Hücre Tabakası Mühendisliğinde Kalınlık ve Vaskülerizasyon Problemlerine Yönelik Stratejilerin Geliştirilmesi
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Date
2019Author
Tevlek, Atakan
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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.