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- ItemDefect-free Naphthalene Diimide Bithiophene Copolymers with Controlled Molar Mass and High Performance via Direct Arylation Polycondensation(Washington, DC : ACS Publications, 2015) Matsidik, Rukiya; Komber, Hartmut; Luzio, Alessandro; Caironi, Mario; Sommer, MichaelA highly efficient, simple, and environmentally friendly protocol for the synthesis of an alternating naphthalene diimide bithiophene copolymer (PNDIT2) via direct arylation polycondensation (DAP) is presented. High molecular weight (MW) PNDIT2 can be obtained in quantitative yield using aromatic solvents. Most critical is the suppression of two major termination reactions of NDIBr end groups: nucleophilic substitution and solvent end-capping by aromatic solvents via C–H activation. In situ solvent end-capping can be used to control MW by varying monomer concentration, whereby end-capping is efficient and MW is low for low concentration and vice versa. Reducing C–H reactivity of the solvent at optimized conditions further increases MW. Chain perfection of PNDIT2 is demonstrated in detail by NMR spectroscopy, which reveals PNDIT2 chains to be fully linear and alternating. This is further confirmed by investigating the optical and thermal properties as a function of MW, which saturate at Mn ≈ 20 kDa, in agreement with controls made by Stille coupling. Field-effect transistor (FET) electron mobilities μsat up to 3 cm2/(V·s) are measured using off-center spin-coating, with FET devices made from DAP PNDIT2 exhibiting better reproducibility compared to Stille controls.
- 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.
- ItemAssessing agreement between preclinical magnetic resonance imaging and histology: An evaluation of their image qualities and quantitative results(San Francisco, California, US : PLOS, 2017) Elschner, Cindy; Korn, Paula; Hauptstock, Maria; Schulz, Matthias C.; Range, Ursula; Jünger, Diana; Scheler, UlrichOne consequence of demographic change is the increasing demand for biocompatible materials for use in implants and prostheses. This is accompanied by a growing number of experimental animals because the interactions between new biomaterials and its host tissue have to be investigated. To evaluate novel materials and engineered tissues the use of nondestructive imaging modalities have been identified as a strategic priority. This provides the opportunity for studying interactions repeatedly with individual animals, along with the advantages of reduced biological variability and decreased number of laboratory animals. However, histological techniques are still the golden standard in preclinical biomaterial research. The present article demonstrates a detailed method comparison between histology and magnetic resonance imaging. This includes the presentation of their image qualities as well as the detailed statistical analysis for assessing agreement between quantitative measures. Exemplarily, the bony ingrowth of tissue engineered bone substitutes for treatment of a cleft-like maxillary bone defect has been evaluated. By using a graphical concordance analysis the mean difference between MRI results and histomorphometrical measures has been examined. The analysis revealed a slightly but significant bias in the case of the bone volume ðbiasHisto MRI: Bonevolume = 2: 40 %, p < 0: 005) and a clearly significant deviation for the remaining defect width ðbiasHisto MRI: Defectwidth = 6: 73 %, p 0: 005Þ: But the study although showed a considerable effect of the analyzed section position to the quantitative result. It could be proven, that the bias of the data sets was less originated due to the imaging modalities, but mainly on the evaluation of different slice positions. The article demonstrated that method comparisons not always need the use of an independent animal study, additionally.
- 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.
- ItemAnti-Prion Drug mPPIg5 Inhibits PrPC Conversion to PrPSc(San Francisco, CA : Public Library of Science, 2013) McCarthy, J.M.; Franke, M.; Resenberger, U.K.; Waldron, S.; Simpson, J.C.; Tatzelt, J.; Appelhans, D.; Rogers, M.S.Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrPSc, an abnormal isoform of the cellular protein PrPC, is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrPSc in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrPC to PrPSc conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future.
- ItemIn vitro model of metastasis to bone marrow mediates prostate cancer castration resistant growth through paracrine and extracellular matrix factors(San Francisco, CA : Public Library of Science, 2012) Lescarbeau, R.M.; Seib, F.P.; Prewitz, M.; Werner, C.; Kaplan, D.L.The spread of prostate cancer cells to the bone marrow microenvironment and castration resistant growth are key steps in disease progression and significant sources of morbidity. However, the biological significance of mesenchymal stem cells (MSCs) and bone marrow derived extracellular matrix (BM-ECM) in this process is not fully understood. We therefore established an in vitro engineered bone marrow tissue model that incorporates hMSCs and BM-ECM to facilitate mechanistic studies of prostate cancer cell survival in androgen-depleted media in response to paracrine factors and BM-ECM. hMSC-derived paracrine factors increased LNCaP cell survival, which was in part attributed to IGFR and IL6 signaling. In addition, BM-ECM increased LNCaP and MDA-PCa-2b cell survival in androgen-depleted conditions, and induced chemoresistance and morphological changes in LNCaPs. To determine the effect of BM-ECM on cell signaling, the phosphorylation status of 46 kinases was examined. Increases in the phosphorylation of MAPK pathway-related proteins as well as sustained Akt phosphorylation were observed in BM-ECM cultures when compared to cultures grown on plasma-treated polystyrene. Blocking MEK1/2 or the PI3K pathway led to a significant reduction in LNCaP survival when cultured on BM-ECM in androgen-depleted conditions. The clinical relevance of these observations was determined by analyzing Erk phosphorylation in human bone metastatic prostate cancer versus non-metastatic prostate cancer, and increased phosphorylation was seen in the metastatic samples. Here we describe an engineered bone marrow model that mimics many features observed in patients and provides a platform for mechanistic in vitro studies.
- ItemGeometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization(San Francisco, CA : Public Library of Science, 2013) Joly, P.; Duda, G.N.; Schöne, M.; Welzel, P.B.; Freudenberg, U.; Werner, C.; Petersen, A.To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.
- ItemGrowth induction and low-oxygen apoptosis inhibition of human CD34 + progenitors in collagen gels(New York, NY : Hindawi, 2013) Avitabile, D.; Salchert, K.; Werner, C.; Capogrossi, M.C.; Pesce, M.Various reports have indicated low survival of injected progenitors into unfavorable environments such as the ischemic myocardium or lower limb tissues. This represents a major bottleneck in stem-cell-based cardiovascular regenerative medicine. Strategies to enhance survival of these cells in recipient tissues have been therefore sought to improve stem cell survival and ensure long-term engraftment. In the present contribution, we show that embedding human cord blood-derived CD34+ cells into a collagen I-based hydrogel containing cytokines is a suitable strategy to promote stem cell proliferation and protect these cells from anoxia-induced apoptosis.
- ItemDiscovery of chitin in skeletons of non-verongiid Red Sea demosponges(San Francisco, California, US : PLOS, 2018) Ehrlich, Hermann; Shaala, Lamiaa A.; Youssef, Diaa T. A.; Żółtowska- Aksamitowska, Sonia; Tsurkan, Mikhail; Galli, Roberta; Meissner, Heike; Wysokowski, Marcin; Petrenko, Iaroslav; Tabachnick, Konstantin R.; Ivanenko, Viatcheslav N.; Bechmann, Nicole; Joseph, Yvonne; Jesionowski, TeofilMarine demosponges (Porifera: Demospongiae) are recognized as first metazoans which have developed over millions of years of evolution effective survival strategies based on unique metabolic pathways to produce both biologically active secondary metabolites and biopolymer-based stiff skeletons with 3D architecture. Up to date, among marine demosponges, only representatives of the Verongiida order have been known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within skeletons of non-verongiid demosponges Acarnus wolffgangi and Echinoclathria gibbosa collected in the Red Sea. Calcofluor white staining, FTIR and Raman analysis, ESI-MS, SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm, with strong evidence, the finding of α-chitin in the skeleton of both species. We suggest that, the finding of chitin within these representatives of Poecilosclerida order is a promising step in the evaluation of these sponges as novel renewable sources for both biologically active metabolites and chitin, which are of prospective application for pharmacology and biomedicine.
- ItemA novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization(San Francisco, Calif. : Public Library of Science, 2010) Sebinger, D.D.R.; Unbekandt, M.; Ganeva, V.V.; Ofenbauer, A.; Werner, C.; Davies, J.A.Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle. © 2010 Sebinger et al.