Indiana University-Purdue University Indianapolis (IUPUI) / A variety of polymers of synthetic origins (e.g., poly(ethylene glycol) or PEG) and naturally derived macromolecules (e.g., silk fibroin or gelatin) have been explored as the backbone materials for hydrogel crosslinking. Purely synthetic hydrogels are usually inert, covalently crosslinked, and have limited degradability unless degradable macromers are synthesized and incorporated into the hydrogel network. Conversely, naturally derived macromers often contain bioactive motifs that can provide biomimicry to the resulting hydrogels. However, hydrogels fabricated from a single macromer often have limitations inherent to the macromer itself. For example, to obtain high modulus PEG-based hydrogels requires an increase in macromer and crosslinker content. This is associated with an increase in radical concentration during polymerization which may cause death of encapsulated cells. Pure gelatin (G) hydrogels have weak mechanical properties and gelatin undergoes thermo-reversible physical gelation. Covalent crosslinking is usually necessary to produce stable gelatin hydrogels, particularly at physiological temperatures. The limitations of these hydrogels may be circumvented by combining them with another macromer (e.g., silk fibroin) to form hybrid hydrogels. Silk fibroin (SF) from Bombyx mori silkworms offers high mechanical strength, slow enzymatic degradability, and can easily form physical hydrogels. The first objective of this thesis was to evaluate the effect of sonication and the presence of synthetic polymer (e.g., poly (ethylene glycol) diacrylate or PEGDA) or natural macromer (e.g., gelatin) on SF physical gelation kinetics. SF physical gelation was assessed qualitatively via tilt tests. Gelation of pure SF solutions was compared to mixtures of SF and PEGDA or G, both with or without sonication of SF prior to mixing. The effect of gelatin on SF gelation was also evaluated quantitatively via real time in situ rheometry. Sonication accelerated gelation of SF from days to hours or minutes depending on SF concentration and sonication intensity. Both PEGDA and G were shown to accelerate SF physical gelation when added to SF and sonicated SF (SSF) solutions. The second objective was to develop a simple strategy to modulate covalently crosslinked PEG-based hydrogel properties by physically entrapping silk fibroin. The physical entrapment of silk fibroin provides an alternative method to increase gel storage modulus (G’) without the cytotoxic effect of increasing macromer and crosslinker concentration, or altering degradation kinetics by increasing co-monomer concentration. The effect of SF entrapment on gel physical and mechanical properties, as well as hydrolytic degradation and chemical gelation kinetics were characterized. SF physical crosslinking within the PEG-based network was shown to increase gel storage moduli by two days after gel fabrication. There was no change hydrolytic degradation rate associated with the increased moduli. SF entrapment did not affect gelation efficiency, but did alter gel physical properties. The third objective of this thesis was to develop a silk-gelatin in situ forming hybrid hydrogel for affinity-based growth factor sequestration and release and in vitro cell culture. SF provides mechanical strength and stability, whereas G contains bioactive motifs that can provide biomimicry to the gel network. Hydrogel G’ and its dependency on temperature, SF processing conditions, and secondary in situ chemical crosslinking (i.e., genipin crosslinking) were studied. Gelatin can be conjugated with heparin, a glycosaminoglycan, to impart growth factor (GF) binding affinity. Growth factor sequestration and release were evaluated in a pair of designed experiments. The hybrid gels were evaluated as substrates for human mesenchymal stem cell proliferation.

Indiana University-Purdue University Indianapolis (IUPUI) / Dynamic changes to the extracellular matrix (ECM) impact many cell fate pro- cesses. The ECM can experience changes in sti ness as well as changes in composi- tion in response to injury, development, and diseases. To better understand the role that these dynamic processes have on the cells residing within the environment, re- searchers have turned towards 4-dimensional (4D) hydrogel designs. These 4D hydro- gels re-capitulate not only 3-dimensional (3D) matrix architectures, but also temporal changes in the physicochemical properties. The goal of this thesis was to design a unify chemistry (i.e., Sortase A (SrtA)-mediated transpeptidation) for dynamic tun- ing hydrogel sti ness and the presence of bioactive ligands. The rst objective was to establish a tunable and cytocompatible enzymatic scheme for softening cell-laden hydrogels. Brie y, the e ects of SrtA-mediated matrix cleavage were investigated us- ing poly(ethylene glycol) (PEG)-peptide hydrogels crosslinked by SrtA-sensitive and insensitive peptides. Initially, the e ects of various parameters with respect to cat- alytic reactions of SrtA were characterized rheologically, including enzyme and sub- strate concentrations, macromer content, peptide composition, and treatment time. Gel moduli pre- and post-enzyme treatment were measured to verify SrtA-mediated hydrogel softening. The cytocompatibility of SrtA-mediated gel softening system was investigated using human mesenchymal stem cell (hMSC). Upon treatment with SrtA and an oligoglycine substrate, encapsulated hMSCs exhibited extensive spreading in comparison to those within statically sti matrices. The second objective was to es- tablish a reversible ligand exchange system utilizing SrtA-mediated transpeptidation. SrtA-sensitive pendant ligands were immobilized within PEG hydrogels, which were treated with SrtA and an oligoglycine substrate to a ord tunable removal of the pen- dant ligand. Through measurement of the liberated pendant peptide concentration, it was found that higher concentrations of SrtA or extending treatment times led to higher ligand removal e ciency. Finally, the e ect of peptide ligand removal on cell behaviors were evaluated using NIH 3T3 broblasts. Fibroblasts were culture both on and within hydrogels containing SrtA-cleavable cell adhesion peptide. After treatment, both conditions led to a decrease in broblast spreading in comparison to non-treated gels. Overall, the utility of SrtA as versatile agent for controlling the mechanical properties and the presence of biologically active components within a hydrogel system was demonstrated. These systems could be further explored with natural-based materials to better mimic the physiological environment experienced by cells.

The diploma thesis focuses on utilization of diffusion cells in reactivity mapping study on humic acids. In the experimental part, samples 6 humic acids of different origin or chemical modification were studied. All the samples were characterized by basic analytical and physico-chemical methods (elemental analysis, thermogravimetry, determination of acidity, spectrometry). Methylene blue was utilized as a model reactive probe and agarose gel as an inert support medium. An effect of interactions between humic acids and methylene blue on diffusivity of the dye in the hydrogel was determined and discussed.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19668号 / 薬科博第56号 / 新制||薬科||7(附属図書館) / 32704 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 橋田 充, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20074号 / 医博第4167号 / 新制||医||1018(附属図書館) / 33190 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 羽賀 博典, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21421号 / 医博第4411号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 瀬原 淳子, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Gewebe sind nicht nur durch ihre biochemische Zusammensetzung definiert, sondern auch durch ihre individuellen mechanischen Eigenschaften. Inzwischen ist es weithin akzeptiert, dass Zellen ihre mechanische Umgebung spüren und darauf reagieren. Zum Beispiel werden Zellmigration und die Differenzierung von Stammzellen durch die Umgebungssteifigkeit beeinflusst. Um diese Effekte in vitro zu untersuchen, wurden viele Zellkulturstudien auf 2D Hydrogelsubstraten durchgeführt. Zusätzlich dazu steigt die Anzahl von Studien an, die hydrogelbasierte 3D-Scaffolds nutzen, um 2D Studien zu validieren und die experimentellen Bedingungen der Situation in vivo anzunähern. Jedoch erweist es sich weiterhin als schwierig den Effekt von Mechanik in 3D in vitro zu untersuchen, da in den gemeinhin genutzten 3D Hydrogelsystemen immer eine Kopplung zwischen Gelporosität und Steifigkeit besteht. Zusätzlich hängt die Konzentration der biologisch aktiven Bindungsstellen für Zellen oft ebenfalls von der Steifigkeit ab. Diese Arbeit präsentiert die Entwicklung und Optimierung neuer 3D Hydrogelkugel-Scaffolds, in denen die Steifigkeit von der Porosität schließlich entkoppelt wird. Mit Hydrogelkugeln als Scaffold-Bausteine ist es nun möglich 3D Scaffolds mit definierten mechanischen Eigenschaften und konstanter Porengröße zu generieren. Während der Methodenentwicklung wurden verschiedene Prinzipien und Kultivierungskammern konstruiert und überarbeitet, gefolgt von der theoretischen Betrachtung der Sauerstoffdiffusion, um die Eignung der gewählten Kammer hinsichtlich Zellvitalität und Zellwachstum zu überprüfen. Eine Kombination aus mehreren getesteten Filtern wurde ausgewählt um HydrogelkugelScaffolds erfolgreich in der ausgewählten Kammer zu generieren. Im Weiteren wurden verschiedene Hydrogelmaterialien untersucht hinsichtlich der erfolgreichen Produktion monodisperser Hydrogelkugeln und der Erzeugung stabiler Scaffolds. Hydrogelkugeln aus Polyacrylamid (PAAm) wurden als Scaffold-Bausteine ausgewählt um damit die Eignung des entwickelten Systems zu demonstrieren lebende Zellen zu mikroskopieren. Außerdem wurde das Überleben von Fibroblasten über vier Tage in unterschiedlich steifen HydrogelkugelScaffolds erfolgreich gezeigt. Weiterhin war es möglich erste Zellmigrationsexperimente durchzuführen. Dafür wurden sowohl einfache PAAm-Hydrogelkugeln als auch mit Adhäsionsmolekülen funktionalisierte Hydrogelkugeln genutzt, um unterschiedlich steife Schichten in einem Scaffold zu erzeugen. Dadurch war es möglich nicht nur Zellmigration anhand von Zelladhäsion in 3D Scaffolds mit Steifigkeitsgradienten zu beobachten, sondern auch Zellmigration ohne Zelladhäsion.:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis / Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. For example, cell migration and stem cell differentiation is affected by stiffness. To study these effects in vitro, many cell culture studies have been performed on 2D hydrogel substrates. Additionally, the amount of 3D studies based on hydrogels as 3D scaffold is increasing to validate 2D in vitro studies and adjust experimental conditions closer to the situation in vivo. However, studying the effects of mechanics in vitro in 3D is still challenging as commonly used 3D hydrogel assays always link gel porosity with stiffness. Additionally, the concentration of biologically active adhesion sides often also depends on the stiffness. This work presents the development and optimization of novel 3D hydrogel bead scaffolds where the stiffness is finally decoupled from porosity. With hydrogel beads as scaffold building blocks it was possible to generate 3D scaffolds with defined mechanical properties and a constant pore size. During the method development, different culture devices were constructed and revised, followed by oxygen diffusion simulations to proof the suitability of the chosen device for cell survival and growth. A combination of different filter approaches was selected to generate hydrogel bead scaffolds in the culture device. Furthermore, different hydrogel materials were investigated regarding successful production of monodisperse beads and stable scaffold generation. Polyacrylamide (PAAm) hydrogel beads were chosen as scaffold building blocks to demonstrate live-cell imaging and successful cell survival over four days in differently compliant hydrogel bead scaffolds. Moreover, first cell migration experiments were performed by using plain PAAm hydrogel beads as well as PAAm hydrogel beads functionalized with adhesion molecules with differently stiff layers in one scaffold. Thereby fibroblast migration was observed not only in adhesion-dependent migration manner, but also in an adhesion-independent mode .:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis

Immune-orchestrating biomaterials that precisely modulate the immune reac-tion to the host could lead the way for improving the implantation outcomein the transplantation field, in comparison to passive biomaterials. The lab-oratory of Dr. Thomas Crouzier has shown hydrogels derived from mucinsare capable of orchestrating the immune response mediated by foreign bodyresponse (FBR), as a result of evading fibrosis. Further, a recent study fromhis group showed sialic acid on mucin hydrogels is essential for the immuno-logical activity of those materials. Mucin glycans transiently activated thendampened macrophages, important orchestrators for material-mediated FBR,in a sialic acid-dependent manner for the majority of cytokines followed. Thematerial properties such as rheological properties, self-healing capacity, andstability, can be governed by the crosslinking chemistry used and have a drasticimpact on the functionalities of the materials. In this project, various cross-linking strategies are applied to tune the hydrogel properties. We show thatthe robust cross-linking formed mucin hydrogels having a 1.5% (wt/v) bettersupported insulin-secreting cells form islet-like organoids, compared to 2.5%mucin hydrogels. We then investigate the self-healing properties of the newmucin hydrogels and their interactions with various cell systems. / Immunologiska biomaterial som specifikt modulerar en immunologisk reak-tion hos värden kan vara vägen till att förbättra resultatet av en transplantationi jämförelse med att använda passiva biomaterial. Inom laboratoriet lett av Dr.Thomas Crouzier har det kunnat påvisas att hydrogels erhållna från mucinerär kapabla till att förändra en immunlogisk respons, Foreign Body Respon-se (FBR) vilket resulterat i att kringgå fibrosis. Utöver detta har ytterligareen studie utförts av hans grupp vilken kunde påvisa att sialinsyra på mucinhydrogel är essentiellt för den immunologiska aktiviteten för dessa biomateri-al. Mucin glykaner aktiveras tillfälligt för att dämpa bildandet av makrofager,vilket är viktigt vid biomaterial FBR i ett sialinsyra-beroende för bildandet avmajoriteten av cytokiner. Materialegenskaperna såsom reologiska egenskaper,självläkande kapacitet, och stabilitet kan regleras genom en krosslänkad kemisom har en drastisk impakt för funktionen för biomaterialen. I detta projekt, ärflera krosslänkande strategier tillämpade för att förstå egenskaperna för hyd-rogelerna. Vi har kunnat påvisa att robusta krosslänkande system av mucinhydrogel bestående av 1.5% (wt/v) ger bättre support till insulin sekreterandeceller att bilda islet liknande organider, att jämföra med 2.5% mucin hydrogel.Vi undersöker sedan den självläkande egenskaperna av nya mucin hydrogeleroch deras interaktioner med ett flertal olika cellsystem.