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- ItemDehydroabietylamine-Based Cellulose Nanofibril Films: A New Class of Sustainable Biomaterials for Highly Efficient, Broad-Spectrum Antimicrobial Effects(Washington, DC : ACS Publications, 2019) Hassan, Ghada; Forsman, Nina; Wan, Xing; Keurulainen, Leena; Bimbo, Luis M.; Johansson, Leena-Sisko; Sipari, Nina; Yli-Kauhaluoma, Jari; Zimmermann, Ralf; Stehl, Susanne; Werner, Carsten; Saris, Per E.J.; Österberg, Monika; Moreira, Vânia M.The design of antimicrobial surfaces as integral parts of advanced biomaterials is nowadays a high research priority, as the accumulation of microorganisms on surfaces inflicts substantial costs on the health and industry sectors. At present, there is a growing interest in designing functional materials from polymers abundant in nature, such as cellulose, that combine sustainability with outstanding mechanical properties and economic production. There is also the need to find suitable replacements for antimicrobial silver-based agents due to environmental toxicity and spread of resistance to metal antimicrobials. Herein we report the unprecedented decoration of cellulose nanofibril (CNF) films with dehydroabietylamine 1 (CNF-CMC-1), to give an innovative contact-active surface active against Gram-positive and Gram-negative bacteria including the methicillin-resistant S. aureus MRSA14TK301, with low potential to spread resistance and good biocompatibility, all achieved with low surface coverage. CNF-CMC-1 was particularly effective against S. aureus ATCC12528, causing virtually complete reduction of the total cells from 10 5 colony forming units (CFU)/mL bacterial suspensions, after 24 h of contact. This gentle chemical modification of the surface of CNF fully retained the beneficial properties of the original film, including moisture buffering and strength, relevant in many potential applications. Our originally designed surface represents a new class of ecofriendly biomaterials that optimizes the performance of CNF by adding antimicrobial properties without the need for environmentally toxic silver. © Copyright 2019 American Chemical Society.
- ItemA hyperbranched dopamine-containing PEG-based polymer for the inhibition of α-synuclein fibrillation(Orlando, Fla. : Academic Press, 2015) Breydo, Leonid; Newland, Ben; Zhang, Hong; Rosser, Anne; Werner, Carsten; Uversky, Vladimir N.; Wang, WenxinAggregation of α-synuclein is believed to play an important role in Parkinson's disease and in other neurodegenerative maladies. Small molecule inhibitors of this process are among the most promising drug candidates for neurodegenerative diseases. Dendrimers have also been studied for anti-fibrillation applications but they can be difficult and expensive to synthetize. Here we show that RAFT polymerization can be used to produce a hyperbranched polyethylene glycol structure via a one-pot reaction. This polymer included a dopamine moiety, a known inhibitor of α-synuclein fibril formation. Dopamine within the polymer structure was capable of aggregation inhibition, although not to the same degree as free dopamine. This result opens up new avenues for the use of controlled radical polymerizations as a means of preparing hyperbranched polymers for anti-fibrillation activity, but shows that the incorporation of functional groups from known small molecules within polymers may alter their biological activity.
- ItemA customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits(Amsterdam [u.a.] : Elsevier Science, 2019) Bellmann, Jessica; Goswami, Ruchi Y.; Girardo, Salvatore; Rein, Nelly; Hosseinzadeh, Zohreh; Hicks, Michael R.; Busskamp, Volker; Pyle, April D.; Werner, Carsten; Sterneckert, JaredNeuromuscular circuits (NMCs) are vital for voluntary movement, and effective models of NMCs are needed to understand the pathogenesis of, as well as to identify effective treatments for, multiple diseases, including Duchenne's muscular dystrophy and amyotrophic lateral sclerosis. Microfluidics are ideal for recapitulating the central and peripheral compartments of NMCs, but myotubes often detach before functional NMCs are formed. In addition, microfluidic systems are often limited to a single experimental unit, which significantly limits their application in disease modeling and drug discovery. Here, we developed a microfluidic platform (MFP) containing over 100 experimental units, making it suitable for medium-throughput applications. To overcome detachment, we incorporated a reactive polymer surface allowing customization of the environment to culture different cell types. Using this approach, we identified conditions that enable long-term co-culture of human motor neurons and myotubes differentiated from human induced pluripotent stem cells inside our MFP. Optogenetics demonstrated the formation of functional NMCs. Furthermore, we developed a novel application of the rabies tracing assay to efficiently identify NMCs in our MFP. Therefore, our MFP enables large-scale generation and quantification of functional NMCs for disease modeling and pharmacological drug targeting. © 2019 The Authors
- ItemMonoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)(San Francisco, California, US : PLOS, 2016) Thamm, Kristina; Graupner, Sylvi; Werner, Carsten; Huttner, Wieland B.; Corbeil, Denis; Nabi, Ivan RThe pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.
- ItemTreatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs(Basel : Molecular Diversity Preservation International, 2019) Bothe, Friederike; Deubel, Anne-Kathrin; Hesse, Eliane; Lotz, Benedict; Groll, Jürgen; Werner, Carsten; Richter, Wiltrud; Hagmann, SebastienDespite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (_CT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemGATA3 promotes the neural progenitor state but not neurogenesis in 3D traumatic injury model of primary human cortical astrocytes(Lausanne : Frontiers Media, 2019) Celikkaya, Hilal; Cosacak, Mehmet Ilyas; Papadimitriou, Christos; Popova, Stanislava; Bhattarai, Prabesh; Biswas, Srijeeta Nag; Siddiqui, Tohid; Wistorf, Sabrina; Nevado-Alcalde, Isabel; Naumann, Lisa; Mashkaryan, Violeta; Brandt, Kerstin; Freudenberg, Uwe; Werner, Carsten; Kizil, CaghanAstrocytes are abundant cell types in the vertebrate central nervous system and can act as neural stem cells in specialized niches where they constitutively generate new neurons. Outside the stem cell niches, however, these glial cells are not neurogenic. Although injuries in the mammalian central nervous system lead to profound proliferation of astrocytes, which cluster at the lesion site to form a gliotic scar, neurogenesis does not take place. Therefore, a plausible regenerative therapeutic option is to coax the endogenous reactive astrocytes to a pre-neurogenic progenitor state and use them as an endogenous reservoir for repair. However, little is known on the mechanisms that promote the neural progenitor state after injuries in humans. Gata3 was previously found to be a mechanism that zebrafish brain uses to injury-dependent induction of neural progenitors. However, the effects of GATA3 in human astrocytes after injury are not known. Therefore, in this report, we investigated how overexpression of GATA3 in primary human astrocytes would affect the neurogenic potential before and after injury in 2D and 3D cultures. We found that primary human astrocytes are unable to induce GATA3 after injury. Lentivirus-mediated overexpression of GATA3 significantly increased the number of GFAP/SOX2 double positive astrocytes and expression of pro-neural factor ASCL1, but failed to induce neurogenesis, suggesting that GATA3 is required for enhancing the neurogenic potential of primary human astrocytes and is not sufficient to induce neurogenesis alone. © 2019 Celikkaya, Cosacak, Papadimitriou, Popova, Bhattarai, Biswas, Siddiqui, Wistorf, Nevado-Alcalde, Naumann, Mashkaryan, Brandt, Freudenberg, Werner and Kizil.
- ItemSutureless fixation of amniotic membrane for therapy of ocular surface disorders(San Francisco, California, US : PLOS, 2015) Kotomin, Ilya; Valtink, Monika; Hofmann, Kai; Frenzel, Annika; Morawietz, Henning; Werner, Carsten; Funk, Richard H. W.; Engelmann, Katrin; Taylor, Andrew WAmniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders.
- ItemPhenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells([London] : Macmillan Publishers Limited, part of Springer Nature, 2015) Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, DenisXenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.
- ItemRecent Progress and Perspectives in the Electrokinetic Characterization of Polyelectrolyte Films(Basel : MDPI, 2015) Zimmermann, Ralf; Werner, Carsten; Duval, Jérôme F LThe analysis of the charge, structure and molecular interactions of/within polymeric substrates defines an important analytical challenge in materials science. Accordingly, advanced electrokinetic methods and theories have been developed to investigate the charging mechanisms and structure of soft material coatings. In particular, there has been significant progress in the quantitative interpretation of streaming current and surface conductivity data of polymeric films from the application of recent theories developed for the electrohydrodynamics of diffuse soft planar interfaces. Here, we review the theory and experimental strategies to analyze the interrelations of the charge and structure of polyelectrolyte layers supported by planar carriers under electrokinetic conditions. To illustrate the options arising from these developments, we discuss experimental and simulation data for plasma-immobilized poly(acrylic acid) films and for a polyelectrolyte bilayer consisting of poly(ethylene imine) and poly(acrylic acid). Finally, we briefly outline potential future developments in the field of the electrokinetics of polyelectrolyte layers.
- ItemThermo-responsive cell culture carriers based on poly(vinyl methyl ether) - The effect of biomolecular ligands to balance cell adhesion and stimulated detachment(Abingdon : Taylor & Francis, 2015) Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V.; Noll, Thomas; Funk, Richard H.W.; Engelmann, Katrin; Werner, CarstenTwo established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.