Tato diplomová práce se zabývá tkáňovým inženýrstvím, zejména tvorbou homogenní, izotropní a planární vrstvy buněk srdečního svalu pomocí dvou technologii:”scaffold-based” a ”scaffold-free”. Nejprve popsaný histologie srdeční stěny, buňky srdečního svalu a buněčné kultury. Následuje popis tkáňového inženýrství, který zahrnuje technologii “cell sheet” a tkáňové inženýrství na bázi scaffoldů. Na konci teoretické části je popsána aplikace tkáňového inženýrství a buněčná vizualizace. Praktická část věnovaná tvorbě planární buněčné vrstvy z kardiomyocytů a fibroblastů s využitím informací z teoretické části.

Isatin-derived 1,2,4-triazines were employed as electron deficient azadienes in inverse electron demand Diels-Alder (IEDDA) chemistry with tethered alkynyl dienophiles, leading to biologically interesting heterocyclic lactam- and lactone-annulated α-carbolines in excellent yields under microwave promotion. The chemistry scope was probed using various alkynyl amines and alcohols, and the impact of the tether length between the triazines and the dienophiles was also investigated. This IEDDA methodology was subsequently applied to the synthesis of an α-carboline library, producing eighty-eight members starting with various isatin-derived triazines, indole-derivatives, and propargylamine derivatives. In a related project, a regioselective Lewis acid catalyzed amidation of dimethyl 5H-pyridazino[4,5-b]indole-1,4-dicarboxylate was established to selectively direct the amidation to occur at either the C1 or C4 ester positions. This chemistry was then applied to tether dienophiles to the pyridazinoindole ring via an amide linkage. Subsequent IEDDA cycloadditions of these pyridazinoindole/dienophile pairs afforded carbazoles in excellent yields under thermal conditions. The scope of this chemistry scope was also thoroughly probed, leading to a library of one hundred and eighty-eight members. The synthesis of a third heterocyclic scaffold using Diels-Alder chemistry was also accomplished. Asymmetric cycloadditions of anthrone with various maleimides were successfully achieved using different chiral organocatalysts. The identity of the optimal catalytic system depended upon the dienophile, with good enantioselectivities achieved (78-83% ee). This chemistry was successfully applied as the stereocontrol element in a Diels-Alder/Functional Group Transformation/retro-Diels-Alder sequence to prepare alkaloidal scaffolds in an optically enriched form.

In order to mimic the extracellular matrix for tissue engineering, recent research approaches often involve 3D printing or electrospinning of fibres to scaffolds as cell carrier material. Within this thesis, a micron fibre printing process, called melt electrospinning writing (MEW), combining both additive manufacturing and electrospinning, has been investigated and improved. Thus, a unique device was developed for accurate process control and manufacturing of high quality constructs. Thereby, different studies could be conducted in order to understand the electrohydrodynamic printing behaviour of different medically relevant thermoplastics as well as to characterise the influence of MEW on the resulting scaffold performance. For reproducible scaffold printing, a commonly occurring processing instability was investigated and defined as pulsing, or in extreme cases as long beading. Here, processing analysis could be performed with the aim to overcome those instabilities and prevent the resulting manufacturing issues. Two different biocompatible polymers were utilised for this study: poly(ε-caprolactone) (PCL) as the only material available for MEW until then and poly(2-ethyl-2-oxazoline) for the first time. A hypothesis including the dependency of pulsing regarding involved mass flows regulated by the feeding pressure and the electrical field strength could be presented. Further, a guide via fibre diameter quantification was established to assess and accomplish high quality printing of scaffolds for subsequent research tasks. By following a combined approach including small sized spinnerets, small flow rates and high field strengths, PCL fibres with submicron-sized fibre diameters (fØ = 817 ± 165 nm) were deposited to defined scaffolds. The resulting material characteristics could be investigated regarding molecular orientation and morphological aspects. Thereby, an alignment and isotropic crystallinity was observed that can be attributed to the distinct acceleration of the solidifying jet in the electrical field and by the collector uptake. Resulting submicron fibres formed accurate but mechanically sensitive structures requiring further preparation for a suitable use in cell biology. To overcome this handling issue, a coating procedure, by using hydrophilic and cross-linkable star-shaped molecules for preparing fibre adhesive but cell repellent collector surfaces, was used. Printing PCL fibre patterns below the critical translation speed (CTS) revealed the opportunity to manufacture sinusoidal shaped fibres analogously to those observed using purely viscous fluids falling on a moving belt. No significant influence of the high voltage field during MEW processing could be observed on the buckling phenomenon. A study on the sinusoidal geometry revealed increasing peak-to-peak values and decreasing wavelengths as a function of decreasing collector speeds sc between CTS > sc ≥ 2/3 CTS independent of feeding pressures. Resulting scaffolds printed at 100 %, 90 %, 80 % and 70 % of CTS exhibited significantly different tensile properties, foremost regarding Young’s moduli (E = 42 ± 7 MPa to 173 ± 22 MPa at 1 – 3 % strain). As known from literature, a changed morphology and mechanical environment can impact cell performance substantially leading to a new opportunity of tailoring TE scaffolds. Further, poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) as well as poly(ε-caprolactone-co-acryloyl carbonate) (PCLAC) copolymers could be used for MEW printing. Those exhibit the opportunity for UV-initiated radical cross-linking in a post-processing step leading to significantly increased mechanical characteristics. Here, single fibres of the polymer composed of 90 mol.% CL and 10 mol.% AC showed a considerable maximum tensile strength of σmax = 53 ± 16 MPa. Furthermore, sinusoidal meanders made of PCLAC yielded a specific tensile stress-strain characteristic mimicking the qualitative behaviour of tendons or ligaments. Cell viability by L929 murine fibroblasts and live/dead staining with human mesenchymal stem cells revealed a promising biomaterial behaviour pointing out MEW printed PCLAC scaffolds as promising choice for medical repair of load-bearing soft tissue. Indeed, one apparent drawback, the small throughput similar to other AM methods, may still prevent MEW’s industrial application yet. However, ongoing research focusses on enlargement of manufacturing speed with the clear perspective of relevant improvement. Thereby, the utilisation of large spinneret sizes may enable printing of high volume rates, while downsizing the resulting fibre diameter via electrical field and mechanical stretching by the collector uptake. Using this approach, limitations of FDM by small nozzle sizes could be overcome. Thinking visionary, such printing devices could be placed in hospitals for patient-specific printing-on-demand therapies one day. Taking the evolved high deposition precision combined with the unique small fibre diameter sizes into account, technical processing of high performance membranes, filters or functional surface finishes also stands to reason. / Um biomimetische extrazelluläre Matrices für das Tissue Engineering herzustellen, bedienen sich aktuelle Forschungsansätze oftmals der Produktion von Faser-Konstrukten durch additive Fertigung oder Elektrospinn-Verfahren. Das sogenannte Melt Electrospinning Writing (MEW) kombiniert Vorteile beider Techniken und weist dadurch ein hohes Applikationspotential auf. Daher bestand das Ziel der vorliegenden Arbeit in der Weiterentwicklung und Erforschung des MEW. Für diesen Zweck wurde eine neuartige Forschungsanlage konzipiert und gebaut, welche mit einzigartiger Verfahrenspräzision und Prozesskontrolle die Fertigung von hochqualitativen Konstrukten ermöglichte. Auf Basis dessen konnten die durchgeführten Studien das Verständnis des elektrohydrodynamischen Druckvorgangs und der untersuchten Prozessparameter vertiefen und letztendlich zur Ausweitung des Verfahrens auf neue medizinisch relevante Thermoplaste beitragen. Um eine reproduzierbare Herstellung von Scaffolds zu ermöglichen, wurde eine häufig auftretende Prozessinstabilität erforscht und als pulsing, oder in stark ausgeprägten Fällen als long beading, klassifiziert. Durch Prozessanalyse konnte zudem eine Methode zur Vermeidung dieser Instabilität entwickelt werden. Dafür wurden zwei unterschiedliche biokompatible Polymere verwendet: Poly(ε-Caprolacton) (PCL) als bis dahin einziger verfügbarer MEW Werkstoff, sowie erstmalig Poly(2-Ethyl-2-Oxazolin). Die aufgestellte Hypothese umfasst eine universelle Abhängigkeit der pulsing Instabilität zu involvierten Massenströmen, welche durch Anpassung des angelegten Prozessdruckes und der elektrischen Feldstärke reguliert werden kann. Um ein optimales Prozessergebnis für nachfolgende Forschungsarbeiten zu erzielen, wurde zusätzlich ein Leitfaden zur quantitativen Bewertung des Grades der Instabilität bereitgestellt. Durch Kombination kleiner Spinndüsen, kleiner Schmelze-Flussraten und hoher elektrischen Feldstärken, konnten erstmalig PCL Fasern mit sub-mikron Durchmessern (fØ = 817 ± 165 nm) zu präzisen Scaffolds verarbeitet werden. Diese wurden anschließend durch materialwissenschaftliche Analytik charakterisiert. Dabei wurde eine molekulare Vorzugsorientierung und isotrope Kristallausrichtung entlang der Faser beobachtet, welche durch den hohen Verstreckungsgrad des erstarrenden Polymerstrahls erklärt werden konnte. Resultierende sub-mikron Fasern konnten zwar für einen akkuraten Druckvorgang verwendet werden, jedoch erwiesen sich die Strukturen als instabil und daher nicht geeignet für die Handhabung bei Zellkulturstudien. Aus diesem Grund wurde ein Beschichtungsansatz mittels hydrophilen und vernetzbaren Sternmolekülen für Substratflächen herangezogen. Während solche modifizierten Oberflächen bekanntermaßen Zelladhäsion verhindern, konnten gedruckte sub-mikron Scaffolds auf der Oberfläche haften und so für biologische Studien verwendet werden. Durch das gezielte Ablegen von Fasern unterhalb der kritischen Translationsgeschwindigkeit (CTS) des Kollektors, konnten sinusförmige Faserstrukturen erzeugt werden. Analog zu rein viskosen Fluiden, welche durch ein bewegliches Band aufgesammelt werden, schien dieser Vorgang dem sogenannten buckling zu unterliegen und daher phänomenologisch nicht oder nur geringfügig vom elektrischen Feld abhängig zu sein. Zudem konnte eine durchgeführte Studie die direkte Abhängigkeit der Fasergeometrie mit der Kollektorbewegung belegen. Unabhängig vom Prozessdruck, führte eine verminderte Kollektorgeschwindigkeit sc in den Grenzen CTS > sc ≥ 2/3 CTS zu erhöhten Amplituden bzw. Spitze-zu-Spitze Werten und verkürzten Wellenlängen. Durch das kontrollierte Ablegen der Fasern bei Geschwindigkeiten von 100 %, 90 % 80 % und 70 % CTS konnten zudem Scaffolds mit unterschiedlichen mechanischen Eigenschaften hergestellt werden. Speziell der Zugmodul wurde dadurch etwa um eine halbe Größenordnung moduliert (Es = 42 ± 7 MPa bis 173 ± 22 MPa bei 1 – 3 % Dehnung). Dies ist in Kombination mit der Strukturierung für maßgeschneiderte TE Scaffolds von großem Interesse, da zelluläre Systeme sensibel auf ihre Umgebung reagieren können. Des Weiteren wurden Poly(L-Lactid-co-ε-Caprolacton-co-Acryloylcarbonat) und Poly(ε-Caprolacton-co-Acryloylcarbonat) (PCLAC) Copolymere hinsichtlich deren MEW Verarbeitbarkeit untersucht. Solche Kunststoffe können nach dem Druckvorgang mit UV-Strahlung radikalisch vernetzt werden und dadurch deutlich erhöhte mechanische Eigenschaften ausbilden. Für Fasern aus 90 mol.% CL und 10 mol.% AC wurden beispielsweise maximale Zugfestigkeiten von σmax = 53 ± 16 MPa ermittelt. MEW gedruckte sinusförmige Faserstrukturen aus PCLAC wiesen darüber hinaus ein biomimetisches Spannungs-Dehnung-Verhalten auf, vergleichbar zu Sehnen- und Ligamentgewebe. Eine Untersuchung der Zellviabilität von L929 murinen Fibroblasten im Eluattest, sowie eine lebend/tot-Färbung von humanen mesenchymalen Stammzellen auf den Scaffolds, ergab vielversprechende Resultate und damit ein relevantes Anwendungspotential solcher Strukturen als Implantat. Neben genannten Vorteilen, weist MEW als Verfahren bislang allerdings geringe Produktionsgeschwindigkeiten auf. Diese sind daher in den Fokus aktueller Forschungsvorhaben gerückt. Einen Ansatz hierfür bieten Spinndüsen mit hohem Innendurchmesser und erhöhter Austragsrate, wobei die optimierte elektrische Feldstärke, sowie ein Verstrecken durch die Kollektorbewegung, zu den erwünschten dünnen Fasern führen können. Dadurch kann die abwärtslimitierte Düsengröße des FDM Verfahrens überwunden werden. Visionär gedacht, könnte eine solche Anlage direkt in Krankenhäusern zur Fertigung von patienten- und defektspezifischen Implantaten eingesetzt werden. Darüber hinaus ermöglicht die hohe Präzision, zusammen mit dem Drucken von Mikro-Fasern, einen technischen Einsatz zur Herstellung von Membranen, Filtern oder funktionalen Oberflächenbeschichtungen.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Root canal disinfection is an essential requirement for the success of regenerative endodontics. Currently, the so-called triple antibiotic paste (TAP) is considered the standard of care. Notwithstanding the good antimicrobial capacity, the high concentration of TAP has shown significant toxicity to human cells, especially dental pulp stem cells. A novel drug release system, i.e., a triple antibiotic paste-mimic electrospun scaffold containing low concentrations of the antibiotics present in the TAP, has emerged as an effective and reliable alternative to fight root canal infections without potential toxic effects on dental stem cells, which are an integral part of the regenerative treatment. Objectives: The aim of this study was to determine the antimicrobial efficacy of an innovative three-dimensional (3D) triple antibiotic paste-mimic tubular scaffold against Actinomyces naeslundii biofilm formed inside human root canal dentinal tubules. Materials and methods: Pure polydioxanone (PDS) polymer solution and PDS loaded with metronidazole, ciprofloxacin and minocycline (35 wt.% of each antibiotic, 3D-TAP-mimic scaffold) were spun into 3D fibrous scaffolds. A. naeslundii (ATCC 43146) was centrifuged to induce biofilm formation inside human root canal dentinal tubules using a dentin slice model (1 mm thickness and 2.5 mm canal diameter). The infected dentin slices were exposed to the 3D-TAP-mimic scaffold, TAP solution (50 mg/mL of each antibiotic), and antibiotic-free PDS. Biofilm elimination was quantitatively and qualitatively analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results: A dense penetration of A. naeslundii biofilm was observed by CLSM throughout the dentinal tubules. 3D-TAP-mimic scaffold significantly reduced the percentage of viable bacteria compared with PDS (p <.05). TAP solution completely eliminated viable bacteria without differing from 3D-TAP-mimic scaffolds. SEM images showed results similar to CLSM. Conclusion: Collectively, the proposed tubular 3D-TAP-mimic scaffold holds significant clinical potential for root canal disinfection strategy prior to regenerative endodontics.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19570号 / 医博第4077号 / 新制||医||1013(附属図書館) / 32606 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Mammary epithelial cells are highly efficient secreting cells. With genetic engineering, the uses of these cells could be endless. Research is being conducted on these cells to determine their full potential to the biotech industry. This paper investigates whether bovine epithelial mammary cells can survive in glutaraldehyde-treated gelatin tubes in an operating bioreactor. Many bioreactors were developed and tested to suit the needs of the cells. Procedures were created and carried out to ensure sterility of the bioreactors. Bovine mammary epithelial cells were implanted in the bioreactors and samples of their growth were taken over time.

“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.

Cardiac disease causes approximately a third of the deaths in the United States. Furthermore, most of these deaths are due to a condition termed atherosclerosis, which is a buildup of plaque in the coronary arteries, leading to occlusion of normal blood flow to the cardiac muscle. Among the methods to treat the condition, stents are devices that are used to restore normal blood flow in the atherosclerotic arteries. Before advancement can be made to these devices and changes can be tested in live models, a reliable testing method that mimics the environment of the native blood vessel is needed. Dr. Kristen Cardinal developed a tissue engineered blood vessel mimic to test intravascular devices. Among the scaffolding material used, electrospun poly (lactide-co-glycolide) (PLGA) has been used as an economic option that can be made in house. PLGA is a biodegradable co-polymer, and when electrospun, creates a porous matrix with tailorable properties. Currently, the standard PLGA electrospinning protocol produces consistent fibrous scaffolds with a mean fiber diameter of 5-6 microns. Research indicates that cell adhesion is more successful in fibrous matrices with a mean fiber diameter at the nanometer level. However, because previous work in the Tissue Engineering Laboratory at Cal Poly sought to ensure a consistent fibrous, there was no model or equation to determine how to change the electrospinning parameter settings to create scaffolds with an optimal mean fiber diameter. To fill this need, biomedical engineering senior Steffi Wong created a design of experiment to systematically approach the electrospinning variables and determine how they interacted with each other, as well as their effect on fiber diameter. The aims of this thesis were to perform the said design of experiments and determine a model to predict the resulting mean fiber diameter of a scaffold based on the electrospinning parameters as well as to determine what combination of parameters would lead to a scaffold with an optimal mean fiber diameter between 100-200 nanometers. The variables tested were solution concentration, gap distance, flow rate, and applied voltage. Each scaffold was imaged and a mean fiber diameter was calculated and used as the predicted variable in a regression analysis, with the variables indicated above as the predictors. The goal of 100-200 nanometer mean fiber diameter was not reached. The smallest mean fiber diameter calculated was 2.74 microns—half of that of the standard protocol. The regression analysis did result in a model to describe how the voltage, gap distance, and flow rate affected the fiber diameter.

<p>Carbon nanotubes (CNTs) are arguably the most widely studied material within the field of nanotechnology. The impressive physical and electronic properties of these materials have led to their investigation in a diverse range of applications. In recent years, the deposition of inorganic nanoparticles upon the surface of CNTs has received much attention. Research within this field has been driven by the desire to develop new composite materials exhibiting novel electronic, optical, and catalytic properties. In addition to the decoration of individual nanotubes, several groups have also investigated the use of CNT thin films as scaffolds for the assembly of inorganic nanostructures as well as other secondary components, including polymers and biomaterials. Nanotube films exhibit a number of physical properties that make them suitable for such applications, displaying impressive electrical conductivity, flexibility, and thermal stability while also possessing a high surface area upon which chemical modification can be conducted.</p><p>This thesis presents work that demonstrates the potential of CNT thin films as flexible conductive scaffolds for the support of a variety of inorganic nanostructures. Procedures are described for the preparation of SWNT -Au nanoparticle composite films that, as subsequently demonstrated, are suitable substrates for the growth of III-V semiconductor nanowires using gas-source molecular beam epitaxy. At the time of writing, the majority of published research within this field focused upon the preparation of SWNT composite films containing spherical metallic or semiconductor nanoparticles. In contrast, the growth of semiconductor nanowires upon nanotube thin films had not been explored. The work described in this thesis therefore represents the development of a novel composite material that combines the flexibility of CNT films with the unique optoeletronic properties exhibited by semiconductor nanowires. The development of functional electronic devices incorporating these materials is also discussed, as is the extension of the methods developed to investigate novel composite materials that combine other inorganic nanostructures with carbon based substrates.</p> / Thesis / Doctor of Philosophy (PhD)