Based on these analyses, it was concluded that PCL and PMeDX were immiscible while miscible blends were obtained with PLLA and PMeDX. Initial degradation of electrospun mats over a period of 5 weeks appears to occur via a surface erosion mechanism. In vitro cell culture studies using HDFs showed that the scaffolds were bioactive and a greater density of viable cells was noted on electrospun PCL/PMeDX and PLLA/PMeDX scaffolds compared to PCL and PLLA mats respectively. HDFs infiltrated through the entire thickness of espun 85/15 PLLA/PMeDX scaffold due to a combination of factors including morphology, porosity, surface characteristics and mechanical properties.

Controlled Surface Chemistry of Diamond/β-SiC Composite Films for Preferential Protein Adsorption

Diamond and SiC both process extraordinary biocompatible, electronic, and chemical properties. A combination of diamond and SiC may lead to highly stable materials, e.g., for implants or biosensors with excellent sensing properties. Here we report on the controllable surface chemistry of diamond/β-SiC composite films and its effect on protein adsorption. For systematic and high-throughput investigations, novel diamond/β-SiC composite films with gradient composition have been synthesized using the hot filament chemical vapor deposition (HFCVD) technique. As revealed by scanning electron microscopy (SEM), the diamond/β-SiC ratio of the composite films shows a continuous change from pure diamond to β-SiC over a length of ∼ 10 mm on the surface. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to unveil the surface termination of chemically oxidized and hydrogen treated surfaces. The surface chemistry of the composite films was found to depend on diamond/β-SiC ratio and the surface treatment. As observed by confocal fluorescence microscopy, albumin and fibrinogen were preferentially adsorbed from buffer: after surface oxidation, the proteins preferred to adsorb on diamond rather than on β-SiC, resulting in an increasing amount of proteins adsorbed to the gradient surfaces with increasing diamond/β-SiC ratio. By contrast, for hydrogen-treated surfaces, the proteins preferentially adsorbed on β-SiC, leading to a decreasing amount of albumin adsorbed on the gradient surfaces with increasing diamond/β-SiC ratio. The mechanism of preferential protein adsorption is discussed by considering the hydrogen bonding of the water self-association network to OH-terminated surfaces and the change of the polar surface energy component, which was determined according to the van Oss method. These results suggest that the diamond/β-SiC gradient film can be a promising material for biomedical applications which require good biocompatibility and selective adsorption of proteins and cells to direct cell migration.

The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells.

In this study, the fabrication, detailed characterization, and application of long-term stable micropatterned bio-interfaces of passivating poly(acrylamide) (PAAm) brushes on transparent gold for application in the study of cell-surface interactions is reported. The micropatterns were fabricated by microcontact printing of an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), SI-ATRP of acrylamide, and subsequently backfilling of the unfunctionalized areas of 400-2500 μm(2) size and systematically altered number of corners with octadecanethiol. As verified by surface plasmon resonance spectroscopy, the physisorption of fibronectin (FN) was restricted to the adhesive areas. Exploiting this platform, the effect of micropattern geometry and size of cell-adhesive FN areas surrounded by passivating PAAm brushes on transparent gold substrates on the attachment of cells and cytoskeleton alignment was investigated at the single-cell level. Exceptional long-term stability of the patterned PAAm brushes and arrays of adhesive areas, in which human pancreatic tumor cells (Patu 8988T) and fibroblast cells (NIH 3T3) were confined for more than one week, was observed. Adhesive areas of 1600 μm(2) or less constrained the cell shape and caused focal adhesions to accumulate in the corners of the pattern. These changes were most obvious for the PatuT cells in adhesive areas of ∼900 μm(2), in which the actin filaments were aligned, following the boundary of the pattern, and merged in the focal adhesions concentrated in the corners of the pattern. NIH 3T3 cells possessed a larger cell area, which led to an optimal cytoskeleton alignment in adhesive patterns of ∼1600 μm(2). The alignment of the cytoskeleton was found to be less pronounced in cells on larger adhesive areas, where the PatuT cells spread similarly to cells on unpatterned substrates. By contrast, the NIH 3T3 cells were found to stretch even on larger adhesive areas, spanning from one corner to the other. The long-term stability under cell culture conditions of the patterns introduced here will also be useful for long-term studies of single and multiple cells, cell motility in toxicity assays, and stem cell differentiation.

Surface nanobubbles studied by atomic force microscopy techniques: Facts, fiction, and open questions

In this review surface nanobubbles, which are presumably gas-filled enclosures found at the solid–liquid interface, are introduced and discussed together with key experimental findings that suggest that these nanoscale features indeed exist and are filled with gas. The most prominent technique used thus far has been atomic force microscopy (AFM). However, due to its potentially invasive nature, AFM data must be interpreted with great care. Owing to their curved interface, the Laplace internal pressure of surface nanobubbles exceeds substantially the outside ambient pressure, and the experimentally observed long term stability is in conflict with estimates of gas transport rates and predicted surface nanobubble lifetimes. Despite recent explanations of both the stability and the unusual nanoscopic contact angles, the development of new co-localization approaches and the adequate analysis of AFM data of surface nanobubbles are important as a means to confirm the gaseous nature and correctly estimate the interfacial curvature.

Molecular Beacon Modified Sensor Chips for Oligonucleotide Detection with Optical Readout

Three different surface bound molecular beacons (MBs) were investigated using surface plasmon fluorescence spectroscopy (SPFS) as an optical readout technique. While MB1 and MB2, both consisting of 36 bases, differed only in the length of the linker for surface attachment, the significantly longer MB3, consisting of 56 bases, comprised an entirely different sequence. For sensor chip preparation, the MBs were chemisorbed on gold via thiol anchors together with different thiol spacers. The influence of important parameters, such as the length of the MBs, the length of the linker between the MBs and the gold surface, the length and nature of the thiol spacers, and the ratio between the MBs and the thiol spacers was studied. After hybridization with the target, the fluorophore of the longer MB3 was oriented close to the surface, and the shorter MBs were standing more or less upright, leading to a larger increase in fluorescence intensity. Fluorescence microscopy revealed a homogeneous distribution of the MBs on the surface. The sensor chips could be used for simple and fast detection of target molecules with a limit of detection in the larger picomolar range. The response time was between 5 and 20 min. Furthermore, it was possible to distinguish between fully complementary and singly mismatched targets. While rinsing with buffer solution after hybridization with target did not result in any signal decrease, complete dehybridization could be carried out by intense rinsing with pure water. The MB modified sensor chips could be prepared in a repeatable manner and reused many times without significant decrease in performance.

Detailed Study of BSA Adsorption on Micro- and Nanocrystalline Diamond/β-SiC Composite Gradient Films by Time-Resolved Fluorescence Microscopy

The adsorption of bovine serum albumin (BSA) on micro- and nanocrystalline diamond/β-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSAFITC conjugate was found to preferentially adsorb on both O-/OH-terminated microcrystalline and nanocrystalline diamond compared to the OH-terminated β-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with an increasing diamond/β-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSAFITC on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66, and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to the fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye-dye and dye-surface distances. The longest decay time was found to correlate linearly with the diamond grain size. The fluorescence of BSAFITC undergoes external dynamic fluorescence quenching on the diamond surface by H- and/or sp2-defects and/or by amorphous carbon or graphite phases. An acceleration of the internal fluorescence concentration quenching in BSAFITC because of structural changes of albumin due to adsorption, is concluded to be a secondary contributor. These results suggest that the micro- and nanocrystalline diamond/β-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)interfaces.

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche.

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyers patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyers patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyers patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research.

Fluorimetric detection of G-quadruplex DNA in solution and adsorbed on surfaces with a selective trinuclear cyanine dye

Quadruplex DNA, which is a relevant target for anticancer therapies, may alter its conformation because of interactions with interfaces. In pursuit of a versatile methodology to probe adsorption-induced conformational changes, the interaction between a fluorescent [2.2.2]heptamethinecyanine dye and quadruplex DNA (G4-DNA) was studied in solution and on surfaces. In solution, the cyanine dye exhibits a strong light-up effect upon the association with G4-DNA without interference from double-stranded DNA. In addition, a terminal π-stacking as a binding mode between the cyanine dye and G4-DNA is concluded using NMR spectroscopy. To unravel the effects of adsorption on the conformation of quadruplex-DNA, G4-DNA, and double-stranded and single-stranded DNA were adsorbed to positively charged poly(allylamine) hydrochloride (PAH) surfaces, both in planar and in constrained 55 nm diameter aluminum oxide nanopore formats. All DNA forms showed a very strong affinity to the PAH surfaces as shown by surface plasmon resonance and reflectometric interference spectroscopy. The significant increase of the fluorescence emission intensity of the cyanine light-up probe observed exclusively for surface immobilized G4-DNA affords evidence for the adsorption of G4-DNA on PAH with retained quadruplex conformation.