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Hücre Tabakası Mühendisliğinde Kalınlık ve Vaskülerizasyon Problemlerine Yönelik Stratejilerin Geliştirilmesi

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10233494.pdf (14.06Mb)
Date
2019
Author
Tevlek, Atakan
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Abstract
In the context of tissue engineering studies, various biomaterials, cells and biosignal molecules have been used to repair and replace diverse tissues either alone or in combination, namely to produce tissue-engineered constructs. Although the tissue engineering journey, which started with cellular therapies, has gone a long way with scaffold-based approaches, the transport of various gases and nutrients that is the basic requirements for cell viability and the removal of metabolic wastes have not been achieved. In this context, the researchers aimed to design the various bioreactors and the three-dimensional biofabrication methods to eliminate the diffusion constraints and to create a vascular network that will provide mass transfer with various techniques in the obtained structures. Within the scope of this thesis studies, it was aimed to produce and vascularize multilayered tissue conjugates by cell sheet engineering which is one of the three dimensional biofabrication methods to be used in various tissue damages. Primarily, MC3T3-E1 mouse pre-osteoblast cells were cultured separately in the temperature responsive culture dishes (Nunc Upcell) in the presence of growth medium and osteogenic medium seperately, and the effect of the culture conditions on cell sheet forming potentials were examined. At this stage, an intact, easily transferable, practically obtainable cell sheets composed of MC3T3-E1 cells and their extracellular matrix were prepared for the first time in the literature. The obtained cell sheets were analysed by different techniques such as microscopic and macroscopic imaging, quantification of the amount of collagen and sulfated glycosaminoglycan (sGAG), determination of the levels of expression of osteogenic genes, investigation of the mineralization process and the calcium content. The results obtained were revealed the superiority of the osteogenic environment on obtaining cell sheets. At the next stage of the thesis study, the thickness of the tissue was increased by laminating the cell sheets obtained by the application of osteogenic medium. At this stage, electrospun poly (L-lactic acid) (PLLA) membranes, which were produced within the scope of the thesis study, were used as a separate method to increase the thickness of the tissue constructs. The process of obtaining final structures was illuminated by various microscopic and macroscopic findings. Later, the in vitro activity of multilayered cell sheets constructed by laminating the cell sheets with PLLA membranes was investigated. In this context, the cell viability and the cell death were investigated by several microscopic and colorimetric methods. Here, the superior survival rate of multilayered structures formed by laminating two cell sheets onto one PLLA membrane and containing a total of 3 PLLA membranes and 4 cell sheets was demonstrated during culture. Thus, within the scope of this thesis study, three-dimensional tissue conjugate structures consisting of cell-biomaterial combinations, which can be transferred in a practical manner, are multi-layered and have a wet state of about 1 mm, are provided. In these structures, the characterization of alkaline phosphatase activity, collagen and sGAG synthesis was carried out during the culture period. In the final part of the thesis, endothelial cell culture studies were carried out to promote the formation of vascular networks in the resulting materials at later stages. For this purpose, surface channel decorated PGS elastomers were produced and characterized and studies were performed to determine the ideal culture conditions of HUVEC cells in parallel. The HUVEC cells cultured with endothelial media (EGM2) for 14 days in the channels of PGS elastomers were exhibited polygonal tubular organization and it was concluded that the angiogenesis was provided. The approaches developed within the scope of this thesis study was suggested to be able to restore craniofacial bone tissue regeneration and it was thought to be potential for tissue engineering applications involving dual or more microenvironment such as osteochondral, cornea or vessel.
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http://hdl.handle.net/11655/6549
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  • Biyomühendislik Bölümü Tez Koleksiyonu [83]
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