Articular cartilage which covers the surfaces of synovial joints is designed to allow smooth contact between long bones and to absorb shock induced during joint movement. Tissue engineering, a means of combining cells, biomaterials, bioreactors and bioactive agents to produce functional tissue replacements suitable for implantation, represents a potential long-term strategy for cartilage repair. The interplay between environmental factors, however, gives rise to complex culture conditions that influence the development of tissue-engineered constructs. A fibrous capsule that is composed of abundant type I collagen molecules and resembles fibrocartilage usually forms at the outer edge of neocartilage, yet the understanding of its modulation by environmental cues is still limited. Therefore, this dissertation was aimed to characterize the capsule formation, development and function through manipulation of biochemical parameters present in a hydrodynamic environment while a chemically reliable media preparation protocol for hydrodynamic cultivation of tissue-engineered cartilage was established. To this end, a novel wavy-wall bioreactor (WWB) that imparts turbulent flow-induced shear stress was employed as the model system and polyglycolic acid scaffolds seeded with bovine primary chondrocytes were cultivated under varied biochemical conditions. The results demonstrated that tissue morphology, biochemical composition and mechanical strength of hydrodynamically engineered cartilage were maintained as the serum content decreased by 80% (from 10% to 2%). Transient exposure of the low-serum constructs to exogenous insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1) further accelerated their development in comparison with continuous treatment with the same bioactive molecules. The process of the capsule formation was found to be activated and modulated by the concentration of serum which contains soluble factors that are able to induce fibrotic processes and the capsule development was further promoted by fluid shear stress. Moreover, the capsule formation in hydrodynamic cultures was identified as a potential biphasic process in response to concentrations of fibrosis-promoting molecules such as TGF-β. Comparison between the capsule-containing and the capsule-free constructs, both of which had comparable tissue properties and were produced by utilizing the WWB system in combination with IGF-1 and TGF-β1, respectively, showed that the presence of the fibrous capsule at the construct periphery effectively improved the ability of engineered cartilage to integrate with native cartilage tissues, but evidently compromised its tissue homogeneity. Characterization of the fibrous capsule and elucidation of the conditions under which it is formed provide important insights for the development of tissue engineering strategies to fabricate clinically relevant cartilage tissue replacements that possess optimized tissue homogeneity and properties while retaining a minimal capsule thickness required to enhance tissue integration.

Compliance mismatch is a significant hurdle to long-term patency in small-diameter arterial bypass grafts. Vascular tissue engineering has the potential to produce compliant, non-thrombogenic small-diameter grafts. However, current engineered grafts are relatively non-compliant, resulting in intimal hyperplasia and graft occlusion when subjected to arterial pressures. This research investigates the mechanical and biological properties of engineered constructs based on a biodegradable synthetic elastomer, poly(glycerol sebacate) (PGS). Several methods for fabricating porous PGS scaffolds in a tubular geometry were developed and compared. Adult baboon vascular cells were cultured in the scaffolds under various in vitro experimental conditions, including variations in initial cell seeding density, the type of scaffold used for culture, culture time, scaffold material, and hydrostatic pressure, and properties of the resultant constructs were compared. Scaffold fabrication using heat-shrinkable mandrels and glass tubes coated with hyaluronic acid significantly decreased tolerances of wall thickness and mechanical properties, improved handling, and decreased culture time required to reach luminal cellular confluence compared to scaffolds made with other fabrication techniques. Altering scaffold material from PGS to poly(lactide-co-glycolide) (PLGA), a benchmark biomaterial, did not affect scaffold yield, porosity, or luminal cellular confluence. Extracellular matrix (ECM) deposition increased with SMC-only culture time, and ECM deposition and remodeling during culture influenced construct compliance. Compared to PLGA scaffolds, PGS scaffolds promoted elastin crosslinking by SMCs and elastic tissue properties but attenuated collagen deposition. Hydrostatic pressure promoted ECM synthesis and deposition by SMCs and decreased construct compliance. Collagen and crosslinked elastin content in constructs correlated positively with construct burst pressure, and a negative correlation dependent on scaffold type was found between collagen content and construct compliance at low pressures. The systematic investigation of culture conditions in this research provides insights into the control of engineered blood vessel properties. The central hypothesis of this work, that grafts engineered from PGS scaffolds and adult vascular cells under biomimetic in vitro culture conditions can possess compliance comparable to autologous vessels, is true at pressures below 60 mmHg and demonstrates potential for PGS-based vascular tissue engineering. Overall, this work provides tools for engineering tubular soft tissues based on porous PGS scaffolds.

Background: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Methods: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Results: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Conclusions: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Heart valve disease (HVD) or a damaged valve can severely compromise the heart's ability to pump efficiently. Balloon valvuloplasty is preferred on neonates with aortic valve stenosis. Even though this procedure decreases the gradient pressure across the aortic valve, restenosis is observed soon after balloon intervention. Tissue engineering heart valves (TEHV), using bone marrow stem cells (BMSCs) and biodegradable scaffolds, have been investigated as an alternative to current non-viable prosthesis. By observing the changes in hemodynamics following balloon aortic valvuloplasty, we could uncover a potential cause for rapid restenosis after balloon intervention. Subsequently, a tissue engineering treatment strategy based on BMSC mechanobiology could be defined. Understanding and identifying the mechanisms by which cytoskeletal changes may lead to cellular differentiation of a valvular phenotype is a first critical step in enhancing the promotion of a robust valvular phenotype from BMSCs.

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.