Determining the correct stoichiometry of Kv2.1/Kv6.4 heterotetramers, functional in multiple stoichiometrical configurations.

The electrically silent (KvS) members of the voltage-gated potassium (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9 selectively modulate Kv2 subunits by forming heterotetrameric Kv2/KvS channels. Based on the reported 3:1 stoichiometry of Kv2.1/Kv9.3 channels, we tested the hypothesis that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. We investigate the Kv2.1/Kv6.4 stoichiometry using single subunit counting and functional characterization of tetrameric concatemers. For selecting the most probable stoichiometry, we introduce a model-selection method that is applicable for any multimeric complex by investigating the stoichiometry of Kv2.1/Kv6.4 channels. Weighted likelihood calculations bring rigor to a powerful technique. Using the weighted-likelihood model-selection method and analysis of electrophysiological data, we show that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. Within this stoichiometry, the Kv6.4 subunits have to be positioned alternating with Kv2.1 to express functional channels. The variability in Kv2/KvS assembly increases the diversity of heterotetrameric configurations and extends the regulatory possibilities of KvS by allowing the presence of more than one silent subunit.

Preparation of thermocleavable conjugates based on ansamitocin and superparamagnetic nanostructured particles by a chemobiosynthetic approach.

A combination of mutasynthesis, precursor-directed biosynthesis and semisynthesis provides access to new ansamitocin derivatives including new nanostructured particle-drug conjugates. These conjugates are based on the toxin ansamitocin and superparamagnetic iron oxide-silica core shell particles. New ansamitocin derivatives that are functionalized either with alkynyl- or azido groups in the ester side chain at C-3 are attached to nanostructured iron oxide core-silica shell particles. Upon exposure to an oscillating electromagnetic field these conjugates heat up and the ansamitocin derivatives are released by a retro-Diels-Alder reaction. For example, one ansamitocin derivative exerts strong antiproliferative activity against various cancer cell lines in the lower nanomolar range while the corresponding nanostructured particle-drug conjugate is not toxic. Therefore, these new conjugates can serve as dormant toxins that can be employed simultaneously in hyperthermia and chemotherapy when external inductive heating is applied.

Towards a biocompatible artificial lung: Covalent functionalization of poly(4-methylpent-1-ene) (TPX) with cRGD pentapeptide.

Covalent multistep coating of poly(methylpentene), the membrane material in lung ventilators, by using a copper-free "click" approach with a modified cyclic RGD peptide, leads to a highly biocompatible poly(methylpentene) surface. The resulting modified membrane preserves the required excellent gas-flow properties while being densely seeded with lung endothelial cells.

Fully defined in situ cross-linkable alginate and hyaluronic acid hydrogels for myocardial tissue engineering.

Despite recent major advances including reprogramming and directed cardiac differentiation of human cells, therapeutic application of in vitro engineered myocardial tissue is still not feasible due to the inability to construct functional large vascularized contractile tissue patches based on clinically applicable and fully defined matrix components. Typical matrices with preformed porous 3D structure cannot be applied due to the obvious lack of migratory capacity of cardiomyocytes (CM). We have therefore developed a fully defined in situ hydrogelation system based on alginate (Alg) and hyaluronic acid (HyA), in which their aldehyde and hydrazide-derivatives enable covalent hydrazone cross-linking of polysaccharides in the presence of viable myocytes. By varying degrees of derivatization, concentrations and composition of blends in a modular system, mechanophysical properties of the resulting hydrogels are easily adjustable. The hydrogel allowed for the generation of contractile bioartificial cardiac tissue from CM-enriched neonatal rat heart cells, which resembles native myocardium. A combination of HyA and highly purified human collagen I led to significantly increased active contraction force compared to collagen, only. Therefore, our in situ cross-linking hydrogels represent a valuable toolbox for the fine-tuning of engineered cardiac tissue's mechanical properties and improved functionality, facilitating clinical translation toward therapeutic heart muscle reconstruction.

Preparation and evaluation of hydrogel-composites from methacrylated hyaluronic acid, alginate, and gelatin for tissue engineering.

Hydrogels are three-dimensional water-insoluble hydrophilic natural or synthetic polymer networks made up of crosslinked water-soluble polymers. The purpose of this study was to develop and directly compare photo crosslinked hydrogels on the basis of pure gelatin, alginate and hyaluronic acid as well as their blends. The functionalization of starting materials with methacrylate moieties was evaluated by 1H-NMR spectroscopy. Hydrogels were prepared from methacrylates by photo cross-linking using UV light. The effect of changing the hydrogel composition was quantified through examination of hydrogel swelling behavior and rheological properties. In addition, the viability and adhesion of neonatal rat cardiomyocytes (NRCM) seeded onto the hydrogels was examined by in vivo imaging of NRCM-mediated scaffold contraction as well as by histological evaluation after immunostaining. Biological testing showed good biocompatibility and cell survival in the presence of all materials discussed. Adhesion of cells could only be observed in the presence of gelatin. Blends of gelatin, alginate and hyaluronic acid are promising candidates for the generation of non-toxic, biocompatible hydrogel scaffolds for tissue engineering. Variation of individual compound ratios in the blends can be used for a precise control of mechanical properties and may allow wide-ranging uses in various tissue engineering applications with different mechanical requirements.

Laser fabrication of three-dimensional CAD scaffolds from photosensitive gelatin for applications in tissue engineering.

In the present work, 3D CAD scaffolds for tissue engineering applications were developed starting from methacrylamide-modified gelatin (GelMOD) using two-photon polymerization (2PP). The scaffolds were cross-linked employing the biocompatible photoinitiator Irgacure 2959. Because gelatin is derived from collagen (i.e., the main constituent of the ECM), the developed materials mimic the cellular microenvironment from a chemical point of view. In addition, by applying the 2PP technique, structural properties of the cellular microenvironment can also be mimicked. Furthermore, in vitro degradation assays indicated that the enzymatic degradation capability of gelatin is preserved for the methacrylamide-modified derivative. An in depth morphological analysis of the 2PP-fabricated scaffolds demonstrated that the parameters of the CAD model are reproduced with great precision, including the ridge-like surface topography on the order of 1.5 µm. The developed scaffolds showed an excellent stability in culture medium. In a final part of the present work, the suitability of the developed scaffolds for tissue engineering applications was verified. The results indicated that the applied materials are suitable to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Upon applying osteogenic stimulation, the seeded cells differentiated into the anticipated lineage. Energy dispersive X-ray (EDX) analysis showed the induced calcification of the scaffolds. The results clearly indicate that 2PP is capable of manufacturing precisely constructed 3D tissue engineering scaffolds using photosensitive polymers as starting material.

Reduced thrombocyte adhesion to endothelialized poly 4-methyl-1-pentene gas exchange membranes­a first step toward bioartificial lung development.

Polymeric materials used in biomedical devices, bioartificial organs, or for the fabrication of tissue engineering scaffolds should completely prevent the activation of the coagulation system and subsequent clot formation. Surface endothelialization is considered an important tool to optimize the blood compatibility of synthetic materials, as a functional endothelial cell layer on an artificial material may help control hemostasis and, therefore, provide a solution to improve the biocompatibility of these materials. Here we report on the endothelialization of poly 4-methyl-1-pentene (PMP) gas exchange membranes using human cord blood-derived late outgrowth endothelial colony forming cells. We achieved complete endothelialization of PMP membranes; and when seeded and cultivated on the membrane, cord blood-derived late outgrowth endothelial colony forming cells maintained both endothelial characteristics and functionality. Endothelialization resulted in significantly lower platelet adhesion and activation compared with unseeded membranes. Of importance, the endothelial layer had no major impact on gas permeability of PMP membranes. This study is a first promising step toward the development of a biofunctionalized surface for the use in gas exchange devices with blood contacting surfaces and a straightforward approach toward a long-term bio-hybrid lung replacement system.