A xilose é um dos principais componentes dos materiais lignocelulósicos, os quais são de grande interesse para produção de bioprodutos como etanol e polihidroxialcanoatos (PHA). Visando melhorar o consumo de xilose em Burkholderia sacchari, uma grande produtora de PHA, os seguintes genes codificadores de xilose isomerase foram nela inseridos e avaliados: xylABs, xylABc, xylAPl, xylABp e xylABx, respectivamente de B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. Foi ainda sintetizado o gene de B. sacchari (xylA*) no qual foram inseridas modificações descritas na literatura como capazes de aumentar o consumo de xilose em outros organismos. As linhagens recombinantes de B. sacchari abrigando os genes xylABs e xylA* tiveram um aumento de aproximadamente 30%, e aquelas abrigando os genes xylABp e xylABx de 23%, no consumo de xilose quando comparadas com a linhagem controle. Essas quatro linhagens recombinantes foram aquelas que conseguiram produzir maior quantidade de P3HB, aproximadamente 70% a mais do que linhagem controle. / Xylose is a major component of lignocellulosic materials, which are of great interest for the production of bio-products, such as ethanol and polyhydroxyalkanoates (PHA). To improve the consumption of xylose in Burkholderia sacchari, a major PHA producer, the following genes, encoding xylose isomerase, were introduced in these bacteria: xylABs, xylABc, xylAPl, xylABp and xylABx, respectively from B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. The gene of B. sacchari (xylA*) was also synthesized with several modifications described in the literature as able to increase the consumption of xylose in other organisms. Recombinant strains harboring B. sacchari xylABs and xylA* gene had an increase of approximately 30% in the xylose consumption compared to the control strain, and those harboring xylABx and xylABp gene an increase of 23%. These four recombinant strains were those that were able to produce more P3HB, approximately 70% more than the control strain.

Os copolímeros de 3-hidroxibutirato e 3-hidroxihexanoato (P3HB-co-3HHx) são da família dos polihidroxialcanoatos (PHA), materiais termoplásticos e biodegradáveis acumulados por bactérias a partir de fontes de carbono renováveis. O P3HB-co-3HHx desperta interesse industrial para frações de 3HHx menores que 20 mol%, assemelhando-se ao polietileno de baixa densidade. Neste projeto, criou-se um sistema genético para controlar a composição do copolímero P3HB-co-3HHx pela indução de genes da biossíntese de precursores 3HB. Baseado no promotor lac foram construídas cinco versões do plasmídeo de controle de copolímero (pCC) utilizando o promotor Lac. Porém, mesmo com a versão mais aprimorada, não foi possível o controle de composição de P3HB-co-3HHx em Pseudomonas sp. LFM461. Através de experimentos de atividade enzimática e RT-qPCR do cDNA do gene phaA e phaB, foi possível indicar que o problema está na impossibilidade do promotor Lac de promover expressão dos genes de biossíntese de 3HB presentes nos pCC. / Polyhydroxyalkanoates (PHA) are polyesters material accumulated by bacteria which has thermoplastic and biodegradable proprieties and can be produced from renewable feedstocks. Copolymers of 3-hydroxybutirate and 3-hydroxyhexanoate (P3HB-co¬-3HHx) which contain less than 20mol% of 3HHx have being researched for its industrial proprieties. Recently, our laboratory has been researching a Pseudomonas sp. strain, LFM461, which can produce high 3HHx content when hosting PHA biosynthesis genes from Aeromonas. In this project, it was design a genetic system for control of P3HB-co-3HHX composition through induction of 3HB monomers biosynthesis. Thus, five versions of a copolymer control plasmid (pCC) was built based on Lac promoter. However, even through improvements on stability and expression profile of pCC it was not possible to stablish an assay of successful PHA composition control in Pseudomonas sp. LFM461. By enzymatic activity and RT-qPCR of cDNA experiments we have indications of problems of Lac promoter on driving the expression of 3HB genes in pCC.

O potencial de bactérias em produzir polihidroxialcanoatos (PHA) a partir de óleo de soja foi avaliado com ênfase em Aeromonas spp. Dez isolados apresentando características de Aeromonas spp. (colônias amarelas em GSP agar e produção de P3HB-co-3HHx) e perfis ARDRA similares foram obtidos. Dois isolados produzindo PHAMCL foram também obtidos e experimentos de identificação adicionais serão necessários para confirmar se pertencem ao gênero Aeromonas. P3HB-co-3HHx contendo diferentes frações molares de 3HHx foram produzidos. Isolados produzindo P3HB-co-3HHx a partir de óleo de soja ou ácido láurico foram incapazes de produzir P3HB a partir de glicose sugerindo sua incapacidade de gerar monômeros 3HB a partir de acetil-CoA. Introdução de plasmídeo abrigando genes de biossíntese de P3HB de Ralstonia eutropha em isolados capazes de utilizar eficientemente óleo de soja como fonte de carbono permitiu a produção de P3HB, mas não PHA contendo monômeros de cadeia média, sugerindo uma transferência ineficiente de intermediários a partir da <font face=\"Symbol\">&#946-oxidação para PHA sintase. / The potential of polyhydroxyalknoates (PHA) production from plant oils was evaluated especially in Aeromonas spp. Ten isolates presenting features of Aeromonas spp. (yellow colonies in GSP agar and production of P3HB-co-3HHx) and similar ARDRA profiles were obtained. Two isolates producing PHAMCL were also obtained and further identification experiments will be needed to confirm their position in Aeromonas genus. P3HB-co-3HHx containing different molar fractions of 3HHx were produced. Isolates producing P3HB-co-3HHx from soybean oil or lauric acid were unable to produce P3HB from glucose suggesting their incapability to generate 3HB monomers from acetyl-CoA. Introduction of a plasmid harboring P3HB biosynthesis genes from Ralstonia eutropha into isolates able to use efficiently soybean oil as carbon source allowed the production of P3HB but not PHA containing HAMCL suggesting an inefficient transfer of intermediates from &#946-oxidation to PHA synthase.

A xilose é um dos principais componentes dos materiais lignocelulósicos, os quais são de grande interesse para produção de bioprodutos como etanol e polihidroxialcanoatos (PHA). Visando melhorar o consumo de xilose em Burkholderia sacchari, uma grande produtora de PHA, os seguintes genes codificadores de xilose isomerase foram nela inseridos e avaliados: xylABs, xylABc, xylAPl, xylABp e xylABx, respectivamente de B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. Foi ainda sintetizado o gene de B. sacchari (xylA*) no qual foram inseridas modificações descritas na literatura como capazes de aumentar o consumo de xilose em outros organismos. As linhagens recombinantes de B. sacchari abrigando os genes xylABs e xylA* tiveram um aumento de aproximadamente 30%, e aquelas abrigando os genes xylABp e xylABx de 23%, no consumo de xilose quando comparadas com a linhagem controle. Essas quatro linhagens recombinantes foram aquelas que conseguiram produzir maior quantidade de P3HB, aproximadamente 70% a mais do que linhagem controle. / Xylose is a major component of lignocellulosic materials, which are of great interest for the production of bio-products, such as ethanol and polyhydroxyalkanoates (PHA). To improve the consumption of xylose in Burkholderia sacchari, a major PHA producer, the following genes, encoding xylose isomerase, were introduced in these bacteria: xylABs, xylABc, xylAPl, xylABp and xylABx, respectively from B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. The gene of B. sacchari (xylA*) was also synthesized with several modifications described in the literature as able to increase the consumption of xylose in other organisms. Recombinant strains harboring B. sacchari xylABs and xylA* gene had an increase of approximately 30% in the xylose consumption compared to the control strain, and those harboring xylABx and xylABp gene an increase of 23%. These four recombinant strains were those that were able to produce more P3HB, approximately 70% more than the control strain.

O potencial de bactérias em produzir polihidroxialcanoatos (PHA) a partir de óleo de soja foi avaliado com ênfase em Aeromonas spp. Dez isolados apresentando características de Aeromonas spp. (colônias amarelas em GSP agar e produção de P3HB-co-3HHx) e perfis ARDRA similares foram obtidos. Dois isolados produzindo PHAMCL foram também obtidos e experimentos de identificação adicionais serão necessários para confirmar se pertencem ao gênero Aeromonas. P3HB-co-3HHx contendo diferentes frações molares de 3HHx foram produzidos. Isolados produzindo P3HB-co-3HHx a partir de óleo de soja ou ácido láurico foram incapazes de produzir P3HB a partir de glicose sugerindo sua incapacidade de gerar monômeros 3HB a partir de acetil-CoA. Introdução de plasmídeo abrigando genes de biossíntese de P3HB de Ralstonia eutropha em isolados capazes de utilizar eficientemente óleo de soja como fonte de carbono permitiu a produção de P3HB, mas não PHA contendo monômeros de cadeia média, sugerindo uma transferência ineficiente de intermediários a partir da <font face=\"Symbol\">&#946-oxidação para PHA sintase. / The potential of polyhydroxyalknoates (PHA) production from plant oils was evaluated especially in Aeromonas spp. Ten isolates presenting features of Aeromonas spp. (yellow colonies in GSP agar and production of P3HB-co-3HHx) and similar ARDRA profiles were obtained. Two isolates producing PHAMCL were also obtained and further identification experiments will be needed to confirm their position in Aeromonas genus. P3HB-co-3HHx containing different molar fractions of 3HHx were produced. Isolates producing P3HB-co-3HHx from soybean oil or lauric acid were unable to produce P3HB from glucose suggesting their incapability to generate 3HB monomers from acetyl-CoA. Introduction of a plasmid harboring P3HB biosynthesis genes from Ralstonia eutropha into isolates able to use efficiently soybean oil as carbon source allowed the production of P3HB but not PHA containing HAMCL suggesting an inefficient transfer of intermediates from &#946-oxidation to PHA synthase.

Burkholderia sacchari é uma espécie de bactéria capaz de acumular polihidroxialcanoatos em condições de limitação de um nutriente essencial e excesso de fonte de carbono. A partir do substrato sacarose, acumula o polímero poli-3-hidroxibutirato (P3HB), poliéster biodegradável de propriedades semelhantes às dos plásticos de origem petroquímica. A partir de sacarose e propionato como fontes de carbono, ela é capaz de acumular o copolímero poli-3-hidroxibutirato-co-3-hidroxivalerato (P3HB-co-3HV), que é mais maleável que o polímero P3HB. No entanto, apenas uma pequena porcentagem do propionato fornecido é convertida em 3HV. Isto se deve à presença de outras vias de catabolismo muito eficientes que transformam o propionato em biomassa, reduzindo a eficiência na produção do copolímero. Estudos em mutantes UV prp-, indicaram que duas vias de catabolismo de propionato podem atuar em B. sacchari: &#945;-oxidação e o ciclo de 2-metilcitrato (2MCC). Esta última teve sua comprovação molecular comprovada, já a outra ainda está sendo estudada, mutantes afetados no consumo de intermediários da &#945;-oxidação foram complementados fragmentos de DNA, obtidos de uma biblioteca genômica de B. sacchari os quais, após sequenciamento e comparação do banco de dados, verificou-se codificarem um regulador transcricional LysR. A análise dos genes adjacentes ao regulador sugeriu que poderiam compor um operon de uma via de &#945;-oxidação. Diante disso, este trabalho busca a comprovação molecular da via da &#945;-oxidação para o catabolismo de propionato em B. sacchari. / Burkholderia sacchari is a species of bacteria capable of accumulating polyhydroxyalkanoates under limiting conditions of an essential nutrient and excess carbon source. From the sucrose substrate, it accumulates polymer poly-3-hydroxybutyrate (P3HB), biodegradable polyester with properties similar to those of petrochemical plastics. From sucrose and propionate as carbon sources, it is able to accumulate the poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV) copolymer, which is more malleable than the polymer P3HB. However, only a small percentage of the supplied propionate is converted into 3HV. This is due to the presence of other very efficient catabolic pathways that transform the propionate into biomass, reducing the production efficiency of the copolymer. Studies on prp- UV mutants have indicated that two pathways of propionate catabolism may act on B. sacchari: the &#945;-oxidation and the 2-methylcitrate cycle (2MCC). The latter had its molecular proof proven, while the other is still being studied, mutants affected in the consumption of &#945;-oxidation intermediates were complemented DNA fragments obtained from a genomic library of B. sacchari which, after sequencing and comparison of the bank Coding for a LysR transcriptional regulator. Analysis of the genes adjacent to the regulator suggested that they could compose an operon of an &#945;-oxidation pathway. In view of this, this work seeks the molecular proof of the &#945;-oxidation pathway for the propionate catabolism in B. sacchari.

Del A: Polyetylentereftalat fibrer I detta arbete presenteras olika metoder för att framställa monofilament av polyetylentereftalat (PET) (diameter: 30-50 µm) med en radiell gradient. Nyutvecklad Raman-spektroskopiteknik har använts för att kartlägga dessa inducerade radiella gradienter i t.ex. kristallinitet. På liknande sätt har FTIR-ATR teknik modifierats och anpassats för att studera ytegenskaperna hos dessa filament. Industriella filamentprover och egna smältspunna PET-filament har framgångsrikt modifierats med användning av olika termiska och kemiska behandlingar för att erhålla fibrer med förbättrade mekaniska egenskaper och minskad fibrillering. De strukturella förändringar som uppträdde i filamenten på mikroskopisk nivå karakteriserades med bl a infraröd analys, termisk analys, Raman-mikroskopi och röntgenteknik (SAXS och WAXD). Tester av fibrilleringsegenskaper utfördes av industriella partners med egenutvecklad teknik följt av testning av masterbatch-fibrer på en vävningssimulator. Resultaten i laboratorieskala avslöjade fibrernas strukturella anisotropi och radiella gradienter, vilka visade en minskad fibrillering med en viss inverkan på de mekaniska egenskaperna.  Del B: Poly(3-hydroxybutyrat) fibrer Detta arbete presenterar studier av poly(3-hydroxybutyrat) (P3HB) fibrer med reversibla strukturförändringar. Tidigare studier har visat att kristallisationen hos P3HB fibrer i huvudsakligen sker i ortorombisk α-kristallform. Stress-anlöpning resulterar dock i en förändring i beteendet hos P3HB-materialet. Strukturen hos P3HB fibrer består av amorfa och kristallina regioner samt en mesofas. Mesofasen antas vara belägen mellan α-kristallerna och uppträder som starkt orienterade bindningskedjor, s k “tie-chains”. Denna studie syftar till att observera effekten av stress-anlöpning på mesofasen och dess beroende av anlöpningsförhållandena. Förändringarna i mesofasen observeras med en anpassad och polariserad Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) samt med Differential Scanning Calorimetry (DSC). Resultaten från ATR-FTIR visar att mesofasen är närvarande i spunna och högt stress-anlöpta fibrer, medan den är frånvarande i fibrer som är lågt stress-anlöpta. Mesofasen kan emellertid återupptas i lågt stress-anlöpta fibrer genom dragning. In situ ATR-FTIR användes för att studera förändringarna i materialbeteendet under en dragningsprocess för att observera periodiciteten i förekomsten av mesofasen. Det visade sig att förekomsten av mesofasen är en starkt reversibel process som observeras som en funktion av topparnas intensitet i ATR-FTIR. / Part A: Poly(ethylene terephthalate) fibres In this work, various methods to produce Poly(ethylene terephthalate) (PET) monofilaments (diameter: 30-50µm) with a radial gradient are presented along with a newly developed Raman spectroscopy technique to map these induced radial gradients in e.g. crystallinity. On similar lines, FTIR-ATR technique has been modified and adapted to study the surface properties of these fine filaments. Industrial filament samples and in-house melt-spun PET filaments have been successfully modified using various thermal and chemical treatments to obtain fibres with improved mechanical properties and reduced fibrillation. The structural changes occurring in the filaments on the microscopic level were characterized using infrared analysis, thermal analysis, Raman microscopy and X-ray techniques (SAXS and WAXD) among others. The fibrillation properties were tested by the industrial partners using a technique developed in-house followed by testing of masterbatch fibres on a weaving simulator. Lab-scale results revealed the structural anisotropy and radial gradient maps of the fibres which also demonstrated reduced fibrillation with some impact on mechanical properties also being observed. Part B: Poly(3-hydroxybutyrate) fibres This work presents studies on poly(3-hydroxybutyrate) (P3HB) fibres with reversible structural changes. Previously reported literature shows that crystallization of P3HB fibres takes place majorly in the orthorhombic α-crystal form. However, the stress-annealing results in a change of the material behaviour of P3HB. P3HB fibres compose of amorphous regions, crystalline regions and mesophase in their structure. The mesophase is supposed to be located in between the α-crystals of the material as highly oriented tie-chains. This study targets to observe the effect of stress-annealing of the mesophase present in the P3HB fibres and its dependence on the annealing conditions. The changes in the mesophase content are observed with the help of a highly adapted polarized Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC). The presented results from polarized ATR-FTIR show that the mesophase is present in as-spun and high stress annealed fibres while it is absent in fibres annealed with low stress. However, the mesophase can be re-obtained in low stress annealed fibres through tensile drawing. In-situ ATR-FTIR was utilized to study the changes in the material behaviour during a tensile drawing process to observe the cyclicity in the occurrence of the mesophase. It was found that the existence of mesophase is a highly reversible process observed as a function of the peak intensities of the polarized ATR-FTIR spectroscopy.

In drei inhaltlich abgeschlossen Teilen werden Fragestellungen bearbeitet, die sich mit dem Einsatz von Polyhydroxybutyraten als Scaffoldmaterialien für das Tissue Engioneering von Knochen beschäftigen. Zunächst wird ein Prozess optimiert, in dem mittels Verpressen und Auslösen von Platzhaltern (Porogen) poröse Träger (Scaffolds) aus Poly-3-hydroxybuttersäure (P3HB) sowie aus P3co4HB hergestellt werden. Diese Scaffolds werden in der Folge mechanisch und strukturell charakterisiert, wobei Druckfestigkeit, Dauerfestigkeit und Viskoelastizität untersucht werden. Im Ergebnis finden sich mehrere Kandidaten, die für die weitere Testung im Tierversuch in Frage kommen. Weiter wird das Abbauverhalten von schmelzgeponnenen P3HB-Fäden untersucht. Dabei wird ein beschleunigtes Modellsystem gewählt, das noch möglichst nahe am physiologischen Fall aber ohne biologisch aktive Komponente (zB. Enzyme) definiert wurde. Die Charakterisierung bedient sich hier der Gelpermeationschromatographie (GPC), des gasgestützten Elektronenrastermikroskops (ESEM), der differentiellen Thermoanalyse (DSC) und der Rasterkraftmikroskopie. Als Ergebnis zeichnete sich ab, dass neben der hydrolytischen Degradation im Gegensatz zu PHB mit kleinerer spezifischer Oberfläche bei den Fäden auch Erosion zum Abbau beiträgt. Eine partikuläre Freisetzung wird nicht beobachtet. Im dritten Teil werden textile Scaffolds aus P3HB mit einer künstlichen extrazellulären Matrix aus Chondroitinsulfaten (CS) und Kollagen versehen. Dem CS kann hier ein positiver Einfluss auf die osteogene Differenzierung von humanen mesenchymalen Stammzellen (hMSC) nachgewiesen werden. Dies wird zum einen durch die verstärkte Expression der alkalischen Phosphatase (ALP) sowie durch die Hochregulation von Proteinen ersichtlich, die bei der osteogenen Differenzierung essentiell sind. In wenigen Gene-Arrays lässt sich ebenfalls erkennen, dass die osteogene Differenzierung durch CS positiv beeinflusst wird. Insbesondere frühe Marker wie ZBTB16 und IGFBPs werden hier identifiziert. Basierend auf den Teilergebnissen wird am Ende ein Beitrag geliefert, der das Tissue Engineering insbesondere für überkritische Röhrenknochendefekte als Methode interessant erscheinen lässt. Dabei werden mechanische Lasten durch konventionelle Fixateure aufgenommen und der Defektraum durch den mehrfachen Einsatz von bio-funktionalisierten flachen Scaffolds gefüllt.

In drei inhaltlich abgeschlossen Teilen werden Fragestellungen bearbeitet, die sich mit dem Einsatz von Polyhydroxybutyraten als Scaffoldmaterialien für das Tissue Engioneering von Knochen beschäftigen. Zunächst wird ein Prozess optimiert, in dem mittels Verpressen und Auslösen von Platzhaltern (Porogen) poröse Träger (Scaffolds) aus Poly-3-hydroxybuttersäure (P3HB) sowie aus P3co4HB hergestellt werden. Diese Scaffolds werden in der Folge mechanisch und strukturell charakterisiert, wobei Druckfestigkeit, Dauerfestigkeit und Viskoelastizität untersucht werden. Im Ergebnis finden sich mehrere Kandidaten, die für die weitere Testung im Tierversuch in Frage kommen. Weiter wird das Abbauverhalten von schmelzgeponnenen P3HB-Fäden untersucht. Dabei wird ein beschleunigtes Modellsystem gewählt, das noch möglichst nahe am physiologischen Fall aber ohne biologisch aktive Komponente (zB. Enzyme) definiert wurde. Die Charakterisierung bedient sich hier der Gelpermeationschromatographie (GPC), des gasgestützten Elektronenrastermikroskops (ESEM), der differentiellen Thermoanalyse (DSC) und der Rasterkraftmikroskopie. Als Ergebnis zeichnete sich ab, dass neben der hydrolytischen Degradation im Gegensatz zu PHB mit kleinerer spezifischer Oberfläche bei den Fäden auch Erosion zum Abbau beiträgt. Eine partikuläre Freisetzung wird nicht beobachtet. Im dritten Teil werden textile Scaffolds aus P3HB mit einer künstlichen extrazellulären Matrix aus Chondroitinsulfaten (CS) und Kollagen versehen. Dem CS kann hier ein positiver Einfluss auf die osteogene Differenzierung von humanen mesenchymalen Stammzellen (hMSC) nachgewiesen werden. Dies wird zum einen durch die verstärkte Expression der alkalischen Phosphatase (ALP) sowie durch die Hochregulation von Proteinen ersichtlich, die bei der osteogenen Differenzierung essentiell sind. In wenigen Gene-Arrays lässt sich ebenfalls erkennen, dass die osteogene Differenzierung durch CS positiv beeinflusst wird. Insbesondere frühe Marker wie ZBTB16 und IGFBPs werden hier identifiziert. Basierend auf den Teilergebnissen wird am Ende ein Beitrag geliefert, der das Tissue Engineering insbesondere für überkritische Röhrenknochendefekte als Methode interessant erscheinen lässt. Dabei werden mechanische Lasten durch konventionelle Fixateure aufgenommen und der Defektraum durch den mehrfachen Einsatz von bio-funktionalisierten flachen Scaffolds gefüllt.:1. Vorwort 3 2. Allgemeine Einführung 5 2.1 Der Knochen 5 2.1.1 Die Knochenbildung 5 2.1.2 Zur Anatomie und Physiologie des Knochens 7 2.2 Tissue Engineering 11 2.2.1 Zelltypen für das Tissue Engineering von Knochen 12 2.2.2 Scaffold Design im Tissue Engineering von Knochen 13 2.3 Polyhydroxyalkanoate 13 2.4 Tissue Engineering am Röhrenknochen 16 2.4.1 Poly(3-hydroxybutyrat)-Scaffolds für das Tissue Engineering von Knochenersatz 17 2.4.2 Matrix Engineering 18 2.5 Ziel der Arbeit 19 3. Mechanik poröser PHB-Scaffolds 21 3.1 Einleitung 21 3.2 Materialien und Methoden 23 3.2.1 Polyhydroxybutyrate und Porogene 23 3.2.2 Uniaxiales Heißpressen 24 3.2.3 Mikrographie 26 3.2.4 Dynamische Differenzkalorimetrie (DSC) 26 3.2.5 Mechanische Druckversuche 26 3.2.6 Mikrocomputertomographie (μCT) 27 3.2.7 Zellviabilität auf den Scaffolds 28 3.3 Ergebnisse 29 3.3.1 Mikrographie 29 3.3.2 Mikrocomputertomographie (μCT) 33 3.3.3 Druckversuche 37 3.3.4 Dynamische Differenzkalorimetrie (DSC) 40 3.3.5 Zellviabilität 40 3.4 Diskussion 40 3.5 Schlussfolgernde Zusammenfassung 46 4. Degradation von P3HB-Fasern 47 4.1 Degradation von Polyhydroxyalkanoaten 47 4.2 Materialien und Methoden 49 4.2.1 Herstellung und Vorbehandlung textiler P3HB-Konstrukte 49 4.2.2 Mechanische Prüfung 50 4.2.3 Beschleunigte Degradation 50 4.2.4 Untersuchung der Oberfläche 50 4.2.5 Dynamische Differenzkalorimetrie (DSC) 51 4.2.6 Gel-Permeations-Chromatographie (GPC) 51 4.3 Ergebnisse 52 4.3.1 Mechanische Tests 52 4.3.2 Die Charakterisierung der Oberfläche 52 4.3.3 Thermische Fasereigenschaften.55 4.3.4 Untersuchung der Molekulargewichte in der GPC 58 4.4 Diskussion 60 4.5 Schlussfolgernde Zusammenfassung 64 5. hMSC auf textilen Scaffolds 67 5.1 Einleitung 67 5.2 Material und Methoden 68 5.2.1 Erzeugung der P3HB-Scaffolds 68 5.2.2 Die Immobilisierung der EZM-Komponenten auf den Scaffolds 69 5.2.3 Isolation, Vorkultur, Besiedlung und Kultur der humanen mesenchymalen Vorläuferzellen 69 5.2.4 Kombinierte Bestimmung von ALP, MTT und Proteingehalt 71 5.2.5 Mikroskopische Untersuchungen 72 5.2.6 Nachweis der Kalziummineralisierung 73 5.2.7 Quantitative real time reverse transcribing polymerase chain reaction (rt-PCR) 73 5.2.8 cRNA Microarray-Untersuchung 74 5.2.9 Zusätzliche Experimente 75 5.3 Ergebnisse 76 5.3.1 Vorhergehende Untersuchung 76 5.3.2 Rasterelektronen-Mikroskopie 77 5.3.3 Konfokale Laser-Scanning-Mikroskopie 79 5.3.4 ALP-Aktivität, SDH-Aktivität und Proteingehalt 82 5.3.5 Mineralisierende Kalziumabscheidung 86 5.3.6 rt-PCR 87 5.3.7 cRNA Microarray-Untersuchung 90 5.3.8 Kulturen von hMSC mit Chondroitinsulfat als gelöstem Zusatz 93 5.4 Diskussion 93 5.5 Schlussfolgernde Zusammenfassung 98 6. Zusammenfassung 101