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This study aims at the development of materials for biodegradable fiducial markers for X-ray based medical imaging and their anchorage in soft tissue. Towards this goal a degradable polymer matrix of poly(l-lactide-co-ε-caprolactone) (P[LAcoCL]) was combined with barium sulfate (BaSO4) and hydroxyapatite (HAp) as radio-opaque fillers. Low pressure plasma treatment was applied to the composite materials to improve cell adhesion and subsequent tissue integration. In particular, the effects of oxygen and ammonia plasmas were evaluated and compared using X-ray photoelectron spectroscopy, atomic force microscopy and dynamic water contact angle measurements as well as in vitro studies using the murine fibroblast cell line L929. To exclude the cytotoxic effects of degradation products of P[LAcoCL] and released BaSO4 or HAp cytotoxicity assays with the degradation products of the composite materials were conducted. The results obtained by this broad range of analytical techniques suggest the application of composites of P[LAcoCL] with BaSO4 and HAp as promising material systems for innovative fiducial markers for soft tissue in X-ray based medical imaging.
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Two 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.
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Functional impairment of the human corneal endothelium can lead to corneal blindness. In order to meet the high demand for transplants with an appropriate human corneal endothelial cell density as a prerequisite for corneal function, several tissue engineering techniques have been developed to generate transplantable endothelial cell sheets. These approaches range from the use of natural membranes, biological polymers and biosynthetic material compositions, to completely synthetic materials as matrices for corneal endothelial cell sheet generation. This review gives an overview about currently used materials for the generation of transplantable corneal endothelial cell sheets with a special focus on thermo-responsive polymer coatings.
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Endothelial cells constitute the natural inner lining of blood vessels and possess anti-thrombogenic properties. This characteristic is frequently used by seeding endothelial cells on vascular prostheses. As the type of anchorage of adhesion ligands to materials surfaces is known to determine the mechanical balance of adherent cells, we investigated herein the behaviour of endothelial cells under physiological shear stress conditions. The adhesion ligand fibronectin was anchored to polymer surfaces of four physicochemical characteristics exhibiting covalent and non-covalent attachment as well as high and low hydrophobicity. The in situ analysis combined with cell tracking of shear stress-induced effects on cultured isolated cells and monolayers under venous (0.5 dyn/cm(2)) and arterial (12 dyn/cm(2)) shear stress over a time period of 24 h revealed distinct differences in their morphological and migratory features. Most pronounced, unidirectional and bimodal migration patterns of endothelial cells in or against flow direction were found in dependence on the type of substrate-matrix anchorage. Combined by an immunofluorescent analysis of the actin cytoskeleton, cell-cell junctions, cell-matrix adhesions, and matrix reorganization these results revealed a distinct balance of laminar shear stress, cell-cell contacts and substrate-matrix anchorage in affecting endothelial cell fate under flow conditions. This analysis underlines the importance of materials surface parameters as well as primary and secondary adhesion ligand anchorage in the context of artificial blood vessels for future therapeutic devices.
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Adhesión Celular/fisiología , Movimiento Celular/fisiología , Uniones Célula-Matriz/fisiología , Células Endoteliales/fisiología , Prótesis Vascular , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Células Endoteliales/citología , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ensayo de Materiales , Polímeros/química , Polímeros/metabolismo , Resistencia al Corte , Estrés Mecánico , Propiedades de SuperficieRESUMEN
The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to Sco1p, the respiratory-deficient yeast sco1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from Sco1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Arabidopsis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas Transportadoras de Cobre , Prueba de Complementación Genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/química , Mitocondrias/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
To evaluate the blood compatibility of new materials, a clear distinction between properties of the materials and effects due to surface contamination by adsorbed endotoxins is essential. This study compares direct contact approaches and elution methods with water, organic solvents, nonionic, and zwitterionic detergents for determination of surface-adsorbed endotoxin by the limulus amoebocyte lysate (LAL) test and determines the blood compatibility of various surfaces with controlled endotoxin contamination in vitro. The LAL test in direct contact with an endotoxin-contaminated surface was concluded to be not practicable for most devices and its sensitivity showed a high dependence on surface characteristics. Among the elution methods, 0.2% Tween-20 showed most stable elution characteristics and appears therefore preferable. Biological reactions at in vitro blood exposure were found to be only minimally influenced by adsorbed endotoxin during the time window of 2 h, allowing for a straightforward discrimination between materials and endotoxin-dependent reactions.