Effect of Nanotopography

In order to examine the effects of nanotopography on the behaviors of cell growth and adhesion, human foreskin fibroblasts were first tested on the surface samples of varying patterns and structural aspect ratios, while their surface conditions were all hydrophilic. Figures 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 and 4.8 show the results of microscopy.

High

Nanotopography

Fig. 4.1 SEM images of 3D sharp-tip nanostructures [28, 30]. Well-ordered silicon nanostruc-tures (a-c 'Nanopillar', d-f 'Nanograting') covered the surface of the sample (1 x 1 cm2) uniformly. While the pattern periodicity was maintained to be 230 nm and the structural tips were sharpened to be <10 nm in apex radius for all the samples, the structural heights were varied from 'Low' (a, d 50-100 nm), 'Mid' (b, e 200-300 nm) to 'High' (c, f 400-500 nm), in order to test the effects of nanotopographical three-dimensionalities on cell adhesions

Fig. 4.1 SEM images of 3D sharp-tip nanostructures [28, 30]. Well-ordered silicon nanostruc-tures (a-c 'Nanopillar', d-f 'Nanograting') covered the surface of the sample (1 x 1 cm2) uniformly. While the pattern periodicity was maintained to be 230 nm and the structural tips were sharpened to be <10 nm in apex radius for all the samples, the structural heights were varied from 'Low' (a, d 50-100 nm), 'Mid' (b, e 200-300 nm) to 'High' (c, f 400-500 nm), in order to test the effects of nanotopographical three-dimensionalities on cell adhesions

Fig. 4.2 Cell viability of human foreskin fibroblast on hydrophilic Nanopillar (a-d) and Nanograting (e-h) of varying aspect ratios. The fluorescence microscopy images were taken in 3 days for Smooth (a, e), Low (b, f), Mid (c, g), and High (d, h) samples. In the images, green color represents viable cells. In (f-h), the arrows (left-right arrow) indicate the direction of the grating patterns on the samples

Thy1 Yfp Mice Ganglia

Fig. 4.2 Cell viability of human foreskin fibroblast on hydrophilic Nanopillar (a-d) and Nanograting (e-h) of varying aspect ratios. The fluorescence microscopy images were taken in 3 days for Smooth (a, e), Low (b, f), Mid (c, g), and High (d, h) samples. In the images, green color represents viable cells. In (f-h), the arrows (left-right arrow) indicate the direction of the grating patterns on the samples

Figure 4.2 shows fluorescent microscope images for cell viability, taken after 3 days. On Nanopillar, smaller cell population and cell size were observed on Nanopillar-Mid (Fig. 4.2c) and Nanopillar-High (Fig. 4.2d) than on Smooth (Fig. 4.2a). Nanopillar-Low (Fig. 4.2b) does not show any significant difference from Smooth. On Nanograting, cells were mostly viable on all the samples. Compared to Smooth (Fig. 4.2e), cells were elongated to the grating direction

Smooth

High

Smooth

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Fig. 4.3 Cell adhesions of human foreskin fibroblast on hydrophilic Nanopillar (a-d) and Nanograting (e-h) [28]. The fluorescence microscope images of immunostaining (nuclei: blue, pFAK: red, in colors) were taken in 3 days for Smooth (a, e), Low (b, f), Mid (c, g), and High (d, h) samples. Each arrow (left-right arrow) in (f-h) represents the direction of grating patterns on the samples

Smooth

Nanoplllar-Low

Nanoplllar-MId

Nan op I liar-High

Nanoplllar-MId

Nan op I liar-High

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Fig. 4.4 Cell morphology of human foreskin fibroblast on hydrophilic Nanopillar topography [28, 30]. The SEM images (top view) were taken in 3 days (a-d) and 1 week (e-h) for Smooth (a, e), Low (b, f), Mid (c, g), and High (d, h) samples. Each inset in Nanopillar (b-d, f-h) represents the orientation of the pillar array on each sample. Detached cells were often observed on Nanopillar-Mid and Nanopillar-High during the sample preparation for SEM, as indicated by arrows (right arrow) in (c, h)

significantly, and the elongation was more pronounced on taller gratings. As reported elsewhere [28], the quantitative measurement of cellular metabolic activity by the alamarBlue™ assay also agreed with the qualitative results of Fig. 4.2.

Figure 4.3 shows the fluorescent microscope images of the immunostaining of phosphorylated focal adhesion kinase (pFAK) taken after 3 days [28]. Human foreskin fibroblasts on Smooth (Fig. 4.3a, e) and Nanopillar (Fig. 4.3b-d)

Smooth Nanograting-Low N a nog rating-Mid N a nog rating-High

Smooth Nanograting-Low N a nog rating-Mid N a nog rating-High

Effects Energy Conservation

Fig. 4.5 Cell morphology of human foreskin fibroblast on hydrophilic Nanograting [28, 30]. The SEM images (top view) were taken in 3 days (a-d) and 1 week (e-h) for Smooth (a, e), Low (b, f), Mid (c, g), and High (d, h) samples. Each inset in Nanograting (b-d, f-h) represents the orientation of the grating array on each sample. Detached cells were often observed on the tall Nanograting during the sample preparation for SEM, as indicated by an arrow (right arrow) in (c)

Fig. 4.5 Cell morphology of human foreskin fibroblast on hydrophilic Nanograting [28, 30]. The SEM images (top view) were taken in 3 days (a-d) and 1 week (e-h) for Smooth (a, e), Low (b, f), Mid (c, g), and High (d, h) samples. Each inset in Nanograting (b-d, f-h) represents the orientation of the grating array on each sample. Detached cells were often observed on the tall Nanograting during the sample preparation for SEM, as indicated by an arrow (right arrow) in (c)

exhibited punctuated adhesion complexes, whereas cells on Nanograting (Fig. 4.6f-h) demonstrated dash-like adhesion complexes clearly aligned to the grating direction. This result suggests that the 3D nanotopographies also induce distinct intracellular processes that may modify the cell's behaviors.

Figure 4.4 shows the SEM images of cell morphology on Nanopillar, taken after 3 days and 1 week [28, 30]. On day 3, cells on Smooth (Fig. 4.4a) exhibited well-spread and flattened cell morphology, which is typical for 2D planar substrates. In contrast, Nanopillar gave rise to significantly different cell morphologies. While being well-spread and flattened (similar to that for Smooth), cell morphology on Nanopillar-Low was elongated (Fig. 4.4b). On Nanopillar-Mid (Fig. 4.4c), an enhanced elongation with a slender morphology was observed, and cell size was significantly smaller. On Nanopillar-High (Fig. 4.4d), fibroblasts were not spread well and exhibited a swollen morphology with much smaller cell size, which is indicative of a poor adhesion and an apoptosis-like process. In 1 week, cell population, spreading, and size increased on all the samples. Cells on Smooth (Fig. 4.4e) and Nanopillar-Low (Fig. 4.4f) maintained the flattened cell morphology with more spreading and larger size. The elongated cell morphology was maintained on Nanopillar-Low (Fig. 4.4f). Compared to day 3, cells on Nanopillar-Mid (Fig. 4.4g) and Nanopillar-High (Fig. 4.4h) after one week were more spread, flattened, and enlarged, with an enhanced elongation and slender morphology. Also, compared to those on Smooth (Fig. 4.4e) and Nanopillar-Low (Fig. 4.4f), cells exhibited a relatively swollen morphology with a smaller cell size, especially on Nanopillar-High (Fig. 4.4h).

Figure 4.5 shows SEM images of cell morphology on Nanograting, taken after 3 days and 1 week [28, 30]. On day 3, although the cell spreading on Nanograting (Fig. 4.5b-d) was not as good as that on Smooth (Fig. 4.5a), fibroblasts spread

Low magnification image High magnification image

Foreskin Elongation

Fig. 4.6 SEM images (tilted view) of human foreskin fibroblast cells cultured on hydrophilic Nanopillar samples: Nanopillar-Low (a, b 3 days), Nanopillar-Mid (c, d 3 days), and Nanopillar-High (e, f 7 days) [30]. The high-magnification image (b) is from the sample cleaved through the silicon substrate and cell to show the cross section. While already discussed with the top-view images in Fig. 4.3, the tilted-view images more clearly show the detachment of cell bodies from the Nanopillar-Mid and Nanopillar-High. Cells detached during the sample preparation for SEM

Fig. 4.6 SEM images (tilted view) of human foreskin fibroblast cells cultured on hydrophilic Nanopillar samples: Nanopillar-Low (a, b 3 days), Nanopillar-Mid (c, d 3 days), and Nanopillar-High (e, f 7 days) [30]. The high-magnification image (b) is from the sample cleaved through the silicon substrate and cell to show the cross section. While already discussed with the top-view images in Fig. 4.3, the tilted-view images more clearly show the detachment of cell bodies from the Nanopillar-Mid and Nanopillar-High. Cells detached during the sample preparation for SEM

relatively well on Nanograting compared to Nanopillars (Fig. 4.4b-d). Fibroblasts on Nanograting were more significantly elongated than on Nanopillar, showing clear alignment along the grating direction. The elongated cell morphology and alignment with the grating direction were more pronounced for taller gratings. Relatively swollen cell morphology was often observed on Nanograting-Mid (Fig. 4.5c) and Nanograting-High (Fig. 4.5d). In 1 week, cell population, spreading, and size increased on all samples. Cells on Nanograting, compared to

Low magnification image High magnification image

Retinal Infarct Oct Image

Fig. 4.7 SEM images (tilted view) of human foreskin fibroblast cells cultured on hydrophilic Nanograting samples: Nanograting-Low (a, b 3 days), Nanograting-Mid (c, d 3 days), and Nanograting-High (e, f 3 days) [30]. The high-magnification images on the right column (b, d, f) are from the sample cleaved through the silicon substrate and cell to show the cross sections. While already discussed with the top-view images in Fig. 4.5, the tilted-view images more clearly show the detachment of cell bodies from the Nanograting-Mid and Nanograting-High. Cells detached during the sample preparation for SEM

Fig. 4.7 SEM images (tilted view) of human foreskin fibroblast cells cultured on hydrophilic Nanograting samples: Nanograting-Low (a, b 3 days), Nanograting-Mid (c, d 3 days), and Nanograting-High (e, f 3 days) [30]. The high-magnification images on the right column (b, d, f) are from the sample cleaved through the silicon substrate and cell to show the cross sections. While already discussed with the top-view images in Fig. 4.5, the tilted-view images more clearly show the detachment of cell bodies from the Nanograting-Mid and Nanograting-High. Cells detached during the sample preparation for SEM

3 days, spread better, with flattened morphology and enlarged size, while the cell elongation and clear alignment to the grating direction was maintained. As reported elsewhere [28, 30], more quantitative data of the cell morphology obtained by image analyses further support the differences between Nanopillar and Nanograting patterns of the varying aspect ratios.

Smooth Low Mid High

Smooth Low Mid High

Fig. 4.8 Optical images (top view) of human foreskin fibroblast cells cultured for 3 weeks on the hydrophilic samples (a and e Smooth samples used as controls for Nanopillar and Nanograting, respectively; b-d Nanopillar-Low, -Mid, and -High, respectively; f-h Nanograting-Low, -Mid, and -High, respectively) [30]. Each image shows the entire surface of the sample of 1 x 1 cm2. Although a cell sheet was formed on all the nanostructure samples after 3 weeks, the cell sheets formed on the tall nanostructures (c, d, h), as indicated by the arrows (right arrow), were detached from the surface and rolled up or removed away in the sample preparation for the images. In (f-h), the arrows (left-right arrow) indicate the grating directions on the samples

Fig. 4.8 Optical images (top view) of human foreskin fibroblast cells cultured for 3 weeks on the hydrophilic samples (a and e Smooth samples used as controls for Nanopillar and Nanograting, respectively; b-d Nanopillar-Low, -Mid, and -High, respectively; f-h Nanograting-Low, -Mid, and -High, respectively) [30]. Each image shows the entire surface of the sample of 1 x 1 cm2. Although a cell sheet was formed on all the nanostructure samples after 3 weeks, the cell sheets formed on the tall nanostructures (c, d, h), as indicated by the arrows (right arrow), were detached from the surface and rolled up or removed away in the sample preparation for the images. In (f-h), the arrows (left-right arrow) indicate the grating directions on the samples

To examine the physical contact between the cells and the nanostructured surfaces, their interfaces were inspected by the SEM, as shown in Figs. 4.6 and 4.7 [30]. Figure 4.6 shows the SEM images of human foreskin fibroblasts on Nanopillar samples. On Nanopillar-Low (Fig. 4.6a, b), it was often shown (Fig. 4.6b) that the short Nanopillar structures were projected through the well-spread, flattened cell body, suggesting that cells accommodated the mild topography by an endocytosis-like process, which would result in a strong cell adherence to the surface. On taller Nanopillar-Mid and Nanopillar-High (Fig. 4.6c-f), as noticed in the top-view images (Fig. 4.4c, h), detached cell bodies were frequently observed after the samples were prepared for SEM. The ends of the detached cells shown in the magnified images (Fig. 4.6d, f) further suggest that the cell bodies were only supported by the Nanopillars' sharp tips, not entering into the valleys between the pillars (i.e., no endocytosis-like process). It also suggests that cell-substrate interactions occurred at the Nanopillars' sharp tips, and that cells' locomotion (e.g., spreading) continued while levitated by the tall, sharp-tip Nanopillar structures. The minimized adhesive area of cells to the surface by the levitation on the sharp tips would result in the weaker cell adherence to the surface, causing the cell detachment during the sample preparations for the SEM images.

Figure 4.7 shows the tilted-view SEM images of human foreskin fibroblasts on Nanograting samples. On Nanograting-Low (Fig. 4.7a, b), unlike on Nano-pillar-Low (Fig. 4.6b), nanogratings did not project through the cells (Fig. 4.7b)

(i.e., no endocytosis-like process). As seen in the top-view images (Fig. 4.5c), detached cell bodies were frequently observed on tall Nanograting samples (Fig. 4.7c, e). Although the clear cell levitation was not observed on Nano-grating-Low (Fig. 4.7b), the magnified tilted-view images of cleaved samples on the right column clearly show that on tall Nanograting samples (Fig. 4.7d, f) cell bodies were only supported by the sharp-tip ridges and did not go down into the valleys. Consistent with the case of Nanopillar samples, it is believed that the minimized cell-solid contact area by tall, sharp-tip Nanograting structures resulted in the self-detachment of the cell bodies during the sample preparation for the SEM images.

Human foreskin fibroblasts were cultured for an extended period (past the 7 days) to see whether they could proliferate enough to form a robust sheet of cell layers and gain good adhesion on the nanostructured substrates. Figure 4.8 shows the optical images of the fibroblasts cultured for 3 weeks [30]. Although the fibroblasts proliferated slower on the tall nanostructures in the beginning, a sheet of cells eventually formed on the nanostructured surfaces after 3 weeks. During the sample preparation for the image (fixation and drying), the cell sheets on Nanopillar-Mid (Fig. 4.8c) and Nanopillar-High (Fig. 4.8d) detached from one side and their edges curled up, while the cell sheets on Smooth (Fig. 4.8a) and Nanopillar-Low (Fig. 4.8b) remained attached. A part of a cell sheet on Nano-grating also detached and curled away from the surface (parallel to the grating direction) in the course of the sample preparation. The detachment was more pronounced on taller Nanogratings (Fig. 4.8f-h), while a whole cell sheet on Smooth remained attached (Fig. 4.8e). In simple mechanical peel tests using tweezers and also jet shear flows, before drying (in a cell culture medium) as well as after drying, the cell sheets formed on the tall nanostructures (Nanopillar-Mid, Nanopillar-High, and Nanograting-High) were also found easier to peel off than those on the other surfaces. This result suggests that the cells on tall nanostructures, even when they formed a 3D sheet, were levitated on the sharp tips so that the cell-solid real contact area was minimized, resulting in a weak overall adherence. It should also be noted that, although less evident compared with the 1-week-old cell bodies, the formed cell sheets on Nanograting still showed the cells' directional elongation to the underlying grating structures, suggesting that the alignment was maintained over time [30].

Renewable Energy Eco Friendly

Renewable Energy Eco Friendly

Renewable energy is energy that is generated from sunlight, rain, tides, geothermal heat and wind. These sources are naturally and constantly replenished, which is why they are deemed as renewable.

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