京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18595号 / 工博第3956号 / 新制||工||1608(附属図書館) / 31495 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 松田 建児, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Motivated by the great potential which is offered by the combination of additive manufacturing and tissue engineering, a novel polymeric bioink platform based on poly(2 oxazoline)s was developed which might help to further advance the young and upcoming field of biofabrication. In the present thesis, the synthesis as well as the characteristics of several diblock copolymers consisting of POx and POzi have been investigated with a special focus on their suitability as bioinks. In general, the copolymerization of 2-oxazolines and 2-oxazines bearing different alkyl side chains was demonstrated to yield polymers in good agreement with the degree of polymerization aimed for and moderate to low dispersities. For every diblock copolymer synthesized during the present study, a more or less pronounced dependency of the dynamic viscosity on temperature could be demonstrated. Diblock copolymers comprising a hydrophilic PMeOx block and a thermoresponsive PnPrOzi block showed temperature induced gelation above a degree of polymerization of 50 and a polymer concentration of 20 wt%. Such a behavior has never been described before for copolymers solely consisting of poly(cyclic imino ether)s. Physically cross linked hydrogels based on POx b POzi copolymers exhibit reverse thermal gelation properties like described for solutions of PNiPAAm and Pluronic F127. However, by applying SANS, DLS, and SLS it could be demonstrated that the underlying gel formation mechanism is different for POx b POzi based hydrogels. It appears that polymersomes with low polydispersity are formed already at very low polymer concentrations of 6 mg/L. Increasing the polymer concentration resulted in the formation of a bicontinuous sponge like structure which might be formed due to the merger of several vesicles. For longer polymer chains a phase transition into a gyroid structure was postulated and corresponds well with the observed rheological data. Stable hydrogels with an unusually high mechanical strength (G’ ~ 4 kPa) have been formed above TGel which could be adjusted over a range of 20 °C by changing the degree of polymerization if maintaining the symmetric polymer architecture. Variations of the chain ends revealed only a minor influence on TGel whereas the influence of the solvent should not be neglected as shown by a comparison of cell culture medium and MilliQ water. Rotationally as well as oscillatory rheological measurements revealed a high suitability for printing as POx b POzi based hydrogels exhibit strong shear thinning behavior in combination with outstanding recovery properties after high shear stress. Cell viability assays (WST-1) of PMeOx b PnPrOzi copolymers against NIH 3T3 fibroblasts and HaCat cells indicated that the polymers were well tolerated by the cells as no dose-dependent cytotoxicity could be observed after 24 h at non-gelling concentrations up to 100 g/L. In summary, copolymers consisting of POx and POzi significantly increased the accessible range of properties of POx based materials. In particular thermogelation of aqueous solutions of diblock copolymers comprising PMeOx and PnPrOzi was never described before for any copolymer consisting solely of POx or POzi. In combination with other characteristics, e.g. very good cytocompatibility at high polymer concentrations and comparably high mechanical strength, the formed hydrogels could be successfully used for 3D bioprinting. Although the results appear promising and the developed hydrogel is a serious bioink candidate, competition is tough and it remains an open question which system or systems will be used in the future. / Motiviert durch das große Potential, das die Kombination aus additiver Fertigung und künstlicher Geweberegeneration bietet, wurde eine neuartige polymerbasierte Biotintenplattform auf Basis von Poly(2 oxazolin)en entwickelt. Diese soll zukünftig dazu beitragen das noch junge, aber aufstrebende Forschungsfeld der Biofabrikation weiterzuentwickeln. In der vorliegenden Arbeit wurden die Synthese sowie die Eigenschaften von mehreren Diblock Copolymeren, bestehend aus POx und POzi, untersucht, wobei der Hauptfokus auf deren Eignung als Biotinte lag. Grundsätzlich konnte gezeigt werden, dass Copolymere, bestehend aus 2 Oxazolinen und 2 Oxazinen, die unterschiedliche Alkylseitenketten besitzen, synthetisiert werden können. Dabei lagen die ermittelten Polymerisationsgrade nahe am zuvor errechneten Wert. Die Polymere wiesen mittlere bis niedrigere Dispersitäten auf. Für jedes der im Rahmen der vorliegenden Arbeit synthetisierten Diblock Copolymere konnte eine mehr oder weniger starke Abhängigkeit der dynamischen Viskosität von der Temperatur gezeigt werden. Allerdings ist es nicht möglich, aus den thermischen Eigenschaften des Bulkmaterials Rückschlüsse auf das temperaturabhängige Verhalten in Lösung zu ziehen. Diblock Copolymere mit einem hydrophilen PMeOx Block und einem thermoresponsiven PnPrOzi Block bildeten oberhalb einer Kettenlänge von 50 Einheiten und einer Polymerkonzentration von 20 Gew% ein physikalisches Gel. Solch ein Verhalten wurde bisher noch nicht für Copolymere, die ausschließlich auf POx oder seinen höheren Homologen basieren, beschrieben. Physikalische Hydrogele, basierend auf POx b POzi Copolymeren, weisen eine umgekehrte thermische Gelierung wie auch wässrige Lösungen von PNiPAAm und Pluronic F127 auf. Allerdings konnte durch die komplementäre Verwendung von SANS, DLS und SLS gezeigt werden, dass sich der zugrundeliegende Gelbildungsmechanismus für POx b POzi basierte Hydrogele deutlich von den beiden zuvor genannten unterscheidet. Es wird davon ausgegangen, dass sich zunächst bei einer sehr geringen Polymerkonzentration von 6 mg/L Vesikel mit geringer Polydispersität ausbilden. Eine Erhöhung der Konzentration resultiert in der Ausbildung eines bikontinuierlichen Netzwerks mit schwammartiger Struktur. Dieses bildet sich vermutlich durch die Fusion mehrerer Vesikel. Des Weiteren wird für höhere Polymerisationsgrade ein Phasenübergang zu einer gyroidalen Struktur postuliert der sich sehr gut mit den gewonnenen rheologischen Daten deckt. Stabile Hydrogele mit außergewöhnlich hoher mechanischer Stärke (G‘ ≈ 4kPa) bildeten sich oberhalb der Tgel, die über eine Temperaturspanne von 20 °C durch Änderung des Polymerisationsgrades eingestellt werden konnte. Veränderung der Kettenenden zeigten nur einen geringen Einfluss auf die TGel, wobei der Einfluss des verwendeten Lösemittels nicht unterschätzt werden sollte. Dies konnte durch den direkten Vergleich von MilliQ Wasser und Zellkulturmedium gezeigt werden. Rheologische Untersuchungen, die sowohl im rotierenden als auch im oszillierenden Modus durchgeführt wurden, zeigten eine gute Eignung der POx b POzi basierten Hydrogele für Extrusion basierte Druckverfahren. Insbesondere aufgrund des stark ausgeprägten scherverdünnenden Verhaltens und der ausgezeichneten Strukturerholung nach hoher Scherbelastung sollten gute Druckergebnisse erzielbar sein. Zellviabilität-Assays (WST-1) von PMeOx b PnPrOzi Copolymeren an NIH 3T3 Fibroblasten und HaCat-Zellen zeigten, dass die Polymere bei Konzentrationen von bis zu 100 g/L und Inkubationszeiten von 24 h keine dosisabhängige Zytotoxizität besitzen. Zusammenfassend kann festgehalten werden, dass die Copolymerisation von POx und POzi den verfügbaren Eigenschaftsbereich von POx basierten Materialien deutlich vergrößert hat. Insbesondere die temperaturinduzierte Gelierung von wässrigen Polymerlösungen wurde noch nie zuvor für ein anderes Copolymer auf Basis von POx und POzi beschrieben. Aufgrund ihrer herausragenden Eigenschaften, wozu unter anderem eine sehr gute Zytokompatibilität bei hohen Polymerkonzentrationen und eine vergleichsweise hohe mechanische Festigkeit zählen, konnten die entwickelten Hydrogele erfolgreich für den 3D Biodruck verwendet werden. Obwohl die beschriebenen Ergebnisse sehr vielversprechend sind und die entwickelte Hydrogelplattform folglich als ernstzunehmender Biotintenkandidat angesehen werden sollte, ist die Konkurrenz sehr groß und es bleibt abzuwarten, welche Tinte bzw. Tinten in Zukunft zum Einsatz kommen.

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Trä är ett biologiskt material som anses vara både förnyelsebart och hållbart byggnadsmaterial vilket har gjort att det har blivit populärare att bygga i trä. En av de största utmaningarna med att använda trä som konstruktionsmaterial i större byggnader och höghus är brandsäkerheten. I det här examensarbetet har provbitar av bok (Fagus sylvatica L.) vakuumimpregnerats med fenolformaldehydharts (PF) och olika nivåer av ammoniumdivätefosfat (ADF). Provbitarna användes sedan för att testa träets dess mekaniska styrka, hygroskopiska förmåga samt brandegenskaper. Resultaten från studien visar att vakuumimpregnering med kemikalierna ammoniumdivätefosfat (ADF) och fenolformaldehydharts (PF) i trä ger ett virke som är starkare, mer formstabilt och som har ett bättre brandskydd jämfört med obehandlat trä.

Hydrogels are high water-content polymers that mimic the extracellular matrix of cells. The polymers can have many sources and be of natural origin from the extracellular matrix (ECM) of cells or be synthetically derived. Two such polymers are hyaluronic acid and gelatin, which can with the help of the release of free radicals from photoinitiators, initiated by UV light, polymerise, and form a hydrogel. In these hydrogels, cells can be encapsulated. The hydrogels can in turn be used to maintain cells as they are in the natural environment. For example, hydrogels can provide an in-vivo-like ECM for stem cells and endothelial cells by supporting “stemness” and cell-to-cell contact; respectively. We aim to establish a protocol for culturing cells in the hydrogelas a first milestone in a project focused on profiling the metabolome of cells grown in hydrogels. To accomplish this, four types of cells, namely mouse brain microvascular endothelial cells (bEnd.3), human umbilical vein endothelial cells (HUVECs), adult human lung fibroblast (hLFs) and mesenchymal stem cells (MSCs), were evaluated for growth in hyaluronic acid methacrylate (HA-ma), hyaluronic acid acrylamide (HA-am) as well as a QuattroGel composed by gelatin methacryloyl (GelMA), HA-ma, fibrinogen and thrombin. It was found that HA-masupported viability and the stemness of mesenchymal stem cells, of which the metabolome can be further studied in order to evaluate the difference between regular 2D maintenance and maintenance in 3D. No sprouting was observed for the other cells encapsulated in the hydrogel, and further experiments are needed to find the source of error.

Despite being one of the most important organs of vertebrates, the material properties of skin are also one of the most poorly understood. In the field of designing medical devices and surgical tools there are significant advantages to having a model that describes the interaction of forces between a blade tip and skin during surgical cutting. In general, skin can best be described as a composite layer consisting of a viscoelastic dermis with interwoven collagen and elastin fibers beneath a superficial epidermis. The purpose of this research is to study the fracture toughness of porcine skin during practical cutting applications, the behavior of skin under quasistatic loads, and viscoelastic behavior of skin during stress relaxation. To fully describe the mechanics of skin in this model tensile test are conducted to determine the material properties of skin. The fracture toughness of the material is calculated by measuring the energy release rate of the material during required during cutting with Number 11 scalpel blade with a tip radius of 12 [micro]m . These results are then compared to a finite element analysis with a debonding interface and a Mooney-Rivlin hyperelastic material model with viscoelastic relaxation in an effort to predict the loads required by tools during surgical applications. The main outcome of this research is the development of a testing protocol and material model of skin that can be used in finite element simulations of uniaxial loads and surgical cutting.

“Blood vessel mimics” (BVMs) are tissue-engineered constructs that serve as in vitro preclinical testing models for intravascular devices. The Cal Poly Tissue Engineering lab specifically uses BVMs to test the cellular response to stent implantation. PLGA scaffolds are electrospun in-house using the current “Standard Protocol” and used as the framework for these constructs. The performance of BVMs greatly depends on material and mechanical properties of the scaffolds. It is desirable to create BVMs with reproducible properties so that they can be consistent models that ultimately generate more reliable results for intravascular device testing. Reproducibility stems from the consistency of the scaffolds. Thus, scaffolds with consistent material and mechanical properties are necessary for creating reproducible BVMs. The aim of this thesis was to characterize the reproducibility of the electrospun PLGA scaffolds using fiber diameter measurements and compliance testing. Initial work in this investigation involved designing and testing several experimental electrospinning protocols to obtain smaller fiber diameters, which have been shown to elicit more ideal cellular responses. The most successful protocol in that regard was then analyzed for the reproducibility of fiber diameters and compared to the reproducibility of the Standard Protocol. After determining that the Standard Protocol produced scaffolds with more consistent fibers, a large-scale reproducibility study was performed using this protocol. In this expanded study, both fiber diameter and compliance were analyzed and used to characterize the scaffolds. It was established that the scaffolds demonstrated inconsistent mean fiber diameter and mean compliance. The current standard electrospinning protocol therefore does not create PLGA scaffolds with statistically reproducible properties. Future modifications should be made to the electrospinning parameters in order to reduce variability between the scaffolds and future studies should be performed to determine the acceptable range of properties.

Medical implants constitute an essential advancement in modern medicine, often restoring or replacing functionality to failed organs. Whether a medical implant is temporary or permanent, medical implants carry the risk of implant failure due to an infection. Implant-associated infections (IAI) are challenging to treat and often result in increased medical costs, prolonged hospital stays, implant failure, and, in some instances, severe infections that can lead to amputations, sepsis, or mortality. Eradicating an IAI can be challenging since bacteria can form biofilms on the implant’s surface. The biofilms comprise an extracellular matrix protecting the bacterial cells against systemic antibiotics and the host’s immune system. Treating an IAI usually entails a broad range of antibiotic treatment and surgical procedures for tissue debridement or implant replacement. For the reasons stated above, scientists and engineers continue to develop technologies to protect the surface of medical implants against infections. Amongst the new technologies, Liquid-Infused Surfaces (LIS) are renowned for their repellent and anti-fouling properties created by tethering a stable liquid layer onto the surface. However, many challenges remain to adopt this technology for implantable devices. For instance, the high repellent properties can hinder implant-tissue interaction and discourage proper integration with the body. Furthermore, the stable liquid layer is contingent on the surface properties of the coated material. In other words, the long-term stability of these coatings may be compromised if the surface chemistry is covered by biological processes such as biofilm formation from adherent bacteria. This thesis aims to expand on the applications of LIS coatings and enhance their properties for implantable materials. This thesis reviews different types of antibiotic surface coatings and further examines LIS technologies as a viable antibacterial coating for medical implants. Then, three novel multifunctional LIS coatings are presented. The first developed coating enhanced the antibacterial properties of the coating by adding bactericidal agents within the LIS coating. The developed antibiotic liquid-infused coating not only repelled bacteria but also lysed bacteria upon contact. The second coating was designed to promote tissue integration. This multifunctional coating promoted cell deposition and proliferation while remaining repellent toward bacteria, while the conventional LIS coating displayed poor cell availability. Lastly, a collagen-bacteriophage conjugated liquid-infused coating was developed to promote tissue integration while having a two-tier layer of antibacterial protection. This coating was tested in a mouse sepsis model and prevented mortality of all mice, with other groups as high as 90% mortality. These coatings constitute essential steppingstones to bring LIS technology to medical implants. / Dissertation / Doctor of Philosophy (PhD) / Implant-associated infections (IAI) remain a significant problem in modern medicine. IAIs are challenging to treat and often result in increased medical costs, prolonged hospital stays, implant failure, and, in some instances, severe infections that can lead to sepsis or mortality. For these reasons, new technologies have been developed to protect the surface of medical implants against infections. Amongst the new technologies, Liquid-Infused Surfaces (LIS) are renowned for their repellent and anti-fouling properties created by tethering a stable liquid layer onto the surface. This thesis aims to expand on the applications of LIS coatings and enhance their properties for implantable materials. This thesis reviews different types of antibiotic surface coatings, examines LIS technologies, and presents three novel multifunctional LIS coatings. The newly developed coatings enhance the LIS coatings through the addition of antibacterial properties and biomolecules to promote tissue integration.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24834号 / 医博第5002号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内, 理, 教授 上杉, 志成, 教授 金子, 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM