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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.
- ItemDendritic glycopolymers based on dendritic polyamine scaffolds: view on their synthetic approaches, characteristics and potential for biomedical applications(London : Soc., 2014) Appelhans, Dietmar; Klajnert-Maculewicz, Barbara; Janaszewska, Anna; Lazniewska, Joanna; Voit, BrigitteIn this review we highlight the potential for biomedical applications of dendritic glycopolymers based on polyamine scaffolds. The complex interplay of the molecular characteristics of the dendritic architectures and their specific interactions with various (bio)molecules are elucidated with various examples. A special role of the individual sugar units attached to the dendritic scaffolds and their density is identified, which govern ionic and H-bond interactions, and biological targeting, but to a large extent are also responsible for the significantly reduced toxicity of the dendritic glycopolymers compared to their polyamine scaffolds. Thus, the application of dendritic glycopolymers in drug delivery systems for gene transfection but also as therapeutics in neurodegenerative diseases has great promise.
- ItemUltracompact three-dimensional tubular conductivity microsensors for ionic and biosensing applications(Washington, DC : American Chemical Society, 2014) Martinez-Cisneros, C.S.; Sanchez, S.; Xi, W.; Schmidt, O.G.We present ultracompact three-dimensional tubular structures integrating Au-based electrodes as impedimetric microsensors for the in-flow determination of mono- and divalent ionic species and HeLa cells. The microsensors show an improved performance of 2 orders of magnitude (limit of detection = 0.1 nM for KCl) compared to conventional planar conductivity detection systems integrated in microfluidic platforms and the capability to detect single HeLa cells in flowing phosphate buffered saline. These highly integrated conductivity tubular sensors thus open new possibilities for lab-in-a-tube devices for bioapplications such as biosensing and bioelectronics.
- ItemElectron beam-induced immobilization of laccase on porous supports for waste water treatment applications(Basel : MDPI AG, 2014) Jahangiri, E.; Reichelt, S.; Thomas, I.; Hausmann, K.; Schlosser, D.; Schulze, A.The versatile oxidase enzyme laccase was immobilized on porous supports such as polymer membranes and cryogels with a view of using such biocatalysts in bioreactors aiming at the degradation of environmental pollutants in wastewater. Besides a large surface area for supporting the biocatalyst, the aforementioned porous systems also offer the possibility for simultaneous filtration applications in wastewater treatment. Herein a "green" water-based, initiator-free, and straightforward route to highly reactive membrane and cryogel-based bioreactors is presented, where laccase was immobilized onto the porous polymer supports using a water-based electron beam-initiated grafting reaction. In a second approach, the laccase redox mediators 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and syringaldehyde were cross-linked instead of the enzyme via electron irradiation in a frozen aqueous poly(acrylate) mixture in a one pot set-up, yielding a mechanical stable macroporous cryogel with interconnected pores ranging from 10 to 50 μm in size. The membranes as well as the cryogels were characterized regarding their morphology, chemical composition, and catalytic activity. The reactivity towards waste-water pollutants was demonstrated by the degradation of the model compound bisphenol A (BPA). Both membrane- and cryogel-immobilized laccase remained highly active after electron beam irradiation. Apparent specific BPA removal rates were higher for cryogel-than for membrane-immobilized and free laccase, whereas membrane-immobilized laccase was more stable with respect to maintenance of enzymatic activity and prevention of enzyme leakage from the carrier than cryogel-immobilized laccase. Cryogel-immobilized redox mediators remained functional in accelerating the laccase-catalyzed BPA degradation, and especially ABTS was found to act more efficiently in immobilized than in freely dissolved state.
- ItemThe influence of the Δk280 mutation and N- or C-terminal extensions on the structure, dynamics, and fibril morphology of the tau R2 repeat(London [u.a.] : Royal Society of Chemistry, 2014) Raz, Y.; Adler, J.; Vogel, A.; Scheidt, H.A.; Häupl, T.; Abel, B.; Huster, D.; Miller, Y.Tau is a microtubule-associated protein and is involved in microtubule assembly and stabilization. It consists of four repeats that bind to the microtubule. The ΔK280 deletion mutation in the tau R2 repeat region is directly associated with the development of the frontotemporal dementia parkinsonism linked to chromosome 17 (FTDP-17). This deletion mutation is known to accelerate tau R2 repeat aggregation. However, the secondary and the tertiary structures of the self-assembled ΔK280 tau R2 repeat mutant aggregates are still controversial. Moreover, it is unclear whether extensions by one residue in the N- or the C-terminus of this mutant can influence the secondary or the tertiary structure. Herein, we combine solid-state NMR, atomic force microscopy, electron microscopy and all-atom explicit molecular dynamics simulations to investigate the effects of the deletion mutation and the N- and the C-terminal extension of this mutant on the structure. Our main findings show that the deletion mutation induces the formation of small aggregates, such as oligomers, and reduces the formation of fibrils. However, the extensions in the N- or the C-terminus revealed more fibril formation than small aggregates. Further, in the deletion mutation only one structure is preferred, while the N- and the C-terminal extensions strongly lead to polymorphic states. Finally, our broad and combined experimental and computational techniques provide direct structural information regarding ΔK280 tau R2 repeat mutant aggregates and their extensions in the N- and C-terminii by one residue.
- ItemBio-responsive polymer hydrogels homeostatically regulate blood coagulation(London : Nature Publishing Group, 2013) Maitz, Manfred F.; Freudenberg, U.; Tsurkan, M.V.; Fischer, M.; Beyrich, T.; Werner, C.Bio-responsive polymer architectures can empower medical therapies by engaging molecular feedback-response mechanisms resembling the homeostatic adaptation of living tissues to varying environmental constraints. Here we show that a blood coagulation-responsive hydrogel system can deliver heparin in amounts triggered by the environmental levels of thrombin, the key enzyme of the coagulation cascade, which - in turn - becomes inactivated due to released heparin. The bio-responsive hydrogel quantitatively quenches blood coagulation over several hours in the presence of pro-coagulant stimuli and during repeated incubation with fresh, non-anticoagulated blood. These features enable the introduced material to provide sustainable, autoregulated anticoagulation, addressing a key challenge of many medical therapies. Beyond that, the explored concept may facilitate the development of materials that allow the effective and controlled application of drugs and biomolecules.
- ItemNon-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways(San Francisco, Calif. : Lightbinders, 2015) Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, KristianNon-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.
- ItemEvaluation of osseointegration of titanium alloyed implants modified by plasma polymerization(Basel : MDPI AG, 2014) Gabler, C.; Zietz, C.; Göhler, R.; Fritsche, A.; Lindner, T.; Haenle, M.; Finke, B.; Meichsner, J.; Lenz, S.; Frerich, B.; Lüthen, F.; Nebe, J.B.; Bader, R.By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V) coated with plasma-polymerized allylamine (PPAAm) and plasma-polymerized ethylenediamine (PPEDA) versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC) was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%). Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5%) and implants with PPEDA a significantly increased BIC (63.7%). In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.
- ItemInteraction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells(Auckland : DOVE Medical Press, 2014) Woltmann, B.; Torger, B.; Müller, M.; Hempel, U.Background: Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis). For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles' composition and surface net charge. Materials and methods: Polyelectrolyte complex nanoparticles (PECNPs) composed of the polycations poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) in combination with the polyanions dextran sulfate (DS) or cellulose sulfate (CS) were prepared. PECNPs' physicochemical properties (size, net charge) were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs) cultured on immobilized PECNP films (5-50 nmol·cm-2) by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay, as well as cell morphology (phase contrast microscopy). Results: PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC+NP) or negative (PEC-NP) net charge were obtained. The PECNP composition significantly affected cell morphology and metabolic activity, whereas the net charge had a negligible influence. Therefore, we classified PECNPs into "variant systems" featuring a significant dose dependency of metabolic activity (DEAE/CS, PEI/DS) and "invariant systems" lacking such a dependency (DEAE/DS, PEI/CS). Immunofluorescence imaging of fluorescein isothiocyanate isomer I (FITC)-labeled PECNPs suggested internalization into hMSCs remaining stable for 8 days. Conclusion: Our study demonstrated that PECNP composition affects hMSC behavior. In particular, the PEI/CS system showed biocompatibility in a wide concentration range, representing a suitable system for local drug delivery from PECNP-functionalized bone substitute materials.
- ItemDipA, a pore-forming protein in the outer membrane of lyme disease spirochetes exhibits specificity for the permeation of dicarboxylate(San Francisco, CA : Public Library of Science, 2012) Thein, Marcus; Bonde, Mari; Bunikis, Ignas; Denker, Katrin; Sickmann, Albert; Bergström, Sven; Benz, RolandLyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a singlechannel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.