Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

No description available.

Die klinisch relevantesten Knieverletzungen betreffen Läsionen oder Rupturen der Bänder im Kniegelenk mit einer Häufigkeit von etwa 40%, wobei allein 46% der Verletzungen das vordere Kreuzband (ACL) betreffen. Bei einer Verletzung des ACL kommt es, aufgrund mangelnder Vaskularisierung und verletzter Synovialmembran, nicht zu einer selbst induzierten Regeneration. Deshalb besteht bei einer ausbleibenden Therapie langfristig ein erhöhtes Risiko für Arthrose verbunden mit chronischen Schmerzen und Einschränkungen der Gelenkbeweglichkeit. Der Goldstandard liegt in der Transplantation von patienteneigenem Gewebe der Patellar- oder Semitendinosussehne, wobei die Gründe für das 3-10%-ige Implantatversagen z. B. in der falschen Platzierung und Fixierung des Implantates oder in einer falsch bemessenen Implantatgröße verbunden mit einer verminderten Festigkeit liegen. Ebenso sind die Nachbildung der typischen Gewebestrukturzonen vom Ligament zur knöchernen Integration sowie die damit verbundenen spezifischen mechanischen Eigenschaften nicht umsetzbar. Die genannten Nachteile legen nahe, dass ein anhaltend großer Forschungs- und Entwicklungsbedarf hinsichtlich neuartiger Therapiemethoden besteht. Im Tissue Engineering wird hierbei eine Behandlungsstrategie mit hohem Erfolgspotential gesehen. Dazu ist die Entwicklung eines temporären Zellträgers (Scaffold), der als artifizielle Matrix für die Zellen dient und die spezifischen strukturellen und mechanischen Anforderungen des nativen Gewebes erfüllt, essentiell für das Behandlungsgelingen. Das Ziel muss dabei stets die mechanische und strukturelle Wiederherstellung des ACL bei möglichst komplett vermiedener Entnahmemorbidität sein. Eine angepasste Porosität und zellspezifische Porengrößenverteilung der Scaffolds sind für eine gleichmäßige Zellproliferation in vivo erforderlich. Weiterhin müssen auch die mechanischen Eigenschaften über einen bekannten und im Idealfall definiert einstellbaren Degradationszeitraum stabil sein. Für die Scaffoldherstellung wurden die biokompatiblen und biologisch abbaubaren Materialien Polylactid (PLA) oder Poly(lactic-co-ε-caprolacton) (P(LA-CL)) gewählt. Beide Materialien gelten als medizinisch gut verträglich bzw. sind bereits als Medizinprodukt zugelassen. PLA weist eine langsames Degradationsverhalten auf und wird deshalb als potentiell geeignetes Material für das Tissue Engineering von Bändern und Sehnen gesehen. Aus zellbiologischer Sicht konnte P(LA-CL) als Optimum herausgestellt werden. Es konnte im Rahmen der Arbeit gezeigt werden, dass die Sticktechnik im Vergleich zu anderen textilen Herstellungsverfahren, wie dem Stricken oder Flechten, einen großen Gestaltungsspielraum zur Entwicklung einer mechanisch und strukturell angepassten Scaffoldstruktur für das Tissue Engineering von Ligamentgewebe bietet. Die Sticktechnik ermöglichte somit die Kombination beider Fadenmaterialien in einem Gestick. Ober- und Unterfaden können zudem aus unterschiedlichen Materialklassen sowie –typen bestehen. Neben den mechanischen Eigenschaften wurden damit auch die Porosität der Scaffoldstruktur, das Abbauverhalten und die zellbiologischen Erfordernisse wesentlich beeinflusst. Die Strukturzonen Ligament, Knorpel und Knochen konnten nach dem Vorbild des nativen Gewebeübergangs durch unterschiedliche Stickmuster gestaltet werden. Die Umsetzung war ohne zusätzliche Prozessschritte oder Maschinenmodifikationen möglich. Bei der Gestaltung der Ligamentzone zeigte sich, dass der Stickparameter Duplizierverschiebung die mechanischen Kennwerte Steifigkeit und Toe-Region wesentlich beeinflusst. Weiterhin konnte ein gradueller Musterübergang von Ligament- zu Knochenzone gestaltet sowie eine temporäre Barriere aus kollagenen Materialien in das Scaffold integriert werden. Die Steifigkeit des bereits etablierten Stickmusters für die knöcherne Integration konnte durch eine additive Modifizierung auf das Sechsfache des Ausgangswertes gesteigert werden. Die beiden Methoden „Übereinandersticken“ und „Stapeln/Verriegeln“ zur Herstellung von 3D-Gesticken ermöglichten es sowohl homogene als auch graduelle Porengrößenverteilungen zu generieren. Die damit hergestellten Gesticke in lapinem und humanem Maßstab wurden zudem den mechanischen Ansprüchen nativer ACL durch das Nachempfinden des spezifischen Kraft-Dehnungsverhaltens gerecht. Die umfangreiche Charakterisierung der mechanischen Eigenschaften konnte bei statischen und dynamischen Belastungszuständen realisiert werden, sodass durch die ermittelten Daten nicht nur Aussagen zum Versagensverhalten, sondern auch zu typischen alltäglichen Bewegungsvorgängen, wie Gehen oder Treppen steigen, getroffen werden können. Unter zyklischer Belastung wurden signifikante Unterschiede der Verlustarbeit zwischen den Gesticken und den lapinen ACL deutlich, die durch die Strukturbesonderheiten der Gesticke erklärt sowie durch eine passend gewählte Vorkonditionierung verringert werden können. Das Relaxationsverhalten der Gesticke war hingegen mit dem nativer ACL-Gewebe vergleichbar. Aufgrund der eingeschränkten mitotischen Aktivität des ACL-Gewebes wird mehrheitlich eine Gerüststruktur aus langsam degradierenden Materialien gefordert. Über einen Zeitraum von 168 Tagen wurde das hydrolytische Abbauverhalten untersucht. Die erzielten Ergebnisse lassen den Schluss zu, dass eine ausreichende Stabilität der Scaffolds vorliegt. Auf dieser Grundlage wurde ein die Arbeit abschließender in vitro Versuch mit funktionalisierten und zellbesiedelten Gesticken über 28 Tage durchgeführt. Die Ausrichtung des Zellwachstums geschah dabei entlang der Fadenmaterialien und somit in die für das Gestick vorgesehene Belastungsrichtung. Es ist deshalb davon auszugehen, dass die entwickelte Stickmusterstruktur einen essentiellen Einfluss auf das Zellverhalten hat. Aus wissenschaftlicher Sicht wurde mit der Arbeit ein wesentlicher Beitrag zur Charakterisierung gestickter 3D-Strukturen, die als Scaffolds für Tissue Engineering Anwendungen im Bereich mechanisch stark belasteter Weichgewebe dienen könnten, geleistet. Die Ergebnisse bieten den Anreiz für fortführende Arbeiten in ersten in vivo Studien.

During heart valve remodeling, and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype which is characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-containing stress fibers, the hallmark of activated myofibroblasts, are also observed when VICs are placed under tension due to altered mechanical loading in vivo or during in vitro culture on stiff substrates or under high mechanical loads and in the presence of transforming growth factor-beta1 (TGF-β1). The work presented herein describes three distinct model systems for application of controlled mechanical environment to VICs cultured in vitro. The first system uses polyacrylamide (PA) gels of defined stiffness to evaluate the response of VICs over a large range of stiffness levels and TGF-β1 concentration. The second system controls the boundary stiffness of cell-populated gels using springs of defined stiffness. The third system cyclically stretches soft or stiff two-dimensional (2D) gels while cells are cultured on the gel surface as it is deformed. Through the use of these model systems, we have found that the level of 2D stiffness required to maintain the quiescent VIC phenotype is potentially too low for a material to both act as matrix to support cell growth in the non-activated state and also to withstand the mechanical loading that occurs during the cardiac cycle. Further, we found that increasing the boundary stiffness on a three-dimensional (3D) cell populated collagen gel resulted in increased cellular contractile forces, αSMA expression, and collagen gel (material) stiffness. Finally, VIC morphology is significantly altered in response to stiffness and stretch. On soft 2D substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates. Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. These studies provide critical information for characterizing how VICs respond to mechanical stimuli. Characterization of these responses is important for the development of tissue engineered heart valves and contributes to the understanding of the role of mechanical cues on valve pathology and disease onset and progression. While this work is focused on valvular interstitial cells, the culture conditions and methods for applying mechanical stimulation could be applied to numerous other adherent cell types providing information on the response to mechanical stimuli relevant for optimizing cell culture, engineered tissues or fundamental research of disease states.

Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.

No description available.

No description available.

<p>Mesenchymal stem cells have shown potential for success in gene therapy due to their ability to differentiate and their immunomodulatory properties <em>in vivo</em>. Although they have many inherent characteristics that are suitable for use within gene therapy, genetic modification of these cells is more difficult. Since MSCs are available in limited quantities and cannot be expanded indefinitely, the modification technique must ensure efficient expression of the transgene, a high cell survival rate and an intact ability to differentiate to various cell lineages. We optimized electroporation conditions for the genetic engineering of bone marrow-derived and umbilical cord blood-derived mesenchymal stem cells. MSCs engineered using electroporation conditions produced more transgene expression than cells engineered with cationic lipids in bone marrow-derived mesenchymal stem cells, but produced similar amounts in umbilical cord blood-derived mesenchymal stem cells. Optimal electroporation conditions also expressed more transgene than polymer based transfection reagent in umbilical cord blood-derived mesenchymal stem cells. Cell survival after optimal electroporation conditions was 67% in umbilical cord blood-derived mesenchymal stem cells. Most importantly, cells maintained their ability to differentiate into osteogenic, chondrogenic and adipogenic cell lineages. Electroporating umbilical cord blood-derived mesenchymal stem cells with a Factor IX containing plasmid lead to the FIX protein being expressed for over 12 days <em>in vitro</em>. This optimized electroporation protocol has created a fast, easy, economic and efficient method for genetically modifying mesenchymal stem cells without altering their ability to differentiate.</p> / Master of Applied Science (MASc)

Wound dressing plays an important role in wound recovery and collagen interacts with the human body in such a way that it has specific advantages compared to synthetic materials. The aim of the present study was to get an optimal crosslinking agent for collagen and so the mechanical, chemical and biochemical properties of crosslinked collagen materials were investigated. Fibroblast cells are important in the process of wound healing, so the interaction of human fibroblast cells with crosslinked collagen films were investigated as well. Collagen I was isolated from bovine achilles tendons and collagen films were formed using the isolated collagen I solution. Collagen films were crosslinked with glutaraldehyde (GA), genipin, hexamethylenediisocyanate (HMDC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) at the equal concentration of 0.02 M and these crosslinked collagen films were compared with uncrosslinked collagen films (control). The surfaces of the crosslinked films were investigated using scanning electron microscopy (SEM). There was observable fibre structure on GA- and genipin-crosslinked collagen films. The tensile strength, elongation at break and low strain modulus of the crosslinked collagen films were investigated. The results showed that GA-, genipin- and HMDC-crosslinked collagen films obtained higher tensile strength than the control. Elongation at break of all the crosslinked collagen films became lower than the control. GA- and genipin-crosslinked collagen films obtained higher low strain modulus than other crosslinked collagen films and the control. The denaturation temperatures of all crosslinked collagen films were significantly higher than the control and the denaturation temperatures of GA- and genipin-crosslinked films were much higher than those of HMDC- and EDC-crosslinked films. All the crosslinked collagen films were resistant to the digestion of collagenase. These results suggest that all the crosslinking agents are effective and GA- and genipin-crosslinked films obtained more extensive crosslinking. The interaction of crosslinked collagen films with fibroblast cells was investigated, e.g. adhesion, proliferation and migration of fibroblast cells. The results demonstrated that the control, genipin- and EDC-crosslinked collagen films were conducive to cell adhesion. Fibroblast cells on the control, genipin- and EDC-crosslinked collagen films were able to proliferate after 24 hours, with increased growth after 48 hours. The fibroblast cells on the control, genipin- and EDC-crosslinked collagen films migrated directionally. The cells on genipin-crosslinked film initiated directional migration earlier than those on control- and EDC-crosslinked films. In summary, genipin crosslinked collagen films show high denaturation temperature, higher tensile strength and good biocompatibility for fibroblast cells adhesion, proliferation and migration. Genipin should be regarded as a suitable crosslinking agent for reconstituted collagen for use in wound dressing.

No description available.