Date

2012

Author

Özçelik, Hayriye

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Micro and nanopatterned surfaces are powerful experimental platforms for investigating the mechanisms of cell adhesion, cell orientation, differentiation and they enable significant contributions to the fields of basic cell and stem cell biology, and tissue engineering. In this study, interaction between micro and nanopatterned polymeric surfaces and different cell types was investigated. Three types of micropillars were produced by photolithography (Type 1-3), while nanometer sized pillars were produced in the form of an array by electron beam lithography (EBL). Replica of silicon masters were made of polydimethylsiloxane (PDMS). Polymeric [P(L-D,L)LA and a P(L-D,L)LA:PLGA blend] replica were prepared by solvent casting of these on the PDMS template and used in in vitro studies. The final substrates were characterized by various microscopic methods such as light microscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to investigate deformation of the nucleus in response to the physical restrictions imposed by micropillars, Type 1 and Type 2 pillars were used. These substrates were covered with pillars with different interpillar distances. While Type 1 is covered with symmetrically (in X-Y directions) distributed pillars, Type 2 pillars were distributed asymmetrically and the inter-pillar distances were increased. Nuclei deformation of five cell v types, two cancer cell lines (MCF7 and Saos-2), one healthy bone cell (hFOB1.19), one stem cell (bone marrow origined mesemchymal stem cells, BMSCs) and one standard biomaterial test cell type, (L929) fibroblasts was examined by using fluorescence microscopy and SEM. The nuclei of Saos-2 and MCF7 cells were found to be deformed most drastically. Nucleus deformation and intactness of nuclear membrane was examined by Anti- Lamin A staining. The interaction of the cells with micropillars was visualized by labelling focal adhesion complexes (FAC). Wettabilities of patterned and smooth surfaces were determined. As the patterns become denser (closer micropillars, Type 1) the hydrophobicity increased. Similar to water droplets, the cells were mostly spread at the top of the Type 1 pillars. The number of cells spread on the substrate surface was much higher on Type 2 patterned films. In order to support these qualitative findings, nucleus deformation was quantified by image analysis. Frequency of nucleus deformation was determined as the ratio of deformed to the total number of nuclei (%). In order to quantify the intensity of nuclei deformation, their circularity was evaluated. In addition to nucleus deformation, alterations in the ratio of cell area-to-nucleus area in response to micropillars were determined by image analysis. The results indicated that cancerous cells were more deformable. The qualitative microscopic evaluation and the data obtained by quantification of the nucleus and cellular deformation were in good agreement. In addition, the findings were consistent with expectations which suggest that cancerous cells are “softer”. In the second part of the research the force applied by the cells on arrays of micropillars with high aspect ratios (Type 3 substrates) during tugging at the pillars was investigated. Micropillars were produced using P(L-D,L)LA as well as a 60:40 blend of P(L-D,L)LA with PLGA. The blend is a material with lower stiffness than P(L-D,L)LA. The mechanical properties of the two materials were determined by tensile testing of solvent cast films. Deformation of Type 3 micropillars by the cellular tugging force of Saos-2 and L929 was studied by fluorescence and SEM microscopy, both on stiff and softer substrates. Displacements of the centers nodes of the pillars were evaluated from SEM micrographs. On the stiff surface, the two cell types bent the pillars to the same extent. On the other softer substrate (blends), however, the maximum displacements observed with Saos-2 cells were higher than the ones caused on the stiffer substrate or the ones caused by L929 cells. It is reported that stiffness of the substrate can determine stem cell lineage commitment. In order to examine the effects of change of substrate stiffness on osteogenic differentiation of BMSCs, osteopontin (OPN) expression was determined microscopically. It was found that osteogenic differentiation is enhanced when BMSCs are cultured on P(L-D,L)LA Type 3 pillars. vi In the last part of research, arrays of nanopillars whose interpillar distances systematically varied to form different fields were examined in terms of adhesion and alignment in order to determine the differential adhesion of BMSCs and Saos-2 cells. The difference in their adhesion preference on nanopillar arrays was quantified by image analysis. It was observed that BMSCs and Saos-2 cells behaved in an opposite manner with respect to each other on the fields with the highest density of nanopillars. The BMSCs avoided the most densely nanopillar covered fields and occupied the pattern free regions. The Saos-2, on the other hand, occupied the most densely nanopillar covered fields and left the pattern free regions almost unpopulated. It was also found that both BMSCs and Saos-2 cells aligned in the direction of the shorter distance between the pillars. Both BMSCs and Saos-2 cells started to align on the pillars if the distance in any direction was >1.5 μm. To better understand the effects of chemical and physical cues, protein coating and material stiffness were tested as two additional parameters. After fibronectin coating, the surfaces of P(L-D,L)LA films with the highly dense pillar covered fields, which were avoided when uncoated, were highly populated by the BMSC. Similarly, decreasing the stiffness of a surface which was normally avoided by the BMSCs made it more acceptable for the cells to attach.

Subject Keywords

Cell adhesion., Cytology., Polymerization., Polymers.

URI

http://etd.lib.metu.edu.tr/upload/12614798/index.pdf
https://hdl.handle.net/11511/22069

Collections

Graduate School of Natural and Applied Sciences, Thesis