Tissue engineering is an emerging field aimed to combine biological, engineering and material methods to create a biomimetic three dimensional (3D) environment to control cells proliferation and functional tissue formation. In such an artificial structural environment, a scaffold, made from biomaterial(s), plays an essential role by providing a mechanical support and biological guidance platform. Hence, fabrication of tissue scaffolds is of a fundamental importance, yet a challenging task, in tissue engineering. This task becomes more challenging if living cells need to be encapsulated in the scaffolds so as to fabricate scaffolds with structures to mimic the native ones, mainly due to the issue of process-induced cell damage. This research aims to develop novel methods to model the process of fabricating cell-encapsulated scaffolds and process-induced cell damage. Particularly, this research focuses on the scaffold fabrication process based on the dispensing-based rapid prototyping technique - one of the most promising scaffold fabrication methods nowadays, by which a 3D scaffold is fabricated by laying down multiple, precisely formed layers in succession. In the dispensing-based scaffold fabrication process, the flow behavior of biomaterials solution can significantly affect the flow rate of material dispensed, thus the structure of scaffold fabricated. In this research, characterization of flow behavior of materials was studied; and models to represent the flow behaviour and its influence on the scaffold structure were developed. The resultant models were shown able to greatly improve the scaffold fabrication in terms of process parameter determination. If cells are encapsulated in hydrogel for scaffold fabrication, cell density can affect the mechanical properties of hydrogel scaffolds formed. In this research, the influence of cell density on mechanical properties of hydrogel scaffolds was investigated. Furthermore, finite element analysis (FEA) of mechanical properties of scaffolds with varying cell densities was performed.The results show that the local stress and strain energy on cells varies at different cell densities. The method developed may greatly facilitate hydrogel scaffolds design to minimize cell damage in scaffold and promote tissue regeneration. . In the cell-encapsulated scaffold fabrication process, cells inevitably suffer from mechanical forces and other process-induced hazards. In such a harsh environment, cells deform and may be injured, even damaged due to mechanical breakage of cell membrane. In this research, three primary physical variables: shear stress, exposure time, and temperature were examined and investigated with regard to their effects on cell damage. Cell damage laws through the development phenomenal models and computational fluidic dynamic (CFD) models were established; and their applications to the cell-encapsulated scaffold fabrication process were pursued. The results obtained show these models and modeling methods not only allow one to optimize process parameters to preserve cell viability but also provide a novel strategy to probe cell damage mechanism in microscopic view.

Fabrication of Tissue Engineering Scaffolds Using Stereolithography Benita M. Comeau 226 Pages Directed by Dr. Clifford L. Henderson New methods and materials for the fabrication of hierarchically structured, 3D tissue scaffolds using stereolithography (SL) are presented. The ability to chemically modify selected areas on a scaffold is one way to direct cell growth in deliberate patterns; which is necessary for the engineering of complex, functioning tissues. SL will allow for the building of complex 3D structures with well defined geometries, and a second level of order is created by subsequent modification of chemical groups via catalyzing a de-protection event through exposure to another wavelength of light. The investigated system utilizes an acid-catalyzed de-protection event to change the surface chemistry of an SL-made polymer, analogous to conventional chemically amplified photoresists. The chemical modification alters the surface energy, affecting how proteins interact with the material. This allows selective areas to be more favorable towards cell adhesion. The results of this work include the identification of cytocompatible photo-acid generators that are necessary for the acid-catalyzed de-protection, the demonstration that traditional photolithographic materials may be used for cell patterning, quartz crystal microbalance studies which illuminate why these patterning methods work, the design and performance of a mirror array based stereolithographic apparatus capable of multi-wavelength exposures, and the synthesis and formulation of a novel stereolithographic resin for use in this system. The findings suggest that this system has great potential for use in cell and tissue studies, and possibilities for future use and research are discussed.

Thesis (M.S.)--University of Louisville, 1995. / School of Dentistry, Department of Biological and Biophysical Sciences. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Wang, Yadong; Committee Member: Bao, Gang; Committee Member: Bellamkonda, Ravi; Committee Member: Boyan, Barbara; Committee Member: Chaikof, Elliot; Committee Member: Meredith, J. Carson.

Thesis (M.S.)--University of Louisville, 1995. / School of Dentistry, Department of Biological and Biophysical Sciences. Includes bibliographical references.

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

Recent advances in immunological technology have made it feasible to investigate the diagnosis and treatment of cancer with radiolabelled anti-tumor antibodies. The red bone marrow and endosteal cells of bone are likely to be the dose limiting tissues for systemic applications. Therefore, it is of clinical importance to quantitate their dose. Due to the small size of the marrow cavities in trabecular bone, it is experimentally difficult to measure the electron dose distribution. A computer simulation of electron transport is used to determine the dose distribution inside the marrow cavity. Electrons are backscattered more from bone than soft tissue, thereby increasing the dose to the radiosensitive endosteum and red bone marrow. A point source of beta activity (204Tl and 147pm) sandwiched between planar slabs of bone and red bone marrow equivalent plastics and 7LiF thermoluminescent dosimeters (TLD's) were used to determine the dose increase at various distances from the interface. Experimental results were compared with calculations using the Monte Carlo codes EGS (Electron Gamma Shower, SLAC) and CYLTRAN (Oak Ridge National Laboratory). The planar geometry was used as a benchmark geometry to compare the computer codes with experiment. After checking the accuracy of the codes for low energy electron transport, ACCEPT, a version of CYLTRAN, was used to investigate the radiation dose increase due to a point source of beta activity inside a polystyrene sphere bounded by aluminum. Spheres with radii of 200 and 500 microns were used. / Thesis / Master of Science (MS)

The popular use of in vitro systems for toxicity studies has increased dramatically over the past decade. Among the in vitro systems used, primary hepatocyte cultures are the most widely employed. However, in addition to being difficult to obtain and maintain in culture, the functional heterogeneity of liver is absent. Primary organ cultures of thin liver slices should overcome these limitations but the lack of a reproducible method for the rapid preparation of thin, consistent slices, combined with the difficulty in maintaining adult liver tissue in culture, has hindered their use for in vitro hepatotoxicity studies. Using a recently-developed tissue slicer, thin, consistent liver slices were prepared rapidly under minimally traumatic conditions. Subsequent culture of these slices in a novel dynamic organ culture system (DOCS) resulted in a maintenance of hepatocyte functional integrity. Slice adenosine triphosphate (ATP) and K⁺ content were maintained at in vivo levels, following an initial recovery period (2-4h) for up to 20h. Protein synthesis and secretion were linear for 20h and 16h respectively. Slices also synthesized glycogen between 4 and 12h in culture and were hormonally-responsive during the 20h culture period as demonstrated by a two-fold stimulation of glucose production by glucagon (10⁻⁷ M). Bromobenzene and allyl alcohol hepatotoxicity were studied in this system of organ culture. The slices retained their biotransformation ability for at least 6h based on maintenance of cytochrome P-450 content and O-deethylase activity. Either compound caused dose (.01-1.0 mM) and time (0-6h) dependent cytotoxicity as indicated by the loss of slice K⁺, inhibition of protein synthesis and leakage of lactate dehydrogenase (LDH). By 2h, a significant (p < 0.05) inhibition of protein synthesis was observed in allyl alcohol (.05 mM) treated slices. At 4h and 6h, significant loss of slice K('+), LDH, and inhibition of protein synthesis were evident in slices exposed to allyl alcohol (0.25 mM) or bromobenzene (0.5 mM). This toxicity was blocked by co-treatment with pyrazole (1.0 mM) or SKF 525-A (100 μM) in slices exposed to allyl alcohol or bromobenzene, respectively. Therefore, this system provides a new tool for the in vitro study of hepatotoxicity under conditions where hepatocellular functional integrity and biotransformation are maintained.

The objective of the present study was to develop a comprehensive and reproducible regeneration system for spinach (Spinacia oleracea L. ) from commercially important cultivars and to assess the potential use of spinach for Agrobacterium tumefaciensmediated transformation. Tissue cultures of spinach were initiated from seed material. Axenic shoot cultures of spinach were established on MS-based medium containing 1.0 VM NAA at a temperature of 15°C and under a 16 h photoperiod. These three parameters were found most suitable for the establishment of shoot cultures and the encouragement of axillary shoot growth. Attempts to enhance axillary shoot production of spinach were investigated by the use of a double phase culture system, employing semi-solid and liquid culture media. The application of liquid medium was feasable with a volume of 5 ml for a duration of 7 or 14 d or with a volume of 10 ml for a duration of 7 d, but the multiplication rate of spinach was not increased. Adventitious shoot production was initiated from cultured spinach root explants derived from axenic shoots or hypocotyl explants. Sections from root tips and middle segments exhibited the highest shoot regeneration capacity when cultured on Nitsch and Nitsch (1969) medium supplemented with 20 μM NAA and 5.0 QCM GA3. Histological analysis demonstrated that the regenerating shoots originated directly from the root explants. Adventitious shoots were rooted on MS-based medium containing 1.0 μM NAA and transferred to the glasshouse, where the plants were grown to maturity. Seeds collected from regenerated plants were 95 % viable, producing a homgenous, fertile Rl-generation. Flow cytometric analysis was used to determine ploidy levels of regenerated plants and their progenies and showed that spinach leaf tissue from all generations displayed an even proportion of Go/G1 cells and G2/M cells, which may be characteristic for this species. Transformation studies using in vitro derived spinach explants demonstrated a positive response using two strains of Agrobacterium tumefaciens. The highest transformation rate was achieved with 25 % of explants being GUS-positive, therefore confirming susceptibility of spinach to the binary vector containing both T-DNA border sequences. It was found that best results were obtained with root explants which had been incubated for 8 weeks prior to co-cultivation with Agrobacterium and in vitro material which had been maintained in culture for up to 2 years. This reproducible regeneration system for spinach and the demonstration that spinach is amenable to Agrobacterium-mediated transformation provides the basis for potential commercial application within spinach breeding, aiming to generate an improved crop plant.

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