Ligament and tendon injuries remain a persistent clinical challenge, accounting for up to 45% of the 32 million musculoskeletal injuries reported in the U.S. each year. However, current soft tissue repair and reconstruction techniques are limited by insufficient integration with subchondral bone, potentially leading to graft failure and suboptimal functional outcomes. Therefore, there is a pressing clinical need for functional solutions that can enable integrative soft tissue reconstruction via regeneration of the fibrocartilaginous insertion present at the junction between bone and major ligaments and tendons. This fibrocartilaginous enthesis consists of compositionally distinct but structurally continuous tissue regions (non-calcified and calcified fibrocartilage), and it plays a critical role in mediating complex load transfer between soft tissue and bone while minimizing the formation of stress concentrations at the insertion. Given the functional significance of the insertion site and using the anterior cruciate ligament (ACL) as a model tissue, the objective of this thesis is identify and optimize tissue engineering strategies for regeneration of the fibrocartilaginous interface. Thus, the studies detailed in this thesis consist of elucidation of key interface characteristics that can inform interface scaffold design, identification of an optimal cell source, and optimization of chemical and physical stimuli for fibrocartilage formation. To guide biomimetic scaffold design, this thesis began with quantitative mapping of the compositional and structural properties of the native ligament-to-bone interface. As both the aligned collagen matrix structure and distinctive mineral distribution pattern across the insertion were shown to be highly conserved over time, an ideal scaffold for fibrocartilage interface regeneration should therefore consist of aligned fibers and must be able to support the formation of both non-mineralized and mineralized fibrocartilage tissues. Additionally, evaluation of ex vivo behavior of insertion fibrochondrocytes cultured on aligned nanofiber scaffolds indicated that an ideal system for fibrocartilage regeneration should also support cell-mediated deposition of both types I and II collagen as well as proteoglycans. Comparison of potential cell sources for fibrocartilage tissue engineering showed that synovium-derived mesenchymal stem cells (SDSCs) exhibited higher proliferative and fibrochondrogenic differentiation potential compared to bone marrow-derived mesenchymal stem cells. Thus, subsequent studies focused on optimization of culture parameters for SDSC-mediated fibrocartilage formation. Nanofiber scaffolds that provided controlled release of transforming growth factor (TGF)-β3, which is known to play a critical role in development of the insertion as well as in scarless healing, were developed to guide SDSC differentiation. Scaffold-mediated TGF-β3 delivery enhanced cell proliferation and matrix synthesis in a dose-dependent manner, resulting in synthesis of fibrocartilaginous matrix consisting of both type I and II collagen as well as proteoglycans. As mechanical loading is known to also play a critical role in insertion development, a custom bioreactor that mimics the complex loads sustained at the interface was also developed. It was shown that the bioreactor simultaneously generated both tensile and compressive stresses and modulated SDSC matrix synthesis, where deposition of fibrocartilaginous matrix was observed on mechanically loaded scaffolds without any additional chemical co-stimulation. Finally, as a functional scaffold for integrative ACL repair must support the establishment of both non-mineralized and mineralized tissue regions, the combined effects of TGF-β3 and hydroxyapatite (HA) on MSC-mediated formation of mineralized fibrocartilage were also explored. The addition of HA nanoparticles to the scaffold was shown to enhance cell proliferation and matrix synthesis and represents a promising strategy for formation of mineralized fibrocartilage. Collectively, these observations delineate the importance of bioinspired chemical and physical stimuli in fibrochondrogenic differentiation, and how they can be optimized for stem cell-mediated interface regeneration. These studies yield valuable scaffold design criteria and establish in vitro culture parameters that can be applied to functional integration of soft connective tissue with bone at various critical attachments throughout the musculoskeletal system, including the ligament and tendon-to-bone entheses, as well as for regeneration of other important fibrocartilaginous tissues.

Collagen II and the proteoglycan aggrecan are key extracellular matrix (ECM) proteins in cartilaginous tissues such as the intervertebral disc (IVD). Given the functional role that these structural and functional proteins have in the IVD, ECM in tissue engineered intervertebral disc (TE IVD) constructs needs to recapitulate native tissue. As such, there is a need to understand the structure and mechanical function of these molecules in native tissue to inform TE strategies. The aims here were to characterise aggrecan and collagen II using atomic force microscopy (AFM), size-exclusion chromatography multi angle light scattering (SEC-MALS), histology, quantitative PCR, nanomechanical and computational modelling in: (i) skeletally immature and mature bovine articular cartilage (AC) and nucleus pulposus (NP), (ii) TE IVD constructs cultured in hypoxia or treated with transforming growth factor beta [TGFÎ23] or growth differentiation factor [GDF6]), and (iii) porcine AC and NP tissue. No variation in collagen II structure was observed although the proportion of organised fibrillar collagen varied between tissues. Both intact (containing all three globular domains) and non-intact (fragmented) aggrecan monomers were isolated from both AC and IVD and TE IVD constructs. Mature intact native NP aggrecan was ~60 nm shorter (core protein length) compared to AC. In skeletally mature bovine NP and AC tissue, most aggrecan monomers were fragmented (99% and 95%, respectively) with fragments smaller and more structurally heterogeneous in NP. Similar fragmentation was observed in skeletally immature bovine AC (99.5%), indicating fragmentation occurs developmentally at an early age. Fragmentation was not a result of enhanced gelatinase activity. Aggrecan monomers isolated from notochordal cell rich porcine NP were also highly fragmented, similar to bovine NP. Application of a computational packing model suggested fragmentation may affect porosity and nutrient transfer. The reduced modulus was greater in AC than NP (497 kPa and 76.7 kPa, respectively) with the difference likely due to the organisation and abundance of ECM molecules, rather than individual structure. Growth factors (GDF6 and TGFÎ23), and not oxygen tension treated TE IVD constructs were structurally (with >95% fragmented monomers), histologically and mechanically (GDF6: 60.2 kPa; TGFÎ23; 69.9 kPa) similar to native NP tissue (76.7 kPa) and there was evidence of gelatinase activity. To conclude, these results show that the ultrastructure of intact aggrecan was tissue and cell dependent, and could be modified by manipulation of cell culture conditions, specifically GDF6 which may play a role in aggrecan glycosylation.

Damaged cartilage can cause severe pain and restricted mobility, with few long term treatments available. The developing field of tissue engineering offers an alternative to the currently used full joint replacement. Restoring damaged cartilage through tissue engineering would enable an active lifestyle to be recovered and retained, without restrictions to joint mobility. This is increasingly important as the prevalence of osteoarthritis rises. Tissue engineering requires biomaterial scaffolds that mimic the function of the tissue while cells develop, and so the scaffold must provide the appropriate biological, chemical and mechanical stimuli. In this work, methods were developed to enable the design of scaffolds that mimic the microstructure and mechanical properties of articular cartilage. Electrospinning was investigated as a method to mimic the nanoscale collagen fibres within cartilage extracellular matrix. A parametric study was conducted to determine how changes to a gelatin solution affect the mechanical properties of the non-woven fibrous mesh. The solution properties had a clear impact on the morphology of the fibres, but the effect on the mesh mechanical properties was convoluted. The results demonstrated the need for greater understanding of the 3D morphology of electrospun meshes, to establish how these may be altered in order to design scaffolds with desirable mechanical properties. The fracture mechanics of soft materials are complex, and are generally overlooked when designing tissue engineering scaffolds. The complexities have led to a lack of standardised testing, making comparisons between studies impractical. In this work, fracture testing methods were compared, using a viscoelastic polymer to mimic some of the complexities of soft tissue mechanics. Mode III trouser tear tests and mode I pure shear tests were found to provide reliable measurements. Due to the ease of testing small samples, trouser tear testing was concluded to be the most advantageous for determining the fracture resistance of soft tissue engineering scaffolds. Finally, electrospun meshes were combined with hydrogels to create biomimetic scaffolds, which were characterised using tensile and trouser tear fracture tests. Fibre-reinforcement was shown to enhance the mechanical properties of a weak hydrogel, but diminished those of a strong, tough polyacrylamide (PAAm)-alginate hydrogel. The PAAm-alginate hydrogel exhibited mechanical properties close to those of natural articular cartilage, but without the microstructure that would enhance its suitability for use as a cartilage tissue engineering scaffold. An alternative method for reinforcing PAAm-alginate was proposed, which shows promise for producing a biocompatible scaffold that mimics both the mechanics and the microstructure of articular cartilage. Ultimately, this thesis aimed to improve the design of biomimetic scaffolds for cartilage tissue engineering, and advance mechanical characterisation techniques within this field.

Articular cartilage is the connective tissue lining the ends of long bones, providing a dynamic surface that bears load while providing a smooth surface for articulation. When damaged, however, this tissue exhibits a poor capacity for repair, lacking the lymphatics and vasculature necessary for remodeling. Osteoarthritis (OA), a growing health and economic burden, is the most common disease afflicting the knee joint. Impacting nearly thirty million Americans and responsible for approximately $90 billion in total annual costs, this disease is characterized by a progressive loss of cartilage accompanied by joint pain and dysfunction. Moreover, while generally considered to be a disease of the elderly (65 years and up), evidence suggests the disease may be traced to joint injuries in young, active individuals, of whom nearly 50% will develop signs of OA within 20 years of the injury. For these reasons, significant research efforts are directed at developing tissue-engineered cartilage as a cell-based approach to articular cartilage repair. Clinical success, however, will depend on the ability of tissue-engineered cartilage to survive and thrive in a milieu of harsh mechanical and chemical agents. To this end, previous work in our laboratory has focused on growing tissues appropriate for repair of focal defects and entire articular surfaces, thereby investigating the role of mechanical and chemical stimuli in tissue development. While we have had success at producing replacement tissues with certain qualities appropriate for clinical function, engineered cartilage capable of withstanding the full range of insults in vivo has yet to be developed. For this reason, and in an effort to address this shortcoming, the work described in this dissertation aims to (1) further characterize and (2) optimize the response of tissue-engineered cartilage to physical loading and the concomitant chemical insult found in the injured or diseased diarthrodial joint, as well as (3) provide a clinically relevant strategy for joint resurfacing. Together, this holistic approach maximizes the chances for in vivo success of tissue-engineered cartilage. Regular joint movement and dynamic loads are important for the maintenance of healthy articular cartilage. Extensive work has been done demonstrating the impact of mechanical load on the composition of the extracellular matrix and the biosynthetic activity of resident chondrocytes in explant cultures as well as in tissue-engineered cartilage. In further characterizing the response of tissue-engineered cartilage to mechanical load, the work in this dissertation demonstrated the impact of displacement-controlled and load-controlled stimulation on the mechanical and biochemical properties of engineered cartilage. Additionally, these studies captured tension-compression nonlinearity in tissue-engineered cartilage, highlighting the role of the proteoglycan-collagen network in the ability to withstand dynamic loads in vivo, and optimized a commercial bioreactor for use with engineered cartilage. The deleterious chemical environment of the diseased joint is also well investigated. It is therefore essential to consider the impact of pro-inflammatory cytokines on the mechanical and biochemical development of tissue-engineered cartilage, as chemical injury is known to promote degradation of extracellular matrix constituents and ultimately failure of the tissue. Combining expertise in interleukin-1\alpha, dexamethasone, and drug delivery systems, a dexamethasone drug delivery system was developed and demonstrated to provide chondroprotection for tissue-engineered cartilage in the presence of supraphysiologic doses of pro-inflammatory cytokines. These results highlight the clinical relevance of this approach and indicate potential success as a therapeutic strategy. Clinical success, however, will not only be determined by the mechanical and biochemical properties of tissue-engineered cartilage. For engineered cartilage to bear loads in vivo, it is necessary to match the natural topology of the articular surface, recapitulating normal contact geometries and load distribution across the joint. To ensure success, a method for fabricating a bilayered engineered construct with biofidelic cartilage and subchondral bone curvatures was developed. This approach aims to create a cell-based cartilage replacement that restores joint congruencies, normalizes load distributions across the joint, and serves as a potential platform for the repair of both focal defects and full joint surfaces. The research described in this dissertation more fully characterizes the benefits of mechanical stimulation, prescribes a method for chondroprotection in vivo, and provides a strategy for creating a cartilage replacement that perfectly matches the native architecture of the knee, thus laying the groundwork for clinical success of tissue-engineered cartilage.

Being the most abundant protein in the body, by mass, type I collagen provides the building blocks for tissues such as bone, extra-cellular matrix, tendons, cornea, etc[1-3]. The ability of a single protein to create structures with such various mechanical properties is not fully understood. Before one can engineer and assemble a complex tissue, such as cornea, the mechanisms underlying the formation and assembly, mechanical properties, and structure must be investigated and quantified. The work presented herein contains an extensive study of Type I collagen from the molecular to the tissue level. The engineering of collagenous tissues that mimic the mechanical and optical properties of native human cornea have been performed by a number of groups[4-7]. In all of these studies, the corneal-mimicking tissues have been created using a number of methods including repeated flow casting. To date, the ability to create self-assembled corneal tissue has not been achieved. Understanding the mechanisms of formation of native cornea will not only bring us closer to achieving self-assembled transplantable corneal tissue but will also aid in the engineering of all collagenous tissues and other structures comprised of filamentous units. Recently, the study of type I collagen has primarily focused on the tissue, fiber, and fibril scale[2, 8-21]. Grant, et al.[20] measured the elastic modulus of collagen fibrils in various solutions and found that by increasing ion concentration, in the solution around the fibril, the elastic modulus increased. The solution dependent behavior of the elastic modulus of collagen fibrils was measured but the cause of the dependence was unknown. Grant et al. state that due to the complex nature of the interactions between collagen fibrils and aqueous solutions, the exact cause of this effect is difficult to determine. Through work presented herein, not only do we show that this behavior is seen at the molecular level but also quantify the relationship between ionic concentration and molecular stiffness for a variety of ionic species. Studies of collagen mechanics, on the molecular level, are brief[22-26]. The most prominent of these studies in recent years was performed by Sun, et al.[27] wherein a persistence length of 14.5nm, for human type I procollagen, was measured. The persistence length of the molecule, which is a measure of flexibility, is a highly debated topic with quoted values of 14.5nm[27], 57nm[28], 130nm[29], 175nm[30], 308nm[31], and 544nm[32]. The broad range of values indicates that the flexibility of the collagen molecule is a complex question. It became apparent that the disagreement of the persistence length of molecular collagen in the literature may be due to the use of different ionic solutions. To address this, an initial atomic force microscope, AFM, study of the persistence length of molecular collagen diluted in DI water and two ionic solutions was conducted. This study showed that there is a strong solution dependence to the flexibility of the molecule. The ionic solutions presented molecules with a large persistence length, a straightened configuration, while the DI water dilution resulted in a persistence length that was a factor of 10 smaller. Because two different complex ionic solutions in the initial study showed different persistence lengths, an evaluation of the effect of each individual salt was performed. To elucidate the effects of individual ionic species on the conformations and persistence length of Type I collagen varying concentration of monovalent and divalent salts with different cations and anions were tested. It was found that increasing ionic concentration for all species types resulted in a higher persistence length but the rate of change in persistence length as a function of concentration is unique to each species. In 2002 Leikina, et at.[33] suggested that Type I molecular collagen is unstable at body temperature using differential scanning calorimetry. To examine these results, an AFM study was performed that imaged the collagen molecules after being held at body temperature for varying times. The density of molecules deposited onto mica, above a 200nm length cutoff, was calculated and it shows that the number of molecules above 200nm in length decreases with increasing incubation time. These environmental studies were performed with an aim to understanding the role of environment in creating a corneal mimicking tissue. Currently, the most promising method of collagen membrane fabrication for corneal replacement was developed by Tanaka, et al.[4]. This unique repeated flow casting method allows for the manufacturing of transparent collagen membranes with controllable thickness and fibrillar alignment. Using the repeated flow casting technique, orthogonally oriented collagen membranes were created and their optical properties were measured using the Generalized High Accuracy Universal Polarimeter, G-HAUP. When engineering a tissue for the eye, the optical properties of the tissue are of the utmost importance. Appropriately for corneal tissues, the measurements for linear birefringence and linear dichroism were negligible. It was clear, from the literature, that a fundamental understanding of molecular type I collagen was not available. In this work, the mechanical properties and environmentally sensitive behavior of bovine dermal type I molecular collagen is studied. The exploration into the unique behavior of these systems begins with documenting the rich ionic species and concentration dependent flexibility of molecular type I collagen and the temperature dependence on the stability of the molecule is tested. The study concludes with the construction of corneal mimicking tissues using the repeated flow casting method and measuring the complex optical properties of these tissues.

Carbon nanotubes (CNTs) are one of the most popular nanomaterials. There has been increasing interest in the development and applications of carbon nanotubes due to their huge potential in industrial and medical applications. Recent applications of carbon nanotubes include development of scaffolds and drug delivery systems. Despite rapidly emerging applications of CNTs, little is known about the impact of CNTs on cellular processes, especially mesenchymal stem cell (MSC)'s differentiation. Also, the effects of nanoparticle exposure under different conditions on cellular responses have not been well characterized yet. To characterize the effects of CNTs on creating nanoscale scaffolds for tissue engineering, we incorporated multi-walled CNTs (MWCNTs) into reconstituted type I collagen, and evaluated proliferation, differentiation, mineralization and inflammatory response of MSC on those scaffolds. MWCNTs were homogeneously distributed in collagen matrix, and strongly entrapped in collagen at the concentrations below 100 ppm. Alkaline phosphatase (AP) activity and mineralized nodules of extracellular matrix (ECM) were monitored as osteogenic differentiation markers. AP activity was significantly increased in 12 days after being replaced by differentiating media. Collagen enhanced AP activity, and MWCNT-collagen scaffolds induced additional increase in AP activity. The MSC released a significantly higher level of AP on MWCNT-collagen scaffolds than the plastic surface did at day 16. An increasing percentage of ECM mineralization was seen at day 16 after being replaced by differentiating media in the presence of MWCNT-collagen scaffolds. This study indicated the possibility of enhancement in MSC differentiation in the MWCNT-collagen scaffolds. The increased level of differentiation markers was due to the increased stiffness of the scaffolds for MSC. Our data indicated that the collagen-MWCNT scaffolds might have the potential application to create nanoscale scaffold materials for tissue engineering. To illustrate the effects of interleukin-8 (IL-8) expression in human alveolar epithelial cells (A549) under various exposure conditions of CNT, we measured the level of IL-8 expression in the presence and absence of serum following exposure of SWCNTs. The results demonstrated that the IL-8 expression was enhanced in the presence of serum. The IL-8 expression kept increasing at low concentration even after removing SWCNTs from the media. Further studies are required to characterize biological functions and toxicological potentials of nanomaterials.

Le besoin clinique de nouvelles stratégies pour pallier les limites des techniques actuelles dans le cas de régénération osseuse a permis l’émergence de l’ingénierie tissulaire osseuse. Les stratégies basées sur les techniques d’ingénierie tissulaire semblent être une alternative à l’utilisation de greffes et ainsi de s’affranchir des limites qu’elles présentent. L’approche adoptée dans le cadre de cette thèse consiste en le développement et l’utilisation d’hydrogels comme matériaux d’échafaudage pour le comblement et la régénération de tissus osseux. De nombreuses approches utilisant elles aussi des hydrogels existent, chacune possède ses avantages et limites. Dans ce contexte, nos travaux ont consisté en l’utilisation d’un hydrogel non-polymérique comme matériau de base dans le développement des stratégies. Brièvement, plusieurs types cellulaires sont présents au sein du tissu osseux et vont participer aux processus de formation et de régénération osseuse. L’objectif de nos stratégies a été l’apport de cellules souches exogènes puis leur différenciation en cellules ostéoformatrices, ou le recrutement et la différenciation des cellules de l’hôte en cellules ostéoformatrices. Le gel de GNF a été utilisé comme matrice tridimensionnelle pour ses propriétés d’injectabilité, de gélification en l’absence d’agent de réticulation toxique et son potentiel ostéoinducteur. Ce travail a consisté au développement de stratégies pour l’ingénierie tissulaire osseuse en associant le gel de GNF à une matrice naturelle de collagène cellularisée ou à des molécules bioactives pour promouvoir la régénération de lésions osseuses. Ces travaux ont permis de développer et caractériser des stratégies pertinentes pour la régénération de lésions osseuses basées sur l’utilisation d’hydrogels. / New strategies to overcome the clinical limitations of current techniques for bone defect filling and regeneration has led to the involvement of bone tissue engineering. Indeed, strategies based on tissue engineering techniques seem to be an alternative to the use of grafts and thus to defeat their limits. The approach employed in this thesis consists in development and use of hydrogels as scaffold materials for bone defect filling and regeneration. There are many approaches that also use hydrogels, each one with its advantages and limitations. In this context, our work consisted in the use of a non-polymeric hydrogel as basic material in the development of strategies for bone tissue engineering. Briefly, several cell types are present within bone tissue and will participate in the processes of bone formation and regeneration. The objective of our strategies was the contribution of exogenous stem cells and then their differentiation into osteogenic cells or the recruitment and differentiation of the host cells into osteogenic cells within the material. The GNF gel was used as a three-dimensional matrix considering its properties of injectability, gelation in the absence of toxic crosslinking agent and its osteoinductive potential. The goal was to develop strategies for bone tissue engineering by combining the GNF gel with a natural matrix of cellular collagen or bioactive molecules to promote the regeneration of bone lesions. This work allowed to develop and characterize strategies relevant to the regeneration of bone lesions based on the use of hydrogels.

acase@tulane.edu

Die Epithelzellen des renalen proximalen Tubulus resorbieren große Mengen an Wasser, Glucose und weiteren wertvollen Substanzen aus dem Primärharn, um deren Ausscheidung zu verhindern. Weiterhin sekretieren sie harnpflichtige Substanzen in den Primärharn und sind in der Lage, in die Zelle aufgenommene Substanzen enzymatisch umzusetzen. Diese Funktionen machen den renalen proximalen Tubulus zu einer wichtigen Einheit für die Nie-renfunktion. Sie führen aber auch zu einer hohen Empfindlichkeit gegenüber toxischen Effek-ten von Fremdstoffen. Daher ist ein In-vitro-Modell des renalen proximalen Tubulusepithels sowohl für die Erforschung physiologischer und pathologischer Mechanismen als auch zur Testung der Toxizität von Substanzen, insbesondere neuen Arzneimitteln, bedeutend. Ein weiteres Forschungsfeld, für das ein In-vitro-Gewebe von großem Nutzen wäre, ist die Ent-wicklung von bioartifiziellen Nierenersatzsystemen. Aufgrund Spezies-spezifischer Unterschiede, z.B. in der Expression von Transportproteinen und Enzymen, ist ein Modell mit humanen Zellen anzustreben. Bisher besteht jedoch ein Mangel an Modellen, die das renale proximale Tubulusepithel für die oben genannten An-wendungsbereiche adäquat abbilden. Das Ziel dieser Arbeit war deshalb der Aufbau eines humanen In-vitro-Modells des renalen proximalen Tubulus unter Verwendung von humanen Nierenzellen (human kidney-derived cells, hKDCs), die Eigenschaften renaler Vorläuferzellen aufweisen. In Kombination mit die-sen Zellen wurden verschiedene Kultursubstrate getestet. Dabei zeigte sich, dass die Zellen sowohl in Zellkulturplatten als auch auf Kollagen-Typ-I-beschichteten Insertmembranen mehrschichtig wachsen, ohne die typische Morphologie renaler proximaler Tubuluszellen auszubilden. In einem dreidimensionalen Kollagen-Typ-I-Hydrogel bildeten die hKDCs hin-gegen tubuläre bzw. zystäre Strukturen mit einer kubischen bis hochprismatischen Morpho-logie. Da für die oben erwähnten Anwendungsbereiche jedoch eine planare Zellschicht benö-tigt wird, erfolgte die Testung weiterer biologischer Matrices. Diese waren die Small intestinal submucosa (SIS) und das Biological vascularized scaffold (BioVaSc). Beide ließen sich aus porcinem Dünndarm herstellen, wobei bei der SIS die Mucosa sowie das Mesenterium ent-fernt wurden. Bei der BioVaSc handelt es sich um ein Darmsegment mit erhaltenem Ge-fäßsystem, dass zur Perfusion genutzt wird. Nach ihrer Kultur auf der SIS wiesen die hKDCs das typische Wachstum und die charakteris-tische Morphologie des renalen proximalen Tubulusepithels auf. Dazu gehören die Kontakt-hemmung, die das einschichtige Wachstum ermöglicht, die kubisch bis hochprismatische Morphologie sowie die Bildung eines Bürstensaums an der apikalen Zellmembran. Anhand einer Kollagen-Typ-IV- und einer Alcianblau-Färbung ließ sich die Bildung einer Basalmemb-ran an der Grenze zur SIS nachweisen. Bürstensaum- und Basalmembranbildung zeigten die zelluläre Polarisierung. Weiterhin waren typische Markerproteine renaler proximaler Tu-buluszellen wie N-Cadherin und Aquaporin-1 immunhistochemisch, zum Teil deutlich stärker als bei den Ausgangszellen, nachweisbar. Dies belegt einen positiven Einfluss der extrazellu-lären Matrixkomponenten der SIS auf die Ausbildung von Charakteristika des renalen proxi-malen Tubulusepithels. Die Albuminaufnahme als spezifische Funktion war ebenfalls nach-weisbar. Die molekularen Veränderungen der hKDCs während der Kultivierung auf der SIS ließen sich weiterhin mittels Raman-Spektroskopie bestätigen. Aufgrund der starken Interak-tion zwischen Tubulusepithel und umgebenden Kapillarnetzwerk wurde weiterhin die Co-Kultur mit Endothelzellen etabliert. Für den Vergleich der hKDCs mit einer etablierten humanen Zelllinie renaler proximaler Tu-buluszellen wurde die HK-2-Zelllinie verwendet. Mit dieser Zelllinie ließen sich die Ergebnisse der hKDCs jedoch nicht reproduzieren, was auf die fehlende Sensitivität der transformierten Zelllinie auf die Substrateigenschaften zurückzuführen ist. In der dynamischen Kultur mit der BioVaSc als Matrix waren ein inhomogenes Wachstum sowie eine variierende Markerexpression zu beobachten. Die ließ sich vor allem auf den starken Einfluss der Aussaatdichte sowie die Festigkeit der Matrix zurückführen. Bei einer erfolgreichen Optimierung der Kultur kann dieses Modell jedoch für komplexere Studien in der pharmakologischen Entwicklung nützlich sein. Mit der Kombination aus hKDCs und SIS ist es gelungen, eine einzelne, durchgängige Zell-schicht zu generieren, die wichtige Charakteristika des renalen proximalen Tubulusepithels aufweist. Weitere Untersuchungen sind nun nötig, um die Funktionalität des Modells weiter-gehend zu charakterisieren (z.B. der Transport von Substanzen und Sensitivität gegenüber toxischen Substanzen). Anschließend kann es für die spezifischen Anwendungen weiterentwickelt werden. / The epithelial cells of the renal proximal tubule resorb high amounts of water, glucose and other valuable substances from the primary urine to prevent their excretion. Furthermore, they secrete metabolic waste products into the primary urine and are able to enzymatically alter absorbed substances. These functions make the renal proximal tubule an important unit for kidney function, but also lead to a high sensitivity towards toxicity of xenobiotics. There-fore, an in vitro model of the renal proximal tubular epithelium is important not only for the investigation of physiological and pathological processes, but also for toxicity testing of sub-stances, in particular, new pharmaceuticals. A further research area, for which an in vitro tis-sue would be of great value, is the development of bioartificial kidney assist devices. Due to species-specific differences, e.g. regarding the expression of transport proteins and enzymes, a model with human cells should be aimed. Until recently, there has been a lack of models that adequately simulate the renal proximal tubular epithelium for the above men-tioned fields. Therefore, the aim of this work was to develop a human in vitro model of the renal proximal tubule using human kidney-derived cells (hKDCs), which exhibit renal progenitor cell charac-teristics. Different culture substrates were tested in combination with these cells. hKDCs in cell culture plates as well as on collagen type I-coated insert membranes grew in multilayers without developing the typical morphology of renal proximal tubular cells. In contrast, in a three-dimensional collagen type I hydrogel, hKDCs formed tubular and cystic structures with a cubic to high-prismatic morphology. However, since a planar cell layer is required for the above mentioned research fields, small intestinal submucosa (SIS) and the biological vascu-larized scaffold (BioVaSc) were tested, which are both made of porcine small intestine. For SIS production, the mucosa and the mesenterium were removed, whereas the BioVaSc is a segment of the small intestine with a preserved vascular system, which can be used for per-fusion. Following their culture on the SIS, hKDCs featured the typical growth and characteristic mor-phology of the renal proximal tubule epithelium. hKDCs were contact-inhibited, which allows monolayered growth; they had a cubic to high-prismatic morphology and developed a brush border at their apical cell membrane. By collagen type IV and alcian blue staining, the for-mation of a basement membrane at the cell-matrix border was detectable. Brush border and basement membrane formation showed cellular polarization. Furthermore, marker proteins of renal proximal tubular cells such as aquaporin-1 and N-cadherin were shown by immuno-histochemistry, which were partially stronger than before SIS culture. This demonstrates a positive influence of the extracellular matrix components of the SIS on the development of characteristics of the renal proximal tubular epithelium by hKDCs. Albumin uptake as a spe-cific function was likewise detectable. The molecular changes of hKDCs during their culture on the SIS were also identified by Raman spectroscopy. Due to the strong interaction of the tubule epithelium with the peritubular capillaries, the co-culture of hKDCs with endothelial was established as well. For comparison of hKDCs with a well-established human cell line of renal proximal tubular origin, the HK-2 cell line was used. With this cell line, the results of hKDCs were not repro-ducible, which can be explained by the lacking sensitivity of the transformed cell line towards the substrate properties. In the dynamic culture with the BioVaSc scaffold, an inhomogeneous growth and a varying marker expression were observed. This was ascribed to the strong influence of the cell seed-ing density and the low matrix stiffness. If successfully optimized, this culture model can be useful for more complex studies in the pharmacological development. In summary, with the combination of hKDCs and the SIS, a single, continuous cell layer could be generated that exhibits essential characteristics of the renal proximal tubular epithelium. More studies are required to further characterize the functionality of the model (e.g. transport of substances and sensitivity towards toxic substances). Subsequently, it can be further de-veloped for specific applications.

Each year millions of plastic and reconstructive procedures are performed to regenerate soft tissue defects after, for example, traumata, deep burns or tumor resections. Tissue engineered adipose tissue grafts are a promising alternative to autologous fat transfer or synthetic implants to meet this demand for adipose tissue. Strategies of tissue engineering, especially the use of cell carriers, provide an environment for better cell survival, an easier positioning and supplemented with the appropriate conditions a faster vascularization in vivo. To successfully engineer an adipose tissue substitute for clinical use, it is crucial to know the actual intended application. In some areas, like the upper and lower extremities, only a thin subcutaneous fat layer is needed and in others, large volumes of vascularized fat grafts are more desirable. The use and interplay of stem cells and selected scaffolds were investigated and provide now a basis for the generation of fitted and suitable substitutes in two different application areas. Complex injuries of the upper and lower extremities, in many cases, lead to excessive scarring. Due to severe damage to the subcutaneous fat layer, a common sequela is adhesion formation to mobile structures like tendons, nerves, and blood vessels resulting in restricted motion and disabling pain [Moor 1996, McHugh 1997]. In order to generate a subcutaneous fat layer to cushion scarred tissue after substantial burns or injuries, different collagen matrices were tested for clinical handling and the ability to support adipogenesis. When testing five different collagen matrices, PermacolTM and StratticeTM showed promising characteristics; additionally both possess the clinical approval. Under culture conditions, only PermacolTM, a cross-linked collagen matrix, exhibited an excellent long-term stability. Ranking nearly on the same level was StratticeTM, a non-cross-linked dermal scaffold; it only exhibited a slight shrinkage. All other scaffolds tested were severely compromised in stability under culture conditions. Engineering a subcutaneous fat layer, a construct would be desirable with a thin layer of emerging fat for cushioning on one side, and a non-seeded other side for cell migration and host integration. With PermacolTM and StratticeTM, it was possible to produce constructs with ASC (adipose derived stem cells) seeded on one side, which could be adipogenically differentiated. Additionally, the thickness of the cell layer could be varied. Thereby, it becomes possible to adjust the thickness of the construct to the surrounding tissue. In order to reduce the pre-implantation time ex vivo and the costs, the culture time was varied by testing different induction protocols. An adipogenic induction period of only four days was demonstrated to be sufficient to obtain a substantial adipogenic differentiation of the applied ASC. Thus, seeded with ASC, PermacolTM and StratticeTM are suitable scaffolds to engineer subcutaneous fat layers for reconstruction of the upper and lower extremities, as they support adipogenesis and are appropriately thin, and therefore would not compromise the cosmesis. For the engineering of large-volume adipose tissue, adequate vascularization still represents a major challenge. With the objective to engineer vascularized fat pads, it is important to consider the slow kinetics of revascularization in vivo. Therefore, a decellularized porcine jejunum with pre-existing vascular structures and pedicles to connect to the host vasculature or the circulation of a bioreactor system was used. In a first step, the ability of a small decellularized jejunal section was tested for cell adhesion and for supporting adipogenic differentiation of hASC mono-cultures. Cell adhesion and adipogenic maturation of ASC seeded on the jejunal material was verified through histological and molecular analysis. After the successful mono-culture, the goal was to establish a MVEC (microvascular endothelial cells) and ASC co-culture; suitable culture conditions had to be found, which support the viability of both cell types and do not interfere with the adipogenic differentiation. After the elimination of EGF (epidermal growth factor) from the co-culture medium, substantial adipogenic maturation was observed. In the next step, a large jejunal segment (length 8 cm), with its pre-existing vascular structures and arterial/venous pedicles, was connected to the supply system of a custom-made bioreactor. After successful reseeding the vascular structure with endothelial cells, the lumen was seeded with ASC which were then adipogenically induced. Histological and molecular examinations confirmed adipogenic maturation and the existence of seeded vessels within the engineered construct. Noteworthily, a co-localization of adipogenically differentiating ASC and endothelial cells in vascular networks could be observed. So, for the first time a vascularized fat construct was developed in vitro, based on the use of a decellularized porcine jejunum. As this engineered construct can be connected to a supply system or even to a patient vasculature, it is versatile in use, for example, as transplant in plastic and reconstruction surgery, as model in basic research or as an in vitro drug testing system. To summarize, in this work a promising substitute for subcutaneous fat layer reconstruction, in the upper and lower extremities, was developed, and the first, as far as reported, in vitro generated adipose tissue construct with integrated vascular networks was successfully engineered. / Jedes Jahr werden Millionen von plastischen und wiederherstellenden Eingriffe durchgeführt, um zum Beispiel nach Traumata, hochgradigen Verbrennungen oder Tumorekonstruktionen, die natürliche Erscheinung und Funktion im Bereich von Weichgewebsdefekt wiederherzustellen. Gezüchtete Fettgewebskonstrukte sind eine vielversprechende Alternative zu autologen Fettgewebstransfers oder synthetischen Implantaten, um dem Bedarf an Fettgewebe gerecht zu werden. Die Strategien der Gewebezüchtung, besonders das Verwenden von Zellträgern, schaffen eine Umgebung für besseres Zellüberleben, eine einfachere Positionierung und - versehen mit den entsprechenden Eigenschaften - eine schnellere Vaskularisierung in vivo. Um erfolgreich einen Fettgewebe-Ersatz für die klinische Anwendung herzustellen, ist es notwendig das spätere Anwendungsgebiet zu kennen. In manchen Bereichen, wie in den oberen und unteren Extremitäten, braucht man nur eine dünne Unterhautfettschicht, und in anderen Bereichen wiederum ist ein großes Volumen an vaskularisiertem Fettgewebskonstrukt anzustreben. Die Nutzung und das Zusammenspiel von Stammzellen und ausgewählten Zellträgern wurden untersucht und legen nun eine Basis für die Herstellung von passendem und zweckmäßigem Ersatzgewebe zweier unterschiedlicher Anwendungsgebiete. Komplexe Verletzungen der oberen und unteren Extremitäten führen oftmals zu beträchtlicher Narbenbildung. Eine häufige Folgeerscheinung, hervorgerufen durch eine schwere Beschädigung des Unterhautfettgewebes, ist die Adhäsion zwischen mobilen Strukturen wie Sehnen, Nerven und Blutgefäßen. Dies resultiert dann in eingeschränkter Beweglichkeit und lähmenden Schmerzen [Moor 1996, McHugh 1997]. Um eine subkutane Fettschicht herzustellen, die das vernarbte Gewebe nach schwerer Verbrennung oder Verletzung polstert, wurden verschiedene Kollagenmaterialien auf die klinische Handhabung und die Unterstützung der Adipogenese untersucht. In der Untersuchung von fünf verschiedenen Kollagenmatrices zeigten PermacolTM und StratticeTM vielversprechende Eigenschaften. Beide besitzen außerdem die klinische Zulassung. PermacolTM, eine chemisch quervernetzte Kollagenmatrix, zeigte unter Kulturbedingungen hervorragende Langzeitstabilität. Fast ebenso gute Eigenschaften konnten bei StratticeTM, einem nicht vernetzten dermalen Gerüstmaterial, beobachtet werden; es zeigte lediglich leichte Schrumpfung. Alle sonst getesteten Kollagenmaterialien waren unter Kulturbedingungen stark in ihrer Stabilität beeinträchtigt. Zur Herstellung einer subkutanen Fettschicht wäre ein Konstrukt wünschenswert mit einer dünnen, gerade entstehenden Fettschicht für die Polsterung auf der einen Seite und einer nicht besiedelten anderen Seite für die Zelleinwanderung und die Integration in das umliegende Gewebe. Mit PermacolTM und StratticeTM war es möglich Konstrukte herzustellen, welche auf einer Seite mit ASC (aus dem Fettgewebe isolierte Stammzellen) besiedelt und anschließend adipogen differenziert werden konnten. Zusätzlich konnte die Dicke der Zellschicht hierbei variiert werden. Somit ist es möglich die Dicke des Konstruktes an das umliegende Gewebe anzupassen. Um die Preimplantationszeit ex vivo zu verkürzen und damit auch die Kosten zu senken, wurde die Kulturzeit variiert, indem verschiedene Induktionsprotokolle getestet wurden. Eine adipogene Induktionsperiode von nur vier Tagen erwies sich als ausreichend, um eine substantielle adipogene Differenzierung der eingesetzten ASC zu erreichen. Das heißt, die mit ASC besiedelten PermacolTM und StratticeTM Matrices sind zweckdienliche Zellträgermaterialien, um eine subkutane Fettschicht für die oberen und unteren Extremitäten herzustellen, da sie die Adipogenese unterstützen und durch die nur geringe und anpassbare Dicke die Kosmesis nicht beeinträchtigen. Für die Entwicklung von großvolumigem Fettgewebe stellt die adäquate Vaskularisierung noch immer eine große Herausforderung dar. Mit dem Ziel ein vaskularisiertes Fettkonstrukt herzustellen, ist es wichtig die langsame Kinetik der Revaskularisierung in vivo zu berücksichtigen. Daher wurde hier ein dezellularisiertes Schweinedarmsegment mit schon vorhandenen Gefäßstrukturen und Gefäßanschlüssen für die Verbindung zum Kreislaufsystem des Patienten oder eines Bioreaktor-Systems verwendet. Im ersten Schritt wurden auf einem kleinen dezellularisierten Schweinedarm-Stück die Zelladhäsion und die adipogene Differenzierung der ASC in Monokultur getestet. Die Zelladhäsion und die adipogene Reifung konnte mittels histologischer und molekularer Analysen auf dem jejunalen Material nachgewiesen werden. Nach der erfolgreichen Monokultur musste die Co-Kultur von MVEC (micro vaskuläre Endothelzellen) und ASC etabliert werden. Um dieses Ziel zu erreichen, wurden geeignete Kulturbedingungen gesucht, die die Lebensfähigkeit beider Zelltypen unterstützen und gleichzeitig die adipogene Differenzierung nicht beeinträchtigen. Nach dem Ausschluss von EGF (epidermaler Wachstumsfaktor) aus dem Co-Kulturmedium wurde eine substantielle adipogene Reifung der ASC beobachtet. Im nächsten Schritt wurde ein großes dezellularisiertes jejunales Darmsegment (Länge 8 cm) mit der schon existenten Gefäßstruktur und dem arteriellen und venösen Gefäßstiel an den spezialangefertigten Bioreaktor angeschlossen. Nach der erfolgreichen Wiederbesiedelung der Gefäßstrukturen mit Endothelzellen wurde das Darmlumen mit ASC besiedelt, welche anschließend adipogen induziert wurden. Histologische und molekulare Untersuchungen konnten die adipogenen Reifung und die Existenz von besiedelten Gefäßen im hergestellten Konstrukt bestätigen. Besonders erwähnenswert ist die Beobachtung der Co-Lokalisierung von adipogen differenzierenden ASC und Endothelzellen in vasculären Netzwerken. Somit wurde zum ersten Mal - basierend auf einem dezellularisierten Schweinedarm - ein vaskularisiertes Fettgewebskonstrukt in vitro hergestellt. Da dieses Konstrukt an das Versorgungssystem angeschlossen oder mit dem Blutkreislauf des Patienten verbunden werden kann, ist es vielfältig einsetzbar, zum Beispiel in der plastisch-rekonstruktiven Chirurgie, als Modell in der Grundlagenforschung oder als ein in vitro Medikamenten-Testsystem. Zusammengefasst, wurde in der vorgelegten Arbeit ein vielversprechendes Ersatzmaterial für die Rekonstruktion des Unterhautfettgewebes für die unteren und oberen Extremitäten entwickelt, und zum ersten Mal erfolgreich, so weit in der Literatur bekannt, ein Fettgewebskonstrukt mit integriertem vaskularisiertem Netzwerk in vitro generiert.