Voice disorders affect up to 9% of the population, and can be caused by vocal fold scarring. They can reduce the ability of a person to participate in the workplace and can also cause depression in individuals. Vocal fold scarring changes the organization and composition of the lamina propria, and affects the biomechanical properties of the tissue. These changes cause an inability of the lamina propria to produce a normal mucosal wave during speech, resulting in hoarseness or complete voice loss. Currently there is a clinical need for treatment options for vocal fold scarring. This work focuses on developing a novel, elastomeric electrospun biomaterial to be used as a model system to evaluate the response of immortalized human vocal fold fibroblast cells (HVFF) to scaffold architecture and to the presence of an elastin polypeptide. The scaffold was developed by electrospinning Tecoflex™. Electrospun scaffolds were successfully made with aligned and unaligned fibers, and they were characterized using scanning electron microscopy (SEM) and uniaxial tensile testing. The aligned scaffolds had initial elastic moduli of ~14 MPa and ~0.3 MPa in the preferred and cross-preferred direction respectively. The unaligned scaffolds had initial elastic moduli of ~5 MPa and ~0.6 MPa in the preferred and cross-preferred direction respectively. An elastin-like polypeptide (ELP) developed in the Woodhouse lab, ELP4, was successfully adsorbed onto the scaffolds to investigate the effect of ELP’s on the HVFF cells. HVFF cells were seeded onto the scaffolds and their viability, proliferation, morphology, and gene expression were characterized. Over the culture period the cells remained viable, and showed signs of proliferation. The scaffold topography had a significant impact on the orientation of the cells, with very aligned cultures on the aligned scaffold, and randomly oriented cells on the unaligned scaffold. The scaffold alignment and the ELP4 coating impacted the extracellular matrix gene expression. The ELP4 coating, and the aligned scaffolds promoted elastin synthesis when tested on day 7, and may also reduce collagen-3 expression on day 3. These results signify that aligned electrospun scaffolds, as well as an ELP4 coating, may be promising to use in future biodegradable vocal fold constructs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-26 09:49:22.891

The treatment of bone defects is facing the situation of lacking donations for autotransplantation. As a valid approach, scaffold-based tissue engineering combines the construction of well-defined porous scaffolds with advanced cell culturing technology to guide tissue regeneration. The role for the scaffold is to provide a suitable environment with a sufficient mechanical stiffness, supports for cell attachment, migration, nutrients and metabolite transport and space for cell remodeling and tissue regeneration. The random copolymers poly(L-lactide-co-ɛ-caprolactone) (poly(LLA-co-CL)) and poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) have been successfully incorporated into 3D porous scaffolds to induce specific interactions with cells and direct osteogenic cell differentiation. In this thesis, these scaffolds have been modified in chemical and physical ways to map and understand requirements for bone regeneration. Scaffold functionalities and properties, such as hydrophilicity, stiffness, size/shape, and reproducibility, were studied. The hydrophilicity was varied by adding 3–20 % (w/w) Tween 80 to poly(LLA-co-CL) and poly(LLA-co-DXO) respectively, which resulted in contact angles from 35° to 15°. With 3 % Tween 80, the resultant mechanical and thermal properties were similar to pristine polymer materials. Tween 80 did not significantly influence cell attachment or proliferation but did stimulate the mRNA expression of osteogenetic markers. The surface functionality and mechanical properties were altered by introducing nanodiamond particles (n-DP) into poly(LLA-co-CL) scaffolds by means of surface physisorption or hybrid blending. Scaffold with n-DP physisorbed showed improved cell attachment, differentiation, and bone reformation. Hybrid n-DP/poly(LLA-co-CL) composites were obtained by direct blending of polylactide modified n-DP (n-DP-PLA) with poly(LLA-coCL). The n-DP-PLA was prepared by sodium hydride-mediated anionic polymerization using n-DP as the initiator. Prepared n-DP-PLA could be dispersed homogenously in organic solvents and blended with poly(LLA-coCL) solution. The n-DP-PLA particles were homogenously distributed in the composite material, which significantly improved mechanical properties. For comparison, the addition of benzoquinone-modified n-DP (n-DP-BQ) did not reinforce poly(LLA-co-CL). This indicated the importance of specific surface grafting, which determined different particle-polymer interactions. For the treatment of critical size defects, a large porous poly(LLA-co-CL) scaffold (12.5 mm diameter × 25 mm thickness) was developed and produced by molding and salt-leaching methods. The large porous scaffolds were evaluated in a scaffold-customized perfusion-based bioreactor system. It was obvious that the scaffold could support improved cell distribution and support the stimulation of human mesenchymal stem cell (hMSC) especially with dynamic flow in a bioreactor. To improve the scaffolding technique, a three-dimensional fiber deposition (3DF) technique was employed to build layer-based scaffolds. Poly(LLA-coCL) scaffolds produced by the 3DF method showed enhanced mechanical properties and a homogeneous distribution of human osteoblasts (hOBs) in the scaffolds. Although poly(LLA-co-CL) was thermally degraded, the degradation did not influence the scaffold mechanical properties. Based on the computerized design, a 3DF scaffold of amorphous copolymer poly(LLAco-CL) provides high-precision control and reproducibility. In summary, the design of porous scaffolds is one of the essential factors in tissue engineering as to mimicking the intrinsic extracellular environment. For bone tissue engineering, an optimized scaffold can maintain a contact angle greater than 35 degrees. Pristine or modified n-DP, introduced as an additive by surface physisorption or direct blending, can improve scaffold mechanical properties and cell response. Various sizes of scaffolds can be easily produced by a mold-mediated salt-leaching method. However, when 100 % reproducibility is required, the 3DF method can be used to create customizable scaffolds. / <p>QC 20140929</p>

Coronary heart disease is a leading cause of death among Americans for which coronary artery bypass graft (CABG) surgery is a standard surgical treatment. The success of CABG surgery is impaired by the compliance mismatch between vascular grafts and native vessels. Tissue engineered vascular grafts (TEVGs) have the potential to be compliance matched and thereby reduce the risk of graft failure. Glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen constructs were fabricated and mechanically tested in a previous study by our research group at 2, 8, and 24 hours of GLUT vapor exposure. Constructs electrospun with tropoelastin in addition to gelatin and fibrinogen fibers were also fabricated and tested for the same amounts of GLUT vapor exposure. The current study details a computational method that was developed to predict the material properties of our constructs for crosslinking times between 2 and 24 hours by interpolation and regression of the 2, 8, and 24 hour crosslinking time data. Matlab and Abaqus were used to determine the optimal combination of fabrication parameters to produce compliance matched constructs. The validity of the method was first tested on a 16 hour crosslinked gelatin/fibrinogen construct of 130μm thickness. The predicted compliance was 0.00059 mmHg-1 while the experimentally determined compliance was 0.00065 mmHg-1, a relative difference of 9.2%. Prior data in our laboratory has shown the compliance of the left anterior descending porcine coronary (LADC) artery to be 0.00071 ± 0.0003 mmHg-1. The optimization algorithm predicts that a 258μm thick construct that is GLUT vapor crosslinked for 8.1 hours would match LADC compliance. The algorithm was expanded to predict the compliance of constructs consisting of alternating layers of tropoelastin/gelatin/fibrinogen and gelatin/fibrinogen. A four layered graft was designed and fabricated using this optimization routine. The layered construct was found to have a compliance of 0.00051 mmHg-1 while the predicted compliance was 0.00061 mmHg-1, a difference of 16%. This is a promising method for matching the compliance of our TEVGs with the native tissue of various specimens.

The development of bone regeneration materials to support new bone formation is an active field of research. This report describes the development and characterization of a novel composite scaffold made of a chitosan-alginate co-polymer hydrogel matrix and eggshell (ES) particles. Scaffolds with ES particles or with nanotextured ES (NTES) particles following treatment with phosphoric acid were compared to scaffolds without particles. The scaffolds with particles exhibited a higher porosity and a larger median pore size. Their mechanical strength remained low, but both scaffold types were more resistant to deformation following compression than the scaffolds without particles. The osteogenic potential of the scaffolds was then evaluated with human bone-marrow derived mesenchymal stem cells (MSCs) from four different donors. Results showed that the inclusion of ES or NTES particles significantly increased MSC adherence and viability, as well as alkaline phosphatase activity in the scaffolds. A change of cell morphology and a small, although not statistically significant, increase of osteogenic protein expression (RUNX2 and osteopontin) were also observed at later time points (days 14 and 21). Overall, this research highlights the potential of ES for bone regeneration applications, opening the door for a high-value repurposing of a current industrial waste product.

Der Ersatz von Knochengewebe durch die Methode des Tissue Engineerings stellt eine viel versprechende Alternative zu konventionellen Therapieformen dar. Jedoch müssen die bisherigen Kulturbedingungen verbessert werden, um das Differenzierungsverhalten von Zellen optimal steuern zu können. Dabei spielt nicht nur die Wahl eines geeigneten Scaffolds und der zu verwendenden Zellen, sondern auch die des Kultursystems eine entscheidende Rolle. In einem dynamischen Kultursystem zirkuliert Medium und bietet gegenüber einem statischen Kultursystem veränderte Bedingungen bezüglich Nährstoffversorgung und Stimulation durch Flüssigkeitsscherstress. Um die Einflüsse der veränderten Bedingungen zu analysieren, wird in dieser Arbeit ein dynamisches Kultursystem etabliert. Dazu werden Calciumphosphat(CaP)-Scaffolds mit dem 3D Powder Printing System gedruckt und mit Zellen der Osteosarkomzelllinie MG63 oder der Fibroblastenzelllinie L-929 besiedelt. In 17 Versuchsreihen werden die zellbesiedelten Scaffolds bei unterschiedlichen Fließgeschwindigkeiten und über unterschiedliche Kultivierungszeiträume kontinuierlich perfundiert. Anhand der Wachstumsparameter Zellzahl und Zellviabiltät, sowie der Morphologie und räumlichen Verteilung der Zellen werden die Qualitäten der Kultursysteme untersucht und mit statischen Kultursystemen verglichen. Die mit dem 3D Powder Printing System gedruckten Scaffolds erweisen sich als geeignet: Nach 6-tägiger Kultur können unter dem Rasterelektronenmikroskop auf den CaP-Scaffolds eine reichliche Zellbesiedelung mit morphologisch gesunden Zellen, die in das Porensystem hineinwachsen, beobachtet werden. Bei beiden Zelllinien nehmen in beiden Kultursystemen die Wachstumsparameter über einen 6-tägigen Kultivierungszeitraum stetig zu und eine Langzeitkultur über 30 Tage kann in beiden Kultursystemen am Leben erhalten werden. Die kontinuierliche Perfusion in einem dynamischen Kultursystem wirkt sich auf das Zellwachstum günstig aus. Im Vergleich von dynamischen zu statischem Kultursystem über einen 6-tägigen Kultivierungszeitraum wachsen beide Zelllinien im dynamischen Kultursystem besser. Dabei spielt die Fließgeschwindigkeit im dynamischen Kultursystem auf die verbesserte Nährstoffversorgung und Stimulation durch Flüssigkeitsscherstress eine Rolle. Außerdem ist zu beachten, dass der Einfluss der Fließgeschwindigkeit des Mediums auf die einzelnen Scaffolds innerhalb des Kulturcontainers unterschiedlich ist. Dies hängt vom Strömungsprofil im Container ab und macht sich durch eine erhöhte Standardabweichung der Messwerte gegenüber der statischen Kultur bemerkbar. / The replacement of bone tissue by the method of tissue engineering represents a promising alternative to conventional forms of therapy. However, current culture conditions have to be optimized in order to control the differentiation behavior of cells. In this context, the choice of the appropriate scaffolds and cells as well as of a suitable culture system play a crucial role. In contrast to static culture systems, medium circulates in a dynamic culture system which offers changed conditions regarding nutrition and stimulation by fluid induced shear stress. To analyze the effects of changed conditions, a dynamic culture system is established in this work. For this purpose, calcium phosphate (CaP) scaffolds printed by the 3D Powder Printing System were populated with cells of the MG63 osteosarcoma cell line or of the fibroblast cell line L-929. In 17 experiments, the cultured scaffolds were perfused continuously at different flow rates and for different cultivation periods. Based on the growth parameters cell number and cell viability, as well as on the morphology and the spatial distribution of the cells, the qualities of the dynamic culture systems were compared to static culture systems. The scaffolds printed by the 3D Powder Printing System proved to be suitable: After a 6-day culture period, the CaP scaffolds showed an abundant cell colonization with morphologically healthy cells growing into the pore system which was observed under a scanning electron microscope. Using both cell lines in both culture systems, the growth parameters increased continuosly during a 6-day cultivation period and it was possible to keep a long-term culture over 30 days in both culture systems alive. The continuous perfusion in a dynamic culture system has a favorable effect on cell growth. In comparison of dynamic to static culture systems over a 6-day culture period, both cell lines grew better in the dynamic culture system. Here the flow rate in the dynamic culture system plays a major role controlling the improved nutrition and stimulation by fluid induced shear stress. Furthermore, the influence of the flow rate of the medium on the individual scaffolds within the culture container varies for the different scaffold positions. This depends on the flow profile of the container and is indicated by an increased standard deviation of the measured values when compared to the static culture.

Chitosan was widely studied for applications in tissue regeneration, because of its biodegradability and biocompatibility. However, its insolubility in a neutral solution and long gelation time limit its wide application in tissue engineering. In this thesis, a new chitosan-based biomaterial was synthesized, and its chemical structure and solubility were characterized. Afterwards, the gelation properties (crosslinker, crosslink time, swelling ratio, drug release and biocompatibility) of TMC material was investigated. Results show that TMC has higher water solubility than chitosan. The TMC liquid solution can transform to a hydrogel quickly at body temperature. The formed hydrogel controlled the release of the model protein. Cytotoxicity result shows the cationic TMC hydrogel brings a toxic effect on stromal cells but it may have the potential to inhibit bacteria or cancer cells, although more studied are required to confirm its potential functions. In summary, this new TMC hydrogel has a promising potential in biomedical fields. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Scaffold materials play an essential role in tissue engineering field due to its function of accommodate and guide cell proliferation. In this study, I investigated different types of crosslinked protein membranes that can be produced in microfluidic channels and a number of various types of PLGA electrospun composite nanofiber scaffold to examine their potentials as scaffold materials in tissue engineering. A simplified fabrication technique has been developed to produce a large surface area of crosslinked protein membranes to fulfill the purpose of cell culture experiments. Bovine serum albumin is used along with two acyl chloride crosslinkers, i.e. TCL and IDCL, respectively to accomplish the cross-linking. On the other hand, PLGA is dissolved in HFIP and enhanced with silk fibroin and carbon nanotubes to make composite electrospun materials. The morphology, physicochemical properties and biocompatibility of the membranes are studied. The biocompatibility of the membranes is investigated using cell proliferation of the PC12, ADSCs and neurons cultured on the membranes. Our results show that compared to crosslinked protein membranes, the electrospun materials are easier to prepare, less toxic and more suitable for mass production. Moreover, the electrospun materials are seen to have better biocompatibility in our cell culture study. Furthermore, the composite electrospun materials with high CNTs concentrations demonstrate positive effects on the proliferation of neurons.

Strong, durable terminal regions that can be easily handled by researchers and surgeons are a key factor in the successful fabrication of tissue engineered blood vessels (TEBV). The goal of this study was to fabricate and characterize electrospun cuffs made of poly-caprolactone (PCL) combined with gelatin that reinforce and strengthen each end of cell-derived vascular tissue tubes. PCL is ideal for vascular tissue engineering applications due to its mechanical properties; however, PCL alone does not support cell attachment. Therefore, we introduced gelatin, a natural matrix-derived protein, into the electrospun material to promote cell adhesion. This work compared the effects of two different methods for introducing gelatin into the PCL materials: gelatin coating and gelatin co-electrospinning. Porosity, pore size, fiber diameter, and mechanical properties of the electrospun materials were measured in order to compare the features of gelatin PCL composites that have the greatest impact on cellular infiltration. Porosity was quantified by liquid intrusion, fiber diameter and pore size were measured using scanning electron microscopy, and tensile mechanical testing was used to evaluate strength, elastic modulus, and extensibility. Attachment and outgrowth of smooth muscle cells onto cuff materials was measured to evaluate differences in cellular interactions between materials by using a metabolic attachment assay and a cellular outgrowth assay. Finally, cuffs were fused with totally cell-derived TEBV and the integration of cuffs with tissue was evaluated by longitudinal pull to failure testing and histological analysis. Overall, these cuffs were shown to be able to add length and increase strength to the ends of TEBV for tube cannulation and manipulation during in vitro culture. In particular, PCL:gelatin cospun cuffs were shown to improve cellular attachment and cuff fusion compared to pure PCL cuffs, while still increasing the strength of the TEBV terminal ends.

archives@tulane.edu / 1 / Jessica Margaret Mary Motherwell

Das Arbeitsgebiet Tissue Engineering befasst sich mit der Klärung der Mechanismen, die der Funktionen verschiedener Gewebearten zu Grunde liegen sowie mit der Entwicklung alternativer Strategien zur Behandlung von Organversagen bzw. Organverlusten. Einer der kritischsten Punkte im Tissue Engineering ist die ausreichende Versorgung der Zellen mit Nährstoffen und Sauerstoff. Bioartifizielle Gewebe mit einer Dicke von bis zu 200 µm können mittels Diffusion ausreichend versorgt werden. Für dickere Transplantate ist die Versorgung der Zellen alleine durch Diffusion jedoch nicht gegeben. Hierfür müssen Mechanismen und Strategien zur Prävaskularisierung der artifiziellen Gewebekonstrukte entwickelt werden, damit die Nährstoff- und Sauerstoffversorgung aller Zellen, auch im Inneren des Transplantates, von Anfang an gewährleistet ist. Eine wichtige Rolle bei der Prävaskularisierung spielt die Angiogenese. Dabei ist die Wahl einer geeigneten Zellquelle entscheidend, da die Zellen die Basis für die Angiogenese darstellen. Mikrovaskuläre Endothelzellen (mvEZ) sind maßgeblich an der Angiogenese beteiligt. Das Problem bei der Verwendung von humanen primären mvEZ ist ihre geringe Verfügbarkeit, ihre limitierte Proliferationskapazität und der schnelle Verlust ihrer typischen Endothelzellmarker in-vitro. Der Aufbau standardisierter in-vitro Testsysteme ist durch die geringe Zellausbeute auch nicht möglich. Die upcyte® Technologie bietet hierfür einen Lösungsansatz. In der vorliegenden Arbeit konnten upcyte® mvEZ als Alternative zu primären mvEZ generiert werden. Es konnte gezeigt werden, dass die Zellen eine erweiterte Proliferationsfähigkeit aufweisen und im Vergleich zu primären mvEZ durchschnittlich 15 zusätzliche Populationsverdopplungen leisten können. Dadurch ist es möglich 3x104-fach mehr upcyte® mvEZ eines Spenders zu generieren verglichen mit den korrespondierenden Primärzellen. Die gute und ausreichende Verfügbarkeit der Zellen macht sie interessant für die Standardisierung von in-vitro Testsystemen, ebenso können die Zellen zur Prävaskularisierung von Transplantaten eingesetzt werden. Upcyte® mvEZ zeigen zahlreiche Primärzellmerkmale, die in der Literatur beschrieben sind. Im konfluenten Zustand zeigen sie die für primäre mvEZ spezifische pflastersteinartige Morphologie. Darüber hinaus exprimieren upcyte® mvEZ typische Endothelzellmarker wie CD31, vWF, eNOS, CD105, CD146 und VEGFR-2 vergleichbar zu primären mvEZ. Eine weitere endothelzellspezifische Eigenschaft ist die Bindung von Ulex europaeus agglutinin I Lektin an die alpha-L-Fucose enthaltene Kohlenhydratstrukturen von mvEZs. Auch hier wurden upcyte® Zellen mit primären mvEZ verglichen und zeigten die hierfür charkteristischen Strukturen. Zusätzlich zu Morphologie, Proliferationskapazität und endothelzellspezifischen Markern, zeigen upcyte® mvEZ auch mehrere funktionelle Eigenschaften, welche in primären mvEZ beobachtet werden können, wie beispielsweise die Aufnahme von Dil-markiertem acetyliertem Low Density Lipoprotein (Dil-Ac-LDL) oder die Fähigkeit den Prozess der Angiognese zu unterstützen. Zusätzlich bilden Sphäroide aus upcyte® mvEZ dreidimensionale luminäre Zellformationen in einer Kollagenmatrix aus. Diese Charakteristika zeigen den quasi-primären Phänotyp der upcyte® mvEZs. Upcyte® mvEZ stellen darüber hinaus eine neuartige mögliche Zellquelle für die Generierung prävaskularisierter Trägermaterialien im Tissue Engineering dar. In der vorliegenden Arbeit konnte die Wiederbesiedlung der biologisch vaskularisierte Matrix (BioVaSc) mit upcyte® mvEZ vergleichbar zu primären mvEZ gezeigt werden. Der Einsatz von upcyte® mvEZ in der BioVaSc stellt einen neuen, vielversprechenden Ansatz zur Herstellung eines vaskularisierten Modells für Gewebekonstrukte dar, wie beispielsweise einem Leberkonstrukt. Zusammenfassend konnte in der vorliegenden Arbeit gezeigt werden, dass upcyte® mvEZ vergleichbar zu primären mvEZs sind und somit eine geeignete Alternative für die Generierung prävaskulierter Trägermaterialien und Aufbau von in-vitro Testsystemen darstellen. Darüber hinaus wurde ein neues, innovatives System für die Generierung einer perfundierten, mit Endothelzellen wiederbesiedelten Matrix für künstliches Gewebe in-vitro entwickelt. / The scope of tissue engineering includes researching mechanisms underlying the function of different types of tissue, as well as the development of alternative strategies for the treatment of organ failure or organ loss. One of the critical aspects of tissue engineering is the adequate supply of cells with nutrition and oxygen. Bioartificial tissue up to a thickness of 200µm can be supplied sufficiently via diffusion. For thicker transplants, the supply of cells, only via diffusion is not sufficient. For this purpose, mechanisms and strategies for pre-vascularization of artificial tissue constructs need to be developed in order to ensure the supply of nutrition and oxygen to the inside of a transplant from the beginning. An important part of pre-vascularization is angiogenesis. Thereby, the selection of a suitable cell source is crucial, as these cells form the basis of angiogenesis. Microvascular endothelial cells (mvEC) are an important part of angiogenesis. Using human primary mvEC is critical due to the quick loss of their endothelial cell marker in-vitro but their limited availability and capacity of proliferation presents a problem. Additionally, the number of cells is also not sufficient for setup a standardized in-vitro test system. Upcyte® technology provides an approach to solving this problem. This work focused on the generation of upcyte® mvEC as alternative to primary mvEC. It was shown that cells treated with upcyte® technology have an enhanced capability of proliferation, resulting in 15 additional population doublings, compared to primary mvEC. Thus, it is possible to generate 3x104-fold more upcyte® mvEC from one donor compared to corresponding primary cells. The sufficient cell availability is important for the standardization of in-vitro test systems, as well as the usage for pre-vascularization of transplants. Upcyte® mvEC show many primary cell-like characteristics, which are described in literature. In the confluent state, upcyte® mvEC show a primary cell-specific cobblestone-like morphology. Furthermore, upcyte® mvEC express typical endothelial cell markers, such as CD31, vWF, eNOS, CD105, CD146 and VEGFR-2, at a similar level to primary mvEZ. An additional endothelial cell-specific attribute is the linkage of Ulex europaeus agglutinin I lectin to carbohydrate-structures of mvEC, which contain alpha-L-fucose. These data showed that there was a good comparison between the characteristics of upcyte® and primary mvEC. In addition to morphology, proliferation capacity and endothelial cell-specific markers, upcyte® mvEC showed functional characteristics, which are also observed in primary mvEC. Examples include the uptake of Dil-marked acetylated low density lipoprotein (Dil-Ac-LDL) and the ability to support the process of angiogenesis. In addition, spheroids formed from upcyte® mvEC formed three dimensional luminal cell formations in a collagen matrix. These characteristics show the quasi-phenotype of upcyte® mvEC. Upcyte® mvEC also represents a new promising cell source for the generation of pre-vascularized scaffolds in the tissue engineering context. The use of upcyte® mvEC in BioVaSc represents a promising new approach for producing a model for vascularized tissue constructs, such as a liver constructs. In summary, this work focussed on the development of upcyte® mvEC which were shown to be comparable to primary mvEC and therefore represent a sufficient and reliable alternative cell source for the generation of pre-vascularized scaffolds and the construction of in-vitro test systems. Moreover, a new and innovative system for the generation of a perfusable, endothelialized matrix for artificial tissue in-vitro has been developed.