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Human bestrophin-1 (hBest1) is a transmembrane Ca2+- dependent anion channel, associated with the transport of Cl−, HCO3- ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report:
1) Ca2+, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as “fluidizing”, hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state;
2) in stably transfected MDCK-hBest1 cells, bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (Lo) and liquid-disordered (Ld) fractions, quantifying protein association up to 35 % and 65 % with Lo and Ld. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.
Melamine-formaldehyde resins are widely used for decorative paper impregnation. Resin properties relevant for impregnation are mainly determined already at the stage of resin synthesis by the applied reaction conditions. Thus, understanding the relationship between reaction conditions and technological properties is important. Response surface methodology based on orthogonal parameter level variations is the most suitable tool to identify and quantify factor effects and deduce causal correlation patterns. Here, two major process factors of MF resin synthesis were systematically varied using such a statistical experimental design. To arrive at resins having a broad range of technological properties, initial pH and M:F ratio were varied in a wide range (pH: 7.9–12.1; M:F ratio: 1:1.5–1:4.5). The impregnation behavior of the resins was modeled using viscosity, penetration rate and residual curing capacity as technological responses. Based on the response surface models, nonlinear and synergistic action of process factors was quantified and a suitable process window for preparing resins with favorable impregnation performance was defined. It was found that low M:F ratios (~1:2–1:2.5) and comparatively high starting pHs (~pH 11) yield impregnation resins with rapid impregnation behavior and good residual curing capacity.
Hypothesis
The origin of negative surface charge at water/air interface is still not clear. The most probable origin is specific adsorption of OH− ions. From diffuse layer potential, we can evaluate the surface density of ions in the Stern layer which can be a measure for the specific adsorption of ions and determines whether the surface charge is solely due to the specific adsorption of OH− ions.
Experiments
Equilibrium thickness of foam films of pure water and aqueous solutions of NaCl, HCl, and NaOH was measured as a function of disjoining pressure for water and as a function of concentration for the aqueous solutions at 298.15 K. Quartz-glass cells thoroughly cleaned and immersed in pure water before use were used for the measurement.
Findings
Application of a modified Poisson-Boltzmann equation to the equilibrium film thickness gave the diffuse layer potential and the surface density of ions in the Stern layer. From the concentration dependence of the surface density, it was concluded that not only OH− ions but also Cl− ions and HCO3− and/or CO32− ions adsorb specifically at the water/air interface.
Introduction: Bioresorbable collagenous barrier membranes are used to prevent premature soft tissue ingrowth and to allow bone regeneration. For volume stable indications, only non-absorbable synthetic materials are available. This study investigates a new bioresorbable hydrofluoric acid (HF)-treated magnesium (Mg) mesh in a native collagen membrane for volume stable situations. Materials and Methods: HF-treated and untreated Mg were compared in direct and indirect cytocompatibility assays. In vivo, 18 New Zealand White Rabbits received each four 8 mm calvarial defects and were divided into four groups: (a) HF-treated Mg mesh/collagen membrane, (b) untreated Mg mesh/collagen membrane (c) collagen membrane and (d) sham operation. After 6, 12 and 18 weeks, Mg degradation and bone regeneration was measured using radiological and histological methods. Results: In vitro, HF-treated Mg showed higher cytocompatibility. Histopathologically, HF-Mg prevented gas cavities and was degraded by mononuclear cells via phagocytosis up to 12 weeks. Untreated Mg showed partially significant more gas cavities and a fibrous tissue reaction. Bone regeneration was not significantly different between all groups. Discussion and Conclusions: HF-Mg meshes embedded in native collagen membranes represent a volume stable and biocompatible alternative to the non-absorbable synthetic materials. HF-Mg shows less corrosion and is degraded by phagocytosis. However, the application of membranes did not result in higher bone regeneration.
Controlling the surface properties and structure of thin nanosized coatings is of primary importance in diverse engineering and medical applications. Here we report on how the nanostructure, growth mechanism, thickness, roughness, and hydrophilicity of nanocomposites composed of weak natural or strong synthetic polyelectrolytes (PE) can be tailored by graphene oxide (GO) doping. GO reverses the build‐up mechanism affecting the internal structure and the hydrophilicity in a way depending on the type of the PE‐matrix. The extent of GO‐adsorption and its impact on the surface morphology was found to be independent on the type of the underlying PE‐matrix. The nanostructure of the hybrid films is not significantly altered when a single surface‐exposed GO‐layer is deposited, while increasing the number of embedded GO‐layers leads to pronounced surface heterogeneity. These results are expected to have valuable impact on the construction strategies of coatings with tunable surface properties.
Thermoplastic polymers like ethylene-octene copolymer (EOC) may be grafted with silanes via reactive extrusion to enable subsequent crosslinking for advanced biomaterials manufacture. However, this reactive extrusion process is difficult to control and it is still challenging to reproducibly arrive at well-defined products. Moreover, high grafting degrees require a considerable excess of grafting reagent. A large proportion of the silane passes through the process without reacting and needs to be removed at great expense by subsequent purification. This results in unnecessarily high consumption of chemicals and a rather resource-inefficient process. It is thus desired to be able to define desired grafting degrees with optimum grafting efficiency by means of suitable process control. In this study, the continuous grafting of vinyltrimethoxysilane (VTMS) on ethylene-octene copolymer (EOC) via reactive extrusion was investigated. Successful grafting was verified and quantified by 1H-NMR spectroscopy. The effects of five process parameters and their synergistic interactions on grafting degree and grafting efficiency were determined using a face-centered experimental design (FCD). Response surface methodology (RSM) was applied to derive a causal process model and define process windows yielding arbitrary grafting degrees between <2 and >5% at a minimum waste of grafting agent. It was found that the reactive extrusion process was strongly influenced by several second-order interaction effects making this process difficult to control. Grafting efficiencies between 75 and 80% can be realized as long as grafting degrees <2% are admitted.
Here, we report the continuous peroxide-initiated grafting of vinyltrimethoxysilane (VTMS) onto a standard polyolefin by means of reactive extrusion to produce a functionalized liquid ethylene propylene copolymer (EPM). The effects of the process parameters governing the grafting reaction and their synergistic interactions are identified, quantified and used in a mathematical model of the extrusion process. As process variables the VTMS and peroxide concentrations and the extruder temperature setting were systematically studied for their influence on the grafting and the relative grafting degree using a face-centered central composite design (FCD). The grafting degree was quantified by 1H NMR spectroscopy. Response surface methodology (RSM) was used to calculate the most efficient grafting process in terms of chemical usage and graft yield. With the defined processing window, it was possible to make precise predictions about the grafting degree with at the same time highest possible relative degree of grafting.
Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.
In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 μm (as indicated by the mean d90 and d10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the mean coating thickness was found to be 33.2 ± 7.3 μm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where the biotin–streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.
Here, we study resin cure and network formation of solid melamine formaldehyde pre-polymer over a large temperature range viadynamic temperature curing profiles. Real-time infrared spectroscopy is used to analyze the chemical changes during network formation and network hardening. By applying chemometrics (multivariate curve resolution,MCR), the essential chemical functionalities that constitute the network at a given stage of curing are mathematically extracted and tracked over time. The three spectral components identified by MCR were methylol-rich, ether linkages-rich and methylene linkages-rich resin entities. Based on dynamic changes of their characteristic spectral patterns in dependence of temperature, curing is divided into five phases: (I) stationary phase with free methylols as main chemical feature, (II) formation of flexible network cross-linked by ether linkages, (III) formation of rigid, ether-cross-linked network, (IV) further hardening via transformation of methylols and ethers into methylene-cross-linkages, and (V) network consolidation via transformation of ether into methylene bridges. The presented spectroscopic/chemometric approach can be used as methodological basis for the functionality design of MF-based surface films at the stage of laminate pressing, i.e., for tailoring the technological property profile of cured MF films using a causal understanding of the underlying chemistry based on molecular markers and spectroscopic fingerprints.