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.

"The MCNP6 radiation transport code was used to quantify changes in the absorbed dose tissue conversion factors for LiF, Al2O3, and silicon-based electronic dosimeters. While normally calibrated in-air and applied to all general geometric measurements, tissue conversion factors for each dosimeter were obtained at various depths in a simulated water phantom and compared against the standard in-air calibration method. In these experiments, a mono-energetic photon source was modeled at energies between 30 keV and 300 keV for a point-source placed at the center of a water phantom, a point-source placed at the surface of the phantom, and for a 10-cm radial field geometry. Again, mono-energetic photon source was modeled up to 1300 keV for a disk-source placed at the surface of the phantom and dosimetric calculations were obtained for water, LiF, Al2O3, and silicon at depths of 1 mm to 35 cm from the source. The dosimeter’s absorbed dose conversion factor was calculated as a ratio of the absorbed dose to water to that of the dosimeter measured at a specified phantom depth. The dosimeter’s calibration value also was obtained for both mono and polyenergetic source and the calibration value from poly-energetic source was compared with the intensity weighted average calibration value from mono-energetic photon. The calculated changes in the tissue conversion factors are significant because the American Association of Physicists in Medicine (AAPM) recommend that measurements of a brachytherapy or diagnostic source be made with an overall uncertainity of 5% or better. Yet, based on results, the absorbed dose tissue conversion factor for a LiF dosimeter was found to deviate from its calibration value by up to 9%, an Al2O3 dosimeter by 43%, and a silicon dosimeter by 61%. These uncertainties are in addition to the normal measurement uncertainties. By applying these tissue correction factors, these data may be used to meet the AAPM measurement requirements for mono-energetic and poly-energetic sources at measurement depths up to 35 cm under the irradiation geometries investigated herein. "

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.

"The large number of patients suffering from cardiovascular diseases has led to a high demand for functional arterial replacements. A variety of approaches to vascular graft tissue engineering have shown promise, including seeding cells onto natural and synthetic scaffolds or by culturing cell sheets which are subsequently rolled into a tube without exogenous scaffolds. The goal of this project is to develop and characterize cell-derived, fully biological small diameter tissue engineered tubes by seeding and culturing cells directly on tubular supports. Rat aortic smooth muscle cells were seeded onto collagen-coated silicone mandrels and cultured for 14 days. Cells proliferated on the mandrels to form tubes (1.19 mm inner diameter, 1.68 +/- 0.1 mm outer diameter and 230 +/- 63 microns thick; n=72). Histological analysis of the developed tissue tubes demonstrated circumferential alignment of smooth muscle cells, abundant glycosaminoglycan production and some amount of collagen production. On inflating at a constant rate, it was observed that the tissue tubes dilated to an average burst pressure of 256 +/- 76 mmHg; (n=11). In order to observe the effects of addition of soluble factors on extracellular matrix synthesis and mechanical properties, tissue tubes were grown in culture medium supplemented with 50 microgram/ml sodium ascorbate. A significant decrease in outer diameter and wall thickness (1.57 +/- 0.02 mm and 189 +/- 10 microns; n=6 respectively) in the treated groups was observed as compared to (1.66 +/- 0.06 mm and 234 +/- 32 microns; n=6; p<0.05) for the untreated control groups. A slight increase in collagen production was observed by visual assessment of histological images of the ascorbate-treated tissue tubes. This suggests that by using a direct cell seeding approach, it is possible to develop completely biologic small diameter cell-derived tissue tubes that can withstand handling, and it may also possible to modulate matrix synthesis by optimizing cell culture conditions. "

Peripheral artery disease (PAD) and the consecutive build-up of an atherosclerotic plaque restricting blood flow to the lower limbs lead to critical limb ischaemia, one of the most common circulation problems in the world. Although a small number of interventions (such as surgery or revascularization treatments) are available, patients with this condition are often too ill for these procedures, giving a poor prognosis for the disease. Several strategies to promote neovascularization using different stem cell populations with angiogenic potential have been proposed as plausible therapies. Perivascular cells (PCs), key structural components of the wall of small and large blood vessels have numerous advantages over other cell types since they are highly abundant, easy to obtain from the stromal vascular fraction (SVF) of human adipose tissue (an ethically approved source) and have mesenchymal and angiogenic properties. The work described in this thesis addressed the hypothesis that PCs isolated from human white adipose tissue would promote the recovery of blood flow in an ischaemic hindlimb by increasing blood vessel number and blood perfusion to the foot. To investigate whether PCs from human white adipose tissue could rapidly increase neovascularization and, therefore, be used as a possible therapeutic treatment for PAD and critical limb ischaemia, the initial aim was to validate, characterise and demonstrate the properties of the murine equivalent of these cells, in order to establish a direct link between the injected cells and the ones natively found in the mouse. This was then followed by the use of murine models of angiogenesis to determine whether transplanted human PCs stimulate angiogenesis in vivo. Initial studies using immunohistochemistry, fluorescence-activated cell sorting (FACS) and in vitro mesodermal differentiation demonstrated that perivascular cells (namely pericytes and adventitial cells) are present in multiple mouse organs, can be sorted to purity, and have mesenchymal stem cell (MSC) properties. These cells had similar characteristics to their human counterparts, thus validating the mouse as a suitable model for determining whether transplanted human PCs could stimulate angiogenesis. Using in vitro and two in vivo (sponge implantation and hindlimb ischaemia) models, it was shown that human PCs have angiogenic properties being capable of tube formation and interaction with endothelial cells, as well as promoting angiogenesis within sponges. Contrary to expectations, PCs did not increase blood perfusion to the mouse ischaemic hindlimb, despite increasing microcirculation within the skeletal muscle and myofibre regeneration. This work showed that PCs obtained from human adipose tissue have important therapeutic implications in promoting angiogenesis and skeletal muscle regeneration but failed to increase arteriogenesis which is the key mechanism allowing the restoration of blood perfusion.

Ductal Carcinoma in Situ (DCIS) does not have a clinically useful indicator of malignancy, and it is often benign, except in 20% of cases. Even more important, it has a cure – removal of the affected breast. DCIS patients overwhelmingly elect for invasive therapies to escape that 20% malignant chance. Overtreatment such as this costs the patients, and it highlights the need for a DCIS model capable of distinguishing the 20% in need of treatment. Some labs have taken steps toward three-dimensional, complex, and biomimetic models of mammary tissues using a variety of endogenous and synthetic gels and 3D printing. We developed FRESH (Freeform Reversible Embedding of Suspended Hydrogels) as the first method capable of 3D printing highly biomimetic shapes from endogenous gels. Utilizing FRESH, we aim to rapid prototype models of mammary duct epithelia that are biomimetic, parametric, and capable of iterative evolution. First, we investigate the principles of 3D printers modified for extruding fluids and construct a comprehensive hardware and software platform for printing gelling fluids. Second, we apply the FRESH method to 3D print collagen and alginate hydrogels, demonstrating patency of printed vascular models, topological fidelity, and the synergistic combination of hydrogel properties in multi-material prints. Finally, we rapid prototype an epithelial monolayer by seeding a 3D printed collagen manifold, and we demonstrate maintenance of the tissue’s geometry across a week of culture. We provide evidence of fidelity in prints such as an epithelial tree printed at 200% scale using unmodified collagen type I, and we investigate the combination of hydrogel properties in multi-material prints by utilizing a second hydrogel (alginate) to reinforce and preserve the fidelity of this collagen tree during handling. Our approach utilizes faster (>40 mm/s), cheaper (

A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Medicine in Therapeutic Radiology. / Soft tissue sarcomas of the extremities were previously treated either with wide excision alone, leading to a 30 to 50% local recurrence rate, or amputation, which although lead to a recurrence rate of less than 5%, caused significant physical and psychological morbidity. In addition to the :risk of local recurrence, distant metaatases occur in 30 to 50% of high grade lesions.(Abbreviation abstract) / Andrew Chakane 2018

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