Investigation of cell morphology using scanning electron microscopy. 46

Figure 4.6 Morphology of human epithelial carcinoma HeLa cells cultured on Petri dish (A), smooth bare silicon (native oxide) surface (B), 1D periodic line silicon (native oxide) surface, and 2D periodic pillar silicon (native oxide) surface for 24hr imaged by scanning electron microscopy. Areas of lower cell density were selected to facilitate observation of individual cell shapes. The images of the cells shown in the selected micrographs are typical of cells throughout the culture.

The cells exhibited different shapes on Petri dish, patterned and non-patterned silicon surfaces with native oxide (Fig. 4.6). By way of parenthesis, in the preparation procedures of SEM samples with cells adhered on the surface, it was hard to dehydrate the samples without harming the PDMS substrate (the organic PDMS go through carbonization process and become carbon dust) so here we used silicon oxide

as substrate for observing the cell morphology on patterned and non-patterned surface.

Scanning electron micrographs of the cells on the 2D periodic pillar substrates showed that after 24hr seeding, HeLa cells attached with a spherical morphology around 20 μm diameter (Fig. 4.6D). Besides, comparing with the morphology and orientation of cells cultured on smooth substrates and on 2D periodic pillar, cells elongate and align on 1 μm grooves/ridges of 1D periodic line (Fig. 4.6C). The results similar with the cells which adhered on patterned PDMS surface.

In agreement with aligned nanofibers were sufficient to induce neurite elongation and outgrowth and greatly promoted cell migration [79], our results also found that when the external topography induce cells elongation, it is helpful for cell adhesion and proliferation. Moreover the bovine aortic endothelial cells on the nanotube surface facilitate a polarized distribution of contact with a lamellipodial protrusion in the front and the detachment of the tail in the back for accelerated cellular locomotion [80], it is clear that becoming more elongated and mobile form promote cells crawling toward each other and regulate cell to cell communication. On the contrary, similarly to the report of Dalby et al. [81], cells were inhibited from becoming fully flattened to the islands and even expressed a cell cycle arrest, but took on normal morphologies and were able to proliferate and become confluent on the flat controls.

Future research with these topographies should concentrate on casting them into approved polymers and degradable polymers and integrating with normal cell types to improve their usefulness as tissue-engineering scaffolds. Having the ability to control cell adhesion to a material may help in orthopedic and wound-healing procedures, where increased cell adhesion is required, and in applications such as heart valves and catheters, where reduced adhesion is required.

Chapter 5: Conclusions

With the use of an inexpensive and quick method to produce nanotopography, this study has shown a significant cell response in spreading, morphology, cytoskeleton, and proliferation of fibroblasts on the test surfaces. In these results, we found that cell elongation and alignment on grooves and ridges on 1D periodic line/space patterned surface. By contrast, when the cells cultured on the substrate of 2D periodic pillar pattern, they showed poor adhesion and spreading properties accompanying retardation of growth and proliferation. In order to clarify the effects of micro-patterned substrates on DNA content, we chose the substrate with the size of 1 μm periodic line/space pattern and 1 μm periodic pillar pattern for cell cycle analysis.

A slight increase of G1 phase population was observed when cells grew on 2D periodic pillar patterned substrate. And a decreased percentage of S phase as cells adhered to flat PDMS and 2D periodic pillars PDMS surface. The reduced S phase with cells which adhered to flat PDMS and 2D periodic pillars PDMS surface were further confirmed by measuring the BrdU incorporation. In contrast, accompanying with S phase increased, a significant decreased of G2/M phase population was observed when cells grew on substrate patterned with 1D periodic line. Moreover, as the focal adhesion proteins decreased, there was an increased expression of tumor suppressor p53, and especially with active MMP-9 released. In conclusion, substrate with 1D periodic line/space pattern promotes cells to appear significant response to elongation and alignment, while substrate with 2D periodic pillar pattern reduces cell adhesion, spreading, growth, proliferation and even promotes metastasis.

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