The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

<p>The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence. </p><p>Major results of the dissertation are as follows. Culture conditions of 100 &#956;g/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.</p> / Dissertation

As an atomic layer of sp2-hybridized carbon atoms closely packed in a honeycomb lattice, graphene has been attracting increasing attention since its discovery in 2004 due to its extraordinary physicochemical properties. Graphene oxide (GO), a non-stoichiometric graphene derivative with the carbon plane abundantly decorated with hydroxyl, epoxide and carboxylic groups, can be massively and cost-effectively produced from natural graphite following Hummers method. GO has greater aqueous solubility than pristine graphene due to its oxygen-functionalities. Various solution-based chemical methods can be applied to GO, which has stimulated a new research area called ???wet chemistry of grahene???. Among them, chemical reduction of GO provides a facile route for large-scale synthesis of graphene. With abundant oxygen-functionalities in its structure, GO can potentially act as a suitable precursor for chemical modifications of graphene through methods used in organic chemistry. Special attention should be paid to that the hydroxyl groups in GO belong to tertiary alcohols, and steric hindrance should be considered when performing chemical modifications. Diethylaminosulfur trifluoride (DAST), a fluorinating reagent, is ineffective in fluorinating GO due to the steric hindrance of tertiary hydroxyls. However, DAST is effective in reducing GO. The capability of DAST for GO reduction is close to hydrazine, but the reduction reaction can be performed at lower temperature for DAST. As a two-dimensional (2D) nanomaterial with good aqueous solubility, biocompatibility and excellent intrinsic mechanical properties, GO is particularly useful in preparing 3D hybrid hydrogel scaffolds for tissue engineering applications.

Tissue engineering approaches have attempted to address some of the problems associated with articular cartilage defect repair, but grafts with sufficient functional properties have yet to reach clinical practice. Mechanical loads are properly controlled in the body to maintain the functional properties of articular cartilage. This inspires the inclusion of mechanical stimulation in any in vitro production of tissue engineered constructs for defect repair. This mechanical stimulation must improve the functional properties (both biochemical and structural) of engineered articular cartilage tissue. Only a few studies have applied more than two loading types to mimic the complex in vivo load/flow conditions. The general hypothesis of the present thesis proposes that the generation of functional articular cartilage substitute tissue in vitro benefits from load and fluid flow conditions similar to those occurring in vivo. It is specifically hypothesized that application of compression, shear and perfusion on chondrocyte-seeded constructs will improve their properties. It is also hypothesized that protein production of the cell-seeded constructs can be improved in a depth-dependent manner with some loading combinations. Thus, a hydromechanical stimulator system was developed that was capable of simultaneously applying compression, shear and perfusion. Functionality of system was tested by series of short-term pilot studies to optimize some of the system parameters. In these studies, agarose-chondrocytes constructs were stimulated for 2 weeks. Then, longer-term (21- 31 days) studies were performed to examine the effects of both mechanical (compression and dynamic shear) and hydromechanical (compression, dynamic shear and fluid flow) stimulation on glycosaminoglycan and collagen production. The effects of these loading conditions were also investigated for three layers of construct to find out if protein could be localized differently depth-wise. In one of the longer-term studies, the chosen mechanical and hydromechanical stimulation conditions increased total collagen production, with higher amount of collagen for hydromechanical compared with mechanical loading condition. However, their effectiveness in increasing total glycosaminoglycan production was inconclusive with the current loading regimes. The hydromechanically stimulated construct could localize higher collagen production to the top layer compared with middle and bottom layers. Some effectiveness of hydromechanical stimulation was demonstrated in this thesis. Future studies will be directed towards further optimization of parameters such as stimulation frequency and duration as well as fluid perfusion rate to produce constructs with more glycosaminoglycan and collagen.

This study consists of two projects related to Tissue Engineering: Engineering biomimetic scaffolds for bone regeneration and ear reconstruction, and bioreactor design for ex-vivo bioengineered scaffold. The co-electrospinning method was used to produce composite membranes with different layers from gelatin and polycaprolactone (PCL) nanofibers, followed by paper-stacking cell seeded membranes to mimic the twisted plywood structure found in lobster cuticles. 3D laser scanner was used to capture the precise shape of a human ear model; and the negative molds were fabricated to compress scaffolds into the shape of human ear. Design for assembly (DFA) method was used to analyze and improve the design of the current bioreactor. A new design is proposed to ease operation, save time and increase the application efficiency. The proposed solution is evaluated in a virtual environment using 3D assembly modeling and simulation.

Tissue engineering of the annulus fibrosus (AF) is challenging due to its complex lamellar structure. Polyurethane scaffolds have shown promise in AF tissue engineering. The current study examines whether matrix protein coatings (collagen type I, fibronectin, or vitronectin) would enhance cell attachment and promote cell and collagen orientation that more closely mimics native AF. The results demonstrate that the greatest cell attachment occurred with fibronectin (Fn)-coated scaffolds. Cells on Fn-coated scaffolds were also aligned parallel to scaffold fibers, a process that involved α5β1 integrin, determined by integrin-specific blocking antibodies. The inhibition of this integrin reduced AF cell spreading and alignment and the changes in cell shape were regulated by the actin cytoskeleton, demonstrated using cytochalasin D inhibitor. Cells on Fn-coated scaffolds formed fibrillar Fn, synthesized significantly more collagen, and showed alignment of type I collagen that more closely mimics native AF therefore facilitating the development of the tissue in vitro.

Eine traumatische Läsion am artikulären Knorpel stellt eine noch ungelöste Herausforderung für den behandelnden Arzt dar. Bei den betroffenen Patienten handelt es sich häufig um junge, sportlich aktive Menschen im Arbeitsprozess {Hjelle et al. 2002}, bei denen eine längerfristige Belastungs- und Bewegungseinschränkung oder sogar eine Verminderung der Erwerbsfähigkeit zwingend vermieden werden muss. Jedoch existiert für den traumatischen Gelenkknorpelschaden derzeit noch keine Therapie mit sehr gutem, funktionellem Langzeitergebnis {Richter 2005}. Die konservativen Therapieformen haben immer eine narbige Ausheilung zur Folge. Aber auch mit der chirurgischen Basisversorgung, bestehend aus Debridement und Lavage des betroffenen Gelenks, kann keine Ausheilung erreicht werden. Regenerierende Verfahren, die auf der Grundlage der Penetration des subchondralen Knochens basieren, sollen durch das Einspülen von körpereigenen Stammzellen in den Defekt eine autologe Regeneration induzieren. Langzeitstudien zeigen, dass trotz der oft erreichten, guten funktionellen Ergebnisse histomorphologisch keine Wiederherstellung von intaktem, hyalinem Gelenkknorpel erreicht werden kann {Gaissmaier et al. 2003, Bernholt/Höher 2003}. Vielversprechende neue Therapiekonzepte liefert derzeit das „Tissue Engineering“ am Gelenkknorpel. Vor allem die Autologe Chondrozytentransplantation (ACT) eröffnet vollkommen neue Behandlungsstrategien. Bei der ACT werden arthroskopisch patienteneigene Chondrozyten gewonnen, die in dreidimensionale Gele eingesät und in Zellkultur gebracht werden. Durch verschiedene Stimulationstechniken werden die Zellen zur Proliferation und Produktion von Extrazellulärer Matrix (ECM), insbesondere Kollagen und Proteoglykan, angeregt. Nach drei bis sechs Wochen Kultivierung erfolgt die Implantation in den aufbereiteten Knorpeldefekt des Patienten. Aktuelle Studien konnten zeigen, dass das Transplantat mittelfristig ohne Narbenbildung in den bestehenden Gelenkknorpel einwächst und so die Inkongruenz des Gelenks, welche präarthrotisch wirkt, aufhebt {Brittberg et al. 1996, Peterson et al. 2000, Horas et al. 2000}. Um dieses neuartige Verfahren schnell im klinischen Alltag zu etablieren, bedarf es ausgereifter analytischer Methoden, die eine Qualitätsprüfung des biotechnologisch hergestellten Knorpels ermöglichen. MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) ist eine schnelle und sehr sensitive Methode, um die molaren Massen von Stoffen genau zu bestimmen und komplex zusammengesetzte Proben zu analysieren. Ziel dieser Arbeit war es, MALDI-TOF Massenspektrometrie als Analyseverfahren zu nutzen, um die Zusammensetzung des natürlichen Knorpels zu bestimmen und die hier gewonnenen Erkenntnisse auf biotechnologisch hergestellten Knorpel anzuwenden. Somit war die Überprüfung der Anwendbarkeit dieser massenspektrometrischen Untersuchungsmethode auf das komplexe biologische System Knorpel die erste Fragestellung in dieser Arbeit. Es erfolgte daher zunächst die Anpassung und Optimierung der Präparations- und Messmethoden mit dem Ziel, standardisierte Protokolle festzulegen, welche zu reproduzierbaren Spektren führen. Es schloss sich die Analyse der kommerziell verfügbaren Hauptbestandteile der ECM - Proteoglykane und Kollagene – mittels MADI-TOF MS an. Hierzu wurden Chondroitinsulfat und Kollagen verschiedener Typen enzymatisch verdaut und analysiert. So konnten Referenzspektren erstellt werden, welche die Grundlage für die Analyse des wesentlich komplexeren Systems des natürlichen Knorpels bildeten. Durch den Einsatz von Trypsin zur Hydrolyse nach thermischer Denaturierung des Kollagens wurde die Differenzierung zwischen den verschiedenen Kollagentypen ermöglicht. Es schlossen sich Studien zur Quantifizierung der enzymatischen Verdauungsprodukte der ECM an, welche das Ziel verfolgten, neben der stofflichen Zusammensetzung Aussagen zu den relativen Anteilen der einzelnen Bestandteile zu erlauben. Nachdem die grundsätzliche Eignung der Methode gezeigt werden konnte, diente der zweite Teil der hier dargelegten Forschungsarbeit der Beantwortung der Frage, ob die Erkenntnisse, welche bei der Analyse der Einzelbestandteile gewonnen wurden, auf natürliches Knorpelgewebe anwendbar sind. Der artikuläre Schweineknorpel ähnelt im Aufbau und den biomechanischen Eigenschaften dem menschlichen Knorpel. Da er zudem noch günstig und in adäquaten Mengen zur Verfügung gestellt werden kann, wurde der Gelenkknorpel des Schweins als Modellgewebe für die Anwendbarkeit der Methode auf natürlichen Knorpel genutzt. Ziel war es, die Hauptbestandteile der ECM einwandfrei zu identifizieren, Untersuchungen zur Quantifizierung anzustellen und Unterschiede zwischen den Knorpeltypen verschiedener Spezies nachzuweisen. Im letzten Abschnitt dieser Arbeit erfolgte die Analyse von biotechnologisch hergestelltem Knorpel und somit die Anwendung der bisher erworbenen, vor allem einem theoretisch-wissenschaftlichem Interesse folgenden Erkenntnisse auf eine praktisch-klinische Fragestellung nach der tatsächlichen Beschaffenheit des zu transplantierenden Konstrukts. Dazu wurden Konstrukte, welche zum einem aus dreidimensionalen Agarosegele bestückt mit Chondrozyten, zum anderen aus Kollagengel mit eingesäten Stammzellen oder Chondrozyten bestanden, untersucht. Dieser Schritt erfolgte in Zusammenarbeit mit dem Biotechnologisch-Biomedizinischen Zentrum Leipzig. Ziel war es, die bis dahin etablierten Probenpräparations- und Messbedingungen auf den künstlichen Knorpel anzuwenden und mit Referenzspektren von Bestandteilen der ECM sowie des natürlichen Schweineknorpels zu vergleichen. Durch die Analyse des Materials zu verschiedenen Kultivierungszeitpunkten sollte eine Aussage über die Qualität und die Menge der de novo synthetisierten ECM erreicht werden. Mit dieser Arbeit wurde die Grundlage geschaffen, die MALDI-TOF MS als geeignetes analytisches Verfahren zur Bestimmung der Konstruktqualität einzuführen. Perspektivisch sollte es so möglich sein, die Qualität der Konstrukte, welche für die Autologe Chondrozytentransplantation bestimmt sind, zu bewerten und zu überwachen.

This thesis hypothesizes that the introduction of facile functional handles on the periphery and cores of dendrimers can lead to novel highly functional dendrimers useful for modular surface modifications of dendrimers with biological units of choice for tunable delivery devices and for high-end imaging applications respectively. The first functional handles introduced were on the periphery of poly(amide)-based dendrimers. The dendrimers were built by convergent strategies and were equipped with one and/or two selective, robust, and orthogonal functional handles for modular attachment to and transformation of dendrimer surface. By using azides, alkynes and aldehydes as robust functional handles and investigating their orthogonality and activity by high yielding couplings with small organic and biologically significant molecules a strategy and methodology for development of tunable dendrimer surfaces for numerous future applications was facilitated. Our second functional handles were introduced in the core of poly(amidoamine) based dendrimers. Raman labels such as triple bonds and carbon-deuterium bonds with vibrational frequencies in the background free vibrational zone of 2100-2500 cm-1 were introduced in the core of dendrimers for scaffold specific labeling. By encapsulationg Ag nanodots within these dendrimers, high fluorescence and scaffold specific Raman labeling could be achieved. This strategy lays the foundation for the creation of ultrabright, scaffold specific information containing biological labels for studying single cell dynamics. Finally, the use of dendritic frameworks in heterogeneous solid supported catalysis for enhanced cooperativity in reactions involving bimetallic transition states is explored and applied. Prior to this thesis work, heterogeneous resin supported catalysts for HKR reactions suffered from high catalyst loadings and low enantioselectivities induced by the solid support. With the use of flexible linker and dendron framework supporting the catalysts, both of these problems were addressed. This method opens up new routes for creation of highly active heterogeneous solid catalysts involving a bimetallic intermediate. In the end, the current status of dendritic frameworks is reviewed and methodological extensions to this work are suggested. Conceptions of how our functional dendritic architectures would be useful for future biological and catalytic applications are explored and detailed.

Articular cartilage disorders are a leading cause of human disability in many countries around the world. In this work, new techniques and strategies were developed to improve the quality of cartilage produced in vitro by methods of tissue engineering. Chondrocytes were isolated from the hip and knee joints of aborted human foetuses. The cells were expanded and seeded into scaffolds and the seeded scaffolds were cultured in perfusion bioreactors. The quality of the final cartilage constructs was assessed biochemically by measuring their content of glycosaminoglycan (GAG), total collagen and collagen type II and histologically by staining cross-sections of the constructs for GAG, collagen type I and collagen type II. The amount of proteoglycan released in the culture medium was also measured at regular intervals. Proteoglycans from tissue-engineered cartilage and spent culture medium were compared and analysed for degradation and capability of aggregation. During monolayer expansion, the chondrocyte differentiation indices decreased, the cell size increased and the percentage of cells present in G2/S??M phase decreased with the greatest changes occurring during the first passage. Expanding chondrocytes in PGA or PGA??alginate scaffolds produced cells with a higher level of differentiation than monolayer-expanded cells. However, PGA and PGA??alginate could not be justified as suitable systems for the routine expansion of chondrocytes mainly because of the relatively low cell proliferation obtained. Two new methods for seeding of cells into scaffolds were investigated using PGA and PGA??alginate as scaffold materials. Both methods produced high seeding efficiencies and homogeneous distribution of cells. When seeded PGA??alginate scaffolds were cultured in perfusion bioreactors, they produced good quality constructs with higher concentrations of extracellular matrix (ECM) components compared with previously described methods. However, when seeded PGA scaffolds were cultured in perfusion bioreactors, they produced small constructs of poor quality. Investigation of the effect of medium flow rate on the PGA scaffolds showed that a low flow rate was needed at the beginning of the culture to enable the cells to form a framework onto which other synthesised elements could deposit. Applying a gradual increase in medium flow rate to PGA scaffolds cultured in perfusion bioreactors solved the shrinkage problem and produced constructs with quality similar to those produced using PGA??alginate scaffolds. A novel compression bioreactor that mimicked the physiological stimulation of cartilage by joint movement was constructed. Using this bioreactor, compressed constructs showed significantly higher wet weight and higher concentrations of GAG, total collagen and collagen type II compared with non-compressed constructs.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.