Date

2013

Author

Antmen, Ezgi

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Cell and substrate interactions are important in tissue engineering products especially on the behavior of the cells such as adhesion, migration, proliferation, and differentiation. These have been widely studied using substrates with different physical, chemical, and mechanical properties and form. In this study, elastin-like recombinamers (ELRs) were used blended with collagen or only collagen as the surface material. The ELR used in this study has Valine-Proline-Glycine-X-Glycine aminoacid sequences in its primary structure as the repeating sequence. Collagen was used because it is a biodegradable and biocompatible polymer. The films used for this study were designed to have micropillar covered surfaces with the pillar dimensions involving 4 regions covered with 8x8 µm² and 16x16 µm² pillars separated by either 4 or 8 µm gaps with a height of 5 µ. Saos-2 human osteosarcoma cell line was used to study cell behavior (proliferation, adhesion and conformational change) on these films. Films were observed after 1 day and 14 days of culture by using fluorescence microscopy and SEM and ALP activity of the cells on the micropillar covered surfaces were determined by alkaline phosphatase (ALP) assay after 14 days of culture. Deformation extent and fraction of deformed cells were measured. In terms of adhesion, there was no significant difference in between the different surfaces as a result of ELR presence whereas the micropillar dimensions affected the number of cells and cell numbers were the least at area B which has the lowest pillar size with highest gap (8x8 µm², gap 8 µm) where it was higher for unpatterned surfaces on the 1st day of the culture. Also there was no consistent difference between the three types of films considering the contribution of ELR to the stiffness of the surface. For the 14th day of the culture, it was observed that there is almost no cell on the micropillar surfaces but there were a number of cells on the middle of the films which has no micropillar probably due to the deformation in the micropillar geometries of the films stored in PBS. In terms of ALP concentration, results showed that the highest ALP activity on the films with the highest ELR ratio and higher ALP activity with the cells on the B area but the highest activity with cells on TCP. Lastly, in terms of conformational changes of the cells, it was observed that cells and their nuclei are deformed on the micropillar covered surfaces on all types of films. ELR and collagen content of the films did not seem to affect nuclei deformation. However, pillar placement and dimensions seem to be effective on the nuclei shapes that nuclei of the cells fall in the gaps when the gaps are large enough and when the gaps were smaller, nuclei were observed mostly on the pillar surfaces instead of the gaps. Moreover, there was a consistency between the nucleus and cell deformations in terms of shape. Also there was no cell deformation on the smooth, unpatterned surfaces and the reason of the deformations was the pillars on the film surfaces. In deformation quantification analysis, nuclei deformation frequency, nuclei circularity and nuclei perimeter showed no significant difference between the different films with changing ELR contents. However, considering the pillar dimensions, the highest deformation frequency was on the smallest pillar with largest gap, the least circularity meaning the highest deformation can be seen for the design B (8x8 µm², gap 8 µm) and the highest perimeter for the nuclei was for areas having the highest pillar gaps as area B (8x8 µm², gap 8 µm) and D (16x16 µm², gap 8 µm).

Subject Keywords

Cytology., Cell nuclei., Collagen., Tissue engineering.

URI

http://etd.lib.metu.edu.tr/upload/12616256/index.pdf
https://hdl.handle.net/11511/22918

Collections

Graduate School of Natural and Applied Sciences, Thesis