Im Rahmen dieser Arbeit wurde für ein magnetisches Hydrogel auf Alginat-Methylcellulose-Basis und Mikropartikeln aus Magnetit über einen Beobachtungszeit-raum von mindestens 14 Tagen eine umfassende Charakterisierung des Deformationsverhaltens auf makroskopischer Ebene und der Partikelanalyse auf mikrostruktureller Ebene in Abhängigkeit von Partikelgehalt, Alterung und Lagerungsbedingungen durchgeführt. Für diese Untersuchungen wurde ein, den Maxwell-Aufbau verwendender Versuchsstand konstruiert, der ein definiertes Feld mit einem annähernd konstanten Feldgradient in axialer Richtung aufweist. Die Hauptuntersuchungen wurden an Tag 0, 1 und 14 für Proben mit 5, 15, 25 und 35 wt% Magnetit durchgeführt. Schwerpunkte der Arbeit sind zum einen die Bestimmung von E-Modul, Zeitverhalten und Biegeverhalten und zum anderen die Erstellung von Modellen zur Beschreibung des Deformationsverhaltens. Als weitere Eigenschaften wurden das Fließverhalten, die Formstabilität, die Dichte und die magnetischen Eigenschaften analysiert. Hinsichtlich einer zyklischen Stimulation für eine medizinische Anwendung wurden Proben mit 25 wt% Magnetit über 14 Tage täglich einer Frequenz von 1 Hz über 3 h ausgesetzt. Dafür wurde im Rahmen dieser Arbeit der 9-Kammern-Bioreaktor CyMAD entwickelt und nach erfolgter Stimulation Untersuchungen zum Biegeverhalten und eine Partikelanalyse durchgeführt.:Inhaltsverzeichnis I Abkürzungsverzeichnis V Symbolverzeichnis VI 1 Einleitung 1 1.1 Medizinischer Hintergrund und Motivation 1 1.2 Lösungsansatz 2 1.3 Ziele und Inhalt der Arbeit 3 2 Grundlagen 5 2.1 Matrixmaterial 5 2.1.1 Alginat 5 2.1.2 Methylcellulose 6 2.1.3 Alginat-MC-Scaffolds 8 2.2 Magnetismus 9 2.3 Tissue Engineering 12 2.3.1 Additive Fertigung von Hydrogelen 13 2.3.2 Gestaltungs- und Einsatzmöglichkeiten von Alginatscaffolds 15 2.3.3 Smart Materials im Tissue Engineering - Ermittlung des Deformationsvermögens und Anwendungen von magnetischen Druckpasten 16 2.3.4 Stimulation von Zellen – applizierte Dehnungen und Versuchsaufbauten 17 3 Materialien und Probenherstellung 21 3.1 Wahl der magnetischen Partikel 21 3.2 Herstellung der Druckpaste 22 3.3 Probenherstellung 22 3.3.1 Versuche zum Biegeverhalten 22 3.3.2 Zugversuche 22 3.3.3 VSM-Messungen 23 3.4 Lagerungsbedingungen 24 4 Verwendete und angepasste Messtechnik 25 4.1 Rotationsviskosimeter und Zugprüfmaschine 25 4.1.1 Definition der mechanischen Eigenschaften 25 a) Fließverhalten 25 b) Elastizitätsmodul 26 4.1.2 Ermittlung der mechanischen Eigenschaften 27 a) Messgerät Physica MCR 301von Anton Paar 27 b) Aufbau als Rotationsviskosimeter und Versuchsdurchführung 28 c) Aufbau, Durchführung und Adaptionen der Zugversuche hinsichtlich der Messung von Hydrogelen 29 4.2 Vibrating Sample Magnetometer 30 4.2.1 Aufbau und Funktionsweise 31 4.2.2 Messgerät VSM 7407 32 4.3 Röntgencomputertomografie 32 4.3.1 Grundlagen der Erzeugung von Röntgenstrahlung 32 4.3.2 Messprinzip und Datenverarbeitung der Röntgencomputertomografie 33 4.3.3 Experimentelle Aufbauten für die Röntgencomputertomografie 36 a) Messtechnische Gegebenheiten der Labortomografieanlage TomoTU 36 b) Versuchsstand „Magnetmesszelle“ 37 5 Verarbeitung der Bilddaten 40 5.1 Makroskopische Betrachtungen 40 5.1.1 Ermittlung der Biegelinie 40 5.1.2 Ermittlung des Zeitverhaltens 42 5.1.3 Ermittlung der Formstabilität 42 5.2 Mikroskopische Betrachtungen 43 5.2.1 Ermittlung der Partikelverteilung mittels Watershed-Algorithmus 43 5.2.2 Ermittlung der richtungsabhängigen Paarkorrelationsfunktion 44 6 Modellierung des Deformationsverhaltens 46 6.1 Beschreibung des Zeitverhaltens 46 6.1.1 PTn-Glieder (Regelungstechnik) 46 6.1.2 Retardation von Kunststoffen 47 6.2 Modellierung der Durchbiegung mittels Balkentheorie 48 6.2.1 Annahmen und Vereinfachungen 48 6.2.2 Gestaltung der Probenaufnahme 49 6.2.3 Berechnungen der Biegelinie und der benötigten Kraft 51 6.3 Berechnung der magnetischen Kraft 52 6.4 Modellbereich 54 7 Grundcharakterisierung und Vorbetrachtungen 55 7.1 Reproduzierbarkeit der Alginat-PBS-Lösung 55 7.2 Charakterisierung des verwendeten Magnetits 57 7.2.1 Oberflächenbeschaffenheit und Form 57 7.2.2 Partikelgrößenverteilung 57 7.3 Sensitivitätsanalyse 58 7.3.1 Wahl der Parameter und deren Kombination 58 7.3.2 Einfluss von Temperatur und Lagermedium auf den Probendurchmesser 59 7.3.3 Zusammenfassung 62 7.4 Formstabilität der Proben 62 7.5 Dichte 63 7.6 Magnetische Eigenschaften 64 8 Experimentelle Untersuchungen zum Deformationsverhalten 67 8.1 Elastizitätsmodul 67 8.1.1 Grundlegendes zur Auswertung 67 8.1.2 E-Moduln nach der Vernetzung 70 8.1.3 E-Modul in Abhängigkeit von Zeit und Partikelkonzentration 73 8.1.4 E-Modul für 25 wt% Magnetit in Abhängigkeit von der Zeit 75 8.1.5 E-Modul für einen beschleunigten Alterungsprozess 76 8.2 Zeitverhalten 77 8.2.1 Sprungantwort 77 a) Fitfunktion zur Beschreibung des zeitabhängigen Deformationsverhaltens 77 b) Definition und Bestimmung der Parameter 81 8.2.2 Schlussfolgerungen für die Untersuchungen zum Deformationsverhalten 82 a) Bedeutung für die Erfassung der Biegelinie 82 b) Bedeutung für zyklische Belastungen 83 8.3 Biegeverhalten 83 8.3.1 Validierung des Fastscans 85 8.3.2 Biegelinien 88 a) Abhängigkeit der Biegelinie von Alter und Partikelkonzentration 88 b) Vergleich nicht stimulierter und stimulierter Proben mit 25 wt% Magnetit 91 8.3.3 Vergleich von Theorie und Praxis 92 8.3.4 Fehlerdiskussion 98 a) Korrektur der Biegelinie 98 b) Eignung des Modells 100 8.4 Partikelanalyse 101 8.4.1 Partikelverteilung 102 8.4.2 Paarkorrelationsfunktion 108 8.5 Bedeutung für das TE 114 8.5.1 Vergleich zu etablierten Verfahren im TE 114 8.5.2 Übertragbarkeit auf autoklaviertes Material 115 8.5.3 Gestaltungsansatz für Scaffolds 115 9 Zusammenfassung 118 10 Ausblick 121 Tabellenverzeichnis X Abbildungsverzeichnis XI Literaturverzeichnis XVI

Polysaccharides are known as among the most abundant natural polymers on the Earth. As this class of material is usually renewable, biodegradable, biocompatible in many contexts and environmentally friendly, it is of great interest to use these benign polymers to design and prepare materials, especially for applications with green and biomedical purposes. In this dissertation we will discuss novel pathways to two different types of polysaccharide-based materials: block copolymers and hydrogels. Block copolymers are composed of two or even more covalent bonded polymer blocks that have quite distinct properties. Synthesis of polysaccharide-based block copolymers is an attractive and challenging research topic, opening up promising application potential and requiring advances in polysaccharide regio- and chemoselectivity. Herein, we report two independent approaches to prepare these interesting and potential useful materials. In one approach, trimethyl cellulose was modified regiospecifically at the reducing end anomeric carbon to create an ω-unsaturated alkyl acetal by solvolysis with an ω-unsaturated alcohol. Then, olefin cross-metathesis, a known versatile and mild tool for polysaccharide chemical modification, was used to couple the trimethyl cellulose block with various polymer blocks containing acrylates. To demonstrate the method, trimethyl cellulose-b-poly(tetrahydrofuran), cellulose-b-poly(ethylene glycol), and cellulose-b-poly(lactic acid) were synthesized by this coupling strategy. In another approach, we introduced a simple and novel method to prepare dextran-based block copolymers. In this strategy, N-bromosuccinimide (NBS)/triphenyl phosphine (PPh3) was chosen to regioselectively brominate the only primary alcohol of linear unbranched dextran. The resulting dextran, bearing a terminal C-6 bromide, was coupled with several amine terminated polymers via SN2 substitution to obtain block copolymers, including dextran-b-polystyrene, dextran-b-poly(N-isopropylacrylamide) and dextran-b-poly(ethylene glycol). Dextran-b-poly(N-isopropylacrylamide) exhibits thermally-induced micellization as revealed by dynamic light scattering, forming micelles with 155 nm diameter at 40 °C. Dextran-b-polystyrene film was analyzed by small angle X-ray scattering, suggesting the existence of microphase separation. This dissertation also introduces a novel, simple and effective strategy to prepare polysaccharide-based hydrogels. Hydrogels are typically crosslinked hydrophilic polymers that have high water affinity and no longer dissolve in water. Polysaccharide-based hydrogels are of great interest to for biomedical applications due to their benefits including biocompatibility, polyfunctionality, and biodegradability. Recently the Edgar group has discovered that chemoselective oxidation of oligo(hydroxypropyl)-substituted polysaccharides impairs ketone groups at the termini of the oligo(hydroxypropyl) side chains. These ketones can condense with amines to form imines, leading hydrogel formation., Based on this concept, we prepared oxidized hydroxypropyl polysaccharide/chitosan hydrogels. This class of all-polysaccharide hydrogels exhibits a series of interesting properties such as tunable moduli (300 Pa to 13 kPa), self-healing, injectability, and high swelling ratios. To further explore imine-crosslinked hydrogels, we designed thermally responsive hydrogels by using a Jeffamine, a polyethylene oxide-b-polypropylene oxide-b-polyethylene oxide triblock copolymer with two terminal amines. As the Jeffamine has a lower critical solution temperature, oxidized hydroxypropyl cellulose/Jeffamine hydrogels display moduli that are tunable by controlling the temperature. / Doctor of Philosophy / Polysaccharide are natural polymers that are among the most abundant polymers on Earth. It is greatly in society's interest to extend the scope of their applications, due to the benign nature of polysaccharides. This dissertation mainly focuses on two polysaccharides: cellulose and dextran. Cellulose is a long linear polymer of linked glucose molecules. As cellulose is sustainable, biodegradable, non-toxic, affordable and accessable for chemical modification, it is a suitable polymer for biomedical and environmentally friendly application. Dextran is also a polymer chain made up only of glucose but connected with each other differently from cellulose by, bacterial fermentation, and it may be lightly branched. It is biocompatible in many situations and is biodegradable both in vivo and in the environment, thus it has been investigated for drug delivery and many other medical applications. Using these two polysaccharides, we designed and prepared two quite different classes of materials: block copolymers and hydrogels. Block copolymers consist of two or more different types of polymer blocks connected by strong covalent bonds. As block copolymerization enables construction of a single polymer comprising segments with distinct properties, it is appealing to synthesize a block copolymer which preserves the properties of natural polymers coupled to very different polymers, such as polyolefins (e.g. the polyethylene that is used for milk bottles). In order to prepare polysaccharide-based block copolymers, we developed two different synthetic routes to end-functionalize methyl cellulose and dextran , and these resulting products were used to prepare two independent series of polysaccharide-based block copolymers via combination (in other words, sticking the polysaccharide and, e.g., the polyethylene together end to end). This study confirms the feasibility of this method to make methyl cellulose-based and dextran-based block copolymers. We expect these classes of materials will have significant potential in applications including drug delivery, as compatibilizers for polymer blends of materials that otherwise cannot be mixed (polyolefin/polysaccharide), membrane and adhesive. Hydrogels are crosslinked polymer networks with high water affinity, and they have been heavily investigated in the field of tissue engineering, drug delivery, agriculture and 3D printing. Polysaccharide-based hydrogels are attractive materials for these applications because they are biocompatible, biodegradable and have polyfunctionality. However, any use of toxic small molecules to crosslink the hydrogels diminishes their usefulness in biomedical applications. In this work, we demonstrate a simple, green and efficient method for preparation of all-polysaccharide-based hydrogels. The starting materials, oxidized hydroxypropylpolysaccharide, were simply prepared by using household bleach (NaOCl) as the oxidation reagent. We discovered that oxidized hydroxypropyl polysaccharides readily form hydrogels with hydrophilic amine-containing polymers like chitosan (a natural polysaccharide that comes from shells of crustaceans like crabs or shrimp) and Jeffamines, affording interesting properties including tunable stiffness, self-healing, injectability, and responsiveness to acidity and temperature. We expect that this new class of hydrogel will be very promising for biomedical-related applications.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25188号 / 医博第5074号 / 新制||医||1072(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 永井 純正, 教授 椛島 健治, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Partial or complete tears of the anterior cruciate ligament (ACL) can greatly afflict quality of life and often require surgical reconstruction with autograft or allograft tissue to restore native knee biomechanical function. However, limitations exist with these treatments that include donor site pain and weakness found with autografts, and longer "ligamentization" and integration times due to the devitalization of allograft tissue. Alternatively, a tissue engineering approach has been proposed for the fabrication of patient-specific grafts that can more rapidly and completely heal after ACL reconstruction. Electrospun micro-fiber networks have been widely utilized as biomaterial scaffolds to support the growth and differentiation of mesenchymal stem cells toward many tissue lineages including ligament. However, these micro-fiber networks do not possess suitable sizes and shapes for a ligament application and cannot support cell infiltration. The objective of this work was to develop techniques to 1) rapidly cellularize micro-fiber networks, 2) assemble micro-fiber networks into cylindrical composites, 3) provide cues to mesenchymal stem cells (MSCs) to guide their differentiation toward a ligament phenotype. The cellularization of micro-fiber networks was performed utilizing a co-electrospinning/electrospraying technique. Cells deposited within a cell culture medium solution remained where they were deposited and did not proliferate. The inclusion of space-filling hydrogel network such as collagen was necessary to reduce the density of the micro-fiber network to facilitate spreading. However, it became apparent that the incorporation of significant collagen phase was necessary for long-term MSC survival within the micro-fiber network. Next, two approaches were developed to fabricate large cylindrical, composites. The first approach utilized a co-electrospinning/electrospraying technique to generate micro-fiber/collagen composites that were subsequently rolled into cylinders. These cylindrical composites exhibited greater diameters and water weight percentages as collagen content increased. However, the high micro-fiber content of these composites was inhibitory to cell survival. In the second approach, thin layers (~5-10 fibers) of aligned electrospun PEUR fibers were encapsulated within a collagen gel and subsequently rolled the composites into cylinders. These sparse-fiber composites were nearly 98% by weight water and confocal imaging revealed the presence of sparse fiber layers (~5 fibers thick) separated by approximately 200 μm thick collagen layers. We hypothesize that the proliferation and migration of MSCs within these micro-fiber/collagen composites may not be restricted by the presence of a dense, non-manipulatable electrospun fiber network present in traditionally rolled fiber composites. Simple model platforms were then developed to study the influence of sparse micro-fibers on MSCs differentiation within a collagen hydrogel. MSCs in the presence of the softest (5.6 MPa) micro-fibers elongated and oriented to the underlying network and exhibited greater expression of scleraxis, and α-smooth muscle actin compared to the stiffest (31 MPa) fibers. Additionally, preliminary results revealed that the incorporation of fibroblast growth factor-2 and growth and differentiation factor-5 onto micro-fibers through chemical conjugation enhanced expression of the ligamentous markers collagen I, scleraxis, and tenomodulin. In conclusion, micro-fiber/collagen composite materials must possess sufficient space to support the infiltration and differentiation of MSCs. The strategies described in this document could be combined to fabricate large, micro-fiber/collagen composites that can support cell infiltration and provide relevant cues to guide the formation of an engineered ligament tissue. / Ph. D.

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. / Master of Science / Tendon injuries are common in horses and are painful and can be career- and life-ending. Tendons have a special structure and organization that enables them to withstand high tensile forces without permanent deformation. Injured tendons heal by forming stiff, disorganized scar tissue that makes the tendon more prone to re-injury. The lining of urinary bladders from pigs (UBM) provides a physical mesh and signaling factors that help heal injuries in a variety of tissues to a more normal state. However, UBM has not been evaluated in a laboratory model of tendon tissue formation to determine how it can help heal tendon injuries. Three-dimensional models of new tendon tissue formation (neotendons) were made with rat tail tendon matrix and stem cells collected from horse bone marrow. The neotendons were placed under steady tension for 3 weeks. The models were collected after 1 and 3 weeks to measure their width, numbers of live cells, cell and matrix organization, levels of tendon matrix components and expression of genes found in tendons. Most cells in the neotendons remained alive during the study period. Over time, UBM-treated and untreated neotendons became narrower compared to their starting width. The width of UBM-treated neotendons decreased faster than non-treated neontendons in the first week of the study. Cells became longer, narrower, and oriented along lines of tension. Expression of genes important in tendon development and structure either increased or was constant over time. UBM treatment did not change cell shape or increase levels of tendon-associated genes, DNA, or tendon matrix components. Our novel tendon model successfully created organized tendon-like tissue when placed under tension. However, UBM treatment did not improve formation of tendon-like tissue to a greater extent than controls.

The research presented in this thesis includes the development of designs, materials, and fabrication processes and the results of characterization experiments for a meso-scale hydrogel-based lipid bilayer array system. Two design concepts are investigated as methods for forming Droplet Interface Bilayer (DIB) arrays. Both concepts use a base of patterned silver with Ag/AgCl electrodes patterned onto a flat polymer substrate. In one technique, photopolymerizable hydrogel is cured through a mask to form an array of individual hydrogels on top of the patterned electrodes. The other technique introduces a second type of polymer substrate that physically supports an array of hydrogels using a set of microchannels. This second substrate is fitted onto the first to contact the hydrogels to the electrodes. The hydrogels are used to support and shape droplets of water containing phospholipids, which self-assemble at the surface of the droplet when submerged in oil. Two opposing substrates can then be pushed together, and a bilayer will form at the point where each pair of monolayers come into contact. The photopatterning technique is used to produce small arrays of hydrogels on top of a simple electrode pattern. Systems utilizing the microchannel substrate are used to create mesoscale hydrogel arrays of up to 3x3 that maintained a low resistance (~50-150 kΩ) connection to the substrate. Up to three bilayers are formed simultaneously and verified through visual observation and by recording the current response behavior. Arrays of varying sizes and dimensions and with different electrode patterns can be produced quickly and inexpensively using basic laboratory techniques. The designs and fabrication processes for both types of arrays are created with an eye toward future development of similar systems at the microscale. / Master of Science

Keratins are a family of structural proteins that can be extracted from a variety of sources including wool, nails, skin, hooves, and hair. Keratin can be processed into different constructs such as coatings, scaffolds, and hydrogels, and has shown favorable results when placed in in vitro and in vivo settings for different tissue regeneration applications. Over three decades, keratin extraction technology has been continuously modified, and these differences in extraction processes have distinct effects on the characteristics of the end product. In this work, we examine the effect of keratin aggregation during a widely-used purification step, dialysis ultra-filtration, on material characteristics of the final keratin product when fabricated into a hydrogel. Two distinct dialysis procedures were applied during the extraction of oxidized keratin (keratose): one promoting protein aggregation and the other mitigating it. Analyses of material properties such as mechanical and enzymatic stability were conducted in addition to observing the differences in solution behavior between products. Data revealed that protein aggregation during the extraction process has a profound effect on keratose hydrogel material properties. After determination of the effect of protein aggregation during extraction on keratose hydrogels, investigation of how a blended material comprised of said keratose and type I collagen was undertaken. It was hypothesized that a blend would result in mixing at the molecular level, resulting in improved properties compared to either pure material alone. A protocol was created to make stable keratose/type I collagen blends and material characterization techniques were applied to determine the inherent properties of samples with differing ratios. Crosslinking density, mechanical properties, enzymatic degradation properties, water uptake capacity, structural architecture, and thermal properties were all assessed. In addition, the ability of this material to maintain cell viability was conducted. Results showed that the addition of type I collagen has a significant effect on the properties of hydrogel blends with keratose compared to the pure keratose system. This was mostly evident with hydrogel mechanical stability and material architecture. Finally, the ability to use this hybrid material as a luminal filler for a nerve conduit during peripheral nerve regeneration was explored in an in vitro setting. The ability of this blend to promote Schwann cell viability was assessed in addition to determining the ability of these cells to attach and migrate through the material matrix. These experiments demonstrate proof-of-concept for the application of using keratose/type I collagen matrices as a luminal filler in peripheral nerve guidance conduits. / Doctor of Philosophy / Keratins are a family of structural proteins that can be extracted from wool, skin, nails, and hair, and that have been investigated in the field of tissue regeneration. Humans make several types of keratins, so it has a natural acceptance by the body and its inflammatory and immune systems. However, keratins can be hard to make and process into useful products. Many methods for producing keratin biomaterials have been developed over the past 30 years, but most of them are not ideal. This work sought to explore a production method that addresses a particular problem, that of protein aggregation during purification. In so doing, methods can be optimized to create more useful keratin biomaterials. Experiments comparing preparation methods that maximize and minimize protein aggregation were compared. Data showed that minimizing aggregation leads to better biomaterial characteristics, thus demonstrating the potential impact of targeting this processing step. However, even after optimization of purification, keratins still have limitations. Most notably their mechanical strength is not as great as some other materials. A typical approach to address this in other systems has been by blending. In the present work, we explored a blend made from keratin and type 1 collagen. A method was developed to effectively blend keratin and collagen and create stable mixtures that yielded protein-to-protein coordination. Such interactions typically yield beneficial material characteristics such as increased strength. Data showed that intimate mixing of the two proteins was achieved, and resulting characteristics were improved compared to either pure material. Finally, studies were conducted to assess the potential for keratin/collagen blends to be used to regenerate injured nerves. A common method is to enclose the ends of a cut nerve into a tube and let the nerve re-grow through the tube to its target muscle. An important characteristic is an ability for cells to populate the interior of the tube and help the nerve fibers grow. In the present study, we investigated the behavior of a particularly important cell, the Schwann cell, to attach, move and grow through a keratin/collagen biomaterial. Data showed good cell behavior, suggesting that the material could be used in a medical product for nerve repair.

<p dir="ltr">PDAC, the most common type of pancreatic cancer, is a highly metastatic cancer that has a low survival rate. It is histologically characterized by a thick desmoplastic stroma. Counterintuitively, PCCs can still manage to survive in such a restrictive environment and even metastasize to distant organs. Over the years, efforts have been made to find out the mechanisms underlying these perplexing behaviors. However, questions about the role of ECM accumulation and enhanced stiffness in PCC dissemination remained unanswered. In this dissertation, we aim to advance the material design for tumor modeling, and propose an explanation for the malignant cell behavior in the PDAC TME. This is achieved through the use of hydrogel-based tumor models that recapitulate the elevated stiffness of the tumor tissue. Specifically, hydrogel stiffness was tuned to mimic the PDAC TME to understand how PCCs and CAFs respond to various substrate stiffnesses temporally. Next, we employ bio-orthogonal click chemistries to create hydrogels with on-demand stiffening capabilities, as well as hyaluronic acid deposition in the hydrogel, to investigate the effect of dynamic change in matrix stiffness and composition on PDAC cells and CAFs. Lastly, by leveraging thiol-norbornene, aldehyde-hydrazide, and tetrazine-norbornene click chemistries, we created a microporous hydrogel system with a conformation that combines both the advantage of 3D cell culture and the non-restricting nature of 2D cell culture. Additionally, the system allows the application of modularized user-defined factors, including, but not limited to stiffness and HA deposition to the system. Stiff gel in 2D facilitated cell spreading of Pa03C in the presence of CAF. Despite being more restrictive on cell spreading, stiff gelatin gel in 3D induced cytokines that promote matrix remodeling and spreading cell morphology can be restored by stiffening with HA. Overall, this dissertation demonstrated that ECM component (i.e., HA), culture dimensionality, and cell-cell interaction play a huge role in cell behavior, and these factors may interact with each other and result in synergistic effects.</p>

Irreversible electroporation has recently emerged as an effective focal ablation technique. When performed clinically, the procedure involves placing electrodes into, or around, a target tissue and applying a series of short, but intense, pulsed electric fields. Oftentimes, patient specific treatment plans are employed to guide procedures by merging medical imaging with algorithms for determining the electric field distribution in the tissue. The electric field dictates treatment outcomes by increasing a cell's transmembrane potential to levels where it becomes energetically favorable for the membrane to shift to a state of enhanced permeability. If the membrane remains permeabilized long enough to disrupt homeostasis, cells eventually die. By utilizing this phenomenon, irreversible electroporation has had success in killing cancer cells and treating localized tumors. Additionally, if the pulse parameters are chosen to limit Joule heating, irreversible electroporation can be performed safely on surgically inoperable tumors located next to major blood vessels and nerves. As with all technologies, there is room for improvement. One drawback associated with therapeutic irreversible electroporation is that patients must be temporarily paralyzed and maintained under general anesthesia to prevent intense muscle contractions occurring in response to pulsing. The muscle contractions may be painful and can dislodge the electrodes. To overcome this limitation, we have developed a system capable of achieving non-thermal irreversible electroporation without causing muscle contractions. This progress is the main focus of this dissertation. We describe the theoretical basis for how this new system utilizes alterations in pulse polarity and duration to induce electroporation with little associated excitation of muscle and nerves. Additionally, the system is shown to have the theoretical potential to improve lesion predictability, especially in regions containing multiple tissue types. We perform experiments on three-dimensional in vitro tumor constructs and in vivo on healthy rat brain tissue and implanted tumors in mice. The tumor constructs offer a new way to rapidly characterize the cellular response and optimize pulse parameters, and the tests conducted on live tissue confirm the ability of this new ablation system to be used without general anesthesia and a neuromuscular blockade. Situations can arise in which it is challenging to design an electroporation protocol that simultaneously covers the targeted tissue with a sufficient electric field and avoids unwanted thermal effects. For instance, thermal damage can occur unintentionally if the applied voltage or number of pulses are raised to ablate a large volume in a single treatment. Additionally, the new system for inducing ablation without muscle contractions actually requires an elevated electric field. To ensure that these procedures can continue to be performed safely next to major blood vessels and nerves, we have developed new electrode devices that absorb heat out of the tissue during treatment. These devices incorporate phase change materials that, in the past, have been reserved for industrial applications. We describe an experimentally validated numerical model of tissue electroporation with phase change electrodes that illustrates their ability to reduce the probability for thermal damage. Additionally, a parametric study is conducted on various electrode properties to narrow in on the ideal design. / Ph. D.

No / Larval biotherapy is a debridement tool used in wound management. The mechanism of action involves degradation of eschar by serine proteases including chymotrypsin within the alimentary fluids of first instar Lucilia sericata. With the rationale of obviating some limitations of biotherapy, including cost, complexity of use, and patient reticence, the present study describes a mobile hydrogel formulation containing freeze-dried recombinant L. sericata chymotrypsin designed for topical application. Neither freeze-drying nor formulation into the hydrogel significantly attenuated the measured activity of released enzyme compared to fresh-frozen enzyme in aqueous solution. Gel electrophoresis confirmed qualitatively that the chymotrypsin/hydrogel formulation both with and without supplementary urea at 10% w/v degraded human chronic wound eschar ex vivo. Mindful that the hallmark of intractability of chronic wounds is aberrant biochemistry, the pH activity profile for the enzyme/hydrogel formulation was compared with exudate pH in chronic wounds of mixed aetiology in a cohort of 48 hospital in-patients. Five patients' wounds were acidic, however, the remainder were predominantly alkaline and coincided with the pH optimum for the insect enzyme. Thus, a recombinant L. sericata chymotrypsin and hydrogel formulation could represent a pragmatic alternative to larval therapy for the management of chronic wounds.