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- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
- ItemPolymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings(Columbus, Ohio : American Chemical Society, 2017) Buzzacchera, Irene; Vorobii, Mariia; Kostina, Nina Yu; de Los Santos Pereira, Andres; Riedel, Tomáš; Bruns, Michael; Ogieglo, Wojciech; Möller, Martin; Wilson, Christopher J.; Rodriguez-Emmenegger, CesarImplantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.
- ItemReconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics(Weinheim : Wiley-VCH, 2021) Jain, Puja; Nishiguchi, Akihiro; Linz, Georg; Wessling, Matthias; Ludwig, Andreas; Rossaint, Rolf; Möller, Martin; Singh, SmritiAlveolar-capillary basement membrane (BM) is ultra-thin (<2 µm) extracellular matrix that maintains integral epithelial-endothelial cell layers. In vitro reconstructions of alveolar-capillary barrier supported on synthetic scaffolds closely resembling the fibrous and ultra-thin natural BM are essential in mimicking the lung pathophysiology. Although BM topology and dimensions are well known to significantly influence cellular behavior, conventionally used BM mimics fail to recreate this natural niche. To overcome this, electrospun ultra-thin 2 µm poly(caprolactone) (PCL) nanofibrous mesh is used to establish an alveolar-capillary barrier model of lung endothelial/epithelial cells. Transepithelial electrical resistance (TEER) and permeability studies reveal integral tight junctions and improved mass transport through the highly porous PCL meshes compared to conventional dense membranes with etched pores. The chemotaxis of neutrophils is shown across the barrier in presence of inflammatory response that is naturally impeded in confined regions. Conventional requirement of 3 µm or larger pore size can lead to barrier disruption due to epithelial/endothelial cell invasion. Despite high porosity, the interconnected BM mimic prevents barrier disruption and allows neutrophil transmigration, thereby demonstrating the physiological relevance of the thin nanofibrous meshes. It is envisioned that these bipolar cultured barriers would contribute to an organ-level in vitro model for pathological disease, environmental pollutants, and nanotoxicology. © 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH
- ItemMulti-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures(London : BioMed Central, 2016) Viell, Jörn; Inouye, Hideyo; Szekely, Noemi K.; Frielinghaus, Henrich; Marks, Caroline; Wang, Yumei; Anders, Nico; Spiess, Antje C.; Makowski, LeeBackground: The valorization of biomass for chemicals and fuels requires efficient pretreatment. One effective strategy involves the pretreatment with ionic liquids which enables enzymatic saccharification of wood within a few hours under mild conditions. This pretreatment strategy is, however, limited by water and the ionic liquids are rather expensive. The scarce understanding of the involved effects, however, challenges the design of alternative pretreatment concepts. This work investigates the multi length-scale effects of pretreatment of wood in 1-ethyl-3-methylimidazolium acetate (EMIMAc) in mixtures with water using spectroscopy, X-ray and neutron scattering. Results: The structure of beech wood is disintegrated in EMIMAc/water mixtures with a water content up to 8.6 wt%. Above 10.7 wt%, the pretreated wood is not disintegrated, but still much better digested enzymatically compared to native wood. In both regimes, component analysis of the solid after pretreatment shows an extraction of few percent of lignin and hemicellulose. In concentrated EMIMAc, xylan is extracted more efficiently and lignin is defunctionalized. Corresponding to the disintegration at macroscopic scale, SANS and XRD show isotropy and a loss of crystallinity in the pretreated wood, but without distinct reflections of type II cellulose. Hence, the microfibril assembly is decrystallized into rather amorphous cellulose within the cell wall. Conclusions: The molecular and structural changes elucidate the processes of wood pretreatment in EMIMAc/water mixtures. In the aqueous regime with >10.7 wt% water in EMIMAc, xyloglucan and lignin moieties are extracted, which leads to coalescence of fibrillary cellulose structures. Dilute EMIMAc/water mixtures thus resemble established aqueous pretreatment concepts. In concentrated EMIMAc, the swelling due to decrystallinization of cellulose, dissolution of cross-linking xylan, and defunctionalization of lignin releases the mechanical stress to result in macroscopic disintegration of cells. The remaining cell wall constituents of lignin and hemicellulose, however, limit a recrystallization of the solvated cellulose. These pretreatment mechanisms are beyond common pretreatment concepts and pave the way for a formulation of mechanistic requirements of pretreatment with simpler pretreatment liquors. © 2016 Viell et al.
- ItemA scalable bubble-free membrane aerator for biosurfactant production(New York, NY : Wiley, 2021) Bongartz, Patrick; Bator, Isabel; Baitalow, Kristina; Keller, Robert; Tiso, Till; Blank, Lars Mathias; Wessling, MatthiasThe bioeconomy is a paramount pillar in the mitigation of greenhouse gas emissions and climate change. Still, the industrialization of bioprocesses is limited by economical and technical obstacles. The synthesis of biosurfactants as advanced substitutes for crude-oil-based surfactants is often restrained by excessive foaming. We present the synergistic combination of simulations and experiments towards a reactor design of a submerged membrane module for the efficient bubble-free aeration of bioreactors. A digital twin of the combined bioreactor and membrane aeration module was created and the membrane arrangement was optimized in computational fluid dynamics studies with respect to fluid mixing. The optimized design was prototyped and tested in whole-cell biocatalysis to produce rhamnolipid biosurfactants from sugars. Without any foam formation, the new design enables a considerable higher space-time yield compared to previous studies with membrane modules. The design approach of this study is of generic nature beyond rhamnolipid production.
- ItemProspects and challenges of translational corneal bioprinting(Basel : MDPI AG, 2020) Fuest, Matthias; Yam, Gary Hin-Fai; Mehta, Jodhbir S.; Campos, Daniela F.DuarteCorneal transplantation remains the ultimate treatment option for advanced stromal and endothelial disorders. Corneal tissue engineering has gained increasing interest in recent years, as it can bypass many complications of conventional corneal transplantation. The human cornea is an ideal organ for tissue engineering, as it is avascular and immune-privileged. Mimicking the complex mechanical properties, the surface curvature, and stromal cytoarchitecure of the in vivo corneal tissue remains a great challenge for tissue engineering approaches. For this reason, automated biofabrication strategies, such as bioprinting, may offer additional spatial control during the manufacturing process to generate full-thickness cell-laden 3D corneal constructs. In this review, we discuss recent advances in bioprinting and biomaterials used for in vitro and ex vivo corneal tissue engineering, corneal cell-biomaterial interactions after bioprinting, and future directions of corneal bioprinting aiming at engineering a full-thickness human cornea in the lab. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemBiadhesive Peptides for Assembling Stainless Steel and Compound Loaded Micro-Containers(Weinheim : Wiley-VCH, 2019) Apitius, Lina; Buschmann, Sven; Bergs, Christian; Schönauer, David; Jakob, Felix; Pich, Andrij; Schwaneberg, UlrichBiadhesive peptides (peptesives) are an attractive tool for assembling two chemically different materials—for example, stainless steel and polycaprolactone (PCL). Stainless steel is used in medical stents and PCL is used as a biodegradable polymer for fabrication of tissue growth scaffolds and drug delivering micro-containers. Biadhesive peptides are composed of two domains (e.g., dermaseptin S1 and LCI) with different material-binding properties that are separated through a stiff peptide-spacer. The peptesive dermaseptin S1-domain Z-LCI immobilizes antibiotic-loaded PCL micro-containers on stainless steel surfaces. Immobilization is visualized by microscopy and field emission scanning electron microscopy analysis and released antibiotic from the micro-containers is confirmed through growth inhibition of Escherichia coli cells.
- ItemUnraveling the Mechanism and Kinetics of Binding of an LCI-eGFP-Polymer for Antifouling Coatings(Weinheim : Wiley-VCH, 2021) Söder, Dominik; Garay-Sarmiento, Manuela; Rahimi, Khosrow; Obstals, Fabian; Dedisch, Sarah; Haraszti, Tamás; Davari, Mehdi D.; Jakob, Felix; Heß, Christoph; Schwaneberg, Ulrich; Rodriguez-Emmenegger, CesarThe ability of proteins to adsorb irreversibly onto surfaces opens new possibilities to functionalize biological interfaces. Herein, the mechanism and kinetics of adsorption of protein-polymer macromolecules with the ability to equip surfaces with antifouling properties are investigated. These macromolecules consist of the liquid chromatography peak I peptide from which antifouling polymer brushes are grafted using single electron transfer-living radical polymerization. Surface plasmon resonance spectroscopy reveals an adsorption mechanism that follows a Langmuir-type of binding with a strong binding affinity to gold. X-ray reflectivity supports this by proving that the binding occurs exclusively by the peptide. However, the lateral organization at the surface is directed by the cylindrical eGFP. The antifouling functionality of the unimolecular coatings is confirmed by contact with blood plasma. All coatings reduce the fouling from blood plasma by 8894% with only minor effect of the degree of polymerization for the studied range (DP between 101 and 932). The excellent antifouling properties, combined with the ease of polymerization and the straightforward coating procedure make this a very promising antifouling concept for a multiplicity of applications.
- ItemDecreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress(Washington, DC : Soc., 2019) Liu, Boqun; Hasrat, Zarief; Poolman, Bert; Boersma, Arnold J.; Mullineaux, Conrad W.Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.
- ItemPolymer-based controlled-release fed-batch microtiter plate - diminishing the gap between early process development and production conditions(Berlin ; Heidelberg : Springer, 2019) Keil, T.; Dittrich, B.; Lattermann, C.; Habicher, T.; Büchs, J.Background: Fed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates. Results: Immobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations. Conclusions: The polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible. © 2019 The Author(s).