Am Herzzentrum Leipzig konnte bereits unter Einsatz von neonatalen Kardiomyozyten ein dreidimensionales vaskularisiertes Engineered Heart Tissue (EHT) etabliert und in Rat- ten mit dilatativer Kardiomyopathie implantiert werden. In Hinblick auf einen möglichen klinischen Einsatz zur Behandlung von fortgeschrittenen kardialen Erkrankungen ist es notwendig die neonatalen Kardiomyozyten der etablierten EHTs durch eine alternative Zellsorte zu ersetzen. In der vorliegenden Arbeit wurden mesenchymale Stammzellen (MSCs) aus dem Kno- chenmark der Ratte verwendet, da sie autolog aus fast jedem Körpergewebe gewonnen werden können und somit ethisch und immunologisch unbedenklich sind. Es ist gelungen formstabile, transplantationsfähige Engineered Tissues sowohl aus selbst isolierten MSCs (sMSC-ET) als auch aus kommerziell erworbenen MSCs (cMSC-ET) herzustellen. Bereits in vitro hatten sich in den künstlich hergestellten sMSC-ETs und cMSC-ETs Mikrogefäße entwickelt. Nach Implantation der MSC-ETs um ein Rattenherz verbesserte sich deren Vas- kularisierung signifikant. Zusätzlich konnte in vivo eine De-novo-Synthese von elastischen Fasern als Zeichen eines Anpassungsprozesses nachgewiesen werden. Das Hauptziel dieser Arbeit, nämlich die kardiale Differenzierung der MSCs, wurde jedoch verfehlt. Entspre- chend diesem Ergebnis steht bis heute ein endgültiger Beweis aus, ob MSCs fähig sind sich in funktionelle echte Kardiomyozyten zu differenzieren. Überdies entwickelte sich aus einem der drei implantierten cMSC-ETs ein hochmalignes undifferenziertes pleomorphes Sarkom, welches infiltrierend in das Rattenherz einwuchs. Diese Beobachtung wurde nicht für sMSC-ETs gemacht. Bei der histologischen Analyse des pleomorphen Sarkoms zeigte sich, dass dieses keine gewebespezifischen Connexine an Stellen des invasiven Wachstums exprimierte und nahezu keine am Übergang vom Tumor- zum Herzgewebe. Gleichwohl bestand zwischen proliferativer Aktivität und Connexin- Expression eine negative Korrelation. Diese Beobachtungen unterstützen zwei bekannte Theorien. Zum einen könnte das invasive Wachstum von Tumoren durch eine gestörte bzw. fehlende Kommunikation von Gap-Junction-Kanälen zwischen Tumor- und gesunden Gewebe ermöglicht worden sein. Zum anderen könnten Connexine ihrerseits über zellge- bundene molekulare Wechselwirkungen die Tumorprogression beeinträchtigen. Zusammenfassend lässt sich feststellen, dass MSCs nicht für die Herstellung von artifi- ziellem Herzgewebe geeignet sind, wohl aber für die Herstellung von künstlichen Blutge- fäßsystemen. Als sinnvolle Zellalternative bieten sich induzierte pluripotente Stammzellen (IPS-Zellen) an, da deren Differenzierbarkeit zu funktionellen Kardiomyozyten glaubhaft bewiesen werden konnte. Auch IPS-Zellen bergen ein onkogenes Potential. Daher gilt es einheitliche Kontrollen und Sicherheitsmessungen in der Herstellung von pluripotenten wie auch multipotenten Stammzellen für zellbasierte Therapien zu entwickeln und verpflichtend einzuführen.

The application of wound dressings is the most common method of wound management. In the past two decades, a novel type of wound dressing has been introduced that functions according to tissue engineering principles and provides an implantable platform for wound regeneration. The focus of this thesis was to develop such a wound dressing with multi-layer architecture that would be capable of absorbing wound exudates, be flexible with adequate contact with the wound bed, and have desirable porosity to allow cell migration. This thesis concludes with the development of a wound dressing which is comprised of three separate layers bonded together. The first layer, which would be directly in contact with the wound bed, was a gelatin scaffold of uniform porosity produced through an optimised gas foaming method. In this part of the research, in addition to optimising the gas foaming process parameters, a comprehensive comparison between applying four different crosslinking agents (glutaraldehyde, hexamethylene diisocyante, poly ether epoxide, and genipin) was carried out. The scaffolds, although showing a uniform porosity, had the tensile strength (240 kPa) lower than the reported value for natural skin (850 kPa). To strengthen the porous scaffold, a middle layer was applied and bonded to it. The middle layer with a thickness of 120m was adhered to the gelatin scaffold, functioning as a mechanical support and exudate absorbent. This layer comprised of a chitosan-gelatin composite which exhibited a tensile strength of 26 MPa. The chitosangelatin membrane bonded to the gelatin scaffold had a combined tensile strength of 644 kPa, approaching natural skin tensile properties. The wound dressing assembly was completed by applying a plasticised gelatin membrane as the third and final layer above the chitosan-gelatin composite. This membrane with a thickness of 130m, was plasticised using glycerol. It was designed with the primary function of covering the wound against debris, bacteria, and excessive manipulation, but also safeguarding the chitosan-gelatin membrane from disintegration once the wound exudate had been absorbed. The presented multi-layer design architecture provides a combination of a conventional wound dressing occlusive functionality with a modern tissue engineering approach in one product. Application of gas foaming resulted in a pore system that had an optimised porosity in comparison with commercially available wound dressings, by providing a more spherical pore system with pore size distribution closer to desirable values for skin tissue engineering (125m). It is anticipated that the design of the biomaterial would result in accelerated wound healing and reducing long term care in a cost-effective manner.

Poly[lactic co-glycolic] acid (PLGA) is a biocompatible polymer commonly used in the field of tissue engineering, but its mechanical properties tend to be less than ideal for most orthopedic applications. Five PLGA composites, reinforced with 0 to 1% nonfunctionalized single-walled carbon nanotubes, were prepared and tested for tensile strength. In order to achieve consistent nanotube dispersions, sodium dodecyl sulfate was incorporated as a surfactant. The polymer scaffold fabrication methods were successful at creating suitable samples for tensile testing. After the tests were performed, scanning electron microscope images were taken to examine the fractured edges and determine the cause of failure. Analysis of fractured surfaces indicated good nanotube dispersions in all composite samples, and an increase in tensile strength, with respect to the control (0.532 MPa), was found for composites at the 0.07% nanotube and 0.09% nanotube concentrations (0.570 MPa and 0.643 MPa respectively). Total length at failure decreased as carbon nanotube concentration increased. This experiment showed a promising trend toward increasing the mechanical properties of PLGA/carbon nanotube composites and represented a prospective foundation for future research.

Made available in DSpace on 2016-12-09T13:52:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-06. Added 1 bitstream(s) on 2016-12-09T13:55:23Z : No. of bitstreams: 1 000870881_20170306.pdf: 367778 bytes, checksum: db82c4998c7d49050765cd4b14882f61 (MD5) Bitstreams deleted on 2017-03-10T13:07:30Z: 000870881_20170306.pdf,. Added 1 bitstream(s) on 2017-03-10T13:08:15Z : No. of bitstreams: 1 000870881.pdf: 6408119 bytes, checksum: a00dbe5b7026255d78fde75fe5e150aa (MD5) / Estruturas tridimensionais (scaffolds) construídas por técnicas de impressão tridimensional vêm ganhando espaço em reconstrução e regeneração de tecidos, pois permitem a fabricação de scaffolds com formas complexas. O objetivo deste trabalho foi caracterizar físico-química e biologicamente matrizes de poli (3-hidroxibutirato) (PHB), confeccionadas por impressão tridimensional via sinterização seletiva a laser (selective laser sinterization - SLS), funcionalizadas com fibroína da seda (FB), apatitas (HA) e/ou peptídeo de crescimento osteogênico (osteogenic growth peptide - OGP), com finalidade de regeneração óssea. Os resultados de caracterização demonstraram que a impressão via SLS produziu scaffolds de PHB com percentual de porosidade de 55,8±0,7% e com poros regulares de 500-700 µm. A adição de FB e HA mantiveram resultados de porosidade semelhantes e aumentaram a taxa de absorção de água do PHB no período inicial de 2 h. A incorporação de FB melhorou a resistência à compressão e o módulo de elasticidade, sendo este também melhorado por FB-HA. Nos ensaios in vitro observou-se uma boa adesão, espraiamento e proliferação celular em todos os grupos. Na avaliação indireta da precipitação de cálcio sobre as amostras, observou-se um melhor resultado no grupo FB-HA, principalmente em 3 dias. In vivo, os grupos experimentais falharam em estimular a osteogênese de contato, entretanto mantiveram a espessura da área de implantação, o que não ocorreu no grupo controle coágulo. Conclui-se que os scaffolds avaliados possuem algumas propriedades favoráveis a aplicação em regeneração óssea, entretanto alguns mecanismos de funcionamento dos mesmos in vivo, principalmente em longo prazo, ainda precisam ser entendidos e melhorados. / Three-dimensional structures (scaffolds) fabricated by three-dimensional printing techniques (3DP), have been widely used in tissues reconstruction and regeneration. The main advantage of that technique is the possibility of manufacturing scaffolds with complex shapes. The aim of this study was to perform physicochemical and biological characterization of three-dimensional matrices of poly (3-hydroxybutyrate) (PHB), made by 3DP via selective laser sintering (SLS), functionalized with silk fibroin (FB), apatites (HA) and / or osteogenic growth peptide (OGP), to favor bone regeneration. The results demonstrated that SLS 3DP produced PHB scaffolds with porosity percentage of 55.8 ± 0.7% with regular pores of 500-700 µm. The addition of HA and FB results in similar porosity and enhanced PHB water absorption rate, at the 2h timepoint. The incorporation of FB improved compressive strength and elastic modulus, which was also improved by FB-HA. In vitro tests showed good cell adhesion, spreading and proliferation in all. In the indirect evaluation of calcium precipitation a better result was observed in FB-HA group mainly in 3 days. In vivo experimental groups failed to stimulate contact osteogenesis. However, the scaffolds preserved the thickness of implantation area, which did not occur in the control clot group. It was concluded that the scaffolds evaluated in this study have some favorable properties for bone regeneration. However some of their action mechanisms in vivo, especially in long-term periods, still have to be better understood.

Orientador: Joni Augusto Cirelli / Banca: Ana Paula Faloni de Souza / Banca:Paulo Tambasco de Oliveira / Banca: Roberto Henrique Barbeiro / Banca: Elcio Marcantonio Junior / Resumo: Estruturas tridimensionais (scaffolds) construídas por técnicas de impressão tridimensional vêm ganhando espaço em reconstrução e regeneração de tecidos, pois permitem a fabricação de scaffolds com formas complexas. O objetivo deste trabalho foi caracterizar físico-química e biologicamente matrizes de poli (3-hidroxibutirato) (PHB), confeccionadas por impressão tridimensional via sinterização seletiva a laser (selective laser sinterization - SLS), funcionalizadas com fibroína da seda (FB), apatitas (HA) e/ou peptídeo de crescimento osteogênico (osteogenic growth peptide - OGP), com finalidade de regeneração óssea. Os resultados de caracterização demonstraram que a impressão via SLS produziu scaffolds de PHB com percentual de porosidade de 55,8±0,7% e com poros regulares de 500-700 µm. A adição de FB e HA mantiveram resultados de porosidade semelhantes e aumentaram a taxa de absorção de água do PHB no período inicial de 2 h. A incorporação de FB melhorou a resistência à compressão e o módulo de elasticidade, sendo este também melhorado por FB-HA. Nos ensaios in vitro observou-se uma boa adesão, espraiamento e proliferação celular em todos os grupos. Na avaliação indireta da precipitação de cálcio sobre as amostras, observou-se um melhor resultado no grupo FB-HA, principalmente em 3 dias. In vivo, os grupos experimentais falharam em estimular a osteogênese de contato, entretanto mantiveram a espessura da área de implantação, o que não ocorreu no grupo controle coágulo. Conclui-se que os scaffolds avaliados possuem algumas propriedades favoráveis a aplicação em regeneração óssea, entretanto alguns mecanismos de funcionamento dos mesmos in vivo, principalmente em longo prazo, ainda precisam ser entendidos e melhorados. / Abstract: Three-dimensional structures (scaffolds) fabricated by three-dimensional printing techniques (3DP), have been widely used in tissues reconstruction and regeneration. The main advantage of that technique is the possibility of manufacturing scaffolds with complex shapes. The aim of this study was to perform physicochemical and biological characterization of three-dimensional matrices of poly (3-hydroxybutyrate) (PHB), made by 3DP via selective laser sintering (SLS), functionalized with silk fibroin (FB), apatites (HA) and / or osteogenic growth peptide (OGP), to favor bone regeneration. The results demonstrated that SLS 3DP produced PHB scaffolds with porosity percentage of 55.8 ± 0.7% with regular pores of 500-700 µm. The addition of HA and FB results in similar porosity and enhanced PHB water absorption rate, at the 2h timepoint. The incorporation of FB improved compressive strength and elastic modulus, which was also improved by FB-HA. In vitro tests showed good cell adhesion, spreading and proliferation in all. In the indirect evaluation of calcium precipitation a better result was observed in FB-HA group mainly in 3 days. In vivo experimental groups failed to stimulate contact osteogenesis. However, the scaffolds preserved the thickness of implantation area, which did not occur in the control clot group. It was concluded that the scaffolds evaluated in this study have some favorable properties for bone regeneration. However some of their action mechanisms in vivo, especially in long-term periods, still have to be better understood. / Doutor

Tissue engineering merges the disciplines of study like cell biology, materials science, engineering and surgery to enable growth of new living tissues on scaffolding constructed from implanted polymeric materials. One of the most important aspects of tissue engineering related to material science is design of the polymer scaffolds. The polymer scaffolds needs to have some specific mechanical strength over certain period of time. In this work bioresorbable aliphatic polymers (PCL and PLLA) were blended using extrusion and solution methods. These blends were then extruded and electrospun into fibers. The fibers were then subjected to FDA standard in vitro immersion degradation tests where its mechanical strength, water absorption, weight loss were observed during the eight weeks. The results indicate that the mechanical strength and rate of degradation can be tailored by changing the ratio of PCL and PLLA in the blend. Processing influences these parameters, with the loss of mechanical strength and rate of degradation being higher in electrospun fibers compared to those extruded. A second effort in this thesis addressed the potential separation of the scaffold from the tissue (loss of apposition) due to the differences in their low strain responses. This hypothesis that using knit with low tension will have better compliance was tested and confirmed.

Articular cartilage lesions that occur upon intensive sport, trauma or degenerative disease represent a severe therapeutic problem. At present, osteoarthritis is the most common joint disease worldwide, affecting around 10% of men and 18% of women over 60 years of age (302). The poor self-regeneration capacity of cartilage and the lack of efficient therapeutic treatment options to regenerate durable articular cartilage tissue, provide the rationale for the development of new treatment options based on cartilage tissue engineering approaches (281). The integrated use of cells, biomaterials and growth factors to guide tissue development has the potential to provide functional substitutes of lost or damaged tissues (2,3). For the regeneration of cartilage, the availability of mesenchymal stromal cells (MSCs) or their recruitment into the defect site is fundamental (281). Due to their high proliferation capacity, the possibility to differentiate into chondrocytes and their potential to attract other progenitor cells into the defect site, bone marrow-derived mesenchymal stromal cells (BMSCs) are still regarded as an attractive cell source for cartilage tissue engineering (80). However, in order to successfully engineer cartilage tissue, a better understanding of basic principles of developmental processes and microenvironmental cues that guide chondrogenesis is required. / Verletzungen des Gelenkknorpels, die durch intensiven Sport, Trauma oder degenerative Krankheiten induziert wurden, stellen ein großes therapeutisches Problem dar. Heutzutage ist Arthrose die weltweit häufigste Gelenkerkrankung, die etwa 10% der männlichen und 18% der weiblichen Bevölkerung über 60 Jahre betrifft (302). Die geringe intrinsische Heilungskapazität von Knorpelgewebe und das Fehlen effizienter Behandlungsmethoden, um dauerhaften Gelenkknorpel zu erzeugen, bilden die Grundlage für die Entwicklung neuartiger Behandlungsmethoden auf Basis des Tissue Engineering (281). Hierbei verfügt speziell der integrierte Einsatz von Zellen, Biomaterialien und Wachstumsfaktoren über das Potential zerstörtes oder geschädigtes Gewebe zu ersetzen bzw. die Regeneration von neuem Gewebe zu fördern (2,3). Für die Regeneration von Knorpelgewebe ist vor allem die Verfügbarkeit von mesenchymalen Stammzellen (MSC) und deren Rekrutierung in die Defektzone von großer Bedeutung (281). Aufgrund ihrer hohen Proliferationsrate, der Fähigkeit in Chondrozyten zu differenzieren und des Potentials andere Vorläuferzellen in die Defektzone zu rekrutieren bilden MSCs auch heute noch einen attraktiven Ansatz im Knorpel-Tissue Engineering (80). Eine wichtige Voraussetzung für die erfolgreiche Entwicklung von Knorpelgewebe ist jedoch ein besseres Verständnis der grundlegenden Entwicklungsprozesse und der Einflussfaktoren der Mikroumgebung, die die Chondrogenese regulieren.

Organ-on-chip models are a rapidly evolving and promising tool for studying human physiology and disease and developing therapeutics. However, due to the lack of fabrication processes of pertinent precision to deliver well-defined architectural and mechanical elements, organ-on-chip models have been limited in recapitulating structural and biomechanical features of many tissues, which has impeded the modeling power and clinical relevance of these tools. The elusive in vitro replication of the pumping function and mechanical loading of the human heart, an outstanding instance of a structurally and mechanically complex physiological system, exemplifies the need for stronger fabrication processes. In this work, we investigated the potential of two-photon direct laser writing (TPDLW), an emerging high-precision fabrication technique, in enabling the generation of structurally and biomechanically complex organ-on-chip models. We first identify the functional principles, advantages and limitations of TPDLW, and review existing applications of TPDLW for in vitro studies. Inspired by the fabrication versatility of TPDLW, we then engineer a microfluidic cardiac pump powered by human stem-cell-derived cardiomyocytes (hiPSC-CM), aiming to replicate the ventricular pumping function on a chip by constructing miniaturized analogues of the functional elements of the human heart. We specifically fabricate a microscale metamaterial scaffold with fine-tuned mechanical properties to support the formation and cyclic contraction of an unprecedentedly miniaturized induced pluripotent stem cell derived ventricular chamber. Furthermore, we fabricate microfluidic valves with extreme sensitivity to rectify the flow generated by the ventricular chamber. The integrated microfluidic system recapitulates ventricular fluidic function and exhibits for the first time in vitro all phases of the ventricular hemodynamic loading pattern. Finally, we demonstrate a technique of increasing the fabrication output of TPDLW that could enable its broader adoption. Together, our results highlight the potential of high-precision fabrication in expanding the accessible spectrum of organ-on-a-chip models towards structurally and biomechanically sophisticated tissue architectures. This dissertation is accompanied by a set of supplementary videos depicting the results of our experimental efforts. Movie 1 shows a cardiac tissue beating on an inverted hexagon scaffold. Movie 2 shows a compressive test on helical scaffold that is later embedded in a cardiac tissue. Movie 3 show a beating cardiac chamber on helical scaffold that can generate measurable flow. Movie 4 shows a functional suspension valve that is later embedded in the device with the cardiac chamber. Movie 5 shows the function of a suspension valve that rectifies oscillating flow. Movie 6 shows that the same suspension valve can rectify flow of increasing frequency. Movie 7 shows that the combined chamber and valves exhibit directional flow. Finally, movie 8 shows that the addition of afterload in the combined system leads to the emergence of isovolumetric phases. / 2023-09-24T00:00:00Z

archives@tulane.edu / Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow the attachment and proliferation of several different cell types. Decellularized tissue-derived scaffolds are considered close replicates of the tissue microenvironment. Decellularized adipose tissue (DAT) hydrogel has proven to be a useful tool for tissue engineering applications in pre-clinical models. The first aim of the present study was to characterize the biochemical composition of DAT hydrogel. The DAT hydrogel was prepared by processing adipose tissue acquired from three female human donors, and subsequently quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS). The enriched and depleted proteins were determined in DAT hydrogel and further analyzed by gene ontology (GO) analysis. Extracellular matrix proteins were found to be enriched, while cellular proteins were depleted relative to native adipose tissue. Furthermore, GO analysis identified that the enriched proteins could affect various biological processes via the regulation of a range of cellular pathways. The second aim was focused on the analysis of the effect of adipose-derived stromal/stem cells (ASCs) and DAT hydrogel interaction on cell morphology, proliferation, differentiation, and hydrogel microstructure. The ASCs seeded in DAT hydrogel remained viable and displayed proliferation. The adipogenic and osteogenic differentiation of ASCs seeded in DAT hydrogel was confirmed by marker gene expression and histochemical staining. Moreover, ASC attachment and differentiation altered the fibril arrangement, which indicated remodeling of the DAT hydrogel. The third aim was to analyze the regenerative potential of DAT hydrogel in a critical-sized mouse femoral defect model. The DAT hydrogel alone, or its composites with ASCs, osteo-induced ASCs (OIASC), and hydroxyapatite were tested for the ability to mediate repair of the femoral defect. The data indicated that DAT hydrogel promoted bone regeneration alone, while the regeneration was enhanced in the presence of OIASCs and hydroxyapatite. In summary, the current findings confirm that DAT hydrogel is a cytocompatible and bio-active scaffold, with potential utility as an off-the-shelf product for tissue engineering applications. In future, the analysis of DAT hydrogel using a wider range of donors representing different body mass index, age, gender, and ethnicity will provide a more comprehensive characterization. / 0 / Omair A. Mohiuddin

The structural organization of cells and their associated extracellular matrix (ECM) is critical to overall tissue function. Recapitulating the complex, highly organized structure of a target tissue is a key to achieve the unique functional characteristics of native tissue. However, achieving this goal requires a system in which substrate physicochemical properties such as modulus, topology and surface chemistry can be modulated. Here, we developed a cell sheet-based harvest & transfer system that can rapidly produce patterned 2D cell sheets in any physiologically relevant size and shape for various cell types. We further show that these cell sheets can be stacked one on top of the other with high cell viability while preserving the patterns, and that they remain sufficiently intact in vivo to allow neovascularization. We can thus use this system to mimic both the 2D and 3D structure of native tissue structure. A further advantage of our system is its substrate modulus tuning capability, which allows us to provide an optimal biomechanical environment for the differentiation and phenotypic stabilization of specific cell types. Because hydrogels theoretically have no limit in 2D shape and size, this system is scalable for producing quality controlled multiple cell sheets in a short period of time. Our model should also aid in understanding the mechanisms that underlie cell-cell and cell-ECM communication in 3D environments, which will be imperative to improving engineered tissue design. We thus ultimately envision that our system could allow the rapid fabrication of functionalized three dimensional thick tissues from multiple stacks of cell sheets derived from autologous cells, which would be an important step forward in both tissue modeling and regenerative medicine in general. Finally, this system can also potentially serve as a powerful model to study in vivo tissue formation and growth as well as cancer cell behavior.