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Polyvinyl alcohol hydrogel (PVA) is a synthetic polymer with an increasing application in the biomedical field that can potentially be used for vascular grafting. However, the tissue and blood–material interactions of such gels and membranesare unknown in detail. The objectives of this study were to: (a) assess the biocompatibility and (b) hemocompatibility of PVA-based membranes in order to get some insight into its potential use as a vascular graft. PVA was evaluated isolated or in copolymerization with dextran (DX), a biopolymer with known effects in blood coagulation homeostasis.

Mesenchymal Stem Cells from Extra-Embryonic Tissues for Tissue Engineering – Regeneration of the Peripheral Nerve

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton's jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each: Group 1, sciatic axonotmesis injury without any other intervention (Group 1-Crush); Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250–1 500 human mesenchymal stem cells (total volume of 50 μL) (Group 2-CrushCell); Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane covered with a monolayer of non-differentiated human mesenchymal stem cells (Group 3-CrushChitIIICell) and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane (Group 4-CrushChitIII). Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carried out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may represent a very promising clinical tool in peripheral nerve reconstructive surgery. Yet, umbilical cord human mesenchymal stem cells, that can be expanded in culture and induced to form several different types of cells, may prove, in future experiments, to be a new source of cells for cell therapy, including targets such as peripheral nerve and muscle.

Due to the high re-discovery rate of already known active compounds in recent drug research it appears reasonable to expand the search on unexplored environments with unique living conditions and yet undiscovered organisms. The deep sea demonstrates such a marginally investigated environment harboring presumably undiscovered bacterial taxa which are adapted to the extreme living conditions in the deep sea environment and which might possess unknown metabolites. The aim of the present thesis was therefore the characterization of deep-sea bacteria with the purpose to find novel and bioactive substances produced by these bacteria. Heterotrophic mesophilic bacterial strains were recovered and phylogenetically characterized from two totally different deep-sea habitats: the extremely oligotrophic Eastern Mediterranean and the Logatchev hydrothermal vent field (LHF) located at 15°N along the Mid Atlantic Ridge. The antimicrobially active bacteria isolated from the hydrothermal environments were mainly assigned to the Gammaproteobacteria. One bioactive strain, M41T, revealed to be a representative of a novel genus and species, Amphritea atlanticaT. This strain was isolated from a mussel field at LHF and physiological analysis showed that it is well adapted to the mesophilic temperatures of hydrothermally influenced environments. In addition, the bacterial community of diffusive fluids emanating out of a mussel field at LHF was investigated. A 16S rRNA gene library was analyzed in combination with functional genes involved in biochemical pathways of CO2 fixation (aclB, cbbM, cbbL) and sulfur oxidation/reduction (soxB, aprA). It turned out, that Epsilonproteobacteria and Gammaproteobacteria comprise a considerable part of the microbial community in diffuse fluids and have the genetic potential to use different pathways for carbon fixation and sulfur oxidation. The bacterial strains obtained from the Eastern Mediterranean deep sea were also analyzed for specifically adapted bacterial strains and antimicrobial activities. Predominantly Gram-positive strains were isolated from the untreated sediment, while incubation of sediment at in situ pressure revealed that Gammaproteobacteria were enriched at the simulated deep-sea conditions. Bacterial strains affiliating to the genus Micromonospora were selected for further analysis in order to investigate their potential to produce bioactive substances. This led to the discovery and structure elucidation of the novel cytotoxic macrolide levantilide A and a derivative thereof, levantilide B, both of which are produced by a deep-sea Micromonospora strain, strain A77. Thus, the results of the present study demonstrate that deep-sea habitats are a promising source for novel bacterial taxa and for the discovery of new natural products as well. Im Zuge der hohen Wiederentdeckungsrate bekannter Verbindungen in der heutigen Wirkstoffforschung erscheint es sinnvoll, die Suche auf neue Habitate mit einzigartigen Lebensbedingungen und bislang unbekannten Organismen auszudehnen. Ein solches bislang nur marginal untersuchtes Gebiet stellt die Tiefsee dar. Es ist zu erwarten, dass in der Tiefsee noch viele neue Bakterien-Taxa auf ihre Entdeckung warten, die physiologisch an die extremen Lebensbedingungen angepasst sind und über neue Metabolite verfügen, die wiederum für die biotechnologische oder medizinische Forschung von Interesse sein können. Ziel der vorliegenden Arbeit war es daher, Tiefseebakterien näher zu charakterisieren und sie hinsichtlich ihres Potenzials zur Wirkstoffproduktion zu untersuchen. Mesophile Bakterien wurden von zwei sehr unterschiedlichen Tiefseestandorten isoliert und charakterisiert: die extrem nährstoffarmen Tiefseesedimente des östlichen Mittelmeeres sowie das Logatchev-Hydrothermalfeld (LHF) am Mittelatlantischen Rücken. Die vom LHF isolierten Stämme mit antimikrobieller Aktivität waren hauptsächlich den Gammaproteobakterien zuzuordnen. Unter diesen befand sich auch der Stamm M41T, welcher im Zuge dieser Studien als Repräsentant einer neuen Gattung, Ampritea atlanticaT, beschrieben wurde. Physiologische Untersuchungen dieses von einem Muschelfeld isolierten Stammes zeigten, dass dieser gut an die mesophilen Temperaturen von hydrothermal beeinflussten Gebieten angepasst ist. Des Weiteren wurde die Bakteriengemeinschaft an einer diffusen Fluidaustrittstelle über einem Muschelfeld des LHF untersucht. Die Ergebnisse aus einer 16S rRNA Genbank und die kombinierte Untersuchung von Schlüsselenzymen für verschiedene CO2-Fixierungswege und den Schwefelkreislauf ergaben, dass Epsilonproteobakterien und Gammaproteobakterien einen beachtlichen Teil der Bakteriengemeinschaften auszumachen scheinen und darüber hinaus das genetische Potenzial haben, unterschiedliche Biosynthesewege für die CO2-Fixierung und die Schwefeloxidation zu verwenden. Auch die Isolate aus dem östlichen Mittelmeer wurden hinsichtlich des Vorkommens neuer Arten, spezieller Anpassungen sowie antimikrobieller Aktivitäten untersucht. Während aus den unbehandelten Sedimenten in erster Linie Gram-positive Isolate gewonnen wurden, hatte eine Inkubation des Sedimentes bei in situ Druck eine Verschiebung der Bakteriengemeinschaft, hauptsächlich hin zu Vertretern der Gammaproteobakterien, zur Folge. Von den gewonnenen Isolaten wurden die Vertreter der Gattung Micromonospora für die Suche nach bioaktiven Wirkstoffen ausgewählt. So konnte ein neues zytotoxisch wirksames Makrolid, levantilide A, und ein Derivat, levantilide B, aus einem der Micromonospora-Stämme, Isolat A77, isoliert und beschrieben werden. Die Ergebnisse der vorliegenden Arbeit zeigen, dass aus Tiefsee-Standorten eine Vielzahl unbekannter Bakterien-Taxa kultiviert werden können und dass Tiefseebakterien eine vielversprechende Quelle für neue Wirkstoffe darstellen.

Marine environments like surfaces of macroorganisms (i.e. algae, sponges, bryozoa, corals) or deep sea habitats provide excellent ecological niches for bacteria and fungi producing new natural products. Therefore, major goals of our projects are the isolation, identification and fermentation of marine-derived bacteria and fungi as well as extraction, structure elucidation and the determination of the biological activity of their metabolites. In addition, studies concerning the interactions between marine microorganisms and the microbial interactions with their hosts are in our focus. We will present recent results from our work. Two recently described new species Tenacibaculum adriaticum isolated from a bryozoan and Amphritea atlantica (representing a new genus) derived from a deep sea mussel, both display antibiotic activities. Sponge-associated bacteria like Bacillus subtilis or Streptomyces sp. were identified as producers of new bacillaenes and phenazines, respectively. Interestingly, subinhibitory concentrations of microbial metabolites induced the production of new phenazines, which inhibited the growth of bacteria and different tumoral cell lines. The examination of fungal isolates revealed Penicillium sp. as the producer of new pentaens. The biosynthesis of these substances was studied by feeding experiments with 13C labelled substances. Genetic approaches were used to select bacterial strains showing genes encoding for polyketide-synthase (PKS) and non-ribosomal peptide-synthetase (NRPS). These strains were selected for analysis of biological active new metabolites. In conclusion, the exploitation of our unique strain collections offers great chances to find new natural products with possible application in human health, crop protection and other applied fields.